WO2025240909A1

WO2025240909A1 - Hybridization-based capture and enrichment of target nucleic acid sequences

Info

Publication number: WO2025240909A1
Application number: PCT/US2025/029840
Authority: WO
Inventors: Xiaodong QI; Junhua Zhao; Andrew Boddicker; Kelly WISEMAN; Michael Previte; Hui Zhen MAH; Shawn Levy; Kyle METCALFE
Original assignee: Element Biosciences Inc
Current assignee: Element Biosciences Inc
Priority date: 2024-05-17
Filing date: 2025-05-16
Publication date: 2025-11-20
Anticipated expiration: 2026-11-17

Abstract

The present disclosure provides methods for conducting hybridization-based enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences. In some embodiments, library molecules having target sequences can be enriched from a mixture of target and non-target library molecules by employing target-specific baits/probes in a hybridization-based workflow. In some embodiments, the methods comprise preparing a plurality of closed circle library bait complexes using linear library molecules or covalently closed circular library molecules. In some embodiments, the closed circle library bait complexes can be prepared by conducting on-support hybridization to generate immobilized closed circle library bait complexes, or by conducting in-solution hybridization followed by immobilization.

Description

HYBRIDIZATION-BASED CAPTURE AND ENRICHMENT

OF TARGET NUCLEIC ACID SEQUENCES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and benefit of, U.S. Provisional Application No. 63/649,212 filed on May 17, 2024, U.S. Provisional Application No. 63/682,025, filed on August 12, 2024, U.S. Provisional Application No. 63/719,980, filed on November 13, 2024, U.S. Provisional Application No. 63/730,903, filed on December 11, 2024, U.S. Provisional Application No. 63/780,736, filed on March 31, 2025 and U.S. Provisional Application No. 63/789,063, filed on April 15, 2025, the contents of each of which are incorporated by reference in their entirety herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (ELEM_035_007WO_SeqList_ST26.xml; Size: 364,880 bytes; and Date of Creation: May 15, 2025) are herein incorporated by reference in their entireties.

BACKGROUND

[0003] Massively parallel sequencing methods have applications in biomedical research and healthcare setting as they allow for analyzing large quantities of nucleic acids with different sequences from biological samples. However, for sequencing samples with mixtures of sequences, sequences that are present at low frequency in the mixture can be challenging to sequence. There thus exists a need in the art for improved methods of enriching target sequences from samples comprising mixtures of sequences, in a manner compatible with downstream sequencing workflows.

SUMMARY

[0004] The disclosure provides methods for enriching target polynucleotides from a mixture of polynucleotides comprising target and non-target polynucleotides, the method comprising: (a) providing a plurality of closed circle library bait complexes immobilized to a capture support, wherein individual closed circle library bait complexes comprise: (i) a covalently closed circular library molecule comprising a polynucleotide comprising a target sequence and at least one universal adaptor sequence, and (ii) a target-specific bait/probe hybridized to at least a portion of the target sequence; (b) conducting a rolling circle amplification reaction using the target-specific bait/probe to initiate amplification, thereby generating a plurality of immobilized concatemer template molecules; and (c) sequencing at least a portion of the plurality of immobilized concatemer template molecules.

[0005] The disclosure provides methods for enriching target polynucleotides from a mixture of polynucleotides comprising target and non-target polynucleotides, comprising (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) forming a first plurality of closed circle library bait complexes immobilized to the capture support, wherein individual closed circle library bait complexes in the first plurality comprise (i) a covalently closed circular library molecule comprising a polynucleotide comprising a target sequence and at least one universal adaptor sequence, and (ii) a target-specific bait/probe hybridized to the target sequence, wherein an individual target-specific bait/probe comprises an (i) oligonucleotide comprising a targetspecific sequence that can selectively hybridize to at least a portion of the target sequence of the covalently closed circular library molecule, (ii) an affinity moiety at the 5’ end of the target-specific oligonucleotide, and (iii) an extendible 3’ end, and wherein the forming of step (b) comprises binding the affinity moieties of the individual target-specific baits/probes of the first plurality to the embedded receptor moieties of the capture support, thereby generating a first plurality of circle-bait complexes immobilized to the capture support; (c) conducting a rolling circle amplification reaction using the extendible 3’ ends of the targetspecific bait/probes to initiate the rolling circle amplification reaction, thereby generating a plurality of immobilized concatemer template molecules; and (d) sequencing at least a portion of individual immobilized concatemer template molecules.

[0006] In some embodiments of the methods of the disclosure, individual receptor moieties comprise streptavidin or avidin. In some embodiments, the affinity moiety comprises biotin, desthiobiotin or iminobiotin.

[0007] In some embodiments of the methods of the disclosure, a melting temperature of the target-specific bait/probe hybridized to the target sequence is between about 80 °C and about 85 °C, between about 85 °C and about 90 °C, between about 90 °C and about 95 °C, or between about 95 °C and about 98 °C. In some embodiments, selective hybridization of the target-specific bait/probe to the at least a portion of the target sequence is conducted at a temperature that is: between about 5 °C and about 10 °C less than the Tm of the targetspecific bait/probe hybridized to the target sequence, between about 10 °C and about 20 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence, between about 20 °C and about 30 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence, between about 30 °C and about 40 °C less than the Tm of the targetspecific bait/probe hybridized to the target sequence, or between about 40 °C and about 50 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence.

[0008] In some embodiments of the methods of the disclosure, the first plurality of closed circle library bait complexes are immobilized to the capture support at pre-determined locations, thereby forming a pre-determined pattern of immobilized circle-bait complexes. In some embodiments, the first plurality of closed circle library bait complexes are immobilized to the capture support at random and non-predetermined locations. In some embodiments, the density of the first plurality of closed circle library bait complexes immobilized to the support is between 10² and 10¹⁵ closed circle library bait complexes per mm². In some embodiments, the first plurality of closed circle library bait complexes comprises target-specific bait/probes having between 2 and 10,000 different target-specific sequences. In some embodiments, the density of the plurality of immobilized concatemer template molecules is between about 10² and about 10¹⁵ immobilized concatemer template molecules per mm².

[0009] In some embodiments of the methods of the disclosure, the plurality of immobilized concatemer template molecules comprises between 2 and 10,000 different target-specific sequences. In some embodiments, at least some of the immobilized concatemer template molecules comprise nearest neighbor concatemer molecules that touch each other and/or overlap each other when viewed from any angle of the capture support including above, below or side views of the capture support. In some embodiments, the capture support comprises 200 million - 1.5 billion immobilized concatemer template molecules immobilized thereto.

[0010] In some embodiments of the methods of the disclosure, the rolling circle amplification reaction comprises contacting the first plurality of closed circle library bait complexes with a rolling circle amplification reagent comprising a plurality of stranddisplacing polymerases, and a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and dUTP, and wherein the rolling circle amplification reaction generates a plurality of immobilized concatemer template molecules, individual immobilized concatemer template molecules comprising at least one uracil nucleobase.

[0011] In some embodiments of the methods of the disclosure, the rolling circle amplification reagent comprises a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball.

[0012] In some embodiments of the methods of the disclosure, the capture support comprises a plurality of pinning primers immobilized to the support, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide, wherein the affinity moiety of individual pinning primers binds an embedded receptor moiety of the capture support. In some embodiments, individual pining primers comprise a blocking group at the 3’ end of the oligonucleotide, wherein the blocking group inhibits polymerase-catalyzed extension of the 3’ end of the pinning primer. In some embodiments, the density of the plurality of pinning primers is between 10² and 10¹⁵ pinning primers per mm².

[0013] In some embodiments of the methods of the disclosure, individual immobilized concatemer template molecules comprise a universal sequence for binding a pinning primer. In some embodiments, at least a portion of individual pinning primers hybridize to a portion of an immobilized concatemer template molecule to pin down a portion of the immobilized concatemer template molecules to the capture support.

[0014] In some embodiments of the methods of the disclosure, the methods further comprise (i) conducting a re-seeding reaction comprising forming a second plurality of closed circle library bait complexes immobilized to the capture support by binding the affinity moiety of individual target-specific baits/probes of a second plurality of target-specific baits/probes to receptor moieties of the capture support, thereby generating a second plurality of closed circle library bait complexes immobilized to the capture support, wherein the reseeding reaction is conducted after the rolling circle amplification of step (c) and prior to the sequencing of step (d); (ii) conducting a second rolling circle amplification reaction using the extendible 3’ ends of the second plurality of target-specific bait/probes to initiate the second rolling circle amplification reaction, thereby generating a second plurality of immobilized concatemer template molecules; and (iii) sequencing at least a portion of individual immobilized concatemer template molecules from the first and second pluralities of concatemer template molecules.

[0015] In some embodiments of the methods of the disclosure, the sequencing comprises sequencing the first and second pluralities of immobilized concatemer template molecules essentially simultaneously. In some embodiments, the sequencing comprises sequencing the first plurality of immobilized concatemer template molecules and then sequencing the second plurality of immobilized concatemer template molecules.

[0016] In some embodiments of the methods of the disclosure, the methods comprise (i) conducting a re-seeding reaction comprising forming a second plurality of closed circle library bait complexes immobilized to the capture support by binding the affinity moiety of individual target-specific baits/probes of a second plurality of target-specific baits/probes to receptor moieties of the capture support thereby generating a second plurality of closed circle library bait complexes immobilized to the capture support, wherein the re-seeding reaction is conducted after sequencing the first plurality of immobilized concatemer template molecules of step (d); (ii) conducting a second rolling circle amplification reaction using the extendible 3’ ends of the second plurality of target-specific bait/probes to initiate the second rolling circle amplification reaction, thereby generating a second plurality of immobilized concatemer template molecules; and (iii) sequencing at least a portion of individual immobilized concatemer template molecules from the second plurality of immobilized concatemer template molecules.

[0017] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) forming a plurality of closed circle library bait complexes by contacting a plurality of target-specific baits/probes to a plurality of covalently closed circular library molecules , wherein the plurality of covalently closed circular library molecules comprises a mixture of covalently closed circular library molecules comprising target sequences and covalently closed circular library molecules comprising non-target sequences, wherein the contacting is conducted in-solution under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual covalently closed circular library molecules to corresponding target-specific baits/probes, thereby generating a plurality of closed circle library bait complexes that are enriched for target sequences; (c) contacting the capture support with the plurality of closed circle library bait complexes, thereby generating a plurality of immobilized closed circle library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of an individual target-specific baits/probe to a receptor moiety of the capture support; and (d) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target-specific baits/probes, thereby generating the plurality of immobilized concatemer template molecules.

[0018] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating, (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating, and (iii) a plurality of target-specific baits/probes immobilized to the capture support; (b) forming a plurality of immobilized closed circle library bait complexes by contacting a plurality of covalently closed circular library molecules to the plurality of target-specific baits/probes, wherein the plurality of covalently closed circular library molecules comprises a mixture of covalently closed circular library molecules comprising target sequences and covalently closed circular library molecules comprising non-target sequences, wherein the contacting is conducted on the capture support under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual covalently closed circular library molecules to corresponding target-specific baits/probes, thereby generating the plurality of immobilized closed circle library bait complexes that are enriched for polynucleotides having target sequences; and (c) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

[0019] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) forming a plurality of library bait complexes by contacting a plurality of target-specific baits/probes to a plurality of linear library molecules, wherein the plurality of linear library molecules comprises a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted in-solution under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of library bait complexes that are enriched for polynucleotides having target sequences; (c) contacting the capture support with the plurality of library bait complexes, thereby generating a plurality of immobilized library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes to a receptor moiety; (d) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of singlestranded top strand circularization oligonucleotides under a condition suitable for hybridizing the ends of individual linear library molecules with individual single-stranded top strand circularization oligonucleotides to form individual open circle library complexes each having one nick, and enzymatically ligating the one nick, thereby generating a plurality of covalently closed circular library molecules each being hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and (e) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

[0020] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) forming a plurality of library bait complexes by contacting a plurality of target-specific baits/probes to a plurality of linear library molecules, wherein the plurality of linear library molecules comprises a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted in-solution under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of library bait complexes that are enriched for polynucleotides having target sequences; (c) contacting the capture support with the plurality of library bait complexes, thereby generating a plurality of immobilized library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes to a receptor moiety; (d) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of doublestranded top strand circularization oligonucleotides, individual double-stranded top strand circularization oligonucleotides comprising a long strand and a short strand, wherein the long and short strands are hybridized together to form the double-stranded molecule having a double-stranded region and two flanking single-stranded regions, wherein the contacting is conducted under a condition suitable for hybridizing one end of a linear library molecule to one end of the long splint strand and hybridizing the other end of the linear library molecule to the other end of the long splint strand, thereby forming an open circle library bait complex having two nicks, and enzymatically ligating the two nicks thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and (e) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

[0021] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating, (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating, and (iii) a plurality of target-specific baits/probes immobilized to the capture support; (b) forming a plurality of immobilized library bait complexes by contacting a plurality of linear library molecules to the plurality of targetspecific baits/probes, wherein the plurality of linear library molecules comprise a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted on the capture support under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences; (c) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of single-stranded top strand circularization oligonucleotides under a condition suitable for hybridizing the ends of individual linear library molecules with individual singlestranded top strand circularization oligonucleotides to form individual open circle library splint complexes, individual open circle library splint complexes having one nick, and enzymatically ligating the one nick thereby generating a plurality of covalently closed circular library molecules hybridized to immobilized target-specific bait/probes, thereby forming a plurality of immobilized closed circle library bait complexes; and (d) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules. [0022] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating, (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating, and (iii) a plurality of target-specific baits/probes immobilized to the capture support; (b) forming a plurality of immobilized library bait complexes by contacting a plurality of linear library molecules to the plurality of targetspecific baits/probes, wherein the plurality of linear library molecules comprise a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted on the capture support under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences; (c) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of double-stranded top strand circularization oligonucleotides, individual doublestranded top strand circularization oligonucleotides comprising a long strand and a short strand, wherein the long and short strands are hybridized together to form the double-stranded molecule having a double-stranded region and two flanking single-stranded regions, wherein the contacting is conducted under a condition suitable for hybridizing one end of a linear library molecule to one end of the long strand and hybridizing the other end of the linear library molecule to the other end of the long strand, thereby forming an open circle library bait complex having two nicks, and enzymatically ligating the two nicks thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized targetspecific bait/probe thereby forming a plurality of immobilized closed circle library bait complexes; and (d) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules. [0023] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) contacting in-solution a plurality of linear library molecules with a plurality of top strand circularization oligonucleotides, wherein individual linear library molecules comprise an insert region having a target sequence or a non-target sequence, wherein the insert region is flanked on either side by one or more universal adaptor sequences, wherein the one or more universal adaptor sequences at one side of the insert region are not the same as the one or more universal adaptor sequences at the other side of the insert region, wherein individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence, a bridging sequence, and a terminal 3’ extendible moiety, wherein the anchor sequence can hybridize to one or more universal adaptor sequences at one side of the insert region, wherein the bridging sequence can hybridize to one or more universal adaptor sequences at the other end of the same linear library molecule; (c) contacting in-solution the plurality of linear library molecules with a plurality of target-specific baits/probes, wherein the contacting of step (c) is conducted under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of captured linear library bait complexes that are enriched for polynucleotides having target sequences; (d) forming a plurality of open circle library bait complexes by hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the one side of the insert region, and hybridizing the bridging sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the other end of the insert region of an individual linear library molecule, thereby forming a plurality of open circle library bait complexes having one nick; (e) contacting the capture support with the plurality of open circle library bait complexes, thereby generating a plurality of immobilized open circle library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of an individual target-specific bait/probe to a receptor moiety; (f) contacting the capture support with a ligation reagent, thereby ligating the one nick of individual immobilized open circle library bait complexes, thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific baits/probes, thereby forming a plurality of immobilized closed circle library bait complexes; and (g) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the immobilized target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

[0024] In some embodiments of the methods of the disclosure, the methods further comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) contacting in-solution a plurality of linear library molecules with a plurality of top strand circularization oligonucleotides, wherein individual linear library molecules comprise an insert region having a target sequence or a non-target sequence, wherein the insert region is flanked on either side by one or more universal adaptor sequences, wherein the one or more universal adaptor sequences at one side of the insert region are not the same as the one or more universal adaptor sequences at the other side of the insert region, wherein individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence, an additional sequence, a bridging sequence, and a 3’ non-extendible blocking group, wherein the bridging sequence can hybridize to the one or more universal adaptor sequences at the one side of the insert region, wherein the bridging sequence can hybridize to the one or more universal adaptor sequences at the other side of the insert region, wherein the additional sequence of individual top strand circularization oligonucleotides does not hybridize to a portion of individual linear library molecules; (c) contacting in-solution the plurality of linear library molecules with a plurality of target-specific baits/probes wherein the contacting of step (c) is conducted under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of captured linear library bait complexes that are enriched for polynucleotides having target sequences; (d) forming a plurality of open circle library bait complexes by hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the one side of the insert region, and hybridizing the bridging sequence to at least a portion of the one or more universal adaptor sequences at the other end of the insert region of an individual linear library molecule, thereby forming the plurality of individual open circle library bait complexes having a gap; (e) contacting the capture support with the plurality of open circle library bait complexes, thereby generating a plurality of immobilized open circle library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of an individual target-specific bait/probe to a receptor moiety; (f) contacting the plurality of immobilized open circle library bait complexes on the capture support with gap fill-in reagent for conducting a polymerase-catalyzed gap fill-in reaction, thereby generating a plurality of immobilized open circle library bait complexes having a nick, and contacting the plurality of immobilized open circle library bait complexes with a ligation reagent for ligating the nicks, thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and (g) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the immobilized target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

[0025] In some embodiments of the methods of the disclosure, the methods comprise (a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) contacting in-solution a plurality of linear library molecules with a plurality of top strand circularization oligonucleotides, wherein individual linear library molecules comprise an insert region having a target sequence or a non-target sequence, wherein the insert region is flanked on either side by one or more universal adaptor sequences, wherein the one or more universal adaptor sequences at one side of the insert region are not the same as the one or more universal adaptor sequences at the other side of the insert region, wherein individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence, a bridging sequence, and a terminal 3’ non-extendible blocking group, wherein the anchor sequence can hybridize to the one or more universal adaptor sequences at one side of the insert region, wherein the bridging sequence can hybridize to the one or more universal adaptor sequences at the other side of the insert region, and wherein a 5’ portion of individual linear library molecules forms a 5’ overhang flap structure upon hybridization with the bridging sequence which is cleavable by a 5’ flap endonuclease, thereby generating a newly cleaved 5’ end having a phosphate group; (c) contacting in-solution the plurality of linear library molecules with a plurality of target-specific baits/probes, wherein the contacting of step (c) is conducted under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of captured linear library bait complexes that are enriched for polynucleotides having target sequences; (d) forming a plurality of open circle library bait complexes comprising a 5’ overhang flap structure by hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the one side of the insert region, and hybridizing the bridging sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the other end of the insert region of the individual linear library molecule, thereby forming a plurality of open circle library bait complexes having a 5’ overhang flap structure; (e) contacting the capture support with the plurality of open circle library bait complexes, thereby generating a plurality of immobilized open circle library bait complexes with 5’ overhang flap structures, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes to a receptor moiety, wherein the plurality of immobilized open circle library bait complexes are enriched for polynucleotides carrying target sequences; (f) contacting the capture support with a flap cleavage reagent under a condition suitable for cleaving the 5’ overhang flap structures, thereby forming a plurality of cleavage products, wherein individual cleavage products comprise an open circle library molecule with a newly cleaved 5’ end and a noncleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same library molecule form an open circle library molecule having a nick while being hybridized to the top strand circularization oligonucleotide, and ligating the nick to generate a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and (g) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the immobilized target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

[0026] In some embodiments of the methods of the disclosure, the sequencing comprises (a) contacting a first plurality of polymerases to (i) the plurality of immobilized concatemer template molecules and (ii) a plurality of nucleic acid primers, wherein the contacting is conducted under a condition suitable to bind the first plurality of polymerases to the plurality of immobilized concatemer template molecules and the plurality of nucleic acid primers, thereby forming a first plurality of complexed polymerases each comprising a polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises an immobilized concatemer template molecule hybridized to a nucleic acid primer; (b) contacting the first plurality of complexed polymerases with a plurality of multivalent molecules to form a plurality of multivalent-binding complexes, wherein individual multivalent molecules in the plurality comprise a core attached to multiple nucleotide arms and individual nucleotide arms are attached to a nucleotide moiety, wherein the contacting is conducted under a condition suitable for binding complementary nucleotide moieties of the multivalent molecules to at least two of the first plurality of complexed polymerases thereby forming a plurality of multivalent-binding complexes, and the condition is suitable for inhibiting incorporation of the complementary nucleotide moieties into the nucleic acid primers of the plurality of multivalent-binding complexes; (c) detecting the plurality of multivalent-binding complexes; and (d) identifying the nucleobase of the complementary nucleotide moieties in the plurality of multivalent-binding complexes, thereby determining the sequence of the nucleic acid template molecules. In some embodiments, the sequencing comprises (e) dissociating the plurality of multivalent-binding complexes by removing the first plurality of polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; (f) contacting the plurality of the nucleic acid duplexes retained at step (e) with a second plurality of a polymerases under a condition suitable for binding the second plurality of polymerases to the plurality of the nucleic acid duplexes, thereby forming a second plurality of complexed polymerases, individual complexed polymerases comprising a polymerase bound to a nucleic acid duplex; and (g) contacting the second plurality of second polymerases with a plurality of nucleotides, wherein the contacting is conducted under a condition suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the complexed polymerases, thereby forming a plurality of nucleotide-binding complexes, and the condition is suitable for promoting nucleotide incorporation of the bound complementary nucleotides into the nucleic acid primers of the nucleotide-binding complexes. In some embodiments, the sequencing further comprises (h) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases. In some embodiments, the sequencing further comprises (i) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases; and (j) identifying the nucleobases of the complementary nucleotides which are incorporated into the primers of the nucleotide-complexed polymerases. In some embodiments, the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases are not detected or identified.

[0027] In some embodiments of the methods of the disclosure, the contacting the first plurality of complexed polymerases with the plurality of multivalent molecules of step (b) is conducted in the presence of a non-catalytic divalent cation that inhibits polymerase- catalyzed nucleotide incorporation, optionally wherein the non-catalytic divalent cation comprises strontium or barium. In some embodiments, the contacting the second plurality of complexed polymerases with the plurality of nucleotides of step (g) is conducted in the presence of a catalytic divalent cation that promotes polymerase-catalyzed nucleotide incorporation, optionally wherein the catalytic divalent cation comprises magnesium or manganese.

[0028] In some embodiments of the methods of the disclosure, the plurality of immobilized concatemer template molecules in step (a) comprise clonally amplified immobilized concatemer template molecules. In some embodiments, individual immobilized concatemer template molecules in the plurality of step (a) comprise a concatemer template molecule having two or more tandem copies of a target sequence. In some embodiments, the immobilized concatemer template molecules in the plurality of immobilized concatemer template molecules in step (a) comprise the same target of interest sequence or different target of interest sequences.

[0029] In some embodiments of the methods of the disclosure, individual multivalent molecules in the plurality of multivalent molecules comprise: (a) a core; and (b) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide moiety, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide moiety. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits. In some embodiments, the plurality of nucleotide arms attached to a given core have the same type of nucleotide moieties, and wherein the types of nucleotide moieties comprise dATP, dGTP, dCTP, dTTP or dUTP. In some embodiments, the plurality of multivalent molecules comprise one type of a multivalent molecule wherein each multivalent molecule in the plurality has the same type of nucleotide moiety selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments of the methods of the disclosure, the plurality of multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules each type having nucleotide moieties selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a core that is labeled with a fluorophore. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide moieties that are labeled with a fluorophore.

[0030] In some embodiments of the methods of the disclosure, individual nucleotides in the plurality of nucleotides in step (g) comprise an aromatic base, a five carbon sugar, and 1- 10 phosphate groups. In some embodiments, the plurality of nucleotides of step (g) comprise one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP, or comprise a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, at least one of the nucleotides in the plurality of nucleotides in step (g) is labeled with a fluorophore. In some embodiments, the plurality of nucleotides in step (g) lack a fluorophore label. In some embodiments, at least one of the nucleotides in the plurality of nucleotides of step (g) comprises a removable chain terminating moiety attached to the 3’ carbon position of the sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3 ’OH moiety on the sugar group.

[0031] In some embodiments of the methods of the disclosure, the sequencing further comprises forming a plurality of binding complexes, comprising the steps: (a) binding a first nucleic acid primer, a first polymerase, and a first multivalent molecule to a first portion of an immobilized concatemer template molecule, thereby forming a first binding complex, wherein a first nucleotide moiety of the first multivalent molecule binds to the first polymerase; and (b) binding a second nucleic acid primer, a second polymerase, and the first multivalent molecule to a second portion of the immobilized concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide moiety of the first multivalent molecule binds to the second polymerase, wherein the first and second binding complexes which include the same multivalent molecule form an avidity complex.

[0032] In some embodiments of the methods of the disclosure, the sequencing further comprises (a) contacting the first plurality of polymerases and the plurality of nucleic acid primers with different portions of an immobilized concatemer template molecule to form at least first and second complexed polymerases on the immobilized concatemer template molecule; (b) contacting a plurality of multivalent molecules to the at least first and second complexed polymerases , under conditions suitable to bind a single multivalent molecule from the plurality to the first and second complexed polymerases, wherein at least a first nucleotide moiety of the single multivalent molecule is bound to the first complexed polymerase which includes a first primer hybridized to a first portion of the immobilized concatemer template molecule, thereby forming a first binding complex, and wherein at least a second nucleotide moiety of the single multivalent molecule is bound to the second complexed polymerase which includes a second primer hybridized to a second portion of the concatemer template molecule, thereby forming a second binding complex, and wherein the contacting is conducted under a condition suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide moieties in the first and second binding complexes, and wherein the first and second binding complexes which are bound to the same multivalent molecule form an avidity complex; (c) detecting the first and second binding complexes on the immobilized concatemer template molecule; and (d) identifying the first nucleotide moiety in the first binding complex thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide moiety in the second binding complex thereby determining the sequence of the second portion of the immobilized concatemer template molecule.

DESCRIPTION OF THE DRAWINGS

[0033] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0034] FIG. 1 is a schematic of various exemplary configurations of multivalent molecules. Left (Class I): schematics of multivalent molecules having a “starburst” or “helter-skelter” configuration. Center (Class II): a schematic of a multivalent molecule having a dendrimer configuration. Right (Class III): a schematic of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide moieties are designated ‘N’, biotin is designated ‘B’, and streptavidin is designated ‘SA’. [0035] FIG. 2 is a schematic of an exemplary multivalent molecule comprising a generic core attached to a plurality of nucleotide-arms.

[0036] FIG. 3 is a schematic of an exemplary multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.

[0037] FIG. 4 shows a schematic of an exemplary multivalent molecule comprising a core attached to a plurality of nucleotide-arms, where the nucleotide arms comprise biotin, spacer, linker and a nucleotide moiety.

[0038] FIG. 5 is a schematic of an exemplary nucleotide-arm comprising a core attachment moiety, spacer, linker and nucleotide moiety. [0039] FIG. 6 shows the chemical structure of an exemplary spacer.

[0040] FIG. 7 shows the chemical structures of various exemplary linkers, including an 11- atom Linker, 16-atom Linker, 23 -atom Linker and an N3 Linker.

[0041] FIG. 8 shows the chemical structures of various exemplary linkers, including Linkers 1-9.

[0042] FIG. 9 shows the chemical structures of various exemplary linkers joined/attached to nucleotide moieties.

[0043] FIG. 10 shows the chemical structures of various exemplary linkers joined/attached to nucleotide moieties.

[0044] FIG. 11 shows the chemical structures of various exemplary linkers joined/attached to nucleotide moieties.

[0045] FIG. 12 shows the chemical structure of an exemplary biotinylated nucleotide-arm. In this example, the nucleotide moiety is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0046] FIG. 13 is a schematic of an exemplary low binding support comprising a glass substrate and alternating layers of hydrophilic coatings which are covalently or non- covalently adhered to the glass, and which further comprises chemically-reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides). In alternative embodiments, the support can be made of any material such as glass, plastic or a polymer material.

[0047] FIG. 14 is a schematic showing an exemplary linear single stranded library molecule (100) hybridizing with a double-stranded splint molecule (200) thereby circularizing the library molecule to form a library-splint complex (500) with two nicks. The linear single stranded library molecule (100) can comprise: a universal adaptor sequence for binding a second universal surface primer (120), such as a surface pinning primer; a left sample index sequence (160); a universal adaptor sequence for binding a forward sequencing primer (140); a sequence of interest (also referred to herein as an “insert”, 110); a universal adaptor sequence for binding a reverse sequencing primer (150); an optional unique identification sequence (180) (e.g., UMI); a right sample index sequence (170); and a universal adaptor sequence for binding a first universal surface primer (130), such as a surface capture primer. The double-stranded splint molecule (200) can comprise a first splint strand (also referred to as the “long strand,” (300)) hybridized to a second splint strand (also referred to as the “short strand,” (400)). The first splint strand can comprise a first region (320) that hybridizes with a sequence on one end of the linear single stranded library molecule, and a second region (330) that hybridizes with a sequence on the other end of the linear single stranded library molecule, thereby circularizing the library molecule to form a library-splint complex (500) with two nicks. The internal region (310) of the first splint strand can hybridize to the second splint strand (400). The second splint strand (400) can include three sub-regions which are designated 1st, 2nd and 3rd in FIG. 14. The first subregion can comprise a universal adaptor sequence for binding a third universal surface primer, the second sub-region can comprise a universal adaptor sequence for binding a fourth surface primer, and the third sub-region can comprise a sample index sequence optionally having 5-20 bases and/or a unique identification sequence having 2-10 or more bases (e.g., NN). The second splint strand (400) can be designed to carry new adaptor sequences to be introduced into the library molecule upon circularization. The internal region (310) of the first splint strand (300) can comprise three sub-regions, where the fourth sub-region hybridizes to the first sub-region of the second splint strand (400), and the fifth sub-region hybridizes to the second sub-region of the second splint strand (400), and the sixth sub-region hybridizes to the third sub-region of the second splint strand (400).

[0048] FIG. 15 is a schematic showing an exemplary workflow for generating a covalently closed circular library molecule. In (A), a library-splint complex (500) comprising two nicks can be subjected to an enzymatic ligation reaction to close the two nicks to form a covalently closed circular library molecule (600) which is hybridized to a first splint strand (300) as shown in (B). The first splint strand (300) can be used as an amplification primer to conduct a rolling circle amplification reaction. The first splint strand (300) can be removed or enzymatically degraded while retaining the covalently closed circular library molecule (600) as shown in (C). Alternatively, the rolling circle amplification reaction can be conducted by hybridizing a soluble amplification primer to the covalently closed circular library molecule (600) and conducting a rolling circle amplification reaction using a plurality of strand displacing polymerases and a plurality of nucleotides. As a further alternative, the rolling circle amplification reaction can be conducted by hybridizing the covalently closed circular library molecule (600) to a primer immobilized to a support, and conducting a rolling circle amplification reaction using a plurality of strand displacing polymerases and a plurality of nucleotides.

[0049] FIG. 16 shows the nucleotide sequences of an exemplary double-stranded splint molecule (200) having a first splint strand (300) and a second splint strand (400). The exemplary first splint strand comprises a first region (320; SEQ ID NO: 4), a second region (330; SEQ ID NO: 5), and an internal region (310) having a fourth sub-region (SEQ ID NO: 6) and fifth sub-region (SEQ ID NO: 7). The first splint strand (300) comprises the first and second regions ((320) and (330)) and the internal region (310) (SEQ ID NO: 8). The exemplary second splint strand (400) comprises a first sub-region (SEQ ID NO: 1) and second sub-region (SEQ ID NO: 2). The second splint strand (400) comprises the first and second sub-regions (SEQ ID NO: 3).

[0050] FIG. 17 is a schematic showing an exemplary linear single stranded library molecule (700) hybridizing with a single-stranded splint molecule (also referred to herein as a “single-stranded splint strand,” (800)) thereby circularizing the library molecule to form a library-splint complex (900) with a nick. The exemplary linear single stranded library molecule (700) comprises: a universal adaptor sequence for binding a second universal surface primer (720) (e.g., a universal surface pinning primer); a left sample index sequence (760); a universal adaptor sequence for binding a forward sequencing primer (740); a sequence of interest (also referred to herein as an “insert,” 710); a universal adaptor sequence for binding a reverse sequencing primer (750); an optional unique identification sequence (780) (e.g., UMI); a right sample index sequence (770); and a universal adaptor sequence for binding a first universal surface primer (730) (e.g., a universal capture primer). The singlestranded splint molecule (800) can comprise a first region (810) that hybridizes with a sequence on one end of the linear single stranded library molecule, and a second region (820) that hybridizes with a sequence on the other end of the linear single stranded library molecule thereby circularizing the library molecule to form a library-splint complex (900) with a nick. [0051] FIG. 18 is a schematic showing an exemplary workflow for generating a covalently closed circular library molecule. In (A), a library-splint complex (900) comprising one nick undergoes a ligation reaction to close the nick to form a covalently closed circular library molecule (1000) which is hybridized to a single-stranded splint molecule (800) as shown in (B). The single-stranded splint molecule (800) can be used as an amplification primer to conduct a rolling circle amplification reaction. The single-stranded splint molecule (800) can be removed or enzymatically degraded while retaining the covalently closed circular library molecule (1000) as shown in (C). Alternatively, a rolling circle amplification reaction can be conducted by hybridizing a soluble amplification primer to the covalently closed circular library molecule (1000) and conducting a rolling circle amplification reaction using a plurality of strand displacing polymerases and a plurality of nucleotides.

[0052] FIG. 19 shows the nucleotide sequences of an exemplary single-stranded splint molecule (800). The exemplary single-stranded splint strand comprises a first region (810; SEQ ID NO: 9), a second region (820; SEQ ID NO: 10). [0053] FIG. 20 is a schematic showing a double-stranded linear library molecule (700) comprising a top strand and a bottom strand. Both strands of the double-stranded linear library molecule can comprise a sequence of interest (also referred to as an insert region) comprising a target sequence flanked on both sides by universal adaptor sequences. The linear library molecule can comprise any arrangement of insert region and universal adaptor sequences.

[0054] FIG. 21 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a support (also referred to as a “capture support”) for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0055] FIG. 22 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0056] FIG. 23 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0057] FIG. 24 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0058] FIG. 25 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to single pass sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0059] FIG. 26 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0060] FIG. 27 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0061] FIG. 28 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0062] FIG. 29 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0063] FIG. 30 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0064] FIG. 31 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0065] FIG. 32 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic. [0066] FIG. 33 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to single pass sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0067] FIG. 34 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to pairwise sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0068] FIG. 35 shows an embodiment of a linear library molecule (1100) that can be used to generate a covalently closed circular library molecule which can be hybridized to a targetspecific bait/probe to generate a closed circle library bait complex. The closed circle library bait complex can be immobilized to a capture support for rolling circle amplification to generate a concatemer template molecule immobilized to the capture support. The immobilized concatemer template molecule can be subjected to single pass sequencing. Different portions of the immobilized concatemer template molecules can be sequenced in the order shown in the schematic.

[0069] FIG. 36A is a schematic showing an open circle library bait complex comprising a linear library molecule hybridized to a single-stranded top strand circularization oligonucleotide and a target-specific bait/probe. Hybridization of the ends of the linear library molecule with the ends of the single-stranded top strand circularization oligonucleotide forms an open circle library bait complex with a nick.

[0070] FIG. 36B is a schematic showing an open circle library bait complex comprising a linear library molecule hybridized to a single-stranded top strand circularization oligonucleotide and a target-specific bait/probe. Hybridization of the ends of the linear library molecule with the ends of the single-stranded top strand circularization oligonucleotide forms an open circle library bait complex with a gap. [0071] FIG. 36C is a schematic showing an open circle library bait complex comprising a linear library molecule hybridized to a double-stranded top strand circularization oligonucleotide and a target-specific bait/probe. The double-stranded top strand circularization oligonucleotide comprises a long strand and a short strand hybridized together. Hybridization of the ends of the linear library molecule with the ends of the long strands forms an open circle library bait complex with two nicks.

[0072] FIG. 36D is a schematic showing an open circle library bait complex comprising a linear library molecule hybridized to a single-stranded top strand circularization oligonucleotide and a target-specific bait/probe. One end of the single-stranded top strand circularization oligonucleotide can hybridize to one end of the linear library molecule, and the other end of the single-stranded top strand circularization oligonucleotide can hybridize near the other end of the linear library molecule to form an open circle library bait complex with a 5’ overhang flap.

DETAILED DESCRIPTION

Introduction

[0073] The present disclosure provides methods for conducting hybridization-based enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences. The mixture of polynucleotides can comprise library molecules having target and non-target sequences. The library molecules comprising target sequences can be enriched from the mixture of library molecules by employing targetspecific baits/probes, which contain sequences that are complementary to and hybridize with the target sequences in the mixture of molecules, in a hybridization-based workflow which is suitable for downstream applications, such as next generation sequencing of the enriched target sequences.

[0074] The methods of the disclosure include preparing a plurality of closed circle library bait complexes using a variety of suitable starting samples, such as linear library molecules or covalently closed circular library molecules. The closed circle library bait complexes can be prepared by conducting on-support hybridization of the target-specific baits/probes and the mixture of library molecules to generate closed circle library bait complexes which are immobilized to a capture support. Alternatively, the closed circle library bait complexes can be prepared by conducting in-solution hybridization of the target-specific baits/probes and the mixture of library molecules, followed by immobilization to a capture support. As a nonlimiting example, closed circle library bait complexes can be immobilized to a capture support which comprises (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. The target-specific baits/probes comprise an affinity moiety that can bind to the receptor moiety embedded in the polymer coating of the capture support. The capture support and target-specific baits/probes are designed to permit the skilled artisan to conduct customizable workflows to enrich for the desired target polynucleotide sequences, and to analyze the enriched target polynucleotides via massively parallel sequencing.

Definitions

[0075] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole. [0076] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art. Techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature utilized in connection with, and the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[0077] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.

[0078] It is understood the use of the alternative term (e.g., “or”) is taken to mean either one or both or any combination thereof of the alternatives.

[0079] The term “and/or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0080] As used herein and in the appended claims, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided.

[0081] As used herein, the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg.

Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Also, where ranges and/or subranges of values are provided, the ranges and/or subranges can include the endpoints of the ranges and/or subranges.

[0082] As used herein, “corresponding to” or “corresponds to” and similar terms refer to two or more entities whose identities are sufficiently related such that the identity of one entity can be used to determine the identity, position and/or other properties of the other entity. As a non-limiting example, a barcode sequence can be said to correspond to a particular sequence of interest if the barcode sequence can be used to determine the identity of the sequence of interest.

[0083] The term “polymerase” and its variants, as used herein, comprises an enzyme comprising a domain that binds a nucleotide (or nucleoside) where the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities including, but not limited to, base analog detection activities, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. A polymerase can be any enzyme that can catalyze polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically but not necessarily such nucleotide polymerization can occur in a template-dependent fashion. Typically, a polymerase comprises one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes other enzymatic activities, such as for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacing activity. A polymerase can include without limitation naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze nucleotide polymerization (e.g., catalytically active fragment). The polymerase includes catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes comprising a nucleotide binding domain. In some embodiments, a polymerase can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase can be expressed in prokaryote, eukaryote, viral, or phage organisms. In some embodiments, a polymerase can be post-translationally modified proteins or fragments thereof. A polymerase can be derived from a prokaryote, eukaryote, virus or phage. A polymerase comprises DNA-directed DNA polymerase and RNA-directed DNA polymerase. Exemplary suitable polymerases are described in U.S. Patent No. 11,859,241, the contents of which are incorporated by reference in their entirety herein.

[0084] As used herein, the term “strand displacing” refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acids and synthesize a new strand in a template-based manner. Strand displacing polymerases displace a complementary strand from a template strand and catalyze new strand synthesis. Strand displacing polymerases include mesophilic and thermophilic polymerases. Strand displacing polymerases include wild type enzymes, and variants including exonuclease minus mutants, mutant versions, chimeric enzymes and truncated enzymes. Examples of strand displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), KI enow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi® from Expedeon), or variant EquiPhi29™ DNA polymerase (e.g., from Thermo Fisher Scientific®), or chimeric QualiPhi® DNA polymerase (e.g., from 4basebio®).

[0085] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically- synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and/or sugars. Nucleic acids comprise naturally-occurring internucleosidic linkages, for example phosphodiester linkages. Nucleic acids can lack a phosphate group. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise one type of polynucleotides or a mixture of two or more different types of polynucleotides.

[0086] The term “operably linked” and “operably joined” or related terms as used herein refers to juxtaposition of components. The juxtapositioned components can be linked together covalently. For example, two nucleic acid components can be enzymatically ligated together where the linkage that joins together the two components comprises phosphodiester linkage. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function on a second nucleic acid component. For example, linkage between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) can be ligated to a vector where the linkage permits expression or functioning of the transgene sequence contained in the vector. In some embodiments, a transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, enhancer, transcription and/or translation initiation sequence, transcription and/or translation termination sequence, polypeptide secretion signal sequences, and the like. In some embodiments, the host cell regulatory sequence controls expression of the level, timing and/or location of the transgene. [0087] The terms “linked”, “joined”, “attached”, “appended” and variants thereof comprise any type of fusion, bond, adherence or association between any combination of compounds or molecules that is of sufficient stability to withstand use in the particular procedure. The procedure can include but are not limited to: nucleotide binding; nucleotide incorporation; de-blocking (e.g., removal of chain-terminating moiety); washing; removing; flowing; detecting; imaging and/or identifying. Such linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such linkage occurs intramolecularly, for example linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule. In some embodiments,, such linkage can occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like. Some examples of linkages can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0088] The term “primer” and related terms used herein refers to an oligonucleotide that is capable of hybridizing with a DNA and/or RNA polynucleotide template to form a duplex molecule. Primers comprise natural nucleotides and/or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers may have any length, but typically range from 4-50 nucleotides. A typical primer comprises a 5’ end and 3’ end. The 3’ end of the primer can include a 3’ OH moiety which serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3’ end of the primer can lack a 3’ OH moiety, or can include a terminal 3’ blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer can be labeled with a detectable reporter moiety. A primer can be in solution (e.g., a soluble primer) or can be immobilized to a support (e.g., a capture primer).

[0089] The term “template nucleic acid”, “template polynucleotide”, “target nucleic acid” “target polynucleotide”, “template strand,” “template molecule” and other variations refer to a nucleic acid strand that serves as the basis nucleic acid molecule for any of the methods described herein, e.g. sequencing or amplification methods. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from a naturally- occurring source, recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other forms. The template nucleic acids can include an insert portion having an insert sequence. The template nucleic acids can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin embedded tissue, needle biopsies, circulating tumor cells, cell free circulating DNA, or any type of nucleic acid library. The insert portion can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses, cells, tissues, normal or diseased cells or tissues, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, semen, environmental samples, culture samples, or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods.

The insert portion can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and composition analysis. The template molecules disclosed herein can be concatemer template molecules, which comprise two or more copies of a particular sequence. For example, a concatemer template molecule can comprise two or more tandem copies of a sequence of interest and at least one other sequence feature, such as any of the barcode sequences, index sequences, or sequencing, surface capture or surface pinning primer binding sequences disclosed herein.

[0090] The term “adaptor” and related terms refers to oligonucleotides that can be operably linked to a target polynucleotide, where the adaptor confers a function to the cojoined adaptor-target molecule. Adaptors comprise DNA, RNA, chimeric DNA/RNA, or analogs thereof. Adaptors can include at least one ribonucleoside residue. Adaptors can be single-stranded, double-stranded, or have single-stranded and/or double-stranded portions. Adaptors can be configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors can be any length, including 4-100 nucleotides or longer. Adaptors can have blunt ends, overhang ends, or a combination of both. Overhang ends include 5’ overhang and 3’ overhang ends. The 5’ end of a single-stranded adaptor, or one strand of a double-stranded adaptor, can have a 5’ phosphate group or lack a 5’ phosphate group. Adaptors can include a 5’ tail that does not hybridize to a target polynucleotide (e.g., tailed adaptor), or adaptors can be non-tailed. An adaptor can include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., soluble or immobilized capture primers). Adaptors can include a random sequence or degenerate sequence. Adaptors can include at least one inosine residue. Adaptors can include at least one phosphorothioate, phosphorothiolate and/or phosphoramidate linkage. Adaptors can include a barcode sequence which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. Adaptors can include a unique identification sequence (e.g., unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify a nucleic acid molecule to which the adaptor is appended. In some embodiments, a unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls and/or increase sensitivity of variant detection. Adaptors can include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from a group consisting of type I, type II, type III, type IV, type Hs or type IIB.

[0091] In some embodiments, primer sequences, such as any of the amplification primer sequences, sequencing primer sequences, surface capture primer sequences, surface pinning primer sequences, target-specific oligonucleotide sequences, sample barcode sequences, or spatial barcode sequences can be about 3-200 nucleotides in length, or about 3-150 nucleotides in length, about 3-100 nucleotides in length, or about 3-50 nucleotides in length, or about 3-25 nucleotides in length.

[0092] The term “universal sequence” and related terms refers to a sequence in a nucleic acid molecule that is common among two or more polynucleotide molecules. For example, an adaptor having a universal sequence (a “universal adaptor”) can be operably joined to a plurality of polynucleotides so that the population of co-joined molecules carry the same universal adaptor sequence. Examples of universal adaptor sequences include an amplification primer sequence, a sequencing primer sequence or a capture primer sequence (e.g., soluble or immobilized capture primers).

[0093] As used herein “target sequence” refers to a sequence that is selectively enriched and optionally sequenced using the methods described herein. Target sequences can be enriched by selective hybridization target-specific bait/probes which include oligonucleotides comprising target-specific sequences that are complementary to, or substantially complementary target sequences, and capable of selectively hybridizing to target sequences. [0094] As used herein, the terms “selectively hybridizing” and “selectively binds” in the context of any binding agent, e.g., an oligonucleotide or oligonucleotide complex of the disclosure, refers to a binding agent that hybridizes or binds specifically to a target, e.g., a target sequence, such as with a high specificity, and does not significantly hybridize or bind other unrelated targets or sequences. The person of ordinary skill in the art will appreciate that a binding agent that binds specifically to a target sequence with high specificity, but binds to non-targets (off-targets) with suitably low specificity can still be said to selectively bind to the target.

[0095] When used in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refers to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a duplex region. Hybridization can comprise Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, may be wholly complementary, or partially complementary. Complementary nucleic acid strands need not hybridize with each other across their entire length. The complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides. Two sequences can be said to “selectively hybridize” when the two sequences hybridize using complementary base pairing to form a double stranded nucleic acid in which only the desired sequences, or substantially only the desired sequences, are bound to each other. The skilled artisan will appreciate that two sequences need not be perfectly complementary in order to selectively hybridize to one another.

[0096] When used in reference to nucleic acids, the terms “extend”, “extending”, “extension” and other variants, refers to incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3’ OH end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be conducted with natural nucleotides and/or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent fashion. Any suitable method of extending a nucleic acid molecule may be used, including primer extension catalyzed by a DNA polymerase or RNA polymerase. [0097] The term “nucleotides” and related terms refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Canonical or non-canonical nucleotides are consistent with use of the term. The phosphate in some embodiments comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog. The term “nucleoside” refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be non-labeled or labeled with a detectable reporter moiety.

[0098] Nucleotides (and nucleosides) typically comprise a hetero cyclic base including substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants, or analogs of the same. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and/or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N⁶-A²- isopentenyladenine (6iA), N⁶-A²-isopentenyl-2-methylthioadenine (2ms6iA), N⁶- methyladenine, guanine (G), isoguanine, N²-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O⁶-methylguanine; 7- deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4- thiothymine (4sT), 5,6-dihydrothymine, O⁴-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0099] Nucleotides (and nucleosides) typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). The sugar moiety comprises: ribosyl; 2'-deoxyribosyl; 3 '-deoxyribosyl; 2', 3 '-dideoxyribosyl; 2', 3'- didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3 '-alkoxyribosyl; 3 '-azidoribosyl; 3 '-aminoribosyl; 3 '-fluororibosyl; 3'-mercaptoriboxyl; 3 '-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[00100] In some embodiments, nucleotides comprise a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[00101] The term “reporter moiety”, “reporter moieties” or related terms refers to a compound that generates, or causes to generate, a detectable signal. A reporter moiety is sometimes called a “label”. Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. A reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other. It is well known to one skilled in the art to select reporter moieties so that each absorbs excitation radiation and/or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. Reporter moieties can be linked (e.g., operably linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or support (e.g., surfaces).

[00102] A reporter moiety (or label) comprises a fluorescent label or a fluorophore. Exemplary fluorescent moieties which may serve as fluorescent labels or fluorophores include, but are not limited to fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo- NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY 530/550 C3 hydrazide, BODIPY 493/503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium based cyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates, Terbium chelates, Alexa Fluor® dyes, DyLight® dyes, Atto™ dyes, LightCycler® Red dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Green™ dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes and others known in the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and consist of two indolenin, benzo-indolium, pyridium, thiozolium, and/or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise 1 - [6-(2, 5-dioxopyrrolidin- 1 -yloxy)-6-oxohexyl]-2-(3 - { 1 - [6-(2, 5-dioxopyrrolidin- 1 - yloxy)-6-oxohexyl]-3,3-dimethyl-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3- dimethyl-3H-indolium or l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{ l-[6-(2,5- dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2- ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5- dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3-dien-l- yl)-3 ,3 -dimethyl-3H-indol- 1 -ium or 1 -(6-((2, 5-dioxopyrrolidin- 1 -yl)oxy)-6-oxohexyl)-2- ((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5- sulfoindolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and Cy7 (which may comprise l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H- indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium or l-(5-carboxypentyl)-2- [(lE,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]- 3H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule. Additional suitable dyes are described, for example, in U.S. 2024/0240249A1, the contents of which are incorporated by reference in their entirety herein.

[00103] In some embodiments, the reporter moiety can be a FRET pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[00104] When used in reference to nucleic acids, the terms “amplify”, “amplifying”, “amplification”, and other related terms include producing multiple copies of an original polynucleotide template molecule, where the copies comprise a sequence that is complementary to the template sequence, or the copies comprise a sequence that is the same as the template sequence. In some embodiments, the copies comprise a sequence that is substantially identical to a template sequence, or is substantially identical to a sequence that is complementary to the template sequence.

[00105] The term “rolling circle amplification” generally refers to an amplification method that employs a circular nucleic acid template molecule containing a target sequence of interest, an amplification primer binding sequence, and optionally one or more adaptor sequences, such as a sequencing primer binding sequence and/or a sample index sequence. The circularized nucleic acid template molecule can comprise a covalently closed circular library molecule as described herein. The rolling circle amplification reaction can be conducted under isothermal amplification conditions, and includes the circularized nucleic acid template molecule, an amplification primer, a strand-displacing polymerase and a plurality of nucleotides, to generate a concatemer template molecule containing tandem repeat sequences of the circular template molecule. The concatemer template molecule can self-collapse to form a nucleic acid nanoball. The shape and size of the nanoball can be further compacted by including a pair of inverted repeat sequences in the circular template molecule, or by conducting the rolling circle amplification reaction in the presence of one or more compaction oligonucleotides, for example compaction oligonucleotides having at least four consecutive guanines. The rolling circle amplification reaction generates concatemer template molecules comprising repeat copies of a sequence that can form a guanine tetrad, and the resulting concatemer template molecule can fold to form an intramolecular G- quadruplex structure. Without wishing to be bound by theory, formation of the guanine tetrads and G-quadruplexes in the DNA nanoballs may increase the stability of the nanoballs, allowing the nanoballs to retain their compact size and shape, which can withstand repeated flows of reagents for conducting any of the sequencing workflows described herein. An additional advantage of using rolling circle amplification to generate clonal amplicons for a sequencing workflow is that the repeat copies of the target sequence in the nanoball can be simultaneously sequenced to increase signal intensity.

[00106] The rolling circle amplification reaction can optionally include a positively charged metal complex, including for example hexamine (e.g., cobalt hexamine III) which can interact electrostatically with the negatively charged phosphate backbone of DNA and condense the concatemer template molecules into a compact structure. The concatemer template molecules can collapse into a DNA nanoball having a more compact size and/or shape compared to a nanoball generated from a rolling circle amplification reaction conducted without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). Inclusion of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the rolling circle amplification reaction can improve FWHM (full width half maximum) of a spot image of the DNA nanoball. The spot image can be represented as a Gaussian spot and the size can be measured as a FWHM. A smaller spot size as indicated by a smaller FWHM typically correlates with an improved image of the spot. In some embodiments, the FWHM of a DNA nanoball spot can be about 10 um or smaller.

[00107] In some embodiments, the rolling circle amplification reaction of can be conducted in the presence of a plurality of compaction oligonucleotides that can bind portions of concatemer template molecules generated by conducting a rolling circle amplification region (e.g., see U.S. Published Application No. 2024/0084380, filed August 15, 2023, and published March 14, 2024, the contents of which are incorporated by reference in their entirety herein).

[00108] The term “support” as used herein refers to a substrate that is designed for deposition of biological molecules or biological samples for assays and/or analyses. Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), polypeptides, saccharides, lipids, a single cell or multiple cells. Examples of biological samples include but are not limited to saliva, phlegm, mucus, blood, plasma, serum, urine, stool, sweat, tears and fluids from tissues or organs. [00109] The term “capture support” refers to a support as described herein, which includes a plurality of receptor moieties immobilized to the support. The receptor moieties are capable of binding an affinity moiety conjugated to a target-specific oligonucleotide (a “targetspecific bait/probe”) and/or a surface pinning primer. The support can be coated with at least one layer of hydrophilic polymer coating and the receptor moieties can embedded in the at least one layer of hydrophilic polymer coating. However, the skilled artisan will appreciate that any suitable method of immobilizing the receptor moieties to the support can be used to generate a capture support.

[00110] When used in reference to immobilized proteins, the term “immobilized” and related terms refer to proteins (e.g., receptor moieties) that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support.

[00111] When used in reference to immobilized nucleic acids, the term “immobilized” and related terms refer to nucleic acid molecules that are attached to a support, either directly or indirectly, through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support. Exemplary nucleic acid molecules include surface capture primers, nucleic acid template molecules and extension products of capture primers, sequencing primers and/or amplification primers.

[00112] When used in reference to immobilized nucleic acid template molecules, the term “immobilized” and related terms refer to nucleic acid template molecules that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support. In some embodiments, the nucleic acid template molecules include concatemer template molecules.

[00113] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example comprising a capillary or interior surface of a capillary.

[00114] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can be regularly or irregularly textured, including bumps, etched, pores, three-dimensional scaffolds, or any combination thereof.

[00115] In some embodiments, the support comprises a bead having any shape, including spherical, hemi-spherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-like, conical, triangular, cubical, polygonal, tubular or wire-like. [00116] The support can be fabricated from any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[00117] In some embodiments, the support comprises: (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the plurality of receptor moieties can be embedded at random locations in the at least one layer of hydrophilic polymer coating. In some embodiments, the plurality of receptor moieties can be embedded at pre-determined locations in the at least one layer of hydrophilic polymer coating. In some embodiments, individual receptor moieties can bind an affinity moiety. [00118] The support can have a plurality (e.g., two or more) of nucleic acid template molecules immobilized thereon. The plurality of immobilized nucleic acid template molecules have the same sequence or have different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid template molecules are immobilized to different sites on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid template molecules are immobilized to a site on the support. In some embodiments, the nucleic acid template molecule comprises a concatemer template molecule.

[00119] The term “array” refers to a support comprising a plurality of sites located at predetermined locations on the support to form an array of sites. The sites can be discrete and separated by interstitial regions. In some embodiments, the pre-determined sites on the support can be arranged in one dimension in a row or a column, or arranged in two dimensions in rows and columns. In some embodiments, the plurality of pre-determined sites is arranged on the support in an organized fashion. In some embodiments, the plurality of pre-determined sites is arranged in any organized pattern, including rectilinear, hexagonal patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. The pitch between different pairs of sites can be the same or can vary. In some embodiments, the support comprises at least 10² sites, at least 10³ sites, at least 10⁴ sites, at least 10⁵ sites, at least 10⁶ sites, at least 10⁷ sites, at least 10⁸ sites, at least 10⁹ sites, at least 10¹⁰ sites, at least 10¹¹ sites, at least 10¹² sites, at least 10¹³ sites, at least 10¹⁴ sites, at least 10¹⁵ sites, or more, where the sites are located at pre-determined locations on the support. In some embodiments, the support comprises between about 10² sites and about 10¹⁵ sites, between about 10⁵ sites and about 10¹⁵ sites, between about IO¹⁰ sites and about 10¹⁵ sites, between about 10³ sites and about 10¹⁴ sites, between about 10⁴ sites and about 10¹³ sites, between about 10⁵ sites and about 10¹² sites, between about 10⁶ sites and about 10¹¹ sites, between about 10⁷ sites and about IO¹⁰ sites, between about 10⁸ sites and about IO¹⁰ sites, or any range therebetween located at pre-determined locations on the support. In some embodiments, a plurality of pre-determined sites on the support (e.g., 10² - 10¹⁵ sites or more) are immobilized with nucleic acid template molecules to form a nucleic acid template array. In some embodiments, the nucleic acid template molecules that are immobilized at a plurality of pre-determined sites. In some embodiments, the nucleic acid template molecules that are immobilized at a plurality of pre-determined sites, for example immobilized at 10² - 10¹⁵ sites or more. In some embodiments, the immobilized nucleic acid template molecules can be clonally-amplified (e.g., via rolling circle amplification) to generate immobilized concatemer template molecules at the plurality of pre-determined sites.

[00120] In some embodiments, a support comprising a plurality of sites located at random locations on the support is referred to herein as a support having randomly located sites thereon. The location of the randomly located sites on the support are not pre-determined. The plurality of randomly-located sites is arranged on the support in a disordered and/or unpredictable fashion. In some embodiments, the support comprises at least 10² sites, at least 10³ sites, at least 10⁴ sites, at least 10⁵ sites, at least 10⁶ sites, at least 10⁷ sites, at least 10⁸ sites, at least 10⁹ sites, at least IO¹⁰ sites, at least 10¹¹ sites, at least 10¹² sites, at least 10¹³ sites, at least 10¹⁴ sites, at least 10¹⁵ sites, or more, where the sites are randomly located on the support. In some embodiments, the support comprises between about 10² sites and about 10¹⁵ sites, between about 10⁵ sites and about 10¹⁵ sites, between about IO¹⁰ sites and about 10¹⁵ sites, between about 10³ sites and about 10¹⁴ sites, between about 10⁴ sites and about 10¹³ sites, between about 10⁵ sites and about 10¹² sites, between about 10⁶ sites and about 10¹¹ sites, between about 10⁷ sites and about IO¹⁰ sites, or between about 10⁸ sites and about IO¹⁰ sites, or any range therebetween located at random locations on the support. In some embodiments, a plurality of randomly located sites on the support (e.g., 10² - 10¹⁵ sites or more) are immobilized with nucleic acid template molecules. In some embodiments, the immobilized nucleic acid template molecules are clonally-amplified (e.g., via rolling circle amplification) to generate immobilized concatemer template molecules at the plurality of randomly located sites. [00121] The support can comprise a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating (a “capture support”). In some embodiments, plurality of receptor moieties are in fluid communication with each other to permit flowing a solution of reagents (e.g., nucleic acid template molecules, soluble primers, oligonucleotides, enzymes, nucleotides, divalent cations, buffers, and the like) onto the support so that the plurality of embedded receptor moieties can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of embedded receptor moieties can be used to conduct nucleic acid amplification reactions (e.g., RCA, MDA, PCR and bridge amplification) essentially simultaneously on the plurality of embedded receptor moieties. In some embodiments, the fluid communication of the plurality of embedded receptor moieties can be used to conduct nucleic acid sequencing reactions essentially simultaneously on the plurality of embedded receptor moieties. In some embodiments, the support comprises receptor moieties located at at least 10² sites, at least 10³ sites, at least 10⁴ sites, at least 10⁵ sites, at least 10⁶ sites, at least 10⁷ sites, at least 10⁸ sites, at least 10⁹ sites, at least IO¹⁰ sites, at least 10¹¹ sites, at least 10¹² sites, at least 10¹³ sites, at least 10¹⁴ sites, at least 10¹⁵ sites, or more, on the support. In some embodiments, the support comprises receptor moieties located at between about 10² sites and about 10¹⁵ sites, between about 10⁵ sites and about 10¹⁵ sites, between about IO¹⁰ sites and about 10¹⁵ sites, between about 10³ sites and about 10¹⁴ sites, between about 10⁴ sites and about 10¹³ sites, between about 10⁵ sites and about 10¹² sites, between about 10⁶ sites and about 10¹¹ sites, between about 10⁷ sites and about IO¹⁰ sites, or between about 10⁸ sites and about IO¹⁰ sites, or any range therebetween on the support.

[00122] In some embodiments, the plurality of concatemer template molecules immobilized to the at least one coating layer on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., enzymes, nucleotides, multivalent molecules, catalytic divalent cations, non-catalytic divalent cations, and the like) onto the support so that the plurality of immobilized concatemer template molecules on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized concatemer template molecules can be used to conduct nucleotide binding assays and/or conduct nucleotide polymerization reactions (e.g., primer extension or sequencing) essentially simultaneously on the plurality of immobilized concatemer template molecules, and optionally to conduct detection and imaging for massively parallel sequencing. [00123] In some embodiments, one or more nucleic acid template molecules are immobilized on the support, for example immobilized at the sites on the support. In some embodiments, the one or more nucleic acid template molecules are clonally-amplified. In some embodiments, the one or more nucleic acid template molecules are clonally-amplified off the support (e.g., in-solution) and then deposited onto the support and immobilized on the support. In some embodiments, the clonal amplification reaction of the one or more nucleic acid template molecules is conducted on the support resulting in immobilization on the support. In some embodiments, the one or more nucleic acid template molecules are clonally- amplified (e.g., in solution or on the support) using a nucleic acid amplification reaction, including any one or any combination of: polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), realtime SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinasedependent amplification, and/or single-stranded binding (SSB) protein-dependent amplification.

[00124] As used herein, the term “binding complex” refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or a nucleotide unit of a multivalent molecule, where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In the binding complex, the free nucleotide or nucleotide unit may or may not be bound to the 3’ end of the nucleic acid primer at a position that is opposite a complementary nucleotide in the nucleic acid template molecule. A “ternary complex” is an example of a binding complex which is formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, where the free nucleotide or nucleotide unit is bound to the 3’ end of the nucleic acid primer (as part of the nucleic acid duplex) at a position that is opposite a complementary nucleotide in the nucleic acid template molecule.

[00125] The term “persistence time” and related terms refer to the length of time that a binding complex, which is formed between the target nucleic acid, a primer, a polymerase, a conjugated or unconjugated nucleotide, remains stable without any binding component dissociates from the binding complex. The persistence time is indicative of the stability of the binding complex and strength of the binding interactions. Persistence time can be measured by observing the onset and/or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in a binding complex, thus allowing the signal from the label to be detected during the persistence time of the binding complex. One exemplary label is a fluorescent label.

[00126] The present disclosure provides various reagents, and methods that employ the reagents for conducting a trapping reaction, an imaging reaction, a nucleic acid denaturation (de-hybridization) and/or a stepping reaction. The various reagents can include at least one pH buffering agent. The full name of the pH buffering agents is listed herein.

[00127] The term “Tris” refers to a pH buffering agent Tris(hydroxymethyl)- aminomethane. The term “Tris-HCl” refers to a pH buffering agent Tris(hydroxymethyl)- aminomethane hydrochloride. The term “Tris-acetate” refers to a pH buffering agent comprising an acetate salt of Tris (hydroxymethyl)-aminomethane.

[00128] The term “Tricine” refers to a pH buffering agent N-[tris(hydroxymethyl) methyl]glycine.

[00129] The term “Bicine” refers to a pH buffering agent N,N-bis(2-hydroxyethyl)glycine.

[00130] The term “Bis-Tris propane” refers to a pH buffering agent 1,3 Bis[tris(hydroxymethyl)methylamino]propane.

[00131] The term “HEPES” refers to a pH buffering agent 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid.

[00132] The term “MES” refers to a pH buffering agent 2-(7V-morpholino)ethanesulfonic acid).

[00133] The term “MOPS” refers to a pH buffering agent 3-(N- morpholino)propanesulfonic acid.

[00134] The term “MOPSO” refers to a pH buffering agent 3-(N-morpholino)-2- hydroxypropanesulfonic acid.

[00135] The term “BES” refers to a pH buffering agent N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid.

[00136] The term “TES” refers to a pH buffering agent 2-[(2-Hydroxy-

1, lbis(hydroxymethyl)ethyl)amino]ethanesulfonic acid).

[00137] The term “CAPS” refers to a pH buffering agent 3 -(cyclohexylamino)- 1- propanesuhinic acid.

[00138] The term “TAPS” refers to a pH buffering agent N-[Tris(hydroxymethyl)methyl]- 3 -amino propane sulfonic acid.

[00139] The term “TAPSO” refers to a pH buffering agent N- [Tris(hydroxymethyl)methyl]-3-amino-2-hyidroxypropansulfonic acid. [00140] The term “ACES” refers to a pH buffering agent 7V-(2-Acetamido)-2- aminoethanesulfonic acid.

[00141] The term “PIPES” refers to a pH buffering agent piperazine- l,4-bis(2- ethanesulfonic acid.

[00142] The term “ethanolamine” refers to a pH buffering agent that is also known as 2- aminoethanol.

[00143] The term “sequencing” and related terms refers to a method for obtaining nucleotide sequence information from a nucleic acid molecule, typically by determining the identity of at least some nucleotides (including their nucleobase components) within the nucleic acid molecule. In some embodiments, the sequence information of a given region of a nucleic acid molecule includes identifying each and every nucleotide within a region that is sequenced. In some embodiments, sequencing information determines only some of the nucleotides a region, while the identity of some nucleotides remains undetermined or incorrectly determined. Any suitable method of sequencing may be used in combination with the capture methods described herein. In an exemplary embodiment, sequencing can include label-free or ion based sequencing methods. In some embodiments, sequencing can include labeled or dye-containing nucleotide or fluorescent based nucleotide sequencing methods. In some embodiments, sequencing can include polony-based sequencing or bridge sequencing methods. In some embodiments, the sequencing employs polymerases and multivalent molecules for generating at least one avidity complex, wherein individual multivalent molecules comprise a plurality of nucleotide moieties tethered to a core. In some embodiments, the sequencing employs polymerases and free nucleotides for performing sequencing-by-synthesis. In some embodiments, the sequencing employs a ligase enzyme and a plurality of sequence-specific oligonucleotides for performing sequence-by-ligation.

[00144] Throughout this application various publications, patents, and/or patent applications are referenced. The disclosures of the publications, patents and/or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

Methods for Enriching Target Polynucleotides

[00145] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising: (a) preparing a plurality of closed circle library bait complexes immobilized to a capture support, wherein individual closed circle library bait complexes comprise (i) a covalently closed circular library molecule comprising a polynucleotide having a target sequence and at least one universal adaptor sequence, and (ii) a target-specific bait/probe that is selectively hybridized to at least a portion of a corresponding target sequence of a covalently closed circular library molecule; (b) conducting a rolling circle amplification reaction using the target-specific bait/probe to initiate amplification, thereby generating a plurality of immobilized concatemer template molecules; and (c) sequencing at least a portion individual immobilized concatemer template molecules of the plurality of immobilized concatemer template molecules. In some embodiments, individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of a target sequence of a covalently closed circular library molecule, an affinity moiety at the 5’ end, and an extendible 3’ end.

[00146] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): preparing a plurality of immobilized closed circle library bait complexes by binding a plurality of closed circle library bait complexes to a capture support, wherein the capture support comprises (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, individual closed circle library bait complexes comprise (i) a covalently closed circular library molecule comprising a polynucleotide having a target sequence and at least one universal adaptor sequence, and (ii) a target-specific bait/probe that is selectively hybridized to a corresponding target sequence of a covalently closed circular library molecule. In some embodiments, individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of the target sequence of the covalently closed circular library molecule, an affinity moiety at the 5’ end, and an extendible 3’ end. In some embodiments, individual targetspecific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support. In some embodiments, the affinity moiety of individual target-specific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual target-specific baits/probes can be located at the 5’ end or at an internal position.

[00147] In some embodiments, the methods further comprise step (b): conducting a rolling circle amplification reaction using the extendible 3’ end of the immobilized closed circle library bait complexes thereby generating a plurality of immobilized concatemer template molecules.

[00148] In some embodiments, the methods further comprise step (c): sequencing at least a portion of the plurality of immobilized concatemer template molecules. In some embodiments, the sequencing identifies the target sequences.

[00149] In some embodiments, the mixture of target and non-target polynucleotides comprises a plurality of target polynucleotides with different sequences. In some embodiments, the mixture of target and non-target polynucleotides comprises at least 10, at least 10², at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰ target polynucleotides comprising different sequences. In some embodiments, the mixture of target and non-target polynucleotides comprises between about 10 and about 10¹⁰, between about 10² and about 10⁹, between about 10³ and about 10⁸, between about 10² and about 10⁶, between about 10² and about 10⁵, between about 10³ and about 10⁶, or between about 10³ and about 10⁵ target polynucleotides with different sequences, or any range therebetween.

[00150] In some embodiments, preparing the plurality of immobilized closed circle library bait complexes comprises contacting the mixture of target and non-target polynucleotides with a plurality of target-specific baits/probes comprising oligonucleotide comprising a plurality of target-specific sequences, in which the target-specific sequences are different. In some embodiments, the target-specific baits/probes comprise at least 10, at least 10², at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰ different target-specific sequences. In some embodiments, the target-specific baits/probes comprise between about 10 and about 10¹⁰, between about 10² and about 10⁹, between about 10³ and about 10⁸, between about 10² and about 10⁶, between about 10² and about 10⁵, between about 10³ and about 10⁶, or between about 10³ and about 10⁵ different target-specific sequences, or any range therebetween.

(1) Enrichment by Conducting In-Solution Capture of Circularized Library Molecules [00151] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00152] In some embodiments, in step (a), the capture support comprises a plurality of pinning primers immobilized to the capture support, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end. In some embodiments, individual pinning primers comprise a blocking group at the 3’ end. In some embodiments, the blocking group inhibits polymerase-catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind a receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture support lacks a plurality of immobilized pinning primers.

[00153] In some embodiments, the methods comprise step (b): forming a plurality of closed circle library bait complexes by contacting a plurality of target-specific baits/probes to a plurality of covalently closed circular library molecules. In some embodiments, the plurality of covalently closed circular library molecules comprises a mixture of covalently closed circular library molecules comprising target sequences and covalently closed circular library molecules comprising non-target sequences. In some embodiments, individual covalently closed circular library molecules comprise (i) an insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, the contacting is conducted in-solution. In some embodiments, the contacting is conducted under a condition suitable for selectively hybridizing individual target-specific baits/probes to at least a portion of a target sequence, thereby generating a plurality of closed circle library bait complexes that are enriched for polynucleotides having target sequences.

[00154] In some embodiments, the plurality of covalently closed circular library molecules comprising non-target sequences do not selectively hybridize to the plurality of targetspecific baits/probes. In some embodiments, individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of a target sequence of the covalently closed circular library molecule, an affinity moiety at the 5’ end, and an extendible 3’ end. In some embodiments, the targetspecific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual targetspecific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual target-specific baits/probes comprises biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moieties of individual targetspecific baits/probes can be located at the 5’ ends or at internal positions. In some embodiments, the plurality of covalently closed circular library molecules comprising nontarget sequences do not selectively hybridize to the plurality of target-specific baits/probes. In some embodiments, the 3’ ends of individual target-specific baits/probes comprise a moiety that promotes polymerase-catalyzed extension of the 3’ end.

[00155] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual covalently closed circular library molecules to corresponding target-specific baits/probes comprises conducting an insolution hybridization reaction for about 1-15 minutes, about 15-30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, or about 12-16 hours.

[00156] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual covalently closed circular library molecules to corresponding target-specific baits/probes comprises conducting an insolution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, about 55- 60 degrees C, about 60-65 degrees C, about 65-70 degrees C, about 70-75 degrees C, or about 75-80 degrees C.

