WO2025129194A2

WO2025129194A2 - Systems and methods for amplification procedures for sequencing

Info

Publication number: WO2025129194A2
Application number: PCT/US2024/060410
Authority: WO
Inventors: Dina Le-Hien PHAN; Ashley Bryn FIANCE; Karl FROHLICH; Edward BATEH; Alice Wang Yum LEE; Evan HANING; Joseph Anthony; Robert ONO; Tommie Lincecum; Itai RUSINEK; Shay SHILO; Gilad Almogy
Original assignee: Ultima Genomics Inc
Current assignee: Ultima Genomics Inc
Priority date: 2023-12-15
Filing date: 2024-12-16
Publication date: 2025-06-19
Anticipated expiration: 2026-06-15
Also published as: WO2025129194A3

Abstract

Provided are systems and methods for enrichment of positive supports subsequent to amplification. Provided are systems and methods for pre-sequencing treatment that improve sequencing quality.

Description

SYSTEMS AND METHODS FOR AMPLIFICATION PROCEDURES FOR SEQUENCING

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent App. Nos. 63/610,829 filed December 15, 2023 and 63/632,508 filed April 10, 2024, each of which is entirely incorporated by reference herein for all purposes.

BACKGROUND

[0002] Biological sample processing has various applications in the fields of molecular biology⁷ and medicine (e.g., diagnosis). For example, nucleic acid sequencing may provide information that may be used to diagnose a certain condition in a subject and in some cases tailor a treatment plan. Sequencing is widely used for molecular biology applications, including vector designs, gene therapy, vaccine design, industrial strain design and verification. Biological sample processing may involve a fluidics system and/or a detection system.

SUMMARY

[0003] Provided herein are systems and methods for amplification procedures for sequencing. The present disclosure may be advantageous to improve sequencing results. [0004] In an aspect, provided is a method is a method for sequencing data generation. The method comprises (a) loading a plurality of beads comprising a plurality of double-stranded template nucleic acid molecules attached thereto, onto a substrate; (b) on the substrate, denaturing the plurality of double-stranded template nucleic acid molecules to generate a plurality of single-stranded template nucleic acid molecules attached to the plurality of beads and hybridizing a plurality of sequencing primers to the plurality of single-stranded template nucleic acid molecules; and (c) generating the sequencing data on the plurality of doublestranded template nucleic acid molecules by extending the plurality of sequencing primers. [0005] In another aspect, provided is a method for sequencing data generation, comprising: (a) loading a plurality⁷ of beads comprising a plurality⁷ of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to the plurality of single-stranded template nucleic acid molecules; (b) on the substrate, denaturing the plurality of first sequencing primers from the plurality of singlestranded template nucleic acid molecules and re-hybridizing a plurality⁷ of second sequencing primers to the plurality of single-stranded template nucleic acid molecules; and (c) generating the sequencing data on the plurality of single-stranded template nucleic acid molecules by extending the plurality of second sequencing primers.

[0006] In some embodiments, (c) comprises performing sequencing-by -synthesis. In some embodiments, (c) comprises repeating a plurality of cycles of (i) extending the plurality of sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of sequencing primers to generate the sequencing data.

[0007] In some embodiments, the plurality of nucleotides are non-terminated. In some embodiments, the plurality of nucleotides are reversibly terminated. In some embodiments, the plurality of nucleotides comprises a mixture of the labeled nucleotides and unlabeled nucleotides. In some embodiments, the plurality of nucleotides are nucleotides of a single base type.

[0008] In some embodiments, the substrate is rotated prior to, during, or subsequent to loading the plurality of beads onto the substrate. In some embodiments, the substrate is rotated prior to, during, or subsequent to the denaturing of the plurality of double-stranded template nucleic acid molecules. In some embodiments, the substrate is rotated prior to, during, or subsequent to the hybridizing the plurality of sequencing primers to the plurality of single-stranded template nucleic acid molecules. In some embodiments, the substrate is rotated prior to, during, or subsequent to the extending of the plurality of sequencing primers. [0009] In some embodiments, the denaturing in (b) comprises treating the plurality of double-stranded template nucleic acid molecules with sodium hydroxide (NaOH).

[0010] In some embodiments, the plurality of beads are loaded onto a plurality of individually addressable locations on the substrate. In some embodiments, a bead of the plurality of beads comprises at least 1000 double-stranded template nucleic acid molecules of the plurality of double-stranded template nucleic acid molecules. In some embodiments, the at least 1000 double-stranded template nucleic acid molecules are substantially identical copies.

[0011] In some embodiments, (c) comprises performing sequencing-by -synthesis. In some embodiments, (c) comprises repeating a plurality of cycles of (i) extending the plurality of second sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of second sequencing primers to generate the sequencing data. [0012] In another aspect, provided is a method for sequencing data generation, comprising: (a) loading a plurality⁷ of beads comprising a plurality of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to the plurality of single-stranded template nucleic acid molecules; (b) generating a first set of sequencing data on the plurality' of single-stranded template nucleic acid molecules by extending the plurality' of first sequencing primers; (c) on the substrate, denaturing extension products of the plurality of first sequencing primers from the plurality' of single-stranded template nucleic acid molecules and re-hybridizing a plurality of second sequencing primers to the plurality’ of single-stranded template nucleic acid molecules; and (d) generating a second set of sequencing data on the plurality⁷ of single-stranded template nucleic acid molecules by extending the plurality⁷ of second sequencing primers.

[0013] In some embodiments, (d) comprises performing sequencing-by-synthesis. In some embodiments, (d) comprises repeating a plurality of cycles of (i) extending the plurality of second sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of second sequencing primers to generate the sequencing data.

[0014] In some embodiments, the plurality of nucleotides are non-terminated. In some embodiments, the plurality of nucleotides are reversibly terminated. In some embodiments, the plurality of nucleotides comprises a mixture of the labeled nucleotides and unlabeled nucleotides. In some embodiments, the plurality of nucleotides are nucleotides of a single base type.

[0015] In some embodiments, the substrate is rotated prior to, during, or subsequent to the loading of the plurality⁷ of beads onto the substrate. In some embodiments, the substrate is rotated prior to, during, or subsequent to the denaturing of the extension products of the plurality of first sequencing primers from the plurality' of single-stranded template nucleic acid molecules. In some embodiments, the substrate is rotated prior to, during, or subsequent to the re-hybridizing the plurality of second sequencing primers to the plurality of singlestranded template nucleic acid molecules. In some embodiments, the substrate is rotated prior to, during, or subsequent to the extending the plurality' of second sequencing primers.

[0016] In some embodiments, the denaturing in (c) comprises treating the extension products of the plurality of first sequencing primers hybridized to the plurality of single-stranded template nucleic acid molecules with sodium hydroxide (NaOH). [0017] In some embodiments, the plurality of beads are loaded onto a plurality of individually addressable locations on the substrate. In some embodiments, a bead of the plurality of beads comprises at least 1000 single-stranded template nucleic acid molecules of the plurality of single-stranded template nucleic acid molecules. In some embodiments, the at least 1000 single-stranded template nucleic acid molecules are substantially identical copies. [0018] In another aspect, provided is a method for amplification, comprising: (a) providing a first plurality of supports, each having coupled thereto a plurality of a first type of surface primers; (b) hybridizing a template molecule to a support of the first plurality of supports; (c) amplifying the template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the template molecule; (d) contacting the first amplified support with a substrate, wherein the substrate comprises a second plurality of supports coupled thereto, wherein a support of the second plurality of supports comprises a plurality of a second type of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer; (e) hybridizing one or more copies of the template molecule to a support of the second plurality of supports; and (f) amplify ing the one or more copies of the template molecule to provide a second amplified support coupled to the substrate.

[0019] In some embodiments, the method further comprises analyzing the second amplified support to determine a sequence of the template molecule.

[0020] In some embodiments, the second amplified support comprises a plurality' of nucleic acid molecules having substantially 100% sequence identity.

[0021] In some embodiments, the plurality of nucleic acid molecules are copies of copies of the template molecule.

[0022] In some embodiments, the amplifying (f) is performed in the absence of solution primers.

[0023] In another aspect, provided is a method for amplification, comprising: (a) providing a first plurality of supports, each having coupled thereto a plurality of a first type of surface primers; (b) hybridizing a template molecule to a support of the first plurality' of supports; (c) contacting the first plurality' of supports to a substrate, thereby coupling the support to the substrate; (d) amplifying the template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the template molecule; (e) contacting the substrate with a second plurality of supports comprising a plurality of a second ty pe of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer; (f) hybridizing one or more copies of the template molecule to a support of the second plurality of supports; and (g) amplify ing the one or more copies of the template molecule to provide a plurality of copies of the template molecule coupled to the first amplified support.

[0024] In some embodiments, the method further comprises analyzing the plurality of copies of the template molecule to determine a sequence of the template molecule.

[0025] In some embodiments, the plurality of copies of the template molecule have substantially 100% sequence identity.

[0026] In some embodiments, the amplifying (g) is performed in the absence of solution primers.

[0027] In another aspect, provided is a method of amplification, comprising: (a) providing a first plurality of supports, each having coupled thereto a plurality of a first type of surface primers; (b) hybridizing and ligating a double-stranded template molecule to a support of the first plurality of supports, wherein a first strand of the double stranded template molecule is ligated to the support and a second strand of the double-stranded template molecule is hybridized to a surface primer of the first type of surface primers; (c) amplifying the doublestranded template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the second strand of the template molecule; (d) contacting the first amplified support with a substrate, wherein the substrate comprises a second plurality of supports coupled thereto, wherein a support of the second plurality of supports comprises a plurality of a second type of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer; (e) hybridizing one or more copies of the second strand of the double-stranded template molecule to a support of the second plurality of supports; and (I amplifying the one or more copies of the second strand of the double-stranded template molecule and the first strand of the template molecule to provide a second amplified support coupled to the substrate.

[0028] In some embodiments, the coupling (c) comprises covalent coupling.

[0029] In some embodiments, the second amplified support comprises copies of copies of the second strand of the double-stranded template molecule and copies of the first strand of the template molecule. In some embodiments, the second amplified support comprises a plurality of nucleic acid molecules having substantially 100% sequence identity.

[0030] In some embodiments, at least one nucleic acid molecule does not have 100% sequence identity to the plurality of nucleic acid molecules. [0031] In some embodiments, the amplifying (f) is performed in the absence of solution primers.

[0032] In some embodiments, the method further comprises analyzing the plurality of copies of the template molecule to determine a sequence of the template molecule.

[0033] In another aspect, provided is a method for post-amplification enrichment, comprising: (a) subsequent to amplification of a plurality of library molecules, receiving a mixture of positive supports and negative supports, wherein each of the positive supports comprises at least one template strand derived from the plurality of library molecules, and wherein each of the negative supports does not comprise a template strand derived from the plurality of library molecules; (b) contacting the mixture with a plurality of desthiobiotinylated sequencing primers to generate a plurality' of desthiobiotin-bound complexes, each desthiobiotin-bound complex comprising a positive support of the positive supports, wherein a desthiobiotinylated sequencing primer of the plurality of desthiobiotinylated sequencing primers is hybridized to a template strand of the at least one template strand in the positive support; (c) isolating the desthiobiotin-bound complexes from the negative supports in the mixture by contacting the mixture with (i) a plurality of magnetic beads comprising streptavidin and (ii) a magnet, and eluting; and (d) isolating the positive supports from the plurality of magnetic beads by contacting the isolated desthiobiotin-bound complexes with (i) a plurality of biotin moieties and (ii) a magnet, and eluting, wherein the plurality' of biotin moieties binds to the plurality' of magnetic beads to displace the desthiobiotinylated sequencing primer from the plurality’ of magnetic beads.

[0034] In some embodiments, the contacting in (b) comprises contacting the mixture with a primer mixture, the primer mixture comprising the plurality' of desthiobiotiny dated sequencing primers and a plurality' of sequencing primers.

[0035] In some embodiments, less than 30% of the primer mixture is the plurality' of desthiobiotinylated sequencing primers. In some embodiments, less than 15% of the primer mixture is the plurality of desthiobiotinylated sequencing primers.

[0036] In some embodiments, prior to the contacting in (b), any double-stranded nucleic acid molecules on the positive supports are denatured to generate single-stranded positive supports. In some embodiments, the double-stranded nucleic acid molecules are denatured via a denaturing agent, heating, or both. In some embodiments, the denaturing agent comprises sodium hydroxide. [0037] In some embodiments, the method further comprises loading the isolated positive supports onto a substrate and sequencing the at least one template strand on the substrate. [0038] In some embodiments, the method further comprises denaturing any desthiobiotinylated sequencing primers of the plurality of desthiobiotinylated sequencing primers bound to template strands on the positive supports prior to sequencing. In some embodiments, the denaturing is performed on the substrate. In some embodiments, the denaturing is performed prior to loading the isolated positive supports on the substrate. [0039] In some embodiments, the method further comprises hybridizing a plurality of sequencing primers to template strands of the at least one template strand prior to sequencing. In some embodiments, the hybridizing is performed on the substrate. In some embodiments, the hybridizing is performed prior to loading the isolated positive supports on the substrate. [0040] In some embodiments, the positive support comprises a plurality of template strand having substantially 100% sequence identity.

[0041] In some embodiments, two respective template strands on two different positive supports of the positives supports are derived from two different library' molecules of the plurality of library molecules.

[0042] In some embodiments, the positive support comprises a bead.

[0043] In another aspect, provided is a method of amplification, comprising: (a) providing a support comprising a first plurality' of primers and a second plurality of primers, wherein a first primer of the first plurality' of primers comprising a first sequence and is coupled directly to a primer attachment site of the support and wherein a second primer of the second plurality of primers comprises a second sequence and is coupled to a primer attachment site of the support via a tether; (b) hybridizing a template molecule to the first primer and extending the first primer to generate a first extended strand coupled to the support; and (c) hybridizing the second primer to the first extended strand and extending the second primer to generate a second extended strand couple to the support, wherein the second extended strand comprises a sequence capable of hybridizing to another primer of the first plurality of primers.

[0044] In some embodiments, the first and second sequences do not have sequence complementarity.

[0045] In some embodiments, the method further comprises repeating (b) and (c) to generate an amplified support comprising a first plurality of molecules that are copies of the first extended strand, wherein each of the first plurality' of molecules is coupled to the amplified support via a first primer. [0046] In some embodiments, the amplified support further comprises a second plurality of molecules that are copies of the second extended strand, wherein each of the second plurality of molecules is coupled to the amplified support via a second primer.

[0047] In some embodiments, the second primer comprises a cleavage site comprising one or more cleavable moieties.

[0048] In some embodiments, the method further comprises cleaving the cleavage site and removing the second extended strand from the support.

[0049] In some embodiments, the support comprises a polymer mesh, wherein a polymer of the polymer mesh comprises a primer attachment site.

[0050] In some embodiments, the primer attachment site comprises a coupling moiety. In some embodiments, the primer attachment site comprises one of a click chemistry pair. [0051] In some embodiments, the primer attachment site comprises a cyclooctyne.

[0052] In another aspect, provided is a method of post-amplification enrichment, comprising

(a) receiving an emulsion amplification product mixture comprising a plurality of positive supports, a plurality of negative supports, and a plurality of solution amplification primers;

(b) subjecting the amplified product mixture to conditions sufficient to break the emulsion, and providing one or more oligos to hybridize to one or more support-coupled primers in the amplification product mixture; (c) providing a plurality of single stranded binding proteins to bind to the plurality of solution amplification primers, and removing the plurality' of single stranded binding proteins; and (d) isolating the plurality of positive supports from the plurality of negative supports.

[0053] In some embodiments, the conditions sufficient to break the emulsion in (b) comprising, heating, agitation, the application of electrostatic force, or a combination thereof. [0054] In some embodiments, the one or more oligos hybridize to at least 25% of the support-coupled primers.

[0055] In some embodiments, at least one support-coupled primer is coupled to a positive support. In some embodiments, at least one support-coupled primer is couple to a negative support.

[0056] In some embodiments, the plurality of single stranded binding proteins bind to at least 25% of the plurality of solution amplification primers.

[0057] In some embodiments, the emulsion amplification product mixture is a result of the amplification of a plurality' of library molecules. [0058] In some embodiments, each of the positive supports comprises at least one template strand derived from the plurality of library’ molecules, and wherein each of the negative supports does not comprise a template strand derived from the plurality of library' molecules. [0059] In some embodiments, the isolating (d) comprises: (e) contacting the amplification product mixture with a plurality of desthiobiotinylated sequencing primers to generate a plurality of desthiobiotin-bound complexes, each desthiobiotin-bound complex comprising a positive support of the positive supports, wherein a desthiobiotinylated sequencing primer of the plurality of desthiobiotinylated sequencing primers is hybridized to a template strand of the at least one template strand in the positive support; (!) isolating the desthiobiotin-bound complexes from the negative supports in the amplification product mixture by contacting the amplification product mixture with (i) a plurality' of magnetic beads comprising streptavidin and (ii) a magnet, and eluting; and (g) isolating the positive supports from the plurality of magnetic beads by contacting the isolated desthiobiotin-bound complexes with (i) a plurality of biotin moieties and (ii) a magnet, and eluting, wherein the plurality of biotin moi eties binds to the plurality' of magnetic beads to displace the desthiobiotinylated sequencing primer from the plurality' of magnetic beads.

[0060] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative instances of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different instances, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0061] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] 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 (also “Figure” and “FIG.” herein) of which:

[0063] FIG. 1 illustrates an example workflow for processing a sample for sequencing.

[0064] FIG. 2 illustrates examples of individually addressable locations distributed on substrates, as described herein.

[0065] FIG. 3 shows an example image of a substrate with a hexagonal lattice of beads, as described herein.

[0066] FIG. 4 illustrates example systems and methods for loading a sample or a reagent onto a substrate, as described herein.

[0067] FIGs. 5A-5B illustrate multiplexed stations in a sequencing system.

[0068] FIG. 6 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.

[0069] FIGs. 7A and 7B illustrate post-amplification enrichment workflows that use biotin capture moieties.

[0070] FIGs. 7C and 7D illustrate post-amplification enrichment workflow that use desthiobiotin capture moieties.

[0071] FIG. 7E illustrates a post-amplification enrichment workflow that isolates positive supports via stripping.

[0072] FIG. 7F illustrates a post-amplification enrichment workflow that isolates positive supports via cleaving.

[0073] FIGs. 8A-8C illustrate an exemplary' amplification workflow.

[0074] FIGs. 9A and 9B illustrate an exemplary amplification workflow.

[0075] FIGs. 10A and 10B illustrate an exemplary amplification workflow.

[0076] FIGs. 10C and 10D illustrate an exemplary amplification workflow.

[0077] FIG. 10E illustrates an example amplification workflow on a bead.

[0078] FIG. HA illustrates a non-limiting schematic of library molecule preparation.

[0079] FIG. 11B illustrates a non-limiting schematic of library molecule preparation using methylation-specific adapters. [0080] FIG. 11C illustrates a non-limiting example of library molecule preparation using methylated, partially single-stranded adapters.

[0081] FIGs. 12A and 12B illustrate a non-limiting example of end repair and library' molecule preparation.

[0082] FIG. 13 illustrates sequencing metrics from rehybridization workflows.

[0083] FIG. 14 illustrates mean signal vs. homopolymer length for three sequencing runs.

[0084] FIG. 15 illustrates a cross-sectional view of an exemplary physical cap for a centrifugation tube.

[0085] FIGs. 16A-16D illustrate the use of a chemical cap in sample preparation. A tube comprising sample (FIG. 16A) may have a chemical cap added (FIG. 16B). After centrifugation, a chemical cap may dissipate into the supernatant (FIG. 16C) or remain layered over the supernatant (FIG. 16D).

DETAILED DESCRIPTION

[0086] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0087] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0001] When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range is encompassed within the scope of the present disclosure. Where the stated range includes upper or lower limits, ranges excluding either of those included limits are also included in the present disclosure.

[0088] The term “analyte,” as used herein, generally refers to an object that is the subject of analysis, or an object, regardless of being the subject of analysis, that is directly or indirectly analyzed during a process. An analyte may be synthetic. An analyte may be, originate from, and/or be derived from, a sample, such as a biological sample. In some examples, an analyte is or includes a molecule, macromolecule (e.g., nucleic acid, carbohydrate, protein, lipid, etc.), nucleic acid, carbohydrate, lipid, antibody, antibody fragment, antigen, peptide, polypeptide, protein, macromolecular group (e.g., glycoproteins, proteoglycans, ribozy mes, liposomes, etc.), cell, tissue, biological particle, or an organism, or any engineered copy or variant thereof, or any combination thereof. The phrase “processing an analyte,” as used herein, generally refers to one or more stages of interaction with one more samples. Processing an analyte may comprise conducting a chemical reaction, biochemical reaction, enzymatic reaction, hybridization reaction, polymerization reaction, physical reaction, any other reaction, or a combination thereof with, in the presence of, or on, the analyte. Processing an analyte may comprise physical and/or chemical manipulation of the analyte. [0089] The term “biological sample,” as used herein, generally refers to any sample derived or extracted from a subject or specimen. The biological sample can be a fluid (e.g.. blood (e.g., whole blood), saliva, urine, or sweat), tissue (e.g., from an organ or a mass of cellular material (e.g., a tumor)), collection of cells (e.g., cheek swab), hair sample, or feces sample. The biological sample can be a cellular sample or cell-free sample. Examples of biological samples include nucleic acid molecules, amino acids, polypeptides, proteins, carbohydrates, fats, or viruses. In an example, a biological sample is a nucleic acid sample including one or more nucleic acid molecules, such as deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). The nucleic acid sample may comprise cell-free nucleic acid molecules, such as cell- free DNA or cell-free RNA. Non-limiting examples of nucleic acids include DNA. RNA, genomic DNA or synthetic DNA/RNA or coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA (rRNA), short interfering RNA (siRNA), shorthairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, and isolated DNA or isolated RNA of any sequence. Cell free polynucleotides may be fetal in origin (e g., via fluid taken from a pregnant subject) or may be derived from tissue of the subject itself. A biological sample may also refer to a sample engineered to mimic one or more properties (e.g., nucleic acid sequence properties, e.g., sequence identity, length, GC content, etc.) of a sample derived from a subject or specimen.

[0002] As used herein, the term “template nucleic acid” generally refers to the nucleic acid to be sequenced. The template nucleic acid may be an analyte or be associated with an analyte. For example, the analyte can be a mRNA, and the template nucleic acid is the mRNA or a cDNA derived from the mRNA. or other derivative thereof. In another example, the analyte can be a protein, and the template nucleic acid is an oligonucleotide that is conjugated to an antibody that binds to the protein, or derivative thereof. A template nucleic acid may be amplified to produce a colony of nucleic acid molecules attached to a support (e.g., a bead) to produce amplified sequencing signals. The substrate surface may immobilize multiple beads at distinct locations, each bead containing distinct colonies of nucleic acids, and upon detecting the substrate surface, multiple sequencing signals may be simultaneously or substantially simultaneously processed from the different immobilized beads at the distinct locations to generate multiple sequencing reads. In some sequencing methods, the nucleotide flows may comprise non-terminated nucleotides, terminated nucleotides, or a combination thereof.

[0090] The term “nucleotide,” as used herein, generally refers to any nucleotide or nucleotide analog. The nucleotide may be naturally occurring or non-naturally occurring. The nucleotide may be a non-standard, modified, synthesized, or engineered nucleotide. The nucleotide may include a canonical base or a non-canonical base. The nucleotide may comprise an alternative base. The nucleotide may include a modified polyphosphate chain (e.g., triphosphate coupled to a fluorophore). The nucleotide may comprise a label. The nucleotide may be terminated (e.g., reversibly terminated). The nucleotide may be non-terminated (e.g., natural or modified). In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Nucleic acids may also be modified at the base moiety (e.g., at one or more atoms that ty pically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety' or phosphate backbone. Nucleic acids may also contain amine-modified groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide esters (NHS). Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety' (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photo- programmed polymerases, or lower secondary' structure. Nucleotides may be capable of reacting or bonding with detectable moieties for detection.

[0091] The term “sequencing,” as used herein, generally refers to a process for generating or identifying a sequence of a biological molecule, such as a nucleic acid. The sequence may be a nucleic acid sequence which comprises a sequence of nucleic acid bases. Examples of sequencing include single molecule sequencing or sequencing by synthesis. Sequencing may comprise generating sequencing signals and/or sequencing reads. Sequencing may be performed on template nucleic acids immobilized on a support, such as a flow cell, substrate, and/or one or more beads.