[00157] In some embodiments, in step (b), the plurality of target-specific baits/probes and the plurality of covalently closed circular library molecules can be hybridized in-solution in a hybridization reagent under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual covalently closed circular library molecules to corresponding target-specific baits/probes, thereby forming a plurality of closed circle library bait complexes.

[00158] In some embodiments, in step (b) the mixture of covalently closed circular library molecules comprises target-specific sequences and non-target sequences. In some embodiments, in step (b), the mixture of covalently closed circular library molecules comprises 2-500,000 different target sequences, 2-100,000 different target sequences, 100- 100,000 different target sequences, 500-10,000 different target sequences, 2-500 different target sequences, or 1,000-50,000 different target sequences, or any range therebetween. In some embodiments, in step (b), the mixture of covalently closed circular library molecules comprises up to 1,000,000 different target sequences. In some embodiments, the plurality of target-specific baits/probes comprises 2-1,000,000 different target-specific sequences, 2- 500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, in the plurality of closed circle library bait complexes, the plurality of target-specific baits/probes comprise 2-10,000 different target-specific sequences.

[00159] In some embodiments, in step (b), individual covalently closed circular library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual covalently closed circular library molecules comprise an insert region comprising a target or non-target sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. Without limitation, any of the covalently closed circular library molecules shown in FIGS. 15 or 18 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be circularized using the methods disclosed herein or any suitable methods known in the art to form covalently closed circular library molecules which can be used to conduct step (b).

[00160] In some embodiments, in step (b), the plurality of covalently closed circular library molecules can be generated from double-stranded input nucleic acids comprising top strands and complementary bottom strands. In some embodiments, the plurality of covalently closed circular library molecules comprises at least a first and a second sub-population of covalently closed circular library molecules. In some embodiments, the insert regions of individual covalently closed circular library molecules of the first sub-population comprise a target or non-target sequence from a top strand of the input nucleic acid. In some embodiments, the insert regions of individual covalently closed circular library molecules of the second sub-population comprise a target or non-target sequence from a bottom strand of the input nucleic acid.

[00161] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of individual covalently closed circular library molecules of the first sub-population (e.g., top strand insert sequence).

[00162] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of individual covalently closed circular library molecules of the second sub-population (e.g., bottom strand insert sequence).

[00163] In some embodiments, in step (b), at least one of the covalently closed circular library molecules in the plurality comprises at least one deaminated nucleotide base. In some embodiments, covalently closed circular library molecules comprising the at least one deaminated nucleotide base can be removed from the plurality by subjecting the plurality of covalently closed circular library molecules to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (b) comprises contacting the plurality of covalently closed circular library molecules with a reagent that removes deaminated bases and generates gaps, thereby converting the at least one deaminated nucleotide base in the at least one covalently closed circular library molecule into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity, as described herein. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (b) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from covalently closed circular library molecules.

[00164] In some embodiments, step (b) generates a plurality of closed circle library bait complexes comprising a mixture of closed circle library bait complexes comprising target sequences and a plurality of covalently closed circular library molecules comprising non- target sequences. In some embodiments, the mixture of closed circle library bait complexes and covalently closed circular library molecules comprising non-target sequences can be contacted with paramagnetic beads coated with receptor moieties that can bind to the affinity moieties on the closed circle library bait complexes comprising target sequences, thereby separating the closed circle library bait complexes from the covalently closed circular library molecules. In some embodiments, the paramagnetic beads that are bound to the closed circle library bait complexes can be washed to remove the plurality of covalently closed circular library molecules. In some embodiments, the closed circle library bait complexes can be released from the paramagnetic beads, thereby generating a plurality of closed circle library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the plurality of covalently closed circular library molecules comprising nontarget sequences are not removed in step (b). In some embodiments, the plurality of nontarget covalently closed circular library molecules are not removed in step (b) using paramagnetic beads that are coated with receptor moieties. In some embodiments, paramagnetic beads coated with receptor moieties are not used at step (b). In some embodiments, the plurality of covalently closed circular library molecules can be removed in step (c) as described herein.

[00165] In some embodiments, the method comprises step (c): contacting the capture support with the plurality of closed circle library bait complexes, thereby generating a plurality of closed circle library bait complexes immobilized to the capture support, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes (e.g., which are part of individual closed circle library bait complexes) to a receptor moiety of the capture support. In some embodiments, the plurality of closed circle library bait complexes immobilized to the capture support are enriched for polynucleotides carrying target sequences. In some embodiments, the plurality of closed circle library bait complexes can be distributed/contacted with the capture support in a loading reagent under a condition suitable for binding the affinity moiety of individual closed circle library bait complexes to individual receptor moieties embedded in the hydrophilic polymer coating of the capture support, thereby generating a plurality of closed circle library bait complexes immobilized to the capture support.

[00166] In some embodiments, in step (c), the density of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁵ closed circle library bait complexes per mm². In some embodiments, density of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library bait complexes per mm², or any range therebetween.

[00167] In some embodiments, the plurality of closed circle library bait complexes are immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library bait complexes are immobilized to the capture support at predetermined sites and arranged in a pattern.

[00168] In some embodiments, in step (c), the capture support is contacted with the plurality of closed circle library bait complexes and residual non-target covalently closed circular library molecules from step (b). In some embodiments, step (c) comprises contacting the support with a wash reagent to remove the plurality of non-target covalently closed circular library molecules and retain the plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the washing of step (c) can generate a plurality of immobilized closed circle library bait complexes that are enriched for polynucleotides having target sequences.

[00169] In some embodiments, in step (c), the capture support comprises a plurality of pinning primers. In some embodiments, in step (c), the capture support lacks pinning primers. In some embodiments, step (c) comprises contacting the capture support separately with a plurality of pinning primers and the plurality of closed circle library bait complexes in any order. In some embodiments, step (c) comprises contacting the capture support with a plurality of pinning primers and the plurality of closed circle library bait complexes essentially simultaneously.

[00170] In some embodiments, the method comprises step (d): contacting the plurality of closed circle library bait complexes immobilized to the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes (e.g., which are part of the immobilized closed circle library bait complexes) and use the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support, also referred to herein as “immobilized concatemer template molecules”). In some embodiments, individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region (the target sequence) and any universal adaptor sequences present in the covalently closed circular library molecule. In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers. In some embodiments, individual pinning primers hybridize to a portion of an immobilized concatemer template molecule, thereby pinning down a portion of a concatemer template molecule.

[00171] In some embodiments, in step (d), the rolling circle amplification reagent further comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. The rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

[00172] In some embodiments, the methods comprise step (e): sequencing at least a portion of the plurality of concatemer template molecules immobilized to the capture support. In some embodiments, at least a portion of the concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two-stage sequencing, sequencing- by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein. [00173] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis) and U.S. published application No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of step (b) and the distributing of step (c) (the contents of both documents are hereby incorporated by reference in their entireties).

[00174] In some embodiments, after the sequencing of step (e) described above, additional closed circle library bait complexes can be distributed and immobilized to the capture support by conducting a re-seeding workflow. For example, steps (6) - (8) of a re-seeding workflow in which closed circle library bait complexes are generated in-solution and distributed onto a capture support can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding a capture support are described herein.

(2) Enrichment by Conducting On-Support Capture of Circularized Library Molecules [00175] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00176] In some embodiments, in step (a), the capture support comprises a plurality of immobilized target-specific baits/probes, wherein individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of a target sequence, an affinity moiety at the 5’ end, and an extendible 3’ end. In some embodiments, the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, the plurality of target-specific baits/probes comprises DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support (e.g., see FIGS. 40A and 40B). In some embodiments, the affinity moiety of individual target-specific baits/probes comprises biotin, desthiobiotin or iminobiotin.

[00177] In some embodiments, in step (a), the plurality of target-specific baits/probes can be distributed/contacted with the capture support in a loading reagent under a condition suitable for binding the affinity moieties of individual target-specific baits/probes to individual receptor moieties, thereby generating a capture support comprising a plurality of target-specific baits/probes immobilized to the capture support.

[00178] In some embodiments, in step (a), the capture support comprises a plurality of pinning primers immobilized to the capture support. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end. In some embodiments, individual pinning primers comprise a blocking group at the 3’ end. In some embodiments, the blocking group at the 3’ end inhibits polymerase-catalyzed extension of the 3’ end of the pinning primer. In some embodiments, the individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind a receptor moiety of the capture support (e.g., see FIGS. 40A and 40B). In some embodiments, the affinity moieties of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture support lacks a plurality of pinning primers immobilized thereto.

[00179] In some embodiments, the methods comprise step (b): forming a plurality of closed circle library bait complexes immobilized to the capture support by contacting a plurality of covalently closed circular library molecules to the plurality of target-specific baits/probes that are immobilized to the capture support. In some embodiments, the plurality of covalently closed circular library molecules comprises a mixture of covalently closed circular library molecules comprising target sequences and covalently closed circular library molecules comprising non-target sequences. In some embodiments, individual covalently closed circular library molecules comprise (i) an insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, the contacting is conducted on the capture support under a condition suitable for selectively hybridizing individual target-specific baits/probes to at least a portion of a target sequence, thereby generating a plurality of closed circle library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the covalently closed circular library molecules comprising non-target sequences do not selectively hybridize to the immobilized target-specific baits/probes.

[00180] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequences of individual covalently closed circular library molecules to corresponding target-specific baits/probes comprises conducting an on- support hybridization reaction for about 1-15 minutes, about 15-30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, or about 12-16 hours.

[00181] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequences of individual covalently closed circular library molecules to corresponding target-specific baits/probes comprises conducting an on- support hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, about 55- 60 degrees C, about 60-65 degrees C, about 65-70 degrees C, about 70-75 degrees C, or about 75-80 degrees C.

[00182] In some embodiments, in step (b), the plurality of covalently closed circular library molecules can be distributed/contacted with the plurality of target-specific baits/probes that are immobilized to the capture support in a loading reagent and/or a hybridization reagent, under a condition suitable for hybridizing at least a portion of the target sequences of individual covalently closed circular library molecules to corresponding target-specific baits/probes, thereby generating a plurality of immobilized closed circle library bait complexes.

[00183] In some embodiments, in step (b) the mixture of covalently closed circular library molecules comprises target-specific sequences and non-target sequences. In some embodiments, in step (b), the mixture of covalently closed circular library molecules comprises 2-10,000 different target sequences. In some embodiments, in step (b), the mixture of covalently closed circular library molecules comprises 2-500,000 different target sequences, 2-100,000 different target sequences, 100-100,000 different target sequences, 500- 10,000 different target sequences, 2-500 different target sequences, or 1,000-50,000 different target sequences, or any range therebetween. In some embodiments, in step (b), the mixture of covalently closed circular library molecules comprises up to 1,000,000 different target sequences. In some embodiments, the plurality of target-specific baits/probes comprises 2- 1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, the plurality of target-specific baits/probes comprises 2-10,000 different target-specific sequences. In some embodiments, in the plurality of closed circle library bait complexes, the plurality of target-specific baits/probes comprises 2-10,000 different targetspecific sequences. In some embodiments, in the plurality of closed circle library bait complexes, the plurality of target-specific baits/probes comprises 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[00184] In some embodiments, in step (b), individual covalently closed circular library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual covalently closed circular library molecules comprise an insert region comprising a target or non-target sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the covalently closed circular library molecules shown in FIGS. 15 and 18 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be circularized to form covalently closed circular library molecules which can be used to conduct step (b) using the methods described herein or any suitable methods known in the art.

[00185] In some embodiments, in step (b), the plurality of covalently closed circular library molecules can be generated from double-stranded input nucleic acids comprising top strands and complementary bottom strands. In some embodiments, the plurality of covalently closed circular library molecules comprise at least a first and a second sub-population of covalently closed circular library molecules. In some embodiments, the insert regions of individual covalently closed circular library molecules of the first sub-population comprise a target or non-target sequence from a top strand of an input nucleic acid. In some embodiments, the insert regions of individual covalently closed circular library molecules of the second sub-population comprise a target or non-target sequence from a bottom strand of an input nucleic acid.

[00186] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual covalently closed circular library molecule of the first sub-population (e.g., top strand insert sequence).

[00187] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual covalently closed circular library molecule of the second sub-population (e.g., bottom strand insert sequence).

[00188] In some embodiments, prior to step (b), at least one of the covalently closed circular library molecules immobilized to the capture support comprises at least one deaminated nucleotide base. In some embodiments, the covalently closed circular library molecules carrying at least one deaminated nucleotide base can be removed from the plurality by subjecting the plurality of covalently closed circular library molecules to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (b) comprises contacting in-solution the plurality of covalently closed circular library molecules with a reagent that removes deaminated bases and generates gaps, thereby converting the at least one deaminated nucleotide base in the at least one covalently closed circular library molecule into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity, as described herein. In some embodiments, prior to contacting a plurality of covalently closed circular library molecules to the plurality of target-specific baits/probes that are immobilized to the capture support, the plurality of covalently closed circular library molecules can be contacted with the reagent that removes deaminated bases and generates gaps. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (b) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from covalently closed circular library molecules.

[00189] In some embodiments, in step (b) the density of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁵ closed circle library bait complexes per mm². In some embodiments, density of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library bait complexes per mm², or any range therebetween.

[00190] In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00191] In some embodiments, the selective hybridization of step (b) generates a mixture comprising a plurality of immobilized closed circle library bait complexes which carry target sequences and a plurality of covalently closed circular library molecules which carry nontarget sequences. In some embodiments, step (b) comprises removing the plurality of covalently closed circular library molecules from the capture support by contacting the capture support with a wash reagent, thereby removing the plurality of covalently closed circular library molecules and retaining the plurality of immobilized closed circle library bait complexes. In some embodiments, the washing of step (b) can generate a plurality of immobilized closed circle library bait complexes that are enriched for polynucleotides having target sequences.

[00192] In some embodiments, in step (b), the capture support comprises a plurality of pinning primers. In some embodiments, in step (b), the capture support lacks a plurality of pinning primers. In some embodiments, step (b) comprises contacting the capture support with the plurality of pinning primers and the plurality of covalently closed circular library molecules in any order. In some embodiments, step (b) comprises contacting the capture support with the plurality of pinning primer and the plurality of covalently closed circular library molecules essentially simultaneously.

[00193] In some embodiments, the method comprises step (c): contacting the plurality of closed circle library bait complexes immobilized to the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target-specific baits/probes (e.g., which are part of the immobilized closed circle library bait complexes) and using the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support, also referred to as “immobilized concatemer template molecules”). In some embodiments, individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region (target sequence) and any universal adaptor sequences present in the covalently closed circular library molecule. In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers. In some embodiments, individual pinning primers hybridize to a portion of an immobilized concatemer template molecule, thereby pinning down a portion of a concatemer template molecule.

[00194] In some embodiments, in step (d), the rolling circle amplification reagent further comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. The rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

[00195] In some embodiments, prior to the sequencing of step (d) described below, additional target-specific baits/probes and/or additional covalently closed circular library molecules can be immobilized to the capture support by conducting a re-seeding workflow. For example, re-seeding steps (1) - (3) can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding a capture support are described herein.

[00196] In some embodiments, the methods comprise step (d): sequencing at least a portion of individual immobilized concatemer template molecules in the plurality of concatemer template molecules immobilized to the capture support. In some embodiments, at least a portion of the concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein.

[00197] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis) and U.S. published application No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the distributing of step (a) and the hybridizing of step (b) ( the contents of both documents are hereby incorporated by reference in their entireties).

[00198] In some embodiments, after the sequencing of step (d) described above, additional covalently closed circular library molecules can be hybridized to target-specific baits/probes that are immobilized to the capture support by conducting a re-seeding workflow. For example, steps (5) - (6) of a re-seeding workflow in which covalently closed circular library molecules can be distributed onto a capture support having immobilized target-specific baits/probes can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding are described herein.

(3) Enrichment by Conducting In-Solution Capture of Linear Library Molecules

[00199] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00200] In some embodiments, in step (a), the capture support comprises a plurality of pinning primers immobilized to the capture support. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end. In some embodiments, individual pinning primers comprise blocking group at the 3’ end. In some embodiments, the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise non-extendible 3’ ends. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture surface lacks a plurality of immobilized pinning primers.

[00201] In some embodiments, the methods comprise step (b): forming a plurality of library bait complexes by contacting a plurality of target-specific baits/probes to a plurality of linear library molecules. In some embodiments, the plurality of linear library molecules comprises a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences. In some embodiments, individual linear library molecules comprise (i) an insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, the contacting is conducted in-solution under a condition suitable for selectively hybridizing individual target-specific baits/probes to at least a portion of a target sequence, thereby generating a plurality of library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of a target sequence of a corresponding linear library molecule, an affinity moiety at the 5’ end, and an extendible 3’ end. In some embodiments, the 3’ ends of individual targetspecific baits/probes comprise moieties that promote polymerase-catalyzed extension of the 3’ end. In some embodiments, the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual targetspecific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual target-specific baits/probes can be located at the 5’ end or at an internal position. In some embodiments, the plurality of linear library molecules comprising non-target sequences do not selectively hybridize to the target-specific baits/probes.

[00202] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, or about 12-16 hours.

[00203] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, about 55-60 degrees C, about 60- 65 degrees C, about 65-70 degrees C, about 70-75 degrees C, or about 75-80 degrees C.

[00204] In some embodiments, in step (b), the plurality of target-specific baits/probes and the plurality of linear library molecules can be hybridized in-solution in a hybridization reagent under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes thereby forming a plurality of library bait complexes.

[00205] In some embodiments, in step (b), individual linear library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual linear library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b).

[00206] In some embodiments, in step (b), the plurality of linear library molecules can be generated from double-stranded input nucleic acids comprising top strands and complementary bottom strands. In some embodiments, the plurality of linear library molecules comprises at least a first and a second sub-population of linear library molecules. In some embodiments, the inert regions of individual linear library molecules of the first subpopulation comprise a target or non-target sequence from a top strand of an input nucleic acid. In some embodiments, the inert regions of individual linear library molecules of the second sub-population comprise a target or non-target sequence from a bottom strand of an input nucleic acid.

[00207] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of individual linear library molecules of the first sub-population (e.g., top strand insert sequence).

[00208] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of individual linear library molecules of the second sub-population (e.g., bottom strand insert sequence). [00209] In some embodiments, in step (b) the mixture of linear library molecules comprises target-specific sequences and non-target sequences. In some embodiments, in step (b), the mixture of linear library molecules comprises 2-10,000 different target sequences. In some embodiments, in step (b), the mixture of linear library molecules comprises 2-1,000,000 different target sequences, 2-500,000 different target sequences, 2-100,000 different target sequences, 100-100,000 different target sequences, 500-10,000 different target sequences, 2- 500 different target sequences, or 1,000-50,000 different target sequences, or any range therebetween. In some embodiments, in step (b), the mixture of linear library molecules comprises up to 1,000,000 target sequences. In some embodiments, the plurality of targetspecific baits/probes comprise 2-10,000 different target-specific sequences. In some embodiments, the plurality of target-specific baits/probes comprises 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, in the plurality of library bait complexes, the plurality of target-specific baits/probes comprise 2-10,000 different target-specific sequences. In some embodiments, in the plurality of library bait complexes, the plurality of targetspecific baits/probes comprises 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500- 10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[00210] In some embodiments, the selective hybridization of step (b) generates a mixture comprising a plurality of library bait complexes comprising target sequences and a plurality of linear library molecules comprising non-target sequences. In some embodiments, the mixture of library bait complexes and linear library molecules can be contacted with paramagnetic beads coated with receptor moieties that can bind to the affinity moieties on the library bait complexes, thereby separating the target library bait complexes from the linear library molecules. In some embodiments, the paramagnetic beads that are bound to the library bait complexes can be washed to remove the plurality of non-target linear library molecules. In some embodiments, the target library bait complexes can be released from the paramagnetic beads, thereby generating a plurality of library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the plurality of linear library molecules are not removed in step (b). In some embodiments, the plurality of non- target linear library molecules are not removed in step (b) using paramagnetic beads that are coated with receptor moieties. In some embodiments, paramagnetic beads coated with receptor moieties are not used at step (b). In some embodiments, the plurality of non-target linear library molecules can be removed in step (c) as described below.

[00211] In some embodiments, the method comprises step (c): contacting the capture support with the plurality of library bait complexes thereby generating a plurality of library bait complexes immobilized to the capture support. In some embodiments, the contacting is conducted under a condition suitable for binding an affinity moiety of individual targetspecific baits/probes to a receptor moiety of the capture support. In some embodiments, the plurality of immobilized library bait complexes are enriched for polynucleotides carrying target sequences. In some embodiments, the plurality of library bait complexes can be distributed/contacted with the capture support in a loading reagent and/or in a hybridization reagent under a condition suitable for binding the affinity moieties of individual library bait complexes to individual receptor moieties embedded in the hydrophilic polymer coating of the capture support, thereby generating a plurality of library bait complexes immobilized to the capture support.

[00212] In some embodiments, in step (c), the capture support is contacted with the plurality of library bait complexes and residual non-target linear library molecules from step (b). In some embodiments, step (c) comprises contacting the capture support with a wash reagent to remove the residual non-target linear library molecules and retain the plurality of library bait complexes immobilized to the capture support (“immobilized library bait complexes”). In some embodiments, the washing of step (c) can generate a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences.

[00213] In some embodiments, in step (c) the density of library bait complexes immobilized to the capture support is about 10² - 10¹⁵ library bait complexes per mm². In some embodiments, density of library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ library bait complexes per mm², or any range therebetween.

[00214] In some embodiments, the plurality of library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00215] In some embodiments, in step (c), the capture support comprises a plurality of pinning primers. In some embodiments, in step (c), the capture support lacks pinning primers. In some embodiments, in step (c), the capture support can be contacted separately with a plurality of pinning primers and the plurality of library bait complexes in any order. In some embodiments, the capture support can be contacted with a plurality of pinning primers and the plurality of library bait complexes essentially simultaneously.

[00216] In some embodiments, the method comprises forming a plurality of immobilized circle bait complexes using top strand circularization oligonucleotides (e.g., step (dl) or step (d2) both of which are described below).

[00217] In some embodiments, the method comprises circularizing the linear library molecules of the library bait complexes by contacting the library bait complexes immobilized to the capture support with single-stranded or double-stranded circularization oligonucleotides as described in steps (dl), (d2) or (d3).

[00218] In some embodiments, the method comprises step (dl): forming a plurality of closed circle library bait complexes immobilized to the capture support by contacting the plurality of library bait complexes with a plurality of single-stranded top strand circularization oligonucleotides. In some embodiments, the contacting is conducted under a condition suitable for hybridizing the ends of individual linear library molecules (e.g., which are part of individual library bait complexes) with individual single-stranded top strand circularization oligonucleotides to form individual open circle library bait complexes each having one nick (e.g., see FIG. 36A). In some embodiments, the one nick is enzymatically ligatable. In some embodiments, the 5’ ends of individual linear library molecules comprise a phosphate group. In some embodiments, step (dl) comprises enzymatically ligating the nicks, thereby generating a plurality of covalently close circular library molecules hybridized to a target-specific bait/probe immobilized to the capture support, thereby forming a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, individual top strand circularization oligonucleotides comprise a first region that can hybridize with a sequence at one end of a linear library molecule (e.g., FIG. 36 A, region (730)), and a second region that can hybridize with a sequence at the other end of the linear library molecule (e.g., FIG. 36A, region (720)). In some embodiments, the one nick can be contacted with a ligation reagent to close the nick. In some embodiments, the plurality of closed circle library bait complexes immobilized to the capture support that are generated in step (dl) can be subjected to a rolling circle amplification reaction as described in step (e) below.

[00219] In some embodiments, in step (dl), the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36A) that can bind a receptor moiety of the capture support.

[00220] In some embodiments, in step (dl), individual single-stranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that inhibits polymerase-catalyzed extension. In some embodiments, in step (dl), individual singlestranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that promotes polymerase-catalyzed extension.

[00221] In some embodiments, in step (dl), after enzymatically ligating the nicks, at least one of the closed circle library bait complexes comprises a covalently closed circular library molecule having at least one deaminated nucleotide base. In some embodiments, the covalently closed circular library molecule having at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (dl) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps, thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity as described herein. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (dl) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules. In some embodiments, the method comprises step (d2): forming a plurality of closed circle library bait complexes immobilized to the capture support by contacting the plurality of library bait complexes immobilized to the capture support with a plurality of double-stranded top strand circularization oligonucleotides comprising a long strand and a short strand, wherein the long and short strands are hybridized together to form double-stranded top strand circularization oligonucleotides having a double-stranded region and two flanking single-stranded regions. In some embodiments, the contacting is conducted under a condition suitable for hybridizing individual library bait complexes to individual double-stranded top circularization oligonucleotides, wherein one end of the linear library molecule (e.g. which is part of individual library bait complexes) hybridizes to one end of the long strand and the other end of the linear library molecule hybridizes to the other end of the long strand, thereby forming an open circle library bait complex having two nicks (e.g., see FIG. 36C). In some embodiments, the two nicks are enzymatically ligatable. In some embodiments, the 5’ ends of the linear library molecules comprise a phosphate group. In some embodiments, step (d2) comprises enzymatically ligating the two nicks, thereby generating a plurality of covalently closed circular library molecules hybridized to immobilized target-specific baits/probes, thereby forming a plurality of closed circle library bait complexes immobilized to the capture support.

[00222] In some embodiments, in step (d2), individual double-stranded top strand circularization oligonucleotides comprise a long strand having a first region that can hybridize with a sequence at one end of a linear library molecule (e.g., FIG. 36C, region (730)), and a second region that can hybridize with a sequence at the other end of the linear library molecule (e.g., FIG. 36C, region (720)). In some embodiments, the two nicks of step (d2) can be contacted with a ligation reagent to close the nicks. In some embodiments, the plurality of closed circle library bait complexes immobilized to the capture support that are generated in step (d2) can be subjected to a rolling circle amplification reaction as described in step (e) below.

[00223] In some embodiments, in step (d2), the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36C) that can bind a receptor moiety of the capture support.

[00224] In some embodiments, the ligation reagent of steps (dl) and (d2) comprise a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1). In some embodiments, the ligation reagent of steps (dl) and (d2) comprise a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North® DNA ligase (e.g., from New England Biolabs, catalog No. M0238S). In some embodiments the ligation reagent of steps (dl) and (d2) comprise a recombinant thermal tolerant T4 DNA ligase including Hi-T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S). In some embodiments, the ligation reagent of steps (dl) and (d2) comprise a DNA ligase from Thermococcus nautili (e.g., NCBI No.

WP 042693257.1). In some embodiments, the ligation reagent of steps (dl) and (d2) comprises a T4 polynucleotide kinase.

[00225] In some embodiments, in step (d2), the long strand of individual double-stranded top strand circularization oligonucleotides comprises a 3’ end comprising a moiety that inhibits polymerase-catalyzed extension. In some embodiments, in step (d2), the long strand of individual double-stranded top strand circularization oligonucleotides comprises a 3’ end comprising a moiety that promotes polymerase-catalyzed extension.

[00226] In some embodiments, in step (d2), after enzymatically ligating the two nicks, at least one of the closed circle library bait complexes comprises a covalently closed circular library molecule having at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule having at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (d2) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps, thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (d2) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules. In some embodiments, the method comprises forming a plurality of closed circle library bait complexes immobilized to the capture support using single-stranded top strand circularization oligonucleotides carrying additional sequences (e.g., step (d3)). In some embodiments, the method comprises step (d3): forming a plurality of closed circle library bait complexes immobilized to the capture support by contacting the plurality of library bait complexes immobilized to the capture support with a plurality of single-stranded top strand circularization oligonucleotides. In some embodiments, the contacting is conducted under a condition suitable for hybridizing the ends of individual linear library molecules (e.g., which are part of individual library bait complexes) with individual single-stranded top strand circularization oligonucleotides to form individual open circle library bait complexes having a gap between the 5’ and 3’ ends of individual linear library molecules (e.g., see FIG. 36B). In some embodiments, the gap can be 2-100 nucleotides in length. In some embodiments, the gap can be 10-100 nucleotides in length, 2- 50 nucleotides in length, 10-30 nucleotides in length, 2-15 nucleotides in length, 20-80 nucleotides in length, or any range therebetween. In some embodiments, the gap can be subjected to a polymerase-catalyzed fill-in reaction to generate a nick. In some embodiments, the nick can be contacted with a ligation reagent to close the nick. In some embodiments, the 5’ end of individual linear library molecules comprise a phosphate group.

[00227] In some embodiments, in step (d3), individual single-stranded top strand circularization oligonucleotides comprise (i) a first region at one end having a sequence that can hybridize with at least a portion of a universal adaptor sequence at one end of a linear library molecule (e.g., FIG. 36B, region (730)), (ii) a second region (2^nd region) comprising at least one index sequence and/or an additional universal adaptor sequence, and (iii) a third region at the other end having a sequence that can hybridize with at least a portion of a universal adaptor sequence at the other end of the linear library molecule (e.g., FIG. 36B, region (720)). In some embodiments, the single-stranded top strand circularization oligonucleotides comprise first and third regions that flank the second region. In some embodiments, the linear library molecule lacks a sequence that can hybridize with the second region of the single-stranded top strand circularization oligonucleotide. In some embodiments, the second region of individual single-stranded top strand circularization oligonucleotides comprises a sample index sequence, a unique molecular index sequence and/or an additional universal adaptor sequence. In some embodiments, the additional universal adaptor sequence comprises a universal adaptor sequence for binding a compaction oligonucleotide, a universal adaptor sequence for binding a forward sequencing primer, or a universal adaptor sequence for binding a reverse sequencing primer.

[00228] In some embodiments, step (d3) comprises contacting the gap of individual open circle library bait complexes with a fill-in reagent and conducting a polymerase-catalyzed fill-in reaction using the second region of the single-stranded top strand circularization oligonucleotide as a template sequence. In some embodiments, the polymerase-catalyzed fill- in reaction generates open circle library bait complexes each having a nick between the 5’ and 3’ ends of individual linear library molecules. In some embodiments, the fill-in reaction can generate an open circle library bait complex comprising a sequence that is complementary to the second region (2^nd region) of the single-stranded top strand circularization oligonucleotide. In some embodiments, step (d3) comprises contacting the nick of individual open circle library bait complexes with a ligation reagent and enzymatically ligating the nick thereby generating a plurality of covalently close circular library molecules hybridized to immobilized target-specific baits/probes, thereby forming a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the plurality of closed circle library bait complexes immobilized to the capture support that are generated in step (d3) can be subjected to a rolling circle amplification reaction as described in step (e) below.

[00229] In some embodiments, in step (d3), the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36B) that can bind a receptor moiety of the capture support.

[00230] In some embodiments, the fill-in reagent of step (d3) comprises a plurality of nucleotides and a plurality of DNA polymerases. In some embodiments, the plurality of polymerases comprises: a Phusion® High-Fidelity DNA Polymerase; the Stoffel fragment of the AmpliTaq DNA polymerase (e.g., from Applied Biosystems); a Q5® High-Fidelity DNA Polymerase; a Hercules II fusion DNA polymerase (a fusion polymerase); an iProof High Fidelity DNA polymerase (a fusion polymerase); a Taq DNA polymerase; a Pfu DNA polymerase; a Pwo DNA polymerase; an Omni Klentaq LA DNA polymerase; an Omni Klentaq DNA polymerase; or a Kapa HiFi DNA polymerase. In some embodiments, the plurality of nucleotides of the fill-in reagent of step (d3) comprises any combination of two or more of dATP, dGTP, dCTP, dTTP and/or dUTP.

[00231] In some embodiments, the ligation reagent of step (d3) comprises a ligase enzyme. In some embodiments, the ligase enzyme comprises a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1). In some embodiments, the ligase enzyme comprises a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs®, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S). In some embodiments the ligase enzyme comprises a recombinant thermal tolerant T4 DNA ligase including Hi-T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S). In some embodiments, the ligase enzyme comprises a DNA ligase from Thermococcus nautili (e.g., NCBI No. WP_042693257.1). In some embodiments, the ligation reagent of step (d3) comprises a T4 polynucleotide kinase.

[00232] In some embodiments, in step (d3), individual single-stranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that inhibits polymerase-catalyzed extension. In some embodiments, in step (d3), individual singlestranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that promotes polymerase-catalyzed extension.

[00233] In some embodiments, in step (d3), after conducting the polymerase-catalyzed fill- in reaction and enzymatically ligating the nick to generate a plurality of closed circle library bait complexes comprising covalently closed circular library molecules, at least one of the covalently closed circular library molecules comprises at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule comprising at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (d3) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps, thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (d3) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules. In some embodiments, the method comprises step (e): contacting the plurality of closed circle library bait complexes immobilized to the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes (e.g., which are part of the closed circle library bait complexes immobilized to the capture support) and use the covalently closed circular library molecules as template molecules thereby generating a plurality of concatemer template molecules which are immobilized to the capture support ( “immobilized concatemer template molecules”). In some embodiments, individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region and any universal adaptor sequences present in a covalently closed circular library molecule. In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers, wherein individual pinning primers hybridize to a portion of an immobilized concatemer template molecule thereby pinning down a portion of a concatemer template molecule. [00234] In some embodiments, in step (e), the rolling circle amplification reagent comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. The rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

In some embodiments, the methods comprise step (f): sequencing at least a portion of individual concatemer template molecules in the plurality of concatemer template molecules immobilized to the capture support. In some embodiments, at least a portion of the concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two- stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein.

[00235] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis”) and U.S. Application Publication No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of step (b) and the distributing of step (c) (the contents of both documents are hereby incorporated by reference in their entireties).

[00236] In some embodiments, after the sequencing of step (f) described above, the capture support can be subjected to a re-seeding workflow in which additional linear library molecules can be hybridized in-solution to target-specific baits/probes to generate a plurality of library bait complexes, and the library bait complexes can be circularized to generate a plurality of closed circle library bait complexes that are distributed onto the capture support. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (c) and (dl) at least once. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (c) and (d2) at least once. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (c) and (d3) at least once. In some embodiments, steps (6) - (8) of a re-seeding workflow can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding are described herein. In some embodiments, in steps (dl) and (d3), individual single-stranded top strand circularization oligonucleotides comprise a single stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. The ends of a single-stranded top strand circularization oligonucleotide can hybridize to the ends of a top strand of a double stranded linear library molecule from a double stranded input nucleic acid dissociated into a top strand and a bottom strand, to circularize the top strand of the linear library molecule and generate an open circle library complex having a nick between the ends of the linear library molecule. The single-stranded top strand circularization oligonucleotides exhibit little or no hybridization to a bottom strand linear library molecule. In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences. In some embodiments, the single-stranded top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a left sample index sequence of the top strand of a linear library molecule. In some embodiments, the singlestranded top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a right sample index sequence of the top strand of a linear library molecule. In some embodiments the anchor sequence can be located at the 3’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at the other end of the same top strand of the linear library molecule. In some embodiments the anchor sequence can be located at the 5’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at another end of the top strand of the linear library molecule. In some embodiments, the 3’ end of the single-stranded top strand circularization oligonucleotide comprises a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the single-stranded top strand circularization oligonucleotide. In some embodiments, the 3’ end of the single-stranded top strand circularization oligonucleotide comprises a moiety that promotes polymerase- catalyzed extension of the 3’ end of the single-stranded top strand circularization oligonucleotide. In some embodiments, the single-stranded top strand circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-99. In some embodiments, the sequence of the single-stranded top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to the sequences according to any of SEQ ID NOS: 45-99.

[00237] In some embodiments, in an alternative workflow, the plurality of library bait complexes at step (b) can be contacted in-solution with the plurality of single-stranded or double-stranded top strand circularization oligonucleotides, thereby generating a plurality of open circle library bait complexes having one nick (e.g., FIG. 36A), or generating a plurality of open circle library bait complexes having a gap (e.g., FIG. 36B), or generating a plurality of open circle library bait complexes having two nicks (e.g., FIG. 36C). In some embodiments, the plurality of open circle library bait complexes can be contacted/deposited with the capture support to generate a plurality of open circle library bait complexes immobilized to the capture support. In some embodiments, the immobilized open circle library bait complexes can be subjected to nick ligation as described in steps (dl) and (d2), or can be subjected to gap fill-in and nick ligation reactions as described in step (d3), to generate a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the plurality of closed circle library bait complexes can be subjected to rolling circle amplification as described in step (e) to generating a plurality of concatemer template molecules immobilized to the capture support. In some embodiments, the plurality of immobilized concatemer template molecules can be subjected to sequencing reaction as described in step (f).

(4) Enrichment by Conducting On-Support Capture of Linear Library Molecules

[00238] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00239] In some embodiments, in step (a), the capture support comprises a plurality of immobilized target-specific baits/probes, wherein individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of a target sequence of a linear library molecule, an affinity moiety at the 5’ end, and an extendible 3’ end. In some embodiments, the 3’ ends of individual target-specific baits/probes comprise a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, the plurality of target-specific baits/probes comprises DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moieties of individual target-specific baits/probes can bind to a receptor moiety of the capture support. In some embodiments, in step (a), the affinity moiety of individual target-specific baits/probes comprises biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moieties of individual target-specific baits/probes can be located at the 5’ ends or at an internal positions.

[00240] In some embodiments, the plurality of target-specific baits/probes can be distributed/contacted with the capture support in a loading reagent under a condition suitable for binding the affinity moieties of individual target-specific baits/probes to individual receptor moieties embedded in the hydrophilic polymer coating of the capture support, thereby generating a capture support comprising a plurality of target-specific baits/probes immobilized to the capture support.

[00241] In some embodiments, in step (a), the capture support comprises a plurality of immobilized pinning primers immobilized to the capture support. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence, and an affinity moiety at the 5’ end. In some embodiments, individual pinning primers comprise blocking group at the 3’ end. In some embodiments, the blocking group inhibits polymerase-catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprises a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture surface lacks a plurality of immobilized pinning primers.

[00242] In some embodiments, the methods comprise step (b): forming a plurality of library bait complexes immobilized to the capture support by contacting a plurality of linear library molecules to the plurality of target-specific baits/probes that are immobilized to the capture support, wherein the plurality of linear library molecules comprise a mixture of linear library molecules comprising target sequences and linear library molecules comprising nontarget sequences. In some embodiments, individual linear library molecules comprise (i) an insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, the contacting is conducted on the capture support under a condition suitable for selectively hybridizing individual target-specific baits/probes to at least a portion of a target sequence thereby generating a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the plurality of linear library molecules comprising nontarget sequences do not selectively hybridize to the immobilized target-specific baits/probes. In some embodiments, the plurality of linear library molecules can be distributed/contacted with the plurality of target-specific baits/probes that are immobilized to the capture support in a loading reagent and/or a hybridization reagent, under a condition suitable for hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes that are immobilized to the capture support, thereby generating a plurality of immobilized library -bait complexes.

[00243] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual linear library molecules to their cognate target-specific baits/probes that are immobilized to the capture support comprises conducting an on-support hybridization reaction for about 1-15 minutes, about 15- 30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, or about 12-16 hours.

[00244] In some embodiments, in step (b), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual linear library molecules to corresponding target-specific baits/probes that are immobilized to the capture support comprises conducting an on-support hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about SO- 55 degrees C, about 55-60 degrees C, about 60-65 degrees C, about 65-70 degrees C, about 70-75 degrees C, or about 75-80 degrees C. [00245] In some embodiments, in step (b), the mixture of linear library molecules comprises target sequences and non-target sequences. In some embodiments, in step (b), the mixture of linear library molecules comprises 2-10,000 different target sequences. In some embodiments, in step (b), the mixture of linear library molecules comprises 2-500,000 different target sequences, 2-100,000 different target sequences, 100-100,000 different target sequences, 500-10,000 different target sequences, 2-500 different target sequences, or 1,000- 50,000 different target sequences, or any range therebetween. In some embodiments, in step (b), the mixture of linear library molecules comprises up to 1,000,000 different target sequences. In some embodiments, the plurality of target-specific baits/probes comprise 2- 10,000 different target-specific sequences. In some embodiments, the plurality of targetspecific baits/probes comprises 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100- 100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2- 500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, in the plurality of library -bait complexes, the plurality of target-specific baits/probes comprise 2-10,000 different target-specific sequences. In some embodiments, in the plurality of library -bait complexes, the plurality of target-specific baits/probes comprises 2-500,000 different target-specific sequences, 2- 100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[00246] In some embodiments, in step (b), individual linear library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual linear library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b).

[00247] In some embodiments, in step (b), the plurality of linear library molecules can be generated from double-stranded input nucleic acids comprising top strands and complementary bottom strands. In some embodiments, the plurality of linear library molecules comprises at least a first and a second sub-population of linear library molecules. In some embodiments, the inert regions of individual linear library molecules of the first subpopulation comprise a target or non-target sequence from a top strand of an input nucleic acid. In some embodiments, the inert regions of individual linear library molecules of the second sub-population comprise a target or non-target sequence from a bottom strand of an input nucleic acid.

[00248] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual linear library molecule of the first sub-population (e.g., top strand insert sequence). [00249] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual linear library molecule of the second sub-population (e.g., bottom strand insert sequence).

[00250] In some embodiments, in step (b) the density of library bait complexes immobilized to the capture support is between about 10² and about 10¹⁵ library bait complexes per mm². In some embodiments, density of library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ library bait complexes per mm², or any range therebetween.

[00251] In some embodiments, the plurality of library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00252] In some embodiments, the selective hybridization of step (b) generates a mixture comprising a plurality of immobilized library bait complexes which carry target sequences and a plurality of linear library molecules which carry non-target sequences. In some embodiments, step (b) comprises removing the plurality of linear library molecules from the capture support by contacting the capture support with a wash reagent, thereby removing the plurality of linear library molecules and retaining the plurality of immobilized library bait complexes. In some embodiments, the washing of step (b) can generate a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences.

[00253] In some embodiments, in step (b), the capture support comprises a plurality of pinning primers or lacks a plurality of pinning primers. In some embodiments, in step (b), the capture support can be contacted separately with the plurality of pinning primers and the plurality of linear library molecules in any order. In some embodiments, the capture support can be contacted with the plurality of pinning primers and the plurality of linear library molecules essentially simultaneously.

[00254] In some embodiments, the method comprises circularizing the linear library molecules of the library bait complexes immobilized to the capture support by contacting the library bait complexes with single-stranded or double-stranded circularization oligonucleotides as described in steps (cl), (c2) or (c3).

[00255] In some embodiments, the method comprises step (cl): forming a plurality of closed circle library bait complexes immobilized to the capture support by contacting the plurality of library bait complexes with a plurality of single-stranded top strand circularization oligonucleotides. In some embodiments, the contacting can be conducted under a condition suitable for hybridizing the ends of individual linear library molecules (e.g., which are part of individual library -bait complexes) with individual top strand circularization oligonucleotides to form individual open circle library bait complexes having one nick (e.g., see FIG. 36A). In some embodiments, the one nick is enzymatically ligatable. In some embodiments, the 5’ end of individual linear library molecules comprises a phosphate group. In some embodiments, step (cl) comprises enzymatically ligating the nicks, thereby generating a plurality of covalently closed circular library molecules hybridized to targetspecific baits/probes immobilized to the capture support, thereby forming a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, individual top strand circularization oligonucleotides comprise a first region that can hybridize with a sequence at one end of a linear library molecule (e.g., FIG. 36A, region (730)), and a second region that can hybridize with a sequence at the other end of the linear library molecule (e.g., FIG. 36A, region (720)). In some embodiments, the one nick can be contacted with a ligation reagent to close the nick. In some embodiments, the plurality of closed circle library bait complexes immobilized to the capture support that are generated in step (cl) can be subjected to a rolling circle amplification reaction as described in step (d) below.

[00256] In some embodiments, in step (cl), the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36A) that can bind a receptor moiety of the capture support.

[00257] In some embodiments, in step (cl), individual single-stranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that inhibits polymerase-catalyzed extension. In some embodiments, in step (cl), individual singlestranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that promotes polymerase-catalyzed extension.

[00258] In some embodiments, in step (cl), after enzymatically ligating the nicks, at least one of the closed circle library bait complexes comprises a covalently closed circular library molecule having at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule having at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (cl) comprises contacting the plurality of closed circle library bait complexes with a reagent that removes deaminated bases and generates gaps, thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (cl) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules.

[00259] In some embodiments, the method comprises step (c2): forming a plurality of closed circle library bait complexes immobilized to the capture support by contacting the plurality of immobilized library bait complexes with a plurality of double-stranded top strand circularization oligonucleotide each comprising a long strand and a short strand (e.g., FIG. 36C). In some embodiments, the long and short strands are hybridized together to form the double-stranded top strand circularization oligonucleotide having a double-stranded region and two flanking single-stranded regions. In some embodiments, the contacting is conducted under a condition suitable for hybridizing one end of the linear library molecule to one end of the long strand and suitable for hybridizing the other end of the linear library molecule to the other end of the long strand thereby forming an open circle library bait complex having two nicks (e.g., see FIG. 36C). In some embodiments, the two nicks are enzymatically ligatable. In some embodiments, step (c2) comprises enzymatically ligating the two nicks, thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, individual doublestranded top strand circularization oligonucleotides comprise a long strand having a first region that can hybridize with a sequence at one end of a linear library molecule (e.g., FIG. 36C, region (730)), and a second region that can hybridize with a sequence at the other end of the linear library molecule (e.g., FIG. 36C, region (720)). In some embodiments, the two nicks can be contacted with a ligation reagent to close the nicks. In some embodiments, the plurality of closed circle library bait complexes immobilized to the capture support that are generated in step (c2) can be subjected to a rolling circle amplification reaction as described in step (d) below.

[00260] In some embodiments, in step (c2), the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36C) that can bind a receptor moiety of the capture support.

[00261] In some embodiments, the ligation reagent of steps (cl) and (c2) comprise a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1). In some embodiments, the ligation reagent of steps (cl) and (c2) comprise a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S). In some embodiments the ligation reagent of steps (cl) and (c2) comprise a recombinant thermal tolerant T4 DNA ligase including Hi-T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S). In some embodiments, the ligation reagent of steps (cl) and (c2) comprise a DNA ligase from Thermococcus nautili (e.g., NCBI No.

WP 042693257.1). In some embodiments, the ligation reaction of steps (cl) and (c2) comprise a T4 polynucleotide kinase. [00262] In some embodiments, in step (c2), the long strand of individual double-stranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that inhibits polymerase-catalyzed extension. In some embodiments, in step (c2), the long strand of individual double-stranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that promotes polymerase-catalyzed extension.

[00263] In some embodiments, in step (c2), after enzymatically ligating the two nicks, at least one of the closed circle library bait complexes comprises a covalently closed circular library molecule having at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule having at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (c2) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (c2) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules.

[00264] In some embodiments, the method comprises forming a plurality of closed circle library bait complexes immobilized to the capture support using top strand circularization oligonucleotides carrying additional sequences (e.g., step (c3)). In some embodiments, the method comprises step (c3): forming a plurality of closed circle library bait complexes immobilized to the capture support by contacting the plurality of library bait complexes immobilized to the capture support with a plurality of single-stranded top strand circularization oligonucleotides. In some embodiments, the contacting is conducted under a condition suitable for hybridizing the ends of individual linear library molecules (e.g., which are part of individual library bait complexes) with individual single-stranded top strand circularization oligonucleotides to form individual open circle library bait complexes having a gap between the 5’ and 3’ ends of individual linear library molecules (e.g., see FIG. 36B). In some embodiments, the gap can be 2-100 nucleotides in length. In some embodiments, the gap can be 10-100 nucleotides in length, 2-50 nucleotides in length, 10-30 nucleotides in length, 2-15 nucleotides in length, or 20-80 nucleotides in length, or any range therebetween. In some embodiments, the gap can be subjected to a polymerase-catalyzed fill-in reaction to generate a nick. In some embodiments, the nick can be contacted with a ligation reagent to close the nick thereby generating the plurality of closed circle library bait complexes. In some embodiments, the 5’ end of individual linear library molecules comprise a phosphate group. [00265] In some embodiments, in step (c3), individual top strand circularization oligonucleotides comprise (i) a first region at one end having a sequence that can hybridize with at least a portion of a universal adaptor sequence at one end of a linear library molecule (e.g., FIG. 36B, region (730)), (ii) a second region (2^nd region) comprising at least one index sequence and/or an additional universal adaptor sequence, and (iii) a third region at the other end of the top strand circularization oligonucleotide having a sequence that can hybridize with at least a portion of a universal adaptor sequence at the other end of the linear library molecule (e.g., FIG. 36B, region (720)). In some embodiments, the single-stranded top strand circularization oligonucleotides comprise first and third regions that flank the second region. In some embodiments, the linear library molecule lacks a sequence that can hybridize with the second region of the single-stranded top strand circularization oligonucleotide. In some embodiments, the second region of individual top strand circularization oligonucleotides comprises a sample index sequence, a unique molecular index sequence and/or an additional universal adaptor sequence. In some embodiments, the additional universal adaptor sequence comprises a universal adaptor sequence for binding a compaction oligonucleotide, a universal adaptor sequence for binding a forward sequencing primer, or a universal adaptor sequence for binding a reverse sequencing primer.

[00266] In some embodiments, step (c3) comprises contacting the gap of individual open circle library bait complexes with a fill-in reagent and conducting a polymerase-catalyzed fill-in reaction using the second region of the single-stranded top strand circularization oligonucleotide as a template sequence. In some embodiments, the polymerase-catalyzed fill- in reaction generates open circle library bait complexes each having a nick between the 5’ and 3’ ends of individual linear library molecules. In some embodiments, the fill-in reaction can generate an open circle library bait complex comprising a sequence that is complementary to the second region (2^nd region) of the single-stranded top strand circularization oligonucleotide. In some embodiments, step (c3) comprises contacting the nick of individual open circle library bait complexes with a ligation reagent and enzymatically ligating the nick, thereby generating a plurality of covalently close circular library molecules each being hybridized to an immobilized target-specific bait/probe thereby forming a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the plurality of closed circle library bait complexes immobilized to the capture support that are generated in step (c3) can be subjected to a rolling circle amplification reaction as described in step (d) below.

[00267] In some embodiments, in step (c3), the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36B) that can bind a receptor moiety of the capture support.

[00268] In some embodiments, the fill-in reagent of step (c3) comprises a plurality of nucleotides and a plurality of DNA polymerases. In some embodiments, the plurality of polymerases comprises: a Phusion High-Fidelity DNA Polymerase; the Stoffel fragment of the AmpliTaq DNA polymerase (e.g., from Applied Biosystems); a Q5 High-Fidelity DNA Polymerase; a Hercules II fusion DNA polymerase (a fusion polymerase); an iProof High Fidelity DNA polymerase (a fusion polymerase); a Taq DNA polymerase; a Pfu DNA polymerase; a Pwo DNA polymerase; an Omni Klentaq LA DNA polymerase; an Omni Klentaq DNA polymerase; or a Kapa HiFi DNA polymerase. In some embodiments, the plurality of nucleotides of the fill-in reagent of step (c3) comprises any combination of two or more of dATP, dGTP, dCTP, dTTP and/or dUTP.

[00269] In some embodiments, the ligation reagent of step (c3) comprises a ligase enzyme. In some embodiments, the ligase enzyme comprises a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1). In some embodiments, the ligase enzyme comprises a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S). In some embodiments, the ligase enzyme comprises a recombinant thermal tolerant T4 DNA ligase including Hi-T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S). In some embodiments, the ligase enzyme comprises a DNA ligase from Thermococcus nautili (e.g., NCBI No. WP_042693257.1). In some embodiments, the ligase enzyme comprises a T4 polynucleotide kinase.

[00270] In some embodiments, in step (c3), individual single-stranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that inhibits polymerase-catalyzed extension. In some embodiments, in step (c3), individual single- stranded top strand circularization oligonucleotides comprise a 3’ end comprising a moiety that promotes polymerase-catalyzed extension.

[00271] In some embodiments, in step (c3), after conducting the polymerase-catalyzed fill- in reaction and enzymatically ligating the nick to generate a plurality of closed circle library bait complexes comprising covalently closed circular library molecules, at least one of the covalently closed circular library molecules comprises at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule carrying at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gapgeneration. In some embodiments, step (c3) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (c3) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules.

[00272] In some embodiments, the method comprises step (d): contacting the plurality of closed circle library bait complexes immobilized to the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes (e.g., which are part of the closed circle library bait complexes immobilized to the capture support) and use the covalently closed circular library molecules as template molecules thereby generating a plurality of concatemer template molecules which are immobilized to the capture support ( “immobilized concatemer template molecules”). In some embodiments, individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region (target sequence) and any universal adaptor sequences present in a given covalently closed circular library molecule. In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers wherein individual pinning primers hybridize to a portion of an immobilized concatemer template molecule thereby pinning down a portion of a concatemer template molecule.

[00273] In some embodiments, in step (d), the rolling circle amplification reagent comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. In some embodiments, the rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

[00274] In some embodiments, the methods comprise step (e): sequencing at least a portion of individual concatemer template molecules of the plurality of concatemer template molecules immobilized to the capture support. In some embodiments, at least a portion of the concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described below, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein. [00275] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis”) and U.S. Application Publication No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the distributing of step (a) and the hybridizing of step (b) ( the contents of both documents are hereby incorporated by reference in their entireties).

[00276] In some embodiments, after the sequencing of step (e) described above, the capture support can be subjected to a re-seeding workflow in which additional linear library molecules can be hybridized to target-specific baits/probes immobilized to a capture support to generate a plurality of library bait complexes immobilized to the capture support, and the immobilized library bait complexes can be circularized to generate a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (b) and (cl) at least once. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (b) and (c2) at least once. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (b) and (c3) at least once. In some embodiments, steps (6) - (8) of a re-seeding workflow can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding are described herein. In some embodiments, in steps (cl) and (c3), individual single-stranded top strand circularization oligonucleotides comprise a single stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. The ends of a single-stranded top strand circularization oligonucleotide can hybridize to the ends of a top strand of a linear library molecule produced by dissociating a double stranded input nucleic acid into a top strand and a bottom strand, to circularize the linear library molecule and generate an open circle library complex having a nick between the ends of the linear library molecule. The single-stranded top strand circularization oligonucleotides exhibit little or no hybridization to a bottom strand linear library molecule. In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences. In some embodiments, the singlestranded top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a left sample index sequence of the top strand of a linear library molecule. In some embodiments, the single-stranded top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a right sample index sequence of the top strand of a linear library molecule. In some embodiments the anchor sequence can be located at the 3’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at the other end of the same top strand of the linear library molecule. In some embodiments the anchor sequence can be located at the 5’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the single-stranded top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at another end of the top strand of the linear library molecule. In some embodiments, the 3’ end of the single-stranded top strand circularization oligonucleotide comprises a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the single-stranded top strand circularization oligonucleotide. In some embodiments, the 3’ end of the single-stranded top strand circularization oligonucleotide comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end of the single-stranded top strand circularization oligonucleotide. In some embodiments, the single-stranded top strand circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-99. In some embodiments, the sequence of the single-stranded top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to the sequences set forth in SEQ ID NOS: 45-99.

(5) Enrichment by Conducting In-Solution Capture of Top Strands of Linear Library Molecules and Circularization Using Top Strand Circularization Oligonucleotides [00277] The present disclosure provides methods for enriching target polynucleotides from a mixture of double-stranded linear library molecules having target or non-target sequences. Individual double-stranded library molecules comprise complementary top and bottom strands. For the sake of clarity, methods for enriching top and bottom strand linear library molecules having target sequences are described separately. Methods for enriching top strand library molecules having target sequences is described herein in workflow (5). Methods for enriching bottom strand library molecules having target sequences is described below in workflow (10). In some embodiments, methods for enriching top and bottom strands having target sequences can be conducted together in the same hybridization reaction (e.g., step (b)) by employing top strand circularization oligonucleotides for enriching the target top strands, and by employing bottom strand blocker oligonucleotides for enriching the target bottom strands, and the resulting enriched target top strands and target bottom strands can be distributed onto the same capture support for conducting rolling circle amplification and sequencing. In some embodiments, the terminal 5’ end of individual top strand linear library molecules include a phosphate group, therefore the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules. In some embodiments, the terminal 5’ end of individual bottom strand linear library molecules lack a phosphate group, therefore the bottom strand linear library molecules cannot undergo intramolecular ligation to form covalently closed circular library molecules. Thus, conducting enrichment workflows (5) and (10) together will yield strand specific circularization of the top strand linear library molecules.

[00278] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00279] In some embodiments, in step (a), the capture support comprises a plurality of pinning primers immobilized to the capture support. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence, and an affinity moiety at the 5’ end. In some embodiments, individual pinning primers comprise a blocking group at the 3’ end. In some embodiments, the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture surface lacks a plurality of immobilized pinning primers. [00280] In some embodiments, the methods comprise step (b): forming a plurality of open circle library complexes by contacting in-solution a plurality of top strand circularization oligonucleotides to a plurality of linear library molecules (e.g., FIG. 36A). In some embodiments, the plurality of linear library molecules comprises a mixture of linear library molecules having insert regions comprising target or non-target sequences. In some embodiments, the plurality of linear library molecules comprise a mixture of linear library molecules generated from double-stranded input nucleic acids comprising insert regions having top strand sequences or complementary bottom strand sequences. In some embodiments, individual top strand linear library molecules comprise (i) a top strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual bottom strand linear library molecules comprise (i) a bottom strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual top strand circularization oligonucleotides can hybridize to at least a portion of a top strand linear library molecule thereby generating an open circle library complex. In some embodiments, individual top strand circularization oligonucleotides do not hybridize to at least a portion of a bottom strand linear library molecule.

[00281] In some embodiments, in step (b), individual top strand circularization oligonucleotides comprise a single stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. The ends of a top strand circularization oligonucleotide can hybridize to the ends of a top strand of a linear library molecule to circularize the linear library molecule and generate an open circle library complex having a nick between the ends of the linear library molecule (e.g., FIG. 36A). The top strand circularization oligonucleotides exhibit little or no hybridization to a bottom strand linear library molecule.

[00282] In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences.

[00283] In some embodiments, the top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a left sample index sequence of the top strand of a linear library molecule. In some embodiments, the top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a right sample index sequence of the top strand of a linear library molecule. [00284] In some embodiments the anchor sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at another end of the top strand of the linear library molecule (e.g., FIG. 36A).

[00285] In some embodiments the anchor sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at another end of the top strand of the linear library molecule (e.g., FIG. 36A).

[00286] In some embodiments, the 3’ end of the top strand circularization oligonucleotide comprises a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the top strand circularization oligonucleotide. In some embodiments, the 3’ end of the top strand circularization oligonucleotide comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end of the top strand circularization oligonucleotide.

[00287] In some embodiments, the top strand circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-99. In some embodiments, the sequence of the top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to the sequences set forth in SEQ ID NOS: 45-99.

[00288] In some embodiments, the contacting of step (b) is conducted under a condition suitable for hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at one end of an individual top strand linear library molecule, and hybridizing the bridging sequence of the same top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at another end of the individual linear library molecule, thereby forming individual open circle library complexes having one nick. In some embodiments, the nicks are enzymatically ligatable.

[00289] In some embodiments, in step (b), the terminal 5’ ends of individual linear library molecules (e.g., top strands) include a phosphate group. In some embodiments, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules. [00290] In some embodiments, in step (b), the plurality of top strand linear library molecules can be hybridized in-solution with a plurality of top strand circularization oligonucleotides in a hybridization reagent.

[00291] In some embodiments, in step (b), the condition suitable for hybridizing the top strand circularization oligonucleotides to an individual top strand linear library molecule comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15- 30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4- 6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours. [00292] In some embodiments, in step (b), the condition suitable for hybridizing the top strand circularization oligonucleotides to an individual top strand linear library molecule comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about SO- 55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00293] In some embodiments, in step (b), individual top strand linear library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual covalently closed circular library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b).

[00294] In some embodiments, the methods comprise step (c): forming a plurality of open circle library bait complexes by contacting in-solution the plurality of open circle library complexes with a plurality of target-specific baits/probes. In some embodiments, the contacting is conducted in-solution under a condition suitable for selectively hybridizing individual target-specific baits/probes to at least a portion of a target sequence of an open circle library complex, thereby generating a plurality of open circle library bait complexes that are enriched for polynucleotides having target sequences (e.g., FIG. 36A). In some embodiments, the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36 A) that can bind a receptor moiety of the capture support. [00295] In some embodiments, in step (c), the linear library molecules comprising nontarget sequences do not selectively hybridize to the target-specific baits/probes. In some embodiments, individual target-specific baits/probes comprise (i) an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of a target sequence of a linear library molecule, (ii) an affinity moiety at the 5’ end, and (iii) an extendible 3’ end. In some embodiments, the oligonucleotide of the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moieties of individual target-specific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual target-specific baits/probes comprises biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual targetspecific baits/probes can be located at the 5’ end or at an internal position.

[00296] In some embodiments, in step (c), the plurality of target-specific baits/probes and the plurality of open circle library complexes can be hybridized in-solution in a hybridization reagent under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual top strand linear library molecules to corresponding target-specific baits/probes, thereby forming a plurality of open circle library bait complexes.

[00297] In some embodiments, in step (c), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual open circle library complexes to corresponding target-specific baits/probes comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4-6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours.

[00298] In some embodiments, in step (c), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual open circle library complexes to corresponding target-specific baits/probes comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00299] In some embodiments, in step (c), the plurality of target-specific baits/probes comprise 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-250,000 different target-specific sequences, 2-100,000 different target-specific sequences, 2-10,000 different target-specific sequences. In some embodiments, the plurality of open circle library bait complexes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of open circle library bait complexes comprises 2- 500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, in step (c), the plurality of open circle library bait complexes comprises up to 1,000,000 different target-specific sequences. [00300] In some embodiments, in step (c), the plurality of open circle library complexes comprise individual linear library molecules hybridized to a top strand circularization oligonucleotide, wherein the linear library molecules can be generated from double-stranded input nucleic acids comprising top strands and complementary bottom strands. In some embodiments, the plurality of linear library molecules comprises at least a first and a second sub-population of linear library molecules. In some embodiments, the insert regions of individual linear library molecules of the first sub-population comprise a target or non-target sequence from a top strand input nucleic acid. In some embodiments, the insert regions of individual linear library molecules of the second sub-population comprise a target or nontarget sequence from a bottom strand input nucleic acid.

[00301] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual linear library molecule of the first sub-population (e.g., top strand insert sequence). [00302] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual linear library molecule of the second sub-population (e.g., bottom strand insert sequence). [00303] In some embodiments, step (b) can be conducted prior to step (c). In some embodiments, step (c) can be conducted prior to step (b). In some embodiments, steps (b) and

(c) can be conducted essentially simultaneously.

[00304] In some embodiments, the hybridizing of step (c) can generate a mixture comprising a plurality of open circle library bait complexes comprising target sequences and a plurality of linear library molecules comprising non-target sequences. In some embodiments, the mixture of open circle library bait complexes and linear library molecules from step (c) can be contacted with paramagnetic beads coated with receptor moieties that can bind to the affinity moieties on the open circle library bait complexes thereby separating the open circle library bait complexes from the linear library molecules. In some embodiments, the paramagnetic beads that are bound to the open circle library bait complexes can be washed to remove the plurality of linear library molecules. In some embodiments, the open circle library bait complexes can be released from the paramagnetic beads thereby generating a plurality of open circle library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the plurality of non-target linear library molecules are not removed in step (c). In some embodiments, the plurality of non-target linear library molecules are not removed in step (c) using paramagnetic beads that are coated with receptor moieties. In some embodiments, paramagnetic beads coated with receptor moieties are not used at step (c). In some embodiments, the method comprises step

(d): contacting the capture support with the plurality of open circle library bait complexes, thereby generating a plurality of open circle library bait complexes immobilized to the capture support. In some embodiments, the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes (e.g., which are part of the open circle library bait complexes) to a receptor moiety of the capture support. In some embodiments, the plurality of open circle library bait complexes immobilized to the capture support are enriched for polynucleotides carrying target sequences. In some embodiments, the plurality of immobilized open circle library bait complexes comprises top strand library molecules in open circle form.

[00305] In some embodiments, in step (d), the plurality of open circle library bait complexes can be distributed/contacted with the capture support in a loading reagent and/or a hybridization reagent under a condition suitable for binding the affinity moiety of individual open circle library bait complexes to individual receptor moieties embedded in the hydrophilic polymer coating of the capture support, thereby generating a plurality of open circle library bait complexes immobilized to the capture support. [00306] In some embodiments, in step (d) the density of the open circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ open circle library bait complexes per mm². In some embodiments, density of the open circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ open circle library bait complexes per mm², or any range therebetween.

[00307] In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00308] In some embodiments, in step (d), the capture support is contacted with the plurality of open circle library bait complexes and residual non-target linear library molecules from steps (b) and/or (c). In some embodiments, step (d) comprises contacting the support with a wash reagent to remove the residual linear library molecules and retain the plurality of open circle library bait complexes immobilized to the capture support. In some embodiments, the washing of step (d) can generate a plurality of immobilized open circle library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, in step (d), the plurality of open circle library bait complexes immobilized to the capture support comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of open circle library bait complexes immobilized to the support comprises 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different targetspecific sequences, 500-10,000 different target-specific sequences, 2-500 different targetspecific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[00309] In some embodiments, in step (d), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (d) comprises contacting the capture support separately with a plurality of pinning primers and the plurality of open circle library bait complexes in any order. In some embodiments, the capture support can be contacted with a plurality of pinning primers and the plurality of open circle library bait complexes essentially simultaneously.

[00310] In some embodiments, the method comprises step (e): contacting the capture support with a ligation reagent for ligating the one nick of individual immobilized open circle library bait complexes, thereby generating a plurality of covalently closed circular library molecules hybridized to immobilized target-specific baits/probes, thereby forming a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the plurality of immobilized closed circle library bait complexes comprises top strand library molecules that have undergone intramolecular ligation to form covalently closed circular library molecules. In some embodiments, the ligation reagent comprises a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1). In some embodiments, the ligation reagent comprises a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S). In some embodiments the ligation reagent comprises a recombinant thermal tolerant T4 DNA ligase including Hi-T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S). In some embodiments, the ligation reagent comprises a DNA ligase from Thermococcus nautili (e.g., NCBI No. WP 042693257.1). In some embodiments, the ligation reaction comprises a T4 polynucleotide kinase (e.g., from New England Biolabs, catalog # M0201S).

[00311] In some embodiments, in step (e) the density of closed circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ closed circle library bait complexes per mm². In some embodiments, density of the closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library bait complexes per mm², or any range therebetween.

[00312] In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00313] In some embodiments, step (e) comprises contacting the support with a wash reagent to remove any linear library molecules and retain the plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the washing of step (e) can generate a plurality of immobilized closed circle library bait complexes that are enriched for polynucleotides having target sequences. [00314] In some embodiments, in step (e), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (e) comprises contacting the capture support with a plurality of pinning primers prior to or after conducting the ligation reaction. In some embodiments, step (e) comprises contacting the capture support with the ligation reagent and a plurality of pinning primers essentially simultaneously.

[00315] In some embodiments, in step (e), after enzymatically ligating the nicks, at least one of the closed circle library bait complexes comprises a covalently closed circular library molecule having at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule having at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (e) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (e) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules. [00316] In some embodiments, the method comprises step (f): contacting the plurality of closed circle library bait complexes immobilized to the capture support with rolling circle amplification reagents and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes (e.g., which are part of the closed circle library bait complexes immobilized to the capture support) and use the covalently closed circular library molecules as template molecules thereby generating a plurality of concatemer template molecules which are immobilized to the support (“immobilized concatemer template molecules”). In some embodiments, individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region and any universal adaptor sequences present in a covalently closed circular library molecule. In some embodiments, individual immobilized concatemer template molecules comprise sequences from a top strand covalently closed circular library molecule. In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers, wherein individual pinning primers hybridize to a portion of an immobilized concatemer template molecule thereby pinning down a portion of a concatemer template molecule.

[00317] In some embodiments, in step (f), the rolling circle amplification reagent comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. In some embodiments, the rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

[00318] In some embodiments, the methods comprise step (g): sequencing at least a portion of the plurality of immobilized concatemer template molecules. In some embodiments, at least a portion of the concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein.

[00319] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis) and U.S. published application No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of steps (b), (c) and (dl), and the distributing of step (d) (the contents of both documents are hereby incorporated by reference in their entireties).

[00320] In some embodiments, after the sequencing of step (g) described above, the capture support can be subjected to a re-seeding workflow in which additional linear library molecules can be hybridized in-solution to top strand circularization oligonucleotides to generate a plurality of open circle library complexes, and the open circle library complexes can be hybridized to target-specific baits/probes to generate a plurality of open circle library bait complexes that are distributed onto the capture support. In some embodiments, the reseeding workflow comprises closing the nicks of the plurality of open circle library bait complexes to generate a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (b) - (e) at least once. In some embodiments, steps (6) - (8) of a re-seeding workflow can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding are described herein.

(6) Blended Workflow for Enriched Top Strand Library Molecules, Enriched Bottom Strand Library Molecules, and Non-Enriched Whole Genomic Library Molecules - Conducted in Separate Reaction Vessels

[00321] The present disclosure provides a method for conducting a blended enriched exome and non-enriched whole genome workflow by conducting circularization reactions of library molecules in separate reaction vessels. In some embodiments, a first circularization reaction can be conducted in a first reaction vessel comprising (i) an enriched exome top strand library and (ii) an enriched exome bottom strand library. In some embodiments, a second circularization reaction can be conducted in a second reaction vessel comprising a non-enriched whole genomic library. In some embodiments, after conducting separate circularization reactions, the contents of the first and second reaction vessels can be mixed together to generate a mixture of library molecules comprising target and non-target sequences. The mixture of library molecules can be distributed onto a capture support for immobilization. The immobilized blended libraries can be subjected to rolling circle amplification and sequencing.

[00322] The present disclosure provides methods for conducting a blended enriched exome library and non-enriched whole genomic library, from a mixture of target and nontarget sequences, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00323] In some embodiments, in step (a), the capture support comprises a plurality of pinning primers immobilized to the capture support. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence, and an affinity moiety at the 5’ end. In some embodiments, individual pinning primers comprise a blocking moiety at the 3’ end. In some embodiments, the blocking moiety inhibits polymerase-catalyzed extension of the 3’ end of the pinning primer. In some embodiments, the blocking moiety can be converted to a moiety that promotes polymerase-catalyzed extension. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind a receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture surface lacks a plurality of immobilized pinning primers.

[00324] In some embodiments, the methods comprise step (b): providing a plurality of linear library molecules and apportioning the plurality of linear library molecules into a plurality of reaction vessels, including at least a first and a second reaction vessel. In some embodiments, the plurality of linear library molecules comprises a mixture of linear library molecules having insert regions comprising target or non-target sequences. In some embodiments, the plurality of linear library molecules comprise a mixture of linear library molecules generated from double-stranded input nucleic acids comprising insert regions having top strand sequences or complementary bottom strand sequences. In some embodiments, individual top strand linear library molecules comprise (i) a top strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual bottom strand linear library molecules comprise (i) a bottom strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, the terminal 5’ end of individual top strand linear library molecules include a phosphate group. In some embodiments, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules.

[00325] In some embodiments, in step (b), the plurality of linear library molecules comprise top strand linear library molecules and bottom strand linear library molecules comprising (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual top strand and bottom strand linear library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b).

[00326] In some embodiments, the methods comprise step (c): contacting the linear library molecules in the first reaction vessel with a mixture of oligonucleotides including (i) a plurality of top strand circularization oligonucleotides and (ii) a plurality of bottom strand blocker oligonucleotides. In some embodiments, the contacting can be conducted in-solution. In some embodiments, the contacting can be conducted with a hybridization reagent under a condition suitable for hybridizing the plurality of linear library molecules to the mixture of oligonucleotides. [00327] In some embodiments, the contacting of step (c) can generate a plurality of library molecule-oligonucleotide complexes, individual library molecule-oligonucleotide complexes comprising a top strand linear library molecule and a top strand circularization oligonucleotide (an “open circle library complex”), or a bottom strand linear library molecule and a bottom strand blocker oligonucleotide (a “library blocker complex”). In some embodiments, the plurality of library molecule-oligonucleotide complexes comprises (i) a plurality of open circle library complexes, and (ii) a plurality of library blocker complexes. [00328] In some embodiments, in step (c), individual open circle library complexes comprise a top strand linear library molecule hybridized to a top strand circularization oligonucleotide, wherein one end of individual top strand linear library molecules are hybridized to a first end of a top strand circularization oligonucleotide and wherein the other end of the top strand linear library molecule is hybridized to a second end of the top strand circularization oligonucleotide, thereby generating an open circle library complex having a nick between the ends of the linear library molecule (e.g., FIG. 36 A). In some embodiments, the nick is enzymatically ligatable. In some embodiments, the 3’ end of the top strand circularization oligonucleotides comprise a moiety that blocks polymerase-catalyzed extension. In some embodiments, the top strand circularization oligonucleotides have nonextendible 3’ ends.

[00329] In some embodiments, in step (c), individual library blocker complexes comprise a bottom strand linear library molecule hybridized to a bottom strand blocker oligonucleotide, wherein the bottom strand blocker oligonucleotide can hybridize to at least a portion of the bottom strand linear library molecule. Individual bottom strand blocker oligonucleotides comprise at least one sequence that can hybridize to a universal adaptor sequence of one of the bottom strand linear library molecule. Individual bottom strand blocker oligonucleotides lack a sequence that can hybridize to a target sequence (e.g., insert region) of a bottom strand linear library molecule. Individual library blocker complexes comprise a double-stranded region formed from hybridization between a portion of the bottom strand linear library molecule and the bottom strand blocker oligonucleotide. Individual library blocker complexes do not circularize to form open circle library molecules. In some embodiments, the 3’ ends of the bottom strand blocker oligonucleotides comprise a moiety that blocks polymerase-catalyzed extension. In some embodiments, the bottom strand blocker oligonucleotides have non-extendible 3’ ends.

[00330] In some embodiments, in step (c), the condition suitable for hybridizing the plurality of linear library molecules with the mixture of oligonucleotides (e.g., the mixture comprising a plurality of top strand circularization oligonucleotides and bottom strand blocker oligonucleotides) comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, or about 12-16 hours.

[00331] In some embodiments, in step (c), the condition suitable for hybridizing the plurality of linear library molecules with the mixture of oligonucleotides comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00332] In some embodiments, in step (c), individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. The anchor sequence of a top strand circularization oligonucleotide can hybridize to one end of a top strand linear library molecule and the bridging sequence can hybridize to another end of the top strand linear library molecule to circularize the linear library molecule and generate an open circle library complex having a nick between the ends of the top strand linear library molecule (e.g., FIG. 36A). In some embodiments, individual top strand circularization oligonucleotides can hybridize to at least a portion of a top strand linear library molecule, thereby generating an open circle library complex. In some embodiments, individual top strand circularization oligonucleotides do not hybridize to at least a portion of a bottom strand linear library molecule. In some embodiments the anchor sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to at least a portion of a universal adaptor sequence at the other end of the top strand linear library molecule. In some embodiments the anchor sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to at least a portion of a universal adaptor sequence at the other end of the top strand linear library molecule. In some embodiments, the anchor sequence can hybridize to at least a portion of one or more universal adaptor sequences of the linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences. In some embodiments, individual top strand circularization oligonucleotides lack a sequence that can hybridize with the insert region of a top strand linear library molecule. The top strand circularization oligonucleotide can include or lack a sequence that can hybridize to a left sample index sequence of the top strand linear library molecule. The top strand circularization oligonucleotide can include or lack a sequence that can hybridize to a right sample index sequence of the top strand linear library molecule. In some embodiments, the 3’ end of the top strand circularization oligonucleotide comprises a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the top strand circularization oligonucleotide. In some embodiments, the blocking moiety can be converted to a moiety that promotes polymerase-catalyzed extension of the top strand circularization oligonucleotide. In some embodiments, the top strand circularization oligonucleotide comprises a 3’ non-extendible end. In some embodiments, the 3’ end of the top strand circularization oligonucleotide comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end of the top strand circularization oligonucleotide.

[00333] In some embodiments, the contacting of step (c) is conducted under a condition suitable for hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at one end of a top strand linear library molecule, and suitable for hybridizing the bridging sequence of the same top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at the other end of the same top strand linear library molecule, thereby generating an open circle library complex having a nick between the ends of the top strand linear library molecule.

[00334] In some embodiments, in step (c), the top strand circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-99. In some embodiments, the sequence of the top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of the sequences set forth in SEQ ID NOS: 45-99.

[00335] In some embodiments, in step (c), a terminal 5’ end of individual linear library molecules (e.g., top strand linear library molecules) include a phosphate group. In some embodiments, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules.

[00336] In some embodiments, in step (c), individual bottom strand blocker oligonucleotides comprise single-stranded oligonucleotides that can hybridize to one or more universal adaptor sequences of a bottom strand linear library molecule and inhibit hybridization of another oligonucleotide to the one or more universal adaptor sequences. In some embodiments, the bottom strand blocker oligonucleotide comprises an anchor sequence that can hybridize with one end of a bottom strand linear library molecule, and lacks a bridging sequence that can hybridize with another end of the bottom strand linear library molecule. Thus, the bottom strand blocker oligonucleotide can hybridize to at least a portion of a bottom strand linear library molecule but does not circularize the linear library molecule to generate an open circle library molecule. In some embodiments, individual bottom strand blocker oligonucleotides can hybridize to at least a portion of a bottom strand linear library molecule, thereby generating a library blocker complex. In some embodiments, individual bottom strand blocker oligonucleotides do not hybridize to at least a portion of a top strand linear library molecule. In some embodiments, the bottom strand blocker oligonucleotide can hybridize to at least a portion of one or more universal adaptor sequences located on the 3’ side of the insert sequence. In some embodiments, the bottom strand blocker oligonucleotide can hybridize to at least a portion of one or more universal adaptor sequences located on the 5’ side of the insert sequence. In some embodiments, individual bottom strand blocker oligonucleotides lack a sequence that can hybridize with the insert region of a bottom strand linear library molecule. The bottom strand blocker oligonucleotide can include or lack a sequence that can hybridize to a left sample index sequence of the bottom strand linear library molecule. The bottom strand blocker oligonucleotide can include or lack a sequence that can hybridize to a right sample index sequence of the bottom strand linear library molecule. In some embodiments, the 3’ ends of the bottom strand blocker oligonucleotide comprise a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the bottom strand blocker oligonucleotide. In some embodiments, the blocking group can be converted to a moiety that promotes polymerase-catalyzed extension of the bottom strand blocker oligonucleotide. In some embodiments, the bottom strand blocker oligonucleotides comprise a 3’ non-extendible end. In some embodiments, the 3’ end of the bottom strand blocker oligonucleotide comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end of the bottom strand blocker oligonucleotide.

[00337] In some embodiments, in step (c), the bottom strand blocker oligonucleotides comprise any of the sequences according to SEQ ID NOS: 101-129. In some embodiments, the sequence of the bottom strand blocker oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to the sequences of any of the bottom strand blocker oligonucleotides according to SEQ ID NOS: 101-129. [00338] In some embodiments, the methods comprise step (d): contacting in-solution the plurality of library molecule-oligonucleotide complexes in the first reaction vessel with a plurality of target-specific baits/probes, thereby generating a plurality of library molecule bait complexes. In some embodiments, the contacting can be conducted under a condition suitable for selectively binding the target-specific baits/probes to corresponding target sequences of the library molecule-oligonucleotide complexes. In some embodiments, the plurality of linear library molecules comprising non-target sequences do not selectively hybridize to the targetspecific baits/probes. In some embodiments, the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36A) that can bind a receptor moiety of the capture support.

[00339] In some embodiments, in step (d), the plurality of target-specific baits/probes can selectively bind the top strand insert region of at least two of the open circle library complexes to generate a plurality of open circle library bait complexes that are enriched for polynucleotides having top strand target sequences. In some embodiments, an open circle library bait complex comprises a library molecule bait complex.

[00340] In some embodiments, in step (d), the plurality of target-specific baits/probes can selectively bind the bottom strand insert region of at least two of the library blocker complexes to generate a plurality of library blocker bait complexes that are enriched for polynucleotides having bottom strand target sequences. In some embodiments, a library blocker bait complex comprises a library molecule bait complex.

[00341] In some embodiments, in step (d), the condition suitable for hybridizing the plurality of library molecule-oligonucleotide complexes with the plurality of target-specific baits/probes comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4-6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours.

[00342] In some embodiments, in step (d), the condition suitable for hybridizing the plurality of library molecule-oligonucleotide complexes with the plurality of target-specific baits/probes comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65- 70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00343] In some embodiments, in step (d), individual target-specific baits/probes comprise an oligonucleotide comprising (i) a target-specific sequence that can selectively hybridize to at least a portion of a target sequence of a library molecule, (ii) an affinity moiety at the 5’ end, and (iii) an extendible 3’ end. In some embodiments, individual target-specific baits/probes comprise a moiety at the 3’ end that promotes polymerase-catalyzed extension. In some embodiments, the plurality of target-specific baits/probes comprises extendible 3’ ends. In some embodiments, the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual targetspecific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moi eties of individual target-specific baits/probes can be located at the 5’ end or at an internal position.

[00344] In some embodiments, in step (d), the plurality of target-specific baits/probes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of target-specific baits/probes comprises 2-1,000,000 different target-specific sequences, 2- 500,000 different target-specific sequences, the plurality of target-specific baits/probes comprises 2-250,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different targetspecific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, the plurality of open circle library bait complexes comprise 2-10,000 different top strand insert sequences. In some embodiments, the plurality of open circle library bait complexes comprises 2-1,000,000 different top strand insert sequences, 2-500,000 different top strand insert sequences, 2- 250,000 different top strand insert sequences, 2-100,000 different top strand insert sequences, 100-100,000 different top strand insert sequences, 500-10,000 different top strand insert sequences, 2-500 different top strand insert sequences, or 1,000-50,000 different top strand insert sequences, or any range therebetween. In some embodiments, the plurality of library blocker bait complexes comprises 2-10,000 different bottom strand insert sequences. In some embodiments, the plurality of library blocker bait complexes comprises 2-1,000,000 different bottom strand insert sequences, 2-500,000 different bottom strand insert sequences, 2- 250,000 different bottom strand insert sequences, 2-100,000 different bottom strand insert sequences, 100-100,000 different bottom strand insert sequences, 500-10,000 different

I l l bottom strand insert sequences, 2-500 different bottom strand insert sequences, or 1,000- 50,000 different bottom strand insert sequences, or any range therebetween.

[00345] In some embodiments, step (c) can be conducted prior to step (d). In some embodiments, step (d) can be conducted prior to step (c). In some embodiments, steps (c) and (d) can be conducted essentially simultaneously.

[00346] In some embodiments, the methods comprise step (e): contacting the linear library molecules in the second reaction vessel with a plurality of spike-in circularization oligonucleotides. In some embodiments, the contacting can be conducted in-solution. In some embodiments, the contacting can be conducted with a hybridization reagent under a condition suitable for hybridizing the plurality of linear library molecules to the plurality of spike-in circularization oligonucleotides, thereby generating a plurality of open circle library spike-in complexes. In some embodiments, the linear library molecules in the second reaction vessel are not contacted with a plurality of target-specific baits/probes.

[00347] In some embodiments, in step (e), individual open circle library spike-in complexes comprise a top strand linear library molecule hybridized to a spike-in circularization oligonucleotide, wherein one end of an individual top strand linear library molecule is hybridized to a first end of a spike-in circularization oligonucleotide, and wherein another end of the top strand linear library molecule is hybridized to a second end of one of the spike-in circularization oligonucleotide, thereby generating an open circle library spike-in complex having a nick between the ends of the linear library molecule. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the 3’ ends of the spike-in circularization oligonucleotides comprise moieties that promote polymerase- catalyzed extension. In some embodiments, the spike-in circularization oligonucleotides have extendible 3’ ends. In some embodiments, the 5’ ends of individual spike-in circularization oligonucleotides comprise affinity moieties that can bind one of the receptor moieties embedded in the at least one layer of hydrophilic polymer coating of the capture support. In some embodiments, the affinity moiety of the spike-in circularization oligonucleotide comprises biotin, desthiobiotin or iminobiotin.

[00348] In some embodiments, in step (e), the condition suitable for hybridizing the plurality of linear library molecules with the plurality of spike-in circularization oligonucleotides comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, about 12-16 hours. [00349] In some embodiments, in step (e), the condition suitable for hybridizing the plurality of linear library molecules with the plurality of spike-in circularization oligonucleotides comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00350] In some embodiments, in step (e), individual spike-in circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. The anchor sequence of a spike-in circularization oligonucleotide can hybridize to one end of a top strand linear library molecule, and the bridging sequence can hybridize to another end of the same top strand linear library molecule, to circularize the linear library molecule and generate an open circle library spike-in complex having a nick between the ends of the top strand linear library molecule. In some embodiments, the anchor sequence can hybridize to at least a portion of one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the one or more universal adaptor sequences. In some embodiments, the bridging sequence can hybridize to at least a portion of one or more universal adaptor sequences at another end of the top strand linear library molecule. In some embodiments the anchor sequence can be located at the 3’ end of the spike-in circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the spike-in circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at another end of the same top strand linear library molecule. In some embodiments, the anchor sequence can be located at the 5’ end of the spike-in circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the spike-in circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at the other end of the same top strand linear library molecule. In some embodiments, individual spike-in circularization oligonucleotides lack a sequence that can hybridize with the insert region of a top strand linear library molecule. The spike-in circularization oligonucleotide can include or lack a sequence that can hybridize to a left sample index sequence of the top strand linear library molecule. The spike-in circularization oligonucleotide can include or lack a sequence that can hybridize to a right sample index sequence of the top strand linear library molecule. In some embodiments, the 3’ ends of the spike-in circularization oligonucleotide comprise moi eties that promotes polymerase-catalyzed extension of the 3’ ends of the spike-in circularization oligonucleotides. In some embodiments, individual spike-in circularization oligonucleotides comprise a 3’ extendible end. In some embodiments, the 3’ ends of the spike-in circularization oligonucleotides comprise blocking moieties that inhibit polymerase-catalyzed extension of the 3’ ends of the spike-in circularization oligonucleotides. In some embodiments, the blocking group can be converted to a moiety that promotes polymerase- catalyzed extension of the spike-in circularization oligonucleotide. In some embodiments, the 5’ ends of individual spike-in circularization oligonucleotides comprise affinity moieties that can bind one of the receptor moieties embedded in the at least one layer of hydrophilic polymer coating of the capture support. In some embodiments, the affinity moiety of the spike-in circularization oligonucleotide comprises biotin, desthiobiotin or iminobiotin.

[00351] In some embodiments, the contacting of step (e) is conducted under a condition suitable for hybridizing the anchor sequence of the spike-in circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at one end of a top strand linear library molecule, and hybridizing the bridging sequence of the spike-in circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at the other end of the same top strand linear library molecule, thereby generating an open circle library spike-in complex having a nick between the ends of the top strand linear library molecule. [00352] In some embodiments, in step (e), the spike-in circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-100. In some embodiments, the sequence of the spike-in circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of SEQ ID NOS: 45-100.

[00353] In some embodiments, in step (e), the terminal 5’ ends of individual linear library molecules (e.g., top strand linear library molecules) include a phosphate group. In some embodiments, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules.

[00354] In some embodiments, in step (e), the plurality of top strand linear library molecules can be contacted in-solution with a plurality of spike-in circularization oligonucleotides in a hybridization reagent.

[00355] In some embodiments, the method comprises step (f): contacting the capture support with a mixture of (i) the plurality of open circle library bait complexes and the plurality of library blocker bait complexes from the first reaction vessel of step (d), and (ii) the plurality of open circle library spike-in complexes from the second reaction vessel of step (e), thereby generating a plurality of library complexes immobilized to the capture support. In some embodiments, the contacting can be conducted using a loading reagent and/or a hybridization reagent.

[00356] In some embodiments, in step (f), the capture support can be contacted separately with the contents of the first and second reaction vessels in any order. In some embodiments, in step (f), the capture support can be contacted with the contents of the first and second reaction vessels essentially simultaneously.

[00357] In some embodiments, the contacting of step (f) is conducted under a condition suitable for binding the affinity moiety of individual open circle library bait complexes to a receptor moiety of the capture support, thereby generating a plurality of open circle library bait complexes immobilized to the capture support.

[00358] In some embodiments, the contacting of step (f) is conducted under a condition suitable for binding the affinity moiety of individual library blocker bait complexes to a receptor moiety of the capture support, thereby generating a plurality of library blocker bait complexes immobilized to the capture support.

[00359] In some embodiments, the contacting of step (f) is conducted under a condition suitable for binding the affinity moiety of individual open circle library spike-in complexes to a receptor moiety of the capture support, thereby generating a plurality of open circle library spike-in complexes immobilized to the capture support.

[00360] In some embodiments, the contacting of step (f) generates a mixture of (i) a plurality of open circle library bait complexes that are enriched for top strand target sequences, (ii) a plurality of library blocker bait complexes that are enriched for bottom strand target sequences, and (iii) a plurality of open circle library spike-in complexes that are not enriched for target sequences.

In some embodiments, in step (f), the capture support can be contacted with the plurality of open circle library bait complexes and the plurality of open circle library spike-in complexes at a ratio of about 1 : 1, or about 2:1, or about 3: 1, or about 4: 1, or about 5: 1, or about 6: 1, or about 7:1, or about 8: 1, or about 9: 1, or about 10: 1, or any ratio therebetween. In some embodiments, the capture support can be contacted with the plurality of open circle library bait complexes and the plurality of open circle library spike-in complexes at a ratio of about 100: 1, or about 75: 1, or about 50: 1, or about 25: 1 or about 10: 1, or any ratio therebetween. An exemplary ratio comprises three parts open circle library bait complexes and one part open circle library spike-in complexes. Another exemplary ratio comprises five parts open circle library bait complexes and one part open circle library spike-in complexes.

[00361] In some embodiments, in step (f), the capture support can be contacted with about 0.1 - 50% (e.g., about 1% to about 50%, about 5% to about 50%, about 5% to about 40%, about 10% to about 30%, about 10% to about 20%, about 1% to about 10%, about 20% to about 50%, or any range therebetween) of the plurality of open circle library spike-in complexes compared to the plurality of open circle library bait complexes.

[00362] In some embodiments, in step (f) the density of open circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ open circle library bait complexes per mm². In some embodiments, the density of the open circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ open circle library bait complexes per mm², or any range therebetween.

[00363] In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00364] In some embodiments, in step (f) the density of open circle library spike-in complexes immobilized to the capture support is about 10² - 10¹⁵ open circle library spike-in complexes per mm². In some embodiments, the density of the open circle library spike-in complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ open circle library spike-in complexes per mm², or any range therebetween.

[00365] In some embodiments, the plurality of open circle library spike-in complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of immobilized open circle library spike-in complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00366] In some embodiments, in step (f) the density of immobilized library blocker bait complexes immobilized to the capture support is about 10² - 10¹⁵ per mm². In some embodiments, the density of immobilized library blocker bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ immobilized library blocker bait complexes per mm², or any range therebetween. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00367] In some embodiments, in step (f), contacting the capture support can include contacting with residual linear library molecules from any of steps (c) - (e). In some embodiments, step (f) comprises contacting the support with a wash reagent to remove the residual linear library molecules and retain the plurality of immobilized open circle library bait complexes, open circle library spike-in complexes, and library blocker bait complexes. [00368] In some embodiments, in step (f), the plurality of immobilized open circle library bait complexes comprises 2-10,000 different top strand insert sequences. In some embodiments, the plurality of immobilized open circle library bait complexes comprises 2- 1,000,00 different top strand insert sequences, 2-500,000 different top strand insert sequences, 2-100,000 different top strand insert sequences, 100-100,000 different top strand insert sequences, 500-10,000 different top strand insert sequences, 2-500 different top strand insert sequences, or 1,000-50,000 different top strand insert sequences, or any range therebetween. In some embodiments, the plurality of immobilized open circle library spike-in complexes comprises 2-10,000 different top strand insert sequences. In some embodiments, the plurality of immobilized open circle library spike-in complexes comprises 2-1,000,000 different top strand insert sequences, 2-500,000 different top strand insert sequences, 2- 100,000 different top strand insert sequences, 100-100,000 different top strand insert sequences, 500-10,000 different top strand insert sequences, 2-500 different top strand insert sequences, or 1,000-50,000 different top strand insert sequences, or any range therebetween. In some embodiments, the plurality of immobilized library blocker bait complexes comprises 2-10,000 different bottom strand insert sequences. In some embodiments, the plurality of immobilized library blocker bait complexes comprises 2-1,000,000 different bottom strand insert sequences, 2-500,000 different bottom strand insert sequences, 2-100,000 different bottom strand insert sequences, 100-100,000 different bottom strand insert sequences, 500- 10,000 different bottom strand insert sequences, 2-500 different bottom strand insert sequences, or 1,000-50,000 different bottom strand insert sequences, or any range therebetween.

[00369] In some embodiments, in step (f), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (f) comprises contacting the capture support separately with a plurality of pinning primers, the contents of the first reaction vessel, and the contents of the second reaction vessel, in any order. In some embodiments, the capture support can be contacted with a plurality of pinning primers, the contents of the first reaction vessel, and the contents of the second reaction vessel, essentially simultaneously.

[00370] In some embodiments, the method comprises step (g): contacting the capture support with a ligation reagent thereby generating a plurality of closed circle library bait complexed immobilized to the capture support, and a plurality of closed circle library spikein complexes immobilized to the capture support.

[00371] In some embodiments, in step (g), the ligation reagent closes the nicks of individual open circle library bait complexes to generate a plurality of closed circle library bait complexes comprising covalently closed circular top strand library molecules hybridized to a top strand circularization oligonucleotide and a target-specific bait/probe which is immobilized to the capture support.

[00372] In some embodiments, in step (g), the ligation reagent closes the nicks of individual open circle library spike-in complexes to generate a plurality of closed circle library spike-in complexes comprising covalently closed circular top strand library molecules hybridized to a spike-in circularization oligonucleotide which is immobilized to the capture support.

[00373] In some embodiments, in step (g), the library blocker bait complexes lack a nick and do not form a covalently closed circular library molecule. In some embodiments, individual library blocker bait complexes comprise a linear bottom strand library molecule hybridized to a bottom strand blocker oligonucleotide and a target-specific bait/probe which is immobilized to the capture support. In some embodiments, the library blocker bait complexes do not form open circle library molecules with a nick.

[00374] In some embodiments, in step (g), the ligation reagent comprises a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1). In some embodiments, the ligation reagent comprises a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S). In some embodiments the ligation reagent comprises a recombinant thermal tolerant T4 DNA ligase including Hi- T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S). In some embodiments, the ligation reagent comprises a DNA ligase from Thermococcus nautili (e.g., NCBI No. WP 042693257.1). In some embodiments, the ligation reaction comprises a T4 polynucleotide kinase (e.g., from New England Biolabs, catalog # M0201S).

[00375] In some embodiments, in step (g) the density of the plurality of closed circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ closed circle library bait complexes per mm². In some embodiments, the density of the plurality of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library bait complexes per mm², or any range therebetween. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00376] In some embodiments, in step (g) the density of the plurality of closed circle library spike-in complexes immobilized to the capture support is about 10² - 10¹⁵ closed circle library spike-in complexes per mm². In some embodiments, the density of the plurality of closed circle library spike-in complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library spike-in complexes per mm², or any range therebetween. In some embodiments, the plurality of closed circle library spike-in complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library spike-in complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00377] In some embodiments, in step (g) the density of the plurality of library blocker bait complexes immobilized to the capture support is about 10² - 10¹⁵ library blocker bait complexes per mm². In some embodiments, the density of the plurality of library blocker bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ library blocker bait complexes per mm², or any range therebetween. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00378] In some embodiments, step (g) comprises contacting the support with a wash reagent to remove linear library molecules and retain the pluralities of (i) immobilized closed circle library bait complexes, (ii) immobilized closed circle library spike-in complexes, and (iii) immobilized library blocker bait complexes. In some embodiments, the washing of step (g) can generate a plurality of immobilized library molecules that are enriched for polynucleotides having target sequences.

[00379] In some embodiments, in step (g), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (g) comprises contacting the capture support with a plurality of pinning primers prior to or after conducting the ligation reaction. In some embodiments, step (g) comprises contacting the capture support with the ligation reagent and a plurality of pinning primers essentially simultaneously.

[00380] In some embodiments, step (g) comprises a ligation reaction that generates a plurality of covalently closed circular library molecules immobilized to the capture support, including (i) a plurality of covalently closed circular library molecules that form closed circle library bait complexes, and (ii) a plurality of covalently closed circular library molecules that form closed circle library spike-in complexes. In some embodiments, after conducting the ligation reaction of step (g), at least one of the covalently closed circular library molecules immobilized to the capture support comprises at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule comprising at least one deaminated nucleotide base can be removed by subjecting the plurality of covalently closed circular library molecules to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (g) comprises contacting the plurality of covalently closed circular library molecules that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps thereby converting the at least one deaminated nucleotide base in the at least one covalently closed circular library molecule into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (g) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from covalently closed circular library molecules.

[00381] In some embodiments, the method comprises step (h): contacting the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction.

[00382] In some embodiments, in step (h), the rolling circle amplification reaction is conducted under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes and use the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules from the plurality of closed circle library bait complexes which are immobilized to the support.

[00383] In some embodiments, in step (h), the rolling circle amplification reaction is conducted under a condition suitable to extend the 3’ ends of the immobilized spike-in circularization oligonucleotides and use the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules from the plurality of closed circle library spike-in complexes which are immobilized to the support. [00384] In some embodiments, in step (h), the plurality of immobilized library blocker bait complexes do not undergo rolling circle amplification.

[00385] In some embodiments, in step (h), individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region (target sequence) and any universal adaptor sequences present in a covalently closed circular library molecule. In some embodiments, individual immobilized concatemer template molecules comprise sequences from a top strand linear library molecules used to generate the covalently closed circular library molecules using the methods described herein.

[00386] In some embodiments, in step (h), the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers wherein individual pinning primers hybridize to a portion of an immobilized concatemer template molecule thereby pinning down a portion of a concatemer template molecule.

[00387] In some embodiments, in step (h), the rolling circle amplification reagent comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. In some embodiments, the rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

[00388] In some embodiments, the methods comprise step (i): sequencing at least a portion of the individual immobilized concatemer template molecules in the plurality of immobilized concatemer template molecules. In some embodiments, at least a portion of the concatemers are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing of step (i) determines the sequence of the insert regions. In some embodiments, the sequencing of step (i) identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and nucleotide reagents which include nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two- stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein.

[00389] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis) and U.S. published application No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of any of steps (c) - (e) and the contacting/distributing of step (f) (where the contents of both documents are hereby incorporated by reference in their entireties). (7) Blended Workflow for Enriched Top Strand Library Molecules, Enriched Bottom Strand Library Molecules, and Non-Enriched Whole Genomic Library Molecules - Conducted in the Same Reaction Vessel

[00390] The present disclosure provides a method for conducting a blended enriched exome and non-enriched whole genome workflow by conducting a circularization reaction of library molecules in one reaction vessel. In some embodiments, the one reaction vessel comprises (i) an enriched exome top strand library, (ii) an enriched exome bottom strand library, and (iii) non-enriched whole genomic library. The circularization reaction can generate a mixture of library molecules. After conducting the circularization reaction, the mixture of library molecules can be distributed onto a capture support for immobilization. The immobilized mixture of library molecules can be subjected to rolling circle amplification and sequencing.

[00391] The present disclosure provides methods for conducting a blended enriched exome library and non-enriched whole genomic library, from a mixture of target and nontarget polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00392] In some embodiments, in step (a), the capture support comprises a plurality of immobilized pinning primers, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide. In some embodiments, individual pinning primers comprise a blocking group at the 3’ end of the oligonucleotide wherein the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture support lacks a pinning primer. [00393] In some embodiments, the methods comprise step (b): providing a plurality of linear library molecules and distributing the plurality of linear library molecules into a reaction vessel. In some embodiments, the plurality of linear library molecules comprises a mixture of linear library molecules having insert regions comprising target or non-target sequences. In some embodiments, the plurality of linear library molecules comprise a mixture of linear library molecules generated from double-stranded input nucleic acids comprising insert regions having top strand sequences or complementary bottom strand sequences (“top strand linear library molecules” and “bottom strand linear library molecules”). In some embodiments, individual top strand linear library molecules comprise (i) a top strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual bottom strand linear library molecules comprise (i) a bottom strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, the terminal 5’ ends of individual top strand linear library molecules include a phosphate group. In some embodiments, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules.

[00394] In some embodiments, in step (b), individual top strand and bottom strand linear library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual linear library molecules (top strand and/or bottom strand linear library molecules) comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). [00395] In some embodiments, the methods comprise step (c): contacting the plurality of linear library molecules in the reaction vessel with a mixture of oligonucleotides including (i) a plurality of top strand circularization oligonucleotides, (ii) a plurality of bottom strand blocker oligonucleotides, and (iii) a plurality of spike-in circularization oligonucleotides. In some embodiments, the contacting can be conducted in-solution in the same reaction vessel. [00396] In some embodiments, the contacting of step (c) can be conducted with a hybridization reagent under a condition suitable for hybridizing the plurality of linear library molecules to the mixture of oligonucleotides.

[00397] In some embodiments, the contacting of step (c) can generate a plurality of library molecule-oligonucleotide complexes, individual library molecule-oligonucleotide complexes comprising a top strand linear library molecule and a top strand circularization oligonucleotide (an “open circle library complex”), bottom strand linear library molecule and a bottom strand blocker oligonucleotide (a “library blocker complex”) or a top strand linear library molecule and a spike-in circularization oligonucleotide (a “open circle library spike-in complex”). In some embodiments, the plurality of library molecule-oligonucleotide complexes comprises (i) a plurality of open circle library complexes (ii) a plurality of library blocker complexes and (iii) a plurality of open circle library spike-in complexes.

[00398] In some embodiments, in step (c), individual open circle library complexes comprise a top strand linear library molecule hybridized to a top strand circularization oligonucleotide, wherein one end of individual top strand linear library molecules are hybridized to a first end of a top strand circularization oligonucleotide and wherein the other end of the top strand linear library molecule is hybridized to a second end of the top strand circularization oligonucleotide, thereby generating an open circle library complex having a nick between the ends of the linear library molecule. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the 3’ ends of the top strand circularization oligonucleotides comprise a moiety that blocks polymerase-catalyzed extension. In some embodiments, the top strand circularization oligonucleotides have non-extendible 3’ ends. In some embodiments, the 3’ ends of the top strand circularization oligonucleotides comprise a moiety that promotes polymerase-catalyzed extension. In some embodiments, the top strand circularization oligonucleotides have extendible 3’ ends.

[00399] In some embodiments, in step (c), individual library blocker complexes comprise a bottom strand linear library molecule hybridized to a bottom strand blocker oligonucleotide, wherein the bottom strand blocker oligonucleotide can hybridize to at least a portion of the bottom strand linear library molecule. Individual library blocker complexes comprise a double-stranded region formed from hybridization between a portion of the bottom strand linear library molecule and the bottom strand blocker oligonucleotide. Individual library blocker complexes do not circularize to form open circle library molecules. In some embodiments, the 3’ ends of the bottom strand blocker oligonucleotides comprise a moiety that blocks polymerase-catalyzed extension. In some embodiments, the bottom strand blocker oligonucleotides have non-extendible 3’ ends.

[00400] In some embodiments, in step (c), individual open circle library spike-in complexes comprise a top strand linear library molecule hybridized to a spike-in circularization oligonucleotide, wherein one end of individual top strand linear library molecules are hybridized to a first end of one of the spike-in circularization oligonucleotides and wherein the other end of the same top strand linear library molecule is hybridized to a second end of one of the same spike-in circularization oligonucleotides, thereby generating an open circle library spike-in complex having a nick between the ends of the linear library molecule. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the 3’ ends of the spike-in circularization oligonucleotides comprise a moiety that promotes polymerase-catalyzed extension. In some embodiments, the spike-in circularization oligonucleotides have extendible 3’ ends. In some embodiments, the 3’ ends of the spike-in circularization oligonucleotides comprise a moiety that inhibits polymerase-catalyzed extension. In some embodiments, the spike-in circularization oligonucleotides have non- extendible 3’ ends. In some embodiments, the 5’ end of individual spike-in circularization oligonucleotides comprise an affinity moiety that can bind one of the receptor moieties embedded in the at least one layer of hydrophilic polymer coating of the capture support. In some embodiments, the affinity moiety of the spike-in circularization oligonucleotide comprises biotin, desthiobiotin or iminobiotin.

[00401] In some embodiments, in step (c), the condition suitable for hybridizing the plurality of linear library molecules with the mixture of oligonucleotides (e.g., the mixture comprising a plurality of top strand circularization oligonucleotides, bottom strand blocker oligonucleotides and spike-in circularization oligonucleotides) comprises conducting an insolution hybridization reaction for about 1-15 minutes, about 15-30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4-6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours.

[00402] In some embodiments, in step (c), the condition suitable for hybridizing the plurality of linear library molecules with the mixture of oligonucleotides comprises conducting an in-solution hybridization reaction at a temperature of about 50-55 degrees C, or about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00403] In some embodiments, in step (c), individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. The anchor sequence of a top strand circularization oligonucleotide can hybridize to one end of a top strand linear library molecule and the bridging sequence can hybridize to another end of the top strand linear library molecule to circularize the linear library molecule and generate an open circle library complex having a nick between the ends of the top strand linear library molecule. In some embodiments, individual top strand circularization oligonucleotides can hybridize to at least a portion of a top strand linear library molecule thereby generating an open circle library complex. In some embodiments, individual top strand circularization oligonucleotides do not hybridize to at least a portion of a bottom strand linear library molecule. In some embodiments the anchor sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to at least a portion of a universal adaptor sequence at the other end of the same top strand linear library molecule. In some embodiments the anchor sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to at least a portion of a universal adaptor sequence at another end of the top strand linear library molecule. In some embodiments, the anchor sequence can hybridize to at least a portion of one or more universal adaptor sequences of the linear library molecule and inhibit hybridization of another oligonucleotide to the universal adaptor sequences. In some embodiments, individual top strand circularization oligonucleotides lack a sequence that can hybridize with the insert region of a top strand linear library molecule. The top strand circularization oligonucleotide can include or lack a sequence that can hybridize to a left sample index sequence of the top strand linear library molecule. The top strand circularization oligonucleotide can include or lack a sequence that can hybridize to a right sample index sequence of the top strand linear library molecule. In some embodiments, the 3’ end of the top strand circularization oligonucleotide comprises a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the top strand circularization oligonucleotide. In some embodiments, the top strand circularization oligonucleotide comprises a 3’ non-extendible end. In some embodiments, the 3’ end of the top strand circularization oligonucleotide comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end of the top strand circularization oligonucleotide.

[00404] In some embodiments, the contacting of step (c) is conducted under a condition suitable for hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at one end of a top strand linear library molecule, and suitable for hybridizing the bridging sequence of the same top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at the other end of the same top strand linear library molecule, thereby generating an open circle library complex having a nick between the ends of the top strand linear library molecule.

[00405] In some embodiments, in step (c), the top strand circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-99. In some embodiments, the sequence of the top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of the sequences set forth in SEQ ID NOS: 45-99.

[00406] In some embodiments, in step (c), individual bottom strand blocker oligonucleotides comprise single-stranded oligonucleotides that can hybridize to one or more universal adaptor sequences of a bottom strand linear library molecule and inhibit hybridization of another oligonucleotide to the one or more universal adaptor sequences. In some embodiments, the bottom strand blocker oligonucleotide comprises an anchor sequence that can hybridize with one end of a bottom strand linear library molecule, and lacks a bridging sequence that can hybridize with another end of the bottom strand linear library molecule. Thus, the bottom strand blocker oligonucleotide can hybridize to at least a portion of a bottom strand linear library molecule but does not circularize the linear library molecule to generate an open circle library molecule. In some embodiments, individual bottom strand blocker oligonucleotides can hybridize to at least a portion of a bottom strand linear library molecule thereby generating a library blocker complex. In some embodiments, individual bottom strand blocker oligonucleotides do not hybridize to at least a portion of a top strand linear library molecule. In some embodiments, the bottom strand blocker oligonucleotide can hybridize to least a portion of one or more universal adaptor sequences located on the 3’ side of the insert sequence. In some embodiments, the bottom strand blocker oligonucleotide can hybridize to one or more universal adaptor sequences located on the 5’ side of the insert sequence. In some embodiments, individual bottom strand blocker oligonucleotides lack a sequence that can hybridize with the insert region of a bottom strand linear library molecule. The bottom strand blocker oligonucleotide can include or lack a sequence that can hybridize to a left sample index sequence of the bottom strand linear library molecule. The bottom strand blocker oligonucleotide can include or lack a sequence that can hybridize to a right sample index sequence of the bottom strand linear library molecule. In some embodiments, the 3’ ends of the bottom strand blocker oligonucleotides comprise a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the bottom strand blocker oligonucleotide. In some embodiments, the bottom strand blocker oligonucleotide comprises a 3’ non-extendible end. In some embodiments, the 3’ end of the bottom strand blocker oligonucleotide comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end of the bottom strand blocker oligonucleotide.

[00407] In some embodiments, in step (c), the bottom strand blocker oligonucleotides comprise any of the sequences according to SEQ ID NOS: 101-129. In some embodiments, the sequence of the bottom strand blocker oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of the sequences set forth in SEQ ID NOS: 101-129. [00408] In some embodiments, in step (c), individual spike-in circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. The anchor sequence of a spike-in circularization oligonucleotide can hybridize to one end of a top strand linear library molecule, and the bridging sequence of the same spike-in circularization oligonucleotide can hybridize to another end of the same top strand linear library molecule, to circularize the linear library molecule and generate an open circle library spike-in complex having a nick between the ends of the top strand linear library molecule. In some embodiments, the anchor sequence can hybridize to at least a portion of one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences. In some embodiments, the bridging sequence can hybridize to at least a portion of one or more universal adaptor sequences at another end of the top strand linear library molecule. In some embodiments, individual spike-in circularization oligonucleotides can hybridize to at least a portion of a top strand linear library molecule thereby generating an open circle library spike-in complex. In some embodiments, individual spike-in circularization oligonucleotides do not hybridize to at least a portion of a bottom strand linear library molecule. In some embodiments the anchor sequence can be located at the 3’ end of the spike-in circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the spike-in circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at another end of the top strand linear library molecule. In some embodiments the anchor sequence can be located at the 5’ end of the spike-in circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at one end of the top strand linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the spike-in circularization oligonucleotide and can hybridize to at least a portion of one or more universal adaptor sequences at another end of the same top strand linear library molecule. In some embodiments, individual spike-in circularization oligonucleotides lack a sequence that can hybridize with the insert region of a top strand linear library molecule. The spike-in circularization oligonucleotides can include or lack a sequence that can hybridize to a left sample index sequence of the top strand linear library molecule. The spike-in circularization oligonucleotides can include or lack a sequence that can hybridize to a right sample index sequence of the top strand linear library molecule. In some embodiments, the 3’ ends of the spike-in circularization oligonucleotide comprise a moiety that promotes polymerase- catalyzed extension of the 3’ end of the spike-in circularization oligonucleotide. In some embodiments, individual spike-in circularization oligonucleotides comprise a 3’ extendible end. In some embodiments, the 3’ ends of the spike-in circularization oligonucleotides comprise a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the spike-in circularization oligonucleotide. In some embodiments, the 5’ end of individual spike-in circularization oligonucleotides comprise an affinity moiety that can bind one of the receptor moi eties embedded in the at least one layer of hydrophilic polymer coating of the capture support. In some embodiments, the affinity moiety of the spike-in circularization oligonucleotide comprises biotin, desthiobiotin or iminobiotin.

[00409] In some embodiments, the contacting of step (c) is conducted under a condition suitable for hybridizing the anchor sequence of the spike-in circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at one end of a top strand linear library molecule, and hybridizing the bridging sequence of the spike-in circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at another end of the top strand linear library molecule, thereby generating an open circle library spikein complex having a nick between the ends of the top strand linear library molecule. [00410] In some embodiments, in step (c), the spike-in circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-100. In some embodiments, the sequence of the spike-in circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of SEQ ID NOS: 45-100. In some embodiments, step (c) comprises contacting the plurality of linear library molecules in-solution with a mixture of oligonucleotides including (i) a plurality of top strand circularization oligonucleotides, (ii) a plurality of bottom strand blocker oligonucleotides, and (iii) a plurality of spike-in circularization oligonucleotides, wherein the mixture of top strand circularization oligonucleotides and spike-in circularization oligonucleotides comprises a ratio of about 1 : 1, or about 2: 1, or about 3: 1, or about 4: 1, or about 5: 1, or about 6: 1, or about 7:1, or about 8: 1, or about 9: 1, or about 10: 1, or any ratio in between. In some embodiments, the mixture of top strand circularization oligonucleotides and spike-in circularization oligonucleotides comprises a ratio of about 100: 1, or about 75:1, or about 50: 1, or about 25:1 or about 10: 1, or any ratio therebetween. An exemplary ratio comprises four parts top strand circularization oligonucleotides and one part spike-in circularization oligonucleotides. Another exemplary ratio comprises ten parts top strand circularization oligonucleotides and one part spike-in circularization oligonucleotides.

[00411] In some embodiments, the mixture of top strand circularization oligonucleotides and bottom strand blocker oligonucleotides comprises a ratio of about 1 : 1, or about 2: 1, or about 3: 1, or about 4: 1, or about 5: 1, or about 6: 1, or about 7: 1, or about 8: 1, or about 9: 1, or about 10: 1, or any ratio in between. In some embodiments, the mixture of top strand circularization oligonucleotides and bottom strand blocker oligonucleotides comprises a ratio of about 100: 1, or about 75:1, or about 50: 1, or about 25:1 or about 10: 1, or any ratio therebetween. An exemplary ratio comprises one part top strand circularization oligonucleotides and one part bottom strand blocker oligonucleotides. An exemplary ratio comprises two parts top strand circularization oligonucleotides and one part bottom strand blocker oligonucleotides.

[00412] In some embodiments, the method comprises step (d): contacting in-solution the plurality of library molecule-oligonucleotide complexes with a plurality of target-specific baits/probes, thereby generating a plurality of library molecule bait complexes and a plurality of open circle library spike-in complexes. In some embodiments, the contacting can be conducted under a condition suitable for selectively binding the target-specific baits/probes to their cognate target sequences of the library molecule-oligonucleotide complexes. In some embodiments, the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36 A) that can bind a receptor moiety of the capture support.

[00413] In some embodiments, in step (d), the plurality of target-specific baits/probes can selectively bind the top strand insert region of at least two of the open circle library complexes to generate a plurality of open circle library bait complexes that are enriched for polynucleotides having top strand target sequences. In some embodiments, an open circle library bait complex comprises a library molecule bait complex.

[00414] In some embodiments, in step (d), the plurality of target-specific baits/probes can selectively bind the bottom strand insert region of at least two of the library blocker complexes to generate a plurality of library blocker bait complexes that are enriched for polynucleotides having bottom strand target sequences. In some embodiments, a library blocker bait complex comprises a library molecule bait complex.

[00415] In some embodiments, in step (d), the plurality of target-specific baits/probes do not selectively bind the top strand insert region of the open circle library spike-in complexes. In some embodiments, the open circle library spike-in complexes are not enriched for polynucleotides having target sequences.

[00416] In some embodiments, the contacting of step (d) can generate a mixture of library molecules comprising (i) a plurality of open circle library bait complexes that are enriched for top strand target sequences, (ii) a plurality of library blocker bait complexes that are enriched for bottom strand target sequences, and (iii) a plurality of open circle library spike-in complexes that are not enriched for target sequences.

[00417] In some embodiments, in step (d), the condition suitable for hybridizing the plurality of library molecule-oligonucleotide complexes with the plurality of target-specific baits/probes comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, or about 12-16 hours.

[00418] In some embodiments, in step (d), the condition suitable for hybridizing the plurality of library molecule-oligonucleotide complexes with the plurality of target-specific baits/probes comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65- 70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00419] In some embodiments, in step (d), individual target-specific baits/probes comprise an oligonucleotide comprising (i) a target-specific sequence that can selectively hybridize to at least a portion of the target sequence of a given library molecule, (ii) an affinity moiety at the 5’ end, and (iii) an extendible 3’ end. In some embodiments, individual target-specific baits/probes comprise a moiety at the 3’ end that promotes polymerase-catalyzed extension. In some embodiments, the plurality of target-specific baits/probes comprises extendible 3’ ends. In some embodiments, the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual targetspecific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual target-specific baits/probes can be located at the 5’ end or at an internal position.

[00420] In some embodiments, in step (d), the plurality of target-specific baits/probes comprise 2-10,000 different target-specific sequences. In some embodiments, the plurality of target-specific baits/probes comprises 2-1,000,000 different target-specific sequences, 2- 500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, the plurality of open circle library bait complexes comprise 2-10,000 different top strand insert sequences. In some embodiments, the plurality of open circle library bait complexes comprises 2-1,000,000 different top strand insert sequences, 2-500,000 different top strand insert sequences, 2- 100,000 different top strand insert sequences, 100-100,000 different top strand insert sequences, 500-10,000 different top strand insert sequences, 2-500 different top strand insert sequences, or 1,000-50,000 different top strand insert sequences, or any range therebetween. In some embodiments, the plurality of library blocker bait complexes comprise 2-10,000 different bottom strand insert sequences. In some embodiments, the plurality of library blocker bait complexes comprises 2-1,000,000 different bottom strand insert sequences, 2- 500,000 different bottom strand insert sequences, 2-100,000 different bottom strand insert sequences, 100-100,000 different bottom strand insert sequences, 500-10,000 different bottom strand insert sequences, 2-500 different bottom strand insert sequences, or 1,000- 50,000 different bottom strand insert sequences, or any range therebetween. In some embodiments, the plurality of open circle library spike-in complexes comprise 2-10,000 different top strand insert sequences. In some embodiments, the plurality of open circle library spike-in complexes comprises 2-1,000,000 different top strand insert sequences, 2- 500,000 different top strand insert sequences, 2-100,000 different top strand insert sequences, 100-100,000 different top strand insert sequences, 500-10,000 different top strand insert sequences, 2-500 different top strand insert sequences, or 1,000-50,000 different top strand insert sequences, or any range therebetween.

[00421] In some embodiments, step (c) can be conducted prior to step (d). In some embodiments, step (d) can be conducted prior to step (c).

[00422] In some embodiments, the contacting of steps (c) and (d) can be conducted essentially simultaneously. In some embodiments, the contacting of steps (c) and (d) can be conducted essentially simultaneously wherein the contacting comprises conducting an insolution hybridization reaction for about 1-15 minutes, about 15-30 minutes, about 30-60 minutes, about 60-120 minutes, about 2-4 hours, about 4-6 hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, or about 12-16 hours.

[00423] In some embodiments, the contacting of steps (c) and (d) can be conducted essentially simultaneously. In some embodiments, the contacting of steps (c) and (d) can be conducted essentially simultaneously wherein the contacting comprises conducting an insolution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00424] In some embodiments, the method comprises step (e): contacting the capture support with the plurality of library molecule bait complexes and the plurality of open circle library spike-in complexes under a condition suitable for binding the affinity moiety of individual library molecule bait complexes and individual open circle library spike-in complexes to an embedded receptor moiety of the capture support, thereby generating a plurality of library complexes immobilized to the capture support. In some embodiments, the plurality of library complexes immobilized to the capture support comprises a mixture of (i) a plurality of open circle library bait complexes that are enriched for top strand target sequences, (ii) a plurality of library blocker bait complexes that are enriched for bottom strand target sequences, and (iii) a plurality of open circle library spike-in complexes that are not enriched for target sequences.

[00425] In some embodiments, the contacting of step (e) can be conducted under a condition suitable for binding an affinity moiety of individual open circle library bait complexes to a receptor moiety of the capture support thereby generating a plurality of open circle library bait complexes immobilized to the capture support.

[00426] In some embodiments, the contacting of step (e) can be conducted under a condition suitable for binding an affinity moiety of individual library blocker bait complexes to a receptor moiety of the capture support thereby generating a plurality of library blocker bait complexes immobilized to the capture support.

[00427] In some embodiments, the contacting of step (e) can be conducted under a condition suitable for binding an affinity moiety of individual open circle library spike-in complexes to a receptor moiety of the capture support thereby generating a plurality of open circle library spike-in complexes immobilized to the capture support.

[00428] In some embodiments, in step (e), the density of open circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ per mm². In some embodiments, the density of the open circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ open circle library bait complexes per mm², or any range therebetween. In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00429] In some embodiments, in step (e), the density of library blocker bait complexes immobilized to the capture support is about 10² - 10¹⁵ library blocker bait complexes per mm². In some embodiments, the density of the library blocker bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ library blocker bait complexes per mm², or any range therebetween. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00430] In some embodiments, in step (e), the density of open circle library spike-in complexes immobilized to the capture support is about 10² - 10¹⁵ open circle library spike-in complexes per mm². In some embodiments, the density of the open circle library spike-in complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ open circle library spike-in complexes per mm², or any range therebetween. In some embodiments, the plurality of open circle library spike-in complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of open circle library spike-in complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00431] In some embodiments, in step (e), contacting the capture support can include residual linear library molecules from steps (b), (c) and/or (d). In some embodiments, step (e) comprises contacting the support with a wash reagent to remove residual linear library molecules and retain the plurality of library complexes immobilized to the capture support. In some embodiments, the plurality of library complexes immobilized to the capture support comprise: (i) the plurality of open circle library bait complexes that are enriched for top strand target sequences, (ii) the plurality of library blocker bait complexes that are enriched for bottom strand target sequences, and (iii) the plurality of open circle library spike-in complexes that are not enriched for target sequences.

[00432] In some embodiments, in step (e), the plurality of open circle library bait complexes immobilized to the capture support comprises 2-10,000 different top strand insert sequences. In some embodiments, the plurality of open circle library bait complexes comprises 2-1,000,000 different top strand insert sequences, 2-500,000 different top strand insert sequences, 2-100,000 different top strand insert sequences, 100-100,000 different top strand insert sequences, 500-10,000 different top strand insert sequences, 2-500 different top strand insert sequences, or 1,000-50,000 different top strand insert sequences, or any range therebetween. In some embodiments, the plurality of library blocker bait complexes immobilized to the capture support comprises 2-10,000 different bottom strand insert sequences. In some embodiments, the plurality of library blocker bait complexes comprises 2-1,000,000 different bottom strand insert sequences, 2-500,000 different bottom strand insert sequences, 2-100,000 different bottom strand insert sequences, 100-100,000 different bottom strand insert sequences, 500-10,000 different bottom strand insert sequences, 2-500 different bottom strand insert sequences, or 1,000-50,000 different bottom strand insert sequences, or any range therebetween. In some embodiments, the plurality of open circle library spike-in complexes immobilized to the capture support comprises 2-10,000 different top strand insert sequences. In some embodiments, the plurality of open circle library spike-in complexes comprises 2-1,000,000 different top strand insert sequences, 2-500,000 different top strand insert sequences, 2-100,000 different top strand insert sequences, 100-100,000 different top strand insert sequences, 500-10,000 different top strand insert sequences, 2-500 different top strand insert sequences, or 1,000-50,000 different top strand insert sequences, or any range therebetween.

[00433] In some embodiments, in step (e), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (e) comprises contacting the capture support separately with a plurality of pinning primers and the plurality of library complexes separately in any order. In some embodiments, the capture support can be contacted with a plurality of pinning primers and the plurality of library complexes essentially simultaneously.

[00434] In some embodiments, the method comprises step (f): contacting the capture support with a ligation reagent thereby generating a plurality of closed circle library bait complexed immobilized to the capture support, and a plurality of closed circle library spikein complexes immobilized to the capture support.

[00435] In some embodiments, in step (f), the ligation reagent closes the nicks of individual open circle library bait complexes to generate a plurality of closed circle library bait complexes comprising covalently closed circular top strand library molecules hybridized to a top strand circularization oligonucleotide and a target-specific bait/probe which is immobilized to the capture support.

[00436] In some embodiments, in step (f), the ligation reagent closes the nicks of individual open circle library spike-in complexes to generate a plurality of closed circle library spike-in complexes comprising covalently closed circular top strand library molecules hybridized to a spike-in circularization oligonucleotide which is immobilized to the capture support.

[00437] In some embodiments, in step (f), the library blocker bait complexes lack a nick and do not form a covalently closed circular library molecule. In some embodiments, individual library blocker bait complexes comprise a linear bottom strand library molecule hybridized to a bottom strand blocker oligonucleotide and a target-specific bait/probe which is immobilized to the capture support.

[00438] In some embodiments, in step (f), the ligation reagent comprises a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1). In some embodiments, the ligation reagent comprises a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S). In some embodiments the ligation reagent comprises a recombinant thermal tolerant T4 DNA ligase including Hi- T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S). In some embodiments, the ligation reagent comprises a DNA ligase from Thermococcus nautili (e.g., NCBI No.

WP 042693257.1). In some embodiments, the ligation reaction comprises a T4 polynucleotide kinase (e.g., from New England Biolabs, catalog # M0201S).

[00439] In some embodiments, in step (f) the density of the plurality of closed circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ closed circle library bait complexes per mm². In some embodiments, the density of the plurality of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library bait complexes per mm², or any range therebetween. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00440] In some embodiments, in step (f) the density of the plurality of closed circle library spike-in complexes immobilized to the capture support is about 10² - 10¹⁵ closed circle library spike-in complexes per mm². In some embodiments, the density of the plurality of closed circle library spike-in complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library spike-in complexes per mm², or any range therebetween. In some embodiments, the plurality of closed circle library spike-in complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library spike-in complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00441] In some embodiments, in step (f) the density of the plurality of library blocker bait complexes immobilized to the capture support is about 10² - 10¹⁵ library blocker bait complexes per mm². In some embodiments, the density of the plurality of library blocker bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ library blocker bait complexes per mm², or any range therebetween. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00442] In some embodiments, step (f) comprises contacting the support with a wash reagent to remove linear library molecules and retain the plurality of (i) immobilized closed circle library bait complexes, (ii) immobilized closed circle library spike-in complexes, and (iii) immobilized library blocker bait complexes. In some embodiments, the washing of step (f) can generate a plurality of immobilized library molecules that are enriched for polynucleotides having target sequences.

[00443] In some embodiments, in step (f), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (f) comprises contacting the capture support with a plurality of pinning primers prior to or after conducting the ligation reaction. In some embodiments, step (f) comprises contacting the capture support with the ligation reagent and a plurality of pinning primers essentially simultaneously.

[00444] In some embodiments, step (f) comprises a ligation reaction that generates a plurality of covalently closed circular library molecules immobilized to the capture support, including (i) a plurality of covalently closed circular library molecules that form closed circle library bait complexes, and (ii) a plurality of covalently closed circular library molecules that form closed circle library spike-in complexes. In some embodiments, after conducting the ligation reaction of step (f), at least one of the covalently closed circular library molecules immobilized to the capture support comprises at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule comprising at least one deaminated nucleotide base can be removed by subjecting the plurality of covalently closed circular library molecules to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (f) comprises contacting the plurality of covalently closed circular library molecules that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps thereby converting the at least one deaminated nucleotide base in the at least one covalently closed circular library molecule into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (f) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from covalently closed circular library molecules.

[00445] In some embodiments, the method comprises step (g): contacting the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction.

[00446] In some embodiments, in step (g), the rolling circle amplification reaction is conducted under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes and use the covalently closed circular library molecules as template molecules thereby generating a plurality of concatemer template molecules from the plurality of closed circle library bait complexes which are immobilized to the support (“immobilized concatemer template molecules”).

[00447] In some embodiments, in step (g), the rolling circle amplification reaction is conducted under a condition suitable to extend the 3’ ends of the immobilized spike-in circularization oligonucleotides and use the covalently closed circular library molecules as template molecules thereby generating a plurality of concatemer template molecules from the plurality of closed circle library spike-in complexes which are immobilized to the support (“immobilized concatemer template molecules”).

[00448] In some embodiments, in step (g), the plurality of immobilized library blocker bait complexes do not undergo rolling circle amplification.

[00449] In some embodiments, in step (g), individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region and any universal adaptor sequences present in a covalently closed circular library molecule. In some embodiments, individual immobilized concatemer template molecules comprise sequences from a top strand covalently closed circular library molecule.

[00450] In some embodiments, in step (g), the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers wherein individual pinning primers hybridize to a portion of an immobilized concatemer template molecule thereby pinning down a portion of a concatemer template molecule.

[00451] In some embodiments, in step (g), the rolling circle amplification reagent comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. In some embodiments, the rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

[00452] In some embodiments, the methods comprise step (h): sequencing at least a portion of individual concatemer template molecules in the plurality of immobilized concatemer template molecules. In some embodiments, at least a portion of the individual concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing of step (h) determines the sequence of the insert regions. In some embodiments, the sequencing of step (h) identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and nucleotide reagents which include nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein.

[00453] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis) and U.S. published application No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of steps (c) and (d) and the contacting/distributing of step (e) (where the contents of both documents are hereby incorporated by reference in their entireties).

(8) Enrichment by Conducting In-Solution Capture of Top Strands of Linear Library Molecules and Circularization Using Top Strand Circularization Oligonucleotides and a Flap Endonuclease

[00454] The present disclosure provides methods for enriching target polynucleotides from a mixture of double-stranded linear library molecules having target or non-target sequences. Individual double-stranded library molecules comprise complementary top and bottom strands. For the sake of clarity, methods for enriching top and bottom strand linear library molecules having target sequences are described separately. Methods for enriching top strand library molecules having target sequences are described herein in workflow (8). Methods for enriching bottom strand library molecules having target sequences are described herein in workflow (10). In some embodiments, methods for enriching top and bottom strands having target sequences can be conducted together in the same hybridization reaction (e.g., step (b)) by employing top strand circularization oligonucleotides for enriching the target top strands, and by employing bottom strand blocker oligonucleotides for enriching the target bottom strands, and the resulting enriched target top strands and target bottom strands can be distributed onto the same capture support for conducting rolling circle amplification and sequencing. In some embodiments, the terminal 5’ ends of individual top strand linear library molecules include a phosphate group. Therefore, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules. In some embodiments, the terminal 5’ ends of individual bottom strand linear library molecules lack a phosphate group. Therefore, the bottom strand linear library molecules cannot undergo intramolecular ligation to form covalently closed circular library molecules. Thus, conducting enrichment workflows (8) and (10) together will yield strand specific circularization of the top strand linear library molecules.

[00455] In some embodiments, the 5’ end portion of individual top strand linear library molecules can form a 5’ overhang flap structure upon hybridization with a top strand circularization oligonucleotide. In some embodiments, the 5’ overhang flap structure is cleavable with a structure specific 5’ flap endonuclease which forms a newly cleaved 5’ end which is enzymatically ligatable with the non-cleaved 3’ end. Consequently, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules. The covalently closed circular library molecules can be subjected to rolling circle amplification to generate concatemer template molecules which can be sequenced.

[00456] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00457] In some embodiments, in step (a), the capture support comprises a plurality of immobilized pinning primers, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide. In some embodiments, individual pinning primers comprise a blocking group at the 3’ end of the oligonucleotide wherein the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind a receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture support lacks a pinning primer.

[00458] In some embodiments, the methods comprise step (b): forming a plurality of open circle library complexes by contacting in-solution a plurality of top strand circularization oligonucleotides with a plurality of top strand linear library molecules, wherein individual open circle library complexes in the plurality comprise a 5’ overhang flap structure (e.g., FIG. 36D). In some embodiments, the 5’ flap structure of a top strand linear library molecule comprises a sequence that is not complementary to a sequence in the 5’ end portion of the top strand circularization oligonucleotide. In some embodiments, the 5’ flap structure is 2-10 nucleotides in length. In some embodiments, the 5’ overhang flaps are cleavable with a structure specific 5’ flap endonuclease. In some embodiments, the contacting is conducted under a condition suitable for hybridizing a first region of individual top strand circularization oligonucleotides to a sequence at one end of a given top strand linear library molecule, and suitable for hybridizing a second region of the same individual top strand circularization oligonucleotide to a sequence near the other end of the same top strand linear library molecule thereby forming a 5’ flap structure.

[00459] In some embodiments, in step (b), the plurality of linear library molecules comprise a mixture of linear library molecules having insert regions comprising target or non-target sequences. In some embodiments, the plurality of linear library molecules comprise a mixture of linear library molecules generated from double-stranded input nucleic acids comprising insert regions having top strand sequences or complementary bottom strand sequences (top strand linear library molecules and bottom strand linear library molecules). In some embodiments, individual top strand linear library molecules comprise (i) a top strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual bottom strand linear library molecules comprise (i) a bottom strand insert region comprising a target sequence or a non- target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual top strand circularization oligonucleotides can hybridize to at least a portion of a top strand linear library molecule thereby generating an open circle library complex comprising a 5’ overhang flap structure. In some embodiments, individual top strand circularization oligonucleotides do not hybridize to at least a portion of a bottom strand linear library molecule.

[00460] In some embodiments, in step (b), individual top strand circularization oligonucleotides comprise a single stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. One end of a top strand circularization oligonucleotide can hybridize to one end of a linear library molecule and the other end of the top strand circularization oligonucleotide can hybridize to a sequence near the end of the same linear library molecule, thereby generating an open circle library complex having 5’ overhang flap structure (e.g., FIG. 36D). The top strand circularization oligonucleotides exhibit little or no hybridization to a bottom strand linear library molecule.

[00461] In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences.

[00462] In some embodiments, the top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a left sample index sequence of the top strand of a linear library molecule. In some embodiments, the top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a right sample index sequence of the top strand of a linear library molecule.

[00463] In some embodiments the anchor sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of) the other end of the same top strand of the linear library molecule. In some embodiments, the 5’ overhang flap structure of the top strand linear library molecule comprises a sequence that is not complementary to the bridging sequence of the top strand circularization oligonucleotide. [00464] In some embodiments the anchor sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of) the other end of the same top strand of the linear library molecule. In some embodiments, the 5’ overhang flap structure of the top strand linear library molecule comprises a sequence that is not complementary to the bridging sequence of the top strand circularization oligonucleotide.

[00465] In some embodiments, the 3’ ends of the top strand circularization oligonucleotides comprises a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ ends of the top strand circularization oligonucleotide. In some embodiments, the 3’ ends of the top strand circularization oligonucleotides comprises a moiety that promotes polymerase-catalyzed extension of the 3’ ends of the top strand circularization oligonucleotide. In some embodiments, the top strand circularization oligonucleotides comprise any of the sequences according the SEQ ID NOS: 45-99. In some embodiments, the sequence of the top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of the sequences set forth in SEQ ID NOS: 45-99.

[00466] In some embodiments, the contacting of step (b) is conducted under a condition suitable for hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at one end of an individual top strand linear library molecule, and suitable for hybridizing the bridging sequence of the same top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences near the other end of the same individual linear library molecule, thereby forming individual open circle library complexes each having a 5’ overhang flap structure. In some embodiments, the 5’ flap structure of a top strand linear library molecule comprises a sequence that is not complementary to a sequence in the 5’ end portion of the top strand circularization oligonucleotide.

[00467] In some embodiments, in step (b), the plurality of top strand linear library molecules can be hybridized in-solution with a plurality of top strand circularization oligonucleotides in a hybridization reagent.

[00468] In some embodiments, in step (b), the condition suitable for hybridizing the top strand circularization oligonucleotides to an individual top strand linear library molecule comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15- 30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4- 6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours. [00469] In some embodiments, in step (b), the condition suitable for hybridizing the top strand circularization oligonucleotides to an individual top strand linear library molecule comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about SO- 55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00470] In some embodiments, in step (b), individual top strand linear library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual covalently closed circular library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b).

[00471] In some embodiments, the methods comprise step (c): forming a plurality of open circle library bait complexes wherein individual open circle library bait complexes comprise a 5’ overhang flap structure, by contacting in-solution the plurality of open circle library complexes with a plurality of target-specific baits/probes. In some embodiments, the contacting is conducted in-solution under a condition suitable for selectively hybridizing individual target-specific baits/probes to at least a portion of a target sequence of an insert region of an open circle library complex, thereby generating a plurality of open circle library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36D) that can bind a receptor moiety of the capture support.

[00472] In some embodiments, in step (c), the plurality of non-target linear library molecules do not selectively hybridize to the target-specific baits/probes. In some embodiments, individual target-specific baits/probes comprise (i) an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of the target sequence of a given linear library molecule, (ii) an affinity moiety at the 5’ end, and (iii) an extendible 3’ end. In some embodiments, the oligonucleotide of the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual target-specific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual target-specific baits/probes can be located at the 5’ end or at an internal position.

[00473] In some embodiments, in step (c), the plurality of target-specific baits/probes and the plurality of open circle library complexes can be hybridized in-solution in a hybridization reagent under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual top strand linear library molecules to corresponding target-specific baits/probes thereby forming a plurality of open circle library bait complexes.

[00474] In some embodiments, in step (c), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual top strand linear library molecules to corresponding target-specific baits/probes comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4-6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours.

[00475] In some embodiments, in step (c), the condition suitable for selectively hybridizing at least a portion of the target sequence of individual top strand linear library molecules corresponding cognate target-specific baits/probes comprises conducting an insolution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00476] In some embodiments, in step (c), the plurality of target-specific baits/probes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of target-specific baits/probes comprises 2-1,000,000 different target-specific sequences, 2- 500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, the plurality of open circle library bait complexes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of open circle library bait complexes comprises 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500- 10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[00477] In some embodiments, in step (c), the plurality of open circle library complexes comprises individual linear library molecules hybridized to a top strand circularization oligonucleotide, wherein the linear library molecules can be generated from double-stranded input nucleic acids comprising top strands and complementary bottom strands. In some embodiments, the plurality of linear library molecules comprises at least a first and a second sub-population of linear library molecules. In some embodiments, the insert regions of individual linear library molecules of the first sub-population comprise a target or non-target sequence from a top strand input nucleic acid. In some embodiments, the insert regions of individual linear library molecules of the second sub-population comprise a target or nontarget sequence from a bottom strand input nucleic acid. [00478] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual linear library molecule of the first sub-population (e.g., top strand insert sequence). [00479] In some embodiments, individual target-specific baits/probes can selectively hybridize to at least a portion of an insert region comprising a target sequence of an individual linear library molecule of the second sub-population (e.g., bottom strand insert sequence).

[00480] In some embodiments, step (b) can be conducted prior to step (c). In some embodiments, step (c) can be conducted prior to step (b). In some embodiments, steps (b) and (c) can be conducted essentially simultaneously.

[00481] In some embodiments, the hybridizing of steps (b) and (c) can generate a mixture comprising a plurality of open circle library bait complexes which carry target sequences and a plurality of linear library molecules which carry non-target polynucleotide sequences. In some embodiments, the mixture of open circle library bait complexes and linear library molecules from step (b) and/or step (c) can be contacted with paramagnetic beads coated with receptor moieties that can bind to the affinity moieties on the open circle library bait complexes thereby separating the open circle library bait complexes from the linear library molecules. In some embodiments, the paramagnetic beads that are bound to open circle library bait complexes can be washed to remove the plurality of linear library molecules. In some embodiments, the open circle library bait complexes can be released from the paramagnetic beads thereby generating a plurality of open circle library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the plurality of linear library molecules are not removed in step (b) and/or step (c). In some embodiments, the plurality of non-target linear library molecules are not removed in step (b) and/or step (c) using paramagnetic beads that are coated with receptor moieties. In some embodiments, step (b) and/or step (c) do not use paramagnetic beads coated with receptor moieties.

[00482] In some embodiments, the method comprises step (d): contacting the capture support with the plurality of open circle library bait complexes comprising a 5’ overhang flap structure, thereby generating a plurality of open circle library bait complexes immobilized to the capture support. In some embodiments, the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes (e.g., which are part of the open circle library bait complexes) to a receptor moiety of the capture support. In some embodiments, the plurality of open circle library bait complexes immobilized to the capture support are enriched for polynucleotides carrying target sequences. In some embodiments, the plurality of immobilized open circle library bait complexes comprises top strand library molecules in open circle form and comprising a 5’ overhang flap structure. [00483] In some embodiments, in step (d), the plurality of open circle library bait complexes comprising a 5’ overhang flap structure can be distributed/contacted with the capture support in a loading reagent and/or a hybridization reagent under a condition suitable for binding the affinity moiety of individual open circle library bait complexes to individual receptor moieties embedded in the hydrophilic polymer coating of the capture support, thereby generating a plurality of open circle library bait complexes immobilized to the capture support.

[00484] In some embodiments, in step (d) the plurality of open circle library bait complexes with 5’ overhang flap structures can be immobilized to the capture support at a density of about 10² - 10¹⁵ open circle library bait complexes per mm². In some embodiments, the density of open circle library bait complexes with 5’ overhang flap structures immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ open circle library bait complexes per mm², or any range therebetween. In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at nonpredetermined and random sites. In some embodiments, the plurality of open circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00485] In some embodiments, in step (d), the capture support is contacted with the plurality of open circle library bait complexes with 5’ overhang flap structures and residual non-target linear library molecules from steps (b) and/or (c). In some embodiments, step (d) comprises contacting the support with a wash reagent to remove the residual linear library molecules and retain the plurality of immobilized open circle library bait complexes with 5’ overhang flap structures. In some embodiments, the washing of step (d) can generate a plurality of immobilized open circle library bait complexes with 5’ overhang flap structures that are enriched for polynucleotides having target sequences. In some embodiments, in step (d), the plurality of immobilized open circle library bait complexes with 5’ overhang flap structures comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of open circle library bait complexes with 5’ overhang flap structures comprise 2- 1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. [00486] In some embodiments, in step (d), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (d) comprises contacting the capture support separately with a plurality of pinning primers and the plurality of open circle library bait complexes with 5’ overhang flap structures in any order. In some embodiments, the capture support can be contacted with a plurality of pinning primers and the plurality of open circle library bait complexes with 5’ overhang flap structures essentially simultaneously.

[00487] In some embodiments, the method comprises step (e): contacting the capture support with a flap cleavage reagent, under a condition suitable for cleaving the 5’ overhang flap structures, thereby forming a plurality of cleavage products, wherein individual cleavage products comprise an open circle library bait complex with a newly cleaved 5’ end and a noncleaved 3’ end. In some embodiments, the newly cleaved 5’ ends comprise a phosphate group. In some embodiments, the newly cleaved 5’ end and the non-cleaved 3’ end of the same library molecule form an open circle library molecule having a nick while being hybridized to the top strand circularization oligonucleotide and the target-specific bait/probe. In some embodiments, the nick is enzymatically ligatable.

[00488] In some embodiments, the flap cleavage reagent comprises at least one 5’ flap endonuclease. In some embodiments, the 5’ cleavage reagent comprises at least one 5’ flap endonuclease and a DNA ligase enzyme. In some embodiments, the DNA ligase enzyme in the 5’ cleavage reagent can ligate together the newly cleaved 5’ end and the non-cleaved 3’ end of individual open circle library molecule thereby generating a plurality of covalently closed circular library molecules hybridized to a top strand circularization oligonucleotide and an immobilized target-specific bait/probe thereby forming a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the plurality of immobilized closed circle library bait complexes comprises top strand library molecules that have undergone intramolecular ligation to form covalently closed circular library molecules.

[00489] In some embodiments, the flap cleavage reagent comprises at least one 5’ flap endonuclease. In some embodiments, the 5’ flap endonuclease comprises a structure-specific 5’ flap endonuclease which can cleave off the 5’ flap structure of single-stranded DNA or RNA. The structure-specific 5’ flap endonuclease does not cleave a specific sequence, but instead cleaves a 5’ overhang flap structure. The structure specific 5’ flap endonuclease catalyzes hydrolytic cleavage of the phosphodiester bond at the 5’ flap structure to release the 5’ overhang flap. In some embodiments, the flap cleavage reagent comprises a DNA ligase. In some embodiments, the flap cleavage reagent comprises a T4 polynucleotide kinase. [00490] In some embodiments, in step (e) the density of closed circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ per mm². In some embodiments, the density of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about IO¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library bait complexes per mm², or any range therebetween. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00491] In some embodiments, step (e) comprises contacting the support with a wash reagent to remove any non-target linear library molecules and retain the plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the washing of step (e) can generate a plurality of immobilized closed circle library bait complexes that are enriched for polynucleotides having target sequences.