Sample Processing Methods

[0092] Described herein are devices, systems, methods, compositions, and kits for processing samples, such as to prepare a sample for sequencing, to sequence a sample, and/or to analyze sequencing data. FIG. 1 illustrates an example sequencing workflow 100, according to the devices, systems, methods, compositions, and kits of the present disclosure.

[0093] Supports and/or template nucleic acids may be provided and/or prepared (101) to be compatible with downstream sequencing operations (e.g., 107). A support (e.g., bead) may help immobilize a template nucleic acid to a substrate, such as when the template nucleic acid is coupled to the support, and the support is in turn immobilized to the substrate. The support may further function as a binding entity to retain derivatives molecules (e.g., amplification products) from a same template nucleic acid together for dow nstream processing, such as for sequencing operations. This may be useful in distinguishing a colony from other colonies (e.g., on other supports) and generating amplified sequencing signals corresponding to a template nucleic acid. A support may comprise an oligonucleotide comprising one or more functional nucleic acid sequences. The oligonucleotide may be single-stranded, doublestranded, or partially double-stranded. For example, the oligonucleotide may comprise a capture sequence, a primer sequence, a sequencing primer sequence, a barcode sequence, a sample index sequence, a unique molecular identifier (UMI), a flow cell adapter sequence, an adapter sequence, a target sequence, a random sequence, a binding sequence (e.g., for a splint, primer, template nucleic acid, capture sequence, etc ), or any other functional sequence useful for a downstream operation, a complement thereof, or any combination thereof. The capture sequence may be configured to hybridize to a sequence of a template nucleic acid or derivative thereof. The support may comprise a plurality of oligonucleotides, for example on the order of 10, 10², 10³, 10⁴, 10’, 10⁶, 10⁷, or more molecules. The support may comprise a single species of oligonucleotide which comprise identical sequences. The support may comprise multiple species of oligonucleotides which have varying sequences. In some cases, the support comprises a single species of a primer (e.g., forward primer) for amplification. In some cases, the support comprises two species of primer (e.g., forward primer, reverse primer) for amplification. Devices, systems, methods, compositions, and kits for preparing and using support species are described in further detail in U.S. Patent Pub. No. 20220042072A1 and International Patent Pub. No. W02022040557A2, each of which is incorporated herein by reference in its entirety.

[0094] A support may comprise one or more capture entities, where a capture entity is configured for capture by a capturing entity. A capture entity may be coupled to or be part of an oligonucleotide coupled to the support. A capture entity may be coupled to or be part of the support. Examples of capture entity -capturing entity pairs and capturing entity -capture entity pairs include: streptavidin (SA)-biotin; complementary sequences; magnetic particle- magnetic field system; charged particle-electric field system; azide-cyclooctyne; thiol- maleimide; click chemistry pairs; cross-linking pairs; etc. The capture entity-capturing entitypair may comprise one or more chemically modified bases. A capture entity and capturing entity may bind, couple, hybridize, or otherwise associate with each other. The association may comprise formation of a covalent bond, non-covalent bond, releasable bond (e.g., cleavable bond that is cleavable upon application of a stimulus), and/or no bond. The capture entity may be capable of linking to a nucleotide. In some instances, the capturing entity may comprise a secondary capture entity, for example, for subsequent capture by a secondary capturing entity. The secondary capture entity and secondary capturing entity may comprise any one or more of the capturing mechanisms described elsewhere herein.

[0095] A support may comprise one or more cleavable moieties, also referred to herein as excisable moieties. The cleavable moiety may be coupled to or be part of an oligonucleotide coupled to the support. The cleavable moiety may be coupled to the support. A cleavable moiety may comprise any useful moiety that can be used to cleave an oligonucleotide (or portion thereof) from the support, or otherwise release a nucleic acid strand from the support and/or the oligonucleotide. A cleavable moiety may comprise a uracil, a ribonucleotide, methylated nucleotide, or other modified nucleotide that is excisable or cleavable using an enzyme (e.g., UDG, RNAse, APE1, MspJI, endonuclease, exonuclease, etc.). The cleavable moiety may comprise an abasic site or an analog of an abasic site (e.g., dSpacer). a dideoxyribose, a spacer, e.g., C3 spacer, hexanediol, triethylene glycol spacer (e.g.. Spacer 9), hexa-ethyleneglycol spacer (e.g., Spacer 18), a photocleavable moiety-, or combinations or analogs thereof. Alternatively, or in addition, the cleavable moiety may be cleavable using one or more stimuli, e.g., photo-stimulus, chemical stimulus, thermal stimulus, etc.

[0096] The sequencing workflow 100 may not involve supports, for example when a template nucleic acid and/or its derivatives are directly attached to a substrate and amplified and/or sequenced from the substrate. [0097] A template nucleic acid may include an insert sequence sourced from a biological sample. The template nucleic acid may be derived from any nucleic acid of the biological sample (e.g., endogenous nucleic acid) and result from any number of processing operations, such as but not limited to fragmentation, degradation or digestion, transposition, ligation, reverse transcription, extension, replication, etc. The template nucleic acid may be singlestranded, double-stranded, or partially double-stranded. A template nucleic acid may comprise one or more functional nucleic acid sequences. For example, the template nucleic acid may comprise a capture sequence, a primer sequence, a sequencing primer sequence, a barcode sequence, a sample index sequence, a unique molecular identifier (UM1), a flow cell adapter sequence, an adapter sequence, a target sequence, a random sequence, a binding sequence (e.g., for a splint, primer, template nucleic acid, capture sequence, etc.), or any other functional sequence useful for a downstream operation, a complement thereof, or any combination thereof. The template nucleic acid may comprise an adapter sequence configured to be captured by a capture sequence of an oligonucleotide coupled to a support. The one or more functional nucleic acid sequences may be disposed at one end or at both ends of the insert sequence. A nucleic acid molecule comprising the insert sequence, or complement thereof, may be processed with (e.g.. attached to, extend from, etc.) one or more adapter molecules to generate the template nucleic acid comprising the insert sequence and one or more functional nucleic acid sequences. A template nucleic acid may comprise one or more capture entities and/or one or more cleavable moieties that are described elsewhere herein.

[0098] Optionally, the supports and/or template nucleic acids may be pre-enriched (102). For example, a support comprising a distinct oligonucleotide sequence is pre-enriched to isolate from a mixture comprising support(s) that do not have the distinct oligonucleotide sequence. For example, a template nucleic acid comprising a distinct configuration (e.g., comprising a particular adapter sequence) is pre-enriched to isolate from a mixture comprising template nucleic acids that do not have the distinct configuration. In some cases, the capture entity on the supports and/or template nucleic acids are used for pre-enrichment.

[0099] The supports and template nucleic acids may be attached (103) to generate supporttemplate complexes. A template nucleic acid may be coupled to a support via any method(s) that results in a stable association between the template nucleic acid and the support. For example, the template nucleic acid may hybridize to an oligonucleotide on the support; the template nucleic acid may be ligated to a nucleic acid coupled to the support; the template nucleic acid may hybridize to one or more intermediary molecules, such as a splint, bridge, and/or primer molecule, which hybridizes to an oligonucleotide on the support; and/or the template nucleic acid may be hybridized to an oligonucleotide on a support, which oligonucleotide comprises a primer sequence which is extended. In some cases, the respective concentrations of the supports and template nucleic acids may be adjusted such that a majority of support-template complexes are single template-attached supports (e.g., a support attached to a single template nucleic acid).

[0100] Optionally, support-template complexes may be pre-enriched (104), wherein a support-template complex is isolated from a mixture comprising support(s) and/or template nucleic acid(s) that are not attached to each other. In some cases, the capture entity on the supports and/or template nucleic acids are used for pre-enrichment.

[0101] The template nucleic acids may be subjected to amplification reactions (105) to generate a plurality of amplification products immobilized to the support. Such amplification reactions may comprise performing polymerase chain reaction (PCR) or any other amplification methods described herein, including but not limited to emulsion PCR (ePCR or emPCR), isothermal amplification, recombinase polymerase amplification (RPA), rolling circle amplification (RCA), multiple displacement amplification (MDA), bridge amplification, template walking, etc. Amplification reactions can occur while the support is immobilized to a substrate. Amplification reactions can occur off the substrate, such as in solution, or on a different surface or platform. Amplification reactions can occur in isolated reaction volumes, such as within multiple droplets in an emulsion during emulsion PCR (ePCR or emPCR), or in wells or tubes.

[0102] Optionally, subsequent to amplification, the supports, template nucleic acids, and/or support-template complexes may be subjected to post-amplification processing (106). Often, subsequent to amplification, a resulting mixture may comprise a mix of positive supports (e.g., those comprising a template nucleic acid molecule) and negative supports (e.g., those not attached to template nucleic acid molecules). Enrichment procedure(s) may isolate positive supports from the mixtures. Example methods of enrichment of amplified supports are described in U.S. Patent Nos. 10,900,078, U.S. Patent Pub. No. 20210079464A1, and International Patent Pub. No. W02022040557A2. each of which is entirety incorporated by reference herein.

[0103] The template nucleic acids may be subject to sequencing (107). The template nucleic acid(s) may be sequenced while attached to the support. Alternatively, the template nucleic acid molecules may be free of the support when sequenced and/or analyzed. The template nucleic acids may be sequenced while immobilized to a substrate, such as via a support or otherwise. Examples of substrate-based sample processing systems are described elsewhere herein. Any sequencing method may be used, for example pyrosequencing, single molecule sequencing, sequencing by synthesis (SBS), sequencing by ligation, sequencing by binding, etc.

[0104] For example, sequencing comprises extending a sequencing primer (or growing strand) hybridized to a template nucleic acid by providing labeled nucleotide reagents, washing away unincorporated nucleotides from the reaction space, and detecting one or more signals from the labeled nucleotide reagents which are indicative of an incorporation event or lack thereof. After detection, the labels may be cleaved and the whole process may be repeated any number of times to determine sequence information of the template nucleic acid. One or more intermediary flows may be provided intra- or inter- repeat, such as washing flows, label cleaving flows, terminator cleaving flows, reaction-completing flows (e.g., double tap flow, triple tap flow, etc.), labeled flows (or bright flows), unlabeled flows (or dark flows), phasing flows, chemical scar capping flows, etc. A nucleotide mixture that is provided during any one flow may comprise only labeled nucleotides, only unlabeled nucleotides, or a mixture of labeled and unlabeled nucleotides. The mixture of labeled and unlabeled nucleotides may be of any fraction of labeled nucleotides, such as at least or at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%. 95%. or 99%. A nucleotide mixture that is provided during any one flow may comprise only non-terminated nucleotides, only terminated nucleotides, or a mixture of terminated and non-terminated nucleotides. When using only non-terminated nucleotides, terminator cleaving flows may be omitted from the sequencing process. When using terminated nucleotides, to proceed with the next step of extension, prior to. during, or subsequent to detection, a terminator cleaving flow may be provided to cleave blocking moieties. A nucleotide mixture that is provided during any one flow may comprise any number of canonical base types (e.g., A, T, G, C, U), such as a single canonical base type, two canonical base types, three canonical base types, four canonical base types or five canonical base types (including T and U). Different types of nucleotide bases may be flowed in any order and/or in any mixture of base types that is useful for sequencing. Various flow-based sequencing systems and methods are described in U.S. Pat. Pub. No. 2022/0170089A1, which is entirely incorporated by reference herein for all purposes. Labeled nucleotides may comprise a dye, fluorophore, or quantum dot, multiples thereof, and/or combination thereof. In some cases, nucleotides of different canonical base types may be labeled and detectable at a single frequency (e.g., using the same or different dyes). In other cases, nucleotides of different canonical base types may be labeled and detectable at different frequencies (e.g., using the same or different dyes).

[0105] Subsequent to sequencing, the sequencing signals collected and/or generated may be subjected to data analysis (108). The sequencing signals may be processed to generate base calls and/or sequencing reads. In some cases, the sequencing reads may be processed to generate diagnostics data to the biological sample, or the subject from which the biological sample was derived from. The data analysis may comprise image processing, alignment to a genome or reference genome, training and/or trained algorithms, error correction, and the like.

[0106] While the sequencing workflow 100 with respect to FIG. 1 has been described with respect to the use of supports to bind template molecules, it will be appreciated that the different supports may be effectively replaced by using spatially distinct locations on one or more surfaces, which do not necessarily have to be the surfaces of individual supports (e g., beads). For example, a first spatially distinct location on a surface may be capable of directly immobilizing a first colony of a first template nucleic acid and a second spatially distinct location on the same surface (or a different surface) may be capable of directly immobilizing a second colony of a second template nucleic acid to distinguish from the first colony. In some cases, the surface comprising the spatially distinct locations may be a surface of the substrate on which the sample is sequenced, thus streamlining the amplification-sequencing workflow.

[0107] It will be appreciated that in some instances, the different operations described in the sequencing workflow 100 may be performed in a different order. It will be appreciated that in some instances, one or more operations described in the sequencing workflow 100 may be omitted or replaced with other comparable operation(s). It will be appreciated that in some instances, one or more additional operations described in the sequencing workflow 100 may be performed. The different operations described with respect to sequencing workflow 100 may be performed with the help of open substrate systems described herein.

Open substrate systems [0108] Described herein are devices, systems, and methods that use open substrates or open flow cell geometries to process a sample. The term “open substrate,” as used herein, generally refers to a substrate in which any point on an active surface of the substrate is physically accessible from a direction normal to the substrate. The devices, systems and methods may be used to facilitate any application or process involving a reaction or interaction between two objects, such as between an analyte and a reagent or between two reagents. For example, the reaction or interaction may be chemical (e.g., polymerase reaction) or physical (e.g., displacement). The devices, systems, and methods described herein may benefit from higher efficiency, such as from faster reagent delivery and lower volumes of reagents required per surface area. The devices, systems, and methods described herein may avoid contamination problems common to microfluidic channel flow cells that are fed from multiport valves which can be a source of carryover from one reagent to the next. The devices, systems, and methods may benefit from shorter completion time, use of fewer resources (e g., various reagents), and/or reduced system costs. The open substrates or flow cell geometries may be used to process any analyte from any sample, such as but not limited to, nucleic acid molecules, protein molecules, antibodies, antigens, cells, and/or organisms, as described herein. The open substrates or flow cell geometries may be used for any application or process, such as, but not limited to, sequencing by synthesis, sequencing by ligation, amplification, proteomics, single cell processing, barcoding, and sample preparation, as described herein.

[0109] A sample processing system may comprise a substrate, and devices and systems that perform one or more operations with or on the substrate. The sample processing system may permit highly efficient dispensing of analytes and reagents onto the substrate. The sample processing may permit highly efficient imaging of one or more analytes, or signals corresponding thereto, on the substrate. The sample processing system may comprise an imaging system comprising a detector. Substrates, detectors, and sample processing hardware that can be used in the sample processing system are described in further detail in U.S. Patent Pub. No. 20200326327A1, U.S. Patent Pub. No. 20210079464A1, International Patent Pub. No. WO2022072652A1, U.S. Patent Pub. No. 20210354126A1, and International Patent Pub. No. WO2023192403A2, each of which is entirely incorporated herein by reference for all purposes. Substrates

[0110] The substrate may be a solid substrate. The substrate may entirely or partially comprise one or more of rubber, glass, silicon, a metal such as aluminum, copper, titanium, chromium, or steel, a ceramic such as titanium oxide or silicon nitride, a plastic such as polyethylene (PE), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadiene styrene (ABS), polyacetylene, polyamides, polycarbonates, polyesters, polyurethanes, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamine formaldehyde (MF), urea-formaldehyde (UF), polyetheretherketone (PEEK), polyetherimide (PEI), polyimides, polylactic acid (PLA), furans, silicones, polysulfones, any mixture of any of the preceding materials, or any other appropriate material. The substrate may be entirely or partially coated with one or more layers of a metal such as aluminum, copper, silver, or gold, an oxide such as a silicon oxide (SixOy, where x, y may take on any possible values), a photoresist such as SU8, a surface coating such as an aminosilane or hydrogel, polyacrylic acid, polyacrylamide dextran, polyethylene glycol (PEG), or any combination of any of the preceding materials, or any other appropriate coating. The substrate may comprise multiple layers of the same or different type of material. The substrate may be fully or partially opaque to visible light. The substrate may be fully or partially transparent to visible light. A surface of the substrate may be modified to comprise active chemical groups, such as amines, esters, hydroxyls, epoxides, and the like, or a combination thereof. A surface of the substrate may be modified to comprise any of the binders or linkers described herein. In some instances, such binders, linkers, active chemical groups, and the like may be added as an additional layer or coating to the substrate.

[OHl] The substrate may have the general form of a cylinder, a cylindrical shell or disk, a rectangular prism, or any other geometric form. The substrate may have a thickness (e.g., a minimum dimension) of at least and/or at most about 100 micrometers (pm), 200 pm, 500 pm, 1 millimeter (mm), 2 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 or mm. The substrate may have a first lateral dimension (such as a width for a substrate having the general form of a rectangular prism or a radius or diameter for a substrate having the general form of a cylinder) and/or a second lateral dimension (such as a length for a substrate having the general form of a rectangular prism) of at least and/or at most about 1 mm, 2 mm, 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 100 mm, 150 mm, 200 mm, 300 mm, 400 mm, 500 mm, 1,000 mm, 1,500 mm, 2,000 mm, 2,500 mm, 3,000 mm, 4,000 mm, 5,000 mm or more.

[0112] One or more surfaces of the substrate may be exposed to and accessible from a surrounding open environment. In some cases, the surrounding open environment may be controlled and/or confined in a larger controlled environment.

[0113] The substrate may comprise a plurality of individually addressable locations. The individually addressable locations may comprise locations that are physically accessible for manipulation. The manipulation may comprise, for example, placement, extraction, reagent dispensing, seeding, heating, cooling, or agitation. The manipulation may be accomplished through, for example, localized microfluidic, pipet, optical, laser, acoustic, magnetic, and/or electromagnetic interactions with the analyte or its surroundings. The individually addressable locations may comprise locations that are digitally accessible. For example, each individually addressable location may be located, identified, and/or accessed electronically or digitally for indexing, mapping, sensing, associating with a device (e.g., detector, processor, dispenser, etc.), or otherwise processing. In some cases, the individually addressable locations may be defined by physical features of the substrate (e.g., on a modified surface) to distinguish from each other and from non-individually addressable locations. In some cases, the individually addressable locations may not be defined by physical features of the substrate, and instead may be defined digitally (e.g., by indexing) and/or via the analytes and/or reagents that are loaded on the substrate (e.g., the locations in which analytes are immobilized on the substrate). The plurality of individually addressable locations may be arranged as an array, randomly, or according to any pattern, on the substrate. FIG. 2 illustrates different substrates (from a top view) comprising different arrangements of individually addressable locations 201, with panel A showing a substantially rectangular substrate with regular linear arrays, panel B showing a substantially circular substrate with regular linear arrays, and panel C showing an arbitrarily shaped substrate with irregular arrays.

[0114] The substrate may have any number of individually addressable locations, for example, on the order of 1, 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or more individually addressable locations. Each individually addressable location may have any shape or form, for example the general shape or form of a circle, oval, square, rectangle, polygonal, or non-polygonal shape when viewed from the top. A plurality of individually addressable locations can have uniform shape or form, or different shapes or forms. An individually addressable location may have any size. In some cases, an individually addressable location may have an area of at least and/or at most about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.25, 1.3, 1.4 ,1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2, 2.25, 2.5. 2.75, 3, 3.25. 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5. 5.5, 6, 7, 8. 9, 10 square micron (pm²), or more. The individually addressable locations may be distributed on a substrate with a pitch determined by the distance between the center of a first location and the center of the closest or neighboring individually addressable location. Locations may be spaced with a pitch of at least and/or at most about 0.1, 0.2. 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1. 1.1, 1.2, 1.25, 1.3, 1.4 ,1.5, 1.6, 1.7. 1.75. 1.8. 1.9, 2, 2.25, 2.5. 2.75, 3. 3.25. 3.5, 3.75. 4, 4.25, 4.5, 4.75, 5, 5.5. 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 micron (pm). In some cases, the pitch between two individually addressable locations may be determined as a function of a size of a loading object (e.g., bead). For example, where the loading object is a bead having a maximum diameter, the pitch may be at least about the maximum diameter of the loading object.

[0115] Each of the plurality of individually addressable locations, or each of a subset of the locations, may be capable of immobilizing thereto an analyte (e.g., a nucleic acid, a protein, a carbohydrate, etc.) or a reagent (e.g., a nucleic acid, a probe molecule, a barcode molecule, an antibody molecule, a primer molecule, a bead, etc.). In some cases, an analyte or reagent may be immobilized to an individually addressable location via a support, such as a bead. In an example, a first bead comprising a first colony of nucleic acid molecules each comprising a first template sequence is immobilized to a first individually addressable location, and a second bead comprising a second colony of nucleic acid molecules each comprising a second template sequence is immobilized to a second individually addressable location. A substrate may comprise more than one type of individually addressable location arranged as an array, randomly, or according to any pattern, on the substrate. In some cases, different types of individually addressable locations may have different chemical, physical, and/or biological properties (e.g., hydrophobicity, charge, color, topography, size, dimensions, geometry, etc.). [0116] In some cases, an individually addressable location may comprise a distinct surface chemistry. The distinct surface chemistry may distinguish between different addressable locations and/or distinguish an individually addressable location from surrounding locations. For example, a first location type may comprise a first surface chemistry, and a second location type may lack the first surface chemistry. In another example, the first location type may comprise the first surface chemistry and the second location ty pe may comprise a second, different surface chemistry. A first location type may have a first affinity towards an object (e.g., a bead comprising nucleic acid molecules, e.g., amplicons, immobilized thereto) and a second location type may have a second, different affinity' towards the same object. In other examples, a first location type comprising a first surface chemistry may have an affinity towards a first sample type (e.g.. a bead comprising nucleic acid molecules, e.g., amplicons, immobilized thereto) and exclude a second sample type (e.g., a bead lacking nucleic acid molecules, e.g., amplicons, immobilized thereto). The first location type and the second location type may or may not be disposed on the surface in alternating fashion. A first location type or region type may comprise a positively charged surface chemistry’ and a second location type or region type may comprise a negatively charged surface chemistry. A first location ty pe or region type may comprise a hydrophobic surface chemistry and a second location type or region type may comprise a hydrophilic surface chemistry'. A first location type comprises a binder, as described elsewhere herein, and a second location type does not comprise the binder or comprises a different binder. In some cases, a surface chemistry may comprise an amine. In some cases, a surface chemistry may comprise a silane (e.g., tetramethylsilane). In some cases, the surface chemistry' may comprise hexamethyldisilazane (HMDS). In some cases, the surface chemistry' may comprise (3-aminopropyl)triethoxysilane (APTMS). In some cases, the surface chemistry may comprise a surface primer molecule or any oligonucleotide molecule that has any degree of affinity towards another molecule. In one example, the substrate comprises a plurality' of individually⁷ addressable locations, each defined by APTMS, which are positively charged and has affinity towards an amplified bead (e.g., a bead comprising nucleic acid molecules, e.g., amplicons, immobilized thereto) which exhibits a negative charge. The locations surrounding the plurality of individually addressable locations may comprise HMDS which repels amplified beads.

[0117] In some cases, the individually addressable locations may be indexed, e.g., spatially. Data corresponding to an indexed location, collected over multiple periods of time, may be linked to the same indexed location. In some cases, sequencing signal data collected from an indexed location, during iterations of sequencing-by -synthesis flows, are linked to the indexed location to generate a sequencing read for an analyte immobilized at the indexed location. In some embodiments, the individually addressable locations are indexed by demarcating part of the surface, such as by etching or notching the surface, using a dye or ink, depositing a topographical mark, depositing a sample (e.g.. a control nucleic acid sample), depositing a reference object (e.g., e.g., a reference bead that always emits a detectable signal during detection), and the like, and the individually addressable locations may be indexed with reference to such demarcations. A combination of positive demarcations and negative demarcations (lack thereof) may be used to index the individually addressable locations. In some embodiments, each of the individually addressable locations is indexed. In some embodiments, a subset of the individually addressable locations is indexed. In some embodiments, the individually addressable locations are not indexed, and a different region of the substrate is indexed.