[00492] In some embodiments, in step (e), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (e) comprises contacting the capture support with a plurality of pinning primers and the flap cleavage reagent in any order. In some embodiments, step (e) comprises contacting the capture support with the plurality of pinning primers and the flap cleavage reagent essentially simultaneously. [00493] In some embodiments, in step (e), after cleaving the 5’ overhang flap structures and enzymatically ligating the nick, at least one of the immobilized closed circle library bait complexes comprises a covalently closed circular library molecule having at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule having at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (e) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (e) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules.

[00494] In some embodiments, the method comprises step (f): contacting the plurality of closed circle library bait complexes immobilized to the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes (e.g., which are part of the closed circle library bait complexes immobilized to the capture support) and use the covalently closed circular library molecules as template molecules thereby generating a plurality of concatemer template molecules which are immobilized to the support (“immobilized concatemer template molecules”). In some embodiments, individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region and any universal adaptor sequences present in a given covalently closed circular library molecule. In some embodiments, individual immobilized concatemer template molecules comprise sequences from a top strand covalently closed circular library molecule. In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers wherein individual pinning primers hybridize to a portion of an immobilized concatemer template molecule thereby pinning down a portion of a concatemer template molecule.

[00495] In some embodiments, in step (f), the rolling circle amplification reagent comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. In some embodiments, the rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides. [00496] In some embodiments, the methods comprise step (g): sequencing at least a portion of individual immobilized concatemer template molecules in the plurality of immobilized concatemer template molecules. In some embodiments, at least a portion of the concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two- stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein.

[00497] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis”) and U.S. Application Publication No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of steps (b) and (c), and the contacting/distributing of step (d) (where the contents of both documents are hereby incorporated by reference in their entireties).

[00498] In some embodiments, after the sequencing of step (g) described above, the capture support can be subjected to a re-seeding workflow in which additional linear library molecules can be hybridized in-solution to top strand circularization oligonucleotides to generate a plurality of open circle library complexes comprising 5’ overhang flap structures, and the open circle library complexes can be hybridized target-specific baits/probes to generate a plurality of open circle library bait complexes comprising 5’ overhang flap structures that are distributed onto the capture support. In some embodiments, the re-seeding workflow comprises cleaving the 5’ flap structure and closing the resulting nick to generate a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (b) - (e) at least once. In some embodiments, steps (6) - (8) of a re-seeding workflow can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding are described herein.

[00499] In some embodiments, the flap cleavage reagent of step (e) comprises at least one 5’ archaeal organism. In some embodiments, the 5’ flap endonuclease (FEN1) comprises a thermostable enzyme.

[00500] In some embodiments, the flap cleavage reagent of step (e) comprises at least one 5’ flap endonuclease that originates from an Archaebacterial species including without limitation Archaeoglobus fulgidus (Afu FEN1; Chapados et al., 2004 Cell 116:39-50; Hosfield et al., 1998 J. Biol. Chem. 273:27154-27161; Hosfield 1998 Cell 95; 135-146; Allawi 2003 J. Mol. Biol. 328:537-554), Methanobacterium thermoautotrophicum (Mth FEN1), Pyrococcus furiosus (Pfu FEN1; Kaiser et al., 1999 J. Biol. Chem. 274:21387- 21394), Methanococcus jannaschii (Mja FEN1; Hosfield et al., 1998 J. Biol. Chem. 273:27154-27161; Hwang 1998 Nature Struct. Biol. 5:707-713; Rao 1998 J. Bacteriol. 180:5406-5412; Bae 1999 Mol. Cells 9:45-48), Pyrococcus woesei (Pwo FEN1), Pyrococcus horikoshii (Pho FEN1; Matsui et al., 1999 J. Biol. Chem. 274: 18297-18309; Matsui 2002 J. Biol. Chem. 277:37840-37847; Matsui 2014 Extremophiles 18:415-427), Archaeoglobus veneficus (Ave FEN1), Thermococcus kodakarensis (Tko FEN1; Burkhart 2017 J. Bacteriol. 199:e00141-17; Muzzamal 2020 Folia Microbiol (Praha) 62:407-415), Desulfurococcus amylolyticus (Dam FEN1; Mase 2011 Acta. Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 67:209-213), Aeropyrum pemix (Ape FEN1; Collins 2004 Acta. Crystallogr. D. Biol. Crystallogr. 60: 1674-1678), Sulfolobus tokodaii (Sto FEN1; Horie 2007 Biosci.

Biotechnol. Biochem. 71 :855-865), and Sulfolobus solfataricus (Sso FEN1; Beattie and Bell 2012 EMBO. J. 31 : 1556-1567). The contents of these references are hereby expressly incorporated by reference in their entireties.

[00501] In some embodiments, the flap cleavage reagent of step (e) comprises a 5’ flap endonuclease from Thermococcus sp. 9 degrees North (9°N FEN1) (e.g., from New England Biolabs, catalog # M0645S).

[00502] In some embodiments, the flap cleavage reagent of step (e) comprises at least one 5’ flap endonuclease that originates from a eukaryotic organism including without limitation murine FEN1 (Harrington and Lieber 1994 EMBO J. 13: 1235-1246), yeast FEN1 (Harrington and Lieber 1994 Genes Dev. 8: 1344-1355), and human FEN1 (Hiraoka et al., 1995 Genomics 25:220-225). The contents of these references are hereby expressly incorporated by reference in their entireties.

[00503] In some embodiments, the flap cleavage reagent of step (e) comprises a mixture of two or more different types of flap endonucleases for example selected from any of the flap endonucleases described herein.

[00504] In some embodiments, the flap cleavage reagent of step (e) comprises at least one fusion enzyme comprising a portion of at least one 5’ flap endonuclease for example selected from any of the 5’ flap endonucleases described herein.

[00505] In some embodiments, the flap cleavage reagent of step (e) comprises a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1).

[00506] In some embodiments, the flap cleavage reagent of step (e) comprises a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S).

[00507] In some embodiments, the flap cleavage reagent of step (e) comprises a recombinant thermal tolerant T4 DNA ligase including Hi-T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S).

[00508] In some embodiments, the flap cleavage reagent of step (e) comprises a DNA ligase from Thermococcus nautili (e.g., NCBI No. WP_042693257.1).

[00509] In some embodiments, the flap cleavage reagent of step (e) comprises a T4 polynucleotide kinase (e.g., from New England Biolabs, catalog # M0201S).

(9) Enrichment by Conducting On-Support Capture of Top Strands of Linear Library Molecules and Circularization Using Top Strand Circularization Oligonucleotides and a Flap Endonuclease

[00510] The present disclosure provides methods for enriching target polynucleotides from a mixture of double-stranded linear library molecules having target or non-target sequences. Individual double-stranded library molecules comprise complementary top and bottom strands. For the sake of clarity, methods for enriching top and bottom strand linear library molecules having target sequences are described separately. Methods for enriching top strand library molecules having target sequences are described herein in workflow (9). Methods for enriching bottom strand library molecules having target sequences are described herein in workflow (10). In some embodiments, methods for enriching top and bottom strands having target sequences can be conducted together in the same hybridization reaction (e.g., step (b)) by employing top strand circularization oligonucleotides for enriching the target top strands, and by employing bottom strand blocker oligonucleotides for enriching the target bottom strands, and the resulting enriched target top strands and target bottom strands can be distributed onto the same capture support for conducting rolling circle amplification and sequencing. In some embodiments, the terminal 5’ ends of individual top strand linear library molecules include a phosphate group, therefore the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules. In some embodiments, the terminal 5’ ends of individual bottom strand linear library molecules lack a phosphate group, therefore the bottom strand linear library molecules cannot undergo intramolecular ligation to form covalently closed circular library molecules. Thus, conducting enrichment workflows (9) and (10) together will yield strand specific circularization of the top strand linear library molecules.

[00511] In some embodiments, the 5’ portion of individual top strand linear library molecules can form a 5’ overhang flap structure upon hybridization with a top strand circularization oligonucleotide wherein the 5’ overhang flap structure is cleavable with a structure specific 5’ flap endonuclease which forms a newly cleaved 5’ end which is enzymatically ligatable with the non-cleaved 3’ end. Therefore, the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules. The covalently closed circular library molecules can be subjected to rolling circle amplification to generate concatemer template molecules which can be sequenced.

[00512] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof. [00513] In some embodiments, in step (a), the capture support comprises a plurality of immobilized target-specific baits/probes, wherein individual target-specific baits/probes comprises (i) an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of the target sequence of a linear library molecule, (ii) an affinity moiety at the 5’ end, and (iii) an extendible 3’ end. In some embodiments, the oligonucleotides of the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual target-specific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual target-specific baits/probes can be located at the 5’ end or at an internal position.

[00514] In some embodiments, the plurality of target-specific baits/probes can be distributed/contacted with the capture support in a loading reagent under a condition suitable for binding the affinity moieties of individual target-specific baits/probes to individual receptor moieties embedded in the hydrophilic polymer coating of the capture support, thereby generating a capture support comprising a plurality of target-specific baits/probes immobilized to the capture support.

[00515] In some embodiments, in step (a), the capture support comprises a plurality of immobilized pinning primers, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide. In some embodiments, individual pinning primers comprise a blocking group at the 3’ end of the oligonucleotide wherein the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture surface lacks a plurality of immobilized pinning primers.

[00516] In some embodiments, the methods comprise step (b): forming a plurality of library bait complexes immobilized to the capture support by contacting a plurality of linear library molecules to the plurality of target-specific baits/probes that are immobilized to the capture support. In some embodiments, the contacting is conducted on the capture support under a condition suitable for selectively hybridizing at least a portion of the target insert sequences of individual linear library molecules to corresponding target-specific baits/probes that are immobilized on the capture support, thereby generating a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the plurality of non-target linear library molecules do not selectively hybridize to the immobilized target-specific baits/probes.

[00517] In some embodiments, in step (b), the plurality of linear library molecules can be distributed/contacted with the plurality of target-specific baits/probes that are immobilized to the capture support in a loading reagent and/or a hybridization reagent, under a condition suitable for hybridizing at least a portion of the target insert sequences of individual linear library molecules to corresponding target-specific baits/probes that are immobilized on the capture support, thereby generating a plurality of immobilized library bait complexes.

[00518] In some embodiments, in step (b) the mixture of linear library molecules comprises target-specific sequences and non-target sequences. In some embodiments, in step (b), the mixture of linear library molecules comprises 2-10,000 different target sequences. In some embodiments, the mixture of linear library molecules comprises 2-1,000,000 different target sequence, 2-500,000 different target sequences, 2-100,000 different target sequences, 100-100,000 different target sequences, 500-10,000 different target sequences, 2-500 different target sequences, or 1,000-50,000 different target sequences, or any range therebetween. In some embodiments, the plurality of target-specific baits/probes comprises 2- 10,000 different target-specific sequences. In some embodiments the plurality of targetspecific baits/probes comprises 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100- 100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2- 500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, in the plurality of library -bait complexes, the plurality of target-specific baits/probes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality target-specific baits/probes comprises 2- 1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. [00519] In some embodiments, in step (b), the plurality of linear library molecules comprises linear library molecules having insert regions comprising target or non-target sequences. In some embodiments, the plurality of linear library molecules comprises linear library molecules generated from double-stranded input nucleic acids comprising insert regions having top strand sequences or their complementary bottom strand sequences (top strand linear library molecules and bottom strand linear library molecules, respectively). In some embodiments, individual top strand linear library molecules comprise (i) a top strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual bottom strand linear library molecules comprise (i) a bottom strand insert region comprising a target sequence or a non- target sequence and (ii) at least one universal adaptor sequence.

[00520] In some embodiments, in step (b), individual top strand linear library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual covalently closed circular library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b).

[00521] In some embodiments, in step (b), the condition suitable for hybridizing individual linear library molecules with individual immobilized target-specific baits/probes comprises conducting an on-support hybridization reaction for about 1-15 minutes, 15-30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4-6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours. [00522] In some embodiments, in step (b), the condition suitable for hybridizing individual linear library molecules with individual immobilized target-specific baits/probes comprises conducting an on-support hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00523] In some embodiments, in step (b) the density of the library bait complexes immobilized to the capture support is about 10² - 10¹⁵ library bait complexes per mm². In some embodiments, the density of the library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ library bait complexes per mm², or any range therebetween. In some embodiments, the plurality of library bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00524] In some embodiments, the hybridization of step (b) generates a mixture comprising a plurality of immobilized library bait complexes which carry target sequences and a plurality of non-target linear library molecules which carry non-target sequences. In some embodiments, step (b) comprises removing the plurality of linear library molecules from the capture support by contacting the capture support with a wash reagent thereby removing the plurality of linear library molecules and retain the plurality of library bait complexes immobilized to the capture support. In some embodiments, the washing of step (b) can generate a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences.

[00525] In some embodiments, in step (b), the capture support comprises a plurality of pinning primers or lacks a plurality of pinning primers. In some embodiments, in step (b), the capture support can be contacted separately with the plurality of pinning primers and the plurality of linear library molecules in any order. In some embodiments, the capture support can be contacted with the plurality of pinning primer and the plurality of linear library molecules essentially simultaneously.

[00526] In some embodiments, the method comprises step (c): forming a plurality of open circle library bait complexes immobilized to the capture support by contacting the plurality of immobilized library bait complexes with a plurality of top strand circularization oligonucleotides, wherein individual open circle library bait complexes in the plurality comprise a 5’ overhang flap structure (e.g., FIG. 36D). In some embodiments, the 5’ flap structure of a top strand linear library molecule comprises a sequence that is not complementary to a sequence in the 5’ end portion of the top strand circularization oligonucleotide. In some embodiments, the 5’ flap structure is 2-10 nucleotides in length. In some embodiments, the 5’ overhang flaps are cleavable with a structure specific 5’ flap endonuclease. In some embodiments, the contacting is conducted under a condition suitable for hybridizing a first region of individual top strand circularization oligonucleotides to a sequence at one end of a given top strand linear library molecule (e.g., that is part of a library bait complex), and suitable for hybridizing a second region of the same individual top strand circularization oligonucleotide to a sequence near the other end of the same top strand linear library molecule. In some embodiments, the 5’ end of the target-specific/bait probe comprises an affinity moiety (e.g., the pentagon shape in FIG. 36D) that can bind a receptor moiety of the capture support.

[00527] In some embodiments, in step (c), the condition suitable for hybridizing individual linear library molecules (e.g., which are part of individual immobilized library-bait complexes) with individual top strand circularization oligonucleotides comprises conducting an on-support hybridization reaction for about 1-15 minutes, about 15-30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4-6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours.

[00528] In some embodiments, in step (c), the condition suitable for hybridizing individual linear library molecules (e.g., which are part of individual immobilized library-bait complexes) with individual top strand circularization oligonucleotides comprises conducting an on-support hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00529] In some embodiments, in step (c), individual top strand circularization oligonucleotides comprise a single stranded oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end. One end of a top strand circularization oligonucleotide can hybridize to one end of a linear library molecule and the other end of the top strand circularization oligonucleotide can hybridize to a sequence near the end of the same linear library molecule, thereby generating an open circle library complex having 5’ overhang flap structure (e.g., FIG. 36D). The top strand circularization oligonucleotides exhibit little or no hybridization to a bottom strand linear library molecule.

[00530] In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences.

[00531] In some embodiments, the top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a left sample index sequence of the top strand of a linear library molecule. In some embodiments, the top strand circularization oligonucleotide includes or lacks a sequence that can hybridize to a right sample index sequence of the top strand of a linear library molecule.

[00532] In some embodiments the anchor sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of) the other end of the same top strand of the linear library molecule. In some embodiments, the 5’ overhang flap structure of the top strand linear library molecule comprises a sequence that is not complementary to the bridging sequence of the top strand circularization oligonucleotide. [00533] In some embodiments the anchor sequence can be located at the 5’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences at one end of the top strand of the linear library molecule. In some embodiments, the bridging sequence can be located at the 3’ end of the top strand circularization oligonucleotide and can hybridize to one or more universal adaptor sequences near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of) the other end of the same top strand of the linear library molecule. In some embodiments, the 5’ overhang flap structure of the top strand linear library molecule comprises a sequence that is not complementary to the bridging sequence of the top strand circularization oligonucleotide. [00534] In some embodiments, the 3’ end of the top strand circularization oligonucleotides comprises blocking moi eties that inhibits polymerase-catalyzed extension of the 3’ ends of the top strand circularization oligonucleotide. In some embodiments, the 3’ end of the top strand circularization oligonucleotide comprises a moiety that promotes polymerase- catalyzed extension of the 3’ end of the top strand circularization oligonucleotide. [00535] In some embodiments, the top strand circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 45-99. In some embodiments, the sequence of the top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to any of the sequences set forth in SEQ ID NOS: 45-99.

[00536] In some embodiments, the contacting of step (c) is conducted under a condition suitable for hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences at one end of an individual top strand linear library molecule (e.g., which is part of an immobilized library bait complex), and suitable for hybridizing the bridging sequence of the same top strand circularization oligonucleotide to at least a portion of one or more universal adaptor sequences near the other end of the same individual linear library molecule, thereby forming individual open circle library bait complexes each having a 5’ overhang flap structure. In some embodiments, the 5’ flap structure of a top strand linear library molecule comprises a sequence that is not complementary to a sequence in the 5’ end portion of the top strand circularization oligonucleotide.

[00537] In some embodiments, in step (c), the plurality of immobilized library bait complexes can be hybridized on-support with a plurality of top strand circularization oligonucleotides in a hybridization reagent.

[00538] In some embodiments, the method comprises step (d): contacting the capture support with a flap cleavage reagent, under a condition suitable for cleaving the 5’ overhang flap structures thereby forming a plurality of cleavage products, wherein individual cleavage products comprise an open circle library bait complex with a newly cleaved 5’ end and a noncleaved 3’ end. In some embodiments, the newly cleaved 5’ ends comprise a phosphate group. In some embodiments, the newly cleaved 5’ end and the non-cleaved 3’ end of the same library molecule form an open circle library molecule having a nick while being hybridized to the top strand circularization oligonucleotide and the target-specific bait/probe. In some embodiments, the nick is enzymatically ligatable.

[00539] In some embodiments, the flap cleavage reagent comprises at least one 5’ flap endonuclease. In some embodiments, the 5’ cleavage reagent comprises at least one 5’ flap endonuclease and a DNA ligase enzyme. In some embodiments, the DNA ligase enzyme in the 5’ cleavage reagent can ligate together the newly cleaved 5’ end and the non-cleaved 3’ end of individual open circle library molecule thereby generating a plurality of covalently closed circular library molecules hybridized to a top strand circularization oligonucleotide and a target-specific bait/probe thereby forming a plurality of closed circle library bait complexes immobilized to the capture support (“immobilized closed circle library bait complexes”). In some embodiments, the plurality of immobilized closed circle library bait complexes comprises top strand library molecules that have undergone intramolecular ligation to form covalently closed circular library molecules.

[00540] In some embodiments, the flap cleavage reagent comprises at least one 5’ flap endonuclease. In some embodiments, the flap cleavage reagent comprises a DNA ligase. In some embodiments, the flap cleavage reagent comprises a T4 polynucleotide kinase.

[00541] In some embodiments, in step (d) the density of closed circle library bait complexes immobilized to the capture support is about 10² - 10¹⁵ closed circle library bait complexes per mm². In some embodiments, the density of closed circle library bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ closed circle library bait complexes per mm², or any range therebetween. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at nonpredetermined and random sites. In some embodiments, the plurality of closed circle library bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00542] In some embodiments, step (d) comprises contacting the support with a wash reagent to remove any non-target linear library molecules and retain the plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the washing of step (d) can generate a plurality of immobilized closed circle library bait complexes that are enriched for polynucleotides having target sequences.

[00543] In some embodiments, in step (d), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (d) comprises contacting the capture support with a plurality of pinning primers and the flap cleavage reagent in any order. In some embodiments, step (d) comprises contacting the capture support with the plurality of pinning primers and the flap cleavage reagent essentially simultaneously. [00544] In some embodiments, in step (d), after cleaving the 5’ overhang flap structures and enzymatically ligating the nick, at least one of the immobilized closed circle library bait complexes comprises a covalently closed circular library molecule having at least one deaminated nucleotide base. In some embodiments, the at least one covalently closed circular library molecule carrying at least one deaminated nucleotide base can be removed by subjecting the plurality of closed circle library bait complexes to enzymatic removal of deaminated bases and gap-generation. In some embodiments, step (d) comprises contacting the plurality of closed circle library bait complexes that are immobilized to the capture support with a reagent that removes deaminated bases and generates gaps thereby converting the at least one deaminated nucleotide base in the at least one closed circle library bait complex into at least one abasic site. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity. A library molecule having at least one abasic site cannot retain a covalently closed circular form and consequently cannot undergo rolling circle amplification in a subsequent step. In some embodiments, removing covalently closed circular library molecules carrying deaminated nucleotide bases at step (d) can improve sequencing quality scores compared to omitting the removal of deaminated nucleotide base from the covalently closed circular library molecules.

[00545] In some embodiments, the method comprises step (e): contacting the plurality of closed circle library bait complexes immobilized to the capture support with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the immobilized target-specific baits/probes (e.g., which are part of the closed circle library bait complexes immobilized to the capture support) and use the covalently closed circular library molecules as template molecules thereby generating a plurality of concatemer template molecules which are immobilized to the support (“immobilized concatemer template molecules”). In some embodiments, individual immobilized concatemer template molecules comprise multiple tandem repeat sequences of the insert region and any universal adaptor sequences present in a covalently closed circular library molecule. In some embodiments, individual immobilized concatemer template molecules comprise sequences from a top strand covalently closed circular library molecule. In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the capture support comprises a plurality of pinning primers wherein individual pinning primers hybridize to a portion of an immobilized concatemer template molecule thereby pinning down a portion of a concatemer template molecule.

[00546] In some embodiments, in step (e), the rolling circle amplification reagent comprises: (iii) a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the rolling circle amplification reagent lacks compaction oligonucleotides. In some embodiments, the rolling circle amplification reaction can be conducted in the presence or absence of a plurality of compaction oligonucleotides.

[00547] In some embodiments, the methods comprise step (f): sequencing at least a portion of individual immobilized concatemer template molecules in the plurality of immobilized concatemer template molecules. In some embodiments, at least a portion of the concatemer template molecules are sequenced while they are immobilized to the capture support. In some embodiments, single pass sequencing can be conducted. In some embodiments, pairwise sequencing can be conducted, for example as described herein. In some embodiments, the sequencing identifies the target sequences. In some embodiments, the sequencing comprises contacting the plurality of immobilized concatemer template molecules with a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described herein, including two- stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides. Exemplary sequencing methods are described, for example in WO2022266470, WO2023235865 and US20230203564A1, and compaction oligonucleotides are described in W02024040058, the contents of each of which are incorporated by reference in their entireties herein.

[00548] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis”) and U.S. Application Publication No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of steps (b) and (c), and the contacting/distributing of step (d) (where the contents of both documents are hereby incorporated by reference in their entireties).

[00549] In some embodiments, after the sequencing of step (f) described above, the capture support can be subjected to a re-seeding workflow in which additional linear library molecules can be hybridized to the immobilized target-specific baits/probes to generate a plurality of library bait complexes, and the plurality of library bait complexes can be hybridized on-support to a plurality of top strand circularization oligonucleotides to generate a plurality of open circle library bait complexes comprising 5’ overhang flap structures. In some embodiments, the re-seeding workflow comprises cleaving the 5’ flap structure and closing the resulting nick to generate a plurality of closed circle library bait complexes immobilized to the capture support. In some embodiments, the capture support can be subjected to a re-seeding workflow comprising repeating steps (b) - (d) at least once. In some embodiments, steps (6) - (8) of a re-seeding workflow can be conducted to increase the density of immobilized concatemer template molecules. Methods for re-seeding are described herein.

[00550] In some embodiments, the flap cleavage reagent of step (d) comprises at least one 5’ flap endonuclease that originates from a thermophilic organism, a eukaryotic organism or an archaeal organism. In some embodiments, the 5’ flap endonuclease (FEN1) comprises a thermostable enzyme.

[00551] In some embodiments, the flap cleavage reagent of step (d) comprises at least one 5’ flap endonuclease that originates from an Archaebacterial species including without limitation Archaeoglobus fulgidus (Afu FEN1; Chapados et al., 2004 Cell 116:39-50; Hosfield et al., 1998 J. Biol. Chem. 273:27154-27161; Hosfield 1998 Cell 95; 135-146; Allawi 2003 J. Mol. Biol. 328:537-554), Methanobacterium thermoautotrophicum (Mth FEN1), Pyrococcus furiosus (Pfu FEN1; Kaiser et al., 1999 J. Biol. Chem. 274:21387- 21394), Methanococcus jannaschii (Mja FEN1; Hosfield et al., 1998 J. Biol. Chem. 273:27154-27161; Hwang 1998 Nature Struct. Biol. 5:707-713; Rao 1998 J. Bacteriol. 180:5406-5412; Bae 1999 Mol. Cells 9:45-48), Pyrococcus woesei (Pwo FEN1), Pyrococcus horikoshii (Pho FEN1; Matsui et al., 1999 J. Biol. Chem. 274: 18297-18309; Matsui 2002 J. Biol. Chem. 277:37840-37847; Matsui 2014 Extremophiles 18:415-427), Archaeoglobus veneficus (Ave FEN1), Thermococcus kodakarensis (Tko FEN1; Burkhart 2017 J. Bacteriol. 199:e00141-17; Muzzamal 2020 Folia Microbiol (Praha) 62:407-415), Desulfurococcus amylolyticus (Dam FEN1; Mase 2011 Acta. Crystallogr. Sect. F. Struct. Biol. Cryst.

Commun. 67:209-213), Aeropyrum pemix (Ape FEN1; Collins 2004 Acta. Crystallogr. D. Biol. Crystallogr. 60: 1674-1678), Sulfolobus tokodaii (Sto FEN1; Horie 2007 Biosci. Biotechnol. Biochem. 71 :855-865), and Sulfolobus solfataricus (Sso FEN1; Beattie and Bell 2012 EMBO. J. 31 : 1556-1567). The contents of these references are hereby expressly incorporated by reference in their entireties. [00552] In some embodiments, the flap cleavage reagent of step (d) comprises a 5’ flap endonuclease from Thermococcus sp. 9 degrees North (9°N FEN1) (e.g., from New England Biolabs, catalog # M0645S).

[00553] In some embodiments, the flap cleavage reagent of step (d) comprises at least one 5’ flap endonuclease that originates from a eukaryotic organism including without limitation murine FEN1 (Harrington and Lieber 1994 EMBO J. 13: 1235-1246), yeast FEN1 (Harrington and Lieber 1994 Genes Dev. 8: 1344-1355), and human FEN1 (Hiraoka et al., 1995 Genomics 25:220-225). The contents of these references are hereby expressly incorporated by reference in their entireties.

[00554] In some embodiments, the flap cleavage reagent of step (d) comprises a mixture of two or more different types of flap endonucleases for example selected from any of the flap endonucleases described above.

[00555] In some embodiments, the flap cleavage reagent of step (d) comprises at least one fusion enzyme comprising a portion of at least one 5’ flap endonuclease for example selected from any of the 5’ flap endonucleases described above.

[00556] In some embodiments, the flap cleavage reagent of step (d) comprises a bacteriophage DNA ligase including T3 DNA ligase (e.g., NCBI No. 523305.1), T4 DNA ligase (e.g., NCBI No. 049813.1) or T7 DNA ligase (e.g., NCBI No. 041963.1).

[00557] In some embodiments, the flap cleavage reagent of step (d) comprises a thermal stable DNA ligase including Taq DNA ligase (e.g., from New England Biolabs, catalog No. M0208S), Tfu DNA ligase from Thermococcus fumicolans (e.g., UniProtKB/Swiss No. Q9HH07.1), HiFi DNA ligase, or 9 degrees North DNA ligase (e.g., from New England Biolabs, catalog No. M0238S).

[00558] In some embodiments, the flap cleavage reagent of step (d) comprises a recombinant thermal tolerant T4 DNA ligase including Hi-T4 DNA ligase (e.g., from New England Biolabs, catalog # M2622S).

[00559] In some embodiments, the flap cleavage reagent of step (d) comprises a DNA ligase from Thermococcus nautili (e.g., NCBI No. WP_042693257.1).

[00560] In some embodiments, the flap cleavage reagent of step (d) comprises a T4 polynucleotide kinase (e.g., from New England Biolabs, catalog # M0201S). (10) Enrichment by Conducting In-Solution Capture of Bottom Strands of Linear Library Molecules and Immobilization on a Capture Support

[00561] The present disclosure provides methods for enriching target polynucleotides from a mixture of single-stranded linear library molecules having target or non-target sequences. Individual single-stranded linear library molecules can be generated from double-stranded input nucleic acids comprising top strands and their complementary bottom strands (top strand linear library molecules and bottom strand linear library molecules). For the sake of clarity, methods for enriching top and bottom strand linear library molecules having target sequences are described separately. Methods for enriching top strand library molecules having target sequences are described herein in workflows (5), (6), (7), (8) and (9). Methods for enriching bottom strand library molecules having target sequences are described herein in workflow (10).

[00562] In some embodiments, methods for enriching top and bottom linear library molecules having target insert sequences can be conducted together in the same hybridization reaction by employing top strand circularization oligonucleotides for enriching the target top strand linear library molecules (e.g., workflows (5), (6), (7), (8) and (9)), and by employing bottom strand blocker oligonucleotides for enriching the target bottom strand linear library molecules (e.g., workflow (10)), and the resulting linear library molecules enriched for target sequences can be distributed onto the same capture support for conducting rolling circle amplification and sequencing.

[00563] In some embodiments, any one of the top strand circularization oligonucleotides according to SEQ ID NOS: 45-99 can be used in the same hybridization reaction with any one of the bottom strand blocker oligonucleotides according to SEQ ID NOS: 101-129. [00564] In some embodiments, the terminal 5’ end of individual top strand linear library molecules include a phosphate group, and therefore the top strand linear library molecules can undergo intramolecular ligation to form covalently closed circular library molecules. In some embodiments, the terminal 5’ end of individual bottom strand linear library molecules lack a phosphate group, and therefore the bottom strand linear library molecules cannot undergo intramolecular ligation to form covalently closed circular library molecules. Thus, conducting enrichment workflows (5) and (10) together, or conducting enrichment workflows (6) and (10) together, or conducting enrichment workflows (7) and (10) together, will yield strand specific circularization of the top strand linear library molecules.

[00565] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, comprising step (a): providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, the at least one layer of hydrophilic polymer coating comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof.

[00566] In some embodiments, in step (a), the capture support comprises a plurality of immobilized pinning primers, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide. In some embodiments, individual pinning primers comprise a blocking group at the 3’ end of the oligonucleotide wherein the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture support lacks a pinning primer.

[00567] In some embodiments, the methods comprise step (b): forming a plurality of library blocker complexes by contacting in-solution a plurality of bottom strand blocker oligonucleotides with a plurality of linear library molecules. In some embodiments, the contacting can be conducted under a condition that is suitable for hybridizing individual bottom strand blocker oligonucleotides to at least a portion of one of the bottom strand linear library molecules. In some embodiments, the plurality of bottom strand blocker oligonucleotides can be hybridized in-solution with a plurality of bottom strand linear library molecules in a hybridization reagent under a condition suitable for hybridizing the bottom strand blocker oligonucleotide to at least a portion of one or more universal adaptor sequences at one end of the bottom strand linear library molecule thereby forming individual library blocker complexes.

[00568] In some embodiments, in step (b), the plurality of linear library molecules comprises a mixture of linear library molecules having insert regions comprising target or non-target sequences. In some embodiments, the plurality of linear library molecules comprise a mixture of linear library molecules generated from double-stranded input nucleic acids comprising insert regions having top strand sequences or their complementary bottom strand sequences. In some embodiments, individual top strand linear library molecules comprise (i) a top strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual bottom strand linear library molecules comprise (i) a bottom strand insert region comprising a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual bottom strand blocker oligonucleotides can hybridize to at least a portion of a bottom strand linear library molecule thereby generating a library blocker complex. In some embodiments, individual bottom strand blocker oligonucleotides do not hybridize to at least a portion of a top strand linear library molecule.

[00569] In some embodiments, in step (b), the terminal 5’ ends of individual top strand linear library molecules include a phosphate group. In some embodiments, the top strand linear library molecules can undergo intramolecular ligation in a subsequent step to form covalently closed circular library molecules. In some embodiments, the terminal 5’ ends of individual bottom strand linear library molecules lack a phosphate group. In some embodiments, the bottom strand linear library molecules cannot undergo intramolecular ligation in a subsequent step to form covalently closed circular library molecules.

[00570] In some embodiments, in step (b), individual library blocker complexes comprise a bottom strand linear library molecule hybridized to a bottom strand blocker oligonucleotide, wherein the bottom strand blocker oligonucleotide can hybridize to at least a portion of the bottom strand linear library molecule. Individual library blocker complexes comprise a double-stranded region formed from hybridization between a portion of the bottom strand linear library molecule and the bottom strand blocker oligonucleotide. Individual library blocker complexes do not circularize to form open circle library molecules. In some embodiments, the 3’ ends of the bottom strand blocker oligonucleotides comprise a moiety that blocks polymerase-catalyzed extension. In some embodiments, the bottom strand blocker oligonucleotides have non-extendible 3’ ends. In some embodiments, the 3’ ends of the bottom strand blocker oligonucleotides comprise a moiety that promotes polymerase- catalyzed extension. In some embodiments, the bottom strand blocker oligonucleotides have extendible 3’ ends.

[00571] In some embodiments, in step (b), the condition suitable for hybridizing the plurality of linear library molecules with the bottom strand blocker oligonucleotides comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15- 30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4- 6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours. [00572] In some embodiments, in step (b), the condition suitable for hybridizing the plurality of linear library molecules with the bottom strand blocker oligonucleotides comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about SO- 55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00573] In some embodiments, in step (b), individual bottom strand blocker oligonucleotides comprise single-stranded oligonucleotides that can hybridize to at least a portion of one or more universal adaptor sequences of a bottom strand linear library molecule and inhibit hybridization of another oligonucleotide to the universal adaptor sequences. In some embodiments, the bottom strand blocker oligonucleotide comprises an anchor sequence that can hybridize with one end of a bottom strand linear library molecule, and lacks a bridging sequence that can hybridize with the other end of the same bottom strand linear library molecule. Thus, the bottom strand blocker oligonucleotide can hybridize to at least a portion of a bottom strand linear library molecule but does not circularize the linear library molecule to generate an open circle library molecule. In some embodiments, the bottom strand blocker oligonucleotide can hybridize to at least a portion of one or more universal adaptor sequences located on the 3’ side of the insert sequence. In some embodiments, the bottom strand blocker oligonucleotide can hybridize to at least a portion of one or more universal adaptor sequences located on the 5’ side of the insert sequence. In some embodiments, individual bottom strand blocker oligonucleotides lack a sequence that can hybridize with the insert region of a bottom strand linear library molecule. The bottom strand blocker oligonucleotide can include or lack a sequence that can hybridize to a left sample index sequence of the bottom strand linear library molecule. The bottom strand blocker oligonucleotide can include or lack a sequence that can hybridize to a right sample index sequence of the bottom strand linear library molecule. In some embodiments, the 3’ ends of the bottom strand blocker oligonucleotides comprise a blocking moiety that inhibits polymerase-catalyzed extension of the 3’ end of the bottom strand blocker oligonucleotide. In some embodiments, the bottom strand blocker oligonucleotide comprises a 3’ non-extendible end. In some embodiments, the 3’ end of the bottom strand blocker oligonucleotide comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end of the bottom strand blocker oligonucleotide. [00574] In some embodiments, in step (b), the bottom strand blocker oligonucleotides comprise any of the sequences according to SEQ ID NOS: 101-129. In some embodiments, the sequence of the bottom strand blocker oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of the sequences set forth in SEQ ID NOS: 101-129. [00575] In some embodiments, in step (b), individual strand linear library molecules (e.g., top strand and bottom strand linear library molecules) comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual linear library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (b).

[00576] In some embodiments, the methods comprise step (c): forming a plurality of library blocker bait complexes by contacting in-solution the plurality of library blocker complexes with a plurality of target-specific baits/probes. In some embodiments, the contacting is conducted in-solution under a condition suitable for selectively hybridizing individual target-specific baits/probes to at least a portion of a target sequence of an insert region of a library blocker complex thereby generating a plurality of library blocker bait complexes that are enriched for polynucleotides having target sequences.

[00577] In some embodiments, in step (c), the plurality of non-target linear library molecules do not selectively hybridize to the target-specific baits/probes. In some embodiments, individual target-specific baits/probes comprise (i) an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of the target sequence of a linear library molecule, (ii) an affinity moiety at the 5’ end, and (iii) an extendible 3’ end. In some embodiments, the oligonucleotide of the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moiety of individual target-specific baits/probes can bind to a receptor moiety of the capture support of step (a). In some embodiments, the affinity moiety of individual target-specific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual target-specific baits/probes can be located at the 5’ end or at an internal position.

[00578] In some embodiments, in step (c), the plurality of target-specific baits/probes and the plurality of library blocker complexes can be hybridized in-solution in a hybridization reagent under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual bottom strand linear library molecules (e.g., which are part of the library blocker complexes) to corresponding target-specific baits/probes thereby forming a plurality of library blocker bait complexes.

[00579] In some embodiments, in step (c), the condition suitable for selectively hybridizing at least a portion of the target sequences of individual bottom strand linear library molecules to corresponding target-specific baits/probes comprises conducting an in-solution hybridization reaction for about 1-15 minutes, about 15-30 minutes, or about 30-60 minutes, or about 60-120 minutes, or about 2-4 hours, or about 4-6 hours, or about 6-8 hours, or about 8-10 hours, or about 10-12 hours, or about 12-16 hours.

[00580] In some embodiments, in step (c), the condition suitable for selectively hybridizing at least a portion of the target sequences of individual bottom strand linear library molecules to corresponding target-specific baits/probes comprises conducting an in-solution hybridization reaction at a temperature of about 30-35 degrees C, about 35-40 degrees C, about 40-45 degrees C, about 45-50 degrees C, about 50-55 degrees C, or about 55-60 degrees C, or about 60-65 degrees C, or about 65-70 degrees C, or about 70-75 degrees C, or about 75-80 degrees C.

[00581] In some embodiments, in step (c), the plurality of target-specific baits/probes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of target-specific baits/probes comprises 2-1,000,000 different target-specific sequences, 2- 500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween. In some embodiments, the plurality of library blocker bait complexes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of library blocker bait complexes comprises 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[00582] In some embodiments, step (b) can be conducted prior to step (c). In some embodiments, step (c) can be conducted prior to step (b). In some embodiments, steps (b) and (c) can be conducted essentially simultaneously.

[00583] In some embodiments, the hybridizing of steps (b) and (c) can generate a mixture comprising a plurality of library blocker bait complexes which carry target polynucleotide sequences and a plurality of linear library molecules which carry non-target polynucleotide sequences. In some embodiments, the mixture of library blocker bait complexes and nontarget linear library molecules from step (b) and/or step (c) can be contacted with paramagnetic beads coated with receptor moieties that can bind to the affinity moieties on the library blocker bait complexes thereby separating the library blocker bait complexes from the linear library molecules. In some embodiments, the paramagnetic beads that are bound to library blocker bait complexes can be washed to remove the plurality of linear library molecules. In some embodiments, the library blocker bait complexes can be released from the paramagnetic beads thereby generating a plurality of library blocker bait complexes that are enriched for polynucleotides having target sequences. In some embodiments, the plurality of linear library molecules are not removed in step (b) and/or step (c). In some embodiments, the plurality of non-target linear library molecules are not removed in step (b) and/or step (c) using paramagnetic beads that are coated with receptor moieties. In some embodiments, step (b) and/or step (c) does not use paramagnetic beads coated with receptor moieties.

[00584] In some embodiments, the method comprises step (d): contacting the capture support with the plurality of library blocker bait complexes thereby generating a plurality of library blocker bait complexes immobilized to the capture support. In some embodiments, the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes (e.g., which are part of the library blocker bait complexes) to a receptor moiety of the capture support. In some embodiments, the plurality of library blocker bait complexes immobilized to the capture support are enriched for polynucleotides carrying target sequences. In some embodiments, the plurality of immobilized library blocker bait complexes comprises bottom strand library molecules that cannot form an open circle library molecule.

[00585] In some embodiments, in step (d), the terminal 5’ ends of individual library molecules that form the library blocker bait complexes lack a phosphate group, and thus cannot undergo intramolecular ligation to form covalently closed circular library molecules and cannot undergo rolling circle amplification to generate immobilized concatemer template molecules.

[00586] In some embodiments, in step (d), the plurality of library blocker bait complexes can be distributed on/contacted with the capture support in a loading reagent and/or a hybridization reagent under a condition suitable for binding the affinity moiety of individual library blocker bait complexes to individual receptor moieties embedded in the hydrophilic polymer coating of the capture support, thereby generating a plurality of library blocker bait complexes immobilized to the capture support.

[00587] In some embodiments, in step (d) the density of library blocker bait complexes immobilized to the capture support is about 10² - 10¹⁵ library blocker bait complexes per mm². In some embodiments, the density of the library blocker bait complexes immobilized to the capture support is between about 10² and about 10¹⁴, between about 10³ and about 10¹², between about 10⁴ and about 10¹⁰, between about 10⁵ and about 10¹², between about 10⁵ and about 10¹⁵, or between about 10² and about 10⁵ library blocker bait complexes per mm², or any range therebetween. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at non-predetermined and random sites. In some embodiments, the plurality of library blocker bait complexes is immobilized to the capture support at predetermined sites and arranged in a pattern.

[00588] In some embodiments, in step (d), the capture support is contacted with the plurality of library blocker bait complexes and residual linear library molecules from steps (b) and/or (c). In some embodiments, step (d) comprises contacting the support with a wash reagent to remove the residual linear library molecules and retain the plurality of library blocker bait complexes immobilized to the capture support. In some embodiments, the washing of step (d) can generate a plurality of library blocker bait complexes immobilized to the capture support that is enriched for polynucleotides having target sequences. In some embodiments, in step (d), the plurality of immobilized library blocker bait complexes comprises 2-10,000 different target-specific sequences. In some embodiments, the plurality of immobilized library blocker bait complexes comprises 2-1,000,000 different targetspecific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target- specific sequences, 100-100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2-500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[00589] In some embodiments, in step (d), the capture support comprises a plurality of pinning primers or lacks pinning primers. In some embodiments, step (d) comprises contacting the capture support separately with a plurality of pinning primers and the plurality of library blocker bait complexes in any order. In some embodiments, the capture support can be contacted with a plurality of pinning primers and the plurality of library blocker bait complexes essentially simultaneously.

[00590] In some embodiments, any of the hybridization reagents and any of the loading reagents described in U.S. patent No. 11,781,185 (entitled “Methods and Reagent for Nucleic Acid Analysis”) and U.S. Application Publication No. 2020/0347443 (entitled “Nucleic Acid Hybridization Methods”) can be used for the hybridizing of steps (b) and (c), and the distributing of step (d) (where the contents of both documents are hereby incorporated by reference in their entireties).

Library Bait Complexes

[00591] In some embodiments, in any of the methods described herein, a library bait complex comprises a linear library molecule hybridized to a target-specific bait/probe. In some embodiments, the target-specific bait/probe can be hybridized to an insert region of the linear library molecule. The library bait complex can be in-solution or immobilized to a capture support.

Open Circle Library Complexes

[00592] In some embodiments, in any of the methods described herein, an open circle library complex comprises a top strand linear library molecule hybridized to a top strand circularization oligonucleotide. In some embodiments, one end of individual top strand linear library molecules can hybridize to a first end of the top strand circularization oligonucleotide and the other end of the same top strand linear library molecule can hybridize to a second end of the same top strand circularization oligonucleotide, thereby generating an open circle library complex having a gap or a nick between the ends of the linear library molecule. In some embodiments, the gap can be subjected to a gap fill-in reaction to generate a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, one end of individual top strand linear library molecules can hybridize to a first end of the top strand circularization oligonucleotide and the other end of the same top strand linear library molecule can hybridize to a region at the second end of the same top strand circularization oligonucleotide, thereby generating an open circle library complex having a 5’ overhang flap structure. In some embodiments, the 5’ overhang flap structure can be removed by cleavage with a structure specific 5’ flap endonuclease to generate a newly cleaved 5’ end. The newly cleaved 5’ end and the non-cleaved 3’ end can form a nick which is enzymatically ligatable. In some embodiments, the open circle library complex can be in-solution or immobilized to a capture support.

Open Circle Library Bait Complexes

[00593] In some embodiments, in any of the methods described herein, an open circle library bait complex comprises a top strand linear library molecule hybridized to (i) a top strand circularization oligonucleotide and (ii) a target-specific bait/probe. In some embodiments, one end of individual top strand linear library molecules can hybridize to a first end of the top strand circularization oligonucleotide and the other end of the same top strand linear library molecule can hybridize to a second end of the same top strand circularization oligonucleotide, thereby generating an open circle library bait complex having a nick between the ends of the linear library molecule (e.g., FIG. 36A). In some embodiments, the nick is enzymatically ligatable. In some embodiments, a target-specific bait/probe can selectively bind the insert region of the top strand linear library molecule. In some embodiments, the open circle library bait complex can be in-solution or immobilized to a capture support.

[00594] In some embodiments, in any of the methods described herein, an open circle library bait complex comprises a top strand linear library molecule hybridized to (i) a top strand circularization oligonucleotide (e.g., a single-stranded top strand circularization oligonucleotide) and (ii) a target-specific bait/probe. In some embodiments, one end of individual top strand linear library molecules can hybridize to a first end of the top strand circularization oligonucleotide and the other end of the same top strand linear library molecule can hybridize to a second end of the same top strand circularization oligonucleotide, thereby generating an open circle library bait complex having a gap between the ends of the linear library molecule (e.g., FIG. 36B). In some embodiments, the gap can be subjected to a polymerase-catalyzed fill-in reaction to generate a nick. In some embodiments, the nick can be contacted with a ligation reagent to close the nick. In some embodiments, the singlestranded top strand circularization oligonucleotide comprises (i) a first region at one end having a sequence that can hybridize with at least a portion of a universal adaptor sequence at one end of a given linear library molecule, (ii) a second region (2^nd region) comprising at least one index sequence and/or an additional universal adaptor sequence, and (iii) a third region at the other end having a sequence that can hybridize with at least a portion of a universal adaptor sequence at the other end of the same linear library molecule. In some embodiments, the single-stranded top strand circularization oligonucleotides comprise first and third regions that flank the second region. In some embodiments, the linear library molecule lacks a sequence that can hybridize with the second region of the single-stranded top strand circularization oligonucleotide.

[00595] In some embodiments, in any of the methods described herein, an open circle library bait complex comprises a top strand linear library molecule hybridized to (i) a top strand double-stranded circularization oligonucleotide and (ii) a target-specific bait/probe. In some embodiments, the top strand double-strand circularization oligonucleotide comprises a long strand and a short strand, wherein the long and short strands are hybridized together to form the double-stranded top strand circularization oligonucleotides having a double-stranded region and two flanking single-stranded regions. In some embodiments, one end of the linear library molecule can hybridize to one end of the long strand, and the other end of the same linear library molecule can hybridize to the other end of the same long strand thereby forming an open circle library bait complex having two nicks (e.g., FIG. 36C). In some embodiments, the two nicks are enzymatically ligatable. In some embodiments, a target-specific bait/probe can selectively bind the insert region of the top strand linear library molecule. In some embodiments, the open circle library bait complex can be in-solution or immobilized to a capture support.

[00596] In some embodiments, in any of the methods described herein, an open circle library bait complex comprises a top strand linear library molecule hybridized to (i) a top strand circularization oligonucleotide and (ii) a target-specific bait/probe. In some embodiments, the top strand circularization oligonucleotide comprises a single stranded oligonucleotide. In some embodiments, one end of individual top strand linear library molecules can hybridize to a first end of the top strand circularization oligonucleotide and the other end of the same top strand linear library molecule can hybridize to a second end of the same top strand circularization oligonucleotide, thereby generating an open circle library bait complex having a 5’ overhang flap structure (e.g., FIG. 36D). In some embodiments, the 5’ overhang flap structure can be removed by cleavage with a structure specific 5’ flap endonuclease to generate a newly cleaved 5’ end. The newly cleaved 5’ end and the non- cleaved 3’ end can form a nick which is enzymatically ligatable.. In some embodiments, the nick is enzymatically ligatable. In some embodiments, a target-specific bait/probe can selectively bind the insert region of the top strand linear library molecule. In some embodiments, the open circle library bait complex can be in-solution or immobilized to a capture support.

Closed Circle Library Bait Complexes

[00597] In some embodiments, in any of the methods described herein, a closed circle library bait complex comprises covalently closed circular top strand library molecules hybridized to a target-specific bait/probe. In some embodiments, the closed circle library bait complex can be in-solution or immobilized to a capture support.

[00598] In some embodiments, a closed circle library bait complex comprises covalently closed circular library molecules (e.g., covalently closed circular library molecules from the top strand) hybridized to a top strand circularization oligonucleotide and a target-specific bait/probe. The circle library bait complex can be in-solution or immobilized to a capture support.

Open Circle Library Spike-In Complexes

[00599] In some embodiments, in any of the methods described herein, an open circle library spike-in complex comprises a top strand linear library molecule hybridized to a spikein circularization oligonucleotide, wherein one end of the top strand linear library molecule can be hybridized to a first end of a spike-in circularization oligonucleotide and wherein the other end of the same top strand linear library molecule can be hybridized to a second end of one of the same spike-in circularization oligonucleotide, thereby generating an open circle library spike-in complex having a nick between the ends of the linear library molecule. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library spike-in complex can be in-solution or immobilized to a capture support.

Closed Circle Library Spike-In Complexes

[00600] In some embodiments, in any of the methods described herein, a closed circle library spike-in complex comprises a covalently closed circular top strand library molecule hybridized to a spike-in circularization oligonucleotide. The closed circle library spike-in complex can be in-solution or immobilized to a capture support. Library Blocker Complexes

[00601] In some embodiments, in any of the methods described herein, a library blocker complex comprises a bottom strand linear library molecule hybridized to a bottom strand blocker oligonucleotide. A library blocker complex comprises a double-stranded region formed from hybridization between a non-insert region of the bottom strand linear library molecule and the bottom strand blocker oligonucleotide. A library blocker complex does not circularize to form an open circle library molecule. In some embodiments, a library blocker complex can be in-solution.

Library Blocker Bait Complexes

[00602] In some embodiments, in any of the methods described herein, a library blocker bait complex comprises a bottom strand linear library molecule hybridized to (i) a bottom strand blocker oligonucleotide and (ii) a target-specific bait/probe. A library blocker bait complex comprises a first double-stranded region formed from hybridization between a noninsert region of the bottom strand linear library molecule and the bottom strand blocker oligonucleotide. A library blocker bait complex comprises a second double-stranded region formed from hybridization between an insert region of the bottom strand linear library molecule and the target-specific bait/probe. A library blocker bait complex does not circularize to form open circle library molecules. In some embodiments, a library blocker bait complex can be in-solution or immobilized to a capture support.

Reagents that Remove Deaminated Bases and Generates Gaps

[00603] The present disclosure provides reagents that remove deaminated bases and generates gaps in nucleic acids comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity.

[00604] In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity including any one or any combination of two or more of: formamidopyrimidine DNA glycosylase (fpg); uracil N- glycosylase (UNG); uracil DNA glycosylase (UDG); 8-oxoguanine glycosylase (OGG including thermostable OGG); DNA (apurinic) lyase; DNA (apyrimidinic) lyase; and/or endonuclease III. In some embodiments, the reagent that removes deaminated bases and generates gaps can be washed away or deactivated after completion of the glycosylase reaction. [00605] In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having lyase activity that can that breaks the phosphodiester backbone at the 5’ and 3’ sides of the abasic site to release the base-free deoxyribose and generate a gap, wherein the enzyme having lyase activity includes any one or any combination of two or more of: AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase (formamidopyrimidine DNA glycosylase), Endonuclease VIII glycosylase/AP lyase and/or endonuclease III. In some embodiments, the reagent that removes deaminated bases and generates gaps can be washed away or deactivated after completion of the lyase reaction. [00606] In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme that generates an abasic site in a nucleic acid strand and at least one enzyme having lyase activity. In some embodiments, the reagent that removes deaminated bases comprises a mixture of uracil DNA glycosylase and DNA glycosylase- lyase endonuclease VIII, for example and without limitation, USER™ (Uracil-Specific Excision Reagent Enzyme, such as from New England Biolabs) or thermolabile USER (for example from New England Biolabs). In some embodiments, the reagent that removes deaminated bases and generates gaps can be washed away or deactivated after completion of the glycosylase and lyase reactions.

Nucleic Acid Hybridization Reagents and Methods of Use

[00607] The present disclosure provides nucleic acid hybridization reagents for hybridizing a plurality of nucleic acid molecules to generate a plurality of nucleic acid duplexes. In some embodiments, the nucleic acid hybridization reagent comprises at least one solvent and any combination of two or more of the following: at least one pH buffering agent, at least one monovalent cation, a chaotropic agent, a detergent, a reducing agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and/or a crowding agent.

Loading Reagents and Methods of Use

[00608] The present disclosure provides loading reagents for distributing/loading onto a capture support a plurality of target-specific baits/probes, pinning primers, linear library molecules, covalently closed circular library molecules, the top strand circularization oligonucleotides, the bottom strand blocker oligonucleotides and/or the spike-in circularization oligonucleotides. In some embodiments, the loading reagent is designed to promote binding between the receptor moieties embedded in the at least one layer of hydrophilic polymer coating of the capture support and the affinity moieties of the target- specific baits/probes and pinning primers. In some embodiments, the loading reagent comprises at least one solvent and any combination of two or more of the following compounds: at least one pH buffering agent, at least one monovalent cation, a chaotropic agent, a detergent, a reducing agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and/or a crowding agent.

Solvents

[00609] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein, can include at least one solvent. In some embodiments, the solvent comprises water. In some embodiments, the solvent comprises an alcohol comprising a short chain alcohol having 1-6 carbon backbone, including linear or branched alcohols. The short chain alcohol can be methanol, ethanol, propanol, butanol, pentanol or hexanol. In some embodiments, the solvent comprises a polar aprotic solvent including acetonitrile, diethylene glycol, N,N-dimethylacetamide, dimethyl formamide, dimethyl sulfoxide, ethylene glycol, formamide, glycerin, methanol, 7V-methyl-2-pyrrolidinone, hexamethylphosphoramide, nitrobenzene, or nitromethane. pH Buffering Agents

[00610] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include at least one pH buffering agent which can maintain the pH of the reagent in a range that is suitable for nucleic acid hybridization. The pH buffering agent comprises any one or any combination of two or more of Tris, Tris-HCl, Tris-acetate, Tricine, Bicine, Bis-Tris propane, HEPES, MES, 3-(N- morpholino)propanesulfonic acid (MOPS), 2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-{ [1,3- Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-l-sulfonic acid (TES), 3- (Cyclohexylamino)-l -propanesulfonic acid (CAPS), 3-{[l,3-dihydroxy-2- (hydroxymethyl)propan-2-yl]amino}propane-l -sulfonic acid (TAPS), 3-{[l,3-dihydroxy-2- (hydroxymethyl)propan-2-yl]amino}-2-hydroxypropane-l-sulfonic acid (TAPSO), N-(2- acetamido)-2-aminoethanesulfonic acid (ACES), 1,4-Piperazinedi ethanesulfonic acid (PIPES), ethanolamine (a.k.a 2-amino methanol; MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In some embodiments, the pH buffering agent can be present in any of the reagents described herein at a concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM. In some embodiments, the pH of the pH buffering agent which is present in any of the reagents described herein can be adjusted to a pH of about 4-9.5, or a pH of about 5-9, or a pH of about 5-8, or any range therebetween.

[00611] In some embodiments, the hybridization reagent and/or loading reagent comprises a pH buffering agent which comprises a saline sodium citrate (SSC) which includes sodium citrate and sodium chloride. The SSC can be at a concentration of 20X SSC (e.g., 3 M NaCl and 0.3 M sodium citrate), or at 10X SSC (e.g., 1.5 M NaCl and 0.15 M sodium citrate), or at 5X SSC (e.g., 0.75 M NaCl and 0.075 M sodium citrate), or at 2X SSC (e.g., 0.30 M NaCl and 0.003 M sodium citrate), or at IX SSC (e.g., 0.15 M NaCl and 0.015 M sodium citrate).

[00612] In some embodiments, the hybridization reagent and/or loading reagent comprises at least one pH buffering agent having a pH range of about 4-5, or a pH range of about 5-6, or a pH range of about 6-7, or a pH range of about 7-8, or a pH range of about 8-9, or any range therebetween.

[00613] In some embodiments, the hybridization reagent and/or loading reagent comprises at least one pH buffering agent at a concentration of about 1-10 mM, or about 10-20 mM, or about 20-30 mM, or about 30-40 mM, or about 40-50 mM. In some embodiments, the hybridization reagent and/or loading reagent comprises at least one pH buffering agent at a concentration of about 0.1-0.5 mM, or about 0.5-1 mM, or any range therebetween.

Monovalent Cations

[00614] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a monovalent cation which comprises sodium or potassium. In some embodiments, the monovalent cation is in the form of NaCl or KC1. In some embodiments, the hybridization reagent and/or loading reagent comprises NaCl at a concentration of about 25-200 mM, or about 50-150 mM, or any range therebetween. In some embodiments, the hybridization reagent and/or loading reagent comprises NaftPCh (sodium phosphate, dibasic) at a concentration of about 1-100 mM, or about 100-250 mM, or about 250-500 mM, or any range therebetween. In some embodiments, the hybridization reagent and/or loading reagent comprises KC1 at a concentration of about 1-200 mM, or about 25-150 mM, or about 50-100 mM, or any range therebetween.

[00615] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include at least one salt which comprises potassium acetate (e.g., KCH3CO2), MgCh and/or MgSCh. In some embodiments, the hybridization reagent and/or loading reagent comprises potassium acetate at a concentration of about 10-50 mM, or about 50-100 mM, or about 100-150 mM, or about 150-200 mM, or any range therebetween. In some embodiments, the hybridization reagent and/or loading reagent comprises MgCh or MgSCh at a concentration of about 1-10 mM, or about 10-25 mM, or about 25-50 mM, or about 50-100 mM, or any range therebetween.

Ammonium Ions

[00616] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a source of ammonium ions, for example ammonium sulfate. In some embodiments, ammonium sulfate is included in the reagent at a concentration of about 1-50 mM, or about 10-25 mM, or any range therebetween.

Detergents

[00617] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a detergent. In some embodiments, the detergent comprises an ionic detergent such as SDS (sodium dodecyl sulfate). In some embodiments, the detergent comprises a non-ionic detergent such as Triton X-100, Tween 20, Tween 80 or Nonidet P-40. In some embodiments, the detergent comprises a zwitterionic detergent such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate) or 7V-Dodecyl-7V,7V-dimethyl-3-amonio-l -propanesulfate (DetX). In some embodiments, the detergent comprises LDS ( lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate or sodium cholate. In some embodiments, the detergent is included in a reagent at a concentration of about 0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%, or about 0.2-0.25%, or any range therebetween.

Reducing Agents

[00618] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include at least one reducing agent comprising DTT (dithiothreitol), 2-beta mercaptoethanol, TCEP, (tris(2- carboxyethyl)phosphine), formamide, DMSO (dimethylsulfoxide), sodium dithionite (Na2S2O4), glutathione, methionine, betaine, Tris(3-hydroxypropyl)phosphine (THPP) and/or N-acetyl cysteine. The reagents can include the reducing agent at a concentration of about 0.1-0.5 M, or about 0.5-1 M, or about 1-2 M, or any range therebetween. The reagents can include the reducing agent at a concentration of about 0.01-0.1 mM, or about 0.1-1 mM, or about 1-2.5 mM, or about 2.5-5 mM, or about 5- 7.5 mM, or about 7.5-9 mM, or about 9-12 mM, or about 12-25 mM, or about 25-50 mM, or any range therebetween.

Viscosity Agents

[00619] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a viscosity agent comprising a saccharide such as trehalose, sucrose, cellulose, xylitol, mannitol, sorbitol or inositol. In some embodiments, the viscosity agent comprises glycerol or a glycol compound such as ethylene glycol or propylene glycol. The reagents can include the viscosity agent at a concentration of about 0.1-1%, or about 1-5%, or about 5-10%, or about 10-15% based on volume, or any range therebetween. The reagents can include the viscosity agent at a concentration of about 1-50 mM, or about 50-100 mM, or about 100-150 mM, or about 150-200 mM. The reagents can include the viscosity agent at a concentration of about 0.1-0.5 M, or about 0.5-1 M, or about 1-2 M, or about 2-3 M, or about 3-5 M, or any range therebetween.

Chaotropic Agents

[00620] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a chaotropic agent that can disrupt non- covalent bonds such as hydrogen bonds or van der Waals forces. In some embodiments, the chaotropic agent comprises SDS (sodium dodecyl sulfate), urea, thiourea, guanidinium chloride, guanidine hydrochloride, guanidine thiocyanate, guanidine isothionate, potassium thiocyanate, lithium chloride, sodium iodide or sodium perchlorate. The reagents can include a chaotropic agent at a concentration of about 0.1 -5M, about 0.5-4M, about 0.5-3M, about 0.1-1 M, about 1-2 M, about 2-3 M, about 3-4 M, or about 4-5 M, or any range therebetween.

Chelating Agents

[00621] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a chelating agent that binds metal ions by chelation, coordination or covalent bonding. In some embodiments, the chelating agent comprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxy ethylethylenediaminetriacetic acid), DPTA (diethylene triamine pentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium citrate. In some embodiments, the hybridization region comprises a chelating agent at a concentration of about 0.01 - 50 mM, or about 0.1 - 20 mM, or about 0.2 - 10 mM, or any range therebetween.

Zwitterions

[00622] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a source of zwitterions. In some embodiments, the zwitterionic comprises a cationic zwitterionic compound such as a betaine including

N,N,N-trimethylglycine and cocamidopropyl betaine. In some embodiments, the zwitterion comprises an albuminoids including ovalbumin, and the serum albumins derived from bovine, equine, or human. The reagent can include a zwitterion at a concentration of about

O.1-0.5 M, or about 0.5-1 M, or about 1-2 M, or any range therebetween.

Sugar Alcohols

[00623] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a sugar alcohol, comprising a sugar alcohol comprising sucrose, trehalose, maltose, rhamnose, arabinose, fucose, mannitol, sorbitol or adonitol. The reagents can include the sugar alcohol at a concentration of about 1-50 mM, or about 50-100 mM, or about 100-150 mM, or about 150-200 mM, or any range therebetween. The reagents can include the sugar alcohol agent at a concentration of about 0.1-0.5 M, or about 0.5-1 M, or about 1-2 M, or about 2-3 M, or about 3-5 M, or any range therebetween.

Crowding Agents

[00624] In some embodiments, any of the reagents (e.g., hybridization reagents and/or loading reagents) described herein can include a crowding agent that increases molecular crowding. In some embodiments, the crowding agent comprises polyethylene glycol (PEG, e.g., 200-800 molecular weight, e.g., 200, 400, 600, 800 molecular weight) (PEG, e.g., 1-50K molecular weight, e.g., IK, 2K, 4K, 8K molecular weight), dextran, dextran sulfate, hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methycellulose, and hydroxyl methyl cellulose. The crowding agent can be present in the hybridization reagent and/or loading reagent at about 1-10%, or about 10-25%, or about 25-50%, or higher percentages by volume based on the total volume of the hybridization reagent and/or loading reagent, or any range therebetween. Re-Seeding a Capture Support Having Immobilized Baits/Probes and/or Pinning Primers

[00625] The present disclosure provides methods for conducting any of the in-solution enrichment workflows described herein, and a re-seeding workflow in which a first and second plurality of circle bait complexes are separately distributed/seeded onto a capture support, subjected to rolling circle amplification to generate immobilized concatemer template molecules, and the immobilized concatemer template molecules are sequenced. . [00626] The present disclosure provides methods for re-seeding a capture support comprising step (1): providing a capture support comprising at least one layer of a hydrophilic polymer coating and a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating.

[00627] In some embodiments, the methods for re-seeding a capture support comprise step (2): generating in-solution a plurality of circle bait complexes by selectively hybridizing insolution library molecules to their corresponding target-specific baits/probes to generate a first plurality of circle bait complexes or a plurality of library bait complexes.

[00628] In some embodiments, in step (2), individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence, an affinity moiety at the 5’ end of the target-specific oligonucleotide, and the oligonucleotide comprising the target-specific sequence comprises an extendible 3’ end. In some embodiments, the affinity moiety of the target-specific bait/probe can bind a receptor moiety embedded in the hydrophilic polymer coating of the capture support.

[00629] In some embodiments, in step (2), the first plurality of library molecules comprises a plurality of linear library molecules and/or a plurality of covalently closed circular library molecules.

[00630] In some embodiments, in step (2), individual covalently closed circular library are hybridized to a corresponding target-specific bait/probe to generate a first plurality of circle bait complexes.

[00631] In some embodiments, in step (2), individual linear library molecules that are hybridized to a corresponding target-specific bait/probe can be circularized by contacting the linear library molecule with a splint-type nucleic acid molecule to generate individual open circle library molecules each having one or two nicks, and the nicks can be enzymatically ligated to generate individual covalently closed circular library molecules hybridized to the corresponding target-specific bait/probe, thereby generating a first plurality of circle bait complexes. [00632] In some embodiments, in step (2), individual linear library molecules that are hybridized to a corresponding target-specific bait/probe can be circularized by contacting the linear library molecule with a top strand circularization oligonucleotide to generate individual open circle library molecules having a 5’ flap structure which is cleavable with a flap endonuclease. In some embodiments, the cleavage product has one nick that can be enzymatically ligated to generate individual covalently closed circular library molecules hybridized to the corresponding target-specific bait/probe, thereby generating a first plurality of circle bait complexes.

[00633] In some embodiments, in step (2), the splint-type nucleic acid molecules comprise single stranded splints, double stranded splints or a top strand circularization oligonucleotides. Methods for conducting in-solution circularization of linear library molecules using splint-type nucleic acid molecules to generate immobilized circle bait complexes are described above.

[00634] In some embodiments, in step (2), the concentration of the first plurality of library molecules distributed onto the capture support can be about 0.1-1 ug, or about 1-5 ug, or about 5-10 ug, or about 10-50 ug, or about 50-100 ug, or any range therebetween.

[00635] In some embodiments, in step (2), the concentration of the first plurality of library molecules that are distributed onto the capture support can be about 0.1-1 pg, or about 1-5 pg, or about 5-10 pg, or about 10-50 pg, or about 50-100 pg, or any range therebetween.

[00636] In some embodiments, in step (2), the insert regions of the plurality of library molecules comprise different insert sequences. In some embodiments, the library molecules of the first plurality comprise the same universal sequencing primer binding sites. In some embodiments, the library molecules of the first plurality comprise at least a first and second sub-population. In some embodiments, the first sub-population of library molecules of the first plurality comprises a first batch sequencing primer binding site. In some embodiments, the second sub-population of library molecules of the first plurality comprises a second batch sequencing primer binding site. In some embodiments, the first and second batch sequencing primer binding sites bind different sequencing primers.

[00637] In some embodiments, the methods for re-seeding a capture support further comprise step (3): distributing onto the capture support the first plurality of circle bait complexes under a condition suitable for binding the affinity moiety of individual circle bait complexes with a receptor moiety on the capture support, thereby generating a first plurality of immobilized circle bait complexes. [00638] In some embodiments, in step (3), the first plurality of immobilized circle bait complexes are arranged on the capture support in a pre-determined pattern or in a nonpredetermined and random manner.

[00639] In some embodiments, step (3) further comprises distributing onto the capture support a plurality of pinning primers under a condition suitable for binding the affinity moiety of individual pinning primers with a receptor moiety on the capture support, thereby generating a plurality of immobilized pinning primers. In some embodiments, the plurality of immobilized pinning primers is arranged on the capture support in a pre-determined pattern or in a non-predetermined and random manner. In some embodiments, a subset of the embedded receptor moieties of the capture support are bound to a target-specific bait/probe or a pinning primer.

[00640] In some embodiments, the methods for re-seeding a capture support comprise step (4): contacting the first plurality of immobilized circle bait complexes with a rolling circle reagent under a condition suitable for conducting rolling circle amplification in a templatedependent manner using individual immobilized circle bait complexes in the first plurality, thereby generating a first plurality of nucleic acid concatemer template molecules immobilized to the capture support (“immobilized concatemer template molecules”). In some embodiments, the rolling circle amplification reagent comprises a plurality of strand displacing polymerases and a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the rolling circle amplification reagent comprises a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the density of immobilized concatemer template molecules on the capture support is about 10² - 10¹⁵ per mm².

[00641] In some embodiments, the methods for re-seeding a capture support further comprise step (5): sequencing at least a subset of the first plurality of immobilized concatemer template molecules thereby generating a first plurality of sequencing read products. In some embodiments, the sequencing of step (5) comprises imaging a region of the capture support to detect the sequencing reactions of the first plurality of concatemer template molecules. In some embodiments, the sequencing of step (5) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. Various methods for sequencing are described below, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides.

[00642] In some embodiments, in step (5), the full lengths of the first plurality of immobilized concatemer template molecules are sequenced. In some embodiments, partial lengths of the first plurality of immobilized concatemer template molecules are sequenced. [00643] In some embodiments, the sequencing of step (5) comprises hybridizing sequencing primers to sequencing primers binding sites on the first plurality of immobilized concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the immobilized concatemer template molecules in the first plurality can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles.

[00644] In some embodiments, in step (5), the immobilized concatemer template molecules in the first plurality are sequenced. For example, at least 30-50%, or at least 50- 70%, or at least 70-90% of the immobilized concatemer template molecules in the first plurality are sequenced. In some embodiments, 200 million - 1 billion of the first plurality of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the first plurality of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the first plurality of concatemer template molecules can be sequenced.

[00645] In some embodiments, in step (5), a first sub-population of the immobilized concatemer template molecules in the first plurality are sequenced using first batch sequencing primers and first batch sequencing primer binding sites in a first sub-population of immobilized concatemer template molecules.

[00646] In some embodiments, in step (5), a second sub-population of the immobilized concatemer template molecules in the first plurality are sequenced using second batch sequencing primers and second batch sequencing primer binding sites in a second subpopulation of immobilized concatemer template molecules.

[00647] In some embodiments, the method for re-seeding a capture support comprises omitting step (5) so that the first plurality of immobilized concatemer template molecules are not sequenced. In some embodiments, the capture support can be re-seeded with additional library molecules by conducting steps (6) - (8) (described below) to generate a second plurality of immobilized concatemer template molecules, and the first and second plurality of immobilized concatemer template molecules can be sequenced essentially simultaneously or can be sequenced separately in batches according to the method of step (9).

[00648] In some embodiments, the methods for re-seeding a capture support further comprise step (6): generating in-solution a second plurality of circle bait complexes by selectively hybridizing in-solution individual library molecules of a second plurality to their cognate target-specific baits/probes to generate a second plurality of circle bait complexes or a second plurality of library bait complexes.

[00649] In some embodiments, in step (6) individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence, an affinity moiety at the 5’ end of the target-specific oligonucleotide, and oligonucleotide comprising the target-specific sequence comprises an extendible 3’ end. In some embodiments, the affinity moiety of the target-specific bait/probe can bind a receptor moiety embedded in the hydrophilic polymer coating of the capture support.

[00650] In some embodiments, in step (6), the second plurality of library molecules comprises a plurality of linear library molecules and/or a plurality of covalently closed circular library molecules.

[00651] In some embodiments, in step (6), individual covalently closed circular library molecules in the second plurality are hybridized to a corresponding target-specific bait/probe to generate a second plurality of circle bait complexes.

[00652] In some embodiments, in step (6), individual linear library molecules in the second plurality that are hybridized to their cognate target-specific bait/probe can be circularized by contacting the linear library molecule with a splint-type nucleic acid molecule to generate individual immobilized open circle library molecules each having one or two nicks, and the nicks can be enzymatically ligated to generate individual covalently closed circular library molecules hybridized to their cognate target-specific bait/probe thereby generating a second plurality of circle bait complexes. In some embodiments, the splint-type nucleic acid molecules comprise single stranded splints, double stranded splints or a top strand circularization oligonucleotides. Methods for conducting in-solution circularization of linear library molecules using splint-type nucleic acid molecules to generate immobilized circle bait complexes are described above. [00653] In some embodiments, in step (6), the concentration of the second plurality of library molecules that are distributed onto the capture support can be about 1-5 ug, or about 5-10 ug, or about 10-50 ug, or about 50-100 ug, or any range therebetween.

[00654] In some embodiments, in step (6), the concentration of the second plurality of library molecules that are distributed onto the capture support can be about 1-5 pg, or about 5-10 pg, or about 10-50 pg, or about 50-100 pg, or any range therebetween.

[00655] In some embodiments, in step (6), the insert regions of the plurality of library molecules comprise different insert sequences. In some embodiments, the library molecules of the second plurality comprise the same universal sequencing primer binding sites. In some embodiments, the library molecules of the second plurality comprise at least a first and second sub-population. In some embodiments, the first sub-population of library molecules of the second plurality comprises a first batch sequencing primer binding site. In some embodiments, the second sub-population of library molecules of the second plurality comprises a second batch sequencing primer binding site. In some embodiments, the first and second batch sequencing primer binding sites bind different sequencing primers.

[00656] In some embodiments, the methods for re-seeding a capture support comprise step (7): distributing onto the capture support the second plurality of circle bait complexes under a condition suitable for binding the affinity moiety of individual circle bait complexes with a receptor moiety on the capture support, thereby generating a second plurality of immobilized circle bait complexes.

[00657] In some embodiments, in step (7), the second plurality of immobilized circle bait complexes are arranged on the capture support in a pre-determined pattern or in a nonpredetermined and random manner.

[00658] In some embodiments, step (7) further comprises distributing onto the capture support a second plurality of pinning primers under a condition suitable for binding the affinity moiety of individual pinning primers with a receptor moiety on the capture support thereby generating a second plurality of immobilized pinning primers. In some embodiments, the second plurality of immobilized pinning primers are arranged on the capture support in a pre-determined pattern or in a non-predetermined and random manner.

[00659] In some embodiments, the methods for re-seeding a capture support further comprise step (8): contacting the second plurality of immobilized circle bait complexes with a rolling circle reagent under a condition suitable for conducting rolling circle amplification in a template-dependent manner using individual immobilized circle bait complexes in the second plurality, thereby generating a second plurality of nucleic acid concatemer template molecules immobilized to the capture support. In some embodiments, the rolling circle amplification reagent comprises a plurality of strand displacing polymerases and a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the rolling circle amplification reagent further comprises a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the density of immobilized concatemer template molecules on the capture support is about 10² - 10¹⁵ per mm².

[00660] In some embodiments, the methods for re-seeding a capture support comprise step (9): sequencing at least a subset of the second plurality of immobilized concatemer template molecules thereby generating a second plurality of sequencing read products. In some embodiments, the sequencing of step (9) comprises imaging a region of the capture support to detect the sequencing reactions of the second plurality of concatemer template molecules. In some embodiments, the sequencing of step (9) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. Various methods for sequencing are described below, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides.

[00661] In some embodiments, in step (9), the full lengths of the second plurality of immobilized concatemer template molecules are sequenced. In some embodiments, partial lengths of the second plurality of immobilized concatemer template molecules are sequenced. [00662] In some embodiments, the sequencing of step (9) comprises hybridizing sequencing primers to sequencing primers binding sites on the second plurality of immobilized concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the immobilized concatemer template molecules in the second plurality can be subjected to 5- 25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles. [00663] In some embodiments, in step (9), the immobilized concatemer template molecules in the second plurality are sequenced. For example, at least 30-50%, or at least 50- 70%, or at least 70-90% of the immobilized concatemer template molecules in the second plurality are sequenced. In some embodiments, 200 million - 1 billion of the second plurality of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the second plurality of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the second plurality of concatemer template molecules can be sequenced.

[00664] In some embodiments, in step (9), a first sub-population of the immobilized concatemer template molecules in the second plurality are sequenced using first batch sequencing primers and first batch sequencing primer binding sites in a first sub-population of immobilized concatemer template molecules.

[00665] In some embodiments, in step (9), a second sub-population of the immobilized concatemer template molecules in the second plurality are sequenced using second batch sequencing primers and second batch sequencing primer binding sites in a second subpopulation of immobilized concatemer template molecules.

[00666] In some embodiments, the methods for re-seeding a capture support comprise conducting steps (1) - (4) to generate a first plurality of immobilized concatemer template molecules, omitting step (5), conducting steps (6) - (8) to generate a second plurality of immobilized concatemer template molecules, and sequencing the first and second plurality of immobilized concatemer template molecules essentially simultaneously according to the method of step (9).

[00667] In some embodiments, the methods for re-seeding a capture support comprise conducting steps (1) - (4) to generate a first plurality of immobilized concatemer template molecules, omitting step (5), conducting steps (6) - (8) to generate a second plurality of immobilized concatemer template molecules, and sequencing the first plurality of immobilized concatemer template molecules using first batch sequencing primers, and separately sequencing the second plurality of immobilized concatemer template molecules using second batch sequencing primers, wherein the first and second batch sequencing can be conducted according to the method of step (9). Re-Seeding a Capture Support Having Immobilized Baits/Probes and/or Pinning Primers

[00668] The present disclosure provides methods for conducting any of the on-support enrichment workflows described herein, and a re-seeding workflow in which a first and second plurality of immobilized circle bait complexes are separately generated on a capture support and subjected to rolling circle amplification to generate immobilized concatemer template molecules, and the immobilized concatemer template molecules are sequenced. [00669] The present disclosure provides methods for re-seeding a capture support comprising step (1): providing a capture support comprising at least one layer of a hydrophilic polymer coating and a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating.

[00670] In some embodiments, the capture support comprises a plurality of immobilized target-specific baits/probes. In some embodiments, individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence, an affinity moiety at the 5’ end of the target-specific oligonucleotide, and oligonucleotide comprising the targetspecific sequence comprises an extendible 3’ end. In some embodiments, the affinity moiety of the target-specific bait/probe can bind a receptor moiety embedded in the hydrophilic polymer coating of the capture support. In some embodiments, the plurality of immobilized target-specific baits/probes are arranged on the capture support in a pre-determined pattern or in a non-predetermined and random manner.

[00671] In some embodiments, the capture support comprises a plurality of immobilized pinning primers. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide. In some embodiments, the pinning primer comprises a blocking group at the 3’ end of the oligonucleotide, wherein the blocking group inhibits polymerase-catalyzed extension of the 3’ end of the pinning primer. In some embodiments, the pinning primer comprises a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the plurality of immobilized pinning primers is arranged on the capture support in a pre-determined pattern or in a nonpredetermined and random manner. In some embodiments, a subset of the embedded receptor moieties of the capture support are bound to a target-specific bait/probe or a pinning primer. [00672] In some embodiments, the methods for re-seeding a capture support further comprise step (2): distributing onto the capture support a first plurality of library molecules under a condition suitable for selectively hybridizing individual library molecules to their cognate target-specific baits/probes to generate a first plurality of immobilized library molecules.

[00673] In some embodiments, in step (2), the first plurality of library molecules comprises a plurality of linear library molecules and/or a plurality of covalently closed circular library molecules.

[00674] In some embodiments, in step (2), individual immobilized covalently closed circular library molecules comprise a covalently closed circular library molecule hybridized to a corresponding immobilized target-specific bait/probe to generate a first plurality of immobilized circle bait complexes.

[00675] In some embodiments, in step (2), individual immobilized linear library molecules that are hybridized to the corresponding immobilized target-specific bait/probe can be circularized by contacting the linear library molecule with a splint-type nucleic acid molecule to generate individual immobilized open circle library molecules each having one or two nicks, and the nicks can be enzymatically ligated to generate individual covalently closed circular library molecules hybridized to their cognate immobilized target-specific bait/probe thereby generating a first plurality of immobilized circle bait complexes. In some embodiments, the splint-type nucleic acid molecules comprise single stranded splints, double stranded splints or a top strand circularization oligonucleotides. Methods for conducting on- support circularization of linear library molecules using splint-type nucleic acid molecules to generate immobilized circle bait complexes are described above.

[00676] In some embodiments, in step (2), the concentration of the first plurality of library molecules that are distributed onto the capture support can be about 1-5 ug, or about 5-10 ug, or about 10-50 ug, or about 50-100 ug, or any range therebetween.

[00677] In some embodiments, in step (2), the concentration of the first plurality of library molecules that are distributed onto the capture support can be about 1-5 pg, or about 5-10 pg, or about 10-50 pg, or about 50-100 pg, or any range therebetween.

[00678] In some embodiments, in step (2), the insert regions of the plurality of library molecules comprise different insert sequences. In some embodiments, the library molecules of the first plurality comprise the same universal sequencing primer binding sites. In some embodiments, the library molecules of the first plurality comprise at least a first and second sub-population. In some embodiments, the first sub-population of library molecules of the first plurality comprises a first batch sequencing primer binding site. In some embodiments, the second sub-population of library molecules of the first plurality comprises a second batch sequencing primer binding site. In some embodiments, the first and second batch sequencing primer binding sites bind different sequencing primers.

[00679] In some embodiments, the methods for re-seeding a capture support comprise step

(3): contacting the first plurality of immobilized circle bait complexes with a rolling circle reagent under a condition suitable for conducting rolling circle amplification in a templatedependent manner using individual immobilized circle bait complexes in the first plurality, thereby generating a first plurality of nucleic acid concatemer template molecules immobilized to the capture support (“immobilized nucleic acid template molecules”). In some embodiments, the rolling circle amplification reagent comprises a plurality of strand displacing polymerases and a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the rolling circle amplification reagent further comprises a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the density of immobilized concatemer template molecules on the capture support is about 10² - 10¹⁵ per mm².

[00680] In some embodiments, the methods for re-seeding a capture support comprise step

(4): sequencing at least a subset of the first plurality of immobilized concatemer template molecules, thereby generating a first plurality of sequencing read products. In some embodiments, the sequencing of step (4) comprises imaging a region of the capture support to detect the sequencing reactions of the first plurality of concatemer template molecules. In some embodiments, the sequencing of step (4) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. Various methods for sequencing are described below, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides.

[00681] In some embodiments, in step (4), the full lengths of the first plurality of immobilized concatemer template molecules are sequenced. In some embodiments, partial lengths of the first plurality of immobilized concatemer template molecules are sequenced. [00682] In some embodiments, the sequencing of step (4) comprises hybridizing sequencing primers to sequencing primers binding sites on the first plurality of immobilized concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the immobilized concatemer template molecules in the first plurality can be subjected to 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles.

[00683] In some embodiments, in step (4), the immobilized concatemer template molecules in the first plurality are sequenced. For example, at least 30-50%, or at least 50- 70%, or at least 70-90% of the immobilized concatemer template molecules in the first plurality are sequenced. In some embodiments, 200 million - 1 billion of the first plurality of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the first plurality of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the first plurality of concatemer template molecules can be sequenced.

[00684] In some embodiments, in step (4), a first sub-population of the immobilized concatemer template molecules in the first plurality are sequenced using first batch sequencing primers and first batch sequencing primer binding sites in a first sub-population of immobilized concatemer template molecules.

[00685] In some embodiments, in step (4), a second sub-population of the immobilized concatemer template molecules in the first plurality are sequenced using second batch sequencing primers and second batch sequencing primer binding sites in a second subpopulation of immobilized concatemer template molecules.

[00686] In some embodiments, the methods for re-seeding a capture support comprise omitting step (4) so that the first plurality of immobilized concatemer template molecules are not sequenced. In some embodiments, the capture support can be re-seeded with additional library molecules by conducting steps (5) - (6) (described below) to generate a second plurality of immobilized concatemer template molecules, and the first and second plurality of immobilized concatemer template molecules can be sequenced essentially simultaneously or can be sequenced separately in batches according to the method of step (7).

[00687] In some embodiments, the methods for re-seeding a capture support comprise step (5): distributing onto the capture support a second plurality of library molecules under a condition suitable for selectively hybridizing individual library molecules in the second plurality to their cognate target-specific baits/probes to generate a second plurality of immobilized library molecules.

[00688] In some embodiments, step (5) optionally includes distributing a plurality of target-specific baits/probes onto the capture support, thereby immobilizing additional targetspecific baits/probes and increasing the density of immobilized target-specific baits/probes. In some embodiments, step (5) optionally includes distributing a plurality of pinning primers onto the capture support thereby immobilizing additional pinning primers and increasing the density of immobilized pinning primers.

[00689] In some embodiments, in step (5), the second plurality of library molecules comprise a plurality of linear library molecules and/or a plurality of covalently closed circular library molecules.

[00690] In some embodiments, in step (5), individual immobilized covalently closed circular library molecules in the second plurality comprise a covalently closed circular library molecule hybridized to a corresponding immobilized target-specific bait/probe to generate a second plurality of immobilized circle bait complexes.

[00691] In some embodiments, in step (5), individual immobilized linear library molecules that are hybridized to the corresponding immobilized target-specific bait/probe can be circularized by contacting the linear library molecule with a splint-type nucleic acid molecule to generate individual immobilized open circle library molecules each having one or two nicks, and the nicks can be enzymatically ligated to generate individual covalently closed circular library molecules hybridized to their cognate immobilized target-specific bait/probe thereby generating a second plurality of immobilized circle bait complexes. In some embodiments, the splint-type nucleic acid molecules comprise single stranded splints, double stranded splints or a top strand circularization oligonucleotides. Methods for conducting on- support circularization of linear library molecules using splint-type nucleic acid molecules to generate immobilized circle bait complexes are described above.

[00692] In some embodiments, in step (5), the concentration of the second plurality of library molecules that are distributed onto the capture support can be about 1-5 ug, or about 5-10 ug, or about 10-50 ug, or about 50-100 ug, or any range therebetween.

[00693] In some embodiments, in step (5), the concentration of the second plurality of library molecules that are distributed onto the capture support can be about 1-5 pg, or about 5-10 pg, or about 10-50 pg, or about 50-100 pg, or any range therebetween. [00694] In some embodiments, in step (5), the insert regions of the plurality of library molecules comprise different insert sequences. In some embodiments, the library molecules of the second plurality comprise the same universal sequencing primer binding sites. In some embodiments, the library molecules of the second plurality comprise at least a first and second sub-population. In some embodiments, the first sub-population of library molecules of the second plurality comprises a first batch sequencing primer binding site. In some embodiments, the second sub-population of library molecules of the second plurality comprises a second batch sequencing primer binding site. In some embodiments, the first and second batch sequencing primer binding sites bind different sequencing primers.

[00695] In some embodiments, the methods for re-seeding a capture support comprise step

(6): contacting the second plurality of immobilized circle bait complexes with a rolling circle reagent under a condition suitable for conducting rolling circle amplification in a templatedependent manner using individual immobilized circle bait complexes in the second plurality, thereby generating a second plurality of nucleic acid concatemer template molecules immobilized to the capture support. In some embodiments, the rolling circle amplification reagent comprises a plurality of strand displacing polymerases and a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, the rolling circle amplification reagent comprises a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball. In some embodiments, the density of immobilized concatemer template molecules (e.g., of the second plurality) on the capture support is about 10² - 10¹⁵ per mm².

[00696] In some embodiments, the methods for re-seeding a capture support comprise step

(7): sequencing at least a subset of the second plurality of immobilized concatemer template molecules thereby generating a second plurality of sequencing read products. In some embodiments, the sequencing of step (7) comprises imaging a region of the capture support to detect the sequencing reactions of the second plurality of concatemer template molecules. In some embodiments, the sequencing of step (7) comprises conducting any massively parallel nucleic acid sequencing method that employs a plurality of sequencing primers, a plurality of sequencing polymerases and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprises nucleotides, nucleotide analogs and/or multivalent molecules. Various methods for sequencing are described below, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides.

[00697] In some embodiments, in step (7), the full lengths of the second plurality of immobilized concatemer template molecules are sequenced. In some embodiments, partial lengths of the second plurality of immobilized concatemer template molecules are sequenced. [00698] In some embodiments, the sequencing of step (7) comprises hybridizing sequencing primers to sequencing primers binding sites on the second plurality of immobilized concatemer template molecules and conducting up to 1000 cycles of polymerase-catalyzed sequencing reactions using nucleotide reagents. In some embodiments, the immobilized concatemer template molecules in the second plurality can be subjected to 5- 25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles.

[00699] In some embodiments, in step (7), the immobilized concatemer template molecules in the second plurality are sequenced. For example, at least 30-50%, or at least 50- 70%, or at least 70-90% of the immobilized concatemer template molecules in the second plurality are sequenced. In some embodiments, 200 million - 1 billion of the second plurality of concatemer template molecules can be sequenced. In some embodiments, up to 1 billion, or up to 2 billion, or up to 3 billion, or up to 4 billion, or up to 5 billion of the second plurality of concatemer template molecules can be sequenced. In some embodiments, up to 6 billion, or up to 7 billion, or up to 8 billion, or up to 9 billion, or up to 10 billion of the second plurality of concatemer template molecules can be sequenced.

[00700] In some embodiments, in step (7), a first sub-population of the immobilized concatemer template molecules in the second plurality are sequenced using first batch sequencing primers and first batch sequencing primer binding sites in a first sub-population of immobilized concatemer template molecules.

[00701] In some embodiments, in step (7), a second sub-population of the immobilized concatemer template molecules in the second plurality are sequenced using second batch sequencing primers and second batch sequencing primer binding sites in a second subpopulation of immobilized concatemer template molecules.

[00702] In some embodiments, the methods for re-seeding a capture support comprise conducting steps (1) - (3) to generate a first plurality of immobilized concatemer template molecules, omitting step (4), conducting steps (5) - (6) to generate a second plurality of immobilized concatemer template molecules, and sequencing the first and second plurality of immobilized concatemer template molecules essentially simultaneously according to the method of step (7).

[00703] In some embodiments, the methods for re-seeding a capture support comprise conducting steps (1) - (3) to generate a first plurality of immobilized concatemer template molecules, omitting step (4), conducting steps (5) - (6) to generate a second plurality of immobilized concatemer template molecules, and sequencing the first plurality of immobilized concatemer template molecules using first batch sequencing primers, and separately sequencing the second plurality of immobilized concatemer template molecules using second batch sequencing primers, wherein the first and second batch sequencing can be conducted according to the method of step (7).

Batch Sequencing

[00704] The present disclosure provides methods for conducting any of the enrichment workflows (1) - (10) described above, wherein the sequencing step can be conducted by employing a batch sequencing workflow.

[00705] The present disclosure provides methods for batch sequencing comprising step (a): providing a capture support comprising at least one layer of a hydrophilic polymer coating and a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating.

[00706] In some embodiments, the methods for batch sequencing comprise step (b): generating a plurality of concatemer template molecules immobilized to the capture support (“immobilized concatemer template molecules”), wherein the plurality of concatemer template molecules comprises a plurality of sub-populations of concatemer molecules including at least a first and a second sub-population of concatemer template molecules, wherein individual concatemer template molecules in the first sub-population comprises a first batch sequencing primer binding site and a target sequence, and wherein individual concatemer template molecules in the second sub-population comprises a second batch sequencing primer binding site and a target sequence.

[00707] In some embodiments, in step (b), the plurality of concatemer template molecules immobilized to the capture support can be generated using any of the enrichment workflows described herein including any of workflows (1) - (10). In some embodiments, the plurality of immobilized concatemer template molecules can be generated by (A) preparing a plurality of circle bait complexes immobilized to a capture support, wherein individual circle bait complexes comprise (i) a covalently closed circular library molecule comprising a polynucleotide having a target sequence, at least one universal adaptor sequence for binding a batch-specific sequencing primer, and (ii) a target-specific bait/probe that is selectively hybridized to at least a portion of a corresponding target sequence of a covalently closed circular library molecule, and (B) conducting a rolling circle amplification reaction using the target-specific bait/probe to initiate amplification, thereby generating a plurality of concatemer template molecules immobilized to the capture support. In some embodiments, the rolling circle amplification reaction can be conducted in the presence, or in the absence, of a plurality of compaction oligonucleotides. In some embodiments, individual compaction oligonucleotides can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball.

[00708] In some embodiments, in step (b), the concatemer template molecules within the first sub-population have the same first batch sequencing primer binding site, and have different target sequences. In some embodiments, the sequence of the first batch sequencing primer binding site sequence corresponds to the target sequences of the first sub-population. In some embodiments, a first batch sequencing primer selectively binds to the first batch sequencing primer binding site on a concatemer template molecule of the first subpopulation.

[00709] In some embodiments, in step (b), the concatemer template molecules within the second sub-population have the same second batch sequencing primer binding site, and have target sequences different from the target sequences of the first sub-population. In some embodiments, the sequence of the second batch sequencing primer binding site sequence corresponds to the target sequences of the second sub-population. In some embodiments, a second batch sequencing primer selectively binds to the second batch sequencing primer binding site on a concatemer template molecule of the second sub-population. In some embodiments, the first-batch sequencing primer and the second-batch sequencing primers have different sequences.

[00710] In some embodiments, in step (b), the plurality of concatemer template molecules can be immobilized to the capture support at random and non-pre-determined positions on the capture support. In some embodiments, in step (b), the plurality of concatemer template molecules can be immobilized to the capture support at pre-determined positions on the capture support (e.g., a patterned capture support).

[00711] In some embodiments, in step (b), the capture support comprises a plurality of concatemer template molecules immobilized thereon at a density of about 10² - 10¹⁵ concatemer template molecules per mm², wherein the immobilized concatemer template molecules comprise a mixture of at least two sub-populations of concatemer template molecules including at least a first and second sub-population of concatemer template molecules. In some embodiments, the plurality of sub-populations of concatemer template molecules are immobilized to the capture support at a high density, wherein at least some of the immobilized concatemer template molecules in the first and second sub-populations comprise nearest neighbor concatemer template molecules that touch each other and/or overlap each other when viewed from any angle of the capture support including above, below or side views of the capture support. In some embodiments, the capture support comprises up to 200 million concatemer template molecules immobilized thereon, or up to 1 billion concatemer template molecules immobilized thereon, or up to 2 billion concatemer template molecules immobilized thereon, or up to 3 billion concatemer template molecules immobilized thereon, or up to 4 billion concatemer template molecules immobilized thereon, or up to 5 billion concatemer template molecules immobilized thereon, or up to 6 billion concatemer template molecules immobilized thereon. In some embodiments, the capture support comprises up to 7 billion concatemer template molecules immobilized thereon, or up to 8 billion concatemer template molecules immobilized thereon, or up to 9 billion concatemer template molecules immobilized thereon, or up to 10 billion concatemer template molecules immobilized thereon, or up to 20 billion concatemer template molecules immobilized thereon.

[00712] In some embodiments, in step (b), individual concatemer template molecules in the first sub-population further comprise a first batch barcode sequence. In some embodiments, the first batch barcode sequence corresponds to a particular target sequence, or the first batch barcode sequence corresponds to any of the target sequences in the first subpopulation. In some embodiments, a pre-determined first batch barcode sequence can be linked to a given target sequence in the first sub-population (or can be linked to a plurality of different target sequences in the first sub-population), thus the pre-determined first batch barcode sequence corresponds to a given target sequence or sequences in the first subpopulation.

[00713] In some embodiments, in step (b), individual concatemer template molecules in the second sub-population further comprise a second batch barcode sequence. In some embodiments, the second batch barcode sequence corresponds to a particular target sequence, or the second batch barcode sequence corresponds to any of the target sequences in the second sub-population. In some embodiments, a pre-determined second batch barcode sequence can be linked to a given target sequence or sequences in the second sub-population (or can be linked to a plurality of different target sequences in the second sub-population), thus the pre-determined second batch barcode sequence corresponds to a given target sequence or sequences in the second sub-population.

[00714] In some embodiments, in step (b) the first and second batch barcode sequences can include a short random sequence (e.g., NNN) that is 3-20 in length. In some embodiments, sequencing the short random sequence can provide nucleotide diversity and color balance. In some embodiments, sequencing and imaging the short random sequence can be used for polony mapping and location and template registration because the short random sequence provides sufficient nucleotide diversity and color balance.

[00715] In some embodiments, in step (b), in the first and second sub-population of concatemer template molecules the short random sequence (e.g., NNN) has an overall base composition of about 25% or about 20-30% of all four nucleotide bases (e.g., A, G, C and T/U) to provide nucleotide diversity at each sequencing cycle during sequencing the short random sequence (e.g., NNN).

[00716] In some embodiments, in step (b), in the first and second sub-population of concatemer template molecules the proportion of adenine (A) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the first and second sub-population of concatemer template molecules the proportion of guanine (G) at any given position in the short random sequence is about 20- 30% or about 15-35% or about 10-40%. In some embodiments, in the first and second subpopulation of concatemer template molecules the proportion of cytosine (C) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the first and second sub-population of concatemer template molecules the proportion of thymine (T) or uracil (U) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%.

[00717] In some embodiments, in step (b), in the first and second sub-population of concatemer template molecules the proportion of adenine (A) and thymine (T), or the proportion of adenine (A) and uracil (U), at any given position in the short random sequence is about 10-65%. In some embodiments, in the first and second sub-population of concatemer template molecules the proportion of guanine (G) and cytosine (C) at any given position in the short random sequence is about 10-65%.

[00718] In some embodiments, the methods for batch sequencing further comprise step (c): sequencing the first sub-population of concatemer template molecules using a plurality of first batch sequencing primers, thereby generating a plurality of first batch sequencing read products. In some embodiments, the batch sequencing of step (c) comprises imaging a region of the capture support to detect the sequencing reactions of the first sub-population of concatemer template molecules.

[00719] In some embodiments, the methods for batch sequencing comprise step (d): sequencing the second sub-population of concatemer template molecules using a plurality of second batch sequencing primers, thereby generating a plurality of second batch sequencing read products. In some embodiments, the batch sequencing of step (c) comprises imaging a region of the capture support to detect the sequencing reactions of the second sub -population of concatemer template molecules.

[00720] In some embodiments, the batch sequencing reactions of the first sub-population of concatemer template molecules of step (c) can be stopped before initiating the batch sequencing reactions of the second sub-population of concatemer template molecules of step (d).

[00721] In some embodiments, the first batch sequencing of step (c) and the second batch sequencing of step (d) comprise contacting the plurality of immobilized concatemer template molecules with a plurality of batch-specific sequencing primers, a plurality of sequencing polymerases and nucleotide reagents which include nucleotides, nucleotide analogs and/or multivalent molecules. In some embodiments, the sequencing reactions employ nucleotide reagents comprising detectably labeled nucleotide analogs. In some embodiments, the sequencing reactions employ a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules, and incorporating nucleotide analogs. In some embodiments, the sequencing reactions employ non-labeled nucleotide analogs. Various methods for sequencing are described below, including two-stage sequencing, sequencing-by-binding, sequencing using nucleotide analogs and sequencing using phosphate-chain labeled nucleotides.

Pairwise Sequencing Methods

[00722] The present disclosure provides methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides using any of the enrichment workflows described herein to generate a plurality of immobilized concatemer template molecules wherein the immobilized concatemer template molecules can be subjected to pairwise sequencing. [00723] The present disclosure provides pairwise sequencing methods, comprising step (a): preparing a plurality of circle bait complexes immobilized to a capture support, wherein individual circle bait complexes comprise (i) a covalently closed circular library molecule comprising a polynucleotide having a target sequence and at least one universal adaptor sequence, and (ii) a target-specific bait/probe that is selectively hybridized to at least a portion of a corresponding target sequence of a covalently closed circular library molecule, and wherein the plurality of circle bait complexes are immobilized to a capture support. [00724] In some embodiments, in step (a), the plurality of immobilized circle bait complexes can be prepared by conducting any of the target enrichment workflows described herein.

[00725] In some embodiments, in step (a), the plurality of immobilized circle bait complexes can be prepared by conducting an in-solution target enrichment workflow using covalently closed circular library molecules and target-specific baits/probes as described herein in workflow (1).

[00726] In some embodiments, in step (a), the plurality of immobilized circle bait complexes can be prepared by conducting an on-support target enrichment workflow using covalently closed circular library molecules and target-specific baits/probes as described herein in workflow (2).

[00727] In some embodiments, in step (a), the plurality of immobilized circle bait complexes can be prepared by conducting an in-solution target enrichment workflow using linear library molecules, top strand circularization oligonucleotides (e.g., single-stranded or double-stranded), and target-specific baits/probes as described herein in workflow (3). [00728] In some embodiments, in step (a), the plurality of immobilized circle bait complexes can be prepared by conducting an on-support target enrichment workflow using linear library molecules, top strand circularization oligonucleotide (e.g., single-stranded or double-stranded), and target-specific baits/probes as described herein in workflow (4). [00729] In some embodiments, in step (a), the plurality of immobilized circle bait complexes can be prepared by conducting an in-solution target enrichment workflow using linear library molecules, top strand circularization oligonucleotides and target-specific baits/probes as described herein in workflows (5), (6) and (7).

[00730] In some embodiments, in step (a), the plurality of immobilized circle bait complexes can be prepared by conducting an in-solution target enrichment workflow using linear library molecules, top strand circularization oligonucleotides and target-specific baits/probes, and forming 5’ overhang flap structures as described herein in workflows (8) and (9).

[00731] In some embodiments, step (a) further comprises conducting rolling circle amplification on the plurality of circle bait complexes that are immobilized to the capture support thereby generating a plurality of concatemer template molecules immobilized to the capture support. In some embodiments, the 5’ ends of individual concatemer template molecules are covalently joined to a target-specific bait/probe that is immobilized to the capture support. In some embodiments, the rolling circle amplification reaction employs the target-specific bait/probe to initiate amplification thereby generating a plurality of immobilized concatemer template molecules. In some embodiments, the rolling circle amplification reaction is conducted by contacting the plurality of immobilized circle bait complexes with a rolling circle reagent, wherein the rolling circle reagent comprises a strand displacing polymerase and a plurality of nucleotides comprising a mixture of dATP, dGTP dCTP, dTTP and dUTP. In some embodiments, individual immobilized concatemer template molecules are covalently joined to an immobilized target-specific bait/probe. In some embodiments, the rolling circle amplification reaction can be conducted in the presence or the absence of a plurality of compaction oligonucleotides.

[00732] In some embodiments, individual immobilized concatemer template molecules comprise at least one nucleotide having a scissile moiety, wherein individual concatemer template molecules in the plurality are immobilized to a target-specific bait/probe that is immobilized to a capture support. In some embodiments, the immobilized target-specific bait/probe lacks a nucleotide having a scissile moiety. In some embodiments, the capture support comprises a plurality of immobilized target-specific baits/probes. In some embodiments, the capture support lacks a plurality of pinning primers. In some embodiments, the capture support comprises a plurality of target-specific baits/probes and a plurality of pinning primers.

[00733] In some embodiments, the scissile moieties in the immobilized concatemer template molecules of step (a) can be converted into abasic sites in the immobilized concatemer template molecules. In some embodiments, the scissile moieties in the immobilized concatemer template molecules comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. In the concatemer template molecules, the uridine can be converted to an abasic site using uracil DNA glycosylase (UDG), the 8oxoG can be converted to an abasic site using FPG glycosylase, and the deoxyinosine can be converted to an abasic site using AlkA glycosylase. In some embodiments, the immobilized concatemer template molecules include 1-20, 20-40, 40-60, 60-80, 80-100, or a higher number of nucleotides with a scissile moiety. In some embodiments, about 0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30% or a higher percent of the dTTP in the immobilized concatemer template molecules are replaced with nucleotides having a scissile moiety. In some embodiments, the nucleotides having a scissile moiety are distributed at random positions along individual immobilized concatemer template molecules. In some embodiments, the nucleotides having a scissile moiety are distributed at different positions in the different immobilized concatemer template molecules.

[00734] In some embodiments, the linear library molecules and covalently closed circular library molecules comprise (i) a polynucleotide having a target sequence or a non-target sequence and (ii) at least one universal adaptor sequence. In some embodiments, individual the linear library molecules and covalently closed circular library molecules comprise an insert region comprising a target or non-target polynucleotide sequence and any one or any combination of two or more adaptor sequences arranged in any order including: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and/or a universal adaptor sequence for binding a first universal surface primer (e.g., a universal capture primer). In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises a universal adaptor sequence for binding a batch-specific forward sequencing primer. In some embodiments, any of the linear library molecules and covalently closed circular library molecules shown in FIGS. 14 and 17 which comprise different combinations of two or more adaptor sequences arranged in various orders can be used to conduct step (a). In some embodiments, any of the linear library molecules shown in FIGS. 20-35 which comprise different combinations of two or more adaptor sequences arranged in various orders can be circularized to form covalently closed circular library molecules which can be used to conduct step (a).

[00735] In some embodiments, the universal binding sequence (or a complementary sequence thereof) for the forward sequencing primer can hybridize to at least a portion of the forward sequencing primer. In some embodiments, the universal binding sequence (or a complementary sequence thereof) for the reverse sequencing primer can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence (or a complementary sequence thereof) for the soluble compaction oligonucleotide can hybridize to at least a portion of the soluble compaction oligonucleotide.

[00736] In some embodiments, the capture support comprises at least one layer of a hydrophilic polymer coating and a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating.

[00737] In some embodiments, the capture support further comprises a plurality of immobilized target-specific baits/probes, wherein individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence, an affinity moiety at the 5’ end of the target-specific oligonucleotide, and the target-specific oligonucleotide comprises an extendible 3’ end. In some embodiments, the affinity moiety of the targetspecific bait/probe can bind a receptor moiety embedded in the hydrophilic polymer coating of the capture support. In some embodiments, the 3’ ends of individual target-specific bait/probes are covalently joined to individual concatemer template molecules. In some embodiments, the capture support lacks any universal capture primers (e.g., first universal surface primers). In some embodiments, the capture support comprises a plurality of universal capture primers (e.g., first universal surface primers).

[00738] In some embodiments, the capture support further comprises a plurality of immobilized pinning primers, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide. In some embodiments individual pinning primers comprise a blocking group at the 3’ end of the oligonucleotide, wherein the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprises a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support.

[00739] In some embodiments, the affinity moiety of the target-specific baits/probes is the same type of affinity moiety of the pinning primers. In some embodiments, the affinity moiety of the target-specific baits/probes is a different type of affinity moiety of the pinning primers.

[00740] In some embodiments, the capture support further comprises a plurality of targetspecific baits/probes and a plurality of pinning primers. In some embodiments, the capture support further comprises a plurality of target-specific baits/probes or a plurality of pinning primers. [00741] In some embodiments, individual immobilized concatemer template molecule are joined or immobilized to an immobilized target-specific bait/probe, and at least one portion of the individual concatemer template molecule is hybridized to an immobilized pinning primer. The immobilized pinning primer serves to pin down a portion of the immobilized concatemer template molecules to the capture support.

[00742] In some embodiments, the capture support comprises about 10² - 10¹⁵ immobilized target-specific baits/probes per mm². In some embodiments, the capture support comprises about 10² - 10¹⁵ immobilized pinning primers per mm².

[00743] The immobilized target-specific baits/probes and pinning primers are in fluid communication with each other to permit flowing various solutions of linear or circular nucleic acid molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, and the like, onto the capture support so that the plurality of immobilized targetspecific baits/probes, and the primer extension products generated from the immobilized target-specific baits/probes, react with the solutions in a massively parallel manner.

[00744] In some embodiments, the pairwise sequencing method further comprises step (b): sequencing the plurality of immobilized concatemer template molecules thereby generating a plurality of extended forward sequencing primer strands. The sequencing of step (b) comprises contacting the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers under a condition suitable to hybridize at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatemer template molecules, and conducting forward sequencing reactions using one or more types of sequencing polymerases, a plurality of nucleotides and/or multivalent molecules, and the hybridized first forward sequencing primers. The forward sequencing reactions can generate a plurality of extended forward sequencing primer strands. In some embodiments, individual immobilized concatemer template molecules have multiple copies of the forward sequencing primer binding sites, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites in a given immobilized concatemer template molecule can be hybridized to a forward sequencing primer and can undergo a sequencing reaction. Individual immobilized concatemer template molecules can undergo two or more sequence reactions, where each sequencing reaction is initiated from a first forward sequencing primer that is hybridized to a forward sequencing primer binding site. In some embodiments, the soluble forward sequencing primers comprise 3’ OH extendible ends. In some embodiments, the soluble forward sequencing primers comprise a 3’ blocking moiety which can be removed to generate a 3’ OH extendible end. In some embodiments, the soluble forward sequencing primers lack a nucleotide having a scissile moiety. In some embodiments, the sequencing reactions comprise a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reactions comprise a plurality of multivalent molecules having a plurality of nucleotide arms attached to a core, where the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide moiety of a nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. An exemplary nucleotide arm is shown in FIG. 5, and exemplary multivalent molecules are shown in FIGS. 1-4.

[00745] In some embodiments, the pairwise sequencing method further comprises step (c): retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands that are hybridized to the retained immobilized concatemer template molecules. The plurality of extended forward sequencing primer strands can be removed and replaced with a plurality of forward extension strands by conducting a primer extension reaction.

[00746] In some embodiments, step (c) comprises contacting at least one extended forward sequencing primer strand with a plurality of strand displacing polymerases and a plurality of nucleotides and in the absence of soluble amplification primers, under a condition suitable to conduct a strand displacing primer extension reaction using the at least one extended forward sequencing primers strand to initiate the primer extension reaction thereby generating a forward extension strand that is covalently joined to the extended forward sequencing primer strand, wherein the forward extension strand is hybridized to the immobilized concatemer template molecule. For example, one of the extended forward sequencing primer strands can serve as a primer for the strand displacing polymerase. The strand displacing polymerase can extend the extended forward sequencing primer strand, and displace downstream extended forward sequencing primer strands while synthesizing an extended strand that replaces the downstream extended forward sequencing primer strands. The newly extended strand is covalently joined to an extended forward sequencing primer strand. The immobilized concatemer template molecules are retained.

[00747] Examples of strand displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), KI enow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[00748] In some embodiments, the primer extension reaction of step (c) can optionally include a plurality of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate forward extension strands. Individual forward extension strands can collapse into a nanoball having a more compact size and/or shape compared to a nanoball generated from a primer extension reaction conducted without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). Inclusion of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve FWHM (full width half maximum) of a spot image of the nanoball. The spot image can be represented as a Gaussian spot and the size can be measured as a FWHM. A smaller spot size as indicated by a smaller FWHM typically correlates with an improved image of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 pm or smaller.

[00749] In some embodiments, step (c) comprises: (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules; and (ii) contacting the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers (e.g., a second plurality of soluble forward sequencing primers), a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of primer extension polymerases, under a condition suitable to hybridize the plurality of soluble forward sequencing primers to the plurality of immobilized concatemer template molecules and suitable for conducting polymerase-catalyzed primer extension reactions, thereby generating a plurality of forward extension strands, wherein the soluble sequencing primers hybridize with the forward sequencing primer binding sequence in the retained immobilized concatemer template molecules. The primer extension reaction can optionally include a plurality of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate forward extension strands. Individual forward extension strands can collapse into a nanoball having a more compact size and/or shape compared to a nanoball generated from a primer extension reaction conducted without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). Inclusion of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve FWHM (full width half maximum) of a spot image of the nanoball. The spot image can be represented as a Gaussian spot and the size can be measured as a FWHM. A smaller spot size as indicated by a smaller FWHM typically correlates with an improved image of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 pm or smaller.

[00750] In some embodiments, in step (c), the condition suitable to hybridize the plurality of soluble forward sequencing primers to the plurality of immobilized concatemer template molecules that have been retained comprises hybridizing immobilized concatemer template molecules with the soluble primers in the presence of a primer extension polymerase, a plurality of nucleotides, and a high efficiency hybridization buffer. In some embodiments, the high efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant that is no greater than 40 and having a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant that is no greater than 115 and is present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in a range of about 4-8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25- 50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(A-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high efficiency hybridization buffer further comprises betaine.

[00751] In some embodiments, step (c) comprises: (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules; and (ii) contacting the plurality of immobilized concatemer template molecules with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of primer extension polymerases, under a condition suitable to hybridize the plurality of soluble amplification primers to the plurality of immobilized concatemer template molecules and suitable for conducting polymerase- catalyzed primer extension reactions thereby generating a plurality of forward extension strands, wherein the soluble amplification primers hybridize with the soluble amplification primer binding sequence in the retained immobilized concatemer template molecules. The primer extension reaction can optionally include a plurality of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate forward extension strands. Individual forward extension strands can collapse into a nanoball having a more compact size and/or shape compared to a nanoball generated from a primer extension reaction conducted without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). Inclusion of compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve FWHM (full width half maximum) of a spot image of the nanoball. The spot image can be represented as a Gaussian spot and the size can be measured as a FWHM. A smaller spot size as indicated by a smaller FWHM typically correlates with an improved image of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 pm or smaller.

[00752] In some embodiments, in step (c), the condition suitable to hybridize the plurality of soluble amplification primers to the plurality of immobilized concatemer template molecules that have been retained comprises hybridizing immobilized concatemer template molecules with the soluble primers in the presence of a primer extension polymerase, a plurality of nucleotides, and a high efficiency hybridization buffer. In some embodiments, the high efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant that is no greater than 40 and having a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant that is no greater than 115 and is present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in a range of about 4-8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25- 50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(A-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high efficiency hybridization buffer further comprises betaine.

[00753] In some embodiments, in step (c), the plurality of extended forward sequencing primer strands can be removed using an enzyme or a chemical reagent. For example, the plurality of extended forward sequencing primer strands can be enzymatically degraded using a 5’ to 3’ double-stranded DNA exonuclease, including T7 exonuclease (e.g., from New England Biolabs, catalog # M0263S). In some embodiments, the plurality of extended forward sequencing primer strands can be removed with a temperature that favors nucleic acid denaturation.

[00754] In some embodiments, in step (c), a denaturation reagent can be used to remove the plurality of extended forward sequencing primer strands, wherein the denaturation reagent comprises any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride and/or a pH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like). Optionally, the denaturation reagent can further comprise PEG.

[00755] In some embodiments, in step (c), the plurality of extended forward sequencing primer strands can be removed using an elevated temperature (e.g., heat) with or without a nucleic acid denaturation reagent. The plurality of extended forward sequencing primer strands can be subjected to a temperature of about 45-50 °C, or about 50-60 °C, or about 60- 70 °C, or about 70-80 °C, or about 80-90 °C, or about 90-95 °C, or higher temperature.

[00756] In some embodiments, in step (c), the plurality of extended forward sequencing primer strands can be removed using 100% formamide at a temperature of about 65 °C for about 3 minutes, and washing with a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 - 8.5.

[00757] In some embodiments, the primer extension polymerase of step (c) comprises a high fidelity polymerase. In some embodiments, the primer extension polymerase of step (c) comprises a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-tolerant polymerase). Exemplary polymerases include, but are not limited to, Q5U® Hot Start high-fidelity DNA polymerase (e.g., catalog # M0515S from New England Biolabs®), Taq DNA polymerase, One Taq® DNA polymerase (e.g., mixture of Taq and Deep Vent® DNA polymerases, catalog #M0480S from New England Biolabs), LongAmp® Taq DNA polymerase (e.g., catalog #M0323S from New England Biolabs), Epimark® Hot Start Taq DNA polymerase (e.g., catalog #M0490S from New England Biolabs), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., catalog # M0269S from New England Biolabs), E. coli DNA polymerase (e.g., catalog # M0209S from New England Biolabs), Therminator® DNA polymerase (e.g., catalog #M0261S from New England Biolabs), Vent DNA polymerase and Deep Vent® DNA polymerase.

[00758] The pairwise methods described herein can provide increased accuracy in a downstream sequencing reaction because step (c) replaces the extended forward sequencing primer strands that were generated in step (b) with forward extension strands having reduced base errors. The extended forward sequencing primer strands that are generated in step (b) and may or may not contain erroneously incorporated nucleotides due to polymerase- catalyzed mis-paired bases. When step (c) is conducted with a high fidelity DNA polymerase, the resulting forward extension strands may have reduced base errors compared to the extended forward sequencing primer strands. The forward extension strands will be used as a nucleic acid template for a downstream sequencing step (e.g., see step (e) below). Thus, step (c) can increase the sequencing accuracy of the downstream step (e) and therefore increase the overall sequencing accuracy of the pairwise sequencing workflow.

[00759] In some embodiments, the pairwise sequencing method comprises step (d): removing the immobilized concatemer template molecules that were retained by generating abasic sites in the immobilized concatemer template molecules at the nucleotide(s) having the scissile moiety and generating gaps at the abasic sites to generate a plurality of gapcontaining concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized surface primers.

[00760] In some embodiments, the abasic sites are generated on the immobilized concatemer template molecules that contain nucleotides having scissile moieties. In some embodiments, the scissile moieties in the concatemer template molecules comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. The abasic sites can be removed to generate a plurality of immobilized concatemer template molecules having gaps while retaining the plurality of forward extension strands. The abasic sites can be generated by contacting the immobilized concatemer template molecules with an enzyme that removes the nucleo-base at the nucleotide having the scissile moiety. The uracil in the retained concatemer strands can be converted to an abasic site using uracil DNA glycosylase (UDG). The 8oxoG in the retained concatemer strands can be converted to an abasic site using FPG glycosylase. The deoxyinosine in the retained concatemer strands can be converted to an abasic site using AlkA glycosylase.

[00761] In some embodiments, in step (d), the gaps can be generated by contacting the abasic sites in the immobilized concatemer template molecules with an enzyme or a mixture of enzymes having lyase activity that breaks the phosphodiester backbone at the 5’ and 3’ sides of the abasic site to release the base-free deoxyribose and generate a gap. The abasic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/ AP lyase, Endo VIII glycosylase/ AP lyase. In some embodiments, generating the abasic sites and removal of the abasic sites to generate gaps can be achieved using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, for example USER (Uracil- Specific Excision Reagent Enzyme from New England Biolabs) or thermolabile USER (also from New England Biolabs).

[00762] In some embodiments, step (d) comprises removing the plurality of gapcontaining concatemer template molecules using an enzyme, chemical and/or heat and retaining the plurality of forward extension strands wherein individual forward extension strands are hybridized to one of the immobilized target-specific bait/probes. In some embodiments, the plurality of forward extension strands are retained and are immobilized to the capture support.

[00763] For example, the plurality of gap-containing concatemer template molecules can be enzymatically degraded using a 5’ to 3’ double-stranded DNA exonuclease, including T7 exonuclease (e.g., from New England Biolabs, catalog # M0263S). When a 5’ to 3’ doublestranded DNA exonuclease is used for removing gap-containing concatemer template molecules, then the plurality of soluble amplification primers in step (c) can comprise at least one phosphorothioate diester bond at their 5’ ends which can render the soluble amplification primers resistant to exonuclease degradation. In some embodiments, the plurality of soluble amplification primers in step (c) comprise 2-5 or more consecutive phosphorothioate diester bonds at their 5’ ends. In some embodiments, the plurality soluble amplification primers in step (c) comprise at least one ribonucleotide and/or at least one 2’-O-methyl or 2’-O- methoxy ethyl (MOE) nucleotide which can render the forward sequencing primers resistant to exonuclease degradation.

[00764] In some embodiments, in step (d), the plurality of gap-containing concatemer template molecules can be removed using a chemical reagent that favors nucleic acid denaturation. The denaturation reagent can include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or the like).

[00765] In some embodiments, in step (d), the plurality of gap-containing concatemer template molecules can be removed using an elevated temperature (e.g., heat) with or without a nucleic acid denaturation reagent. The gap-containing concatemer template molecules can be subjected to a temperature of about 45-50 °C, or about 50-60 °C, or about 60-70 °C, or about 70-80 °C, or about 80-90 °C, or about 90-95 °C, or higher temperature.

[00766] In some embodiments, in step (d), the plurality of gap-containing concatemer template molecules can be removed using 100% formamide at a temperature of about 65 °C for about 3 minutes, and washing with a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 - 8.5. [00767] In some embodiments, the pairwise sequencing method comprises step (e): sequencing the plurality of retained forward extension strands thereby generating a plurality of extended reverse sequencing primer strands. In some embodiments, the sequencing of step (e) comprises contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers under a condition suitable to hybridize the reverse sequencing primers to the reverse sequencing primer binding site of the retained forward extension strands, and by conducting sequencing reactions using the hybridized reverse sequencing primers wherein the forward sequencing reactions generates a plurality of extended reverse sequencing primer strands. The extended reverse sequencing primer strands are hybridized to the retained forward extension strand. The retained forward extension strand is hybridized to the immobilized target-specific bait/probe. The extended reverse sequencing primer strands are not hybridized to the target-specific bait/probe, or covalently joined to the target-specific bait/probe. Therefore, the extended reverse sequencing primer strands are not immobilized to the capture support.

[00768] In some embodiments, in step (e), the condition suitable to hybridize the reverse sequencing primers to the reverse sequencing primer binding sequences of the retained forward extension strands comprises contacting the plurality of soluble reverse sequencing primers and the retained forward extension strands with a high efficiency hybridization buffer. In some embodiments, the high efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant that is no greater than 40 and having a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant that is no greater than 115 and is present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in a range of about 4-8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(7V-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high efficiency hybridization buffer further comprises betaine. [00769] In an alternative embodiment, the sequencing of step (e) comprises using the immobilized surface primer as a sequencing primer and conducting sequencing reactions to generate a plurality of reverse sequencing strands.

[00770] In some embodiments, the reverse sequencing reactions of step (e) comprises contacting the plurality of soluble reverse sequencing primers with the reverse sequencing primer binding sequences of the retained forward extension strands, one or more types of sequencing polymerases, and a plurality of nucleotides or a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primers comprise 3’ OH extendible ends. In some embodiments, the soluble reverse sequencing primers comprise a 3’ blocking moiety which can be removed to generate a 3’ OH extendible end. In some embodiments, the soluble reverse sequencing primers lack a nucleotide having a scissile moiety. The sequencing reactions that employ nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reactions can generate a plurality of extended reverse sequencing primer strands. In some embodiments, individual retained forward extension strands have multiple copies of the reverse sequencing primer binding sequences/sites, wherein each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites in a given retained forward extension strand can be hybridized to a reverse sequencing primer and can undergo a sequencing reaction. Thus, an individual retained forward extension strand can undergo two or more sequencing reactions, where each sequencing reaction is initiated from a reverse sequencing primer that is hybridized to a reverse sequencing primer binding site.

[00771] In some embodiments, the reverse sequencing reactions of step (e) comprise a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reactions comprise a plurality of multivalent molecules having a plurality of nucleotide arms attached to a core, where the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide moiety of a nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. An exemplary nucleotide arm is shown in FIG. 5, and exemplary multivalent molecules are shown in FIGS. 1-4.

[00772] In some embodiments, the reverse sequencing reactions of step (e) can be conducted using a dark sequencing method. For example, the first 5-30 bases of the retained forward extension strand can be subjected to dark sequencing using non-labeled nucleotide reagents, and the remaining bases of the retained forward extension strand can be subjected to sequencing using detectably labeled nucleotide reagents. As used herein, “dark sequencing” refers to methods that comprise conducting recursive sequencing cycles using non-labeled nucleotide reagents that can extend the sequencing primer, but nucleotide incorporation is not detected. In some embodiments, the non-labeled nucleotide reagents comprise non-labeled canonical nucleotides and/or non-labeled chain terminator nucleotides.

[00773] In some embodiments, at least one washing step can be conducted after any of steps (a) - (e). The washing step can be conducted with a wash buffer comprising a pH buffering agent, a metal chelating agent, a salt, and a detergent.

[00774] In some embodiments, the pH buffering compound in the wash buffer comprises any one or any combination of two or more of Tris, Tris-HCl, Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol; MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In some embodiments, the pH buffering agent can be present in the wash buffer at a concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM. In some embodiments, the pH of the pH buffering agent which is present in any of the reagents described here in can be adjusted to a pH of about 4-9, or a pH of about 5-9, or a pH of about 5-8.

[00775] In some embodiments, the metal chelating agent in the wash buffer comprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxy ethylethylenediaminetriacetic acid), DPTA (diethylene triamine pentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium citrate. In some embodiments, the wash buffer comprises a chelating agent at a concentration of about 0.01 - 50 mM, or about 0.1 - 20 mM, or about 0.2 - 10 mM.

[00776] In some embodiments, the salt in the wash buffer comprises NaCl, KC1, NH2SO4 or potassium glutamate. In some embodiments, the detergent comprises an ionic detergent such as SDS (sodium dodecyl sulfate). The wash buffer can include a monovalent salt at a concentration of about 25-500 mM, or about 50-250 mM, or about 100-200 mM.

[00777] In some embodiments, the detergent in the wash buffer comprises a non-ionic detergent such as Triton X-100, Tween 20, Tween 80 or Nonidet P-40. In some embodiments, the detergent comprises a zwitterionic detergent such as CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-l -propanesulfonate) or A-Dodecyl-A,A-dimethyl-3- amonio-1 -propanesulfate (DetX). In some embodiments, the detergent comprises LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%, or about 0.2-0.25%.

[00778] Exemplary pairwise sequencing methods are described, for example in U.S. patent No. 11,220,707, the contents of which is incorporated by reference in its entirety.

Order of Sequencing for Single Pass Sequencing and Pairwise Sequencing

[00779] The present disclosure provides various methods for enriching target polynucleotides from a mixture of target and non-target polynucleotides, wherein the methods comprise step (a): preparing a plurality of immobilized circle bait complexes by binding a plurality of circle bait complexes to a capture support, wherein individual circle bait complexes comprise a covalently closed circular library molecule hybridized to a targetspecific bait/probe; step (b): conducting a rolling circle amplification reaction using the extendible 3’ end of the target-specific bait/probe to initiate the amplification reaction and using the covalently closed circular library molecule as a template molecule, thereby generating a plurality of immobilized concatemer template molecules, wherein individual immobilized concatemer template molecules comprise multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise universal adaptor sequences and sample indexes from the covalently closed circular molecule; and step (c): sequencing at least a portion of the plurality of immobilized concatemer template molecules. In some embodiments, the sequencing can be conducted as single pass sequencing or pairwise sequencing. In some embodiments, different portions of the immobilized concatemer template molecules can be sequenced in different order.

[00780] In some embodiments, any of the linear library molecules shown in FIGS. 20-35, can be used to prepare the immobilized circle bait complexes and the immobilized concatemer template molecules. In some embodiments, linear library molecules with different arrangements can be constructed to include an insert region, a plurality of universal adaptor sequences and at least one sample index to accommodate single pass sequencing and/or pairwise sequencing. The schematics of FIGS. 20-35 show different orders of sequencing of individual polynucleotide units of a concatemer template molecule corresponding to the linear library molecules described herein. [00781] FIG. 21 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1500) for binding a forward second sequencing primer; a left sample index sequence (1700); a universal adaptor sequence (1300) for binding a forward third sequencing primer; a sequence-of-interest (e.g., insert region (1200)); a universal adaptor sequence (1400) for binding a forward first sequencing primer or a reverse sequencing primer; an optional unique molecular index sequence (1900); a right sample index sequence (1800); and a universal adaptor sequence (1600) for circularization. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 21 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index (1900) and the right sample index (1800); (2) a forward second sequencing read product comprising the left sample index (1700); and (3) a forward third sequencing read product comprising at least a portion of the insert region (1200). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (4) a reverse first sequencing read product comprising at least a portion of the insert region (1200). In FIG. 21, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00782] FIG. 22 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1500) for binding a forward first sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a universal adaptor sequence (1300) for binding a forward third sequencing primer; a sequence-of- interest (e.g., insert region (1200)); a universal adaptor sequence (1400) for binding a forward second sequencing primer or a reverse sequencing primer; a right sample index sequence (1800); and a universal adaptor sequence (1600) for circularization. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 22 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index (1900) and the left sample index (1700); (2) a forward second sequencing read product comprising the right sample index (1800); and (3) a forward third sequencing read product comprising at least a portion of the insert region (1200). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (4) a reverse first sequencing read product comprising at least a portion of the insert region (1200). In FIG. 22, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products. [00783] FIG. 23 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward first sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a sequence-of- interest (e.g., insert region (1200)); a universal adaptor sequence (1400) for binding a forward second sequencing primer or a reverse sequencing primer; a right sample index sequence (1800); and a universal adaptor sequence (1600) for circularization. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 23 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index (1900), the left sample index (1700) and at least a portion of the insert region (1200); and (2) a forward second sequencing read product comprising the right sample index (1800). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (3) a reverse first sequencing read product comprising at least a portion of the insert region (1200). In FIG. 23, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00784] FIG. 24 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward first sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a sequence-of- interest (e.g., insert region (1200)); a universal adaptor sequence (1400) for binding a reverse second sequencing primer; a right sample index sequence (1800); and a universal adaptor sequence (1600) for circularization which can bind a reverse first sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 24 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index (1900), the left sample index (1700) and at least a portion of the insert region (1200). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (2) a reverse first sequencing read product comprising the right sample index (1800); and (3) a reverse second sequencing read product comprising at least a portion of the insert region (1200). In FIG. 24, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00785] FIG. 25 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward first sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a sequence-of- interest (e.g., insert region (1200)); a universal adaptor sequence (1400) for binding a forward second sequencing primer; a right sample index sequence (1800); and a universal adaptor sequence (1600) for circularization. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 25 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index (1900), the left sample index (1700) and at least a portion of the insert region (1200); and (2) a forward second sequencing read product comprising the right sample index (1800). In FIG. 25, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00786] FIG. 26 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1500) for binding a forward first sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a universal adaptor sequence (1300) for binding a forward second sequencing primer; a sequence-of- interest (e.g., insert region (1200)); a right sample index sequence (1800); a universal adaptor sequence (1400) for binding a reverse sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 26 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index (1900) and the left sample index (1700); and (2) a forward second sequencing product comprising at least a portion of the insert region (1200). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (3) a reverse first sequencing read product comprising the right sample index (1800) and at least a portion of the insert region (1200). In FIG. 26, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00787] FIG. 27 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1500) for binding a forward first sequencing primer; a left sample index sequence (1700); a universal adaptor sequence (1300) for binding a forward second sequencing primer; a sequence-of-interest (e.g., insert region (1200)); a right sample index sequence (1800); an optional unique molecular index sequence (1900); and a universal adaptor sequence (1400) for binding a reverse sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 27 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the left sample index (1700); and (2) a forward second sequencing product comprising at least a portion of the insert region (1200). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (3) a reverse first sequencing read product comprising the optional unique molecular index sequence (1900) and the right sample index (1800). In FIG. 27, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00788] FIG. 28 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward first sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a sequence-of- interest (e.g., insert region (1200)); a right sample index sequence (1800); and a universal adaptor sequence (1400) for binding a reverse sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 28 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index sequence (1900), the left sample index (1700) and at least a portion of the sequence-of-interest (FIG. 28, insert region, (1200)). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (2) a reverse sequencing read product comprising the right sample index (1800) and at least a portion of the sequence-of-interest (FIG. 28, insert region, (1200)). In FIG. 28, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00789] FIG. 29 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1500) for binding a forward first sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a universal adaptor sequence (1300) for binding a forward second sequencing primer; a sequence-of- interest (e.g., insert region (1200)); and a universal adaptor sequence (1400) for binding a reverse sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 29 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index sequence (1900) and the left sample index (1700); and (2) a forward sequence sequencing read product comprising at least a portion of the sequence-of-interest (FIG. 29, insert region, (1200)). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (3) a reverse sequencing read product comprising at least a portion of the sequence-of-interest (FIG. 29, insert region, (1200)). In FIG. 29, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00790] FIG. 30 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward second sequencing primer; a sequence-of-interest (e.g., insert region (1200)); a universal adaptor sequence (1500) for binding a forward first sequencing primer or for binding a reverse sequencing primer; an optional unique molecular index sequence (1900); a right sample index sequence (1800); and a universal adaptor sequence (1600) for circularization. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 30 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index sequence (1900) and the right sample index (1800); and (2) a forward second sequencing read product comprising at least a portion of the sequence-of- interest (FIG. 30, insert region, (1200)). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (3) a reverse sequencing read product comprising at least a portion of the sequence-of-interest (FIG. 30, insert region, (1200)). In FIG. 30, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00791] FIG. 31 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a sequence-of- interest (e.g., insert region (1200)); and a universal adaptor sequence (1400) for binding a reverse sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 31 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index sequence (1900), the left sample index (1700) and at least a portion of the sequence-of- interest (FIG. 31, insert region, (1200)). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (2) a reverse sequencing read product comprising at least a portion of the sequence-of-interest (FIG. 31, insert region, (1200)). In FIG. 31, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00792] FIG. 32 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward sequencing primer; a sequence-of- interest (e.g., insert region (1200)); a right sample index sequence (1800); an optional unique molecular index sequence (1900); and a universal adaptor sequence (1400) for binding a reverse sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 32 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising at least a portion of the sequence-of- interest (FIG. 32, insert region, (1200)). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (2) a reverse sequencing read product comprising the optional unique molecular index sequence (1900), the right sample index (1800) and at least a portion of the sequence- of-interest (FIG. 32, insert region, (1200)). In FIG. 32, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products. [00793] FIG. 33 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward sequencing primer; an optional unique molecular index sequence (1900); a left sample index sequence (1700); a sequence-of- interest (e.g., insert region (1200)); and a universal adaptor sequence (1600) for circularization. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 33 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising the optional unique molecular index sequence (1900), the left sample index (1700) and at least a portion of the sequence-of-interest (FIG. 33, insert region, (1200)). In FIG. 33, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00794] FIG. 34 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward sequencing primer; a sequence-of- interest (e.g., insert region (1200)); and a universal adaptor sequence (1400) for binding a reverse sequencing primer. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 34 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising at least a portion of the sequence-of- interest (FIG. 34, insert region, (1200)). In some embodiments, the concatemer template molecule can be subjected to pairwise sequencing to enable sequencing a complementary concatemer template molecule, wherein the order of sequencing further comprises generating: (2) a reverse sequencing read product comprising at least a portion of the sequence-of-interest (FIG. 34, insert region, (1200)). In FIG. 34, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

[00795] FIG. 35 shows an embodiment of a linear library molecule (1100) comprising: a universal adaptor sequence (1300) for binding a forward sequencing primer; a sequence-of- interest (e.g., insert region (1200)); and a universal adaptor sequence (1600) for circularization. In some embodiments, the linear library molecule (1100) can form a covalently closed circular molecule using any of the circularization methods described herein. In some embodiments, the covalently closed circular molecule can be subjected to rolling circle amplification using the covalently closed circular molecule as a template molecule to generate a concatemer template molecule comprising multiple tandem repeat polynucleotide units, wherein individual polynucleotide units comprise the universal adaptor sequences and sample indexes in the covalently closed circular molecule. FIG. 35 also shows an exemplary order of sequencing of individual polynucleotide units of a concatemer template molecule. In some embodiments, the order of sequencing comprises generating: (1) a forward first sequencing read product comprising at least a portion of the sequence-of-interest (FIG. 35, insert region, (1200)). In FIG. 35, the solid arrows represent a sequencing primer and the dashed arrows represent the sequencing read products.

Sources of Nucleic Acids and Fragmenting Nucleic Acids

[00796] The present disclosure provides methods for preparing nucleic acid library molecules for use in any of the target enrichment methods described herein including library circularization workflows, as well as re-seeding workflows and batch sequencing.

[00797] The present disclosure provides methods for preparing nucleic acid library molecules from input nucleic acids (e.g., DNA and/or RNA), and conducting any of the target enrichment methods described herein, including target enrichment of DNA libraries, RNA libraries, or a mixture of DNA and RNA libraries.

[00798] In some embodiments, the insert region of a nucleic acid library molecule comprises a sequence of interest extracted from any source. The insert region can be prepared using recombinant nucleic acid technology including but not limited to any combination of vector cloning, transgenic host cell preparation, host cell culturing and/or PCR amplification. [00799] In some embodiments, the insert region can be isolated from any source including a biological sample (e.g., fresh or live sample) such as a single cell, a plurality of cells or tissue. In some embodiments, the insert region can be isolated from healthy or diseases cells or tissues. In some embodiments, the insert region can be isolated from an archived sample such as a fresh frozen paraffin embedded (FFPE) sample, or from needle biopsies, tissue resection biopsies, circulating tumor cells, cell free circulating DNA (e.g., from tumor cells or a fetus). In some embodiments, the insert region can be isolated by lysing cells or tissues to release their DNA and RNA, and the desired nucleic acid can be separated from non-desired macromolecules such as proteins.

[00800] In some embodiments, the insert region can be isolated in any form, including chromosomal DNA, genomic DNA (e.g., whole genomic), organellar DNA (e.g., mitochondrial, chloroplast or ribosomal), recombinant DNA molecules, cloned DNA or amplified DNA. In some embodiments, the insert region can be methylated or nonmethylated DNA.

[00801] In some embodiments, the insert region can be isolated from any organism including viruses, fungi, prokaryotes or eukaryotes. In some embodiments, the insert region can be isolated from any organism including human, simian, ape, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insect or bacteria. In some embodiments, the insert region can be isolated from organisms borne in air, water, soil or food.

[00802] In some embodiments, the insert region can be isolated from any biological fluid, including blood, urine, serum, lymph, tumor, saliva, anal secretions, vaginal secretions, amniotic samples, perspiration, semen, environmental samples or culture samples. In some embodiments, the insert region can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs.

[00803] In some embodiments, the insert region can be obtained from any type of plant cells including any plant part, including fruit, tuber, leaf, stem, root, tuber, seed, branch, pubescent, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen or leaf sheath.

[00804] In some embodiments, the insert region can be prepared using recombinant nucleic acid technology including but not limited to any combination of vector cloning, transgenic host cell preparation, host cell culturing and/or PCR amplification.

[00805] In some embodiments, the insert region can be prepared from cDNA which is prepared from any type RNA, including RNA that encodes a polypeptide and non-coding RNA. In some embodiments, the RNA comprises mRNA, poly A RNA, or RNA lacking a polyA tail. In some embodiments, the RNA comprises long non-coding RNA, tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA) or antisense RNA. In some embodiments, the RNA comprises pre-sliced RNA, RNA splice variants, and mature-spliced RNA comprising only exons. In some embodiments, the RNA comprises an exon sequence, an intron sequence, an exon-intron junction sequence, or a mixture of exon sequence and intron sequences. In some embodiments, the RNA comprises at least one region of RNA including a 5’ untranslated region, a 5’ cap region, a region having a start codon, a coding region, a region having a stop codon, and/or a 5’ untranslated region. In some embodiments, the 5' cap site of an RNA comprises an N7-methylated guanosine residue joined to the 5'- most residue of the RNA via a 5 '-5' triphosphate linkage. In some embodiments, the 5' cap region of a RNA includes the 5' cap structure and the first 50 nucleotides adjacent to the 5’ cap site.

[00806] In some embodiments, the insert region can be prepared using chemical synthesis procedures using native nucleotides with or without nucleotide analogs or modified nucleotide linkages that confer certain properties, including resistance to enzymatic digestion, or increased thermal stability. Examples of nucleotide analogs and modified nucleotide linkages that inhibit nuclease digestion include phosphorothioate, 2’-O-methyl RNA, inverted dT, and 2’ 3’ dideoxy-dT. In some embodiments, the insert region can include locked nucleic acids (LNA) having increased thermal stability.

[00807] In some embodiments, the insert region can be in fragmented or un-fragmented form, and can be used to prepare linear nucleic acid library molecules. Fragmented forms of the insert region can be obtained by mechanical force, enzymatic or chemical fragmentation methods. The fragmented insert regions can be generated using procedures that yield a population of fragments having overlapping sequences or non-overlapping sequences. [00808] Mechanical fragmentation typically generates randomly fragmented nucleic acid molecules. Mechanical fragmentation methods include mechanical shearing such as fluid shear, constant shear and pulsatile shear. Mechanical fragmentation methods also include mechanical stress including sonication, nebulization and acoustic cavitation. In some embodiments focused acoustic energy can be used to randomly fragment nucleic acid molecules. A commercially-available apparatus (e.g., Covaris) can be used to fragment nucleic acid molecules using focused acoustic energy.

[00809] Enzymatic fragmentation procedures can be conducted under conditions suitable to generate randomly or non-randomly fragmented nucleic acid molecules. For example, restriction endonuclease enzyme digestion can be conducted to completion to generate non- randomly fragmented nucleic acid molecule. Alternatively, partial or incomplete restriction enzyme digestion can be conducted to generate randomly-fragmented nucleic acid molecules. Enzymatic fragmentation using restriction endonuclease enzymes includes any one or any combination of two or more restriction enzymes selected from a group consisting of type I, type II, type Ils, type IIB, type III, or type IV restriction enzymes. Enzymatic fragmentation includes digestion of the nucleic acid with a rare-cutting restriction enzyme, comprising Not I, Asc I, Bae I, AspC I, Pac I, Fse I, Sap I, Sfi I or Psr I. Enzymatic fragmentation include use of any combination of a nicking restriction endonuclease, endonuclease and/or exonuclease. Enzymatic fragmentation can be achieved by conducting a nick translation reaction.

[00810] In some embodiments, enzymatic fragmentation can be achieved by reacting nucleic acids with an enzyme mixture, for example an enzyme that generates single-stranded nicks and another enzyme that catalyzes double-stranded cleavage. An exemplary enzyme mixture is FRAGMENTASE (e.g., from New England Biolabs).

[00811] Fragments of the insert region can be generated with PCR using sequence-specific primers that hybridize to target regions in genomic DNA samples to generate insert regions having known fragment lengths and sequences.

[00812] Targeted genome fragmentation methods using CRISPR/Cas9 can be used to generate fragmented insert regions.

[00813] Fragments of the insert portion can also be generated using a transposase-based tagmentation method using NEXTERA (from Epicentre®).

[00814] The insert region can be single-stranded or double-stranded. The ends of the double-stranded insert region can be blunt-ended, or have a 5’ overhang or a 3’ overhang end, or any combination thereof. One or both ends of the insert region can be subjected to an enzymatic tailing reaction to generate a non-template poly-A tail by employing a terminal transferase reaction. The ends of the insert region can be compatible for joining to at least one adaptor sequence (e.g., universal adaptor sequence or batch-specific adaptor sequence).

[00815] The insert region can be any length, for example the insert region can be about 50- 250, or about 250-500, or about 500-750, or about 750-1000, or about 1000-1500, or about 1500-2000 bases or base pairs in length, or any range therebetween. In some embodiments, the insert region can be 2000-5000 bases or base pairs in length.

[00816] The fragments containing the insert region can be subjected to a size selection process, or the fragments are not size selected. For example, the fragments can be size selected by gel electrophoresis and gel slice extraction. The fragments can be size selected using a solid phase adherence/immobilization method which typically employs micro paramagnetic beads coated with a chemical functional group that interacts with nucleic acids under certain ionic strength conditions with or without polyethylene glycol or polyalkylene glycol. Commercially-available solid phase adherence beads include SPRI (Solid Phase Reversible Immobilization) beads from Beckman Coulter® (AMPUR® XP paramagnetic beads, catalog No. B23318), MAGNA PURE magnetic glass particles (Roche Diagnostics, catalog No. 03003990001), MAGNASIL paramagnetic beads from Promega (catalog No. MD1360), MAGTRATION paramagnetic beads and system from Precision System Science (catalog Nos. Al 120 and A1060), MAG-BIND from Omega Bio-Tek (catalog No. M1378- 01), MAGPREP silica from Millipore® (catalog No. 101193), SNARE DNA purification systems from Bangs Laboratories (catalog Nos. BP691, BP692 and BP693), and CHEMAGEN M-PVA beads from Perkin Elmer® (catalog No. CMG-200).

[00817] In some embodiments, the fragmented nucleic acids can be subjected to enzymatic reactions for end-repair and/or A-tailing. The fragmented nucleic acids can be contacted with a plurality of enzymes under a condition suitable to generate nucleic acid fragments having blunt-ended 5’ phosphorylated ends. In some embodiments, the plurality of enzymes generates blunt-ended fragment having a non-template A-tail at their 3’ ends. The plurality of enzymes comprise two or more enzymes that can catalyze nucleic acid end-repair, phosphorylation and/or A-tailing. The end-repair enzymes include a DNA polymerase (e.g., T4 DNA polymerase) and Klenow fragment. The 5’ end phosphorylation enzyme comprises T4 polynucleotide kinase. The A-tailing enzyme includes a Taq polymerase (e.g., non-proofreading polymerase) and dATP. In some embodiments, the fragmenting, end-repair, phosphorylation and A-tailing can be conducted in a one-pot reaction using a mixture of enzymes.