[0118] The substrate may comprise a planar or substantially planar surface. Substantially planar may refer to planarity at a micrometer level (e.g., a range of unevenness on the planar surface does not exceed the micrometer scale) or nanometer level (e.g.. a range of unevenness on the planar surface does not exceed the nanometer scale). Alternatively, substantially planar may refer to planarity⁷ at less than a nanometer level or greater than a micrometer level (e.g., millimeter level). Alternatively or in addition, a surface of the substrate may be textured or patterned. For example, the substrate may comprise grooves, troughs, hills, pillars, wells, cavities (e g., micro-scale cavities or nano-scale cavities), and/or channels. The substrate may have regular textures and/or patterns across the surface of the substrate. The substrate may have regular geometric structures (e.g., wedges, cuboids, cylinders, spheroids, hemispheres, etc.) above or below a reference level of the surface. Alternatively, the substrate may have irregular textures and/or patterns across the surface of the substrate. The substrate may be textured or patterned such that all features are at or above a reference level of the surface (no features below a reference level of the surface, such as a well). The substrate may be textured or patterned such that all features are at or below a reference level of the surface (no features below a reference level of the surface, such as a pillar). In some instances, a texture of the substrate may comprise structures having a maximum dimension of at most about 500%, 400%, 300%, 200%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%. 0.0001%, 0.00001% of the total thickness of the substrate or a layer of the substrate. In some instances, the textures and/or patterns of the substrate may define at least part of an individually addressable location on the substrate. A textured and/or patterned substrate may be substantially planar. Alternatively, the substrate may be untextured and unpattemed.

[0119] A binder may be configured to immobilize an analyte or reagent to an individually addressable location. In some cases, a surface chemistry⁷ of an individually addressable location may comprise one or more binders. In some cases, a plurality of individually addressable locations may be coated with binders. The binders may be integral to the substrate. The binders may be added to the substrate. For instance, the binders may be added to the substrate as one or more coating layers. The substrate may comprise an order of magnitude of at least and/or at most about 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, IO¹⁰, IO¹¹, 10¹², 10¹³ or more binders. The binders may immobilize analytes or reagents through nonspecific interactions, such as one or more of hydrophilic interactions, hydrophobic interactions, electrostatic interactions, physical interactions (for instance, adhesion to pillars or settling within wells), and the like. Alternatively or in addition, the binders may immobilize analytes or reagents through specific interactions. For instance, where the analyte or reagent is a nucleic acid molecule, the binders may comprise oligonucleotide adaptors configured to bind to the nucleic acid molecule. In other examples, the binders may comprise one or more of antibodies, oligonucleotides, nucleic acid molecules, aptamers, affinity⁷ binding proteins, lipids, carbohydrates, and the like. The binders may immobilize analytes or reagents through any possible combination of interactions. For instance, the binders may immobilize nucleic acid molecules through a combination of physical and chemical interactions, through a combination of protein and nucleic acid interactions, etc. A single binder may bind a single analyte or single reagent, a single binder may bind a plurality of analytes or a plurality of reagents, or a plurality of binders may bind a single analyte or a single reagent. In some instances, the substrate may comprise a plurality of types of binders, for example to bind different types of analytes or reagents. For example, a first type of binders (e.g., oligonucleotides) are configured to bind a first type of analyte (e.g., nucleic acid molecules) or reagent, and a second type of binders (e.g., antibodies) are configured to bind a second type of analyte (e.g., proteins) or reagent. In another example, a first type of binders (e.g., first type of oligonucleotide molecules) are configured to bind a first type of nucleic acid molecules and a second type of binders (e.g., second ty pe of oligonucleotide molecules) are configured to bind a second type of nucleic acid molecules. For example, the substrate may be configured to bind different types of analytes or reagents in certain fractions or specific locations on the substrate by having the different types of binders in the certain fractions or specific locations on the substrate.

[0120] The substrate may be rotatable about an axis, referred to herein as a rotational axis. The rotational axis may or may not be an axis through the center of the substrate. The systems, devices, and apparatus described herein may further comprise an automated or manual rotational unit configured to rotate the substrate. The rotational unit may comprise a motor and/or a rotor. For instance, the substrate may be affixed to a chuck (such as a vacuum chuck). The substrate may be rotated at a rotational speed of at least about 1 revolution per minute (rpm), at least 2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least 50 rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,000 rpm, at least 5.000 rpm, at least 10,000 rpm. or greater. Alternatively or in addition, the substrate may be rotated at a rotational speed of at most about 10,000 rpm, 5,000 rpm, 2,000 rpm, 1,000 rpm, 500 rpm, 200 rpm, 100 rpm, 50 rpm, 20 rpm, 10 rpm, 5 rpm, 2 rpm, 1 rpm, or less. The substrate may be configured to rotate with different rotational velocities during different operations described herein, for example with higher velocities during reagent dispense and with lower velocities during analyte loading and imaging operations. The substrate may be configured to rotate with a rotational velocity that varies according to a time-dependent function, such as a ramp, sinusoid, pulse, or other function or combination of functions. The time-varying function may be periodic or aperiodic.

[0121] Analytes or reagents may be immobilized to the substrate during rotation. Analytes or reagents may be dispensed onto the substrate prior to or during rotation of the substrate. When the substrate is rotated at a relatively high rotational velocity, high speed coating across the substrate may be achieved via tangential inertia directing unconstrained spinning reagents in a partially radial direction (that is, away from the axis of rotation) during rotation, a phenomenon commonly referred to as centrifugal force. In some cases, the substrate may be rotated at relatively low velocities such that reagents dispensed to a certain location do not move to another location, or moves minimally, because of the rotation, to permit controlled dispensing of reagents to desired locations. For example, bead loading may be performed with controlled dispensing. For controlled dispensing, the substrate may be rotating with a rotational frequency of no more than 60, 50, 40, 30, 25, 20, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 rpm or less. In some cases the substrate may be rotating with a rotational frequency of about 5 rpm during controlled dispensing. A speed of substrate rotation may be adjusted according to the appropriate operation (e.g., high speed for spin-coating, high speed for washing the substrate, low speed for sample loading, low speed for detection, low speed for analyte or reagent incubation, etc.).

[0122] In some cases, the substrate may be movable in any vector or direction. For example, such motion may be non-linear (e.g., in rotation about an axis), linear (e.g.. on a rail track), or a hybrid of linear and non-linear motion. In some instances, the systems, devices, and apparatus described herein may further comprise a motion unit configured to move the substrate. The motion unit may comprise any mechanical component, such as a motor, rotor, actuator, linear stage, drum, roller, pulleys, etc., to move the substrate. Analytes or reagents may be immobilized to the substrate during any such motion. Analytes or reagents may be dispensed onto the substrate prior to, during, or subsequent to motion of the substrate.

Loading reagents onto an open substrate

[0123] The surface of the substrate may be in fluid communication with at least one fluid nozzle (of a fluid channel). The surface may be in fluid communication with the fluid nozzle via a non-solid gap, e.g., an air gap. In some cases, the surface may additionally be in fluid communication with at least one fluid outlet. The surface may be in fluid communication with the fluid outlet via an air gap. The nozzle may be configured to direct a solution to the array. The outlet may be configured to receive a solution from the substrate surface. The solution may be directed to the surface using one or more dispensing nozzles. For example, the solution may be directed to the array using at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20 or more dispensing nozzles. In some cases, different reagents (e.g.. nucleotide solutions of different types, different probes, washing solutions, etc.) may be dispensed via different nozzles, such as to prevent contamination. Each nozzle may be connected to a dedicated fluidic line or fluidic valve, which may further prevent contamination. Alternatively, some nozzles may share a fluidic line or fluidic valve, such as for pre-dispense mixing and/or to dispensing to multiple locations. A type of reagent may be dispensed via one or more nozzles. The one or more nozzles may be directed at or in proximity to a center of the substrate. Alternatively, the one or more nozzles may be directed at or in proximity to a location on the substrate other than the center of the substrate. Alternatively or in combination, one or more nozzles may be directed closer to the center of the substrate than one or more of the other nozzles. For instance, one or more nozzles used for dispensing washing reagents may be directed closer to the center of the substrate than one or more nozzles used for dispensing active reagents. The one or more nozzles may be arranged at different radii from the center of the substrate. The nozzles may be angled towards or away from a center of the substrate, or not angled (e.g., normal to the substrate plane). Two or more nozzles may be operated in combination to deliver fluids to the substrate more efficiently. One or more nozzles may be configured to deliver fluids to the substrate as a jet, spray (or other dispersed fluid), and/or droplets. One or more nozzles may be operated to nebulize fluids prior to delivery to the substrate. For example, the fluids may be delivered as aerosol particles. [0124] In some cases, the solution may be dispensed on the substrate while the substrate is stationary; the substrate may then be subjected to rotation (or other motion) following the dispensing of the solution. Alternatively, the substrate may be subjected to rotation (or other motion) prior to the dispensing of the solution; the solution may then be dispensed on the substrate while the substrate is rotating (or otherwise moving). In some cases, rotation of the substrate may yield a centrifugal force (or inertial force directed away from the axis) on the solution, causing the solution to flow radially outward over the array. In this manner, rotation of the substrate may direct the solution across the array. Continued rotation of the substrate over a period of time may dispense a fluid film of a nearly constant thickness across the array.

[0125] One or more conditions such as the rotational velocity' of the substrate, the acceleration of the substrate (e.g., the rate of change of velocity), viscosity of the solution, angle of dispensing (e.g., contact angle of a stream of reagents) of the solution, radial coordinates of dispensing of the solution (e.g., on center, off center, etc.), temperature of the substrate, temperature of the solution, and other factors may be adjusted and/or otherwise optimized to attain a desired wetting on the substrate and/or a film thickness on the substrate, such as to facilitate uniform coating of the substrate. For instance, one or more conditions may be applied to attain a film thickness of at least and/or at most about 10 nanometers (nm), 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1 micrometer (pm), 2 pm, 5 pm, 10 pm, 20 pm, 50 pm, 100 pm, 200 pm, 500 pm, 1 millimeter (mm), or more. One or more conditions may be applied to attain a film thickness that is within a range defined by any two of the preceding values. In some cases, a surfactant may be added to the solution, or a surfactant may be added to the surface to facilitate uniform coating or to facilitate sample loading efficiency. Alternatively or in conjunction, the thickness of the solution may be adjusted using mechanical, electric, physical, or other mechanisms. For example, the solution may be dispensed onto a substrate and subsequently leveled using, e.g.. a physical scraper such as a squeegee, to obtain a desired thickness of uniformity across the substrate.

[0126] Reagents may be dispensed to the substrate to multiple locations, and/or multiple reagents may be dispensed to the substrate to a single location, via different mechanisms. Reagent dispensing mechanisms disclosed herein may be applicable to sample dispensing. For example, a reagent may comprise the sample. The term “loading onto a substrate,” as used herein, may' refer to dispensing of the reagent or the sample to a surface of the substrate in accordance with any reagent dispensing mechanism described herein. [0127] In some cases, dispensing may be achieved via relative motion of the substrate and the dispenser (e.g., nozzle). For example, a reagent may be dispensed to the substrate at a first location, and thereafter travel to a second location different from the first location due to forces (e.g., centrifugal forces, centripetal forces, inertial forces, etc.) caused by motion of the substrate (e.g., rotational motion of the substrate, linear motion of the substrate, combination thereof, etc.). In another example, a reagent may be dispensed to a reference location, and the substrate may be moved relative to the reference location such that the reagent is dispensed to multiple locations of the substrate. In another example, a dispenser may be moved relative to the substrate to dispense the reagent at different locations, for example moved prior to. during, or subsequent to dispensing. In an example, a reagent is ‘painted’ onto the substrate by moving the dispenser and/or the substrate relative to each other, along a desired path on the substrate. The open substrate geometry' may allow for flexible and controlled dispensing of a reagent to a desired location on the substrate. In some cases, dispensing may be achieved without relative motion between the substrate and the dispenser. For example, multiple dispensers may be used to dispense reagents to different locations, and/or multiple reagents to a single location, or a combination thereof (e.g., multiple reagents to multiple locations).

[0128] In another example, an external force (e.g., involving a pressure differential, involving physical force, involving a magnetic force, involving an electrical force, etc.), such as wind, a field-generating device, or a physical device, may be applied to one or more surfaces of the substrate to direct reagents to different locations across the substrate. In another example, the method for dispensing reagents may comprise vibration. In such an example, reagents may be distributed or dispensed onto a single region or multiple regions of the substrate. The substrate may then be subjected to vibration, which may spread the reagent to different locations across the substrate. Alternatively or in conjunction, the method may comprise using mechanical, electric, physical, or other mechanisms to dispense reagents to the substrate. For example, the solution may be dispensed onto a substrate and a physical scraper (e.g., a squeegee) may be used to spread the dispensed material or spread the reagents to different locations and/or to obtain a desired thickness or uniformity across the substrate. Beneficially, such flexible dispensing may be achieved without contamination of the reagents.

[0129] In some instances, where a volume of reagent is dispensed to the substrate at a first location, and thereafter travels to a second location different from the first location, the volume of reagent may travel in a path or paths, such that the travel path or paths are coated with the reagent. In some cases, such travel path or paths may encompass a desired surface area (e.g., entire surface area, partial surface area(s), etc.) of the substrate. In some instances, two or more reagents may be mixed on the surface of the substrate, such as by being dispensed at the same location and/or by directing a first reagent to travel to meet additional reagent(s). In some instances, the mixture of reagents formed on the substrate may be homogenous or substantially homogenous. The mixture of reagents may be formed at a first location on the substrate prior to dispersing the mixing of reagents to other locations on the substrate, such as at locations to meet other reagents or analytes.

[0130] In some embodiments, one or more solutions may be delivered directly to the reaction site without substantial displacement of the one or more solution from the point of delivery. Methods of direct delivery' of a solution to the reaction site may include aerosol delivery' of the solution, applying the solution using an applicator, curtain-coating the solution, slot-die coating, dispensing the solution from a translating dispense probe, dispensing the solution from an array of dispense probes, dipping the substrate into the solution, or contacting the substrate to a sheet comprising the solution.

[0131] Aerosol delivery may comprise delivering a solution to the substrate in aerosol form by directing the solution to the substrate using a pressure nozzle or an ultrasonic nozzle. Applying the solution using an applicator may comprise contacting the substrate with an applicator comprising the solution and translating the applicator relative to the substrate. For example, applying the solution using an applicator may comprise painting the substrate. The solution may be applied in a pattern by translating the applicator, rotating the substrate, translating the substrate, or a combination thereof. Curtain-coating may comprise dispensing the solution from a dispense probe to the substrate in a continuous stream (e.g., a curtain or a flat sheet) and translating the dispense probe relative to the substrate. A solution may be curtain-coated in a pattern by translating the dispense probe, rotating the substrate, translating the substrate, or a combination thereof. Slot-die coating may comprise dispensing the solution from a dispense probe positioned near the substrate such that the solution forms a meniscus between the substrate and the dispense probe and translating the dispense probe relative to the substrate. A solution may be slot-die coated in a pattern by translating the dispense probe, rotating the substrate, translating the substrate, or a combination thereof. Dispensing the solution from a translating dispense probe may comprise translating the dispense probe relative to the substrate in a pattern (e.g., a spiral pattern, a circular pattern, a linear pattern, a striped pattern, a cross-hatched pattern, or a diagonal pattern). Dispensing the solution from an array of dispense probes may comprise dispensing the solution from an array of nozzles (e.g., a shower head) positioned above the substrate such that the solution is dispensed across an area of the substrate substantially simultaneously. Dipping the substrate into the solution may comprise dipping the substrate into a reservoir comprising the solution. In some embodiments, the reservoir may be a shallow reservoir to reduce the volume of the solution required to coat the substrate. Contacting the substrate to a sheet comprising the solution may comprise bringing the substrate in contact with a sheet of material (e.g., a porous sheet or a fibrous sheet) permeated with the solution. The solution may be transferred to the substrate. In some embodiments, the sheet of material may be a single-use sheet. In some embodiments, the sheet of material may be a reusable sheet. In some embodiments, a solution may be dispensed onto a substrate using the method illustrated in FIG. 5B, where a jet of a solution may be dispensed from a nozzle to a rotating substrate. The nozzle may translate radially relative to the rotating substrate, thereby dispensing the solution in a spiral pattern onto the substrate.

[0132] One or more solutions or reagents may be delivered to a substrate by any of the deliver}' methods disclosed herein. Two or more solutions or reagents may be delivered to the substrate using the same or different delivery methods. Two or more solutions may be delivered to the substrate such that the time between contacting a solution or reagent and a subsequent solution or reagent is substantially similar for each region of the substrate contacted to the one or more solutions or reagents. A solution or reagent may be delivered as a single mixture. The solution or reagent may be dispensed in two or more component solutions. For example, each component of the two or more component solutions may be dispensed from a distinct nozzle. The distinct nozzles may dispense the two or more component solutions substantially simultaneously to substantially the same region of the substrate such that a homogenous solution forms on the substrate. Dispensing of each component of the two or more components may be temporally separated. Dispensing of each component may be performed using the same or different delivery methods. Direct delivery of a solution or reagent may be combined with spin-coating.

[0133] A solution may be incubated on the substrate for any desired duration (e.g., minutes, hours, etc.). In some embodiments, the solution may be incubated on the substrate under conditions that maintain a layer of fluid on the surface. One or more of the temperature of the chamber, the humidity of the chamber, the rotation of the substrate, and the composition of the fluid may be adjusted such that the layer of fluid is maintained during incubation. In some instances, during incubation, the substrate may be rotated at an rotational frequency of no more than 60 rpm, 50 rpm, 40 rpm, 30 rpm, 25 rpm, 20 rpm, 15 rpm, 14 rpm, 13 rpm, 12 rpm, 11 rpm, 10 rpm, 9 rpm, 8 rpm, 7 rpm, 6 rpm. 5 rpm, 4 rpm, 3 rpm, 2 rpm, 1 rpm or less. In some cases, the substrate may be rotating with a rotational frequency of about 5 rpm during incubation.

[0134] The substrate or a surface thereof may comprise other features that aid in solution or reagent retention on the substrate or thickness uniformity of the solution or reagent on the substrate. In some cases, the surface may comprise a raised edge (e.g., a rim) which may be used to retain solution on the surface. The surface may comprise a rim near the outer edge of the surface, thereby reducing the amount of the solution that flow s over the outer edge.

[0135] The dispensed solution may comprise any sample or any analyte disclosed herein. The dispensed solution may comprise any reagent disclosed herein. In some cases, the solution may be a reaction mixture comprising a variety of components. In some cases, the solution may be a component of a final mixture (e.g., to be mixed after dispensing). In non-limiting examples, the solution can comprise samples, analytes, supports, beads, probes, nucleotides, oligonucleotides, labels (e.g., dyes), terminators (e.g., blocking groups), other components to aid, accelerate, or decelerate a reaction (e.g., enzymes, catalysts, buffers, saline solutions, chelating agents, reducing agents, other agents, etc.), washing solution, cleavage agents, combinations thereof, deionized water, and other reagents and buffers.

[0136] A sample may comprise beads, as described elsewhere herein, for example beads comprising nucleic acid colonies bound thereto. In some cases, an order of magnitude of at least and/or at most about I0¹, 10², 10³, 10⁴. 10⁵, 10⁶, 10⁷. 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or more beads may be loaded on the substrate, such as to immobilize to as many individually addressable locations. In some cases, the beads may be distinguishable from one another using a property of the beads, such as color, reflectance, anisotropy, brightness, fluorescence, etc. In some cases, as described elsewhere herein, different beads may comprise different tags (e.g., nucleic acid sequences) coupled thereto. For example, a bead may comprise an oligonucleotide molecule comprising a tag (e.g., barcode) that identifies a bead amongst a plurality⁷ of beads. FIG. 3 illustrates images of a portion of a substrate surface after loading a sample containing beads onto a substrate patterned with a substantially hexagonal lattice of individually addressable locations, where the right panel illustrates a zoomed-out image of a portion of a surface, and the left panel illustrates a zoomed-in image of a section of the portion of the surface. After sample loading, a “bead occupancy” may generally refer to the number of a type of individually addressable locations comprising at least one bead out of the total number of individually addressable locations of the same type. A bead “landing efficiency’' may generally refer to the number of beads that bind to the surface out of the total number of beads dispensed on the surface.

[0137] In some cases, beads may be dispensed to the substrate according to one or more systems and methods shown in FIG. 4. As shown in FIG. 4, a solution comprising beads may be dispensed from a dispense probe 401 (e.g., a nozzle) to a substrate 403 (e.g., a wafer) to form a layer 405. The dispense probe may be positioned at a height (“Z") above the substrate. In the illustrated example, the beads are retained in the layer 405 by electrostatic retention, and may immobilize to the substrate at respective individually addressable locations. A set of beads in the solution may each comprise a population of amplified products (e.g., nucleic acid molecules) immobilized thereto, which amplified products accumulate to a negative charge on the bead. Otherwise, the beads may comprise reagents that have a negative charge. The substrate comprises alternating surface chemistry between distinguishable locations, in which a first location type comprises APTMS carrying a positive charge with affinity towards the negative charge of the amplified bead (e.g., a bead comprising amplified products immobilized thereto, and as distinguished from a negative bead which does not the comprise the same) or other bead comprising the negative charge, and a second location type comprises HMDS which has lower affinity and/or is repellant of the amplified bead or other bead comprising the negative charge. Within the layer 405 a bead may successfully land on a first location of the first location type (as in 407). In the illustrated example, the location size is 1 micron, the pitch between the different locations of the same location type (e.g., first location type) is 2 microns, and the layer has a depth of 15 micron. The top right panel illustrates that a reagent solution may be dispensed from the dispense probe 401 as the layer 405 along a path on an open surface of the substrate 403. The reagent may be dispensed on the surface in any desired pattern or path. The substrate 403 and the dispense probe 401 may move in any configuration with respect to each other to achieve any pattern (e.g., linear pattern, substantially spiral pattern, etc.).

[0138] Dispense mechanisms described herein may be operated by a fluid flow unit which may be controlled by one or more controllers, individually or collectively. The fluid flow unit may comprise any of the hardware and software components described with respect to the dispense mechanisms herein.

Detection [0139] An optical system comprising a detector may be configured to detect one or more signals from a detection area on the substrate prior to, during, or subsequent to, the dispensing of reagents to generate an output. Signals from multiple individually addressable locations may be detected during a single detection event. Signals from the same individually addressable location may be detected in multiple instances.

[0140] A signal may be an optical signal (e.g., fluorescent signal), electronic signal, or any detectable signal. The signal may be detected during rotation of the substrate or following termination of the rotation. The signal may be detected while the analyte is in fluid contact with a solution. The signal may be detected following washing of the solution. In some instances, after the detection, the signal may be muted, such as by cleaving a label from a probe and/or the analyte, and/or modifying the probe and/or the analyte. Such cleaving and/or modification may be performed by one or more stimuli, such as exposure to a chemical, an enzyme, light (e.g., ultraviolet light), or temperature change (e.g.. heat). In some instances, the signal may otherwise become undetectable by deactivating or changing the mode (e g., detection wavelength) of the one or more sensors, or terminating or reversing an excitation of the signal. In some instances, detection of a signal may comprise capturing an image or generating a digital output (e.g., between different images).

[0141] The operations of (i) directing a solution to the substrate and (ii) detection of one or more signals indicative of a reaction betw een a probe in the solution and an analyte immobilized to the substrate, may be repeated any number of times. Such operations may be repeated in an iterative manner. For example, the same analyte immobilized to a given location in the array may interact with multiple solutions in multiple cycles and for each iteration, the additional signals detected may provide incremental, or final, data about the analyte during the processing. For example, where the analyte is a nucleic acid molecule and the processing is sequencing, additional signals detected for each iteration may be indicative of one or more bases in the nucleic acid sequence of the nucleic acid molecule. In some cases, multiple solutions can be provided to the substrate without intervening detection events. In some cases, multiple detection events can be performed after a single flow- of solution. In some instances, a washing solution, cleaving solution (e.g., comprising cleavage agent), and/or other solutions may be directed to the substrate between each operation, between each cycle, or a certain number of times for each cycle.

[0142] The optical system may be configured for continuous area scanning of a substrate during rotational motion of the substrate. The term “continuous area scanning (CAS),” as used herein, generally refers to a method in which an object in relative motion is imaged by repeatedly, electronically or computationally, advancing (clocking or triggering) an array sensor at a velocity that compensates for object motion in the detection plane (focal plane). CAS can produce images having a scan dimension larger than the field of the optical system. TDI scanning may be an example of CAS in which the clocking entails shifting photoelectric charge on an area sensor during signal integration. For a TDI sensor, at each clocking step, charge may be shifted by one row, with the last row being read out and digitized. Other modalities may accomplish similar function by high speed area imaging and co-addition of digital data to synthesize a continuous or stepwise continuous scan.