Appending Adaptors to Fragmented or Unfragmented Nucleic Acids

[00818] In some embodiments, individual fragmented (or unfragmented) nucleic acids can be covalently joined to at least one adaptor sequence for library preparation. In general, a nucleic acid fragment is covalently joined at both ends to one or more adaptors to generate a linear library molecule having the arrangement left adaptor-insert-right adaptor. In some embodiments, at least one fragment in the population of fragmented nucleic acids comprises a sequence-of-interest. Individual library molecules in the population of library molecules can have an insert region that is the same or different as other library molecules in the population. In some embodiments, about 1-10 ng, or about 10-50 ng, or about 50-100 ng, or any range therebetween, of input fragmented nucleic acids can be appended to one or more adaptors to generate a linear library.

[00819] Individual nucleic acid fragments can be appended on one or both ends to at least one adaptor sequence to form a recombinant nucleic acid linear library molecule having the general arrangement left adaptor-insert-right adaptor.

[00820] In some embodiments, the nucleic acid fragments can be appended with any one or any combination of two or more adaptors, and arranged in any order, where the adaptors comprise: a universal adaptor sequence for binding a second universal surface primer; a left sample index sequence; a universal adaptor sequence for binding a forward sequencing primer; an insert region comprising a polynucleotide sequence of interest; a universal adaptor sequence for binding a reverse sequencing primer; an optional unique identification sequence (e.g., UMI); a right sample index sequence; and a universal adaptor sequence for binding a first universal surface primer and/or, an adaptor sequence for binding a compaction oligonucleotide.

[00821] In some embodiments, any of the adaptors comprises universal adaptor sequences or batch-specific adaptor sequences, such as for example a sequence for binding a batchspecific sequencing primer.

[00822] Exemplary linear library molecules are shown in FIGS. 14, 17 and 20-35. The skilled artisan appreciates that many other embodiments of linear library molecules comprising adaptor sequences with other arrangements are possible.

[00823] The adaptors can be prepared using chemical synthesis procedures using native nucleotides with or without nucleotide analogs or modified nucleotide linkages that confer certain properties, including resistance to enzymatic digestion, or increased thermal stability. Examples of nucleotide analogs and modified nucleotide linkages that inhibit nuclease digestion include phosphorothioate, 2’-O-methyl RNA, inverted dT, and 2’ 3’ dideoxy-dT. Insert regions that include locked nucleic acids (LNA) have increased thermal stability.

Universal Adaptor Sequences of a Library Molecule

[00824] The present disclosure provides linear and covalently closed circular library molecules comprising an insert region and at least one universal adaptor sequence. The linear and covalently closed circular library molecules, or adaptor sequences disclosed herein, can be used for any of the compositions and kits and/or any of the methods described herein. [00825] In some embodiments, the universal adaptor sequence for binding a second universal surface primer comprises (120) as shown in FIGS. 14-15, (720) as shown in FIGS. 17-18 and 20, or (1500) as shown in FIGS. 21, 22, 26, 27, 29 and 30.

[00826] In some embodiments, the left sample index sequence comprises (160) as shown in FIGS. 14-15, (760) as shown in FIGS. 17-18 and 20, or (1700) as shown in FIGS. 21-29, 31 and 33.

[00827] In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises (140) as shown in FIGS. 14-15, (740) as shown in FIGS. 17-18 and 20, or (1300) as shown in FIGS. 21-35.

[00828] In some embodiments, the universal adaptor sequence for binding a forward sequencing primer comprises any one or any combination of two or more of sequences (1300), (1400) and/or (1500) as shown in FIGS. 21-30.

[00829] In some embodiments, the universal adaptor sequence for binding the reverse sequencing primer comprises (150) as shown in FIGS. 14-15, (750) as shown in FIGS. 17-18 and 20, or (1400) as shown in FIGS. 21-29, 31, 32 and 34.

[00830] In some embodiments, the universal adaptor sequence for binding a reverse sequencing primer comprises any one or any combination of two or more of sequences (1400), (1500) and/or (1600) as shown in FIGS. 24 and 30.

[00831] In some embodiments, the optional unique identification sequence (e.g., unique molecule index, UMI) comprises (180) as shown in FIGS. 14-15, (780) as shown in FIGS. 17-18 and 20, or (1900) as shown in FIGS. 21-33.

[00832] In some embodiments, the right sample index sequence comprises (170) as shown in FIGS. 14-15, (770) as shown in FIGS. 17-18 and 20, or (1800) as shown in FIGS. 21-28, 30 and 32.

[00833] In some embodiments, the universal adaptor sequence for binding a first universal surface primer comprises (130) as shown in FIGS. 14-15, (730) as shown in FIGS. 17-18 and 20, or (1600) as shown in FIGS. 21-25, 30, 33 and 35.

Appending Adaptors via Ligation and/or Primer Extension

[00834] The insert region can be joined at one or both ends to at least one adaptor sequence using a ligase enzyme and/or primer extension reaction to generate a linear library molecule. Covalent linkage between an insert region and the adaptor(s) can be achieved with a DNA or RNA ligase. Exemplary DNA ligases that can ligate double-stranded DNA molecules include T4 DNA ligase and T7 DNA ligase. An adaptor sequence can be appended to an insert sequence by PCR using a tailed primer having 5’ region carrying an adaptor sequence and a 3’ region that is complementary to a portion of the insert sequence. An adaptor sequence can be appended to an insert sequence which is flanked one side or both sides with first and second adaptor sequences by PCR using a tailed primer having 5’ region carrying a third adaptor sequence and a 3’ region that is complementary to a portion of the first or second adaptor sequence.

[00835] In some embodiments, a linear library molecule can be generated by employing a ligation reaction and an optional primer extension reaction. The library molecule can be generated by joining the first end of a double-stranded insert region to a first double-stranded adaptor, and joining the second end of the double-stranded insert region to a second doublestranded adaptor. The first and second double-stranded adaptors each comprise two nucleic acid strands that are fully complementary along their lengths.

[00836] In some embodiments, individual double-stranded insert regions can be joined to a first and a second double-stranded adaptor using a DNA ligase enzyme to generate a doublestranded recombinant molecule. In some embodiments the first and second double-stranded adaptors carry the same adaptor sequences. In some embodiments the first and second double-stranded adaptors carry different adaptor sequences.

[00837] In some embodiments, the library molecule can be generated by joining the first end of a double-stranded insert region to a first double-stranded adaptor having a having a binding sequence for a forward sequencing primer, and joining the second end of the doublestranded insert region to a second double-stranded adaptor having a binding sequence for a reverse sequencing primer, wherein the joining is conducted using a DNA ligase enzyme to generate a double-stranded recombinant molecule. In some embodiments, the first doublestranded adaptor further comprises a left sample index sequence and/or a binding sequence for a pinning primer. In some embodiments, the second double-stranded adaptor further comprises a right sample index sequence and/or a binding sequence for a capture primer.

[00838] In some embodiments, the ligating end of the first and/or the second doublestranded adaptors comprise a blunt end, or an overhang end (e.g., 5’ or 3’ overhang end). [00839] In some embodiments, a linear library molecule can be generated by employing a ligation reaction and primer extension reaction. The library molecule can be generated by joining the first end of a double-stranded insert region to a first double-stranded Y-shaped adaptor (e.g., a first forked adaptor), and joining the second end of a double-stranded insert region to a second double-stranded Y-shaped adaptor (e.g., a second forked adaptor). The first and second Y-shaped adaptors each comprise two nucleic acid strands, where a portion of the two strands are fully complementary to each other and are annealed together and another portion of the two strands are not complementary to each other and are mismatched. In some embodiments, the ligating end of the first and second Y-shaped adaptors comprise an annealed portion that forms a blunt end or an overhang end (e.g., 5’ or 3’ overhang end). [00840] In some embodiments the first and second Y-shaped adaptors carry the same adaptor sequences. In some embodiments the first and second Y-shaped adaptors carry different adaptor sequences.

[00841] In some embodiments, the first strand of the annealed portion and/or the mismatched portion of the Y-shaped adaptor can include at least a portion of an adaptor sequence having a binding sequence for a forward sequencing primer (or a complementary sequence thereof). In some embodiments, the first strand of the annealed portion and/or the mismatched portion of the Y-shaped adaptor can further include a left sample index sequence. In some embodiments, the first strand of the annealed portion and/or the mismatched portion of the Y-shaped adaptor can further include an adaptor sequence having a binding sequence for a pinning primer.

[00842] In some embodiments, the second strand of the annealed portion and/or the mismatched portion of the Y-shaped adaptor can include at least a portion of an adaptor sequence having a binding sequence for a reverse sequencing primer (or a complementary sequence thereof). In some embodiments, the second strand of the annealed portion and/or the mismatched portion of the Y-shaped adaptor can further include a right sample index sequence. In some embodiments, the second strand of the annealed portion and/or the mismatched portion of the Y-shaped adaptor can further include an adaptor sequence having a binding sequence for a capture primer.

[00843] The double-stranded insert region can be joined to the first and second doublestranded Y-shaped adaptors using a DNA ligase enzyme to generate a double-stranded recombinant molecule.

[00844] In some embodiments, the double-stranded recombinant molecules which are generated by ligating the insert region to double-stranded adaptors or Y-shaped adaptors can be subjected to a denaturing condition to generate single-stranded recombinant molecules, and then a primer extension reaction. At least one additional adaptor sequence can be appended to the recombinant molecules by conducting a primer extension reaction using tailed primers (e.g., tailed PCR primers), by contacting/hybri dizing the single-stranded recombinant molecules with a plurality of first tailed primers and conducting at least one primer extension reaction to generate a first double-stranded tailed extension product. [00845] In some embodiments, an additional adaptor sequence can be appended to the first double-stranded tailed extension product by conducting a primer extension reaction using tailed primers (e.g., tailed PCR primers), by contacting/hybri dizing the first double-stranded tailed extension product with a plurality of second tailed primers and conducting at least one primer extension reaction to generate a second double-stranded tailed extension product.

[00846] In some embodiments, individual first tailed primers comprise a 5’ region carrying an adaptor sequence having a binding sequence for a capture surface primer, and a 3’ region that is complementary to at least a portion of the adaptor sequence having a binding sequence for a reverse sequencing primer of the single-stranded recombinant molecules.

[00847] In some embodiments, individual first tailed primers comprise a 5’ region carrying an adaptor sequence having a binding sequence for a capture surface primer, an internal region comprising a right sample index sequence, and a 3’ region that is complementary to at least a portion of the adaptor sequence having a binding sequence for a reverse sequencing primer of the single-stranded recombinant molecules.

[00848] In some embodiments, individual second tailed primers comprise a 5’ region carrying an adaptor sequence having a binding sequence for a pinning surface primer, and a 3’ region that is complementary to at least a portion of the adaptor sequence having a binding sequence for a forward sequencing primer of the first double-stranded tailed extension product.

[00849] In some embodiments, individual second tailed primers comprise a 5’ region carrying an adaptor sequence having a binding sequence for a pinning surface primer, an internal region comprising a left sample index sequence, and a 3’ region that is complementary to at least a portion of the adaptor sequence having a binding sequence for a forward sequencing primer of the first double-stranded tailed extension product.

[00850] In some embodiments, the first tailed PCR primers can be used to conduct a first primer extension reaction and the second tailed PCR primers can be used conduct a second primer extension to generate library molecules comprising an insert region appended on both sides with at least one adaptor. In some embodiments, the first and second tailed PCR primers can be used to conduct multiple PCR cycles (e.g., about 5-20 PCR cycles) to generate library molecules comprising an insert region appended on both sides with at least one adaptor.

[00851] In some embodiments, the double-stranded recombinant molecules which are generated by ligating the insert region to double-stranded adaptors or Y-shaped adaptors are not subjected to PCR to append additional adaptor sequences. Improving Sequencing Scores by Removing Deaminated Nucleotide Bases

[00852] In massively parallel pairwise sequencing workflows using detectably labeled multivalent molecules, high quality sequencing errors have been observed, which include bases that were assigned a high quality sequencing score but mismatched the reference base. Without wishing to be bound by theory, it was believed that these high quality sequencing errors arise from processes upstream or downstream of the sequencing portion of the workflow. An example of upstream processes includes any one or more step(s) of library preparation and loading the prepared library onto a sequencing flowcell for immobilization and sequencing, z.e., steps prior to sequencing.

[00853] Library preparation workflows include numerous steps, including, but not limited to: fragmentation of input nucleic acids; end repair of double-stranded fragmented nucleic acids; non-template tailing of double-stranded fragmented nucleic acids; appending adaptors via ligation or PCR; PCR amplification; denaturation to generate single-stranded library molecules; circularization of single-stranded library molecules; and/or immobilization of linear library molecules or circularized library molecules. In some embodiments, the library preparation workflows can be PCR-free. In some embodiments, the library preparation workflows include removal of deaminated nucleotide bases and gap-generation of nucleic acid molecules carrying at least one deaminated nucleotide base. In some embodiments, the methods for loading a prepared library onto a sequencing flowcell include removal of deaminated nucleotide bases and gap-generation of nucleic acid molecules carrying at least one deaminated nucleotide base.

[00854] High quality base call errors have been detected for T bases when sequencing the first strand (e.g., R1 reads), and high quality base call errors for A bases when sequencing corresponding positions on the complementary second strand (e.g., R2 reads). Many of the high quality T base call errors on the first strand sequence align with C bases in a known reference sequence. Without wishing to be bound by theory, it is believed that some of the bases in the library molecules are deaminated, which leads to the base substitutions including C:G to T:A transitions.

[00855] Deamination is generally the removal of an amino group from a molecule. With respect to nucleotide bases, cytosine (C) can be deaminated to generate uracil (U) where uracil can base pair with adenine (A), guanine (G) can be deaminated to generate xanthine where xanthine can base pair with cytosine (C), and adenine (A) can be deaminate to generate hypoxanthine where hypoxanthine can base pair with cytosine (C). [00856] Workflows for preparing nucleic acid library molecules involve numerous steps that include conditions that can cause deamination of nucleotide bases. For example, and without limitation, deamination can be caused by the presence of deaminase enzymes at any stage in the library prep workflow. As another non-limiting example, any of the library preparation buffers having a low pH can cause base deamination. In another non-limiting example, high temperatures employed for PCR can cause base deamination. In another nonlimiting example, mechanical shearing forces that are used to fragment input nucleic acids can generate damaging free radicals which leads to deamination. Exemplary mechanical forces can include sonication force, acoustic force, nebulizing force, shearing force and cavitation force. In another example, certain chemicals such as bisulfites can cause deamination. The skilled artisan will recognize that nucleotide base deamination can be generated by many other conditions, as well as any combination of the conditions recited above.

[00857] In some embodiments, library preparation workflows can generate a plurality of library molecules comprising a mixture of first and second sub-populations of library molecules. In some embodiments, individual library molecules in the first sub-population carry at least one deaminated nucleotide base. In some embodiments, individual library molecules in the second sub-population lack a deaminated nucleotide base.

[00858] The present disclosure provides compositions and methods for improving sequencing quality scores comprising removing deaminated bases in any nucleic acid molecules throughout the workflow, including, for example, fragmented input nucleic acid molecules. In some embodiments, fragmented input nucleic acid molecules carrying deaminated bases can be removed while retaining fragmented input nucleic acid molecules that lack deaminated bases. In some embodiments, the compositions and methods for removing deaminated bases in a plurality of fragmented input nucleic acid molecules employ at least one enzyme comprising glycosylase activity and/or employs at least one enzyme comprising lyase activity.

[00859] The present disclosure also provides compositions and methods for improving sequencing quality scores comprising removing deaminated bases in a plurality of nucleic acid library molecules. In some embodiments, library molecules carrying deaminated bases can be removed while retaining library molecules that lack deaminated bases. In some embodiments, the compositions and methods for removing deaminated bases in library molecules employ at least one enzyme comprising glycosylase activity and/or employ at least one enzyme comprising lyase activity. In some embodiments, the compositions and methods for removing deaminated bases in library molecules can be applied to any type of nucleic acid library molecules, including, for example, linear library molecules, open circle library molecules and covalently closed circular library molecules. In some embodiments, the compositions and methods for removing deaminated bases in any of the library molecules can be applied on a support (e.g., capture support) prior to or during a rolling circle amplification reaction.

[00860] High quality base call errors near the terminal ends of the insert regions of library molecules have also been detected. For example, G to T errors are detected in the early sequencing cycles of the read 2 strand (R2), or C to A errors are detected on the reverse strand. Without wishing to be bound by theory, it is believed that these errors occur during end repair steps of a library preparation workflow, for example and without limitation, prior to appending adaptors to the end repaired nucleic acid molecules.

[00861] End repair workflows are typically employed after fragmenting input doublestranded nucleic acids via mechanical shearing, which can generate a mixture of fragmented nucleic acids having 5’ overhang ends, 3’ overhang ends and/or blunt ends. End repair workflows can generate double-stranded fragments having blunt ends at both ends of the fragments. End repair workflows can be performed by conducting enzymatic exonuclease degradation of 5’ or 3’ overhang ends and/or by conducting polymerase-catalyzed extension of 3’ under-hang ends. The exonuclease degradation reactions typically do not generate base errors at the ends of the nucleic acid fragments. By contrast, the polymerase-catalyzed extension reaction can be a relatively low fidelity reaction which can introduce base errors, including G to T base errors, at the ends of the nucleic acid fragments. In some embodiments, the end repair workflows comprises a polymerase-catalyzed extension reaction.

[00862] The present disclosure also provides compositions and methods for improving sequencing quality scores comprising reducing base errors including G to T base errors on one strand by conducting an end repair workflow on a plurality of fragmented input nucleic acids, wherein the end repair workflow lacks a polymerase-catalyzed extension of 3’ underhang ends where the opposite strand comprises a 5’ over-hang end. For example, and without limitation, the end repair workflow comprises employing a nuclease enzyme to degrade the 5’ over-hang ends thereby generating a blunt end. In some embodiments, the end repair workflow can also comprise employing a nuclease enzyme to degrade the 3’ over-hang ends thereby generating a blunt end.

[00863] The present disclosure further provides compositions and methods for improving sequencing quality scores comprising reducing base errors including G to T base errors on one strand by conducting dark sequencing during the initial 2-30 e.g., 5-30, 2-20, 10-30, 10- 20, 15-30 bases or any range therebetween) bases of the second read (read 2, or R2) strand. In some embodiments, the dark sequencing can be conducted on 2-30 consecutive bases in the initial portion of the read 2 strand (R2). As used herein, “dark sequencing” refers to methods that comprise conducting recursive sequencing cycles using non-labeled nucleotide reagents that can extend the sequencing primer, but nucleotide incorporation is not detected. In some embodiments, the non-labeled nucleotide reagents comprise non-labeled canonical nucleotides and/or non-labeled chain terminator nucleotides.

[00864] The present disclosure also provides compositions and methods for improving sequencing quality scores comprising any one or any combination of two or more of: (i) removing deaminated bases in any nucleic acid molecule throughout the library preparation workflow; (ii) conducting library preparation workflows that include end repair methods that reduce introduction of erroneous nucleotide bases; (iii) deactivating ligase enzyme activity with an alkaline reagent (e.g., NaOH or KOH) instead of heat; (iv) deactivating exonuclease enzyme activity with an alkaline reagent (e.g., NaOH or KOH) instead of heat; (v) denaturing linear library molecules using an alkaline reagent (e.g., NaOH or KOH) instead of heat; (vi) removing deaminated nucleotide bases in any library molecule (e.g., linear library molecules, open circle library molecules and covalently closed circular library molecules) that is loaded onto a capture support; and/or (vii) dark sequencing. In some embodiments, any of these compositions and methods can reduce C:G to T:A base transitions which can generate higher quality base calls during downstream sequencing workflows. In some embodiments, a sequencing quality score of Q30 can increase to Q40, Q45, Q50, Q55, Q60 or higher sequencing quality scores.

[00865] In some embodiments, the base calling from sequencing data is assessed for accuracy and quality. “Q-score” is a measure of data quality. The Q-score can be defined as a Phred quality score. The Q-score is based on a logarithmic scale. It is generally defined as Q = -10 log(P) where P is the error probability. For example, Q10 represents 10% error, Q20 represents 1% error, Q30 represents 0.1% error and Q40 represents 0.01% error. In another example, Q10 is one error in 10, Q20 is one error in 100, Q30 is one error in 1000, Q40 is one error in 10,000, Q50 is one error in 100,000, and Q60 is one error in 1,000,000.

Preparing Nucleic Acid Molecules with Reduced Deaminated Nucleotide Bases [00866] In some aspects, the present disclosure provides methods for improving sequencing quality scores comprising conducting a library preparation workflow which includes at least one step for reducing/removing deaminated nucleotide bases in the fragmented input nucleic acids and/or in the library molecules and/or in the circularized library molecules and/or in the immobilized circularized library molecules.

Removing Deaminated Bases from Fragmented Input Nucleic Acid Molecules

[00867] In some embodiments, methods for preparing fragmented input nucleic acid molecules having reduced deaminated nucleotide bases comprise step (a): fragmenting input nucleic acids from any source to generate a plurality of fragmented input nucleic acids comprising a mixture of first and second sub-populations of fragmented input nucleic acid molecules. In some embodiments, individual fragmented input nucleic acid molecules in the first sub-population carry at least one deaminated nucleotide base. In some embodiments, individual fragmented input nucleic acid molecules in the second sub-population lack a deaminated nucleotide base. The input nucleic acids can be fragmented using any mechanical force, enzymatic (e.g., restriction endonuclease or FRAGMENTASE) or chemical fragmentation methods as described herein.

[00868] In some embodiments, the methods comprises step (b): contacting the plurality of fragmented input nucleic acid molecules, including the first and second sub-populations of fragmented input nucleic acid molecules, with a reagent that removes deaminated nucleotide bases and generates gaps, thereby converting the at least one deaminated nucleotide base in the first sub-population into at least one abasic site, and thereby generating a plurality of reagent-treated fragmented input nucleic acid molecules. In some embodiments, the reagent that removes deaminated bases and generates gaps comprises at least one enzyme having glycosylase activity and at least one enzyme having lyase activity.

[00869] In some embodiments, the methods comprise optional step (c): appending at least one adaptor to one or both ends of the plurality of reagent-treated fragmented input nucleic acid molecules, thereby generating a plurality of library molecules (e.g., a plurality of linear library molecules). In some embodiments, individual linear library molecules comprise adaptors appended to both ends of a fragmented input nucleic acid, wherein the fragmented input nucleic acid forms the insert region of a linear library molecule. In some embodiments, the plurality of linear library molecules comprises double-stranded linear molecules. In some embodiments, the double-stranded linear library molecules can be denatured using an alkaline reagent (e.g., NaOH or KOH) instead of heat to generate a plurality of single- stranded linear library molecules comprising top and bottom strands. Removing Deaminated Bases from Library Molecules

[00870] In some embodiments, methods for preparing a plurality of library molecules having reduced deaminated nucleotide bases comprise step (a): appending at least one adaptor to one or both ends of fragmented input nucleic acid molecules, thereby generating a plurality of library molecules (e.g., a plurality of linear library molecules) comprising a mixture of first and second sub-populations of library molecules. In some embodiments, individual library molecules in the first sub-population carry at least one deaminated nucleotide base. In some embodiments, individual library molecules in the second subpopulation lack a deaminated nucleotide base. In some embodiments, individual linear library molecules comprise adaptors appended to both ends of a fragmented input nucleic acid, wherein the fragmented input nucleic acid forms the insert region of a linear library molecule. In some embodiments, the insert region of an individual linear library molecule comprises a target or non-target sequence.

[00871] In some embodiments, the methods comprise optional step (b): contacting the plurality of linear library molecules, including the first and second sub-populations of linear library molecules, with a reagent that removes deaminated bases and generates gaps, thereby generating a plurality of reagent-treated linear library molecules, wherein individual linear library molecules comprise at least one abasic site.

[00872] In some embodiments, the methods comprise optional step (c): circularizing the plurality of linear library molecules, thereby generating a plurality of covalently closed circular library molecules having reduced deaminated nucleotide bases. Any of the methods described herein for circularizing linear library molecules can be used to generate a plurality of covalently closed circular library molecules, including for example, employing CIRCLIGASE™, employing telomerase TelN, circularization using single-stranded splint strands, double-stranded splint adaptors, single stranded top strand circularization oligonucleotides, or double-stranded top strand circularization oligonucleotides. Exemplary, non-limiting single-stranded splint strands and double-stranded splint adaptors, and methods related thereto, are described in WO 2023/168444, WO 2023/168443, WO 2024/011145, WO 2024/059550, and WO 2025/024465, the contents of each of which is incorporated by reference herein in their entireties.

[00873] In some embodiments, the methods comprise optional step (d): contacting the plurality of covalently closed circular library molecules with a plurality of target-specific baits/probes, wherein the plurality of covalently closed circular library molecules comprises a mixture of target and non-target covalently closed circular library molecules, wherein the contacting is conducted in-solution under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual covalently closed circular library molecules to corresponding target-specific baits/probes thereby generating a plurality of circle bait complexes (also referred to herein as “closed circle bait library complexes”) that are enriched for polynucleotides having target sequences. In some embodiments, individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of the target sequence of the covalently closed circular library molecule, an affinity moiety at the 5’ end of the targetspecific oligonucleotide, and the target-specific oligonucleotide comprises an extendible 3’ end. In some embodiments, the oligonucleotides of the target-specific baits/probes can hybridize to a known target sequence (e.g., exon or intron), can hybridize to a known genetic variant sequence and/or can hybridize to a known splice junction. In some embodiments, individual target-specific baits/probes comprise DNA, RNA or chimeric DNA and RNA. In some embodiments, the affinity moieties of individual target-specific baits/probes can bind to an embedded receptor moiety of a capture support. In some embodiments, the affinity moiety of individual target-specific baits/probes comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the affinity moiety of individual target-specific baits/probes can be located at the 5’ end or at an internal position. In some embodiments, the plurality of nontarget covalently closed circular library molecules do not selectively hybridize to the targetspecific baits/probes.

[00874] In some embodiments, the methods comprise optional step (e): distributing the plurality of covalently closed circular library molecules onto a capture support to generate a plurality of covalently closed circular library molecules immobilized to the capture support. In some embodiments, the capture support comprises at least one layer of a hydrophilic polymer coating and a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof. In some embodiments, the receptor moieties of the capture support can bind the affinity moieties of the target-specific baits/probes. In some embodiments, the plurality of immobilized covalently closed circular library molecules can optionally be contacted with a rolling circle amplification reagent for conducting rolling circle amplification reaction to generate a plurality of concatemer template molecules immobilized to the capture support. Generating Covalently Closed Circular Library Molecules using Single-Stranded Splint

Strands

[00875] The present disclosure provides methods for generating covalently closed circular library molecules comprising step (a): providing a plurality of linear single stranded library molecules (700) wherein individual library molecules comprise the following components arranged in any order: (i) a universal adaptor sequence for binding a second universal surface primer (720), such as a pinning primer binding site sequence ; (ii) a unique molecular identification sequence (e.g., UMI) (780); (iii) a left sample index sequence (760); (iv) a universal adaptor sequence for binding a forward sequencing primer (740); (v) a sequence of interest (e.g., insert sequence, 710); (vi) a universal adaptor sequence for binding a reverse sequencing primer (750); (vii) a right sample index sequence (770); (viii) a universal adaptor sequence for binding a first universal surface primer (730), such as a capture primer binding site sequence ; and/or (ix) a compaction oligonucleotide binding site (e.g., FIGS. 17-18). In some embodiments, the unique molecular identification sequence (780) (e.g., a unique molecular tag) can be used to uniquely identify individual nucleic acid library molecules to which the unique identification sequence is appended.

[00876] In some embodiments, the sequences of interest can be about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or up to 2000 bases in length, or any range therebetween.

[00877] In some embodiments, the method comprises step (b): providing a plurality of single-stranded splint molecules (800) wherein individual single-stranded splint molecules (800) comprises regions arranged in any order (i) a first region (810) having a sequence that hybridizes with the universal adaptor sequence for binding a second universal surface primer (720) of the single stranded library molecule, and (ii) a second region (820) having a sequence that hybridizes with the universal adaptor sequence for binding a first universal surface primer (730) (e.g., the capture primer binding site sequence) of the single stranded library molecule. An exemplary single-stranded splint strand (800) is shown in FIG. 17-18. [00878] In some embodiments, the method comprises step (c): contacting the plurality of linear single stranded library molecules (700) with the plurality of single-stranded splint molecules (800), wherein the contacting is conducted under a condition suitable to hybridize individual linear single stranded library molecules (700) with individual single-stranded splint molecules (800) thereby circularizing the library molecule to generate a library-splint complex (900), wherein the first region (810) of the single-stranded splint strand is hybridized to the universal adaptor sequence for binding a second universal surface primer (720) of the single stranded library molecule, and wherein the second region (820) of the single-stranded splint strand is hybridized to the universal adaptor sequence for binding a first universal surface primer (730) (e.g., a capture primer binding site sequence) of the single stranded library molecule, wherein the library-splint complex (900) comprises a nick between the terminal 5’ and 3’ ends of the library molecule, and wherein the nick is enzymatically ligatable (e.g., see FIG. 18).

[00879] In some embodiments, the method comprises step (d): enzymatically ligating the nick in the plurality of library-splint complexes (900) thereby generating a plurality of a plurality of covalently closed circular library molecules (900) each hybridized to a singlestranded splint molecules (800) (e.g., FIG. 18). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. In some embodiments, step (d) further comprises conducting a phosphorylation reaction using T4 polynucleotide kinase enzyme. In some embodiments, step (d) further comprises removing the single-stranded splint by conducting an exonuclease reaction using at least one exonuclease enzyme including any combination of two or more of exonuclease I, thermolabile exonuclease I and/or T7 exonuclease.

[00880] In some embodiments, the plurality of covalently closed circular library molecules (1000) comprise covalently closed circular library molecules that can be used for any of the in-solution or on-support enrichment methods described herein.

[00881] In some embodiments, in any of the library-splint complexes (900) described herein, universal adaptor sequence for binding a second universal surface primer (720) in the linear library molecules comprise the sequence SEQ ID NO: 1 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a second universal surface primer (720) is a first left universal adaptor sequence.

[00882] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a second universal surface primer (720) in the linear library molecules comprise the sequence SEQ ID NO: 31 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a second universal surface primer (720) is a first left universal adaptor sequence.

[00883] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a forward sequencing primer (740) in the linear library molecules comprise the sequence SEQ ID NO: 32 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer (740) is a second left universal adaptor sequence. [00884] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a forward sequencing primer (740) in the linear library molecules comprise the sequence 5’- SEQ ID NO: 33 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer (740) is a second left universal adaptor sequence.

[00885] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a forward sequencing primer (740) in the linear library molecules comprise the sequence SEQ ID NO: 34 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a forward sequencing primer (740) is a second left universal adaptor sequence.

[00886] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a reverse sequencing primer (750) in the linear library molecules comprise the sequence SEQ ID NO: 35 or a complementary sequence thereof. In some embodiments, universal adaptor sequence for binding a reverse sequencing primer (750) is a second right universal adaptor sequence.

[00887] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a reverse sequencing primer (750) in the linear library molecules comprise the sequence SEQ ID NO: 36 or a complementary sequence thereof. In some embodiments, universal adaptor sequence for binding a reverse sequencing primer (750) is a second right universal adaptor sequence.

[00888] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a reverse sequencing primer (750) in the linear library molecules comprise the sequence SEQ ID NO: 37 or a complementary sequence thereof. In some embodiments, universal adaptor sequence for binding a reverse sequencing primer (750) is a second right universal adaptor sequence.

[00889] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a first universal surface primer (730) in the linear library molecules comprise the sequence SEQ ID NO: 2 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a first universal surface primer (730) comprises a first right universal adaptor sequence.

[00890] In some embodiments, in any of the library-splint complexes (900) described herein, the universal adaptor sequence for binding a first universal surface primer (730) in the linear library molecules comprise the sequence SEQ ID NO: 38 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a first universal surface primer (730) comprises a first right universal adaptor sequence.

[00891] In some embodiments, in any of the library-splint complexes (900) described herein, the first region (810) of the single-stranded splint strand includes a universal binding sequence for a universal adaptor sequence for binding a second universal surface primer (720) of a library molecule, where the first region (810) comprises the sequence SEQ ID NO: 9 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a second universal surface primer (720) is a first left universal adaptor sequence.

[00892] In some embodiments, in any of the library-splint complexes (900) described herein, the second region (820) of the single-stranded splint strand includes a universal adaptor sequence for binding a first universal surface primer (730) of a library molecule, where the second region (820) comprises the sequence SEQ ID NO: 10 or a complementary sequence thereof.

[00893] In some embodiments, in any of the library-splint complexes (900) described herein, the single-stranded splint strand comprises the sequence

SEQ ID NO: 39 or a complementary sequence thereof. For example see FIG. 20.

[00894] In some embodiments, in any of the library-splint complexes (900) described herein, the first region (810) of the single-stranded splint strand includes a universal binding sequence for a universal adaptor sequence for binding a second universal surface primer (720) of a library molecule, where the first region (810) comprises the sequence SEQ ID NO: 4 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence for binding a second universal surface primer (720) is a first left universal adaptor sequence.

[00895] In some embodiments, in any of the library-splint complexes (900) described herein, the second region (820) of the single-stranded splint strand includes a universal adaptor sequence for a first universal sequencing primer (730) of a library molecule, where the second region (820) comprises the sequence SEQ ID NO: 5 or a complementary sequence thereof.

[00896] In some embodiments, in any of the library-splint complexes (900) described herein, the single-stranded splint strand comprises the sequence SEQ ID NO: 40 or a complementary sequence thereof. Generating Covalently Closed Circular Library Molecules using Double-Stranded Splint Adaptors

[00897] The present disclosure provides methods for generating covalently closed circular library molecules comprising step (a): providing a plurality of linear single stranded library molecules (100) wherein individual library molecules comprise the following components arranged in any order: (i) a universal adaptor sequence for binding a pinning primer binding site sequence(120); (ii) a left sample index sequence (160); (iii) a universal adaptor sequence for binding a forward sequencing primer (140); (iv) a sequence of interest (e.g., insert region) (110); (v) a universal adaptor sequence for binding a reverse sequencing primer (150); (vi) a unique molecular identification sequence (e.g., UMI) (180); (vii) a right sample index sequence (170); (viii) a capture primer binding site sequence (130); and/or (ix) a compaction oligonucleotide binding site. In some embodiments, the unique molecular identification sequence (180) (e.g., a unique molecular tag) can be used to uniquely identify individual nucleic acid library molecules to which the unique identification sequence is appended. An exemplary single-stranded library molecule is shown in FIG. 14.

[00898] In some embodiments, the sequences of interest can be about 50-250 bases in length, or about 250-500 bases in length, or about 500-800 bases in length, or about 800-1200 bases in length, or up to 2000 bases in length.

[00899] In some embodiments, the pinning primer binding site sequence comprises a binding sequence for a first surface primer P5 or a complementary sequence thereof. In some embodiments, the capture primer binding site sequence (130) comprises a binding sequence for a second surface primer P7 or a complementary sequence thereof.

[00900] In some embodiments, a P5 capture primer comprises the sequence SEQ ID NO: 31.

[00901] In some embodiments, a P7 capture primer comprises the sequence: SEQ ID NO: 5.

[00902] In some embodiments, the methods comprise step (b): hybridizing the plurality of linear single stranded library molecules (100) with a plurality of double-stranded splint molecules (200), wherein individual double-stranded splint molecules (200) in the plurality comprise a first splint strand (300) (e.g., long strand) hybridized to a second splint strand (400) (e.g., short strand), wherein the double-stranded splint adaptor includes a doublestranded region and two flanking single-stranded regions, wherein the first splint strand comprises a first region (320), an internal region (310), and a second region (330), and wherein the internal region (310) of the first splint strand is hybridized to the second splint strand (400). An exemplary double-stranded splint strand is shown in FIG. 16.

[00903] In some embodiments, the second splint strand (400) can be divided into at least two sub-regions. In some embodiments, the sub-region can be arranged in a 5’ to 3’ order (i) a third sub-region comprising a sample index sequence or a unique molecular identification sequence, (ii) a second sub-region having a universal binding sequence for a fourth surface primer, and (iii) a first sub-region having a universal binding sequence for a third surface primer (e.g., FIG. 16).

[00904] In some embodiments, the internal region (310) of the first splint strand (300) can be divided into at least two sub-regions, wherein a fourth sub-region hybridizes to the first sub-region of the second splint strand (400), and a fifth sub-region hybridizes to the second sub-region of the second splint strand (400), and a sixth sub-region hybridizes to the third sub-region of the second splint strand (400).

[00905] In some embodiments, the universal binding sequences for the third surface primer do not bind the first surface primer (e.g., P5) or the second surface primer (e.g., P7). In some embodiments, the universal binding sequences for the fourth surface primer do not bind the first surface primer (e.g., P5) or the second surface primer (e.g., P7).

[00906] In some embodiments, the hybridizing of step (b) is conducted under a condition suitable for hybridizing the first region (320) of the first splint strand to the universal adaptor sequence for binding a second universal surface primer (120), such as a pinning primer, of the library molecule, and the condition is suitable for hybridizing the second region (330) of the first splint strand to the at least first right universal sequence (130) of the library molecule, thereby circularizing the plurality of library molecules to form a plurality of library-splint complexes (500). The library-splint complex (500) comprises a first nick between the 5’ end of the library molecule and the 3’ end of the second splint strand (e.g., see FIG. 15). The library-splint complex (500) also comprises a second nick between the 5’ end of the second splint strand and the 3’ end of the library molecule (e.g., see FIG. 15). In some embodiments, the first and second nicks are enzymatically ligatable.

[00907] In some embodiments, the first region (320) of the first splint strand can hybridize to a sense or anti-sense strand of a double-stranded nucleic acid library molecule. In the library-splint complex (500), the second region (330) of the first splint strand can hybridize to a sense or anti-sense strand of a double-stranded nucleic acid library molecule. The doublestranded nucleic acid library molecule can be denatured to generate the single-stranded sense and anti-sense library strands. [00908] In some embodiments, the second splint strand (400) does not hybridize to the sequence of interest (insert region, 110), and the internal region (310) of the first splint strand does not hybridize to the sequence of interest (insert region, 110).

[00909] In some embodiments, the first region (320) of the first splint strand does not hybridize to the sequence of interest (insert region, 110), and the second region (330) of the first splint strand does not hybridize to the sequence of interest (insert region, 110).

[00910] In some embodiments, the methods comprise step (c): contacting the plurality of library-splint complexes (500) from step (b) with a ligase, under a condition suitable to enzymatically ligate the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600) each hybridized to the first splint strand (300) (e.g., FIGS. 15A-15C). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. The nicks can be enzymatically ligated to generate covalently closed circular molecules (600) in which the second splint strand (400) is covalently joined at both ends to the linear single stranded library molecule (100), thereby introducing the new adaptor sequences into the covalently closed circularized library molecule (600), including for example any one or any combination of two or more: (i) the third sub-region comprising a sample index sequence or a unique molecular identification sequence, (ii) the second subregion having a universal binding sequence for a fourth surface primer, and/or (iii) the first sub-region having a universal binding sequence for a third surface primer (e.g., FIGS. 15A- 15C).

[00911] In some embodiments, the methods comprise optional step (d): enzymatically removing the plurality of first splint strands (300) from the plurality of covalently closed circular library molecules (600) by contacting the plurality of covalently closed circular library molecules (600) with at least one exonuclease enzyme to remove the plurality of first splint strands (300) and retaining the plurality of covalently closed circular library molecules (600). In some embodiments, the at least one exonuclease enzyme comprises any combination of two or more of exonuclease I, thermolabile exonuclease I and/or T7 exonuclease.

[00912] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a second universal surface primer (120) (e.g., a left universal adaptor sequence, for binding a surface pinning primer) which binds the first region (320) of the first splint strand, where the universal adaptor sequence (120) comprises the sequence SEQ ID NO: 31 or a complementary sequence thereof. [00913] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a second universal surface primer (120) (e.g., a left universal adaptor sequence, for binding a surface pinning primer) which binds the first region (320) of the first splint strand, where the universal adaptor sequence for binding the second universal surface primer (120) comprises the sequence SEQ ID NO: 1 or a complementary sequence thereof. [00914] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a forward sequencing primer (140) where the universal adaptor sequence comprises the sequence SEQ ID NO: 33 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence is a left universal adaptor sequence. [00915] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a forward sequencing primer (1 0) where the universal adaptor sequence comprises the sequence SEQ ID NO: 34 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence is a left universal adaptor sequence. In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a forward sequencing primer (140) where the universal binding sequence comprises the sequence SEQ ID NO: 32 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence is a left universal adaptor sequence.

[00916] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a reverse sequencing primer (150) where the universal binding sequence comprises the sequence SEQ ID NO: 36 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence is a right universal adaptor sequence.

[00917] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a reverse sequencing primer (150) where the universal binding sequence comprises the sequence SEQ ID NO: 37 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence is a right universal adaptor sequence.

[00918] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a universal adaptor sequence for binding a reverse sequencing primer (150) where the right universal binding sequence comprises the sequence SEQ ID NO: 35 or a complementary sequence thereof. In some embodiments, the universal adaptor sequence is a right universal adaptor sequence. [00919] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a right universal binding sequence (130) which binds the second region (330) of the first splint strand, where the right universal binding sequence (130) comprises the sequence SEQ ID NO: 38 or a complementary sequence thereof.

[00920] In some embodiments, in any of the library-splint complexes (500) described herein, the linear single stranded library molecule (100) includes a right universal binding sequence (130) which binds the second region (330) of the first splint strand, where the right universal binding sequence (130) comprises the sequence SEQ ID NO: 2 or a complementary sequence thereof.

[00921] In some embodiments, in any of the library-splint complexes (500) described herein, the first sub-region of the second splint strand (400) comprises the sequence SEQ ID NO: 1 or a complementary sequence thereof.

[00922] In some embodiments, the second sub-region of the second splint strand (400) comprises the sequence SEQ ID NO: 2 or a complementary sequence thereof.

[00923] In some embodiments, the second splint strand (400) comprises a first and second sub-region comprising the sequence SEQ ID NO: 3 or a complementary sequence thereof, (e.g., see FIG. 16). In some embodiments, the 5’ end of the second splint strand (400) can be phosphorylated or non-phosphorylated.

[00924] In some embodiments, in any of the library-splint complexes (500) described herein, the first region (320) of the first splint strand includes a first universal adaptor sequence which comprises a universal binding sequence (or a complementary sequence thereof) for a universal first surface capture primer, wherein the first region (320) comprises the sequence SEQ ID NO: 4 or a complementary sequence thereof. For example, the first region (320) of the first splint strand can hybridize to a P5 surface primer or a complementary sequence of the P5 surface primer. For example, the P5 surface primer comprises the sequence SEQ ID NO: 41 (e.g., short P5), or the P5 surface primer comprises the sequence SEQ ID NO: 42 (e.g., long P5).

[00925] In some embodiments, the second region (330) of the first splint strand includes a second universal adaptor sequence which comprises a universal binding sequence (or a complementary sequence thereof) for a second surface primer (e.g., a universal pinning primer), wherein the second region (330) comprises the sequence SEQ ID NO: 5 or a complementary sequence thereof. For example, the second region (330) of the first splint strand can hybridize to a P7 surface primer or a complementary sequence of the P7 surface primer. For example, the P7 surface primer comprises the sequence SEQ ID NO: 5 (e.g., short P7), or the P7 surface primer comprises the sequence SEQ ID NO: 43 (e.g., long P7).

[00926] In some embodiments, the first splint strand (300) includes an internal region (310) which comprises a fourth sub-region having the sequence

SEQ ID NO: 6 or a complementary sequence thereof.

[00927] In some embodiments, the first splint strand (300) includes an internal region (310) which comprises a fifth sub-region having the sequence

SEQ ID NO: 10 or a complementary sequence thereof.

[00928] In some embodiments, the first splint strand (300) comprises a first region (320), an internal region (310) having a fourth and fifth sub-region, and a second region (330), having the sequence SEQ ID NO: 8 or a complementary sequence thereof (e.g., see FIG. 16). [00929] In some embodiments, the 5’ end of the first splint strand (300) can be phosphorylated or non-phosphorylated. In some embodiments, the first sub-region of the second splint strand (400) can hybridize to the fourth sub-region of the first splint strand (300). In some embodiments, the second sub-region of the second splint strand (400) can hybridize to the fifth sub-region of the first splint strand (300).

[00930] In some embodiments, in any of the library-splint complexes (500) described herein, the first region (320) of the first splint strand comprises a sequence that can bind universal adaptor sequence for binding a second universal surface primer (120) (e.g., a first left universal adaptor sequence, for binding a surface pinning primer) of a library molecules, where the first region (320) of the first splint strand comprises the sequence SEQ ID NO: 9 or a complementary sequence thereof.

[00931] In some embodiments, in any of the library-splint complexes (500) described herein, the second region (330) of the first splint strand comprises a sequence that can bind a first right universal adaptor sequence (130) of a library molecules, where the second region (330) of the first splint strand comprises the sequence SEQ ID NO: 10 or a complementary sequence thereof. Improved Base Calling Using Sample Indexes

[00932] Generally, it is desirable to prepare nucleic acid libraries that will be distributed onto a support (e.g., coated flowcell), where the library molecules are converted into template molecules that are immobilized at a high density to the support for massively parallel sequencing. For template molecules that are immobilized at high densities at random locations on the support, the challenge of resolving high density fluorescent images for accurate base calling during sequencing runs can become challenging.

[00933] The nucleotide diversity of a population of immobilized template molecules refers to the relative proportion of nucleotides A, G, C and T that are present in each sequencing cycle. An optimal high diversity library will generally include sequence-of-interest (insert) regions having approximately equal proportions of all four nucleotides represented in each cycle of a sequencing run. A low diversity library will generally include sequence-of-interest (insert) regions having a high proportion of certain nucleotides and low proportion of other nucleotides. To overcome the problem of low diversity libraries, a small amount of a high diversity library prepared from PhiX bacteriophage is typically mixed with the library-of- interest (e.g., PhiX spike-in library) and sequenced together on the same flowcell. While the PhiX spike-in library provides nucleotide diversity, it also occupies space on the flowcell thereby replacing the target libraries carrying the sequence-of-interest and reduces the amount of sequencing data obtainable from the target libraries (e.g., reduces sequencing throughput). Another method to overcome the problem of low diversity libraries is to prepare library molecules having at least one sample index sequence that is designed to be color- balanced. However it may be desirable to design a large number of sample index sets, for example a set of single index sample sequences or paired index sample sequences for 16- plex, 24-plex, 96-plex or larger plexy levels. It is challenging to design sample index sequences, as a single or paired sample indexes, for large sample index sets where all of the sample index sequences are color-balanced.

[00934] An alternative method to overcome the challenges of sequencing low diversity library molecules (e.g., at high density on the support) is to prepare libraries having at least one sample index sequence comprising a short random sequence (e.g., NNN) linked directly to a universal sample index sequence, where the short random sequence provides nucleotide diversity and color balance. Embodiments of library molecules comprising sample index sequences (e.g., (160) and (170) are shown in FIG. 14. Embodiments of library molecules comprising sample index sequences (e.g., (760) and (770) are shown in FIG. 17. Embodiments of library molecules comprising sample index sequences (e.g., (1700) and (1800) are shown in FIGS. 21-33. In a population of library molecules each molecule comprising a sample index sequence (e.g., (170) or (160); (760) or (770); or (1700) or (1800)), the short random sequence (e.g., NNN) provides high nucleotide diversity which includes approximately equal proportions of all four nucleotides (e.g., A, G, C, T and/or U) that will be represented in each cycle of a sequencing run. The high nucleotide diversity of the short random sequence also provide color balance during each cycle of the sequencing run. The advantage of designing sample indexes (e.g., (170) or (160)) to include a short random sequence (e.g., NNN) is that, in a low-plexy population of library molecules (e.g., 2- plex or 4-plex), the universal sample index sequences that identify the two or four different samples need not exhibit nucleotide diversity. Additionally, the nucleotide diversity of the short random sequence (e.g., NNN) can obviate the need to include a PhiX spike-in library, or permits use of a reduced amount of PhiX spike-in library to be distributed onto the flowcell and sequenced.

[00935] The library molecule can include at least one sample index sequence (e.g., (170) or (160); (760) or (770); or (1700) or (1800)) which includes a short random sequence (e.g., NNN). In some embodiments, the sequencing data from the sample index sequence (e.g., (170) or (160); (760) or (770); or (1700) or (1800)) can be used for polony mapping and template registration because the short random sequence (e.g., NNN) provides sufficient nucleotide diversity and color balance. The sequencing data from the universal sample index sequence (e.g., (170) and/or (160); (760) or (770); or (1700) or (1800)) can be used to distinguish sequences of interest obtained from different sample sources in a multiplex assay. [00936] In some embodiments, the library molecule comprises two sample index sequences (e.g., (170) and (160)). In some embodiments, the sequencing data from only one of the sample index sequences can be used for polony mapping and/or template registration because the short random sequence provides sufficient nucleotide diversity and color balance. The sequencing data from the first universal sample index sequence (e.g., (170)) and the second universal sample index sequence (e.g., (160)) can be used as dual sample indexes to distinguish sequences of interest obtained from different sample sources in a multiplex assay. In some embodiments, the second sample index sequence (e.g., (160)) may or may not include a second short random sequence (e.g., NNN).

[00937] The order of sequencing the sequence-of-interest region and the sample index region(s) can also be used to improve the challenges of sequencing low diversity library molecules. For example, the region comprising the sample index sequence (e.g., (170) or (160)) can be sequenced first before sequencing the sequence-of-interest region, and the sample index sequence (e.g., (170) or (160); (760) or (770); or (1700) or (1800)) can be associated with the sequence-of-interest region. For example, the region comprising the sample index sequence (e.g., (170) or (160); (760) or (770); or (1700) or (1800)) can be sequenced first including sequencing the short random sequence (e.g., NNN) and optionally sequencing at least a portion of the universal sample index, and then sequencing the sequence-of-interest region. In a population of library molecules, the short random sequence (e.g., NNN) provides nucleotide diversity which may not be provided the sequence-of- interest regions of the library molecules. The short random sequence (e.g., NNN) provides improved nucleotide diversity and color balance for polony mapping and template registration.

[00938] Additionally, when sequencing the sample index region first, the length of the sequenced sample index region is relatively short (e.g., less than 30 nucleotides in length) so that de-hybridization of the product of the sequenced sample index region is more complete. Gentler de-hybridization conditions can be used to remove most or all of the product of the sequenced sample index region which reduces the level of residual signals from any sequencing products remaining hybridized to the template molecules. By contrast, the sequence-of-interest region is typically much longer than the sample index region (e.g., more than 100 nucleotides in length). When the sequence-of-interest region is sequenced before the sample index region, the product of the sequenced sequence-of-interest region must be subjected to harsher de-hybridization conditions to remove any products remaining hybridized to the template molecules which may damage the template molecules.

[00939] The present disclosure provides linear single stranded library molecules (100) each comprising at least one sample index sequence that can be used to distinguish sequences of interest obtained from different sample sources in a multiplex assay, where the at least one sample index sequence comprises a short random sequence (e.g., NNN) linked to a universal sample index sequence. In some embodiments, the left sample index (160) comprises a short random sequence (e.g., NNN) linked to a universal left sample index sequence and/or the right sample index (170) comprises a short random sequence (e.g., NNN) linked to a right universal sample index sequence. The at least one sample index sequence can include sequence diversity for improved base calling. The at least one sample index sequence can be used to improve base calling accuracy.

[00940] In some embodiments, the short random sequence (e.g., NNN) is positioned upstream of the sample index sequence (e.g., (170) and/or (160); (760) and/or (770); or (1700) and/or (1800)) so that during a sequencing run the random sequence portion is sequenced before the universal sample index sequence. In some embodiments, the short random sequence is positioned downstream of the universal sample index sequence so that during a sequencing run the random portion is sequenced after the universal sample index sequence. In some embodiments, the sample index sequence (e.g., (170) and/or (160); (760) and/or (770); or (1700) and/or (1800)) is a universal sample index sequence.

[00941] In some embodiments, in the random sequence each base “N” at a given position is independently selected from A, G, C, T or U. In some embodiments, the random sequence lacks consecutive repeat sequences having 2 or 3 of the same nucleo-base, for example AA, TT, CC, GG, UU, AAA, TTT, CCC, GGG or UUU. In some embodiments, in a population of library molecules the sample index sequences (e.g., (170) and/or (160); (760) and/or (770); or (1700) and/or (1800)) include a short random sequence having a high diversity sequence which includes approximately equal proportions of all four nucleotides (e.g., A, G, C, T and/or U) that will be represented in each cycle of a sequencing run. In some embodiments, the sample index sequence (e.g., (170) and/or (160); (760) and/or (770); or (1700) and/or (1800)) is a universal sample index sequence.

[00942] In some embodiments, the short random sequence (e.g., NNN) comprises 3-20 nucleotides, or 3-10 nucleotides, or 3-8 nucleotides, or 3-6 nucleotides, or 3-5 nucleotides, or 3-4 nucleotides.

[00943] In some embodiments, the short random sequence (e.g., NNN) includes, but is not limited to, AGC, AGT, GAC, GAT, CAT, CAG, TAG, TAC. The skilled artisan will recognize that many more random sequences can be prepared (e.g., 64 possible combinations) where each base “N” at a given position in the random sequence is independently selected from A, G, C, T or U.

[00944] In some embodiments, the universal sample index sequence comprises 5-20 nucleotides, or 7-18 nucleotides, or 9-16 nucleotides, or any range therebetween.

[00945] In some embodiments, in a population of library molecules the short random sequence (e.g., NNN) has an overall base composition of about 25% or about 20-30% of all four nucleotide bases (e.g., A, G, C and T/U) to provide nucleotide diversity at each sequencing cycle during sequencing the short random sequence (e.g., NNN).

[00946] In some embodiments, in the population of library molecules the proportion of adenine (A) at any given position in the short random sequence is about 20-30% or about 15- 35% or about 10-40%. In some embodiments, in the population of library molecules the proportion of guanine (G) at any given position in the short random sequence is about 20- 30% or about 15-35% or about 10-40%. In some embodiments, in the population of library molecules the proportion of cytosine (C) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%. In some embodiments, in the population of library molecules the proportion of thymine (T) or uracil (U) at any given position in the short random sequence is about 20-30% or about 15-35% or about 10-40%.

[00947] In some embodiments, in the population of library molecules the proportion of adenine (A) and thymine (T), or the proportion of adenine (A) and uracil (U), at any given position in the short random sequence is about 10-65%. In some embodiments, in the population of library molecules the proportion of guanine (G) and cytosine (C) at any given position in the short random sequence is about 10-65%.

[00948] In some embodiments, in the population of library molecules the sequence diversity of the short random sequences ensures that no sequencing cycle is presented with fewer than four different nucleotide bases during sequencing at least the short random sequence (e.g., NNN).

[00949] Exemplary sample index sequences (e.g., (170) and/or (160); (760) and/or (770); or (1700) and/or (1800)) that include a short random sequence NNN linked directly to a universal sample index sequence include but are not limited to: NNNGTAGGAGCC;

NNNCCGCTGCTA; NNNAACAACAAG; NNNGGTGGTCTA; NNNTTGGCCAAC; NNNCAGGAGTGC; and NNNATCACACTA. The skilled artisan will recognize that the universal sample index can be any length and have any sequence that can be used to distinguish sequences of interest obtained from different sample sources in a multiplex assay. In a population of a given sample index, for example NNNGTAGGAGCC, the population contains a mixture of individual sample index molecules each carrying the same universal sample index sequence (e.g., GTAGGAGCC) and a different short random sequence (e.g., NNN) where up to 64 different short random sequences may be present in the population of the given sample index.

[00950] In some embodiments, a sequencing reaction includes use of polymerases and nucleotides (e.g., nucleotide analogs) that are labeled with a different fluorophore that corresponds to the nucleo-base. In some embodiments, sequencing the short random sequence (e.g., NNN) using labeled nucleotides (or the equivalent) provides a balanced ratio of fluorescent colors that correspond to the nucleo-bases adenine, cytosine, guanine, thymine and/or uracil in each cycle of a sequencing run. In some embodiments, sequencing the short random sequence (e.g., NNN) and at least a portion of the universal sample index sequence using labeled nucleotides provides a balanced ratio of fluorescent colors that correspond to nucleo-bases adenine, cytosine, guanine, thymine and/or uracil. The labeled nucleotides emit fluorescent signals during the sequencing reactions. In some embodiments, the sequencing reaction is conducted on a sequencing apparatus having a detector that captures fluorescent images from sequencing reactions on the immobilized template molecules. The sequencing apparatus can be configured to relay the fluorescent imaging data captured by the detector to a computer system that is programmed to determine the location (e.g., mapping) of the immobilized template molecules on the flowcell. The computer system can generate a map of the locations of the immobilized template molecules based on the fluorescent imaging data of only the random sequence (e.g., NNN), or based on the random sequence (e.g., NNN) and at least a portion the universal sample index sequence. Thus the few numbers of sequencing cycles used to sequence the random sequence (e.g., NNN) and optionally a portion of the universal sample index sequence can be used to generate a map of the location of the immobilized template molecules. The computer system can be configured to extract the fluorescent color and intensity of only the random sequence (e.g., NNN), or from the random sequence (e.g., NNN) and at least a portion of the universal sample index sequence. The computer system can be configured to use the location of a given immobilized template molecule and the fluorescent color and intensity associated with the given template molecule (which were established while sequencing the random sequence) for base calling while sequencing the insert region (110). The computer system can be configured to detect phasing and pre-phasing while sequencing the random sequence (e.g., NNN) and the universal sample index sequence, and the insert region (110). In some embodiments, the balanced ratio of fluorescent colors provided by the random sequence (e.g., NNN) at each sequencing cycle can improve the quality of the data which is processed from the fluorescent images captured by the detector, and can in turn improve the capability by the computer system to determine the location of the immobilized template molecules on the flowcell, and the color and intensity, all of which can improve base calling accuracy and quality scores of the sequenced insert region (110).

[00951] In some embodiments, a sequencing reaction includes use of polymerases and multivalent molecules that are labeled with a different fluorophore that corresponds to the nucleo-base (e.g., adenine, guanine, cytosine, thymine or uracil) of the nucleotide units that are attached to the nucleotide arms in a given multivalent molecule. In some embodiments, the core of individual multivalent molecules is attached to a fluorophore which corresponds to the nucleotide units (e.g., adenine, guanine, cytosine, thymine or uracil) that are attached to the nucleotide arms in a given multivalent molecule (e.g., see FIGS. 1-4). In some embodiments, at least one of the nucleotide arms of the multivalent molecule comprises a linker and/or nucleotide base that is attached to a fluorophore, and wherein the fluorophore which is attached to a given linker or nucleotide base corresponds to the nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil) of the nucleotide arm. In some embodiments, sequencing the random sequence (e.g., NNN) by conducting the two-stage sequencing method using labeled multivalent molecules provides a balanced ratio of fluorescent colors that correspond to the nucleo-bases adenine, cytosine, guanine, thymine and/or uracil in each cycle of a sequencing run. In some embodiments, sequencing the random sequence (e.g., NNN) and at least a portion of the universal sample index sequence using labeled multivalent molecules provides a balanced ratio of fluorescent colors that correspond to nucleo-bases adenine, cytosine, guanine, thymine and/or uracil. The labeled multivalent molecules emit fluorescent signals during the sequencing reactions. In some embodiments, the sequencing reaction is conducted on a sequencing apparatus having a detector that captures fluorescent images from sequencing reactions on the immobilized template molecules. The sequencing apparatus can be configured to relay the fluorescent imaging data captured by the detector to a computer system that is programmed to determine the location (e.g., mapping) of the immobilized template molecules (polonies) on the flowcell. The computer system can generate a map of the locations of the immobilized template molecules based on the fluorescent imaging data of only the random sequence (e.g., NNN), or based on the random sequence (e.g., NNN) and at least a portion of the universal sample index sequence. Thus the few numbers of sequencing cycles used to sequence the random sequence (e.g., NNN) and optionally a portion of the universal sample index sequence can be used to generate a map of the location of the immobilized template molecules. The computer system can be configured to extract the fluorescent color and intensity of only the random sequence (e.g., NNN), or from the random sequence (e.g., NNN) and the universal sample index sequence. The computer system can be configured to use the location of a given immobilized template molecule and the fluorescent color and intensity associated with the given template molecule (which were established while sequencing the random sequence) for base calling while sequencing the insert region (110). The computer system can be configured to detect phasing and pre-phasing while sequencing the random sequence (e.g., NNN) and the universal sample index sequence, and the insert region (110). In some embodiments, the balanced ratio of fluorescent colors provided by the random sequence (e.g., NNN) at each sequencing cycle can improve the quality of the data which is processed from the fluorescent images captured by the detector, and can in turn improve the capability by the computer system to determine the location of the immobilized template molecules on the flowcell, and the color and intensity, all of which can improve base calling accuracy and quality scores of the sequenced insert region (110).

Methods for Sequencing

[00952] The present disclosure provides methods for sequencing a plurality of concatemer template molecules which are immobilized to a support, for example any of the immobilized concatemer template molecules described herein. In some embodiments, the sequencing reactions employ a plurality of sequencing primers, a plurality of sequencing polymerases, and nucleotide reagents comprising any one or any combination of nucleotides and/or multivalent molecules. In some embodiments, the nucleotide reagents comprise canonical nucleotides. In some embodiments, the nucleotide reagents comprise nucleotide analogs comprise detectably labeled nucleotides. In some embodiments, the nucleotide reagents comprise nucleotides carrying a removable or non-removable chain terminating moiety. In some embodiments, the nucleotide reagents comprise multivalent molecules each comprising a central core attached to multiple polymer arms each having a nucleotide moiety at the end of the arms (e.g., see FIGS. 1-5). In some embodiments, the sequencing reactions employ binding non-labeled nucleotides without incorporation. In some embodiments, the sequencing reactions employ incorporating non-labeled nucleotide analogs. In some embodiments, the sequencing reactions employ incorporating detectably labeled nucleotides having removable chain terminating moiety. In some embodiments, the sequencing reactions employ a two- stage sequencing reaction comprising binding detectably labeled multivalent molecules without incorporation, and incorporating nucleotide analogs. In some embodiments, the sequence reactions employ incorporating a nucleotide moiety from an arm of a multivalent molecule. In some embodiments, the sequencing reactions employ phosphate chain-labeled nucleotides.

Methods for Sequencing using Chain Terminating Nucleotide Analogs

[00953] The present disclosure provides methods for sequencing concatemer template molecules using reversible chain terminating nucleotides, comprising step (a): contacting (i) a plurality of sequencing polymerases, (ii) a plurality of concatemer template molecules immobilized to a support and (iii) a plurality of nucleic acid sequencing primers, where the contacting is conducted under a condition suitable to form a plurality of sequencing polymerase complexes each complex comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a portion of a concatemer template molecule hybridized to a nucleic acid sequencing primer. In some embodiments, the sequencing polymerases comprise a recombinant mutant sequencing polymerase that can bind and incorporate nucleotide analogs. In some embodiments, the sequencing primers comprise 3’ extendible ends or 3’ blocked end that can be converted into a 3’ extendible end. [00954] In some embodiments, the methods comprise step (b): contacting the plurality of sequencing polymerase complexes with a plurality of nucleotides under a condition suitable for binding at least one nucleotide to one of the sequencing polymerase complexes, and the condition is suitable for promoting polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase complex is contacted with the plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and/or manganese. In some embodiments, the plurality of nucleotides comprises at least one nucleotide analog having a chain terminating moiety at the sugar 2’ or 3’ position. In some embodiments, the chain terminating moiety is removable from the sugar 2’ or 3’ position to convert the chain terminating moiety to an OH or H group. In some embodiments, the plurality of nucleotides comprises at least one nucleotide that lacks a chain terminating moiety. In some embodiments, at least one nucleotide is labeled with a detectable reporter moiety (e.g., fluorophore). In some embodiments, the plurality of nucleotides comprise one type of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP or dUTP. In some embodiments, the plurality of nucleotides comprise a mixture of any two or more types of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP.

[00955] In some embodiments, the methods comprise step (c): incorporating at least one nucleotide into the 3’ end of an extendible sequencing primer of at least one sequencing polymerase complex. In some embodiments, the nucleotide incorporation reaction of step (c) comprises a primer extension reaction.

[00956] In some embodiments, the methods comprise step (d): repeating steps (b) and (c) at least once.

[00957] In some embodiments, in step (b), the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker which is cleavable/removable from the base. In some embodiments, at least one of the nucleotides in the plurality is not labeled with a detectable reporter moiety. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) that is attached to the nucleotide can correspond to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permit detection and identification of the nucleotide base. In some embodiments, the nucleotide analog comprises a fluorophore attached to the nucleotide base with a linker which is cleavable/removable from the base, and the nucleotide analog further comprises a chain terminating moiety attached to the 2’ or 3’ sugar position by a linker which is cleavable/removable using the same condition (e.g., chemical cleaving condition) that cleaves the fluorophore from the base.

[00958] In some embodiments, the method further comprises detecting the at least one incorporated nucleotide at step (c) and/or (d). In some embodiments, the method further comprises identifying the at least one incorporated nucleotide at step (c) and/or (d). In some embodiments, the sequence of the concatemer template molecule can be determined by detecting and identifying the nucleotide that binds the sequencing polymerase, thereby determining the sequence of the concatemer template molecule. In some embodiments, the sequence of the concatemer template molecule can be determined by detecting and identifying the nucleotide that incorporates into the 3’ end of the primer, thereby determining the sequence of the concatemer template molecule.

Two-Stage Methods for Nucleic Acid Sequencing

[00959] The present disclosure provides a two-stage method for sequencing any of the immobilized concatemer template molecules described herein. In some embodiments, the first stage comprises binding multivalent molecules to polymerase complexes to form multivalent-binding polymerase complexes, and detecting the multivalent-binding polymerase complexes. In some embodiments, the second stage comprises nucleotide incorporation and extension of the sequencing primer. In some embodiments, one sequencing cycle comprises completion of a first and second stage. In some embodiments, any of the workflows that employ a two-stage sequencing method comprises conducting 5-25 sequencing cycles, or 25-50 sequencing cycles, or 50-75 sequencing cycles, or 75-100 sequencing cycles, or 100-200 sequencing cycles, or 200-500 sequencing cycles, or 500-750 sequencing cycles, or 750-1000 sequencing cycles, or any range therebetween.

[00960] In some embodiments, the first stage comprises step (a): contacting (i) a first plurality of sequencing polymerases, (ii) a plurality of concatemer template molecules immobilized to a support and (iii) a plurality of nucleic acid sequencing primers, where the contacting is conducted under a condition suitable to form a first plurality of sequencing polymerase complexes each complex comprising a first sequencing polymerase bound to a nucleic acid duplex wherein the nucleic acid duplex comprises a portion of a concatemer template molecule hybridized to a nucleic acid sequencing primer. In some embodiments, the sequencing primers comprise 3’ extendible ends or 3’ non-extendible ends.

[00961] In some embodiments, the methods comprise step (b): contacting the first plurality of polymerase complexes with a plurality of multivalent molecules to form a plurality of multivalent-binding polymerase complexes (e.g., binding complexes). In some embodiments, individual multivalent molecules in the plurality of multivalent molecules comprise a core attached to multiple nucleotide arms, and individual nucleotide arms are attached to a nucleotide moiety (e.g., FIGS. 1-4). In some embodiments, the contacting of step (b) is conducted under a condition suitable for binding complementary nucleotide moieties of the multivalent molecules to at least two of the polymerase complexes in the first plurality, thereby forming a plurality of multivalent-binding polymerase complexes. In some embodiments, the condition is suitable for inhibiting polymerase-catalyzed incorporation of the complementary nucleotide moieties into the primers of the plurality of multivalent- binding polymerase complexes. In some embodiments, the contacting of step (b) is conducted in the presence of at least one non-catalytic cation which inhibits polymerase-catalyzed nucleotide incorporation. In some embodiments, the at least one non-catalytic cation comprises strontium, barium and/or calcium.

[00962] In some embodiments, in step (b), at least one of the multivalent molecules in the plurality of multivalent molecules is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

[00963] In some embodiments, in step (b), individual nucleotide arms of a multivalent molecule comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide moiety, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, wherein the linker is attached to the nucleotide moiety (e.g., see FIG. 5). In some embodiments, the labeled multivalent molecules comprise a fluorophore attached to the core, spacer, linker and/or nucleotide moiety of the multivalent molecules.

[00964] In some embodiments, in step (b), the plurality of multivalent molecules comprise at least one multivalent molecule having multiple nucleotide arms (e.g., FIGS. 1-4) each attached with a nucleotide analog (e.g., nucleotide analog moiety), where the nucleotide analog includes a chain terminating moiety at the sugar 2’ and/or 3’ position. In some embodiments, the plurality of multivalent molecules comprises at least one multivalent molecule comprising multiple nucleotide arms each attached with a nucleotide moiety that lacks a chain terminating moiety. [00965] In some embodiments, the methods comprise step (c): detecting the plurality of multivalent-binding polymerase complexes. In some embodiments, the detecting includes detecting the multivalent molecules that are bound to the polymerase complexes in the first plurality, where the complementary nucleotide moieties of the multivalent molecules are bound to the primers but incorporation of the complementary nucleotide moieties is inhibited. In some embodiments, the multivalent molecules are labeled with a detectable reporter moiety to permit detection.

[00966] In some embodiments, the methods comprise step (d): identifying the nucleo-base of the complementary nucleotide moieties that are bound to the first plurality of polymerase complexes, thereby determining the sequence of the concatemer template molecule. In some embodiments, the multivalent molecules are labeled with a detectable reporter moiety that corresponds to the nucleotide moieties attached to the nucleotide arms to permit identification of the complementary nucleotide moieties (e.g., nucleotide base adenine, guanine, cytosine, thymine or uracil) that are bound to the first plurality of polymerase complexes.

[00967] In some embodiments, the second stage of the two-stage sequencing method comprises nucleotide incorporation. In some embodiments, the methods comprise step (e): dissociating the plurality of multivalent-binding polymerase complexes and removing the first plurality of sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes.

[00968] In some embodiments, the methods comprise step (f): contacting the plurality of the nucleic acid duplexes retained at step (e) with a second plurality of sequencing polymerases, wherein the contacting is conducted under a condition suitable for binding the second plurality of sequencing polymerases to the plurality of the retained nucleic acid duplexes, thereby forming a second plurality of polymerase complexes each complex comprising a second sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the second sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[00969] In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is 100% identical to the amino acid sequence as the plurality of the second sequencing polymerases of step (f). In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that differs from the amino acid sequence of the plurality of the second sequencing polymerases of step (f). [00970] In some embodiments, the methods comprise step (g): contacting the second plurality of polymerase complexes with a plurality of nucleotides, wherein the contacting is conducted under a condition suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second polymerase complexes, thereby forming a plurality of nucleotide-polymerase complexes. In some embodiments, the contacting of step (g) is conducted under a condition that is suitable for promoting polymerase-catalyzed incorporation of the bound complementary nucleotides into the primers of the nucleotidepolymerase complexes. In some embodiments, the incorporating the nucleotide into the 3’ end of the primer in step (g) comprises a primer extension reaction. In some embodiments, the contacting of step (g) is conducted in the presence of at least one catalytic cation which promotes nucleotide incorporation. In some embodiments, the at least one catalytic cation comprises magnesium and/or manganese. In some embodiments, the plurality of nucleotides comprises native nucleotides (e.g., non-analog nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides comprises a 2’ and/or 3’ chain terminating moiety which is removable or is not removable. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with detectable reporter moiety. The detectable reporter moiety can comprise a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker which is cleavable/removable from the base or is not removable from the base. In some embodiments, at least one of the nucleotides in the plurality is not labeled with a detectable reporter moiety. In some embodiments, the plurality of nucleotides are non-labeled nucleotides. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) that is attached to the nucleotide can correspond to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permit detection and identification of the nucleotide base.

[00971] In some embodiments, when the plurality of nucleotides in steps (g) comprises labeled nucleotides, the methods comprise step (h): detecting the complementary nucleotides which are incorporated into the primers of the nucleotide-polymerase complexes. In some embodiments, the plurality of nucleotides are labeled with a detectable reporter moiety to permit detection. In some embodiments, when the plurality of nucleotides in steps (g) comprises non-labeled nucleotides, the detecting of step (h) is omitted.

[00972] In some embodiments, when the plurality of nucleotides in steps (g) comprises labeled nucleotides, the methods comprise step (i): identifying the bases of the complementary nucleotides which are incorporated into the primers of the nucleotide- polymerase complexes. In some embodiments, the identification of the incorporated complementary nucleotides in step (i) can be used to confirm the identity of the complementary nucleotides of the multivalent molecules that are bound to the first plurality of polymerase complexes in step (d). In some embodiments, the identifying of step (i) can be used to determine the sequence of the concatemer template molecules. In some embodiments, when the plurality of nucleotides in steps (g) comprises non-labeled nucleotides, the identifying of step (i) is omitted.

[00973] In some embodiments, when the plurality of nucleotides in step (g) comprise 2’ and/or 3’ chain terminating nucleotides, the methods comprise step (j): removing the chain terminating moiety from the incorporated nucleotides.

[00974] In some embodiments, the methods comprise step (k): contacting the plurality of concatemer template molecules which are hybridized to the retained sequencing primers (and which are now extended) with a first plurality of sequencing polymerases, and repeating steps (a) - (j) at least once. . In some embodiments, the sequence of the concatemer template molecules can be determined by detecting and identifying the multivalent molecules that bind the sequencing polymerases but do not incorporate into the 3 ’ end of the primer at steps (c) and (d). In some embodiments, the sequence of the concatemer template molecule can be determined (or confirmed) by detecting and identifying the nucleotide that incorporates into the 3’ end of the primer at steps (h) and (i).

Forming Avidity Complexes

[00975] In some embodiments, in any of the two-stage sequencing methods, the binding of the first plurality of polymerase complexes with the plurality of multivalent molecules forms at least one avidity complex, and the method comprises the steps: (1) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a concatemer template molecule thereby forming a first binding complex, wherein a first nucleotide moiety of the first multivalent molecule binds to the first sequencing polymerase; and (2) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same concatemer template molecule thereby forming a second binding complex, wherein a second nucleotide moiety of the first multivalent molecule binds to the second sequencing polymerase, wherein the first and second binding complexes which include the same multivalent molecule forms an avidity complex. The concatemer template molecule comprises tandem repeat sequences of a sequence of interest and at least one universal site for binding a sequencing primer. The first and second sequencing primers can bind to a sequencing primer binding site along the concatemer template molecule. Exemplary multivalent molecules are shown in FIGS. 1-4.

Forming Avidity Complexes with Detecting and Identifying

[00976] In some embodiments, in any of the two-stage sequencing methods, wherein the method includes binding the first plurality of polymerase complexes with the plurality of multivalent molecules to form at least one avidity complex, the method comprises the steps: (1) contacting the plurality of sequencing polymerases and the plurality of sequencing primers with different portions of a concatemer template molecule to form at least first and second polymerase complexes on the same concatemer template molecule; (2) contacting a plurality of multivalent molecules to the at least first and second polymerase complexes on the same concatemer template molecule, under conditions suitable to bind a single multivalent molecule from the plurality to the first and second polymerase complexes, wherein at least a first nucleotide moiety of the single multivalent molecule is bound to the first polymerase complex which includes a first sequencing primer hybridized to a first portion of the concatemer template molecule thereby forming a first binding complex (e.g., first ternary complex), and wherein at least a second nucleotide moiety of the single multivalent molecule is bound to the second polymerase complex which includes a second sequencing primer hybridized to a second portion of the concatemer template molecule thereby forming a second binding complex (e.g., second ternary complex), wherein the contacting is conducted under a condition suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide moieties in the first and second binding complexes, and wherein the first and second binding complexes which are bound to the same multivalent molecule forms an avidity complex; (3) detecting the first and second binding complexes on the same concatemer template molecule, and (4) identifying the first nucleotide moiety in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide moiety in the second binding complex thereby determining the sequence of the second portion of the concatemer template molecule.

[00977] The concatemer template molecule comprises tandem repeat sequences of a sequence of interest and at least one universal site for binding a sequencing primer. The plurality of nucleic acid primers can bind to a sequencing primer binding site along the concatemer template molecule. Exemplary multivalent molecules are shown in FIGS. 1-4. Sequencing-by-Binding

[00978] The present disclosure provides methods for sequencing concatemer template molecules comprising a sequencing-by-binding (SBB) procedure which employs non-labeled chain-terminating nucleotides. In some embodiments, the sequencing-by-binding (SBB) method comprises the steps of (a) sequentially contacting a primed template nucleic acid (e.g., a an immobilized concatemer template molecule hybridized to a sequencing primer) with at least two separate mixtures under ternary complex stabilizing conditions, wherein the at least two separate mixtures each include a polymerase and a nucleotide, whereby the sequentially contacting results in the primed template nucleic acid being contacted, under the ternary complex stabilizing conditions, with nucleotide cognates for first, second and third base type base types in the template; (b) examining the at least two separate mixtures to determine whether a ternary complex formed; and (c) identifying the next correct nucleotide for the primed template nucleic acid molecule, wherein the next correct nucleotide is identified as a cognate of the first, second or third base type if ternary complex is detected in step (b), and wherein the next correct nucleotide is imputed to be a nucleotide cognate of a fourth base type based on the absence of a ternary complex in step (b); (d) adding a next correct nucleotide to the primer of the primed template nucleic acid after step (b), thereby producing an extended primer; and (e) repeating steps (a) through (d) at least once on the primed template nucleic acid that comprises the extended primer. Exemplary sequencing-by- binding methods are described in U.S. patent Nos. 10,246,744 and 10,731,141 (where the contents of both patents are hereby incorporated by reference in their entireties).

[00979] In some embodiments, in step (a) of any of the sequencing methods described herein, the plurality of concatemer template molecules is immobilized to a support at a density of about 10² - 10¹⁵ per mm².

[00980] In some embodiments, in step (a) of any of the sequencing methods described herein, the plurality of concatemer template molecules is immobilized to a support at predetermined positions on the support, or immobilized to random positions on the support. [00981] In some embodiments, in step (a) of any of the sequencing methods described herein, the support is passivated/coated with at least one polymer layer.

Methods for Sequencing Using Phosphate Chain-Labeled Nucleotide Analogs

[00982] The present disclosure provides methods for sequencing concatemer template molecules using a plurality of phosphate chain-labeled nucleotides, comprising step (a): contacting (i) a plurality of sequencing polymerases, (ii) a plurality of concatemer template molecules immobilized to a support and (iii) a plurality of nucleic acid sequencing primers, wherein the contacting is conducted under a condition suitable to form a plurality of sequencing polymerase complexes, each complex comprising a sequencing polymerase bound to a nucleic acid duplex, and wherein the nucleic acid duplex comprises a portion of a concatemer template molecule hybridized to a nucleic acid sequencing primer. In some embodiments, the sequencing polymerases comprise a recombinant mutant sequencing polymerase that can bind and incorporate nucleotide analogs. In some embodiments, the sequencing primers comprise 3’ extendible ends or 3’ blocked ends that can be converted into 3’ extendible ends.

[00983] In some embodiments, the methods comprise step (b): contacting the plurality of sequencing polymerase complexes with a plurality of phosphate chain-labeled nucleotides under a condition suitable for binding at least one phosphate chain-labeled nucleotide to one of the sequencing polymerase complexes, and the condition is suitable for promoting polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase complex is contacted with the plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and/or manganese. In some embodiments, individual phosphate chain-labeled nucleotides in the plurality comprise an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and phosphate chain comprising 3-20 phosphate groups, where the terminal phosphate group is linked to a detectable reporter moiety (e.g., a fluorophore). The first, second and third phosphate groups can be referred to as alpha, beta and gamma phosphate groups. In some embodiments, a particular detectable reporter moiety which is attached to the terminal phosphate group corresponds to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permit detection and identification of the nucleo-base. In some embodiments, the sequencing polymerases are capable of binding a complementary phosphate chain labeled nucleotide and incorporating the complementary nucleotide opposite a nucleotide in a template molecule. In some embodiments, the polymerase-catalyzed nucleotide incorporation reaction cleaves between the alpha and beta phosphate groups thereby releasing a multi-phosphate chain linked to the detectable reporter moiety. In some embodiments, the plurality of phosphate chain-labeled nucleotides comprises one type or a mixture of any two or more types of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP.

[00984] In some embodiments, the methods comprise step (c): detecting the fluorescent signal emitted by the phosphate chain labeled nucleotide that is bound by the sequencing polymerase, and incorporated into the terminal end of the sequencing primer. In some embodiments, step (c) further comprises identifying the phosphate chain labeled nucleotide that is bound by the sequencing polymerase, and incorporated into the terminal end of the sequencing primer.

[00985] In some embodiments, the methods comprise step (d): repeating steps (b) - (c) at least once. In some embodiments, sequencing methods that employ phosphate chain labeled nucleotides can be conducted according to the methods described in U.S. patent Nos. 7,170,050; 7,302,146; and/or 7,405,281, the contents of which are incorporated by reference herein.

[00986] In some embodiments, in step (a), the plurality of concatemer template molecules is immobilized to a support which comprises a plurality of separate compartments. In some embodiments, the plurality of sequencing polymerases is in solution in a compartment. In some embodiments, at least one sequencing polymerase is immobilized to the bottom of an individual compartment. In some embodiments, the separate compartments comprise a silica bottom through which light can penetrate. In some embodiments, the separate compartments comprise a silica bottom configured with a nanophotonic confinement structure comprising a hole in a metal cladding film (e.g., aluminum cladding film). In some embodiments, the hole in the metal cladding has a small aperture, for example, approximately 70 nm. In some embodiments, the height of the nanophotonic confinement structure is approximately 100 nm. In some embodiments, the nanophotonic confinement structure comprises a zero mode waveguide (ZMW). In some embodiments, the nanophotonic confinement structure contains a liquid.

Non-Catalytic Divalent Cations

[00987] In some embodiments, any of the sequencing methods described herein can include a non-catalytic divalent cation. The non-catalytic divalent cation can promote formation of a ternary complex. In some embodiments, a non-catalytic divalent cation can promote binding of a complementary nucleotide (e.g., free nucleotide) or a complementary nucleotide unit (e.g., of a multivalent molecule) to the 3’ end of a primer that is hybridized to a library or template molecule in the presence of a polymerase. The non-catalytic divalent cation does not promote polymerase-catalyzed incorporation of the nucleotide or the nucleotide unit into the primer. In some embodiments, the non-catalytic divalent cation comprises strontium, barium, scandium, titanium, calcium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, tin or terbium ion. In some embodiments, the non-catalytic divalent cation comprises strontium chloride, strontium acetate, barium acetate or nickel chloride. In some embodiments, the first stage of a two-stage sequencing method includes a non-catalytic divalent cation at a concentration of about 0.1 - 50 mM, or about 1-25 mM, or about 5-10 mM, or any range therebetween.

Catalytic Divalent Cations

[00988] In some embodiments, any of the sequencing methods described herein can include a catalytic divalent cation. The catalytic divalent cation can promote formation of a ternary complex. In some embodiments, a catalytic divalent cation can promote binding of a complementary nucleotide (e.g., free nucleotide) or a complementary nucleotide unit (e.g., of a multivalent molecule) to the 3’ end of a primer that is hybridized to a library or template molecule in the presence of a polymerase. The catalytic divalent cation promotes polymerase- catalyzed incorporation of the nucleotide or the nucleotide unit into the primer. For example, the second stage of a two-stage sequencing method can include a catalytic divalent cation. In some embodiments, the catalytic divalent cation comprises magnesium and manganese. In some embodiments, the catalytic divalent cation can be present in a reagent at a concentration of about 0.1 - 50 mM, or about 1-25 mM, or about 5-10 mM, or any range therebetween.

Sequencing Polymerases

[00989] The present disclosure provides methods for sequencing any of the immobilized concatemer template molecules described herein, where any of the sequencing methods described herein employ at least one type of sequencing polymerase and a plurality of nucleotides, or employ at least one type of sequencing polymerase and a plurality of nucleotides and a plurality of multivalent molecules. In some embodiments, the sequencing polymerase(s) is/are capable of incorporating a complementary nucleotide opposite a nucleotide in a template molecule. In some embodiments, the sequencing polymerase(s) is/are capable of binding a complementary nucleotide unit of a multivalent molecule opposite a nucleotide in a template molecule. In some embodiments, the plurality of sequencing polymerases comprise recombinant mutant polymerases.

[00990] Examples of suitable polymerases for use in sequencing with nucleotides and/or multivalent molecules include but are not limited to: Klenow DNA polymerase; Thermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidates altiarchaeales archaeon; Candidatus Hadarchaeum Yellowstonense; Hadesarchaea archaeon; Euryarchaeota archaeon; Thermoplasmata archaeon; Thermococcus polymerases such as Thermococcus litoralis, bacteriophage T7 DNA polymerase; human alpha, delta and epsilon DNA polymerases; bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; E. coli DNA polymerase III alpha and epsilon; 9 degree N polymerase; reverse transcriptases such as HIV type M or O reverse transcriptases; avian myeloblastosis virus reverse transcriptase; Moloney Murine Leukemia Virus (MMLV) reverse transcriptase; or telomerase. Further non-limiting examples of DNA polymerases include those from various Archaea genera, such as, Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta and the like or variants thereof, including such polymerases as are known in the art such as 9 degrees N, VENT, DEEP VENT, THERMINATOR, Pfu, KOD, Pfx, Tgo and RB69 polymerases. Additional suitable polymerases are described, for example, in U.S. Patent Nos. 11,859,241, 11,788,075, and 12,139,727, the contents of which are incorporated by reference in their entireties herein.

Nucleotides

[00991] The present disclosure provides methods for sequencing any of the immobilized concatemer template molecules described herein, where any of the sequencing methods described herein employ at least one nucleotide. The nucleotides comprise a base, sugar and at least one phosphate group. In some embodiments, at least one nucleotide in the plurality comprises an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1-10 phosphate groups). The plurality of nucleotides can comprise at least one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. The plurality of nucleotides can comprise at a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, at least one nucleotide in the plurality is not a nucleotide analog. In some embodiments, at least one nucleotide in the plurality comprises a nucleotide analog.

[00992] In some embodiments, at least one nucleotide in the plurality of nucleotides comprises a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, at least one nucleotide in the plurality is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoroamidite groups.

[00993] In some embodiments, at least one nucleotide in the plurality of nucleotides comprises a terminator nucleotide analog having a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the chain terminating moiety can inhibit polymerase-catalyzed incorporation of a subsequent nucleotide unit or free nucleotide in a nascent strand during a primer extension reaction. In some embodiments, the chain terminating moiety is attached to the 3’ sugar position where the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain terminating moiety is removable/cleavable from the 3’ sugar position to generate a nucleotide having a 3 ’OH sugar group which is extendible with a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the chain terminating moiety is cleavable/removable from the nucleotide, for example by reacting the chain terminating moiety with a chemical agent, pH change, light or heat. In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPhs)4) with piperidine, or with 2,3-Dichloro- 5,6-dicyano-l,4-benzo-quinone (DDQ). In some embodiments, the chain terminating moieties aryl and benzyl are cleavable with H2 Pd/C. In some embodiments, the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). In some embodiments, the chain terminating moiety carbonate is cleavable with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the chain terminating moieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride. In some embodiments, the chain terminating moiety may be cleavable/removable with nitrous acid. In some embodiments, a chain terminating moiety may be cleavable/removable using a solution comprising nitrite, such as, for example, a combination of nitrite with an acid such as acetic acid, sulfuric acid, or nitric acid. In some further embodiments, said solution may comprise an organic acid.

[00994] In some embodiments, at least one nucleotide in the plurality of nucleotides comprises a terminator nucleotide analog having a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the chain terminating moiety comprises an azide, azido or azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3’-O-azido or 3’-O- azidomethyl group. In some embodiments, the chain terminating moieties azide, azido and azidomethyl group are cleavable/removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP). In some embodiments, the chain terminating moiety comprising one or more of a 3’-O-amino group, a 3’-O-aminomethyl group, a 3’-O- methylamino group, or derivatives thereof may be cleaved with nitrous acid, through a mechanism utilizing nitrous acid, or using a solution comprising nitrous acid. In some embodiments, the chain terminating moiety comprising one or more of a 3’-O-amino group, a 3’-O-aminomethyl group, a 3’-O-methylamino group, or derivatives thereof may be cleaved using a solution comprising nitrite. In some embodiments, for example, nitrite may be combined with or contacted with an acid such as acetic acid, sulfuric acid, or nitric acid. In some further embodiments, for example, nitrite may be combined with or contacted with an organic acid such as, for example, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, or the like.

[00995] In some embodiments, the nucleotide comprises a chain terminating moiety which is selected from a group consisting of 3’-deoxy nucleotides, 2’,3’-dideoxynucleotides, 3’- methyl, 3 ’-azido, 3 ’-azidomethyl, 3’-O-azidoalkyl, 3’-O-ethynyl, 3’-O-aminoalkyl, 3’-O- fluoroalkyl, 3 ’-fluoromethyl, 3’-difluoromethyl, 3’-trifluoromethyl, 3 ’-sulfonyl, 3 ’-malonyl, 3’-amino, 3’-O-amino, 3’-sulfhydral, 3 ’-aminomethyl, 3’-ethyl, 3’butyl, 3’-tert butyl, 3’- Fluorenylmethyloxycarbonyl, 3’ tert-Butyloxy carbonyl, 3’-O-alkyl hydroxylamino group, 3’- phosphorothioate, and 3-O-benzyl, or derivatives thereof.

[00996] In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with detectable reporter moiety. The detectable reporter moiety can comprise a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker which is cleavable/removable from the base. In some embodiments, at least one of the nucleotides in the plurality is not labeled with a detectable reporter moiety. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) that is attached to the nucleotide can correspond to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permit detection and identification of the nucleotide base.

[00997] In some embodiments, the cleavable linker on the nucleotide base comprises a cleavable moiety comprising an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the cleavable linker on the base is cleavable/removable from the base by reacting the cleavable moiety with a chemical agent, pH change, light or heat. In some embodiments, the cleavable moieties alkyl, alkenyl, alkynyl and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPhs)4) with piperidine, or with 2,3-Dichloro- 5,6-dicyano-l,4-benzo-quinone (DDQ). In some embodiments, the cleavable moieties aryl and benzyl are cleavable with H2 Pd/C. In some embodiments, the cleavable moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). In some embodiments, the cleavable moiety carbonate is cleavable with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the cleavable moieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine- HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[00998] In some embodiments, the cleavable linker on the nucleotide base comprises cleavable moiety including an azide, azido or azidomethyl group. In some embodiments, the cleavable moieties azide, azido and azidomethyl group are cleavable/removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[00999] In some embodiments, the chain terminating moiety (e.g., at the sugar 2’ and/or sugar 3’ position) and the cleavable linker on the nucleotide base have the same or different cleavable moieties. In some embodiments, the chain terminating moiety (e.g., at the sugar 2’ and/or sugar 3’ position) and the detectable reporter moiety linked to the base are chemically cleavable/removable with the same chemical agent. In some embodiments, the chain terminating moiety (e.g., at the sugar 2’ and/or sugar 3’ position) and the detectable reporter moiety linked to the base are chemically cleavable/removable with different chemical agents.

Multivalent Molecules

[001000] The present disclosure provides methods for sequencing any of the immobilized concatemer template molecules described herein, where the sequencing methods employ at least one multivalent molecule. In some embodiments, the multivalent molecule comprises a plurality of nucleotide arms attached to a core and having any configuration including a starburst, helter skelter, or bottle brush configuration (e.g., FIG. 1). An exemplary multivalent molecule comprises: (1) a core; and (2) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, sugar and at least one phosphate group, and the linker is attached to the nucleotide unit through the base. In some embodiments, the linker comprises an aliphatic chain or an oligo ethylene glycol chain where both linker chains having 2-6 subunits. In some embodiments, the linker also includes an aromatic moiety. An exemplary nucleotide arm is shown in FIG. 5. Exemplary multivalent molecules are shown in FIGS. 1-4. An exemplary spacer is shown in FIG. 6 and exemplary linkers are shown in FIG. 7 and FIG. 8. Exemplary nucleotides attached to a linker are shown in FIGS. 9-11. An exemplary biotinylated nucleotide arm is shown in FIG. 12.

[001001] FIG. l is a schematic of various configurations of multivalent molecules. Left (Class I): schematics of multivalent molecules having a “starburst” or “helter-skelter” configuration. Center (Class II): a schematic of a multivalent molecule having a dendrimer configuration. Right (Class III): a schematic of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide units are designated ‘N’, biotin is designated ‘B’, and streptavidin is designated ‘SA’.

[001002] In some embodiments, a multivalent molecule comprises a core attached to multiple nucleotide arms, and wherein the multiple nucleotide arms have the same type of nucleotide unit which is selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

[001003] In some embodiments, a multivalent molecule comprises a core attached to multiple nucleotide arms, where each arm includes a nucleotide unit. The nucleotide unit comprises an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1-10 phosphate groups). The plurality of multivalent molecules can comprise one type multivalent molecule having one type of nucleotide unit selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. The plurality of multivalent molecules can comprise at a mixture of any combination of two or more types of multivalent molecules, where individual multivalent molecules in the mixture comprise nucleotide units selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

[001004] In some embodiments, the nucleotide unit comprises a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, at least one nucleotide unit is a nucleotide analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoroamidite groups.

[001005] In some embodiments, the multivalent molecule comprises a core attached to multiple nucleotide arms, wherein individual nucleotide arms comprise a nucleotide unit which is a nucleotide analog having a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the nucleotide unit comprises a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the chain terminating moiety can inhibit polymerase-catalyzed incorporation of a subsequent nucleotide unit or free nucleotide in a nascent strand during a primer extension reaction. In some embodiments, the chain terminating moiety is attached to the 3’ sugar position where the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain terminating moiety is removable/cleavable from the 3’ sugar position to generate a nucleotide having a 3 ’OH sugar group which is extendible with a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the chain terminating moiety is cleavable/removable from the nucleotide unit, for example by reacting the chain terminating moiety with a chemical agent, pH change, light or heat. In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPhs)4) with piperidine, or with 2,3-Dichloro- 5,6-dicyano-l,4-benzo-quinone (DDQ). In some embodiments, the chain terminating moieties aryl and benzyl are cleavable with H2 Pd/C. In some embodiments, the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). In some embodiments, the chain terminating moiety carbonate is cleavable with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the chain terminating moieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[001006] In some embodiments, the nucleotide unit comprises a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the chain terminating moiety comprises an azide, azido or azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3’-O-azido or 3’-O-azidomethyl group. In some embodiments, the chain terminating moieties azide, azido and azidomethyl group are cleavable/removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[001007] In some embodiments, the nucleotide unit comprising a chain terminating moiety which is selected from a group consisting of 3’-deoxy nucleotides, 2’,3’- dideoxynucleotides, 3 ’-methyl, 3 ’-azido, 3 ’-azidomethyl, 3’-O-azidoalkyl, 3’-O-ethynyl, 3’- O-aminoalkyl, 3’-O-fluoroalkyl, 3 ’-fluoromethyl, 3 ’-difluoromethyl, 3 ’-trifluoromethyl, 3’- sulfonyl, 3’-malonyl, 3’-amino, 3’-O-amino, 3’-sulfhydral, 3 ’-aminomethyl, 3’-ethyl, 3’butyl, 3’-tert butyl, 3’- Fluorenylmethyloxycarbonyl, 3’ tert-Butyloxycarbonyl, 3’-O-alkyl hydroxylamino group, 3’-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

[001008] In some embodiments, the multivalent molecule comprises a core attached to multiple nucleotide arms, wherein the nucleotide arms comprise a spacer, linker and nucleotide unit, and wherein the core, linker and/or nucleotide unit is labeled with detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) that is attached to the multivalent molecule can correspond to the base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide unit to permit detection and identification of the nucleotide base.

[001009] In some embodiments, at least one nucleotide arm of a multivalent molecule has a nucleotide unit that is attached to a detectable reporter moiety. In some embodiments, the detectable reporter moiety is attached to the nucleotide base. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) that is attached to the multivalent molecule can correspond to the base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide unit to permit detection and identification of the nucleotide base.

[001010] In some embodiments, the core of a multivalent molecule comprises an avidin- like or streptavidin-like moiety and the core attachment moiety comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type moiety which includes an avidin protein, as well as any derivatives, analogs and other non-native forms of avidin that can bind to at least one biotin moiety. Other forms of avidin moieties include native and recombinant avidin and streptavidin as well as derivatized molecules, e.g. non-glycosylated avidin and truncated streptavidins. For example, avidin moiety includes de-glycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces avidinii), as well as derivatized forms, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, and the commercially-available products EXTRA VIDIN®, CAPTAVIDIN®, NEUTRA VIDIN® and NEUTRALITE AVIDIN.

[001011] In some embodiments, any of the methods for sequencing any of the immobilized concatemer template molecules described herein can include forming a binding complex, where the binding complex comprises (i) a polymerase, a concatemer template molecule duplexed with a primer, and a nucleotide, or the binding complex comprises (ii) a polymerase, a concatemer template molecule duplexed with a primer, and a nucleotide unit of a multivalent molecule. In some embodiments, the binding complex has a persistence time of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 second. The binding complex has a persistence time of greater than about 0.1-0.25 seconds, or about 0.25-0.5 seconds, or about 0.5-0.75 seconds, or about 0.75-1 second, or about 1-2 seconds, or about 2-3 seconds, or about 3-4 second, or about 4-5 seconds, and/or wherein the method is or may be carried out at a temperature of at or above 15 °C, at or above 20 °C, at or above 25 °C, at or above 35 °C, at or above 37 °C, at or above 42 °C at or above 55 °C at or above 60 °C, or at or above 72 °C, or at or above 80 °C, or within a range defined by any of the foregoing. The binding complex (e.g., ternary complex) remains stable until subjected to a condition that causes dissociation of interactions between any of the polymerase, concatemer template molecule, primer and/or the nucleotide unit or the nucleotide. For example, a dissociating condition comprises contacting the binding complex with any one or any combination of a detergent, EDTA and/or water. In some embodiments, the present disclosure provides said method wherein the binding complex is deposited on, attached to, or hybridized to, a surface showing a contrast to noise ratio in the detecting step of greater than 20. In some embodiments, the present disclosure provides said method wherein the contacting is performed under a condition that stabilizes the binding complex when the nucleotide or nucleotide unit is complementary to a next base of the concatemer template nucleic acid, and destabilizes the binding complex when the nucleotide or nucleotide unit is not complementary to the next base of the concatemer template nucleic acid.

Capture Supports

[001012] The present disclosure provides a capture support for any of the compositions and kits and any of the methods described herein, wherein the capture support comprises: (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating.

[001013] In some embodiments, the capture support further comprises a plurality of immobilized target-specific baits/probes, wherein individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence, an affinity moiety at the 5’ end of the target-specific oligonucleotide, and the target-specific oligonucleotide comprises an extendible 3’ end. In some embodiments, the affinity moiety of the targetspecific bait/probe can bind a receptor moiety embedded in the hydrophilic polymer coating of the capture support.

[001014] In some embodiments, the capture support further comprises a plurality of immobilized pinning primers. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide. In some embodiments, individual pinning primers comprise blocking group at the 3’ end of the oligonucleotide wherein the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, the 3’ end of a pinning primer comprises a moiety that promotes polymerase-catalyzed extension of the 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support.

[001015] In some embodiments, the affinity moiety of the target-specific baits/probes is the same type of affinity moiety of the pinning primers. In some embodiments, the affinity moiety of the target-specific baits/probes is a different type of affinity moiety of the pinning primers.

[001016] In some embodiments, the capture support further comprises a plurality of target-specific baits/probes and a plurality of pinning primers. In some embodiments, the capture support further comprises a plurality of target-specific baits/probes or a plurality of pinning primers.

The Support of the Capture Support

[001017] The present disclosure provides a capture support for any of the compositions and kits and any of the methods described herein wherein the capture support comprises a support. In some embodiments, the support can be solid, semi-solid, or a combination of both. In some embodiments, the support can be porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example comprising a capillary or interior surface of a capillary.

[001018] In some embodiments, the support comprises any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[001019] In some embodiments, the surface of the support can be substantially smooth and lack contours and texture. In some embodiments, the support can be regularly or irregularly contoured or textured, including protrusions, bumps, wells, etchings, pores, three- dimensional scaffolds, or any combination thereof. In some embodiments, the support comprises contours arranged in a pre-determined pattern. In some embodiments, the support comprises contours arranged in a repeating pattern. In some embodiments, the support comprises interstitial regions between the contours, where the interstitial regions are arranged in a pre-determined. In some embodiments, the interstitial regions are arranged in a repeating pattern. [001020] In some embodiments, the contours and interstitial regions can be fabricated using any combination of photo-chemical, photo-lithography, electron beam lithography, micro- or nano-imprint lithography, ink-jet printing, or micron-scale printing and/or nanoscale printing.

[001021] In some embodiments, the contours can be functionalized to promote tethering/immobilizing nucleic acid molecules including for example target-specific baits/probes, pinning primers and/or concatemer template molecules. In some embodiments, the contours can be functionalized to promote tethering/immobilizing an enzyme including for example a polymerase. In some embodiments, the interstitial regions can be modified to inhibit immobilizing nucleic acid molecules (e.g., target-specific baits/probes, pinning primers and/or concatemer template molecules) and/or for inhibiting tethering/immobilizing an enzyme (e.g., a polymerase).

[001022] In some embodiments, the capture support comprises at least one partition/barrier that creates separate regions of the capture support. For example, the partition/barrier can prevent fluid flow on one portion of the capture support. The partition/barrier can inhibit nucleic acid and/or enzyme reactions on a portion of the capture support. In some embodiments, the partition/barrier can be placed on the capture support. In some embodiments, the partition/barrier is not placed on the support but is positioned to block fluid flow onto the capture support. In some embodiments, the capture support lacks partitions/barriers that would create separate regions of the support.

[001023] In some embodiments, the capture support is passivated with at least one polymer coating formed as a continuous layer, and at least one of the polymer layers comprise a plurality of target-specific baits/probes that are randomly distributed throughout and on the polymer layer. The target-specific baits/probes can be used to generate immobilized concatemer template molecules. Thus, the immobilized concatemer template molecules are in fluid communication with each other in a massively parallel manner with no barriers to physically separate different concatemer template molecules.

[001024] In some embodiments, the support comprises at least one region (e.g., a feature) which can be functionalized to tether/immobilize nucleic acid molecules and/or enzymes. In some embodiments, the nucleic acids that can be immobilized to the support comprise target-specific baits/probes, pinning primers and/or concatemer template molecules. In some embodiments, the features are arranged on the support in a predetermined manner (e.g., patterned features). In some embodiments, the features are arranged on the support in repeating pattern. In some embodiments, any of the features for immobilizing nucleic acids and/or enzymes can be positioned on the support in a pre-determined manner using ink-jet printing, or micron-scale or nano-scale printing. In some embodiments, the features can be made in any shape including for example, circular, square, triangular or rectangular.

[001025] In some embodiments, the features are arranged on the support in a nonpredetermined manner (e.g., randomly positioned features).

[001026] In some embodiments, a support comprises a plurality of features located at random and non-predetermined positions on the support, where individual features can immobilize a nucleic acid molecule (e.g., target-specific bait/probe, pinning primer or concatemer template molecule). In some embodiments, the features on the support can be functionalized with a chemical compound or a plurality of receptor moieties for immobilizing a nucleic acid molecule (e.g., target-specific bait/probe, receptor moiety, pinning primer or concatemer template molecule).

[001027] In some embodiments, the capture support is passivated with at least one polymer coating formed as a continuous layer. In some embodiments the at least one polymer layer comprises a plurality of receptor moieties embedded in the at least one polymer layer wherein the plurality of receptor moieties are randomly distributed throughout and on the at least one polymer layer. In some embodiments, individual randomly distributed receptor moieties can bind a target-specific bait/probe or a pinning primer thereby immobilizing the target-specific baits/probes or pinning primers. Thus, the immobilized target-specific baits/probes and/or immobilized pinning primers are randomly distributed throughout and on the polymer layer. Thus, the features on the support are randomly distributed on the support. In some embodiments, the immobilized target-specific baits/probes can be used to generate immobilized concatemer template molecules. Thus, the immobilized concatemer template molecules are located at features that are randomly distributed on the support. In some embodiments, the immobilized concatemer template molecules are in fluid communication with each other in a massively parallel manner.

[001028] In some embodiments, the immobilized target-specific baits/probes can be at a high density so that some of the nearest neighbor target-specific baits/probes touch each other and/or overlap each other when viewed from any angle of the support including above, below or side views of the support.

[001029] In some embodiments, the immobilized concatemer template molecules can be at a high density so that some of the nearest neighbor concatemer template molecules touch each other and/or overlap each other when viewed from any angle of the support including above, below or side views of the support. The Coating Layer on the Capture Support

[001030] The present disclosure provides a capture support for any of the compositions and kits and any of the methods described herein wherein the capture support comprises a support having a surface that can be modified with a chemical compound that enables attachment of a polymer coating to the support. For example, the support can be modified with a silane compound. In some embodiments, the silane compound can bind a polymer coating. In some embodiments, the surface of the support can be passivated with at least one polymer coating layer (e.g., FIG. 13). In some embodiments, the support can be passivated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymer coating layers.

[001031] In some embodiments, at least one of the coating layers can form a predetermined pattern, such that the at least one coating layers is confined to one or more discrete regions on the support. For example, the coating layer may be patterned using photolithographic techniques to create an ordered array or random pattern of chemically- modified regions on the support. Alternately or in combination, the coating layer may be patterned using, for example contact printing and/or ink-jet printing techniques. In some embodiments, the coating layer can be distributed on the support in a pre-determined pattern, for example the pre-determined pattern comprises or spots arranged in rows and/or columns or other pre-determined patterns. In some embodiments, the coating layer having a predetermined pattern comprises at least one interstitial region that lacks a polymer coating. In some embodiments, the coating layer forms a porous layer or semi-porous layer. In some embodiments, the coating layer forms a continuous layer on the support wherein the coating forms no pre-determined pattern.

[001032] In some embodiments, at least one of the polymer coating layers comprises a hydrophilic polymer layer. In some embodiments, at least one polymer coating layer comprises polymer molecules having a molecular weight of 0.5K, IK, 2K, 5K or 10K. In some embodiments, at least one polymer coating layer comprises polymer molecules having a molecular weight of at least 1000 Daltons. The hydrophilic polymer coating layer can comprise polyethylene glycol (PEG). The hydrophilic polymer layer can comprise unbranched PEG. In some embodiments, the unbranched PEG comprises methoxy-PEG- acrylate, methoxy -PEG-methacrylate or acryl-PEG. The hydrophilic polymer layer can comprise branched PEG having at least 4 branches, for example the branched PEG comprises 4-16 branches. In some embodiments, the hydrophilic polymer layer comprises cross-linking or lacks cross-linking. In some embodiments, the hydrophilic polymer layer comprises cross- linking to form a hydrogel. In some embodiments, the hydrophilic polymer layer comprises a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating wherein the hydrophilic polymer layer includes cross-linking. In some embodiments, individual coating layers can be the same or different. For example, at least one of the coating layers comprises a different type of PEG compared to another coating layer. In some embodiments, at least one of the coating layers comprises branched PEG polymer and another coating layer comprises unbranched PEG polymer.

[001033] In some embodiments, the hydrophilic polymer layer comprises a monolayer having unbranched polymers which can form a brush monolayer. In some embodiments, the brush monolayer can form an extended brush monolayer. In some embodiments, the brush monolayer comprises a plurality of unbranched polymers where one end of a given unbranched polymer is attached to the support. In some embodiments, the brush monolayer comprises cross-linking. In some embodiments, the brush monolayer comprises a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating wherein the hydrophilic polymer layer includes cross-linking.

[001034] In some embodiments, the at least one coating layer has a degree of hydrophilicity which can be measured as a water contact angle, where the water contact angle is no more than 50 degrees, no more than 40 degrees, no more than 30 degrees, no more than 25 degrees, no more than 20 degrees, no more than 18 degrees, no more than 16 degrees, no more than 14 degrees, no more than 12 degrees, no more than 10 degrees, no more than 8 degrees, no more than 6 degrees, no more than 4 degrees, no more than 2 degrees, or no more than 1 degree. In some embodiments, the contact angle is no more than 40 degrees or no more than 45 degrees. The skilled artisan will recognize that a given hydrophilic coating layer of the present disclosure may exhibit a water contact angle having a value anywhere within these ranges.

[001035] In some embodiments, the capture support comprises at least one functionalized polymer coating layer. In some embodiments, the at least one functionalized polymer coating layer can be covalently bound at least to a portion of the support via a chemical group on the support. In some embodiments, the functionalized polymer coating comprises a plurality of embedded receptor moieties, and a water-soluble protective coating on the functionalized polymer coating and the embedded receptor moieties. In some embodiments, the functionalized polymer coating comprises a poly(N-(5- azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM). Receptor Moieties on the Capture Support

[001036] The present disclosure provides a capture support for any of the compositions and kits and any of the methods described herein wherein the capture support comprises a plurality of receptor moieties. In some embodiments, the plurality of receptor moieties are embedded in the hydrophilic polymer coating. In some embodiments, the receptor moieties can bind the affinity moieties of the target-specific baits/probes and the pinning primers. In some embodiments, the receptor moieties comprise one member of a binding partner and the affinity moieties comprise the other member of the binding partner. In some embodiments, the receptor moiety comprises a ligand moiety that can selectively bind its cognate affinity moiety. Exemplary binding partners comprise: biotin (and its derivatives) and its binding partners avidin, streptavidin and their derivatives; His-tags which bind nickel, cobalt or copper; cysteine, histidine, or histidine patch which bind Ni-NTA; maltose which binds with maltose binding protein (MBP); lectin-carbohydrate binding partners; calcium-calcium binding protein (CBP); acetylcholine and receptor-acetylcholine; protein A and binding partner anti-FLAG antibody; GST and binding partner glutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) protein; antigen or epitope tags which bind to antibody or antibody fragments, particularly antigens such as digoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and their respective antibodies; mouse immunoglobulin and goat anti-mouse immunoglobulin; IgG bound and protein A; receptorreceptor agonist or receptor antagonist; enzyme-enzyme cofactors; enzyme-enzyme inhibitors; and thyroxine-cortisol.

[001037] In some embodiments, the receptor moieties in the hydrophilic polymer coating comprise a streptavidin-type or avidin-type moiety which includes an avidin protein, as well as any derivatives, analogs and other non-native forms of avidin that can bind to at least one biotin moiety. Other forms of avidin moieties include native and recombinant avidin and streptavidin as well as derivatized molecules, including for example non-glycosylated avidin and truncated streptavidins. For example, avidin moiety includes de-glycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces avidinii), as well as derivatized forms, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, and the commercially-available products EXTRA VIDIN, CAPTAVIDIN, NEUTRA VIDIN and NEUTRALITE AVIDIN. Exemplary streptavidin and avidin sequences suitable for use in the receptor moieties described herein comprise any of the sequences according to SEQ ID NOS: 132-138. [001038] In some embodiments, the receptor moieties in the hydrophilic polymer coating comprise a streptavidin-type or avidin-type moiety which typically, but not necessarily, exists as a tetrameric protein comprising four monomers. In some embodiments, individual monomers can bind at least one biotin moiety. In some embodiments, binding between a streptavidin receptor moiety and a biotin affinity moiety is non-covalent. In some embodiments, the streptavidin receptor moiety can bind a biotin affinity moiety with high affinity having a dissociation constant Kd of about 10'¹⁴ to about 10'¹⁵ mol/L.

Target-Specific Baits/Probes

[001039] The present disclosure provides a plurality of target-specific baits/probes for any of the compositions and kits, and any of the methods, described herein. In some embodiments, individual target-specific baits/probes comprise oligonucleotides comprising DNA, RNA, DNA/RNA chimeric or analogs thereof. In some embodiments, the targetspecific baits/probes can be about 10 - 200 nucleotides in length, or about 20-150 nucleotides in length, or about 30-100 nucleotides in length, or about 40-90 nucleotides in length.

[001040] In some embodiments, individual target-specific baits/probes comprise an oligonucleotide comprising a target-specific sequence. In some embodiments, the targetspecific sequence that can selectively hybridize to at least a portion of a target sequence of a linear library molecule or a covalently closed circular library molecule. The function of individual target-specific baits/probes is to selectively hybridize to a target sequence of a library molecule to enrich target polynucleotides from a mixture of target and non-target polynucleotides.

[001041] In some embodiments, the plurality of target-specific baits/probes comprise oligonucleotides having the same target-specific sequence. In some embodiments, the plurality of target-specific baits/probes comprises a mixture of oligonucleotides having two or more different target-specific sequences. In some embodiments, the plurality of targetspecific baits/probes comprises 2-1,000,000 different target-specific sequences, 2-500,000 different target-specific sequences, 2-100,000 different target-specific sequences, 100- 100,000 different target-specific sequences, 500-10,000 different target-specific sequences, 2- 500 different target-specific sequences, or 1,000-50,000 different target-specific sequences, or any range therebetween.

[001042] In some embodiments, individual the target-specific bait/probes comprise a 5’ end attached to an affinity moiety (e.g., the pentagon shape in FIGS. 36A, 36B, 36C and 36D). In some embodiments, the affinity moiety can bind to a receptor moiety of the capture support. In some embodiments, the affinity moiety comprises biotin, a biotin analog or a derivative of biotin. In some embodiments, biotin analogs and derivatives comprise desthiobiotin; an iminobiotin including for example N-hydroxysuccinimide-iminobiotin (NHS-iminobiotin), amino or sulfhydryl derivatives of 2-iminobiotin, or sulfo-succinimide- iminobiotin; amidobiotin; biotin sulfone; caproylamidobiotin; biocytin; biotinyl-s- aminocaproic acid-N-hydroxysuccinimide ester; 2-biotinamidoethanethiol; biotinbromoacetylhydrazide; p-diazobenzoyl biocytin; 3-(N-maleimidopropionyl)biocytin; and 6-(6-biotinamidohexanamido)hexanoate. In some embodiments, the biotin derivative comprises DSB-X BIOTIN, which is commercially available from Molecular Probes, a division of Thermo Fisher Scientific.

[001043] In some embodiments, individual target-specific baits/probes comprise a moiety at the 3’ end that can promote polymerase-catalyzed extension. In some embodiments, individual target-specific baits/probes comprise an extendible 3’ end. In some embodiments, individual target-specific baits/probes comprise an extendible 3’ end which can be used to initiate nucleic acid amplification.

[001044] In some embodiments, individual target-specific baits/probes comprise a blocking moiety at the 3’ end that can inhibit polymerase-catalyzed extension. In some embodiments, individual target-specific baits/probes comprise a non-extendible 3’ end. Exemplary blocking moieties include a chain terminator nucleotide, a dideoxynucleotide and a phosphate group.

[001045] In some embodiments, the plurality of target-specific baits/probes can be insolution. In some embodiments, the plurality of target-specific baits/probes can be immobilized to a capture support. In some embodiments, any layer of the hydrophilic polymer coating of a capture support can include a plurality of target-specific baits/probes.

Top Strand Circularization Oligonucleotides

[001046] The present disclosure provides a plurality of top strand circularization oligonucleotides for any of the compositions and kits, and any of the methods, described herein. In some embodiments, the top strand circularization oligonucleotides are single stranded oligonucleotides comprising DNA, RNA or DNA and RNA. In some embodiments, the top strand circularization oligonucleotides can be 10-300 nucleotides in length, or 15-250 nucleotides in length, or 20-200 nucleotides in length, or 25-150 nucleotides in length.

[001047] In some embodiments, individual top strand circularization oligonucleotides comprise a blocking moiety at the 3’ end that can inhibit polymerase-catalyzed extension. In some embodiments, individual top strand circularization oligonucleotides comprise a nonextendible 3’ end. Exemplary blocking moi eties include a chain terminator nucleotide, a dideoxynucleotide and a phosphate group.

[001048] In some embodiments, individual top strand circularization oligonucleotides comprise a moiety at the 3’ end that can promote polymerase-catalyzed extension. In some embodiments, individual top strand circularization oligonucleotides comprise an extendible 3’ end. In some embodiments, individual top strand circularization oligonucleotides comprise an extendible 3’ end which can be used to initiate nucleic acid amplification.

[001049] In some embodiments, one end of a top strand circularization oligonucleotide can hybridize to one end of a top strand linear library molecule, and the other end can hybridize to the other end (or near the other end) of the tops strand library molecule. In some embodiments, a top strand library molecule comprises an insert region joined on both sides with at least one universal adaptor sequence (e.g., FIG. 20). In some embodiments, the top strand circularization oligonucleotides exhibit little or no hybridization to the insert region of a top strand linear library molecule.

[001050] In some embodiments, individual top strand circularization oligonucleotides comprise an anchor sequence at one end and a bridging sequence at the other end.

[001051] In some embodiments, the anchor sequence is configured to hybridize at or near one end of a linear library molecule, while the bridging sequence is configured to hybridize at or near an opposite end of the linear library. Selection of appropriate anchor and bridging sequences, based on the terminal sequences of top strand linear library molecules, e.g. comprising one or more universal adaptors, will be with the skill of the person of ordinary skill in the art.

[001052] In some embodiments the anchor sequence can be located near the 3’ or 5’ end of the top strand circularization oligonucleotide. In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences at one end of a top strand linear library molecule. In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences.

[001053] In some embodiments, the top strand circularization oligonucleotides comprise an anchor sequence comprising a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001054] In some embodiments, the anchor sequence comprises a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001055] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001056] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a right sample index sequence (770)).

[001057] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001058] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001059] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a first universal surface primer (730)).

[001060] In some embodiments, the anchor sequence includes or lacks a sequence that is complementary to a sample index of the top strand linear library molecule (e.g., FIG. 20, a right sample index sequence (770)).

[001061] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001062] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a left sample index sequence (760)).

[001063] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001064] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001065] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a second universal surface primer (720)).

[001066] In some embodiments, the anchor sequence includes or lacks a sequence that is complementary to a sample index of the top strand linear library molecule (e.g., FIG. 20, a left sample index sequence (760)).

[001067] Exemplary anchor sequences of a top strand circularization oligonucleotide comprise any sequence according to SEQ ID NOS: 45, 48, 49, 73, 74, 77, 84, 85, 86 and 90. [001068] In some embodiments, the top strand circularization oligonucleotide comprises a bridging sequence which can be located near the 3’ or 5’ end of the top strand circularization oligonucleotide. In some embodiments, the bridging sequence can hybridize to one or more universal adaptor sequences at one end of a top strand of the linear library molecule.

[001069] In some embodiments, the top strand circularization oligonucleotides comprise an bridging sequence comprising a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001070] In some embodiments, the bridging sequence comprises a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001071] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001072] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a right sample index sequence (770)).

[001073] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001074] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001075] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a first universal surface primer (730)).

[001076] In some embodiments, the bridging sequence lacks a sequence that is complementary to a sample index (e.g., FIG. 20, a right sample index sequence (770)).

[001077] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001078] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a left sample index sequence (760)).

[001079] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001080] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001081] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a second universal surface primer (720)).

[001082] In some embodiments, the bridging sequence lacks a sequence that is complementary to a sample index (e.g., FIG. 20, a left sample index sequence (760)). [001083] Exemplary bridging sequences of a top strand circularization oligonucleotide comprise any sequence according to SEQ ID NOS: 46, 75 and 87.

[001084] In some embodiments, the top strand circularization oligonucleotide comprises a sub-region that is designed to bind to at least a portion of a sample index sequence of a top strand linear library molecule, but the sub-region of the top strand circularization oligonucleotide lacks a sequence that is complementary to the sample index sequence. Instead, the top strand circularization oligonucleotide comprises a sub-region comprising a random sequence, at least two deoxyinosine nucleotides or a spacer (e.g., 18-carbon spacer) wherein the sub-region can bind at least a portion of a sample index sequence of a top strand linear library molecule. In some embodiments, top strand circularization oligonucleotides comprising an internal sub-region having at least two inosines (designated with “I”), where the internal sub-region is designed to bind/hybridize to a sample index sequence of a top strand linear library molecule comprise a sequence according to any of SEQ ID NOS: 51, 52, 60, 61, 62, 63, 64, 92 and 94.

[001085] In some embodiments, the ends of a top strand circularization oligonucleotide can hybridize to the ends of a top strand linear library molecule thereby generating an open circle library complex having a nick.

[001086] In some embodiments, the ends of a top strand circularization oligonucleotide can hybridize to the ends of a top strand linear library molecule thereby generating an open circle library complex having a gap.

[001087] In some embodiments, one end of a top strand circularization oligonucleotide can hybridize to one end of a top strand linear library molecule and the other end of the top strand circularization oligonucleotide can hybridize to a region near thereby generating an open circle library bait complex having a 5’ overhang flap structure. In some embodiments, the 5’ flap structure of a top strand linear library molecule comprises a sequence that is not complementary to a sequence at one end of the top strand circularization oligonucleotide. In some embodiments, the 5’ flap structure is 2-10 nucleotides in length.

[001088] In some embodiments, the top strand circularization oligonucleotides comprise any of the sequences according SEQ ID NOS: 47, 50-72, 76, 78-83, 88, 89, and 91-99. In some embodiments, the sequence of the top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of the sequences according to SEQ ID NOS: 47, 50-72, 76, 78-83, 88, 89, and 91-99.

Bottom Strand Blocker Oligonucleotides

[001089] The present disclosure provides a plurality of bottom strand blocker oligonucleotides for any of the compositions and kits, and any of the methods, described herein. In some embodiments, individual bottom strand blocker oligonucleotides comprise oligonucleotides comprising DNA, RNA, DNA/RNA chimeric or analogs thereof. In some embodiments, the bottom strand blocker oligonucleotides can be about 10 - 200 nucleotides in length, or about 20-150 nucleotides in length, or about 30-100 nucleotides in length. [001090] In some embodiments, individual bottom strand blocker oligonucleotides comprise a blocking moiety at the 3’ end that can inhibit polymerase-catalyzed extension. In some embodiments, individual bottom strand blocker oligonucleotides comprise a nonextendible 3’ end. Exemplary blocking moi eties include a chain terminator nucleotide, a dideoxynucleotide and a phosphate group. [001091] In some embodiments, individual bottom strand blocker oligonucleotides comprise a moiety at the 3’ end that can promote polymerase-catalyzed extension. In some embodiments, individual bottom strand blocker oligonucleotides comprise an extendible 3’ end. In some embodiments, individual bottom strand blocker oligonucleotides comprise an extendible 3’ end which can be used to initiate nucleic acid amplification.

[001092] In some embodiments, individual bottom strand blocker oligonucleotides comprise an anchor sequence and lack a bridging sequence. In some embodiments, the anchor sequences of individual bottom strand blocker oligonucleotides comprise a sequence that can hybridize to one or more adaptor sequences of a bottom strand linear library molecule. In some embodiments, a bottom strand library molecule comprises an insert region joined on both sides with at least one universal adaptor sequences (e.g., FIG. 20). In some embodiments, the anchor sequence of individual bottom strand blocker oligonucleotides exhibits little or no hybridization to the insert region of a bottom strand linear library molecule.

[001093] In some embodiments, the anchor sequence of a bottom strand blocker oligonucleotide is configured to hybridize at or near one end of a bottom strand linear library molecule. Selection of appropriate anchor sequences, based on the terminal sequences of a bottom strand linear library molecule, e.g. comprising one or more universal adaptors, will be with the skill of the person of ordinary skill in the art.

[001094] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001095] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)). [001096] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)). [001097] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a right sample index sequence (770)).

[001098] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001099] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001100] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a first universal surface primer (730)).

[001101] In some embodiments, the bottom strand blocker oligonucleotide includes or lacks a sequence that is complementary to a sample index of the top strand linear library molecule (e.g., FIG. 20, a right sample index sequence (770)).

[001102] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001103] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a left sample index sequence (760)).

[001104] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)). [001105] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001106] In some embodiments, the bottom strand blocker oligonucleotide comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a second universal surface primer (720)).

[001107] In some embodiments, the bottom strand blocker oligonucleotide includes or lacks a sequence that is complementary to a sample index of the top strand linear library molecule (e.g., FIG. 20, a left sample index sequence (760)).

[001108] In some embodiments, the bottom strand blocker oligonucleotides comprise any of the sequences according SEQ ID NOS: 101-129. In some embodiments, the sequence of the bottom strand blocker oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to any of the sequences according to SEQ ID NOS: 101-129.

Spike-In Circularization Oligonucleotides

[001109] The present disclosure provides a plurality of spike-in circularization oligonucleotides for any of the compositions and kits, and any of the methods, described herein. In some embodiments, individual spike-in circularization oligonucleotide comprise oligonucleotides comprising DNA, RNA, DNA/RNA chimeric or analogs thereof. In some embodiments, the spike-in circularization oligonucleotides can be about 10 - 200 nucleotides in length, or about 20-150 nucleotides in length, or about 30-100 nucleotides in length, or about 40-90 nucleotides in length.

[001110] In some embodiments, individual spike-in circularization oligonucleotides comprise a 5’ end attached to an affinity moiety. In some embodiments, the affinity moiety can bind to a receptor moiety of the capture support. In some embodiments, the affinity moiety comprises biotin, a biotin analog or a derivative of biotin. In some embodiments, biotin analogs and derivatives comprise desthiobiotin; an iminobiotin including for example N-hydroxysuccinimide-iminobiotin (NHS-iminobiotin), amino or sulfhydryl derivatives of 2- iminobiotin, or sulfo-succinimide-iminobiotin; amidobiotin; biotin sulfone; caproylamidobiotin; biocytin; biotinyl-s-aminocaproic acid-N-hydroxysuccinimide ester; 2- biotinamidoethanethiol; biotinbromoacetylhydrazide; p-diazobenzoyl biocytin; 3-(N- maleimidopropionyl)biocytin; and 6-(6-biotinamidohexanamido)hexanoate. In some embodiments, the biotin derivative comprises DSB-X BIOTIN, which is commercially available from Molecular Probes, a division of Thermo Fisher Scientific.

[001111] In some embodiments, individual spike-in circularization oligonucleotides comprise a moiety at the 3’ end that can promote polymerase-catalyzed extension. In some embodiments, individual target-specific baits/probes comprise an extendible 3’ end. In some embodiments, individual target-specific baits/probes comprise an extendible 3’ end which can be used to initiate nucleic acid amplification.

[001112] In some embodiments, individual spike-in circularization oligonucleotides comprise a reversible blocking moiety at the 3’ end that can inhibits polymerase-catalyzed extension. In some embodiments, the reversible blocking moiety can be removed or converted into a 3 ’OH moiety.

[001113] In some embodiments, one end of a spike-in circularization oligonucleotide can hybridize to one end of a top strand linear library molecule, and the other end can hybridize to the other end of the tops strand library molecule. In some embodiments, a top strand library molecule comprises an insert region joined on both sides with at least one universal adaptor sequences (e.g., FIG. 20). In some embodiments, the spike-in circularization oligonucleotides exhibit little or no hybridization to the insert region of a top strand linear library molecule.

[001114] In some embodiments, individual spike-in circularization oligonucleotides comprise an anchor sequence at one end and a bridging sequence at the other end.

[001115] In some embodiments, the anchor sequence is configured to hybridize at or near one end of a linear library molecule, while the bridging sequence is configured to hybridize at or near an opposite end of the linear library. Selection of appropriate anchor and bridging sequences, based on the terminal sequences of top strand linear library molecules, e.g. comprising one or more universal adaptors, will be with the skill of the person of ordinary skill in the art.

[001116] In some embodiments the anchor sequence of a can be located near the 5’ end of the spike-in circularization oligonucleotide. In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences at one end of a top strand linear library molecule. In some embodiments, the anchor sequence can hybridize to one or more universal adaptor sequences of the top strand linear library molecule and inhibit hybridization of another oligonucleotide to the same universal adaptor sequences. [001117] In some embodiments, the spike-in circularization oligonucleotides comprise an anchor sequence comprising a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001118] In some embodiments, the anchor sequence comprises a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001119] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001120] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a right sample index sequence (770)).

[001121] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001122] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001123] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a first universal surface primer (730)). [001124] In some embodiments, the anchor sequence includes or lacks a sequence that is complementary to a sample index of the top strand linear library molecule (e.g., FIG. 20, a right sample index sequence (770)).

[001125] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001126] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a left sample index sequence (760)).

[001127] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001128] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001129] In some embodiments, the anchor sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a second universal surface primer (720)).

[001130] In some embodiments, the anchor sequence includes or lacks a sequence that is complementary to a sample index of the top strand linear library molecule (e.g., FIG. 20, a left sample index sequence (760)).

[001131] Exemplary anchor sequences of a spike-in circularization oligonucleotide comprise any sequence according to SEQ ID NOS: 45, 48, 49, 73, 74, 77, 84, 85, 86 and 90. [001132] In some embodiments, the spike-in circularization oligonucleotide comprises a bridging sequence which can be located near the 3’ end. In some embodiments, the bridging sequence can hybridize to one or more universal adaptor sequences at one end of a top strand of the linear library molecule. [001133] In some embodiments, the spike-in circularization oligonucleotides comprise an bridging sequence comprising a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001134] In some embodiments, the bridging sequence comprises a sequence that is complementary to the entire length of universal adaptor sequences and sample index sequence on one side of the insert region (e.g., FIG. 20, a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001135] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750), a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001136] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a right sample index sequence (770)).

[001137] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a reverse sequencing primer (750) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001138] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a right sample index sequence (770) and a universal adaptor sequence for binding a first universal surface primer (730)).

[001139] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a first universal surface primer (730)). [001140] In some embodiments, the bridging sequence lacks a sequence that is complementary to a sample index (e.g., FIG. 20, a right sample index sequence (770)).

[001141] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740), a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001142] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a left sample index sequence (760)).

[001143] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a left sample index sequence (760) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001144] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a forward sequencing primer (740) and a universal adaptor sequence for binding a second universal surface primer (720)).

[001145] In some embodiments, the bridging sequence comprises a sequence that is complementary to a portion of a universal adaptor sequence on one side of the insert region (e.g., FIG. 20, at least a portion of a universal adaptor sequence for binding a second universal surface primer (720)).

[001146] In some embodiments, the bridging sequence lacks a sequence that is complementary to a sample index (e.g., FIG. 20, a left sample index sequence (760)).

[001147] Exemplary bridging sequences of a spike-in circularization oligonucleotide comprise any sequence according to SEQ ID NOS: 46, 75 and 87.

[001148] In some embodiments, the spike-in circularization oligonucleotide comprises a sub-region that is designed to bind to at least a portion of a sample index sequence of a top strand linear library molecule, but the sub-region of the spike-in circularization oligonucleotide lacks a sequence that is complementary to the sample index sequence. Instead, the spike-in circularization oligonucleotide comprises a sub-region comprising a random sequence, at least two deoxyinosine nucleotides or a spacer (e.g., 18-carbon spacer) wherein the sub-region can bind at least a portion of a sample index sequence of a top strand linear library molecule.

[001149] In some embodiments, the ends of a spike-in circularization oligonucleotide can hybridize to the ends of a top strand linear library molecule thereby generating an open circle library complex having a nick.

[001150] In some embodiments, the spike-in circularization oligonucleotides comprise a sequence according to SEQ ID NOS: 47, 50-72, 76, 78-83, 88, 89, and 91-100. In some embodiments, the sequence of the spike-in circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end relative to the sequences according to SEQ ID NOS: 47, 50-72, 76, 78-83, 88, 89, and 91-100.

Internucleoside Linkages of Target-Specific Baits/Probes, Pinning Primers, Top Strand Circularization Oligonucleotides and Spike-in Circularization Oligonucleotides

[001151] The present disclosure provides a plurality of target-specific baits/probes, a plurality of pinning primers, a plurality of top strand circularization oligonucleotides, and a plurality of spike-in circularization oligonucleotides for any of the compositions and kits, and any of the methods, described herein, wherein individual target-specific baits/probes, individual of pinning primers, individual top strand circularization oligonucleotides and individual spike-in circularization oligonucleotides comprise an oligonucleotide. In some embodiments, at least one of the target-specific baits/probes and/or pinning primers and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprise an oligonucleotide having one or more naturally occurring internucleoside linkages and one or more naturally occurring furanose sugar moieties. In some embodiments, the one or more naturally occurring internucleoside linkages in the oligonucleotide comprise phosphodiester linkages. In some embodiments, the oligonucleotide comprises at least one modified internucleoside linkages and/or at least one modified furanose sugar moieties. In some embodiments, the oligonucleotide comprising one or more modified internucleoside linkage and/or one or more modified furanose sugar moieties can hybridize to a polynucleotide having a target-specific sequence or a universal adaptor sequence.

[001152] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or a top strand circularization oligonucleotide and/or spike-in circularization oligonucleotides comprises one or more modified internucleoside linkages that retain or lack a phosphorus atom. [001153] In some embodiments, the modified internucleoside linkages that retain a phosphorus atom include phosphorothioates, chiral phosphorothioates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkyl-phosphoramidates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates, 5 '-alkylene phosphonates and chiral phosphonates, phosphinates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, phosphonoacetate and thiophosphonoacetate. In some embodiments, the modified internucleoside linkages that retain a phosphorus atom include selenophosphates and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs thereof, and those linkages having inverted polarity wherein one or more internucleoside linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.

[001154] In some embodiments, the modified internucleoside linkages that lack a phosphorus atom include oligonucleotides comprising backbones formed with short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl internucleoside linkages, mixed heteroatom and cycloalkyl intemucleoside linkages, short chain heteroatom internucleoside linkages, or heterocyclic internucleoside linkages. In some embodiments, the modified internucleoside linkages that lack a phosphorus atom comprise morpholino linkages including thiomorpholino linkage, phosphorodiamidate morpholino linkage and morpholino 3’ phosphoramidate linkages. In some embodiments, the modified intemucleoside linkages that lack a phosphorus atom comprise sulfide linkage, sulfoxide linkage, sulfone linkage; sulfonate linkage, sulfonamide linkage, formacetyl linkage, thioformacetyl linkage, methylene formacetyl linkage, thioformacetyl linkage, riboacetyl linkage, alkene containing linkage, sulfamate linkage; methyleneimino linkage, methylenehydrazino linkage, or amide linkage.

Modified Sugar Moieties of Target-Specific Baits/Probes, Pinning Primers, Top strand Circularization Oligonucleotides and Spike-in Circularization Oligonucleotides [001155] The present disclosure provides a plurality of target-specific baits/probes, a plurality of pinning primers, a plurality of top strand circularization oligonucleotides, and a plurality of spike-in circularization oligonucleotides for any of the compositions and kits and any of the methods described herein wherein individual target-specific baits/probes, pinning primers, top strand circularization oligonucleotides and spike-in circularization oligonucleotides comprise an oligonucleotide. In some embodiments, at least one of the target-specific baits/probes and/or pinning primers and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprises an oligonucleotide having at least one naturally occurring furanose sugar moiety or at least one modified furanose sugar moiety. In some embodiments, the oligonucleotide comprises an oligonucleotide mimetic.

[001156] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or a top strand circularization oligonucleotide and/or spike-in circularization oligonucleotides comprises a peptide nucleic acid (PNA) comprising a sugar- backbone replaced with an amide-containing backbone. In some embodiments, the peptide nucleic acid retains one or more heterocyclic base moiety for base pairing and hybridization. In some embodiments, the peptide nucleic acid has little or no charge and exhibits little or no electrostatic repulsion when hybridizing with target polynucleotides.

[001157] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or a top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprises a cyclohexenyl nucleic acid (CeNA) in which a furanose sugar ring is replaced with a cyclohenyl ring. In some embodiments, phosphoramidite chemistry can be used to prepare cyclohexenyl nucleic acids from CeNA DMT-protected phosphoramidite monomers.

[001158] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or a top strand circularization oligonucleotide and/or spike-in circularization oligonucleotides comprises at least one bicyclic sugar moiety to form a locked nucleic acid (LNA). In some embodiments, the 2’ hydroxyl group of the ribosyl sugar ring can be linked to the 4’ carbon atom of the sugar ring to form an LNA having a 2’-C,4’-C- oxymethylene linkage. In some embodiments, the 2’ -4’ linkage comprises a methylene group. In some embodiments, the 3’ hydroxyl group of the ribosyl sugar ring can be linked to the 4’ carbon atom of the sugar ring to form an LNA having a 3’-C,4’-C-oxymethylene linkage. In some embodiments, the 2’ -4’ linkage comprises an ethylene group to form an ethylene bridged nucleic acid which is referred to as an ENA.

[001159] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or a top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprises a bridged nucleic acid (BNA) comprising a bridge at the 2’, 4’ -position of the ribose sugar. In some embodiments, oligonucleotides that include at least one BNA exhibit increased thermal stability when hybridized to a complementary strand, and increase resistance to exonucleases and endonucleases. Phosphorodiamidate Morpholino Oligonucleotides of Target-Specific Baits/Probes, Pinning Primers, Top Strand Circularization Oligonucleotides and Spike-in Circularization Oligonucleotides

[001160] The present disclosure provides a plurality of target-specific baits/probes, a plurality of pinning primers, a plurality of top strand circularization oligonucleotides, and a plurality of spike-in circularization oligonucleotides for any of the compositions and kits, and any of the methods described herein, wherein individual target-specific baits/probes, pinning primers and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprise an oligonucleotide. In some embodiments, at least one of the target-specific baits/probes and/or pinning primers and/or top strand circularization oligonucleotides and/or a plurality of spike-in circularization oligonucleotides comprises an oligonucleotide having a phosphodiamidate morpholino oligonucleotide (PMO) wherein individual monomers in the PMO comprise DNA bases attached to a backbone of sixmembered methylenemorpholine rings which replace ribose or deoxyribose, where the methylenemorpholine rings are linked through phosphorodiamidate groups. In some embodiments, the DNA bases comprise adenine, cytosine, guanine or thymine. In some embodiments, the phosphodiamidate morpholino oligonucleotides are resistant to nuclease degradation. In some embodiments, the phosphodiamidate morpholino oligonucleotides exhibit increased duplex stability when hybridized to RNA.

Degradation-Resistant Target-Specific Baits/Probes, Pinning Primers, Top Strand Circularization Oligonucleotides and Spike-in Circularization Oligonucleotides [001161] The present disclosure provides a plurality of target-specific baits/probes and/or a plurality of pinning primers and/or a plurality of top strand circularization oligonucleotides and/or a plurality of spike-in circularization oligonucleotides for any of the compositions and kits and any of the methods described herein wherein individual targetspecific baits/probes, individual pinning primers and/or individual top strand circularization oligonucleotides and/or individual spike-in circularization oligonucleotides comprise an oligonucleotide. In some embodiments, at least one of target-specific baits/probes and/or pinning primers and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprise an oligonucleotide having a terminal 5’ and/or 3’ protective moiety that confers resistance to exonuclease degradation. In some embodiments, the oligonucleotide comprises at least one internal nucleoside moiety to confer resistance to endonuclease degradation. In some embodiments, the oligonucleotide lacks a terminal 5’ and 3’ protective moiety, and lacks an internal protective moiety.

[001162] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprises one or more phosphorothioate linkage at or near the 5’ and/or 3’ ends to confer exonuclease resistance. In some embodiments, the oligonucleotide comprises one or more phosphorothioate linkage at an internal position to confer endonuclease resistance. In some embodiments, the oligonucleotide comprises one or more 2’-O-methylcytosine bases at or near the 5’ and/or 3’ ends, or at an internal position.

5’ Protective Moieties of Target-Specific Baits/Probes, Pinning Primers, Top Strand Circularization Oligonucleotides and Spike-in Circularization Oligonucleotides

[001163] The present disclosure provides a plurality of target-specific baits/probes and/or a plurality of pinning primers and/or a plurality of top strand circularization oligonucleotides and/or a plurality of spike-in circularization oligonucleotides for any of the compositions and kits and any of the methods described herein wherein individual targetspecific baits/probes and/or individual pinning primers and/or individual top strand circularization oligonucleotides and/or individual spike-in circularization oligonucleotides comprise an oligonucleotide. In some embodiments, at least one of the target-specific baits/probes and/or pinning primers and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprise an oligonucleotide having a moiety at the 5’ end that confers resistance to exonuclease degradation.

[001164] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or top strand circularization oligonucleotides and/or a plurality of spike-in circularization oligonucleotides comprises a 5’ end that is phosphorylated or nonphosphorylated.

[001165] In some embodiments, the terminal 5’ end of the oligonucleotide comprises an inverted nucleotide moiety including for example, a 3 '-3 '-inverted nucleotide moiety or a 3'- 2'-inverted nucleotide moiety.

[001166] In some embodiments, the terminal 5’ end of the oligonucleotide comprises an inverted abasic nucleotide moiety including for example, a 3 '-3 '-inverted abasic moiety or a 3 '-2 '-inverted abasic moiety. [001167] In some embodiments, the terminal 5’ end of the oligonucleotide comprises an acyclic nucleotide, including for example, an acyclic 3',4'-seco nucleotide, an acyclic 3,4- dihydroxybutyl nucleotide, or an acyclic 3,5-dihydroxypentyl nucleotide.

[001168] In some embodiments, the terminal 5’ end of the oligonucleotide comprises a modified nucleotide including for example, a 4',5'-methylene nucleotide, a l-(beta-D- erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1,5- anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl nucleotide, a 1,4-butanediol phosphate, a 3'- phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a 3 '-phosphate, a 3'- phosphorothioate, a phosphorodithioate, or a bridging or non-bridging methylphosphonate moiety.

3’ Protective Moieties of Target-Specific Baits/Probes, Pinning Primers, Top Strand Circularization Oligonucleotides and Spike-in Circularization Oligonucleotides [001169] The present disclosure provides a plurality of target-specific baits/probes and/or a plurality of pinning primers and/or a plurality of top strand circularization oligonucleotides and/or a plurality of spike-in circularization oligonucleotides for any of the compositions and kits, and any of the methods described herein, wherein individual targetspecific baits/probes and/or individual pinning primers and/or individual top strand circularization oligonucleotides and/or individual spike-in circularization oligonucleotides comprise an oligonucleotide. In some embodiments, at least one of the target-specific baits/probes and/or pinning primers and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprise an oligonucleotide having a moiety at the 3’ end that confers resistance to exonuclease degradation.

[001170] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprises a terminal 3’ end having a 5 '-amino-alkyl phosphate, a l,3-diamino-2-propyl phosphate, a 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecyl phosphate, a hydroxypropyl phosphate, or a 1,4-butanediol phosphate.

[001171] In some embodiments, the terminal 3’ end of the oligonucleotide comprises an acyclic nucleotide, including for example an acyclic 3',4'-seco nucleotide. [001172] In some embodiments, the terminal 3’ end of the oligonucleotide comprises an inverted nucleotide, including for example a 5 '-5 '-inverted nucleotide moiety, or a 5 '-5'- inverted abasic moiety.

[001173] In some embodiments, the terminal 3’ end of the oligonucleotide comprises a modified nucleotide including for example, a 4',5'-methylene nucleotide, a l-(beta-D- erythrofuranosyl) nucleotide, a 4'-thio nucleotide, carbocyclic nucleotide, a 1,5- anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a threo-pentofuranosyl nucleotide, a 3,4-dihydroxybutyl nucleotide, or a 3,5-dihydroxypentyl nucleotide.

Non-Extendible 3’ Ends of Target- Specific Baits/Probes, Pinning Primers, Top Strand Circularization Oligonucleotides and Spike-in Circularization Oligonucleotides [001174] The present disclosure provides a plurality of target-specific baits/probes and/or a plurality of pinning primers and/or a plurality of top strand circularization oligonucleotides and/or a plurality of spike-in circularization oligonucleotides for any of the compositions and kits and any of the methods described herein wherein individual targetspecific baits/probes and/or individual pinning primers and/or individual top strand circularization oligonucleotides and/or individual spike-in circularization oligonucleotides comprise an oligonucleotide. In some embodiments, at least one of the target-specific baits/probes and/or pinning primers and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprises an oligonucleotide having a terminal 3 ’OH group which enables primer extension.

[001175] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer and/or top strand circularization oligonucleotides and/or spike-in circularization oligonucleotides comprises a terminal 3’ end having a 3’ blocking group which inhibits primer extension. In some embodiments, the oligonucleotide comprises a terminal 3’ end having a 3’ dideoxynucleotide (e.g., 3’ddC), an inverted dT, a 3’ C3-spacer, a 3’ amino group, or a 3’ phosphorylated end.