[0143] The optical system may comprise one or more sensors. The sensors may detect an image optically projected from the sample. The optical system may comprise one or more optical elements. An optical element may be, for example, a lens, tube lens, prism, mirror, wave plate, filter, attenuator, grating, diaphragm, beam splitter, diffuser, polarizer, depolarizer, retroreflector, spatial light modulator, or any other optical element. The system may comprise any number of sensors. In some cases, a sensor is any detector as described herein. In some examples, the sensor may comprise image sensors, CCD cameras, CMOS cameras, TDI cameras (e.g., TDI line-scan cameras), pseudo-TDI rapid frame rate sensors, or CMOS TDI or hybrid cameras. The optical system may further comprise any one or more optical sources (e.g., lasers, LED light sources, etc.). In some cases, where there are multiple sensors, the different sensors may image the same or different regions of the rotating substrate, in some cases simultaneously. Each sensor of the plurality of sensors may be clocked at a rate appropriate for the region of the rotating substrate imaged by the sensor, which may be based on the distance of the region from the center of the rotating substrate or the tangential velocity of the region. In some cases, multiple scan heads can be operated in parallel along different imaging paths (e.g., interleaved spiral scans, nested spiral scans, interleaved ring scans, nested ring scans). A scan head may comprise one or more of a detector element such as a camera (e.g., a TDI line-scan camera), an illumination source (e.g., as described herein), and one or more optical elements (e.g., as described herein).

[0144] The system may further comprise one or more controllers operatively coupled to the one or more sensors, individually or collectively programmed to process optical signals from the one or more sensors, such as for each region of the rotating substrate.

[0145] In some cases, the optical system may comprise an immersion objective lens. The immersion objective lens may be in contact with an immersion fluid that is in contact with the open substrate. The immersion fluid may comprise any suitable immersion medium for imaging (e.g., water, aqueous, organic solution). In some cases, an enclosure may partially or completely surround a sample-facing end of the optical imaging objective. The enclosure may be configured to contain the immersion fluid. The enclosure may not be in contact with the substrate; for example, a gap between the enclosure and the substrate may be filled by the fluid contained by the enclosure (e.g., the enclosure can retain the fluid via surface tension). In some cases, an electric field may be used to regulate a hydrophobicity of one or more surfaces of the container to retain at least a portion of the fluid contacting the immersion objective lens and the open substrate. In some cases, the immersion fluid may be continuously replenished or recycled via an inlet and outlet to the enclosure.

High Throughput

[0146] An open substrate may be processed within a modular local sample processing environment. A barrier comprising a fluid barrier may be maintained between a sample processing environment and an exterior environment during certain processing operations, such as reagent dispensing and detecting. Systems and methods comprising a fluid barrier are described in further detail in U.S. Patent Pub. No. 20210354126A1, which is entirely- incorporated herein by reference. A modular local sample processing environment may be defined by a chamber and a lid plate, where the lid plate is not in contact with the chamber, and the gap between the lid plate and the chamber may comprise the fluid barrier. The fluid barrier may comprise fluid (e.g., air) from the sample processing environment and/or the exterior environment and may have lower pressure than the sample processing environment, the external environment, or both. The fluid in the fluid barrier may be in coherent motion or bulk motion.

[0147] The sample processing environment may comprise therein a substrate, such as any substrate described elsewhere herein. Any operation performed on or with the substrate, as described elsewhere herein, may be performed within the sample processing environment while the fluid barrier is maintained. For example, the substrate may be rotated within the sample processing environment during various operations. In another example, fluid may be directed to the substrate while the substrate is in the sample processing environment, via a fluid handler (e.g., nozzle) that penetrates the lid plate into the sample processing environment. In another example, a detector can image the substrate while the substrate is in the sample processing environment, via a detector that penetrates the lid plate into the sample processing environment. Beneficially, the fluid barrier may help maintain temperature(s) and/or relative humidit(ies), or ranges thereof, within the sample processing environment during various processing operations.

[0148] The systems described herein, or any element thereof, may be environmentally controlled. For instance, the systems may be maintained at a specified temperature or humidity. For an operation, the systems (or any element thereof) may be maintained at a temperature of at least and/or at most 20 degrees Celsius (°C), 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 95 °C, 100 °C, or more. Different elements of the system may be maintained at different temperatures or within different temperature ranges, such as the temperatures or temperature ranges described herein. Elements of the system may be set at temperatures above the dew point to prevent condensation. Elements of the system may be set at temperatures below the dew point to collect condensation.

[0149] While examples described herein provide relative rotational motion of the substrates and/or detector systems, the substrates and/or detector systems may alternatively or additionally undergo relative non-rotational motion, such as relative linear motion, relative non-linear motion (e.g., curved, arcuate, angled, etc ), and any other types of relative motion. [0150] An open substrate may be retained in the same or approximately the same physical location during processing of an analyte and subsequent detection of a signal associated with the processed analyte. Alternatively, different operations on or with the open substrate may be performed in different stations disposed in different physical locations. For example, a first station may be disposed above, below, adjacent to, or across from a second station. In some cases, the different stations can be housed within an integrated housing. Alternatively, the different stations can be housed separately. In some cases, different stations may be separated by a barrier, such as a retractable barrier (e.g., sliding door). One or more different stations of a system, or portions thereof, may be subjected to different physical conditions, such as different temperatures, pressures, or atmospheric compositions. The open substrate may transition between different stations by transporting the sample processing environment comprising the chamber containing the open substrate between the different stations. One or more mechanical components or mechanisms, such as a robotic arm, elevator mechanism, actuators, rails, and the like, or other mechanisms may be used to transport the sample processing environment.

[0151] An environmental unit (e.g., humidifiers, heaters, heat exchangers, compressors, etc.) may be configured to regulate one or more operating conditions in each station. In some instances, each station may be regulated by independent environmental units. In some instances, a single environmental unit may regulate a plurality of stations. In some instances, a plurality' of environmental units may, individually or collectively, regulate the different stations. An environmental unit may use active methods or passive methods to regulate the operating conditions. For example, the temperature may be controlled using heating or cooling elements. The humidity may be controlled using humidifiers or dehumidifiers. In some instances, a part of a particular station, such as within a sample processing environment, may be further controlled from other parts of the particular station. Different parts may have different local temperatures, pressures, and/or humidity. In one example, the delivery and/or dispersal of reagents may be performed in a first station having a first operating condition, and the detection process may be performed in a second station having a second operating condition different from the first operating condition. The first station may be at a first physical location in which the open substrate is accessible to a fluid handling unit during the delivery and/or dispersal processes, and the second station may be at a second physical location in which the open substrate is accessible to the detector system.

[0152] One or more modular sample environment systems (each having its own barrier system, e.g., fluid barrier) can be used between the different stations. In some instances, the systems described herein may be scaled up to include two or more of a same station type. For example, a sequencing system may include multiple processing and/or detection stations. FIGs. 5A-5B illustrate a system 300 that multiplexes two modular sample environment systems in a three-station system. In FIG. 5A. a first chemistry station (e g., 320a) can operate (e.g.. dispense reagents, e.g., to incorporate nucleotides to perform sequencing by synthesis) via at least a first operating unit (e g., fluid dispenser 309a) on a first substrate (e.g., 311) in a first sample environment system (e.g., 305a) while substantially simultaneously, a detection station (e.g., 320b) can operate (e.g., scan) on a second substrate in a second sample environment system (e.g., 305b) via at least a second operating unit (e.g., detector 301), while substantially simultaneously, a second chemistry station (e.g., 320c) sits idle. An idle station may not operate on a substrate. An idle station (e.g., 320c) may be recharged, reloaded, replaced, cleaned, washed (e.g., to flush reagents), calibrated, reset, kept active (e.g., power on), and/or otherwise maintained during an idle time. After an operating cycle is complete, the sample environment systems may be re-stationed, as in FIG. 5B, where the second substrate in the second sample environment system (e.g., 305b) is re-stationed from the detection station (e.g., 320b) to the second chemistry station (e.g., 320c) for operation (e g., dispensing of reagents, e.g., to incorporate nucleotides to perform sequencing by synthesis) by the second chemistry station, and the first substrate in the first sample environment system (e.g., 305a) is re-stationed from the first chemistry station (e.g., 320a) to the detection station (e.g., 320b) for operation (e.g.. scanning) by the detection station. An operating cycle may be deemed complete when operation at each active, parallel station is complete. During re-stationing, the different sample environment systems may be physically moved (e.g., along the same track or dedicated tracks, e.g., rail(s) 307) to the different stations and/or the different stations may be physically moved to the different sample environment systems. One or more components of a station, such as modular plates 303a, 303b, 303c of plate 303 (e.g., lid plate) defining a particular station(s), may be physically moved to allow a sample environment system to exit the station, enter the station, or cross through the station. During processing of a substrate at station, the environment of a sample environment region (e.g., 315) of a sample environment system (e.g.. 305a) may be controlled and/or regulated according to the station’s requirements. After the next operating cycle is complete, the sample environment systems can be re-stationed again, such as back to the configuration of FIG. 5A, and this re-stationing can be repeated (e.g., between the configurations of FIGs. 5A and 5B) with each completion of an operating cycle until the required processing for a substrate is completed. In this illustrative re-stationing scheme, the detection station may be kept active (e.g., not have idle time not operating on a substrate) for all operating cycles by providing alternating different sample environment systems to the detection station for each consecutive operating cycle. Beneficially, use of the detection station is optimized. Based on different processing or equipment needs, an operator may opt to run the two chemistry stations substantially simultaneously while the detection station is kept idle.

[0153] Beneficially, different operations within the system may be multiplexed with high flexibility and control. For example, as described herein, one or more processing stations may be operated in parallel with one or more detection stations on different substrates in different modular sample environment systems to reduce or eliminate lag between different sequences of operations (e.g., chemistry first, then detection). The modular sample environment systems may be translated between the different stations accordingly to optimize efficient equipment use (e.g.. such that the detection station is in operation almost 100% of the time). In some examples, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more modules or stations of the sequencing system may be multiplexed. For example, 2 or more of the modules may each perform their intended function simultaneously or according to the methods described elsewhere herein. An example of this may comprise two-station multiplexing of an optics station and a chemistry station as described herein. Another example may comprise multiplexing three or more stations and process phases. For example, the method may comprise using staggered chemistry phases sharing a scanning station. The scanning station may be a high-speed scanning station. The modules or stations may be multiplexed using various sequences and configurations.

[0154] The nucleic acid sequencing systems and optical systems described herein (or any elements thereof) may be combined in a variety of architectures.

Post-Amplification Enrichment

[0155] Provided herein are devices, systems, methods, compositions, and kits that enrich amplified supports from non-amplified supports post-amplification. Such devices, systems, methods, compositions, and kits can be applied alternatively or in addition to the postamplification processing operation (e.g., 106) described with respect to sequencing workflow 100 of FIG. 1. Such devices, systems, methods, compositions, and kits can be used in conjunction with the sample processing systems and methods, or components thereof (e.g., substrates, detectors, reagent dispensing, continuous scanning, etc.) described herein.

[0156] Amplification may output a mixture of amplified supports (also referred to herein as positive supports) and non-amplified supports (also referred to herein as negative supports). A support may be a bead or other object. Any type of amplification method described herein (e.g.. PCR. ePCR, RCA. RPA. MDA, etc.), or combination thereof, may be performed to generate the amplification output.

[0157] In some cases, amplification may comprise providing and subjecting to amplification conditions a plurality of library' molecules and a plurality of supports. The plurality of supports may each comprise a plurality of surface primers. The plurality of library molecules and the plurality of surface primers may be pre-attached before amplification as described elsewhere herein. For example, the attachment may be hybridization, ligation, or other covalent or non-covalent coupling. In some cases, after attachment, a same nucleic acid strand may comprise both a surface primer and a library molecule. In other cases, after attachment, the surface primer and the library molecule may be, or be part of, different strands that are hybridized or otherwise attached together. Alternatively, the plurality of library molecules and the plurality of supports may not be attached before amplification as described elsewhere herein. During amplification, the surface primers may be extended to generate copies, identical and/or reverse complement, of the library molecule that are immobilized to the support.

[0158] Thus, an amplified support may comprise a support comprising at least one template nucleic acid strand immobilized thereto. The amplified support may comprise a support comprising a plurality of template nucleic acid strands immobilized thereto. A template nucleic acid strand may be a copy, identical or reverse complement, of a library molecule that is input for amplification. In some cases, each template nucleic acid strand of a plurality of template nucleic acid strands immobilized to a single support may have sequence identity, or substantially 100% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more). It will be appreciated that an amplified colony of nucleic acid strands may comprise slight discrepancies amongst one or more strands due to amplification errors (e.g., PCR errors, chimeric errors, base mismatch errors, etc.). In other cases, multiple template nucleic acid strands immobilized to a single support may have different sequences. In some cases, the amplified support may comprise the template nucleic acid strand as part of a double-stranded molecule. For example, the template nucleic acid strand is coupled to the support and a second strand is hybridized to the template nucleic acid strand, the second strand being a reverse complement copy of the template nucleic acid strand. In another example, the template nucleic acid strand is hybridized to a second strand which is coupled to the support, the template nucleic acid strand being a reverse complement copy of the second strand. In other cases, the amplified support may comprise the template nucleic acid strand as part of a single-stranded molecule. For example, the single-stranded molecule may be coupled to the support. A nucleic acid strand coupled to a support may be covalently coupled or non-covalently coupled. A nucleic acid strand coupled to a support may be reversibly coupled or irreversibly coupled. A nucleic acid strand coupled to a support may be releasably or cleavably coupled or non-releasably or non-cleavably coupled.

[0159] A non-amplified support may be a support that does not comprise any template nucleic acid strand immobilized thereto. The non-amplified support may comprise a plurality of surface primers which are not extended.

[0160] FIG. 7A illustrates a post-amplification enrichment workflow that uses biotin capture moieties. Amplification may generate a mixture of positive supports 702 and negative supports 701, as shown in the top left panel. The template nucleic acid strands in the positive supports 702 may be part of single-stranded or double-stranded molecules (illustrated as double-stranded molecules). If the positive supports comprise double-stranded molecules, the mixture may be subjected to a stripping procedure to denature the double-stranded nucleic acid molecules, leaving single-stranded molecules on the positive supports. If the positive supports comprise single-stranded molecules, the stripping procedure is optional. Then, the mixture may be contacted with biotinylated sequencing primers (sequencing primers 703 comprising biotin moieties 704). The biotinylated sequencing primers may bind to the template nucleic acid strands to generate biotin-bound complexes 706 in the mixture, as shown in the top right panel. Each template nucleic acid strand may comprise a sequencing primer binding site that is configured to bind to a sequencing primer by sequence complementarity. The negative supports 701 may not be bound to any biotin moieties 704 as the biotinylated sequencing primers are unable to bind to any template nucleic acid strands. The mixture may be contacted with magnetic beads comprising streptavidin moieties 705. The biotin moieties 704 in the biotin-bound complexes 706 may bind to the streptavidin moieties 705 on the magnetic beads to generate magnetic complexes 707. The magnetic complexes 707 may be isolated from the negative supports 701 using a magnet or otherwise subjecting the mixture to a magnetic field and eluting. The isolated magnetic complexes 707 may be subjected to a stripping procedure to denature the biotinylated sequencing primers from the template nucleic acid strands on the positive supports 702 to generate enriched positive supports 712 which comprise single-stranded molecules comprising template nucleic acid strands.

[0161] It will be appreciated that the biotin-streptavidin (SA) pair illustrated in this example can be substituted with any other capture entity-capturing entity pair described elsewhere herein. It will be appreciated that the capturing entity (e.g., streptavidin) may comprise any secondary capture entity (other than a magnetic bead) and complementary secondary' capturing entity (other than magnet). For example, the magnetic bead-magnet pair may be substituted with any other capture entity-capturing entity pair described elsewhere herein. [0162] The stripping procedure may comprise treatment with a denaturing agent, such as sodium hydroxide (NaOH) or ethylene carbonate, heating, and/or a combination thereof. The stripping procedure and hybridization of sequencing primers (e.g., biotinylated sequencing primers) may be performed simultaneously (e g., reagents provided in the same mixture) or separately.

[0163] The enriched positive supports 712 may be loaded onto a substrate, such as in a sample processing system described elsewhere herein, and the template nucleic acid strands sequenced. In some cases, a plurality of sequencing primers (e.g., 703) may be hybridized to the template nucleic acid strands on the enriched positive supports 712 prior to loading onto the substrate. In other cases, a plurality' of sequencing primers (e.g.. 703) may be hybridized to the template nucleic acid strands on the enriched positive supports 712 after loading onto the substrate. Then, the plurality of sequencing primers may be extended, using the template nucleic acid strands as a template, during one or more sequencing reactions.

[0164] FIG. 7B illustrates an additional post-amplification enrichment workflow that uses biotin capture moieties. Amplification may generate a mixture of positive supports 702 and negative supports 701. as shown in the top left panel. The template nucleic acid strands in the positive supports 702 may be part of single-stranded or double-stranded molecules (illustrated as double-stranded molecules). If the positive supports comprise double-stranded molecules, the mixture may be subjected to a stripping procedure to denature the doublestranded nucleic acid molecules, leaving single-stranded molecules on the positive supports. If the positive supports comprise single-stranded molecules, the stripping procedure is optional. Then, the mixture may be contacted with a primer mixture comprising sequencing primers and biotinylated sequencing primers (sequencing primers 703 comprising biotin moieties 704). The primer mixture may comprise any fraction of biotinylated primers, such as about, at least about, and/or at most about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% biotinylated primers. The biotinylated sequencing primers and the sequencing primers may bind to the template nucleic acid strands to generate biotin-bound complexes 706 in the mixture, as shown in the top right panel. Compared to the workflow of FIG. 7A, which binds a biotinylated sequencing primer to each template nucleic acid strand, only a subset of template nucleic acid strands on each positive support may be bound to biotinylated sequencing primers and the remaining template nucleic acid strands bound to sequencing primers (e.g.. non-biotinylated sequencing primers). In some cases, the percentage of biotinylated sequencing primers in the primer mixture may be selected such that statistically at least one template nucleic acid strand on each positive support binds to a biotinylated sequencing primer, such that all positive supports 702 are generated into biotin-bound complexes 706. The negative supports 701 may not be bound to any biotin moieties 704 as the biotinylated sequencing primers are unable to bind to any template nucleic acid strands. The mixture may be contacted with magnetic beads comprising streptavidin moieties 705. The biotin moieties 704 in the biotin-bound complexes 706 may bind to the streptavidin moieties 705 on the magnetic beads to generate magnetic complexes 707. The magnetic complexes 707 may be isolated from the negative supports 701 using a magnet or otherwise subjecting the mixture to a magnetic field and eluting. The isolated magnetic complexes 707 may be subjected to a stripping procedure to denature the sequencing primers and biotinylated sequencing primers from the template nucleic acid strands on the positive supports 702 to generate enriched positive supports 712 which comprise single-stranded molecules comprising template nucleic acid strands.

[0165] The enriched positive supports 712 may be loaded onto a substrate, such as in a sample processing system described elsewhere herein, and the template nucleic acid strands sequenced. In some cases, a plurality of sequencing primers (e.g., 703) may be hybridized to the template nucleic acid strands on the enriched positive supports 712 prior to loading onto the substrate. In other cases, a plurality of sequencing primers (e.g.. 703) may be hybridized to the template nucleic acid strands on the enriched positive supports 712 after loading onto the substrate. Then, the plurality of sequencing primers may be extended, using the template nucleic acid strands as a template, during one or more sequencing reactions.

[0166] It will be appreciated that the biotin-streptavidin (SA) pair illustrated in this example can be substituted with any other capture entity-capturing entity pair described elsewhere herein. It will be appreciated that the capturing entity (e.g., streptavidin) may comprise any secondary capture entity (other than a magnetic bead) and complementary secondary capturing entity (other than magnet). For example, the magnetic bead-magnet pair may be substituted with any other capture entity-capturing entity pair described elsewhere herein. [0167] The stripping procedure may comprise treatment with a denaturing agent, such as sodium hydroxide (NaOH) or ethylene carbonate, heating, and/or a combination thereof. The stripping procedure and hybridization of sequencing primers (e.g., biotinylated sequencing primers) may be performed simultaneously (e.g., reagents provided in the same mixture) or separately.

[0168] FIG. 7C illustrates a post-amplification enrichment workflow that uses desthiobiotin capture moieties. Amplification may generate a mixture of positive supports 702 and negative supports 701, as shown in the top left panel. The template nucleic acid strands in the positive supports 702 may be part of single-stranded or double-stranded molecules (illustrated as double-stranded molecules). If the positive supports comprise double-stranded molecules, the mixture may be subjected to a stripping procedure to denature the double-stranded nucleic acid molecules, leaving single-stranded molecules on the positive supports. If the positive supports comprise single-stranded molecules, the stripping procedure is optional. Then, the mixture may be contacted with desthiobiotinylated sequencing primers (sequencing primers 703 comprising desthiobiotin moieties 708). The desthiobiotinylated sequencing primers maybind to the template nucleic acid strands to generate desthiobiotin-bound complexes 716 in the mixture, as shown in the top right panel. Each template nucleic acid strand may comprise a sequencing primer binding site that is configured to bind to a sequencing primer bysequence complementarity. The negative supports 701 may not be bound to any desthiobiotin moieties 708 as the desthiobiotinylated sequencing primers are unable to bind to any template nucleic acid strands. The mixture may be contacted with magnetic beads comprising streptavidin moieties 705. The desthiobiotin moieties 708 in the desthiobiotin-bound complexes 716 may bind to the streptavidin moieties 705 on the magnetic beads to generate magnetic complexes 717. The magnetic complexes 717 may be isolated from the negative supports 701 using a magnet or otherwise subjecting the mixture to a magnetic field and eluting. The isolated magnetic complexes 717 may be contacted with biotin moieties 704. The biotin moieties 704 may bind to all of the streptavidin moieties 705 in the magnetic complexes 717 to generate biotin-magnetic complexes 718 and in the process displacing any desthiobiotin moieties 708 and releasing the desthiobiotin-bound complexes 716 from the magnetic beads. The desthiobiotin-bound complexes 716 may then be isolated from the biotin-magnetic complexes 718 by using a magnet or otherwise subjecting the mixture to a magnetic field and eluting, as show n in the bottom left panel.

[0169] The desthiobiotin-bound complexes 716 may be loaded onto a substrate, such as in a sample processing system described elsewhere herein, and the template nucleic acid strands sequenced. Tn some cases, the desthiobiotinylated sequencing primers hybridized to the template nucleic acid strands in the desthiobiotin-bound complexes may be used as the sequencing primers for one or more sequencing reactions and extended, using the template nucleic acid strands as a template. In other cases, the desthiotinylated sequencing primers may be stripped from the positive supports via a stripping procedure to generate singlestranded, enriched positive supports (e.g., 712 as shown in FIGs. 7A-7B). The stripping maybe performed prior to loading or subsequent to loading the positive supports on the substrate. In some cases, a plurality- of sequencing primers (e.g.. 703) may be hybridized to the template nucleic acid strands on the enriched positive supports prior to loading onto the substrate. In other cases, a plurality of sequencing primers (e.g., 703) may be hybridized to the template nucleic acid strands on the enriched positive supports after loading onto the substrate. Then, the plurality of sequencing primers may be extended, using the template nucleic acid strands as a template, during one or more sequencing reactions.

[0170] Desthiobioitin-streptavidin binding strength (e.g., with disassociation constant (Kd) on the order of 10 ¹¹ M) is lower than that of biotin-streptavidin binding strength (e.g.. with Kd on the order of 10'¹⁵ M). Upon provision of a high concentration of biotin moieties, the biotin-streptavidin bonds may displace the desthiobiotin-streptavidin bonds.

[0171] It will be appreciated that the capturing entity⁷ (e.g., streptavidin) may comprise any secondary capture entity (other than a magnetic bead) and complementary secondary capturing entity (other than magnet). For example, the magnetic bead-magnet pair may be substituted with any other capture entity-capturing entity pair described elsewhere herein. [0172] The stripping procedure may comprise treatment with a denaturing agent, such as sodium hydroxide (NaOH) or ethylene carbonate, heating, and/or a combination thereof. The stripping procedure and hybridization of sequencing primers (e.g., desthiobiotinylated sequencing primers) may be performed simultaneously (e.g., reagents provided in the same mixture) or separately.