[001176] In some embodiments, the terminal 3’ blocking group on the oligonucleotide comprises a blocking group selected from a group consisting of an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, an acetal group or silyl group. [001177] In some embodiments, the terminal 3’ blocking group comprises a 3’-O- azidomethyl group, 3’-O-methyl group, 3’-O-alkyl hydroxylamino group, a 3’- phosphorothioate group, a 3’-O-malonyl group, or a 3’-O-benzyl group.

[001178] In some embodiments, the terminal 3’ blocking group can be removed by reacting with a chemical reagent. For example, the terminal 3’ blocking groups alkyl, alkenyl, alkynyl and allyl are reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPhs)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-l,4-benzo-quinone (DDQ). The terminal 3’ blocking groups aryl and benzyl are reactive with Pd/C. The terminal 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide are reactive with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). The terminal 3’ blocking group carbonate is reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The terminal 3’ blocking groups urea and silyl are reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[001179] In some embodiments, the oligonucleotide of a target-specific bait/probe and/or a pinning primer comprises a terminal 3’ blocking group comprising an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl terminal 3’ blocking group can be reactive with a chemical reagent. In some embodiments, the chemical agent comprises a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

Bisulfite Sequencing with Target-Specific Baits/Probes

[001180] In some embodiments, at least one of the target-specific baits/probes comprises a sequence that can selectively hybridize to an insert region of a library molecule wherein the insert region comprises DNA having a non-methylated target sequence or a methylated target sequence. In some embodiments, the methylated target sequence comprises at least one modified cytosine base wherein the 5-position of the cytosine base comprises a hydroxymethyl (hm) or formyl (f) group. In some embodiments, the at least one modified cytosine base comprises a methylated cytosine base. In some embodiments, the at least one methylated cytosine base comprises, but is not limited to, a 5-methylcytosine (5mC), a 5- hydroxymethylcytosine (5hmC) or a 5-formylcytosine (5fC). In some embodiments, the at least one methylated cytosine base can be converted to a uracil base upon treatment with a bisulfite reagent, an oxidizing reagent and a bisulfite reagent, or a reducing reagent and a bisulfite reagent.

[001181] In some embodiments, at least one of the target-specific baits/probes comprises a sequence that is partially or fully complementary to the insert region of a library molecule having at least one non-methylated cytosine base that is converted to a uracil base upon treatment with a bisulfite reagent, or treatment with an oxidizing reagent and a bisulfite reagent, or treatment with a reducing reagent and a bisulfite reagent.

[001182] In some embodiments, at least one of the target-specific baits/probes comprises a sequence that is partially or fully complementary to the insert region of a library molecule having at least one methylated cytosine base (e.g., a 5-hydroxymethylcytosine (5hmC)) that is converted to a uracil base upon treatment with an oxidizing reagent and a bisulfite reagent.

[001183] In some embodiments, at least one of the target-specific baits/probes comprises a sequence that is partially or fully complementary to the insert region of a library molecule having at least one methylated cytosine base (e.g., 5-formylcytosine (5fC)) that is converted to a uracil base upon treatment with a bisulfite reagent, or treatment with an oxidizing reagent and a bisulfite reagent.

[001184] In some embodiments, at least one of the target-specific baits/probes comprises a sequence that is partially or fully complementary to the insert region of a library molecule having at least one methylated cytosine base (e.g., a 5-methylcytosine (5mC)) that is retained as a methylated cytosine base upon treatment with a bisulfite reagent, or treatment with an oxidizing reagent and a bisulfite reagent, or treatment with a reducing reagent and a bisulfite reagent.

[001185] In some embodiments, the insert region of the library molecule comprises DNA having a target sequence of interest, wherein the DNA can be obtained from any source. In some embodiments, target DNA sequences carrying non-methylated cytosines can be distinguished from target DNA sequences carrying methylated cytosines by conducting a bisulfite workflow.

[001186] In some embodiments, the bisulfite workflow comprises the step (a) obtaining a DNA sample (e.g., a polynucleotide sample) comprising at least one methylated cytosine base; step (b) dividing the DNA sample into four sub-populations comprising a first, second, third and fourth sub-population of DNA samples; step (c) contacting the first sub-population of DNA sample with a bisulfite reagent under a condition suitable for converting a non- methylated cytosine base into a uracil base, retaining a 5-methylcytosine (5mC) base, retaining a 5-hydroxymethylcytosine (5hmC) base, and suitable for converting a 5- formylcytosine (5fC) base into a uracil base; step (d) contacting the second sub-population with an oxidation reagent and then a bisulfite reagent under a condition suitable for converting a non-methylated cytosine base into a uracil base, retaining a 5-methylcytosine (5mC) base, converting a 5-hydroxymethylcytosine (5hmC) base into a uracil base, and converting a 5-formylcytosine (5fC) base into a uracil base; step (e) contacting the third subpopulation of DNA sample with a reducing reagent and then a bisulfite reagent under a condition suitable for converting a non-methylated cytosine base into a uracil base, retaining a 5-methylcytosine (5mC) base, retaining a 5-hydroxymethylcytosine (5hmC) base, and for retaining a 5-formylcytosine (5fC) base, wherein the fourth sub-population of DNA sample is not contacted with a bisulfite reagent, an oxidizing reagent or a reducing reagent; step (f) generating a first, second, third and fourth sub-population of library molecules by appending universal adaptor sequences to the ends of the first, second, third and fourth sub-populations of DNA samples; step (g) enriching for a plurality of target polynucleotides from the first, second, third and fourth sub-populations of library molecules by hybridizing the first, second, third and fourth sub-populations of library molecules with a plurality of target-specific baits/probes and conducting any of the hybridization-based enrichment workflows disclosed herein (e.g., any of enrichment workflows (1) - (10)) to generate a plurality of circle bait complexes immobilized to a capture support, wherein individual circle bait complexes comprise (i) a covalently closed circular library molecule comprising a polynucleotide having a target sequence and at least one universal adaptor sequence, and (ii) a target-specific bait/probe that is selectively hybridized to at least a portion of a corresponding target sequence of a covalently closed circular library molecule; step (h) conducting a rolling circle amplification reaction using the target-specific bait/probe to initiate amplification thereby generating a plurality of immobilized concatemer template molecules, including concatemer template molecules generated from the first, second, third or fourth sub-population of library molecules; step (i) sequencing the plurality of immobilized concatemer template molecules; step (j) identifying the position of at least one uracil base in any of the immobilized concatemer template molecules thereby identifying the position of a non-methylated cytosine base, and a methylated cytosine base which comprises a 5-hydroxymethylcytosine (5hmC) base or a 5-formylcytosine (5fC) base; and step (k) identifying the position of at least one cytosine base in any of the of the immobilized concatemer template molecules thereby identifying the position of a 5-methylcytosine (5mC) base. [001187] In some embodiments, the bisulfite reagent comprises NaHSCh. In some embodiments, the oxidizing reagent comprises potassium perruthenate (KRuO4) and other metal perruthenates; tetraalkylammonium perruthenates, such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TBAP); or polymer-supported perruthenate (PSP) and tetraphenylphosphonium ruthenate. In some embodiments, the reducing reagent comprises NaBH4, NaCNBEU or LiBH4.

[001188] Methods for treating DNA samples with a bisulfite reagent, an oxidizing reagent and a bisulfite reagent, and a reducing reagent and a bisulfite reagent, are described in U.S. patent No. 9,290,807, which is hereby incorporated by reference in its entirety.

Kits

[001189] The present disclosure provides kits for conducting any of the hybridizationbased methods for capture and enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences.

[001190] In some embodiments, the kits comprise at least one capture support. In some embodiments, the capture support comprises (i) a support coated with at least one coating layer and (ii) a plurality of receptor moieties embedded in the at least one coating layer. In some embodiments, the support comprises a chemical compound that enables attachment of a coating layer to the support. In some embodiments, the support comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 coating layers. In some embodiments, the at least one coating layer comprises a hydrophilic polymer coating. In some embodiments, the hydrophilic polymer coating comprises polyethylene glycol (PEG). In some embodiments, the at least one coating layer comprises branched or non-branched hydrophilic polymers. In some embodiments, the hydrophilic polymer coating is cross-linked or non-cross-linked. In some embodiments, the plurality of receptor moieties is embedded in the at least one coating layer. In some embodiments, individual receptor moieties comprise streptavidin or avidin or derivatives thereof. In some embodiments, the receptor moieties comprise any of the sequences according to SEQ IDS: 132-138. In some embodiments, the kit comprises any of the capture supports described herein.

[001191] In some embodiments, the capture support comprises a plurality of pinning primers. In some embodiments, the plurality of pinning primers can be embedded in the at least one coating layer. In some embodiments, individual pinning primers comprise an oligonucleotide having a universal pinning sequence, and an affinity moiety at the 5’ end. In some embodiments, individual pinning primers comprise a non-extendible 3’ end. In some embodiments, individual pinning primers comprise an extendible 3’ end. In some embodiments, the affinity moiety of individual pinning primers can bind an embedded receptor moiety of the capture support. In some embodiments, the affinity moiety of individual pinning primers comprise biotin, desthiobiotin or iminobiotin. In some embodiments, the capture surface lacks a plurality of immobilized pinning primers. In some embodiments, the pinning primers comprise a sequence according to SEQ ID NO: 1. In some embodiments, the pinning primer sequence can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to the sequence of any of the pinning primers described herein. In some embodiments, the kit comprises any of the pinning primers described herein.

[001192] In some embodiments, the kit comprises a loading reagent for distributing oligonucleotides onto the capture support. In some embodiments, the loading reagent can be used for distributing onto the capture support any one or any combination of oligonucleotides comprising: the covalently closed library molecules, the linear library molecules, the targetspecific baits/probes, the linear library molecules, the top strand circularization oligonucleotides, the bottom strand blocker oligonucleotides and/or the spike-in circularization oligonucleotides. In some embodiments, the loading reagent comprises at least one solvent and any combination of two or more of the following compounds: at least one pH buffering agent, at least one monovalent cation, a chaotropic agent, a detergent, a reducing agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and/or a crowding agent. [001193] In some embodiments, the kit comprises instructions for conducting the hybridization-based methods. In some embodiments, the instructions can be provided in printed form. In some embodiments, the instructions can be as an electronic storage data file present on a suitable computer readable storage medium including but not limited to a CD, DVD or USB. In some embodiments, the instructions can be provided in the form of a website address on the internet where instructions can be obtained. In some embodiments, the instructions can be viewed at the website address or can be downloaded from the internet.

[001194] In some embodiments, the kits comprise a plurality of oligonucleotides for conducting any of the hybridization-based methods for capture and enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences. In some embodiments, plurality of oligonucleotides comprise a plurality of targetspecific baits/probes. Individual target-specific baits/probes comprise a target-specific sequence that can selectively hybridize to a target polynucleotide (e.g., insert region) of a linear library molecule. In some embodiments, the plurality of target-specific baits/probes comprises two or more different target-specific sequences. In some embodiments, individual target-specific baits/probes comprise an affinity moiety at the 5’ end and an extendible 3’ end. In some embodiments, the affinity moiety of the target-specific bait/probe can bind a receptor moiety of the capture support.

[001195] In some embodiments, the kit comprises any one or any combination of two or more target-specific baits/probes described herein. In some embodiments, the kit comprises at least one container comprising the plurality of target-specific baits/probes.

[001196] In some embodiments, the kit comprises a hybridization reagent for hybridizing the target-specific baits/probes to the linear library molecules. In some embodiments, the hybridization reagent comprises at least one solvent and any combination of two or more of the following: at least one pH buffering agent, at least one monovalent cation, a chaotropic agent, a detergent, a reducing agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and/or a crowding agent.

[001197] In some embodiments, the kit comprises at least one container comprising the hybridization reagent. In some embodiments, the kit comprises a single container or separate containers for holding the plurality of the target-specific baits/probes and the hybridization reagent.

[001198] In some embodiments, the kit comprises instructions for conducting the hybridization-based methods. In some embodiments, the instructions can be provided in printed form. In some embodiments, the instructions can be as an electronic storage data file present on a suitable computer readable storage medium including but not limited to a CD, DVD or USB. In some embodiments, the instructions can be provided in the form of a website address on the internet where instructions can be obtained. In some embodiments, the instructions can be viewed at the website address or can be downloaded from the internet.

[001199] In some embodiments, the kit comprises a plurality of oligonucleotides for conducting any of the hybridization-based methods for capture and enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences. In some embodiments, plurality of oligonucleotides comprises a plurality of top strand circularization oligonucleotides. Individual top strand circularization oligonucleotides can comprise a first region that can hybridize with a sequence at one end of a linear library molecule and a second region that can hybridize with a sequence at the other end of the same linear library molecule. In some embodiments, the top strand circularization oligonucleotides comprise a 3’ non-extendible end. In some embodiments, the top strand circularization oligonucleotides comprise a 3’ extendible end. In some embodiments, the kit comprises any one or any combination of two or more top strand circularization oligonucleotides described herein. In some embodiments, the first region of a top strand circularization oligonucleotide comprises any one of sequences according to SEQ ID NOS: 45, 48, 49, 73, 74, 77, 84, 85, 86 or 90. In some embodiments, the second region of a top strand circularization oligonucleotide comprises any one of sequences according to SEQ ID NOS: 46, 75 or 87. In some embodiments, the top strand circularization oligonucleotide comprises any one of sequences according to SEQ ID NOS: 47, 50-72, 76-83, or 88-99. In some embodiments, the sequence of the full length top strand circularization oligonucleotides can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to the sequences of any of the top strand circularization oligonucleotides described herein. In some embodiments, the kit comprises at least one container comprising the plurality of top strand circularization oligonucleotides.

[001200] In some embodiments, the kit comprises a plurality of target-specific baits/probes and a plurality of top strand circularization oligonucleotides. In some embodiments, the kit comprises at least two containers, the first container comprising the plurality of target-specific baits/probes, and the second container comprising the plurality of top strand circularization oligonucleotides. In some embodiments, the kit comprises the plurality of target-specific baits/probes and the plurality of top strand circularization oligonucleotides in the same container. In some embodiments, the kit comprises a hybridization reagent for hybridizing the top strand circularization oligonucleotide to the linear library molecules. In some embodiments, the hybridization reagent comprises at least one solvent and any combination of two or more of the following: at least one pH buffering agent, at least one monovalent cation, a chaotropic agent, a detergent, a reducing agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and/or a crowding agent. In some embodiments, the kit comprises at least one container comprising the hybridization reagent. In some embodiments, the kit comprises a single container or separate containers for holding the plurality of the top strand circularization oligonucleotides and the hybridization reagent. In some embodiments, the kit comprises instructions for conducting the hybridization-based methods. In some embodiments, the instructions can be provided in printed form. In some embodiments, the instructions can be as an electronic storage data file present on a suitable computer readable storage medium including but not limited to a CD, DVD or USB. In some embodiments, the instructions can be provided in the form of a website address on the internet where instructions can be obtained. In some embodiments, the instructions can be viewed at the website address or can be downloaded from the internet.

[001201] In some embodiments, the kits comprise a plurality of oligonucleotides for conducting any of the hybridization-based methods for capture and enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences. In some embodiments, plurality of oligonucleotides comprises a plurality of bottom strand blocker oligonucleotides. Individual bottom strand blocker oligonucleotides can hybridize with a sequence at one end of a linear library molecule. In some embodiments, the kit comprises any one or any combination of two or more bottom strand blocker oligonucleotides described herein. In some embodiments, the bottom strand blocker oligonucleotide comprises any one of sequences according to SEQ ID NOS: 101-129. In some embodiments, the sequence of the bottom strand blocker oligonucleotides can be truncated 1- 10 nucleotides on the 5’ or 3’ end compared to the sequences of any of the bottom strand blocker oligonucleotides described herein. In some embodiments, the kit comprises at least one container comprising the plurality of bottom strand blocker oligonucleotides.

[001202] In some embodiments, the kit comprises a plurality of top strand circularization oligonucleotides and a plurality of bottom strand blocker oligonucleotides. In some embodiments, the kit comprises at least two containers, the first container comprising the plurality of top strand circularization oligonucleotides, and the second container comprising the plurality of bottom strand blocker oligonucleotides. In some embodiments, the kit comprises the plurality of top strand circularization oligonucleotides and the plurality of bottom strand blocker oligonucleotides in the same container. In some embodiments, the kit comprises a hybridization reagent for hybridizing the bottom strand blocker oligonucleotide to the linear library molecules.

[001203] In some embodiments, the hybridization reagent comprises at least one solvent and any combination of two or more of the following: at least one pH buffering agent, at least one monovalent cation, a chaotropic agent, a detergent, a reducing agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and/or a crowding agent.

[001204] In some embodiments, the kit comprises at least one container comprising the hybridization reagent. In some embodiments, the kit comprises a single container or separate containers for holding the plurality of the bottom strand blocker oligonucleotides and the hybridization reagent.

[001205] In some embodiments, the kit comprises instructions for conducting the hybridization-based methods. In some embodiments, the instructions can be provided in printed form. In some embodiments, the instructions can be as an electronic storage data file present on a suitable computer readable storage medium including but not limited to a CD, DVD or USB. In some embodiments, the instructions can be provided in the form of a website address on the internet where instructions can be obtained. In some embodiments, the instructions can be viewed at the website address or can be downloaded from the internet.

[001206] In some embodiments, the kit comprises a plurality of oligonucleotides for conducting any of the hybridization-based methods for capture and enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences. In some embodiments, plurality of oligonucleotides comprise a plurality of spikein circularization oligonucleotides. Individual spike-in circularization oligonucleotides comprise a first region that can hybridize with a sequence at one end of a linear library molecule and a second region that can hybridize with a sequence at the other end of the same linear library molecule. In some embodiments, the spike-in circularization oligonucleotides comprise a 3’ extendible end. In some embodiments, the spike-in circularization oligonucleotides comprise a 3’ non-extendible end. In some embodiments, the spike-in circularization oligonucleotides comprise a 5’ end attached to an affinity moiety. In some embodiments, the affinity moiety can bind to a receptor moiety of the capture support. In some embodiments, the affinity moiety comprises biotin, a biotin analog or a derivative of biotin. In some embodiments, the kit comprises any one or any combination of two or more spike-in circularization oligonucleotides described herein. In some embodiments, the first region of a spike-in circularization oligonucleotide comprises any one of sequences according to SEQ ID NOS: 45, 48, 49, 73, 74, 77, 84, 85, 86 or 90. In some embodiments, the second region of a spike-in circularization oligonucleotide comprises any one of sequences according to SEQ ID NOS: 46, 75 and 87. In some embodiments, the spike-in circularization oligonucleotide comprises any one of sequences according to SEQ ID NOS: 47, 50-72, 76, 78-83, 88, 89, or 91-100. In some embodiments, the sequence of the full length spike-in strand circularization oligonucleotide can be truncated 1-10 nucleotides on the 5’ or 3’ end compared to the sequences of any of the spike-in strand circularization oligonucleotides described herein. In some embodiments, the kit comprises at least one container comprising the plurality of spike-in circularization oligonucleotides.

[001207] In some embodiments, the kit comprises a plurality of top strand circularization oligonucleotides and a plurality of spike-in circularization oligonucleotides. In some embodiments, the kit comprises at least two containers, the first container comprising the plurality of top strand circularization oligonucleotides, and the second container comprising the plurality of spike-in circularization oligonucleotides. In some embodiments, the kit comprises the plurality of top strand circularization oligonucleotides and the plurality of spike-in circularization oligonucleotides in the same container. [001208] In some embodiments, the kit comprises a hybridization reagent for hybridizing the spike-in circularization oligonucleotide to the linear library molecules. In some embodiments, the hybridization reagent comprises at least one solvent and any combination of two or more of the following: at least one pH buffering agent, at least one monovalent cation, a chaotropic agent, a detergent, a reducing agent, a chelating agent, an alcohol, a zwitterion, a sugar alcohol and/or a crowding agent. In some embodiments, the kit comprises at least one container comprising the hybridization reagent. In some embodiments, the kit comprises a single container or separate containers for holding the plurality of the spike-in circularization oligonucleotides and the hybridization reagent.

[001209] In some embodiments, the kit comprises instructions for conducting the hybridization-based methods. In some embodiments, the instructions can be provided in printed form. In some embodiments, the instructions can be as an electronic storage data file present on a suitable computer readable storage medium including but not limited to a CD, DVD or USB. In some embodiments, the instructions can be provided in the form of a website address on the internet where instructions can be obtained. In some embodiments, the instructions can be viewed at the website address or can be downloaded from the internet.

Quality Scores

[001210] In some embodiments, any of the disclosed nucleic acids sequencing methods and systems can be employed and provide an average base-calling accuracy of at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% correct over the course of a sequencing run.

[001211] In some embodiments, any of the disclosed nucleic acids sequencing methods and systems can be employed and provide an average base-calling accuracy of at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% correct per every 1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or 100,000 bases called.

[001212] In some embodiments, the quality or accuracy of a sequencing run may be assessed by calculating a Phred quality score (also referred to as a quality score or “Q- score”), which indicates the probability that a given base is called incorrectly by the sequencing system. For example, in some embodiments base calling accuracy for a specific sequencing chemistry and/or sequencing system may be assessed for a large empirical data set derived from performing sequencing runs on a library of known nucleic acid sequences. The Q-score may then be calculated according to the equation: (2=-10 logio where P is the base calling error probability. A Q-score of 30, for example, indicates a probability of making a base calling error of 1 in every 1000 bases called (or a base calling accuracy of 99.9%).

[001213] In some embodiments, any of the disclosed target enrichment methods and sequencing methods and systems can be employed and provide a more accurate base readout. In some embodiments, for example, the disclosed nucleic acid sequencing methods and systems may provide a Q-score for base-calling accuracy over a sequencing run that ranges from about 20 to about 50. In some embodiments, the average Q-score for the run may be at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50.

[001214] In some embodiments, any of the disclosed nucleic acids sequencing methods and systems can be employed and provide a Q-score of greater than 20 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the nucleotide bases identified. In some embodiments, the disclosed nucleic acid sequencing systems may provide a Q-score of greater than 25 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the nucleotide bases identified. In some embodiments, the disclosed nucleic acid sequencing systems may provide a Q-score of greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the nucleotide bases identified. In some embodiments, the disclosed nucleic acid sequencing systems may provide a Q-score of greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the nucleotide bases identified. In some embodiments, the disclosed nucleic acid sequencing systems may provide a Q-score of greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the nucleotide bases identified. In some embodiments, the disclosed nucleic acid sequencing systems may provide a Q-score of greater than 45 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the nucleotide bases identified. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing may provide a Q-score of greater than 50 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the nucleotide bases identified. [001215] In some embodiments, any of the disclosed target enrichment methods and the disclosed two-stage sequencing methods can achieve a percent Q30 score of at least 90-92%, at least 92-94%, at least 94-96%, at least 96-98%, at least 98-99% or up to 100%.

[001216] In some embodiments, any of the disclosed target enrichment methods and the disclosed two-stage sequencing methods can be employed to conduct 75-300 sequencing cycles (e.g., sequencing insert sizes up to 300 nucleotides in length), and can achieve a percent Q30 score of at least 90-92%, at least 92-94%, at least 94-96%, at least 96-98%, at least 98-99% or up to 100%.

[001217] In some embodiments, any of the disclosed target enrichment methods can be used to generate covalently closed circular library molecules comprising insert regions of 100-1000 bases in length, and the covalently closed circular library molecules can be subjected to rolling circle amplification to generate concatemer template molecules which can be sequenced using any of the disclosed two-stage sequencing methods, to achieve a percent Q30 score of at least 90-92%, at least 92-94%, at least 94-96%, at least 96-98%, at least 98-99% or up to 100% for single pass sequencing and pairwise sequencing.

[001218] In some embodiments, any of the disclosed target enrichment methods and the disclosed two-stage sequencing methods can achieve a percent Q40 score of at least 75-78%, at least 78-81%, at least 81-84%, at least 84-87%, at least 87-90%, at least 90-93%, at least 93-96%, at least 96-99% or up to 100%.

[001219] In some embodiments, any of the disclosed target enrichment methods and the disclosed two-stage sequencing methods can be employed to conduct 75-300 sequencing cycles (e.g., sequencing insert sizes up to 300 nucleotides in length), and can achieve a percent Q40 score of at least 90-92%, at least 92-94%, at least 94-96%, at least 96-98%, at least 98-99% or up to 100%.

[001220] In some embodiments, any of the disclosed target enrichment methods can be used to generate covalently closed circular library molecules comprising insert regions of 100-1000 bases in length, and the covalently closed circular library molecules can be subjected to rolling circle amplification to generate concatemer template molecules which can be sequenced using any of the disclosed two-stage sequencing methods, to achieve a percent Q40 score of at least 90-92%, at least 92-94%, at least 94-96%, at least 96-98%, at least 98-99% or up to 100% for single pass sequencing and pairwise sequencing. Sequencing Coverage

[001221] In some embodiments, the level of sequencing coverage can be determined by the number of called bases from sequencing reads that align to a specific target region in a reference genome. Generally, coverage describes the number of sequencing reads that uniquely map to a reference sequence and cover a known region of the genome. For example, any of the target enrichment and sequencing methods or systems described herein can be used to achieve 30X average coverage which means that a given base position of a genome is covered on average by 30 sequencing reads. In some embodiments, 30X average coverage can yield a sequencing quality score of at least Q30 or Q40. 30X-45X coverage is typically considered to be high coverage. Typically, 30X-50X coverage is used for whole genome sequencing (WGS), and 80X-180X coverage is used for whole exome sequencing (WES).

[001222] In some embodiments, any of the target enrichment and sequencing methods or systems described herein can be used to achieve about 20X-10X average coverage, or about 10X-5X average coverage, or about 5X-2X average coverage, or about 2X-1X average coverage, wherein any of these average coverage levels can yield a sequencing quality score of at least Q30 or Q40 or higher quality scores.

[001223] In some embodiments, any of the target enrichment and sequencing methods or systems described herein can be used to achieve about 1X-0.9X average coverage, or about 0.8X average coverage, or about 0.7X average coverage, or about 0.6X average coverage, or about 0.5X average coverage, or about 0.4X average coverage, or about 0.3X average coverage, or about 0.2X average coverage, or about 0. IX average coverage, wherein any of these average coverage levels can yield a sequencing quality score of at least Q30 or Q40 or higher quality scores.

[001224] In some embodiments, any of the target enrichment and sequencing methods described herein can be used to achieve average coverage that is less than about 0.1X coverage, for example about 0.1X-0.009X average coverage, about 0.008X average coverage, about 0.007X average coverage, about 0.006X average coverage, about 0.005X average coverage, about 0.004X average coverage, about 0.003X average coverage, about 0.002X average coverage, or about 0.001X average coverage, wherein any of these average coverage levels can yield a sequencing quality score of at least Q30 or Q40 or higher quality scores.

[001225] In some embodiments, any of the target enrichment and two-stage sequencing methods described herein can be used to achieve about IX average coverage, or about 2X average coverage, or about 3X average coverage, or about 4X average coverage, or about 5X average coverage, wherein any of these average coverage levels can yield a sequencing quality score of at least Q30 or Q40 or higher quality scores.

On-Target Rates

[001226] An on-target metric can provide information about the specificity of a target enrichment workflow. On-target metrics can be determined using sequenced nucleotide bases or sequencing reads. In some embodiments, on-target rate can be defined as a comparison (e.g., a ratio) of (i) the number of sequenced nucleotide bases that can be mapped to the target region (e.g., genome or RNA), and (ii) the number of sequenced nucleotide bases that map to non-target regions.

[001227] In some embodiments, percent reads on-target can be defined as a comparison (e.g., a ratio) of (i) the number of sequencing reads that can be mapped to the target region (e.g., genome or RNA), and (ii) the number of sequencing reads that map to non-target regions. The percent reads on-target can include all sequencing reads that overlap the targeted region by even by a single base.

[001228] In some embodiments, strong probe specificity and/or efficient hybridizationbased target enrichment can be indicated by a higher number of bases or sequencing reads that fall within the target region.

[001229] In some embodiments, a higher on-target rate may be desirable, for example an on-target rate of at least about 80-85%, or about 85-90%, or about 90-95%, or about 95- 99% or on-target rates that are higher than 99%.

[001230] In some embodiments, any of the target enrichment and two-stage sequencing methods described herein can be used to achieve an on-target rate of about 80-85%, or about 85-90%, or about 90-95%, or about 95-99% or on-target rates that are higher than 99%.

[001231] In some embodiments, any of the target enrichment and two-stage sequencing methods described herein can be used to achieve percent reads on-target of about 80-85%, or about 85-90%, or about 90-95%, or about 95-99% or on-target rates that are higher than 99%.

GC-Bias

[001232] The GC content can be unevenly distributed across the genomes of organisms, including, for example, humans. GC-bias can arise when regions of high or low GC content are unevenly represented during sequencing. GC bias can arise from PCR amplification during a library preparation workflow, hybridization-based target enrichment workflows, and sequencing. Sequencing runs having high GC bias indicate that more sequencing is required to achieve the desired sequencing depth across all of the target sequence regions.

[001233] Any of the hybridization-based target enrichment and two-stage sequencing methods described herein can be used to achieve low or reduced GC bias.

Coverage Uniformity

[001234] The fold-80 base penalty is a measure of non-uniformity of coverage that indicates how much additional sequencing is required to bring 80% of target bases to the mean coverage. A fold-80 base penalty value of 0.8 - 1.0 indicates near perfect or perfect uniformity.

[001235] In some embodiments, any of the hybridization-based target enrichment workflows and two-stage sequencing methods described herein can be used to achieve a fold- 80 base penalty of about 0.8 -1, or about 1 - 1.2, or about 1.2 - 1.4, or about 1.4 - 1.6.

Duplication Rate

[001236] Duplicate sequencing reads include multiple sequencing reads that map to the exact same location on a reference sequence, including the ‘5 and 3’ ends. Typically, duplicate reads offer no additional sequencing information, compared to overlapping sequencing reads. During bioinformatic analysis, duplicate sequencing reads are removed, which is known as de-duplication. High duplication rates can arise from PCR overamplification, low input DNA, and low complexity libraries. High duplicate reads can also arise when the same DNA strand is used to form multiple clusters on a flowcell, for example during bridge amplification.

[001237] In some embodiments, any of the target enrichment workflows which include rolling circle amplification of a circularized library molecule to generate a template concatemer template molecule, and two-stage sequencing methods described herein can be used to achieve low duplication rates of about 0.1 - 0.3%, or about 0.3 - 0.6%, or about 0.6 - 0.9%, or about 0.9 - 1.2%, or about 1.2 - 1.5%, or about 1.5 - 1.8 %, or about 1.8 - 2.2%.

Fold Enrichment

[001238] The present disclosure provides methods for conducting hybridization-based enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences that can achieve at least 2-fold, 3-fold, 4-fold or 5-fold enrichment of the polynucleotides having target sequences when compared to mixtures of polynucleotides that have not undergone the methods for hybridization-based enrichment disclosed herein. In some embodiments, the hybridization-based enrichment methods can achieve 10-25 fold enrichment, or 25-50 fold enrichment, or 50-100 fold enrichment. In some embodiments, the hybridization-based enrichment methods can achieve 100-200 fold enrichment, or 200-300 fold enrichment, or 300-400 fold enrichment, or 400-500 fold enrichment.

Variant Detection

[001239] The present disclosure provides methods for conducting hybridization-based enrichment of target polynucleotide sequences from a mixture of polynucleotides having target and non-target sequences wherein the methods can detect genetic variants present in a nucleic acid sample at about 0.01-0.025%, or about 0.025-0.05%, or about 0.05-0.075%, or about 0.075-0.1%, 0.1-0.25%, or about 0.25-0.5%, or about 0.5 - 1%, or about 1-2%, or about 2-5%, or about 5-10%.

SEQUENCES

Note: nucleotide symbols underlined, bolded, and proceeding “+” (e.g., “+A” in SEQ ID NO: 52) denote locked nucleic acids (LNAs). “I” in nucleic acid sequences denotes inosines. The nucleic acid sequences listed in the Table are oriented in a 5’ to 3’ direction.

EXAMPLES

[001240] The following examples are meant to be illustrative and can be used to further understand embodiments of the present disclosure and should not be construed as limiting the scope of the present teachings in any way.

Example 1: Library circularization using blocker/splint adaptors

[001241] 100 ng of human genomic DNA (NA12878) was enzymatically fragmented using a commercially-available fragmentation kit and following the manufacturer’s instructions (e.g., xGen DNA Lib Prep EZ, from Integrated DNA Technologies (IDT), catalog # 10009863). The resulting DNA fragments included terminal 3’ A-tails.

[001242] Forward and reverse sequencing primer adaptors were appended to the fragmented DNA by ligating stubby Y-adaptors using a commercially-available kit designed for Element Bioscience sequencing platform (e.g., xGen Stubby Adapter UDI Primers Element, from Integrated DNA Technologies (IDT), catalog # 10017036). The stubby Y- adaptor ligation step generated partial library molecules. The stubby Y-adaptors included forward and reverse sequencing primer adaptors designed for Element Biosciences sequencing platform.

[001243] The partial library molecules were subjected to SPRI clean-up using AMPure XP beads and freshly prepared 80% ethanol.

[001244] Universal surface capture primers and dual sample indexes were appended to the ends of the partial library molecules by conducting PCR using commercially-available primer pairs (e.g., xGen Library Amplification Primer Mix for Element, from Integrated DNA Technologies (IDT), catalog # 10016958). The PCR amplification step generated full length library molecules. The PCR primer pairs included universal surface capture primers designed for Element Biosciences sequencing platform. The average total length of the full length library molecules was approximately 400-450 bp. The structure of the full length linear double-stranded library molecules is shown in FIG. 17 (top). The linear doublestranded library molecules lacked unique identification sequences (UMI) (e.g., FIG. 18, (780)).

Example 2: In-solution target capture

[001245] 2 ug of the full length linear library molecules (described in Example 1) was mixed with human Cot DNA and top strand circularization oligonucleotides having internal inosine nucleo-bases, and bottom strand blocker oligonucleotides to generate a plurality of captured linear library-circularization complexes. Exemplary top strand circularization oligonucleotides having internal inosine nucleo-bases comprised SEQ ID NOS: 51, 52, 60, 61, 62, 63, 64, 92 and 94. The human Cot DNA was prepared from human placental DNA and enriched for repetitive DNA sequences such as the Alu and Kpn family members (e.g., from Thermo Fisher Scientific, catalog No. 15279011). The human Cot DNA was used to reduce non-specific hybridization. In a separate experiment, 2 ug of the full length linear library molecules was mixed with human Cot DNA and top strand circularization oligonucleotides lacking internal inosines (e.g., see FIGS. 43A-43B, oligonucleotide (iii) and bottom strand blocker oligonucleotides lacking internal inosines to generate a plurality of captured linear library-circularization complexes. The human Cot DNA was included for hybridization to repetitive human DNA sequences. The mixture was dried down using a SpeedVac system. The dried DNA mixture was wetted with a commercially-available hybridization buffer system (e.g., xGen 2X Hybridization Buffer and xGen Hybridization Buffer Enhancer, both from Integrated DNA Technologies (IDT), catalog #1080577).

[001246] The wetted DNA mixture was mixed with a target-specific DNA bait/probe set (e.g., xGen Exome Research Panel V2, from Integrated DNA Technologies (IDT), catalog #10005151), which was incubated at room temperature for at least 5 minutes, and subjected to hybridization conditions in a thermocycler (e.g., lid temperature set at 100 °C, 30 seconds at 95 °C, 16 hours at 65 °C, then hold at 65 °C). The Tm of the target-specific baits/probes was approximately 95 °C. The target-specific bait/probe set comprised a panel of more than 415,000 DNA oligonucleotides designed to hybridize to protein-coding human genomic sequences. The target-specific bait/probes were 5’ biotin-modified oligonucleotides. The hybridization conditions generated a plurality of open circle library bait complexes each having a nick.

Example 3: Immobilization to a capture support and nick ligation

[001247] The plurality of open circle library bait complexes (8 pM) and PhiX spike-in (0.1 pM) and a plurality of pinning primers were distributed onto a capture support in a loading reagent at 65 °C to generate a plurality of immobilized open circle library bait complexes and a plurality of immobilized pinning primers. Different amounts of open circle library bait complexes were distributed onto the capture support, including for example about 0.5 ug, about 0.75 ug, about 1 ug, about 1.25 ug, about 1.5 ug, about 2 ug, about 2.5 ug, about 3 ug, about 3.3 ug, about 4 ug, about 5 ug, and about 6 ug. The capture support comprised (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moi eties embedded in the at least one layer of hydrophilic polymer coating. The capture support comprised a plurality of streptavidin receptor moieties. The target-specific baits/probes and the pinning primers comprised biotin affinity moieties. The loaded capture support was washed at least once with a wash reagent to remove residual non-target linear library molecules and retain the plurality of immobilized open circle library bait complexes and a plurality of immobilized pinning primers.

[001248] The capture support was contacted with a ligation reagent and incubated at 55 °C for 15 minutes to ligate the nicks of the immobilized open circle library bait complexes thereby generating a plurality of immobilized covalently closed circular library molecules each hybridized to an immobilized target-specific bait/probe thereby generating a plurality of immobilized closed circle library bait complexes. The ligation reagent comprised a DNA ligase.

Example 4: Rolling circle amplification

[001249] The capture support was contacted with a rolling circle amplification reagent comprising a strand displacing polymerase, a plurality of compaction oligonucleotides, and a mixture of nucleotides comprising dATP, dGTP, dCTP, dTTP and dUTP, and a rolling circle amplification reaction was conducted to generate a plurality of immobilized concatemer template molecules. The amplification reagent comprised a strand displacing DNA polymerase, a plurality of compaction oligonucleotides, and a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and dUTP.

Example 5: Two-Stage Sequencing

[001250] The immobilized concatemer template molecules described in Example 4 above were subjected to recursive two-stage sequencing reactions using fluorescently-labeled multivalent molecules in the first stage and un-labeled nucleotide analogs (e.g., 3’ chain terminator blocking group) in the second stage. The immobilized concatemer template molecules served as concatemer template molecules for sequencing. The template concatemer template molecules were subjected to 2x75 pairwise sequencing. The read order of sequencing comprised: Read 1, pairwise turn, Read 2, Index 1, and Index 2.

[001251] The first-stage sequencing reaction was conducted by hybridizing a plurality of a soluble sequencing primers to the immobilized concatemer template molecules to form immobilized primer-concatemer duplexes, which were immobilized on a flowcell. A plurality of a first sequencing polymerase was flowed onto the flowcells (e.g., contacting the immobilized primer-concatemer duplexes) and incubated under a condition suitable to bind the sequencing polymerase to the duplexes to form polymerase complexes. A mixture of fluorescently labeled multivalent molecules was flowed onto the flowcell in the presence of a buffer that included a non-catalytic cation (e.g., strontium, barium and/or calcium) and incubated under conditions suitable to bind complementary nucleotide moieties of the multivalent molecules to the polymerase complexes to form avidity complexes without polymerase-catalyzed incorporation of the nucleotide moieties. The fluorescently labeled multivalent molecules were labeled at their cores. Exemplary multivalent molecules are shown in FIGS. 1-4. The polymerase complexes were washed. An image was obtained of the fluorescently labeled multivalent molecules that remined bound to the polymerase complexes. The first sequencing polymerases and multivalent molecules were removed, while retaining the sequencing primers hybridized to the immobilized concatemers (retained duplexes), by washing with a buffer comprising a detergent.

[001252] The first stage sequencing reaction was suitable for forming a plurality of avidity complexes on the concatemer template molecules (e.g., polonies). For example, the first stage sequencing reaction comprised: (a) binding a first nucleic acid primer, a first polymerase, and a first multivalent molecule to a first portion of a concatemer template molecule thereby forming a first binding complex, wherein a first nucleotide moiety of the first multivalent molecule was bound to the first polymerase; and (b) binding a second nucleic acid primer, a second polymerase, and the first multivalent molecule to a second portion of the same concatemer template molecule thereby forming a second binding complex, wherein a second nucleotide moiety of the first multivalent molecule was bound to the second polymerase, wherein the first and second binding complexes which included the same multivalent molecule formed a first avidity complex.

[001253] The second-stage sequencing reaction was conducted by contacting the retained duplexes with a plurality of second sequencing polymerases to form polymerase complexes. A mixture of non-labeled nucleotide analogs (e.g., 3 ’O-m ethylazido nucleotides) (e.g., at different concentrations of about 1-5 uM) was added to the polymerase complexes in the presence of a buffer that included a catalytic cation (e.g., magnesium and/or manganese) and incubated under conditions suitable to bind complementary nucleotides to the polymerase complexes and promote polymerase-catalyzed incorporation of the nucleotides to generate a nascent extended sequencing primer. The polymerase complexes were washed. No image was obtained. The incorporated non-labeled nucleotide analogs were reacted with a cleaving reagent that removes the 3’ O-methylazido group and generates an extendible 3 ’OH group. [001254] The second sequencing polymerases were removed, while retaining the nascent extended sequencing primers hybridized to the concatemer template molecules (retained duplexes), by washing with a buffer comprising a detergent. Recurring sequencing reactions were conducted by performing multiple cycles of first-stage and second-stage sequencing reactions to generate extended forward sequencing primer strands. One cycle comprised a first-stage sequencing reactions and a second-stage sequencing reaction.

[001255] Table 1 below lists various sequencing metrics from several sequencing runs wherein the target enrichment workflow employed in-solution linear library hybridization with top strand circularization oligonucleotides having internal inosine nucleo-bases (e.g., SEQ ID NOS: 51, 52, 60, 61, 62, 63, 64, 92 and 94).

Table 1:

Example 6: DNA and RNA library enrichment workflow

[001256] Matched RNA and DNA isolated from blood and formalin-fixed paraffin- embedded (FFPE) tissue biopsy samples from prostate cancer patients were used as the input source of nucleic acids to prepare libraries.

[001257] 100 ng of RNA was fragmented and subjected to reverse transcription to generate first strand cDNA. Double-stranded cDNA was generated using standard methods. Universal adaptors were appended to both ends of the double-stranded cDNA to generate the linear RNA library molecules.

[001258] 50 ng of DNA was fragmented. Universal adaptors were appended to both ends of the double-stranded DNA to generate the linear DNA library molecules.

[001259] The universal adaptor sequence for binding a second universal surface primer (720) (e.g., comprising the pinning primer binding site sequence) and the capture primer binding site sequence (730) were the same for the RNA and DNA libraries. [001260] The universal adaptor comprising the forward sequencing primer binding site sequence (740) and the universal adaptor sequence for binding a reverse sequencing primer (750) were different for the RNA and DNA libraries.

[001261] Different top strand circularization oligonucleotides were designed to selectively hybridize to either the linear RNA library molecules or the linear DNA library molecules.

[001262] For example, the 5’ end of the top strand circularization oligonucleotides for the RNA library comprised a sequence that hybridized to the universal adaptor sequence for binding a second universal surface primer (720) (for example, comprising the pinning primer binding site sequence) on one side of an RNA library molecule and the 3’ end of the top strand circularization oligonucleotides hybridized to the universal adaptor sequence for binding a first universal surface primer (730) (e.g., capture primer) and the universal adaptor sequence for binding a reverse sequencing primer (750) on the other side of the same RNA library molecule. An exemplary top strand circularization oligonucleotide for the RNA library comprised the sequence SEQ ID NO: 78. Other suitable top strand circularization oligonucleotides comprise any of the sequences according to SEQ ID NOS: 79-83. The 3’ ends of the top strand circularization oligonucleotides for the RNA library were phosphorylated.

[001263] The 5’ end of the top strand circularization oligonucleotides for the DNA library comprised a sequence that hybridized to the universal adaptor sequence for binding a second universal surface primer (720) (for example, comprising pinning primer binding site sequence) on one side of an DNA library molecule, and the 3’ end of the top strand circularization oligonucleotides hybridized to the universal adaptor sequence for binding a first universal surface primer (730) (e.g., capture primer binding site sequence) and the universal adaptor sequence for binding a reverse sequencing primer (750) on the other side of the same DNA library molecule. Exemplary top strand circularization oligonucleotides for the DNA library comprise SEQ ID NOS: 47, 50-72, 76, 78-83, 88, 89, and 91-99. The 3’ ends of the top strand circularization oligonucleotides for the DNA library were phosphorylated. [001264] The linear RNA and DNA library molecules were mixed together, then mixed with their respective top strand circularization oligonucleotides to generate a plurality of captured linear library-circularization complexes. 24 different RNA libraries were pooled together at approximately 166.6 ng/sample. 8 different DNA libraries were pooled together at approximately 375 ng/sample. The mixture was dried down using a SpeedVac system. The dried DNA mixture was wetted with commercially-available hybridization buffer system (e.g., Fast Hybridization Buffer, from Twist Biosciences).

[001265] The wetted RNA and DNA library mixture was mixed with a target-specific DNA library bait/probe set (e.g., Twist Exome 2.0 panel, from Twist Biosciences) and a target-specific RNA library bait/probe set (e.g., Twist RNA exome panel, from Twist Biosciences), which was incubated at room temperature for at least 5 minutes, and subjected to hybridization conditions in a thermocycler (e.g., lid temperature set at 85 °C, 5 minutes at 95 °C, 1 hour at 71 °C, then hold at 71 °C). The target-specific bait/probes were 5’ biotin- modified oligonucleotides. The hybridization conditions generated a plurality of open circle library bait complexes each having a nick.

[001266] The plurality of open circle library bait complexes and PhiX spike-in and a plurality of pinning primers were distributed onto a capture support in a loading reagent at 65 °C to generate a plurality of immobilized open circle library bait complexes and a plurality of immobilized pinning primers. The capture support comprised (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating. The capture support comprised a plurality of streptavidin receptor moieties. The target-specific baits/probes and the pinning primers comprised biotin affinity moieties. The loaded capture support was washed at least once with a wash reagent to remove residual non-target linear library molecules and retain the plurality of immobilized open circle library bait complexes and a plurality of immobilized pinning primers.

[001267] The capture support was contacted with a ligation reagent and incubated at 55 °C for 15 minutes to ligate the nicks of the immobilized open circle library bait complexes thereby generating a plurality of immobilized covalently closed circular library molecules each hybridized to an immobilized target-specific bait/probe thereby generating a plurality of immobilized closed circle library bait complexes.

[001268] The capture support was contacted with a rolling circle amplification reagent comprising a strand displacing polymerase, a plurality of compaction oligonucleotides, and a mixture of nucleotides comprising dATP, dGTP, dCTP, dTTP and dUTP, and a rolling circle amplification reaction was conducted to generate a plurality of immobilized concatemer template molecules.

[001269] The immobilized concatemers described herein in Example 6 were subjected to recursive two-stage sequencing reactions as described in Example 5 above, using fluorescently-labeled multivalent molecules in the first stage and un-labeled nucleotide analogs (e.g., 3’ chain terminator blocking group) in the second stage.

Claims

CLAIMS What is claimed is:

1. A method for enriching target polynucleotides from a mixture of polynucleotides comprising target and non-target polynucleotides, the method comprising: a) providing a plurality of closed circle library bait complexes immobilized to a capture support, wherein individual closed circle library bait complexes comprise:

(i) a covalently closed circular library molecule comprising a polynucleotide comprising a target sequence and at least one universal adaptor sequence, and

(ii) a target-specific bait/probe hybridized to at least a portion of the target sequence; b) conducting a rolling circle amplification reaction using the target-specific bait/probe to initiate amplification, thereby generating a plurality of immobilized concatemer template molecules; and c) sequencing at least a portion of the plurality of immobilized concatemer template molecules.

2. A method for enriching target polynucleotides from a mixture of polynucleotides comprising target and non-target polynucleotides, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) forming a first plurality of closed circle library bait complexes immobilized to the capture support,

• wherein individual closed circle library bait complexes in the first plurality comprise (i) a covalently closed circular library molecule comprising a polynucleotide comprising a target sequence and at least one universal adaptor sequence, and (ii) a target-specific bait/probe hybridized to the target sequence, • wherein an individual target-specific bait/probe comprises an (i) oligonucleotide comprising a target-specific sequence that can selectively hybridize to at least a portion of the target sequence of the covalently closed circular library molecule, (ii) an affinity moiety at the 5’ end of the target-specific oligonucleotide, and (iii) an extendible 3’ end, and

• wherein the forming of step (b) comprises binding the affinity moieties of the individual target-specific baits/probes of the first plurality to the embedded receptor moieties of the capture support, thereby generating a first plurality of circle-bait complexes immobilized to the capture support; c) conducting a rolling circle amplification reaction using the extendible 3’ ends of the target-specific bait/probes to initiate the rolling circle amplification reaction, thereby generating a plurality of immobilized concatemer template molecules; and d) sequencing at least a portion of individual immobilized concatemer template molecules.

3. The method of claim 2, wherein individual receptor moieties comprise streptavidin or avidin.

4. The method of claim 2 or 3, wherein the affinity moiety comprises biotin, desthiobiotin or iminobiotin.

5. The method of any one of claims 2-4, wherein a melting temperature of the targetspecific bait/probe hybridized to the target sequence is between about 80 °C and about 85 °C, between about 85 °C and about 90 °C, between about 90 °C and about 95 °C, or between about 95 °C and about 98 °C.

6. The method of any one of claims 2-5, wherein selective hybridization of the targetspecific bait/probe to the at least a portion of the target sequence is conducted at a temperature that is:

• between about 5 °C and about 10 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence, • between about 10 °C and about 20 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence,

• between about 20 °C and about 30 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence,

• between about 30 °C and about 40 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence, or

• between about 40 °C and about 50 °C less than the Tm of the target-specific bait/probe hybridized to the target sequence.

7. The method of any one of claims 2-6, wherein the first plurality of closed circle library bait complexes are immobilized to the capture support at pre-determined locations, thereby forming a pre-determined pattern of immobilized circle-bait complexes.

8. The method of any one of claims 2-6, wherein the first plurality of closed circle library bait complexes are immobilized to the capture support at random and nonpredetermined locations.

9. The method of any one of claims 2-8, wherein the density of the first plurality of closed circle library bait complexes immobilized to the support is between 10² and 10¹⁵ closed circle library bait complexes per mm².

10. The method of any one of claims 2-9, wherein the first plurality of closed circle library bait complexes comprises target-specific bait/probes having between 2 and 10,000 different target-specific sequences.

11. The method of any one of claims 2-10, wherein the density of the plurality of immobilized concatemer template molecules is between about 10² and about 10¹⁵ immobilized concatemer template molecules per mm².

12. The method of any one of claims 2-11, wherein the plurality of immobilized concatemer template molecules comprises between 2 and 10,000 different targetspecific sequences.

13. The method of any one of claims 2-12, wherein at least some of the immobilized concatemer template molecules comprise nearest neighbor concatemer molecules that touch each other and/or overlap each other when viewed from any angle of the capture support including above, below or side views of the capture support.

14. The method of any one of claims 2-13, wherein the capture support comprises 200 million - 1.5 billion immobilized concatemer template molecules immobilized thereto.

15. The method of any one of claims 2-14, wherein the rolling circle amplification reaction comprises contacting the first plurality of closed circle library bait complexes with a rolling circle amplification reagent comprising a plurality of strand-displacing polymerases, and a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and dUTP, and wherein the rolling circle amplification reaction generates a plurality of immobilized concatemer template molecules, individual immobilized concatemer template molecules comprising at least one uracil nucleobase.

16. The method of claim 15, wherein the rolling circle amplification reagent comprises a plurality of compaction oligonucleotides.

17. The method of claim 16, wherein individual compaction oligonucleotides comprise single-stranded oligonucleotides that can hybridize to two different locations on the concatemer template molecule to pull together distal portions of the concatemer template molecule causing compaction of the concatemer template molecule to form a DNA nanoball.

18. The method of any one of claims 2-16, wherein the capture support comprises a plurality of pinning primers immobilized to the support, wherein individual pinning primers comprise an oligonucleotide having a universal pinning sequence and an affinity moiety at the 5’ end of the oligonucleotide, wherein the affinity moiety of individual pinning primers binds an embedded receptor moiety of the capture support.

19. The method of claim 18, wherein individual pining primers comprise a blocking group at the 3’ end of the oligonucleotide, wherein the blocking group inhibits polymerase- catalyzed extension of the 3’ end of the pinning primer.

20. The method of claim 18 or 19, wherein the density of the plurality of pinning primers is between 10² and 10¹⁵ pinning primers per mm².

21. The method of any one of claims 18-20, wherein individual immobilized concatemer template molecules comprise a universal sequence for binding a pinning primer.

22. The method of claim 21, wherein at least a portion of individual pinning primers hybridize to a portion of an immobilized concatemer template molecule to pin down a portion of the immobilized concatemer template molecules to the capture support.

23. The method of any one of claims 2-22, further comprising:

(i) conducting a re-seeding reaction comprising forming a second plurality of closed circle library bait complexes immobilized to the capture support by binding the affinity moiety of individual target-specific baits/probes of a second plurality of target-specific baits/probes to receptor moieties of the capture support, thereby generating a second plurality of closed circle library bait complexes immobilized to the capture support, wherein the reseeding reaction is conducted after the rolling circle amplification of step (c) and prior to the sequencing of step (d);

(ii) conducting a second rolling circle amplification reaction using the extendible 3’ ends of the second plurality of target-specific bait/probes to initiate the second rolling circle amplification reaction, thereby generating a second plurality of immobilized concatemer template molecules; and

(iii) sequencing at least a portion of individual immobilized concatemer template molecules from the first and second pluralities of concatemer template molecules.

24. The method of claim 23, wherein the sequencing comprises sequencing the first and second pluralities of immobilized concatemer template molecules essentially simultaneously.

25. The method of claim 23, wherein the sequencing comprises sequencing the first plurality of immobilized concatemer template molecules and then sequencing the second plurality of immobilized concatemer template molecules.

26. The method of any one of claims 2-22, comprising:

(i) conducting a re-seeding reaction comprising forming a second plurality of closed circle library bait complexes immobilized to the capture support by binding the affinity moiety of individual target-specific baits/probes of a second plurality of target-specific baits/probes to receptor moieties of the capture support thereby generating a second plurality of closed circle library bait complexes immobilized to the capture support, wherein the reseeding reaction is conducted after sequencing the first plurality of immobilized concatemer template molecules of step (d);

(iii) sequencing at least a portion of individual immobilized concatemer template molecules from the second plurality of immobilized concatemer template molecules.

27. The of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) forming a plurality of closed circle library bait complexes by contacting a plurality of target-specific baits/probes to a plurality of covalently closed circular library molecules , wherein the plurality of covalently closed circular library molecules comprises a mixture of covalently closed circular library molecules comprising target sequences and covalently closed circular library molecules comprising non-target sequences, wherein the contacting is conducted in-solution under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual covalently closed circular library molecules to corresponding target-specific baits/probes, thereby generating a plurality of closed circle library bait complexes that are enriched for target sequences; c) contacting the capture support with the plurality of closed circle library bait complexes, thereby generating a plurality of immobilized closed circle library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of an individual target-specific baits/probe to a receptor moiety of the capture support; and d) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the targetspecific baits/probes, thereby generating the plurality of immobilized concatemer template molecules.

28. The method of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating, (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating, and (iii) a plurality of target-specific baits/probes immobilized to the capture support; b) forming a plurality of immobilized closed circle library bait complexes by contacting a plurality of covalently closed circular library molecules to the plurality of target-specific baits/probes, wherein the plurality of covalently closed circular library molecules comprises a mixture of covalently closed circular library molecules comprising target sequences and covalently closed circular library molecules comprising non-target sequences, wherein the contacting is conducted on the capture support under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual covalently closed circular library molecules to corresponding target-specific baits/probes, thereby generating the plurality of immobilized closed circle library bait complexes that are enriched for polynucleotides having target sequences; and c) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the targetspecific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

9. The method of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) forming a plurality of library bait complexes by contacting a plurality of targetspecific baits/probes to a plurality of linear library molecules, wherein the plurality of linear library molecules comprises a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted in-solution under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of library bait complexes that are enriched for polynucleotides having target sequences; c) contacting the capture support with the plurality of library bait complexes, thereby generating a plurality of immobilized library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes to a receptor moiety; d) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of single-stranded top strand circularization oligonucleotides under a condition suitable for hybridizing the ends of individual linear library molecules with individual single-stranded top strand circularization oligonucleotides to form individual open circle library complexes each having one nick, and enzymatically ligating the one nick, thereby generating a plurality of covalently closed circular library molecules each being hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and e) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the targetspecific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

0. The method of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) forming a plurality of library bait complexes by contacting a plurality of targetspecific baits/probes to a plurality of linear library molecules, wherein the plurality of linear library molecules comprises a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted in-solution under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of library bait complexes that are enriched for polynucleotides having target sequences; c) contacting the capture support with the plurality of library bait complexes, thereby generating a plurality of immobilized library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of individual target-specific baits/probes to a receptor moiety; d) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of double-stranded top strand circularization oligonucleotides, individual doublestranded top strand circularization oligonucleotides comprising a long strand and a short strand, wherein the long and short strands are hybridized together to form the double-stranded molecule having a double-stranded region and two flanking single-stranded regions, wherein the contacting is conducted under a condition suitable for hybridizing one end of a linear library molecule to one end of the long splint strand and hybridizing the other end of the linear library molecule to the other end of the long splint strand, thereby forming an open circle library bait complex having two nicks, and enzymatically ligating the two nicks thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and e) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the targetspecific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

31. The method of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating, (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating, and (iii) a plurality of target-specific baits/probes immobilized to the capture support; b) forming a plurality of immobilized library bait complexes by contacting a plurality of linear library molecules to the plurality of target-specific baits/probes, wherein the plurality of linear library molecules comprise a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted on the capture support under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences; c) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of single-stranded top strand circularization oligonucleotides under a condition suitable for hybridizing the ends of individual linear library molecules with individual single-stranded top strand circularization oligonucleotides to form individual open circle library splint complexes, individual open circle library splint complexes having one nick, and enzymatically ligating the one nick thereby generating a plurality of covalently closed circular library molecules hybridized to immobilized target-specific bait/probes, thereby forming a plurality of immobilized closed circle library bait complexes; and d) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the targetspecific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

2. The method of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating, (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating, and (iii) a plurality of target-specific baits/probes immobilized to the capture support; b) forming a plurality of immobilized library bait complexes by contacting a plurality of linear library molecules to the plurality of target-specific baits/probes, wherein the plurality of linear library molecules comprise a mixture of linear library molecules comprising target sequences and linear library molecules comprising non-target sequences, wherein the contacting is conducted on the capture support under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of immobilized library bait complexes that are enriched for polynucleotides having target sequences; c) forming a plurality of immobilized closed circle library bait complexes by contacting the plurality of immobilized library bait complexes with a plurality of double-stranded top strand circularization oligonucleotides, individual doublestranded top strand circularization oligonucleotides comprising a long strand and a short strand, wherein the long and short strands are hybridized together to form the double-stranded molecule having a double-stranded region and two flanking single-stranded regions, wherein the contacting is conducted under a condition suitable for hybridizing one end of a linear library molecule to one end of the long strand and hybridizing the other end of the linear library molecule to the other end of the long strand, thereby forming an open circle library bait complex having two nicks, and enzymatically ligating the two nicks thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe thereby forming a plurality of immobilized closed circle library bait complexes; and d) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the target- specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

33. The method of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) contacting in-solution a plurality of linear library molecules with a plurality of top strand circularization oligonucleotides, wherein individual linear library molecules comprise an insert region having a target sequence or a non-target sequence, wherein the insert region is flanked on either side by one or more universal adaptor sequences, wherein the one or more universal adaptor sequences at one side of the insert region are not the same as the one or more universal adaptor sequences at the other side of the insert region, wherein individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence, a bridging sequence, and a terminal 3’ extendible moiety, wherein the anchor sequence can hybridize to one or more universal adaptor sequences at one side of the insert region, wherein the bridging sequence can hybridize to one or more universal adaptor sequences at the other end of the same linear library molecule; c) contacting in-solution the plurality of linear library molecules with a plurality of target-specific baits/probes, wherein the contacting of step (c) is conducted under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding targetspecific baits/probes, thereby generating a plurality of captured linear library bait complexes that are enriched for polynucleotides having target sequences; d) forming a plurality of open circle library bait complexes by hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the one side of the insert region, and hybridizing the bridging sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the other end of the insert region of an individual linear library molecule, thereby forming a plurality of open circle library bait complexes having one nick; e) contacting the capture support with the plurality of open circle library bait complexes, thereby generating a plurality of immobilized open circle library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of an individual target-specific bait/probe to a receptor moiety; f) contacting the capture support with a ligation reagent, thereby ligating the one nick of individual immobilized open circle library bait complexes, thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific baits/probes, thereby forming a plurality of immobilized closed circle library bait complexes; and g) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the immobilized target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

34. The method of any one of claims 2-26, further comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) contacting in-solution a plurality of linear library molecules with a plurality of top strand circularization oligonucleotides, wherein individual linear library molecules comprise an insert region having a target sequence or a non-target sequence, wherein the insert region is flanked on either side by one or more universal adaptor sequences, wherein the one or more universal adaptor sequences at one side of the insert region are not the same as the one or more universal adaptor sequences at the other side of the insert region, wherein individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence, an additional sequence, a bridging sequence, and a 3’ non-extendible blocking group, wherein the bridging sequence can hybridize to the one or more universal adaptor sequences at the one side of the insert region, wherein the bridging sequence can hybridize to the one or more universal adaptor sequences at the other side of the insert region, wherein the additional sequence of individual top strand circularization oligonucleotides does not hybridize to a portion of individual linear library molecules; c) contacting in-solution the plurality of linear library molecules with a plurality of target-specific baits/probes wherein the contacting of step (c) is conducted under a condition suitable for selectively hybridizing at least a portion of the target sequence of individual linear library molecules to corresponding target-specific baits/probes, thereby generating a plurality of captured linear library bait complexes that are enriched for polynucleotides having target sequences; d) forming a plurality of open circle library bait complexes by hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the one side of the insert region, and hybridizing the bridging sequence to at least a portion of the one or more universal adaptor sequences at the other end of the insert region of an individual linear library molecule, thereby forming the plurality of individual open circle library bait complexes having a gap; e) contacting the capture support with the plurality of open circle library bait complexes, thereby generating a plurality of immobilized open circle library bait complexes, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of an individual target-specific bait/probe to a receptor moiety; f) contacting the plurality of immobilized open circle library bait complexes on the capture support with gap fill-in reagent for conducting a polymerase-catalyzed gap fill-in reaction, thereby generating a plurality of immobilized open circle library bait complexes having a nick, and contacting the plurality of immobilized open circle library bait complexes with a ligation reagent for ligating the nicks, thereby generating a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and g) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the immobilized target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

35. The method of any one of claims 2-26, comprising: a) providing a capture support comprising (i) a support coated with at least one layer of a hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) contacting in-solution a plurality of linear library molecules with a plurality of top strand circularization oligonucleotides, wherein individual linear library molecules comprise an insert region having a target sequence or a non-target sequence, wherein the insert region is flanked on either side by one or more universal adaptor sequences, wherein the one or more universal adaptor sequences at one side of the insert region are not the same as the one or more universal adaptor sequences at the other side of the insert region, wherein individual top strand circularization oligonucleotides comprise a single-stranded oligonucleotide comprising an anchor sequence, a bridging sequence, and a terminal 3’ non-extendible blocking group, wherein the anchor sequence can hybridize to the one or more universal adaptor sequences at one side of the insert region, wherein the bridging sequence can hybridize to the one or more universal adaptor sequences at the other side of the insert region, and wherein a 5’ portion of individual linear library molecules forms a 5’ overhang flap structure upon hybridization with the bridging sequence which is cleavable by a 5’ flap endonuclease, thereby generating a newly cleaved 5’ end having a phosphate group; c) contacting in-solution the plurality of linear library molecules with a plurality of target-specific baits/probes, wherein the contacting of step (c) is conducted under a condition suitable for selectively hybridizing at least a portion of the target sequences of individual linear library molecules to corresponding targetspecific baits/probes, thereby generating a plurality of captured linear library bait complexes that are enriched for polynucleotides having target sequences; d) forming a plurality of open circle library bait complexes comprising a 5’ overhang flap structure by hybridizing the anchor sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the one side of the insert region, and hybridizing the bridging sequence of the top strand circularization oligonucleotide to at least a portion of the one or more universal adaptor sequences at the other end of the insert region of the individual linear library molecule, thereby forming a plurality of open circle library bait complexes having a 5’ overhang flap structure; e) contacting the capture support with the plurality of open circle library bait complexes, thereby generating a plurality of immobilized open circle library bait complexes with 5’ overhang flap structures, wherein the contacting is conducted under a condition suitable for binding an affinity moiety of individual targetspecific baits/probes to a receptor moiety, wherein the plurality of immobilized open circle library bait complexes are enriched for polynucleotides carrying target sequences; f) contacting the capture support with a flap cleavage reagent under a condition suitable for cleaving the 5’ overhang flap structures, thereby forming a plurality of cleavage products, wherein individual cleavage products comprise an open circle library molecule with a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same library molecule form an open circle library molecule having a nick while being hybridized to the top strand circularization oligonucleotide, and ligating the nick to generate a plurality of covalently closed circular library molecules hybridized to an immobilized target-specific bait/probe, thereby forming a plurality of immobilized closed circle library bait complexes; and g) contacting the plurality of immobilized closed circle library bait complexes with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend 3’ ends of the immobilized target-specific baits/probes, thereby generating a plurality of immobilized concatemer template molecules.

36. The method of any one of claims 1-35, wherein the sequencing comprises: a) contacting a first plurality of polymerases to (i) the plurality of immobilized concatemer template molecules and (ii) a plurality of nucleic acid primers, wherein the contacting is conducted under a condition suitable to bind the first plurality of polymerases to the plurality of immobilized concatemer template molecules and the plurality of nucleic acid primers, thereby forming a first plurality of complexed polymerases each comprising a polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises an immobilized concatemer template molecule hybridized to a nucleic acid primer; b) contacting the first plurality of complexed polymerases with a plurality of multivalent molecules to form a plurality of multivalent-binding complexes, wherein individual multivalent molecules in the plurality comprise a core attached to multiple nucleotide arms and individual nucleotide arms are attached to a nucleotide moiety, wherein the contacting is conducted under a condition suitable for binding complementary nucleotide moieties of the multivalent molecules to at least two of the first plurality of complexed polymerases thereby forming a plurality of multivalent-binding complexes, and the condition is suitable for inhibiting incorporation of the complementary nucleotide moieties into the nucleic acid primers of the plurality of multivalent-binding complexes; c) detecting the plurality of multivalent-binding complexes; and d) identifying the nucleobase of the complementary nucleotide moieties in the plurality of multivalent-binding complexes, thereby determining the sequence of the nucleic acid template molecules.

37. The method of claim 36, comprising: e) dissociating the plurality of multivalent-binding complexes by removing the first plurality of polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; f) contacting the plurality of the nucleic acid duplexes retained at step (e) with a second plurality of a polymerases under a condition suitable for binding the second plurality of polymerases to the plurality of the nucleic acid duplexes, thereby forming a second plurality of complexed polymerases, individual complexed polymerases comprising a polymerase bound to a nucleic acid duplex; and g) contacting the second plurality of second polymerases with a plurality of nucleotides, wherein the contacting is conducted under a condition suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the complexed polymerases, thereby forming a plurality of nucleotide- binding complexes, and the condition is suitable for promoting nucleotide incorporation of the bound complementary nucleotides into the nucleic acid primers of the nucleotide-binding complexes.

38. The method of claim 37, further comprising: (h) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide- complexed polymerases.

39. The method of claim 38, further comprising: i) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases; and j) identifying the nucleobases of the complementary nucleotides which are incorporated into the primers of the nucleotide-complexed polymerases.

40. The method of claim 37, wherein the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases are not detected or identified.

41. The method of any one of claims 36-40, wherein the contacting the first plurality of complexed polymerases with the plurality of multivalent molecules of step (b) is conducted in the presence of a non-catalytic divalent cation that inhibits polymerase- catalyzed nucleotide incorporation, optionally wherein the non-catalytic divalent cation comprises strontium or barium.

42. The method of any one of claim 37-41, wherein the contacting the second plurality of complexed polymerases with the plurality of nucleotides of step (g) is conducted in the presence of a catalytic divalent cation that promotes polymerase-catalyzed nucleotide incorporation, optionally wherein the catalytic divalent cation comprises magnesium or manganese.

43. The method of any one of claims 36-42, wherein the plurality of immobilized concatemer template molecules in step (a) comprise clonally amplified immobilized concatemer template molecules.

44. The method of any one of claims 36-43, wherein individual immobilized concatemer template molecules in the plurality of step (a) comprise a concatemer template molecule having two or more tandem copies of a target sequence.

45. The method of any one of claims 36-44, wherein the immobilized concatemer template molecules in the plurality of immobilized concatemer template molecules in step (a) comprise the same target of interest sequence or different target of interest sequences.

46. The method of any one of claims 36-45, wherein individual multivalent molecules in the plurality of multivalent molecules comprise: (a) a core; and (b) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide moiety, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide moiety.

47. The method of claim 46, wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits.

48. The method of claim 46 or 47, wherein the plurality of nucleotide arms attached to a given core have the same type of nucleotide moieties, and wherein the types of nucleotide moieties comprise dATP, dGTP, dCTP, dTTP or dUTP.

49. The method of claim 46 or 47, wherein the plurality of multivalent molecules comprise one type of a multivalent molecule wherein each multivalent molecule in the plurality has the same type of nucleotide moiety selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

50. The method of claim 46 or 47, wherein the plurality of multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules each type having nucleotide moieties selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

51. The method of any one of claims 36-50, wherein at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore.

52. The method of any one of claims 36-51, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises a core that is labeled with a fluorophore.

53. The method of any one of claims 36-52, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide moieties that are labeled with a fluorophore.

54. The method of any one of claims 37-53, wherein individual nucleotides in the plurality of nucleotides in step (g) comprise an aromatic base, a five carbon sugar, and 1-10 phosphate groups.

55. The method of any one of claims 37-54, wherein the plurality of nucleotides of step (g) comprise one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP, or comprise a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

56. The method of any one of claims 37-54, wherein at least one of the nucleotides in the plurality of nucleotides in step (g) is labeled with a fluorophore.

57. The method of any one of claims 37-54, wherein the plurality of nucleotides in step (g) lack a fluorophore label.

58. The method of any one of claims 37-55, wherein at least one of the nucleotides in the plurality of nucleotides of step (g) comprises a removable chain terminating moiety attached to the 3’ carbon position of the sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3 ’OH moiety on the sugar group.

59. The method of any one of claims 46-58, further comprising forming a plurality of binding complexes, comprising the steps: a) binding a first nucleic acid primer, a first polymerase, and a first multivalent molecule to a first portion of an immobilized concatemer template molecule, thereby forming a first binding complex, wherein a first nucleotide moiety of the first multivalent molecule binds to the first polymerase; and b) binding a second nucleic acid primer, a second polymerase, and the first multivalent molecule to a second portion of the immobilized concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide moiety of the first multivalent molecule binds to the second polymerase, wherein the first and second binding complexes which include the same multivalent molecule form an avidity complex.

60. The method of any one of claims 46-59, further comprising: a) contacting the first plurality of polymerases and the plurality of nucleic acid primers with different portions of an immobilized concatemer template molecule to form at least first and second complexed polymerases on the immobilized concatemer template molecule; b) contacting a plurality of multivalent molecules to the at least first and second complexed polymerases , under conditions suitable to bind a single multivalent molecule from the plurality to the first and second complexed polymerases, wherein at least a first nucleotide moiety of the single multivalent molecule is bound to the first complexed polymerase which includes a first primer hybridized to a first portion of the immobilized concatemer template molecule, thereby forming a first binding complex, and wherein at least a second nucleotide moiety of the single multivalent molecule is bound to the second complexed polymerase which includes a second primer hybridized to a second portion of the concatemer template molecule, thereby forming a second binding complex, and

• wherein the contacting is conducted under a condition suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide moieties in the first and second binding complexes, and

• wherein the first and second binding complexes which are bound to the same multivalent molecule form an avidity complex; c) detecting the first and second binding complexes on the immobilized concatemer template molecule; and d) identifying the first nucleotide moiety in the first binding complex thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide moiety in the second binding complex thereby determining the sequence of the second portion of the immobilized concatemer template molecule.