[0173] FIG. 7D illustrates an additional post-amplification enrichment workflow that uses desthiobiotin capture moieties. Amplification may generate a mixture of positive supports 702 and negative supports 701, as shown in the top left panel. The template nucleic acid strands in the positive supports 702 may be part of single-stranded or double-stranded molecules (illustrated as double-stranded molecules). If the positive supports comprise double-stranded molecules, the mixture may be subjected to a stripping procedure to denature the double-stranded nucleic acid molecules, leaving single-stranded molecules on the positive supports. If the positive supports comprise single-stranded molecules, the stripping procedure is optional. Then, the mixture may be contacted with a primer mixture comprising sequencing primers and desthiobiotinylated sequencing primers (sequencing primers 703 comprising desthiobiotin moieties 708). The primer mixture may comprise any fraction of desthiobiotinylated primers, such as about, at least about, and/or at most about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% biotinylated primers. The desthiobiotinylated sequencing primers and the sequencing primers may bind to the template nucleic acid strands to generate desthiobiotin-bound complexes 716 in the mixture, as shown in the top right panel. Compared to the workflow of FIG. 7C, which binds a desthiobiotinylated sequencing primer to each template nucleic acid strand, only a subset of template nucleic acid strands on each positive support may be bound to desthiobiotinylated sequencing primers and the remaining template nucleic acid strands bound to sequencing primers (e.g., nondesthiobiotinylated sequencing primers). In some cases, the percentage of desthiobiotinylated sequencing primers in the primer mixture may be selected such that statistically at least one template nucleic acid strand on each positive support binds to a desthiobiotinylated sequencing primer, such that all positive supports 702 are generated into desthiobiotin-bound complexes 716. The negative supports 701 may not be bound to any desthiobiotin moieties 708 as the desthiobiotinylated sequencing primers are unable to bind to any template nucleic acid strands. The mixture may be contacted with magnetic beads comprising streptavidin moieties 705. The desthiobiotin moieties 708 in the desthiobiotin-bound complexes 716 may bind to the streptavidin moieties 705 on the magnetic beads to generate magnetic complexes 717. The magnetic complexes 717 may be isolated from the negative supports 701 using a magnet or otherwise subjecting the mixture to a magnetic field and eluting. The isolated magnetic complexes 717 may be contacted with biotin moieties 704. The biotin moieties 704 may bind to all of the streptavidin moieties 705 in the magnetic complexes 717 to generate biotin-magnetic complexes 718 and in the process displacing any desthiobiotin moieties 708 and releasing the desthiobiotin-bound complexes 716 from the magnetic beads. The desthiobiotin-bound complexes 716 may then be isolated from the biotin-magnetic complexes 718 by using a magnet or otherwise subjecting the mixture to a magnetic field and eluting, as shown in the bottom left panel.

[0174] The desthiobiotin-bound complexes 716 may be loaded onto a substrate, such as in a sample processing system described elsewhere herein, and the template nucleic acid strands sequenced. Tn some cases, the desthiobiotinylated sequencing primers hybridized to the template nucleic acid strands in the desthiobiotin-bound complexes may be used as the sequencing primers for one or more sequencing reactions and extended, using the template nucleic acid strands as a template. In other cases, the desthiotinylated sequencing primers may be stripped from the positive supports via a stripping procedure to generate singlestranded, enriched positive supports (e.g., 712 as shown in FIGs. 7A-7B). The stripping may be performed prior to loading or subsequent to loading the positive supports on the substrate. In some cases, a plurality of sequencing primers (e.g.. 703) may be hybridized to the template nucleic acid strands on the enriched positive supports prior to loading onto the substrate. In other cases, a plurality of sequencing primers (e.g., 703) may be hybridized to the template nucleic acid strands on the enriched positive supports after loading onto the substrate. Then, the plurality of sequencing primers may be extended, using the template nucleic acid strands as a template, during one or more sequencing reactions.

[0175] It will be appreciated that the capturing entity (e.g., streptavidin) may comprise any secondary capture entity (other than a magnetic bead) and complementary secondary capturing entity (other than magnet). For example, the magnetic bead-magnet pair may be substituted with any other capture entity-capturing entity pair described elsewhere herein. [0176] The stripping procedure may comprise treatment with a denaturing agent, such as sodium hydroxide (NaOH) or ethylene carbonate, heating, and/or a combination thereof. The stripping procedure and hybridization of sequencing primers (e.g., desthiobiotinylated sequencing primers) may be performed simultaneously (e.g., reagents provided in the same mixture) or separately.

[0177] In the workflows of FIGs. 7A-7D, in some cases, prior to, during, or subsequent to binding sequencing primers (e.g., biotinylated sequencing primers, non-biotinylated sequencing primers) to the template nucleic acid strands on the positive supports, the mixture may be provided with single-stranded binding proteins (SSBs), which bind to single-stranded regions of the template nucleic acid strands (e g., portions of the strands not hybridized to the sequencing primers). SSB proteins may be useful for stabilizing a single-stranded region of a nucleic acid molecule, such as a DNA molecule. An SSB protein may derive from a bacterium. For example, an SSB protein may derive from Escherichia coli (E. coli). An SSB protein may derive from phage T4 (e.g., a T4 Gene 32 Protein or T4 SSB protein). Additional examples of SSB proteins include human replication protein A (hRPA) SSB protein, human SSB1 protein, and Extreme Thermostable SSB protein (New England BioLabs). In some cases, a portion of an SSB protein may be used, such as a truncated SSB protein. A SSB protein may have any useful features, such as a being tetramer.

[0178] FIG. 7E illustrates a post-amplification enrichment workflow that isolates positive supports via stripping. Amplification may generate a mixture of positive supports 702 and negative supports 701, as shown in the top left panel. The positive supports 702 may comprise double-stranded molecules, a double-stranded molecule comprising a template nucleic acid strand hybridized to a reverse complement copy. In some cases, at least one, at least a subset, or all of the reverse complement copies hybridized to the template nucleic acid strands on a single positive support may each comprise a biotin moiety 704. For example, biotinylated amplification primers (e.g., an amplification primer comprising a biotin moiety 704) may be used during the amplification operation. During amplification, biotinylated amplification primers may bind to template nucleic acid strands (e.g., extended surface primers from the supports) and be extended to generate the reverse complement copies comprising a biotin moiety 704. An amplification primer mixture comprising biotinylated amplification primers and non-biotinylated amplification primers may be used during amplification. The amplification primer mixture may comprise any fraction of biotinylated amplification primers, such as about, at least about, and/or at most about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. or 99% biotinylated amplification primers. In some cases, the percentage of biotinylated amplification primers in the amplification primer mixture maybe selected such that statistically at least one template nucleic acid strand on each positive support is hybridized to a reverse complement copy comprising a biotin moiety 704. The negative supports 701 may not be bound to any biotin moieties 704. The mixture of supports may be contacted with magnetic beads comprising streptavidin moieties 705. The biotin moieties 704 in the biotin-bound complexes 706 may bind to the streptavidin moieties 705 on the magnetic beads to generate magnetic complexes 727. The magnetic complexes 727 may be isolated from the negative supports 701 using a magnet or otherwise subjecting the mixture to a magnetic field and eluting. The isolated magnetic complexes 727 may be subjected to a stripping procedure to denature the reverse complement copies, including those comprising biotin moieties, from the template nucleic acid strands on the positive supports 702 to generate enriched positive supports 712 which comprise single-stranded molecules comprising template nucleic acid strands.

[0179] The enriched positive supports 712 may be loaded onto a substrate, such as in a sample processing system described elsewhere herein, and the template nucleic acid strands sequenced. In some cases, a plurality' of sequencing primers (e.g., 703) may be hybridized to the template nucleic acid strands on the enriched positive supports 712 prior to loading onto the substrate. In other cases, a plurality of sequencing primers (e.g.. 703) may be hybridized to the template nucleic acid strands on the enriched positive supports 712 after loading onto the substrate. Then, the plurality' of sequencing primers may be extended, using the template nucleic acid strands as a template, during one or more sequencing reactions.

[0180] It will be appreciated that the biotin-streptavidin (SA) pair illustrated in this example can be substituted with any other capture entity-capturing entity pair described elsewhere herein. It will be appreciated that the capturing entity (e.g., streptavidin) may' comprise any secondary' capture entity- (other than a magnetic bead) and complementary- secondary- capturing entity (other than magnet). For example, the magnetic bead-magnet pair may be substituted with any other capture entity -capturing entity pair described elsewhere herein. [0181] The stripping procedure may comprise treatment with a denaturing agent, such as sodium hydroxide (NaOH) or ethylene carbonate, heating, and/or a combination thereof. The stripping procedure and hybridization of sequencing primers (e.g., biotinylated sequencing primers) may be performed simultaneously (e.g., reagents provided in the same mixture) or separately.

[0182] FIG. 7F illustrates a post-amplification enrichment workflow that isolates positive supports via cleaving. Amplification may generate a mixture of positive supports 702 and negative supports 701, as shown in the top left panel. The positive supports 702 may comprise double-stranded molecules, a double-stranded molecule comprising a template nucleic acid strand hybridized to a reverse complement copy. In some cases, at least one, at least a subset, or all of the reverse complement copies hybridized to the template nucleic acid strands on a single positive support may each comprise, at or adjacent to a distal end from the support, a cleavable moiety 732 and a biotin moiety' 704. For example, amplification primers comprising a cleavable moiety 732 and a biotin moiety 704 may be used during the amplification operation. During amplification, such biotinylated amplification primers may bind to template nucleic acid strands (e.g., extended surface primers from the supports) and be extended to generate the reverse complement copies comprising a cleavable moiety 732 and a biotin moiety 704. An amplification primer mixture comprising biotinylated amplification primers (also comprising the cleavable moieties) and non-biotinylated amplification primers may be used during amplification. The amplification primer mixture may comprise any fraction of biotinylated amplification primers, such as about, at least about, and/or at most about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% biotinylated amplification primers. In some cases, the percentage of biotinylated amplification primers in the amplification primer mixture may be selected such that statistically at least one template nucleic acid strand on each positive support is hybridized to a reverse complement copy comprising a biotin moiety' 704 and a cleavable moiety 732. The negative supports 701 may not be bound to any biotin moieties 704. The mixture of supports may be contacted with magnetic beads comprising streptavidin moieties 705. The biotin moieties 704 in the biotin-bound complexes 706 may bind to the streptavidin moieties 705 on the magnetic beads to generate magnetic complexes 727. The magnetic complexes 727 may be isolated from the negative supports 701 using a magnet or otherwise subjecting the mixture to a magnetic field and eluting. The isolated magnetic complexes 727 may be subjected to a cleaving procedure to cleave the cleavable moieties 732, in the process removing the biotin moieties 704 from the positive supports 702. The positive supports 702 may be isolated from the cleaved biotin moieties 704 bound to the magnetic beads by using a magnet or otherwise subjecting the mixture to a magnetic field and eluting. The isolated positive supports are enriched positive supports 722, as shown in the bottom left panel, which comprise double-stranded molecules comprising template nucleic acid strands.

[0183] The enriched positive supports 722 may be loaded onto a substrate, such as in a sample processing system described elsewhere herein, and the template nucleic acid strands sequenced. The reverse complement copies hybridized to the template nucleic acid strands (in the double-stranded molecules) may be stripped from the positive supports via a stripping procedure to generate single-stranded, enriched positive supports. The stripping may be performed prior to loading or subsequent to loading the positive supports on the substrate. In some cases, a plurality of sequencing primers (e.g., 703) may be hybridized to the template nucleic acid strands on the enriched positive supports prior to loading onto the substrate. In other cases, a plurality of sequencing primers (e.g., 703) may be hybridized to the template nucleic acid strands on the enriched positive supports 712 after loading onto the substrate. Then, the plurality of sequencing primers may be extended, using the template nucleic acid strands as a template, during one or more sequencing reactions.

[0184] It will be appreciated that the biotin-streptavidin (SA) pair illustrated in this example can be substituted with any other capture entity-capturing entity pair described elsewhere herein. It will be appreciated that the capturing entity (e.g., streptavidin) may comprise any secondary capture entity (other than a magnetic bead) and complementary secondary' capturing entity (other than magnet). For example, the magnetic bead-magnet pair may be substituted with any other capture entity-capturing entity pair described elsewhere herein. [0185] A cleavable moiety may comprise any useful moiety that can be used to cleave an oligonucleotide (or portion thereof) from the support, or otherwise release a nucleic acid strand from the support and/or the oligonucleotide. A cleavable moiety may comprise a uracil, a ribonucleotide, methylated nucleotide, or other modified nucleotide that is excisable or cleavable using an enzyme (e.g., uracil DNA glycosylase (UDG), RNAse, APE1, MspJI, endonuclease, exonuclease, etc.). The cleavable moiety may comprise an abasic site or an analog of an abasic site (e.g., dSpacer), a dideoxyribose, a spacer, e.g., C3 spacer, hexanediol, triethylene glycol spacer (e.g., Spacer 9), hexa-ethyleneglycol spacer (e.g., Spacer 18), a photocleavable moiety, or combinations or analogs thereof. Alternatively, or in addition, the cleavable moiety may be cleavable using one or more stimuli, e.g., photo-stimulus, chemical stimulus, thermal stimulus, etc. In one example, the cleavable moiety may comprise a uracil residue, and the cleaving may comprise use of a USER® (Uracil-Specific Excision Reagent) enzyme comprising a mixture of UDG and the DNA glycosylase-lyase Endonuclease VIII. [0186] Alternatively or in addition to the other methods described herein, provided is another post-amplification workflow to isolate positive supports that uses single stranded capture moieties (e.g., single stranded binding proteins) and solution capture probes. Amplification may generate an emulsion amplification product mixture of positive supports (e.g., amplified supports) and negative supports (e.g., unamplified supports). The positive supports may comprise double-stranded molecules, where a double-stranded molecule comprises a template nucleic acid strand hybridized to a reverse complement copy of the template strand. A support (e g., particle) in the emulsion amplification product mixture may be contained within a compartment such as a droplet (e.g., an oil-based droplet as in ePCR). All or substantially all of the droplets comprising a support may comprise one support (e.g., one positive or one negative support). In some cases, one or more droplets may comprise multiple supports; it is typically advantageous for a droplet to contain only one support to enable the production of monoclonal amplified positive supports. Additional discussion of ePCR methods is provided in U.S. Pat. Appl. No. 17/394,692, which is herein incorporated by reference in its entirety.

[0187] After obtaining the emulsion amplification product mixture, the mixture may be subjected to conditions sufficient to break the emulsion. Such breaking conditions may serve to disrupt and coalesce the droplets and extract or pool the materials therein, which materials may comprise positive beads comprising amplicons (e.g., copies of template nucleic acid molecules, or complements thereof), template nucleic acid molecules free in solution, amplification reagents (e.g., polymerase, nucleotides, solution amplification primers, etc.), etc. In some cases, breaking conditions may comprise heating an amplification product mixture, agitating an amplification product mixture, applying electrostatic force (e.g., continuous or alternating) to an amplification product mixture, or a combination thereof. Breaking methods and conditions are described in U.S. Pat. No. 11.904,322, which is herein incorporated by reference in its entirety. [0188] Prior to, subsequent to, or simultaneous with the breaking, one or more capture probes (e.g., oligos capable of hybridizing to primers coupled to a support) may be contacted to the amplification product mixture. These capture probes may bind to support-coupled primers (e.g., primers that were not extended during amplification to provide a template nucleic acid strand). By converting single-stranded support-coupled primers into double-stranded molecules, clumping of supports (e.g., non-covalent, hybridization interactions between positive supports, negative supports, or combinations thereof). An amount of capture primers added to the amplification product mixture may be sufficient to bind to about, at least about, and/or at most about. 1%, 2%. 3%, 4%, 5%. 6%, 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of singlestranded support-coupled primers present in the amplification product mixture.

[0189] After breaking, the amplification product mixture may be contacted with a plurality of single stranded binding proteins (SSBs) or other single stranded binding agents, as described elsewhere herein. These SSBs may bind to single-stranded solution amplification primers (e.g., primers present in the solution for amplifying template nucleic acid molecules during amplification of supports). SSBs may be useful for preventing free solution amplification primers from binding to template nucleic acid strands present on positive supports. An amount of SSBs added to the amplification product mixture may be sufficient to bind to about, at least about, and/or at most about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of single-stranded solution amplification primers present in the amplification product mixture.

[0190] Concurrent with or subsequent to the binding of single-stranded solution amplification primers, positive supports may be isolated from negative supports in the amplification product mixture. Selection of positive supports may be performed in accordance with any methods described herein.

[0191] One or more, or all operations for enrichment described herein may be automated and completed without user intervention. For example, an amplification instrument comprising robotic handlers may perform such operations after amplification is complete without user intervention to output enriched, positive supports. Alternatively, one or more, or all operations for enrichment described herein may be performed by a user and/or with user instructions. Chemical capping

[0192] Provided herein are devices, systems, methods, compositions, and kits that provide chemical caps and methods of capping a centrifugation tube using chemical capping. Such devices, systems, methods, compositions, and kits can be applied during any one of support or template preparation (e.g., 101), pre-enrichment of supports and/or templates and/or support-template complexes (e.g., 102, 104), attachment of supports and templates (e.g., 103), amplification of templates (e.g., 105), and post-amplification processing (e.g., 106) described with respect to sequencing workflow 100 of FIG. 1. Such devices, systems, methods, compositions, and kits can be used in conjunction with the post-amplification processing described herein (e.g., with respect to FIGs. 7A-7F) and/or the sample processing systems and methods, or components thereof (e.g., substrates, detectors, reagent dispensing, continuous scanning, etc.) described herein.

[0193] Chemical capping is a method that includes introducing a chemical on top of the fluid in the centrifuge tube. The chemical provides an air barrier to prevent the fluid from splashing out of and/or evaporating from the centrifuge tubes. The chemical is specifically selected such that it does not react chemically with the fluid in a centrifuge tube (e.g., the chemical is inert with respect to the fluid). Without a reliable capping system, fluid may splash out of or evaporate from the tubes, particularly in centrifugation systems where the tubes are spun around at a high velocity and approach a 90-degree angle (e.g., a horizontal orientation).

[0194] Other methods of capping include physical caps, or “hats” that are manually placed onto the tubes. However, in practice, these physical caps often allow material to leak out of the tubes and/or splash out of the tubes. For example, the caps may pop off entirely during the rapid spinning, allowing material to easily escape the tubes. In some cases, the caps do not properly fit the tubes and allow material to leak out of the tubes during spinning. In other circumstances, placement robots that are used to place the physical caps onto the tubes may incorrectly place the cap (such that it is not properly applied to the tube) and/or break the cap due to excess force applied to the cap during placement.

[0195] Accordingly, the chemical caps described herein are designed to address the problems of conventional physical caps and provide a more reliable seal and capping mechanism for centrifugation tubes. The chemical caps described herein provide an adequate seal to minimize or prevent material from leaking or splashing out of the tubes. Unlike conventional physical caps, the chemical caps described herein do not pop off of or out of the tubes. Further, they do not run the risk of being broken during robotic placement. By utilized the chemical caps described herein, , material loss can be minimized or entirely prevented during the centrifugation process, thereby minimizing cost and time required to complete the centrifugation process (and yield the desired amount of product). The chemical caps are described herein with respect to a DNA amplification process (e.g., recovery of sample after performance of amplification). However, they may be applied to any separation and/or centrifugation process.

[0196] FIG. 15 shows a cross-sectional view of a physical cap 1522 placed on a tube 1520, according to some embodiments. This is an example of a cap that sits within a tube opening. Other types of caps, e.g., screw on caps, are also possible and typically have similar dimensions. As shown, the physical cap 1522 includes an insertion portion 1524 that is configured to push into an opening of the tube 1520. When the cap is a proper size, friction between an outer surface of the insertion portion 1524 of cap 1522 and an interior surface of tube 1520 can hold the physical cap 1522 in place during centrifugation. However, this permits little room for error in the manufacture/sizing of physical cap 1522. If slightly small, the physical cap 1522 will readily fall out of tube 1520 during centrifugation and/or not provide an adequate seal. If slightly large, the physical cap 1522 may pop out during centrifugation. In addition, robotic manipulation of tube caps (e.g., removal of caps by grippers for the addition/removal of material from centrifugation tubes and replacement preparatory’ for centrifugation) must be extremely precise, and is prone to failure (e.g., crushing of caps, misapplication of caps, etc.). An exterior portion 1526 of physical cap 1522 having a diameter that is greater than that of insertion portion 1524 ensures that physical cap 1522 will not slip into tube 1520, such that it would not be easily removable. A screw on cap can have similar issues with robotic manipulation that may lead to spillage or splashing. For instance, robotic manipulation must have a high accuracy in order to correctly screw on such a cap (e.g., such that the cap correctly catches the threads of the tube). In some case, the robotic manipulation can be inaccurate, where a cap is not securely screwed on (e.g., not fully or askew) or where a cap is damaged (e.g., cracked due to robotic pressure).

[0197] In some cases, a chemical cap material can be selected with respect to the following considerations: boiling point, cohesion, hydrophobicity, density, viscosity, reactivity, and melting point. For instance, a higher boiling point than temperatures required during sample preparation and/or centrifugation is desirable so that the cap does not evaporate prior to or during centrifugation. A material with higher cohesion is desirable to reduce the amount of residual material after removal of the cap. For aqueous fluids, a hydrophobic capping material helps retain layer separation between the cap and the sample fluid. Similarly for a hydrophobic sample, a hydrophilic capping material is desirable. Preferentially, a capping material will be low density (e.g., less than water) to support layer separation so the chemical cap remains afloat the sample fluid. In some cases, a capping material will have sufficiently high viscosity to prevent splashing (e.g., from the sample fluid through the capping layer and out of the tube) and a sufficiently low viscosity⁷ for ease of application and removal of the cap. Importantly, capping material may be non-reactive with the sample fluid. In some case, the melting temperature of a capping material may be such that the cap melts during centrifugation (see e.g., FIG. 16C). In some cases, the melting temperature of a capping material may be high enough that the cap does not melt during centrifugation (see e.g., FIG. 16D). A chemical seal may be a liquid at room temperature. Alternatively, a chemical seal may be a solid at room temperature. In some cases, a chemical capping material may comprise a phase change material, and in such cases, a chemical cap may be solid at room temperature and may change to a liquid during centrifugation, e.g., due to the inherent heating of the process. Alternatively or in addition, when using a phase change capping material, the sample tube may be chilled to allow the capping material to solidify prior to centrifugation. In some cases, a chemical cap may provide an airtight seal and/or a watertight seal. Chemical capping material may comprise any of a variety of chemical compounds. The chemical cap may be a phase change material and may comprise a paraffin or wax material. Suitable chemical compounds for the chemical cap can include cyclooctane, 1,3- diphenylacetone. mineral oil, hydrocarbons, triglycerides, vegetable oil, or liquid silicone.

[0198] FIGs. 16A-16D illustrate the use of a chemical cap. In FIG. 16A, tube 1620 comprises a sample fluid 1632. After the fluid 1632 is placed into tube 1620, a chemical cap 1630 may be placed on top of the fluid 1632, depicted in FIG. 16B. The chemical cap 1630 may have any one or more features as described herein. FIG. 16B shows tube 1620 with a chemical cap 1630 layered on top of the sample fluid 1632. Unlike the physical caps described above, chemical cap 1630 is located entirely within tube 1620 (i.e., a topmost surface of chemical cap 1630 is below that of an upper edge or opening of tube 1620). In some cases, chemical cap 1630 may be liquid at room temperature (e.g., a liquid with a density lower than that of fluid 1632 such that chemical cap 1630 will remain on top of fluid 1632). After the chemical cap 1630 is placed onto fluid 1632, the tube 1620 may be centrifuged. The centrifuge rapidly spins or rotates the tube 1620 to separate components of fluid 1632 by density. FIG. 16C shows two components of fluid 1632 adequately separated into a product pellet 1660 and supernatant/ waste 1662. In this example, the chemical cap material has mixed with the fluid 1632 (e.g., during or subsequent to centrifugation). FIG. 16D illustrates an alternative scenario where chemical cap 1630 is retained after centrifugation, and where fluid 1632 has been adequately separated into a product pellet 1660 and supematant/waste 1662. Once the components are adequately separated by centrifugation, they can be physically separated to recover product pellet 1660. For example supematant/waste 1662 may be pipetted or filtered out of tube 1620, leaving product pellet 1660. Where the chemical cap has been retained, that may first be removed.

[0199] In some cases, a chemical cap comprising viscous material may experience a reduction in viscosity during the centrifugation process. The reduction in viscosity⁷ may occur anytime from 10-100 % completion of the centrifugation process time. For instance, this means that for a 1 -minute centrifugation processing time, the reduction in viscosity occurs anytime between 6 and 60 seconds during the centrifugation process time, where the process starts at 0 seconds. In some cases, the reduction in viscosity occurs from 30-100, 30-80, 50- 100, and 50-80 % completion of the centrifugation process time. In some cases, the reduction in viscosity occurs at a time that is less than or equal to 100, 90, 80, 70. 60. 50. 40, 30, or 20 % completion of the centrifugation process time. In some cases, the reduction in viscosity occurs at a time that is greater than or equal to 10, 20, 30, 40, 50, 60, 70, 80, or 90 % completion of the centrifugation process time. In some cases, the reduction in viscosity may occur prior to the conclusion of the centrifugation process time (i .e., at a time that is less than 100 % completion of the centrifugation process time). When this happens, this can cause some material loss (e.g., splashing) from the centrifugation tube towards the end of the process, after the reduction in viscosity has occurred. In some cases, this may be acceptable, particularly when the desired product is a solid pellet, and the liquid supernatant is an undesired product. In some cases, the reduction in viscosity occurs only once the centrifuge reaches maximum speed. In some cases, the centrifuge reaches maximum speed only once the tubes have reached an approximately horizontal position.

[0200] Chemical capping, as described herein, can be used in accordance with any type of centrifuge and centrifuge tube. This may be particularly useful with high-speed laboratory centrifuges (e.g.. centrifuges for use with sample tube volumes of O. lmL to lOOmL). In some cases, the chemical caps described herein may be designed to be compatible with 2- and 1.5- mL microfuge tubes and/or a microcentrifuge that spins from 200-10,000 rpm. However, it will be appreciated that many different sizes of centrifuges and centrifuge tubes can be used with the methods described herein. For example, chemical capping can be used with tubes of a size about O.lmL, 0.25mL. 0.5mL, 0.75mL, ImL, 2mL, 5mL, lOmL, 20mL, 25mL, 50mL, 75mL, lOOmL. 500mL, IL, 2L, 3L, 4L, 5L, 6L, 7L. 8L. 9L. 10L, or of any size within this range.

Amplification on Surface

[0201] Provided herein are devices, systems, methods, compositions, and kits that provide amplified supports on a substrate. Such devices, systems, methods, compositions, and kits can be applied alternatively or in addition to any one of the pre-enrichment of supports and/or templates and/or support-template complexes (e.g., 102, 104), the attachment of supports and templates (e.g.. 103), and the amplification of templates (e.g.. 105) described with respect to sequencing workflow 100 of FIG. 1. Such devices, systems, methods, compositions, and kits can be used in conjunction with the post-amplification processing described herein (e g., with respect to FIGs. 7A-7F) and/or the sample processing systems and methods, or components thereof (e.g., substrates, detectors, reagent dispensing, continuous scanning, etc.) described herein.

[0202] Amplification on surface may output a mixture of amplified supports (e.g., positive supports) and non-amplified supports (e.g., negative supports), where supports may be beads or other objects. Any type of amplification method described herein, or a combination thereof, may be performed to generate the amplification output; however, isothermal amplification methods such as RPA may be preferable.

[0203] In some cases, multiple types of supports may be involved in on-surface amplification systems, methods, compositions, and kits. For example, a first type of support (e.g., an amplification support) may comprise a first type of surface primers, and a second type of support (e.g.. a sequencing support) may comprise a second type of surface primers. The plurality of library molecules may be pre-attached to either the first or second type of surface primers (e.g., to the first or second type of support) before amplification, as described elsewhere herein. For example, the attachment may be hybridization, ligation, or other covalent or non-covalent coupling. In some cases, after attachment, a same nucleic acid strand may comprise both a surface primer and a library molecule. In other cases, after attachment, the surface primer and the library molecule may be, or be part of, different strands that are hybridized or otherwise attached together. Alternatively, the plurality of library molecules and the plurality of supports (e.g., first or second type of supports) may not be attached before amplification as described elsewhere herein. During amplification, the surface primers may be extended to generate copies, identical and/or reverse complement, of the library molecule that are immobilized to the support.

[0204] FIGs. 8-10 illustrate various methods of on-surface amplification, where a first type of support (e.g., an amplification support) comprises a first type of surface primer and a second type of support (e.g., a sequencing support) comprises a second type of surface primer. The use of substrate-coupled primers for amplification may increase the efficiency of amplification by increasing the local concentration of each type of primers.

[0205] In FIG. 8A, a support of a first plurality of supports (e.g., amplification supports) may be coupled (e.g., hybridized) to a library molecule. The first plurality⁷ of supports may be preenriched for attachment to library molecules or may not be pre-enriched. Supports in the first plurality of supports comprise surface primers coupled thereto. Library-coupled supports may be subjected to conditions for linear amplification in solution. The linear amplification may be RPA or another form of isothermal amplification. For example, during linear amplification, a surface primer of a first type annealed (e.g., hybridized) to a library molecule is extended to create an extension product, where the extension product is covalently coupled to the support. The library molecule diffuses away from the surface primer and may anneal to another surface primer of the first type. As there is only one template for extension, this extension is linear rather than exponential, i.e., only one copy of the first (surface) primers are extended in each cycle. This is because there are no or few solution primers that may extend along the extension products (e.g., to produce copies of the libraiy molecule).

[0206] In FIG. 8B, a second plurality of supports (e.g., negative sequencing beads) may be loaded onto a substrate. The second plurality of supports comprise a second type of surface primer. In some cases, the second plurality of supports may couple to the substrate at locations with distinct surface chemistry (e.g.. APTMS) and the coupling may comprise electrostatic attraction. In some cases, loading the second plurality of supports may comprise support self-assembly, as described in Inti. Pub. No. WO2023/205353, which is hereby incorporated by reference in its entirety⁷.

[0207] In FIG. 8C, the linearly amplified first plurality of supports may be loaded onto the substrate comprising the second plurality of supports. In some cases, a single linearly amplified support from the first plurality⁷ of supports associates with a single support from the second plurality of supports. After loading the linearly amplified first plurality of supports, the linear amplification products may be further amplified (e.g., exponentially amplified), where the second plurality of supports comprise second amplification primers. The exponential amplification products may be coupled (e.g., covalently coupled) to supports in the second plurality of supports.

[0208] Alternatively, in FIG. 9A a first plurality of supports (e.g., sequencing beads) may be loaded onto a substrate. In some cases, the first plurality' of supports may be pre-enriched, such that all or substantially all of the first plurality of supports have coupled thereto (e.g., hybridized to surface primers) a library molecule. The supports may further have coupled thereto surface primers of a first type. Subsequent to loading of the first plurality of supports, the library molecules may be amplified (e.g., linearly amplified via isothermal amplification such as RPA) on the first plurality' of supports. To support linear amplification, there may be a lack of solution primers or a limited supply of solution primers during the amplification. Subsequent to linear amplification, as illustrated in FIG. 9B, a second plurality of supports (e g., empty amplification beads) may be loaded onto the substrate. One or more supports of the second plurality of supports may associate with a single support of the first plurality of supports. In some cases, only a single support of the second plurality of supports may associate with a single support of the first plurality of supports. Supports in the second plurality of supports may have coupled thereto surface primers of a second type. Subsequent to loading of the second plurality of supports, the library molecules may be exponentially amplified (e.g., using the linearly amplified copies of library molecules coupled to the first plurality of supports and the second type of surface primers on the second plurality of supports). In some cases, the exponential amplification takes place in the absence of solution primers or with a limited supply of solution primers. Subsequent to the exponential amplification (e.g., isothermal amplification such as RPA), the second plurality- of supports may be removed (e.g., washed out).

[0209] In some cases, to amplily both strands of double-stranded library molecules, an amplification method such as that illustrated in FIGs. 10A-10D may be used. Using sequencing information from both strands of a double-stranded library molecule may serve to improve the quality of sequencing data, as described in Inti. Pat. Appl. No.

PCT/US2024/013236, which is hereby incorporated by reference in its entirety. In FIG. 10A, a double stranded library molecule may be coupled to a support of a first plurality of supports. The first plurality of supports may be pre-enriched at any stage prior to loading the first plurality of beads onto a substrate. The coupled library' molecule must be ligated to a surface primer to ensure that both strands are retained for downstream processing.

Subsequent to ligation, the first plurality of supports may optionally be subjected to linear amplification. As described herein, this linear amplification will serve to produce reverse complements of a first strand of the double-stranded library molecule. Subsequent to the ligation and (optionally) the linear amplification, as illustrated in FIG. 10B, the first plurality of supports may be loaded onto a substrate. The substrate may comprise a second plurality of supports coupled thereto (e.g., covalently or non-covalently coupled to a surface of the substrate). A support of the first plurality of supports may associate with a substrate-coupled support of the second plurality of supports. Subsequent to loading the first plurality’ of supports, the double-stranded library molecules may be subjected to exponential amplification (e.g., exponential RPA) such that an amplified support in the second plurality’ of supports may comprise a plurality of copies of the first strand of a library molecule and a plurality of copies of copies of the second strand of the library molecule (e.g., such that the template molecules on the amplified support have sequence complementarity).

[0210] FIGs. 10C and 10D illustrate an alternative method of retaining sequencing information from both strands of a double-stranded library' molecule. As in FIG. 10A, double-stranded template may be loaded (e.g.. coupled to a surface primer) onto supports in a first plurality of supports and ligated thereto. After ligation, RPA recombinase may be provided to the first plurality’ of supports. RPA recombinase may associate with surface primers of the first plurality’ of supports. Subsequently, and optionally after pre-enrichment of the supports, the plurality’ of supports may be loaded onto a substrate, as illustrated in FIG. 10D. After the supports are loaded onto the substrate, additional reagents for amplification (e.g., strand-displacing polymerase, solution primers, and dNTPs) are provided and exponential RPA may be performed, such that an amplified support in the second plurality’ of supports may comprise a plurality of copies of the first strand of a library molecule and a plurality of copies of copies of the second strand of the library molecule (e.g., such that the template molecules on the amplified support have sequence complementarity).

Advantageously, the method described with respect to FIGs. 10C and 10D only requires one amplification step and only one type of support.

[0211] In some cases, amplification supports and sequencing supports may be a same size (e.g.. have a same diameter). In some cases, amplification supports may be smaller than sequencing supports (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%). In some cases, amplification supports may be larger than sequencing supports (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%). In some cases, a support may comprise a bead, a dendrimer, a nucleic acid nanoparticle (e.g., DNA origami, or unstructured nucleic acid), or other object.

[0212] A support amplified in accordance with these methods (e.g., an amplified sequencing support) may comprise at least one template nucleic acid strand immobilized thereto. The amplified support may comprise a plurality of template nucleic acid strands immobilized thereto. In some cases, the plurality⁷ of template nucleic acid strands may have sequence complementarity. In some cases, the plurality of template nucleic acid strands may be derived from a first strand of a library molecule (e.g., a double stranded library molecule), the second strand of the library molecule, or a combination thereof. A template nucleic acid strand may be a copy, identical or reverse complement, of a library⁷ molecule that is input for amplification. In some cases, each template nucleic acid strand of a plurality of template nucleic acid strands immobilized to a single support may have sequence identity, or substantially 100% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more). It will be appreciated that an amplified colony of nucleic acid strands may comprise slight discrepancies amongst one or more strands due to amplification errors (e.g., PCR errors, chimeric errors, base mismatch errors, etc.). In other cases, multiple template nucleic acid strands immobilized to a single amplified support may have different sequences. In some cases, the amplified support may⁷ comprise the template nucleic acid strand as part of a double-stranded molecule. For example, the template nucleic acid strand is coupled to the amplified support and a second strand is hybridized to the template nucleic acid strand, the second strand being a reverse complement copy of the template nucleic acid strand. In another example, the template nucleic acid strand is hybridized to a second strand which is coupled to the amplified support, the template nucleic acid strand being a reverse complement copy of the second strand. In other cases, the amplified support may comprise the template nucleic acid strand as part of a single-stranded molecule. For example, the single-stranded molecule may be coupled to the amplified support (e.g., covalently coupled or non-covalently coupled, reversibly coupled or irreversibly coupled, non-releasably or non-cleavably coupled, or a combination thereof).

[0213] FIG. 10E illustrates an example amplification workflow on a bead 1020. A bead may comprise a first type of primers and a second type of primers attached thereto. The first type of primers and second type of primers may correspond to and/or comprise forward and reverse primers, respectively, or vice versa, for amplification. A bead 1020 comprises a first type of primer 1024 attached thereto and a second type of primer 1022 attached thereto. The bead 1020 may comprise a plurality of primer attachment sites. For example, the bead may comprise a polymer mesh that comprises a plurality of primer attachment sites. The first type of primers (e.g., 1024) may be attached to a first subset of primer attachment sites on the bead directly or via a first linker. The second type of primers (e.g., 1022) may be attached to a second subset of primer attachment sites on the bead, different from the first subset of primer attachment sites, via a second linker 1026. The first linker or second linker may comprise any tether or linker. For example, a linker may comprise a polymer, such as a polyethylene glycol (PEG) linker with repeating ethylene oxide units. In the case of a direct coupling, the coupling may be via any attachment mechanism, such as a click chemistry pairing or crosslinking. A linker may be connected to a primer and/or to a primer attachment site via any attachment mechanism, such as a click chemistry pairing or crosslinking. The second linker 1026 may be longer than the first linker, if any. or longer than the direct coupling bond between the first type of primers and the first subset of primer attachment sites. In some cases, a maximum length of the second linker 1026 is about, at least about, and/or at most about 5, 10, 15, 20, 25. 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 nanometers (nm) or more. Each of the first type of primers may be attached to the first subset of primer attachment sites on the bead via the same type of linker or different types of linkers. Each of the second type of primer may be attached to the second subset of primer attachment sites on the bead via the same type of linker or different types of linkers. For example, a set of primers may be attached via the same length linkers or different length linkers. Beneficially, the second linker 1026 may permit more mobile diffusion or movement of the second type of primer 1022 than that of the first type of primer 1024 while still tethering the second ty pe of primer 1022 to a vicinity of the bead 1020. Tethering the second type of primer 1022 to the vicinity of the bead may reduce and prevent potential cross-contamination of amplified strands migrating to a neighboring bead and reduce overall polyclonality resulting from amplification.

[0214] A template strand 1030 may undergo amplification on the bead 1020 using the first type of primers (e.g., 1024) and second type of primers (e.g., 1022). The template strand 1030 may comprise a first primer sequence configured to bind to the first type of primer and a second primer sequence corresponding to (e.g., comprising or identical to) the second type of primer. Such primer sequences may be introduced to a template insert via adapter ligation, for example. The template strand 1030 may hybridize to the first type of primer 1024 coupled to the bead 1020, and the first type of primer 1024 may be extended using the template strand 1030 as a template to generate a first extended strand 1032 coupled to the bead. The first extended strand 1032 may comprise a third primer sequence configured to bind to the second ty pe of primer 1022. The first extended strand 1032 may bind to the second type of primer 1022 that is coupled to the bead 1020 via the second linker 1026 and be extended using the first extended strand 1032 as a template to generate a second extended strand 1034 that is coupled to the bead via the second linker 1026. The second extended strand 1034 may be a reverse complement copy of the first extended strand 1032 that is coupled to the bead (directly or via the first linker). The second extended strand 1034 may then be free to bind to another first type of primer (e.g., 1024b) on the bead 1020, which other first type of primer may be extended using the second extended strand 1034 as a template to generate a third extended strand 1032b that is coupled to the bead (directly or via the first linker). Other primers of the second type of primers coupled to the bead (via the second linker) may bind to the first extended strand 1032 (or copies thereof) and be extended to generate additional reverse complement copies of the first extended strand 1032 (e.g., 1034). Other primers of the first type of primers coupled to the bead (directly or via the first linker) may bind to the second extended strand 1034 (or copies thereof) and be extended to generate additional copies of the first extended strand 1032. After the amplification and/or extension reactions, the bead 1020 may comprise a plurality of amplified molecules derived from the template strand 1030, which plurality⁷ of amplified molecules comprises a first set of amplified molecules which are copies of the first extended strand 1032 (e g., a reverse complement copy of the template strand 1030) each coupled to the bead via the first linker or directly to primer attachment sites and a second set of amplified molecules which are copies of the second extended strand 1034 (e.g., a copy of the template strand 1030) each coupled to the bead via the second linker 1026 to primer attachment sites. The bead comprising the plurality⁷ of amplified molecules, or amplified bead, may be subjected to sequencing such as to generate two sets of reads such as paired end reads corresponding to each of the first and second set of amplified molecules. In some cases, one of the two sets of amplified molecules may be removed from the amplified bead prior to sequencing. For example, the second ty pe of primer 1022 may comprise a cleavage site comprising one or more cleavable moieties, such that upon cleavage, the oligonucleotide molecule segment downstream of the cleavage site is removed. In this case, upon cleavage, the second set of amplified molecules may be removed and/or digested. In another example, the first type of primer 1024 may comprise a cleavage site comprising one or more cleavable moieties, such that upon cleavage, the oligonucleotide molecule segment downstream of the cleavage site is removed. In this case, upon cleavage, the first set of amplified molecules may be removed and/or digested.

Library preparation for Methylation Sequencing

Single adapter species for methylation sequencing

[0215] One issue with the construction of libraries for sequencing is the inevitable loss of some sample material during library preparation, especially due to the attachment of adapters to library molecules. For instance, where template molecules are desired to be coupled to a first type of adapter on one end and a second type of adapter on the other end (e.g., where the first and second adapters serve different downstream purposes, such as bead attachment vs. sequencing primer), only about 50% of the resulting library molecules will be ligated to one of each type of adapter (where about 25% of the resulting library molecules will be ligated to the first type of adapter at each end and about 25% of the resulting library molecules will be ligated to the second type of adapter at each end). Thus, there is a significant advantage in terms of library' preparation efficiency if a single species of adapter can be used to serve each distinct downstream purpose. The devices, systems, methods, compositions, and kits provided herein may allow- for the efficient preparation of template nucleic acid molecules for sequencing (e.g., library preparation for methylation sequencing) by the use of a single adapter species. Example schemes are illustrated in FIGs. 11A-11C.

[0216] As shown in FIG. 11A, a template molecule is provided with a plurality of doublestranded adapters (e.g., adapters with first sequence seql hybridized to second sequence seq2). In some cases, the double-stranded adapters comprise a same nucleic acid sequence (e.g., at least a subset of the plurality of sequencing adapters all comprise a same first sequence seql and a same second sequence seq2, where seql and seq2 are complementary ). The template molecule is coupled to one double-stranded adapter at a first end of the template molecule and another doubled-stranded adapter at a second end of the template molecule. In some cases, the coupling comprises a hybridization between complementary sequences on the template molecule and the double-stranded adapter. For example, in some cases, the doublestranded adapters comprise a first region that is double-stranded and a second region that is single-stranded (e.g.. the second region is an overhang). Similarly, the template molecules may comprise a first region that is double-stranded and a second region that is single-stranded (e.g., where the second region is an overhang). In some cases, the overhang sequence of the double-stranded adapter is complementary to the overhang sequence of the template molecule.

[0217] A ligation reaction may be performed after coupling of the double-stranded adapters and the template molecule. The ligation reaction may be performed using a ligase (and optionally a polymerase). After the ligation reaction, a double-stranded template-adapter complex is formed, where the double-stranded template-adapter complex comprises, in e.g., 5’ to 3‘ orientation, the adapter, the template molecule, and the additional adapter. As there is only one species of adapters in the reaction, nearly 100% of the resulting library molecules will comprise the desired molecular complex.

[0218] In some cases, after formation of the double-stranded template-adapter complex molecules, deamination is performed. In some cases, the deamination is bisulfite conversion. In some case, the deamination is Enzymatic Methyl-sequencing (EM-seq) conversion. As a result of deamination, unmethylated cytosines in a double-stranded template-adapter complex are converted to uracils, in both the template and adapter sequences. After the deamination reaction, a double-stranded template-adapter complex is converted into two single-stranded template-adapter complexes, where the single-stranded template-adapter complexes comprise the converted first sequence (e.g., seql-converted) disposed at the first end of the template molecule, the converted template molecule, and the converted second sequence (e.g., seq2- converted) disposed at the second end of the template molecule. The single-stranded template-adapter complexes arise as a result of the deamination reaction due to the decrease in complementarity between the top and bottom strands of a double-stranded templateadapter complex molecule. That is, the top and bottom strands disassociate or denature from each other as a result of unmethylated cytosines being converted to uracils (e.g., seql- converted is not complementary’ to seq2-converted).

[0219] In some cases, after deamination the single-stranded template-adapter complex molecules are amplified. In some cases, the amplification is performed with an additional set of adapters (e.g., conversion sequences). The first additional adapter comprises adapter sequence Pl and an overhang sequence Ol, where O1 has complementarity to seq2- converted. The second additional adapter comprises adapter sequence P2 and an overhang sequence 02, where 02 has complementarity to seql-converted. In some cases, the amplification reaction results in template-double-adapter molecules comprising Pl, seq2- converted, template, seql-converted, and P2. [0220] In some cases, the unconverted first sequence (e.g., seql) comprises one or more unmethylated cytosines. In some cases, seq2 may comprise one or more unmethylated cytosines. In some cases, seql comprises one or more unmethylated cytosines while seq2 does not comprise unmethylated cytosines. In some cases, seq2 comprises one or more unmethylated cytosines and seql does not comprise unmethylated cytosines. In some cases, the one or more unmethylated cytosines are disposed at a 3’ end of the unconverted first sequence and/or the 3’ end of the second unconverted sequence. In some cases, the template- double-adapter molecules are further analyzed after amplification (e.g.. sequencing reaction(s) are performed).

[0221] FIG. 11B illustrates exemplary sequences of adapters during the stages of library preparation in accordance with the method of FIG. 11A. In FIG. 11B, the adapter sequences comprise unmethylated cytosines. After deamination, these unmethylated cytosines will be converted to uracils, thereby reducing hybridization between the first and second strands of these adapters. FIG. 11C illustrates exemplary partially single-stranded adapter sequences for use in library preparation in accordance with the method of FIG 11A. In FIG. 11C, the adapter sequences comprise 5-methylated cytosines.

[0222] Example adapter sequences for methylation-based library preparation, which may be used as described herein (e.g., seql and seq2), are provided in Table 1. Multiple different adapter pairs, where the top strand and the bottom strand have sequence complementarity can be used. For instance, SEQ ID NO: 5 may be used as seql in conjunction with any one of SEQ ID NOs: 12, 14, and 18 as seq2. As another example, SEQ ID NO: 1 and SEQ ID NO: 9 have sequence complementarity and may be used together as an adapter pair.

[0223] Table 2 includes sequences of the adapter molecules from Table 1 after deamination of the double-stranded template-adapter molecules, where each row in Table 1 corresponds to the same row in Table 2 (e.g., SEQ ID NO: 20 is the deaminated sequence of SEQ ID NO: 1). In some instances, this deamination is performed by bisulfite treatment or by EM-seq. For library conversion (e.g., where the attachment of additional adapter sequences to the library molecules is desired), additional sequences may be disposed 5’ of the primer sequences (e.g., additional adapter sequences).

[0224] For some methods, library molecules may need to be uniquely identifiable. For instance, adapters may further comprise UMls, barcodes, or other unique sequences. One example for such an adapter construct, for instance with reference to FIG. 11 A, would have a unique sequence disposed at the 3' end of seql. Table 1. Adapter sequences for methylation sequencing. C refers to 5 -methylcytosine residues.

Table 2. Adapter sequences from Table 1 post-deamination.

End repair and library preparation for methylation sequencing

[0225] Cell-free DNA is an important diagnostic tool for real time, non-invasive monitoring. In particular, sequencing of cfDNA enables MRD for cancer and other disease. However, cfDNA is inherently fragmentary: there can be a large range of fragment sizes, and doublestranded molecules may require end repair (e.g., to fill in overhangs). The process of end repair relies upon the sequence information from only one strand of a double-stranded molecule. Thus, sequencing information from the ends of end-repaired cfDNA molecules may be less accurate than sequencing information from other loci. There is thus an interest in identifying and filtering out end-repaired sequence information.

[0226] FIGs. 12A and 12B illustrate methods for performing end repair of template molecules, where FIG. 12A provides a schematic and FIG. 12B illustrates a template nucleic acid sequence. One or more template nucleic acid molecules are provided (e.g., from a biological sample). A template molecule may comprise a first strand 1202 and a second strand 1204. One or more cytosines in the first and second strand may be methylated (see FIG. 12B). In step 1220 the template molecule may be end-repaired, where the overhang of the first strand 1202 is filled in with region 1206 on the second strand. Any end repair mechanism known in the art may be used. For example, a polymerase may be used to extend the second strand (e.g., by providing a reverse complement for the overhang of the first strand). End repair may be performed with methylated cytosines. That is, any guanines in the overhang of the first strand will be paired with methylated cytosines/ Thus, end repaired regions may be identified after sequencing (e.g., by the locations of methylated cytosines not in CpG sites). In step 1222, one or more adapters 1208 may be added to the template molecule. In some cases, a single species of adapters may be provided (e.g., methylation adapters such as those illustrated in FIGs. 11A-11C and Tables 1-2 or another suitable adapter species). In some cases, multiple adapter species may be provided. In some cases, adapters may be provided instead after deamination. In step 1224, deamination may be performed (e.g., in preparation for methylation sequencing or methylation site identification). This deamination will convert unmethylated cytosines in the template molecule and the adapters (if present) to uracils (and, after amplification, thymines). The library molecules may be further amplified, sequenced, and/or otherwise processed as described elsewhere herein.

[0227] After sequencing, one or more end-repaired regions may be identified by the presence of cytosines in non-CpG sites (e.g., sites that would be expected to be methylated or, if not methylated, sites that were converted to UG after deamination). In some cases, a sequence read portion corresponding to an identified end-repaired site may be filtered from downstream analysis. This may improve the accuracy of determination of methylation rates and/or the accuracy of calling sites as methylated or unmethylated.

Pre-Sequencing Treatment

[0228] After positive supports are enriched (e.g., isolated from negative supports), whether they comprise single-stranded or double-stranded nucleic acid molecules, the enriched, positive supports may be treated with conditions for stripping and/or conditions for rehybridization of sequencing primers.

[0229] The conditions for stripping may comprise treatment with a denaturing agent, such as sodium hydroxide (NaOH) or ethylene carbonate, heating, and/or a combination thereof. The conditions for re-hybridization of sequencing primers may comprise any conditions for hybridization of nucleic acid molecules. A plurality of sequencing primers may be provided for re-hybridization to the template nucleic acid strands. The stripping and hybndization of sequencing primers may be performed simultaneously (e.g., reagents provided in the same mixture) or separately. The respective conditions for stripping and hybridization may be provided to a reaction space comprising the template nucleic acid molecules simultaneously or separately.

[0230] The enriched, positive supports may be treated to stripping conditions on the substrate, i.e., after loading on the substrate. Alternatively, the enriched, positives supports may be treated to stripping conditions off the substrate, i.e., prior to loading on the substrate. The enriched, positive supports may be treated to rehybridization conditions on the substrate, i.e., after loading on the substrate. Alternatively, the enriched, positives supports may be treated to rehybridization conditions off the substrate, i.e., prior to loading on the substrate, such that when they are loaded, the enriched, positive supports comprise sequencing primers re-hybridized to template strands thereon.

[0231] In some cases, the amplified nucleic acid molecules may be treated with multiple rounds of conditions for stripping, conditions for rehybridization, and/or both in any sequence.

[0232] It was unexpectedly discovered that treating the enriched, amplified nucleic acid molecules (e.g., on the positive supports) with conditions for stripping, conditions for rehybridization of sequencing primers, and/or both, prior to sequencing the amplified nucleic acid molecules resulted in a significant improvement in sequencing quality.

[0233] For example, provided herein is a method for sequencing data generation. The method comprises loading a plurality of beads comprising a plurality of double-stranded template nucleic acid molecules attached thereto, onto a substrate; on the substrate, denaturing the plurality of double-stranded template nucleic acid molecules to generate a plurality of singlestranded template nucleic acid molecules attached to the plurality of beads and hybridizing a plurality of sequencing primers to the plurality of single-stranded template nucleic acid molecules; and generating the sequencing data on the plurality of double-stranded template nucleic acid molecules by extending the plurality of sequencing primers.

[0234] Another method for sequencing data generation may comprise: loading a plurality of beads comprising a plurality of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to the plurality⁷ of single-stranded template nucleic acid molecules; on the substrate, denaturing the plurality of first sequencing primers from the plurality of single-stranded template nucleic acid molecules and re-hybridizing a plurality of second sequencing primers to the plurality of single-stranded template nucleic acid molecules; and generating the sequencing data on the plurality of single-stranded template nucleic acid molecules by extending the plurality of second sequencing primers.

[0235] Another method for sequencing data generation may comprise: loading a plurality of beads comprising a plurality of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to the plurality of single-stranded template nucleic acid molecules; generating a first set of sequencing data on the plurality of single-stranded template nucleic acid molecules byextending the plurality of first sequencing primers; on the substrate, denaturing extension products of the plurality of first sequencing primers from the plurality of single-stranded template nucleic acid molecules and re-hybridizing a plurality of second sequencing primers to the plurality of single-stranded template nucleic acid molecules; and generating a second set of sequencing data on the plurality of single-stranded template nucleic acid molecules by extending the plurality of second sequencing primers.

[0236] In some cases, generating sequencing data comprises performing sequencing-by- synthesis. In some cases, generating sequencing data comprises repeating a plurality of cycles of (i) extending the plurality of sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of sequencing primers to generate the sequencing data.

[0237] The plurality of nucleotides may be non-terminated, reversibly terminated, or a combination thereof. The plurality of nucleotides may be nucleotides of a single base type. [0238] The substrate may be rotated prior to, during, or subsequent to the loading the plurality of beads onto the substrate. The substrate may be rotated prior to, during, or subsequent to the denaturing of the plurality of double-stranded template nucleic acid molecules. The substrate may be rotated prior to, during, or subsequent to the hybridizing of the plurality of sequencing primers to the plurality of single-stranded template nucleic acid molecules. The substrate may be rotated prior to, during, or subsequent to the extending the plurality of sequencing primers.

[0239] In some cases, the denaturing comprises treating the plurality of double-stranded template nucleic acid molecules with sodium hydroxide (NaOH).

[0240] In some cases, the plurality of beads are loaded onto a plurality of individually addressable locations on the substrate. A bead of the plurality of beads may comprise at least 1000 double-stranded template nucleic acid molecules of the plurality of double-stranded template nucleic acid molecules. In some case, the at least 1000 double-stranded template nucleic acid molecules are substantially identical copies.

Computer systems

[0241] The present disclosure provides computer control systems that are programmed to implement methods of the disclosure. FIG. 6 shows a computer system 601 that is programmed or otherwise configured to implement methods of the disclosure, such as to control the systems described herein (e.g., reagent dispensing, detecting, etc.) and collect, receive, and/or analyze sequencing information. The computer system 601 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0242] The computer system 601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 601 also includes memory or memory location 610 (e.g., random-access memory, read-only memory . flash memory'), electronic storage unit 615 (e.g., hard disk), communication interface 620 (e g., network adapter) for communicating with one or more other systems, and peripheral devices 625, such as cache, other memory, data storage and/or electronic display adapters. The memory 610, storage unit 615, interface 620 and peripheral devices 625 are in communication with the CPU 605 through a communication bus (solid lines), such as a motherboard. The storage unit 615 can be a data storage unit (or data repository’) for storing data. The computer system 601 can be operatively coupled to a computer network (“network”) 630 with the aid of the communication interface 620. The network 630 can be the Internet, an isolated or substantially isolated internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 630 in some cases is a telecommunication and/or data network. The network 630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 630, in some cases with the aid of the computer system 601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 601 to behave as a client or a server. The CPU 605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 610. The instructions can be directed to the CPU 605, which can subsequently program or otherwise configure the CPU 605 to implement methods of the present disclosure. Examples of operations performed by the CPU 605 can include fetch, decode, execute, and writeback. The CPU 605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0243] The storage unit 615 can store files, such as drivers, libraries, and saved programs. The storage unit 615 can store user data, e.g., user preferences and user programs. The computer system 601 in some cases can include one or more additional data storage units that are external to the computer system 601, such as located on a remote server that is in communication with the computer system 601 through an intranet or the Internet.

[0244] The computer system 601 can communicate with one or more remote computer systems through the network 630. For instance, the computer system 601 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad. Samsung® Galaxy Tab), telephones. Smart phones (e.g., Apple® iPhone, Android-enabled device. Blackberry®), or personal digital assistants. The user can access the computer system 601 via the network 630.

[0245] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 601, such as, for example, on the memory 610 or electronic storage unit 615. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605. In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610. The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0246] Aspects of the systems and methods provided herein, such as the computer system 601, can be embodied in programming. Various aspects of the technology' may be thought of as “products” or “articles of manufacture” ty pically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine- readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory' (e.g., read-only memory, random-access memory', flash memory') or a hard disk. “Storage” type media can include any or all of the tangible memon of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that cany⁷ such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0247] Hence, a machine-readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory’, such as the main memory’ of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light w aves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a earner wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in cartying one or more sequences of one or more instructions to a processor for execution.

[0248] The computer system 601 can include or be in communication with an electronic display 635 that comprises a user interface (UI) 640 for providing, for example, results of nucleic acid amplification, results of a nucleic acid sequence, an interface for user instructions. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0249] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 605. The algorithm can, for example, execute automation and/or perform sequencing data processing.

NUMBERED EMBODIMENTS

[0250] Embodiment 1 : A method for sequencing data generation, comprising: (a) loading a plurality of beads comprising a plurality of double-stranded template nucleic acid molecules attached thereto, onto a substrate; (b) on said substrate, denaturing said plurality of doublestranded template nucleic acid molecules to generate a plurality of single-stranded template nucleic acid molecules attached to said plurality of beads and hybridizing a plurality of sequencing primers to said plurality of single-stranded template nucleic acid molecules; and (c) generating said sequencing data on said plurality of double-stranded template nucleic acid molecules by extending said plurality of sequencing primers.

[0251] Embodiment : The method of embodiment 1. wherein (c) comprises performing sequencing-by-synthesis.

[0252] Embodiment 3: The method of any one of embodiments 1 -2, wherein (c) comprises repeating a plurality of cycles of (i) extending said plurality of sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of sequencing primers to generate said sequencing data.

[0253] Embodiment 4: The method of embodiment 3, wherein said plurality of nucleotides are non-terminated.

[0254] Embodiment 5: The method of embodiment 3. wherein said plurality of nucleotides are reversibly terminated.

[0255] Embodiment 6: The method of any one of embodiments 3-5, wherein said plurality of nucleotides comprises a mixture of said labeled nucleotides and unlabeled nucleotides. [0256] Embodiment 7: The method of any one of embodiments 3-6, wherein said plurality of nucleotides are nucleotides of a single base type.

[0257] Embodiment 8: The method of any one of embodiments 1-7, wherein said substrate is rotated prior to, during, or subsequent to said loading said plurality of beads onto said substrate.

[0258] Embodiment 9: The method of any one of embodiments 1-8, wherein said substrate is rotated prior to, during, or subsequent to said denaturing said plurality of double-stranded template nucleic acid molecules.

[0259] Embodiment 10: The method of any one of embodiments 1-9. wherein said substrate is rotated prior to, during, or subsequent to said hybridizing said plurality⁷ of sequencing primers to said plurality of single-stranded template nucleic acid molecules.

[0260] Embodiment 11: The method of any one of embodiments 1-10, wherein said substrate is rotated prior to, during, or subsequent to said extending said plurality of sequencing primers.

[0261] Embodiment 12: The method of any one of embodiments 1-11, wherein said denaturing in (b) comprises treating said plurality of double-stranded template nucleic acid molecules with sodium hydroxide (NaOH).

[0262] Embodiment 13: The method of any one of embodiments 1-12, wherein said plurality of beads are loaded onto a plurality of individually addressable locations on said substrate.

[0263] Embodiment 14: The method of any one of embodiments 1-13, wherein a bead of said plurality of beads comprises at least 1000 double-stranded template nucleic acid molecules of said plurality of double-stranded template nucleic acid molecules.

[0264] Embodiment 15: The method of embodiment 14, wherein said at least 1000 doublestranded template nucleic acid molecules are substantially identical copies.

[0265] Embodiment 16: A method for sequencing data generation, comprising: (a) loading a plurality of beads comprising a plurality of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to said plurality⁷ of single-stranded template nucleic acid molecules; (b) on said substrate, denaturing said plurality of first sequencing primers from said plurality of singlestranded template nucleic acid molecules and re-hybridizing a plurality of second sequencing primers to said plurality of single-stranded template nucleic acid molecules; and (c) generating said sequencing data on said plurality of single-stranded template nucleic acid molecules by extending said plurality of second sequencing primers. [0266] Embodiment 17: The method of embodiment 16 wherein (c) comprises performing sequencing-by-synthesis.

[0267] Embodiment 18: The method of any one of embodiments 16-17, wherein (c) comprises repeating a plurality of cycles of (i) extending said plurality of second sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality⁷ of second sequencing primers to generate said sequencing data.

[0268] Embodiment 19: The method of embodiment 18, wherein said plurality of nucleotides are non-terminated.

[0269] Embodiment 20: The method of embodiment 18, wherein said plurality of nucleotides are reversibly terminated.

[0270] Embodiment 21 : The method of any one of embodiments 18-20, wherein said plurality of nucleotides comprises a mixture of said labeled nucleotides and unlabeled nucleotides.

[0271] Embodiment 22: The method of any one of embodiments 18-21, wherein said plurality of nucleotides are nucleotides of a single base type.

[0272] Embodiment 23: The method of any one of embodiments 16-22, wherein said substrate is rotated prior to, during, or subsequent to said loading said plurality of beads onto said substrate.

[0273] Embodiment 24: The method of any one of embodiments 16-23, wherein said substrate is rotated prior to, during, or subsequent to said denaturing said plurality of first sequencing primers from said plurality⁷ of single-stranded template nucleic acid molecules. [0274] Embodiment 25: The method of any one of embodiments 16-24, wherein said substrate is rotated prior to, during, or subsequent to said re-hybridizing said plurality of second sequencing primers to said plurality of single-stranded template nucleic acid molecules.

[0275] Embodiment 26: The method of any one of embodiments 16-25, wherein said substrate is rotated prior to, during, or subsequent to said extending said plurality⁷ of second sequencing primers.

[0276] Embodiment 27: The method of any one of embodiments 16-26, wherein said denaturing in (b) comprises treating said plurality of first sequencing primers hybridized to said plurality of single-stranded template nucleic acid molecules with sodium hydroxide (NaOH). [0277] Embodiment 28: The method of any one of embodiments 16-27, wherein said plurality of beads are loaded onto a plurality of individually addressable locations on said substrate.

[0278] Embodiment 29: The method of any one of embodiments 16-28, wherein a bead of said plurality of beads comprises at least 1000 single-stranded template nucleic acid molecules of said plurality of single-stranded template nucleic acid molecules.

[0279] Embodiment 30: The method of embodiment 29, wherein said at least 1000 singlestranded template nucleic acid molecules are substantially identical copies.

[0280] Embodiment 31 : A method for sequencing data generation, comprising: (a) loading a plurality of beads comprising a plurality of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to said plurality of single-stranded template nucleic acid molecules; (b) generating a first set of sequencing data on said plurality of single-stranded template nucleic acid molecules by extending said plurality of first sequencing primers; (c) on said substrate, denaturing extension products of said plurality of first sequencing primers from said plurality of single-stranded template nucleic acid molecules and re-hybridizing a plurality of second sequencing primers to said plurality of single-stranded template nucleic acid molecules; and (d) generating a second set of sequencing data on said plurality of single-stranded template nucleic acid molecules by extending said plurality of second sequencing primers.

[0281] Embodiment 32: The method of embodiment 31 wherein (d) comprises performing sequencing-by-synthesis.

[0282] Embodiment 33: The method of any one of embodiments 31-32, wherein (d) comprises repeating a plurality’ of cycles of (i) extending said plurality of second sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of second sequencing primers to generate said sequencing data.

[0283] Embodiment 34: The method of embodiment 33, wherein said plurality of nucleotides are non-terminated.

[0284] Embodiment 35: The method of embodiment 33, wherein said plurality of nucleotides are reversibly terminated.

[0285] Embodiment 36: The method of any one of embodiments 33-35, wherein said plurality of nucleotides comprises a mixture of said labeled nucleotides and unlabeled nucleotides. [0286] Embodiment 37: The method of any one of embodiments 33-36, wherein said plurality of nucleotides are nucleotides of a single base type.

[0287] Embodiment 38: The method of any one of embodiments 31-37, wherein said substrate is rotated prior to, during, or subsequent to said loading said plurality of beads onto said substrate.

[0288] Embodiment 39: The method of any one of embodiments 31-38, wherein said substrate is rotated prior to, during, or subsequent to said denaturing said extension products of said plurality of first sequencing primers from said plurality of single-stranded template nucleic acid molecules.

[0289] Embodiment 40: The method of any one of embodiments 31-39, wherein said substrate is rotated prior to, during, or subsequent to said re-hybridizing said plurality of second sequencing primers to said plurality of single-stranded template nucleic acid molecules.

[0290] Embodiment 41: The method of any one of embodiments 31-40, wherein said substrate is rotated prior to, during, or subsequent to said extending said plurality of second sequencing primers.

[0291] Embodiment 42: The method of any one of embodiments 31-41, wherein said denaturing in (c) comprises treating said extension products of said plurality of first sequencing primers hybridized to said plurality of single-stranded template nucleic acid molecules with sodium hydroxide (NaOH).

[0292] Embodiment 43: The method of any one of embodiments 31-42, wherein said plurality of beads are loaded onto a plurality of individually addressable locations on said substrate.

[0293] Embodiment 44: The method of any one of embodiments 31-43, wherein a bead of said plurality of beads comprises at least 1000 single-stranded template nucleic acid molecules of said plurality of single-stranded template nucleic acid molecules.

[0294] Embodiment 45: The method of embodiment 44, wherein said at least 1000 singlestranded template nucleic acid molecules are substantially identical copies.

[0295] Embodiment 46: A method for amplification, comprising: (a) providing a first plurality of supports, each having coupled thereto a plurality of a first type of surface primers; (b) hybridizing a template molecule to a support of the first plurality of supports; (c) amplifying the template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the template molecule; (d) contacting the first amplified support with a substrate, wherein the substrate comprises a second plurality of supports coupled thereto, wherein a support of the second plurality of supports comprises a plurality of a second type of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer; (e) hybridizing one or more copies of the template molecule to a support of the second plurality of supports; (f) and amplifying the one or more copies of the template molecule to provide a second amplified support coupled to the substrate.

[0296] Embodiment 47: The method of embodiment 46, further comprising analyzing the second amplified support to determine a sequence of the template molecule.

[0297] Embodiment 48: The method of embodiment 46, wherein the second amplified support comprises a plurality⁷ of nucleic acid molecules having substantially 100% sequence identity.

[0298] Embodiment 49: The method of embodiment 48, wherein the plurality of nucleic acid molecules are copies of copies of the template molecule.

[0299] Embodiment 50: The method of embodiment 46, wherein the amplify ing (f) is performed in the absence of solution primers.

[0300] Embodiment 51 : A method for amplification, comprising: (a) providing a first plurality of supports, each having coupled thereto a plurality of a first type of surface primers; (b) hybridizing a template molecule to a support of the first plurality of supports; (c) contacting the first plurality⁷ of supports to a substrate, thereby coupling the support to the substrate; (d) amplifying the template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the template molecule; (e) contacting the substrate with a second plurality of supports comprising a plurality of a second ty pe of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer; (f) hybridizing one or more copies of the template molecule to a support of the second plurality of supports; and (g) amplifying the one or more copies of the template molecule to provide a plurality⁷ of copies of the template molecule coupled to the first amplified support.

[0301] Embodiment 52: The method of embodiment 51, further comprising analyzing the plurality of copies of the template molecule to determine a sequence of the template molecule.

[0302] Embodiment 53: The method of embodiment 51, wherein the plurality of copies of the template molecule have substantially 100% sequence identity. [0303] Embodiment 54: The method of embodiment 51, wherein the amplifying (g) is performed in the absence of solution primers.

[0304] Embodiment 55: A method of amplification, comprising: (a) providing a first plurality of supports, each having coupled thereto a plurality of a first type of surface primers; (b) hybridizing and ligating a double-stranded template molecule to a support of the first plurality of supports, wherein a first strand of the double stranded template molecule is ligated to the support and a second strand of the double-stranded template molecule is hybridized to a surface primer of the first type of surface primers; (c) amplifying the doublestranded template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the second strand of the template molecule; (d) contacting the first amplified support with a substrate, wherein the substrate comprises a second plurality of supports coupled thereto, wherein a support of the second plurality of supports comprises a plurality of a second type of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer; (e) hybridizing one or more copies of the second strand of the double-stranded template molecule to a support of the second plurality of supports; and (f) amplifying the one or more copies of the second strand of the double-stranded template molecule and the first strand of the template molecule to provide a second amplified support coupled to the substrate.

[0305] Embodiment 56: The method of embodiment 55, wherein the coupling (c) comprises covalent coupling.

[0306] Embodiment 57: The method of embodiment 55, wherein the second amplified support comprises copies of copies of the second strand of the double-stranded template molecule and copies of the first strand of the template molecule.

[0307] Embodiment 58: The method of embodiment 55, wherein the second amplified support comprises a plurality of nucleic acid molecules having substantially 100% sequence identity.

[0308] Embodiment 59: The method of embodiment 58, wherein at least one nucleic acid molecule does not have 100% sequence identify to the plurality of nucleic acid molecules. [0309] Embodiment 60: The method of embodiment 55, wherein the amplifying (f) is performed in the absence of solution primers.

[0310] Embodiment 61 : The method of embodiment 55, further comprising analyzing the plurality of copies of the template molecule to determine a sequence of the template molecule. [0311] Embodiment 62: A method for post-amplification enrichment, comprising: (a) subsequent to amplification of a plurality of library molecules, receiving a mixture of positive supports and negative supports, wherein each of the positive supports comprises at least one template strand derived from the plurality of library molecules, and wherein each of the negative supports does not comprise a template strand derived from the plurality of library molecules; (b) contacting the mixture with a plurality of desthiobiotinylated sequencing primers to generate a plurality of desthiobiotin-bound complexes, each desthiobiotin-bound complex comprising a positive support of the positive supports, wherein a desthiobiotinylated sequencing primer of the plurality of desthiobiotinylated sequencing primers is hybridized to a template strand of the at least one template strand in the positive support; (c) isolating the desthiobiotin-bound complexes from the negative supports in the mixture by contacting the mixture with (i) a plurality of magnetic beads comprising streptavidin and (ii) a magnet, and eluting; and (d) isolating the positive supports from the plurality of magnetic beads by contacting the isolated desthiobiotin-bound complexes with (i) a plurality’ of biotin moieties and (ii) a magnet, and eluting, wherein the plurality of biotin moieties binds to the plurality of magnetic beads to displace the desthiobiotinylated sequencing primer from the plurality of magnetic beads.

[0312] Embodiment 63: The method of embodiment 62, wherein the contacting in (b) comprises contacting the mixture with a primer mixture, the primer mixture comprising the plurality of desthiobiotinylated sequencing primers and a plurality of sequencing primers. [0313] Embodiment 64: The method of embodiment 63, wherein less than 30% of the primer mixture is the plurality of desthiobiotinylated sequencing primers.

[0314] Embodiment 65: The method of embodiment 64, wherein less than 15% of the primer mixture is the plurality of desthiobiotinylated sequencing primers.

[0315] Embodiment 66: The method of any one of embodiments 62-65, wherein, prior to the contacting in (b). any double-stranded nucleic acid molecules on the positive supports are denatured to generate single-stranded positive supports.

[0316] Embodiment 67: The method of embodiment 66, wherein the double-stranded nucleic acid molecules are denatured via a denaturing agent, heating, or both.

[0317] Embodiment 68: The method of embodiment 67, wherein the denaturing agent comprises sodium hydroxide. [0318] Embodiment 69: The method of any one of embodiments 62-68, further comprising loading the isolated positive supports onto a substrate and sequencing the at least one template strand on the substrate.

[0319] Embodiment 70: The method of embodiment 69, further comprising denaturing any desthiobiotinylated sequencing primers of the plurality of desthiobiotinylated sequencing primers bound to template strands on the positive supports prior to sequencing.

[0320] Embodiment 71: The method of embodiment 70, wherein the denaturing is performed on the substrate.

[0321] Embodiment 72: The method of embodiment 70, wherein the denaturing is performed prior to loading the isolated positive supports on the substrate.

[0322] Embodiment 73: The method of any one of embodiments 69-72, further comprising hybridizing a plurality of sequencing primers to template strands of the at least one template strand prior to sequencing.

[0323] Embodiment 74: The method of embodiment 73, wherein the hybridizing is performed on the substrate.

[0324] Embodiment 75: The method of embodiment 73, wherein the hybridizing is performed prior to loading the isolated positive supports on the substrate.

[0325] Embodiment 76: The method of any one of embodiments 62-75, wherein the positive support comprises a plurality of template strand having substantially 100% sequence identity. [0326] Embodiment 77: The method of any one of embodiments 62-76, wherein two respective template strands on two different positive supports of the positives supports are derived from two different library molecules of the plurality of library molecules.

[0327] Embodiment 78: The method of any one of embodiments 62-77, wherein the positive support comprises a bead.

[0328] Embodiment 79: A centrifugation tube comprising: a fluid; and a phase change material placed on top of the fluid, wherein the phase change material comprises a first phase prior to a centrifugation process and a second phase after a centrifugation process is completed.

[0329] Embodiment 80: The centrifugation tube of embodiment 79, wherein the first phase is a solid phase at room temperature.

[0330] Embodiment 81 : The centrifugation tube of embodiment 79 or 80, wherein the phase change material comprises a liquid phase during or after the centrifugation process. [0331] Embodiment 82: The centrifugation tube of any of embodiments 79-81, wherein the phase change material in a solid phase forms a seal to prevent fluid from evaporating or splashing out of the centrifugation tube.

[0332] Embodiment 83: The centrifugation tube of any of embodiments 79-82, wherein the phase change material comprises one or more of wax or paraffin.

[0333] Embodiment 84: The centrifugation tube of any of embodiments 79-83, wherein the phase change material comprises one or more of cyclooctane or 1,3-diphenylacetone.

[0334] Embodiment 85: The centrifugation tube of any of embodiments 79-84, wherein a density of the phase change material is less than a density of water.

[0335] Embodiment 86: The centrifugation tube of any of embodiments 79-85, wherein the phase change material changes from the first phase to the second phase during the centrifugation process.

[0336] Embodiment 87: The centrifugation tube of any of embodiments 79-85, wherein the phase change material assumes the second phase between completion of 50 % to 100 % of the centrifugation process.

[0337] Embodiment 88: The centrifugation tube of any of embodiments 79-87, wherein the centrifugation tube is configured to be used with a microcentrifuge.

[0338] Embodiment 89: The centrifugation tube of any of embodiments 79-88, wherein the centrifugation tube comprises a 1.5 or a 2 mL centrifugation tube.

[0339] Embodiment 90: The centrifugation tube of any one of embodiments 79-89, wherein the fluid comprises one or more reagents for amplification of nucleic acid molecules.

[0340] Embodiment 91 : A method of centrifugation comprising: placing a phase change material on top of a fluid in a centrifugation tube, wherein the phase change material is in first phase; centrifuging the centrifugation tube comprising the phase change material on top of the fluid to allow (i) the fluid to form a product and a supernatant, and (ii) the phase change material to assume a second phase; and separating the supernatant and phase change material from the product.

[0341] Embodiment 92: The method of embodiment 91, wherein the first phase is a solid phase, at room temperature.

[0342] Embodiment 93: The method of embodiment 91, or 92, wherein the second phase is a liquid phase. [0343] Embodiment 94: The method of any of embodiments 91-93, wherein placing the phase change material on top of the fluid in the centrifugation tube comprises forming a seal to prevent the fluid from evaporating or splashing out of the centrifugation tube.

[0344] Embodiment 95: The method of any of embodiments 91-94, wherein placing the phase change material on top of the fluid in the centrifugation tube comprises placing the phase change material in liquid phase on top of the fluid.

[0345] Embodiment 96: The method of any of embodiments 91-95, comprising cooling the centrifugation tube comprising the phase change material on top of the fluid prior to centrifugation.

[0346] Embodiment 97: The method of any of embodiments 91-96, wherein the phase change material comprises one or more of a wax or paraffin.

[0347] Embodiment 98: The method of any of embodiments 91-97, wherein the phase change material comprises one or more of cyclooctane or 1,3 -diphenylacetone.

[0348] Embodiment 99: The method of any of embodiments 91-98, wherein a density of the phase change material is less than a density of water.

[0349] Embodiment 100: The method of any of embodiments 91-99, wherein the phase change material assumes a second phase between 50 and 100 % completion of the centrifugation.

[0350] Embodiment 101: The method of any of embodiments 91-100, wherein the centrifugation tube is configured to be used with a microcentrifuge.

[0351] Embodiment 102: The method of any of embodiments 91-101, wherein centrifugation tube comprises a 1.5 or a 2 mL centrifugation tube.

[0352] Embodiment 103: The method of any one of embodiments 91-102, wherein: prior to centrifugation, the fluid comprises one or more reagents for amplification of nucleic acid molecules; and after centrifugation, the supernatant comprises the one or more reagents.

[0353] Embodiment 104: A centrifugation tube comprising: a fluid; and a viscous material placed on top of the fluid, wherein the viscous material has a density less than that of the fluid, wherein the viscous material forms a seal to prevent fluid from evaporating or splashing out of the centrifugation tube during centrifugation.

[0354] Embodiment 105: The centrifugation tube of embodiment 104, wherein the viscous material comprises an oil. [0355] Embodiment 106: The centrifugation tube of embodiment 104 or 105, wherein the viscous material comprises one or more of a mineral oil, hydrocarbon, triglyceride, vegetable oil, or liquid silicone.

[0356] Embodiment 107: The centrifugation tube of any of embodiments 104-106. wherein a density of the viscous material is less than a density of water.

[0357] Embodiment 108: The centrifugation tube of any of embodiments 104-107, wherein the centrifugation tube is configured to be used with a microcentrifuge.

[0358] Embodiment 109: The centrifugation tube of any of embodiments 104-108. wherein centrifugation tube comprises one or more of a 1.5 or a 2 mL centrifugation tube.

[0359] Embodiment 110: A method of centrifugation comprising: placing a viscous material on top of a fluid in a centrifugation tube, wherein the viscous material at room temperature is a lower density than the density of the fluid, and the viscous material is not chemically reactive, and wherein the centrifugation tube is not physically capped; centrifuging the centrifugation tube comprising the viscous material on top of the fluid to allow the fluid to form a product and a supernatant; and separating the supernatant and liquid phase change material from the product.

[0360] Embodiment 111: The method of embodiment 110, wherein the viscous material forms a seal to prevent fluid from evaporating or splashing out of the centrifugation tube. [0361] Embodiment 112: The method of embodiment 110 or 111, wherein the viscous material comprises an oil.

[0362] Embodiment 113: The method of any of embodiments 110-112. wherein the viscous material comprises one or more of a mineral oil, hydrocarbon, triglyceride, vegetable oil, or liquid silicone.

[0363] Embodiment 114: The method of any of embodiments 110-113, wherein a density of the viscous material is less than a density' of water.

[0364] Embodiment 115: The method of any of embodiments 110-114. wherein the centrifugation tube is configured to be used with a microcentrifuge.

[0365] Embodiment 116: The method of any of embodiments 110-115, wherein centrifugation tube comprises one or more of a 1.5 or a 2 mL centrifugation tube.

[0366] Embodiment 117: A method of amplification, comprising: (a) providing a support comprising a first plurality of primers and a second plurality of primers, wherein a first primer of the first plurality' of primers comprising a first sequence and is coupled directly to a primer attachment site of the support and wherein a second primer of the second plurality of primers comprises a second sequence and is coupled to a primer attachment site of the support via a tether; (b) hybridizing a template molecule to the first primer and extending the first primer to generate a first extended strand coupled to the support; and (c) hybridizing the second primer to the first extended strand and extending the second primer to generate a second extended strand couple to the support, wherein the second extended strand comprises a sequence capable of hybridizing to another primer of the first plurality of primers.

[0367] Embodiment 118: The method of embodiment 117, wherein the first and second sequences do not have sequence complementarity.

[0368] Embodiment 119: The method of embodiment 117, further comprising repeating (b) and (c) to generate an amplified support comprising a first plurality of molecules that are copies of the first extended strand, wherein each of the first plurality of molecules is coupled to the amplified support via a first primer.

[0369] Embodiment 120: The method of embodiment 119, wherein the amplified support further comprises a second plurality of molecules that are copies of the second extended strand, wherein each of the second plurality of molecules is coupled to the amplified support via a second primer.

[0370] Embodiment 121: The method of embodiment 117, wherein the second primer comprises a cleavage site comprising one or more cleavable moieties.

[0371] Embodiment 122: The method of embodiment 121, further comprising cleaving the cleavage site and removing the second extended strand from the support.

[0372] Embodiment 123: The method of embodiment 117, wherein the support comprises a polymer mesh, wherein a polymer of the polymer mesh comprises a primer attachment site. [0373] Embodiment 124: The method of embodiment 123, wherein the primer attachment site comprises a coupling moiety.

[0374] Embodiment 125: The method of embodiment 123 or embodiment 124. wherein the primer attachment site comprises one of a click chemistry pair.

[0375] Embodiment 126: The method of embodiment 125, wherein the primer attachment site comprises a cyclooctyne.

[0376] Embodiment 127: A method of post-amplification enrichment, comprising (a) receiving an emulsion amplification product mixture comprising a plurality of positive supports, a plurality of negative supports, and a plurality of solution amplification primers: (b) subjecting the amplified product mixture to conditions sufficient to break the emulsion, and providing one or more oligos to hybridize to one or more support-coupled primers in the amplification product mixture; (c) providing a plurality of single stranded binding proteins to bind to the plurality of solution amplification primers, and removing the plurality' of single stranded binding proteins; and (d) isolating the plurality of positive supports from the plurality of negative supports.

[0377] Embodiment 128: The method of embodiment 127, wherein the conditions sufficient to break the emulsion in (b) comprising, heating, agitation, the application of electrostatic force, or a combination thereof.

[0378] Embodiment 129: The method of embodiment 127, wherein the one or more oligos hybridize to at least 25% of the support-coupled primers.

[0379] Embodiment 130: The method of embodiment 129, wherein at least one support- coupled primer is coupled to a positive support.

[0380] Embodiment 131: The method of embodiment 129, wherein at least one support- coupled primer is couple to a negative support.

[0381] Embodiment 132: The method of embodiment 127, wherein the plurality of single stranded binding proteins bind to at least 25% of the plurality' of solution amplification primers.

[0382] Embodiment 133: The method of embodiment 127, wherein the emulsion amplification product mixture is a result of the amplification of a plurality of library molecules.

[0383] Embodiment 134: The method of embodiment 133, wherein each of the positive supports comprises at least one template strand derived from the plurality of library’ molecules, and wherein each of the negative supports does not comprise a template strand derived from the plurality of library molecules.

[0384] Embodiment 135: The method of embodiment 134, wherein the isolating (d) comprises: (e) contacting the amplification product mixture with a plurality of desthiobiotinylated sequencing primers to generate a plurality of desthiobiotin-bound complexes, each desthiobiotin-bound complex comprising a positive support of the positive supports, wherein a desthiobiotinylated sequencing primer of the plurality of desthiobiotinylated sequencing primers is hybridized to a template strand of the at least one template strand in the positive support; (1) isolating the desthiobiotin-bound complexes from the negative supports in the amplification product mixture by contacting the amplification product mixture with (i) a plurality' of magnetic beads comprising streptavidin and (ii) a magnet, and eluting; and (g) isolating the positive supports from the plurality' of magnetic beads by contacting the isolated desthiobiotin-bound complexes with (i) a plurality of biotin moieties and (ii) a magnet, and eluting, wherein the plurality of biotin moi eties binds to the plurality of magnetic beads to displace the desthiobiotinylated sequencing primer from the plurality of magnetic beads.

EXAMPLES

[0385] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

Example 1: Sequencing data for different pre-sequencing treatment protocols

[0386] Sequencing quality metrics for sequencing runs performed with samples treated according to different pre-sequencing treatment protocols, as described elsewhere herein, were collected. The different pre-sequencing treatment protocols were the (1) Control, (2) dsDNA, (3) ssDNA, (4) ssDNA (2^nd). In the Control protocol, beads comprising singlestranded template molecules are pre-hybridized to sequencing primers, loaded to the substrate, and then sequenced. In the dsDNA protocol, beads comprising double-stranded template molecules are loaded to the substrate, second strands stripped from the template molecules and sequencing primers hybridized to the single-stranded template molecules while on the substrate, and the beads are then sequenced. In the ssDNA protocol, beads comprising single-stranded template molecules are pre-hybridized to sequencing primers, beads are loaded to the substrate, the sequencing primers are stripped from and rehybridized to the template molecules on the substrate, and the beads are then sequenced. In the ssDNA (2^nd) protocol, beads comprising single-stranded template molecules are pre-hybridized to sequencing primers, beads are loaded to the substrate, the beads are sequenced for a 1^st time, the sequencing products (e.g., extended sequencing primers) are stripped and sequencing primers are rehybridized to the template molecules, and the beads are sequenced for a 2^nd time (the data shown pertains to this second run). The template molecules were sequenced using flow chemistry, as described elsewhere herein, using non-tenninated, single-base flows. For each of the runs, there was comparable sequencing coverage.

[0387] Table 3 shows for each sequencing run, run type, # of beads (in millions), # of pass filter (PF) reads (in millions), PF% (% of # of PF reads in total # of beads), base error rate for 80% of the reads which are selected based on best RSQ (BER80), indel rate, lag rate, lead rate, droop rate, and misincorporation rate for the T-base. Table 3 - Sequencing Quality Metrics

# PF

R „un „ Type , bead .s read ,s PF% BER Indel Lag ° L „ea .d Droop ' Missin ,g

(M) (M) Rate ^{Rate Rate Rate Rate T} dsDNA 9889 7587 76.7% 0.30 0.36 0.05 0.04 0.24 0.26 dsDNA 9738 7605 78.1% 0.30 0.37 0.05 0.03 0.24 0.23 dsDNA 9195 7047 76.6% 0.28 0.35 0.06 0.04 0.24 0.25 dsDNA 10077 7636 75.8% 0.34 0.42 0.05 0.03 0.26 0.25 dsDNA 10282 7716 75.1% - 0.40 0.05 0.03 0.24 0.22 dsDNA 10316 7538 73.1% - 0.45 0.05 0.03 0.25 0.21 dsDNA 6572 4782 72.8% 0.26 0.36 0.05 0.04 0.24 0.28 dsDNA 9889 7587 76.7% 0.30 0.36 0.05 0.04 0.24 0.26 dsDNA 10432 7495 71.8% 0.38 - 0.06 0.04 0.24 0.22 dsDNA 10500 7640 72.8% 0.37 - 0.05 0.03 0.24 0.23

Ave. 9689 7263 74.9% 0.31 0.38 0.05 0.03 0.24 0.24 ssDNA (2^nd) 8085 5434 67.2% 0.30 0.36 0.05 0.03 0.22 0.21 ssDNA (2^nd) 5661 3729 65.9% 0.32 0.37 0.05 0.03 0.23 0.21 ssDNA (2^nd) 8621 5830 67.6% 0.40 0.45 0.05 0.03 0.27 0.27 ssDNA (2^nd) 6833 4737 69.3% 0.53 0.57 0.05 0.03 0.27 0.28 ssDNA (2^nd) 8169 5650 69.2% 0.49 0.55 0.06 0.04 0.26 0.30 ssDNA (2^nd) 6747 4478 66.4% 0.41 0.47 0.06 0.03 0.25 0.28 ssDNA (2^nd) 7166 4906 68.5% 0.55 0.57 0.06 0.04 0.28 0.31 ssDNA (2^nd) 6380 4282 67.1% 0.43 0.47 0.05 0.04 0.27 0.30 ssDNA 9515 6535 68.7% 0.36 0.43 0.06 0.04 0.26 0.23 ssDNA 8263 5883 71.2% 0.40 0.45 0.06 0.05 0.27 0.24

Ave. 7544 5146 68.1% 0.42 0.47 0.05 0.03 0.26 0.26

Control 9108 6008 66.0% 0.48 0.54 0.08 0.04 0.25 0.63

Control 9826 6685 68.0% 0.63 0.65 0.10 0.05 0.24 0.66

Control 8147 4793 58.8% 0.58 - 0.07 0.05 0.19 0.18

Control 10095 7694 76.2% 0.26 - 0.07 0.03 0.17 0.15

Control 7345 4795 65.3% 0.68 0.59 0.09 0.11 0.25 0.24

Control 6857 4939 72.0% 0.54 0.54 0.06 0.07 0.25 0.32

Ave. 8563 5819 67.7% 0.53 0.58 0.08 0.06 0.23 0.36

[0388] As seen in Table 3, the # of beads, PF% (see FIG. 13 panel C), and droop rate (signal decrease over time) measured for each of dsDNA, ssDNA, and ssDNA (2^nd) treatment protocols were comparable with the Control treatment protocol. Unexpectedly, the sequencing runs with the dsDNA, ssDNA, and ssDNA (2^nd) treatment protocols demonstrated an improvement in phasing quality such as shown by the lag rate and lead rate (see FIG. 13 panel A) and in error quality such as shown by the BER80, indel rate, and misincorporation rate of T nucleotides (see FIG. 13 panel B), compared to the Control treatment protocol. The average lag rate for each of the dsDNA and the two ssDNA treatment protocol runs was 0.05, an unexpected improvement compared to the 0.08 average lag rate for the Control treatment protocol runs. The average lead rate for each of the dsDNA and the two ssDNA treatment protocol runs was 0.03 and an unexpected improvement compared to the 0.06 average lead rate for the Control treatment protocol runs. The average misincorporation rate of thymine for the dsDNA and the two ssDNA treatment protocol runs was 0.24 and 0.26, respectively, an unexpected improvement compared to the 0.36 average thymine misincorporation rate for the Control treatment protocol runs. The average BER80 for the dsDNA and the two ssDNA treatment protocol runs was 0.31 and 0.42, respectively, an unexpected improvement compared to the 0.53 average BER80 for the Control treatment protocol runs. The average indel rate for the dsDNA and the two ssDNA treatment protocol runs was 0.38 and 0.47, respectively, an unexpected improvement compared to the 0.58 average indel rate for the Control treatment protocol runs.

[0389] Table 4 shows the total class error rate for three of the sequencing runs from Table 3, one sequencing run selected from each of the Control, dsDNA, and ssDNA treatment protocol runs for comparison. The total class error rates are broken down into different flow bins to show the error rate progression as the number of sequencing flows progresses in each sequencing run. The total class error rates are indicative of the total error across all four bases called. It can be seen that the total class error rates are higher for the Control treatment protocol run in each flow bin compared to each of the dsDNA and ssDNA treatment protocol runs, demonstrating an unexpected improvement in error quality when sequencing runs follow the dsDNA or ssDNA treatment protocols compared to the Control treatment protocol.

Table 4 - Class Error Rates

Flows

[0390] Table 5 shows the homopolymer base-calling error rate for the same three sequencing runs compared in Table 4. The base-calling error rates are broken down into bins of different homopolymer lengths to show the error rate progression as the length of homopolymers called increases. It can be seen that for up to 8mers (homopolymers of up to 8 bases), the error rates are higher for the Control treatment protocol run compared to each of the dsDNA and ssDNA treatment protocol runs, demonstrating an unexpected improvement in homopolymer base-calling quality when sequencing runs follow the dsDNA or ssDNA treatment protocols. That is, for up to 8-mer homopolymer base-calling, following the dsDNA treatment protocol yielded best results, then the ssDNA treatment protocol, then the Control treatment protocol.

Table 5 - Homopolymer Base-Calling Error Rates H-mer length

[0391] FIG. 14 illustrates a plot of mean signal vs. homopolymer length for the three sequencing runs compared in Tables 4 and 5, showing the plot in panels (A), (B), and (C) for the Control, dsDNA, and ssDNA treatment protocols, respectively. It can be seen that the mean signals for homopolymers were higher for the dsDNA and ssDNA treatment protocol runs compared to the Control treatment protocol, which may be the result of improved homopolymer completion and/or an effect of lower phasing rates demonstrated in the former two treatment protocol runs, as described above.

[0392] While preferred embodiments of the present invention have been show n and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for sequencing data generation, comprising:

(a) loading a plurality⁷ of beads comprising a plurality⁷ of double-stranded template nucleic acid molecules attached thereto, onto a substrate;

(b) on said substrate, denaturing said plurality of double-stranded template nucleic acid molecules to generate a plurality of single-stranded template nucleic acid molecules attached to said plurality⁷ of beads and hybridizing a plurality⁷ of sequencing primers to said plurality⁷ of single-stranded template nucleic acid molecules; and

(c) generating said sequencing data on said plurality⁷ of double-stranded template nucleic acid molecules by extending said plurality⁷ of sequencing primers.

2. The method of claim 1, wherein (c) comprises performing sequencing-by-synthesis.

3. The method of any one of claims 1-2, wherein (c) comprises repeating a plurality of cycles of (i) extending said plurality of sequencing primers using a plurality⁷ of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of sequencing pnmers to generate said sequencing data.

4. The method of claim 3, wherein said plurality of nucleotides are non-terminated.

5. The method of claim 3, wherein said plurality of nucleotides are reversibly terminated.

6. The method of any one of claims 3-5, wherein said plurality of nucleotides comprises a mixture of said labeled nucleotides and unlabeled nucleotides.

7. The method of any one of claims 3-6, wherein said plurality of nucleotides are nucleotides of a single base type.

8. The method of any one of claims 1-7, wherein said substrate is rotated prior to, during, or subsequent to said loading said plurality⁷ of beads onto said substrate.

9. The method of any one of claims 1-8, wherein said substrate is rotated prior to, during, or subsequent to said denaturing said plurality⁷ of double-stranded template nucleic acid molecules.

10. The method of any one of claims 1-9, wherein said substrate is rotated prior to, during, or subsequent to said hybridizing said plurality' of sequencing primers to said plurality of single-stranded template nucleic acid molecules.

11. The method of any one of claims 1-10, wherein said substrate is rotated prior to, during, or subsequent to said extending said plurality of sequencing primers.

12. The method of any one of claims 1-11, wherein said denaturing in (b) comprises treating said plurality of double-stranded template nucleic acid molecules with sodium hydroxide (NaOH).

13. The method of any one of claims 1-12, wherein said plurality of beads are loaded onto a plurality of individually addressable locations on said substrate.

14. The method of any one of claims 1-13, wherein a bead of said plurality of beads comprises at least 1000 double-stranded template nucleic acid molecules of said plurality of double-stranded template nucleic acid molecules.

15. The method of claim 14, wherein said at least 1000 double-stranded template nucleic acid molecules are substantially identical copies.

1 . A method for sequencing data generation, comprising:

(a) loading a plurality of beads comprising a plurality of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to said plurality of single-stranded template nucleic acid molecules;

(b) on said substrate, denaturing said plurality of first sequencing primers from said plurality of single-stranded template nucleic acid molecules and re- hybridizing a plurality of second sequencing primers to said plurality of single-stranded template nucleic acid molecules; and

(c) generating said sequencing data on said plurality of single-stranded template nucleic acid molecules by extending said plurality of second sequencing primers.

17. The method of claim 16 wherein (c) comprises performing sequencing-by-synthesis.

18. The method of any one of claims 16-17, wherein (c) comprises repeating a plurality of cycles of (i) extending said plurality of second sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of second sequencing primers to generate said sequencing data.

19. The method of claim 18, wherein said plurality of nucleotides are non-terminated.

20. The method of claim 18, wherein said plurality of nucleotides are reversibly terminated.

21. The method of any one of claims 18-20, wherein said plurality of nucleotides comprises a mixture of said labeled nucleotides and unlabeled nucleotides.

22. The method of any one of claims 18-21, wherein said plurality of nucleotides are nucleotides of a single base type.

23. The method of any one of claims 16-22, wherein said substrate is rotated prior to, during, or subsequent to said loading said plurality of beads onto said substrate.

24. The method of any one of claims 16-23, wherein said substrate is rotated prior to, during, or subsequent to said denaturing said plurality of first sequencing primers from said plurality of single-stranded template nucleic acid molecules.

25. The method of any one of claims 16-24, wherein said substrate is rotated prior to, during, or subsequent to said re-hybridizing said plurality of second sequencing primers to said plurality of single-stranded template nucleic acid molecules.

26. The method of any one of claims 16-25, wherein said substrate is rotated prior to, during, or subsequent to said extending said plurality of second sequencing primers.

27. The method of any one of claims 16-26, wherein said denaturing in (b) comprises treating said plurality of first sequencing primers hybridized to said plurality of single-stranded template nucleic acid molecules with sodium hydroxide (NaOH).

28. The method of any one of claims 16-27, wherein said plurality of beads are loaded onto a plurality of individually addressable locations on said substrate.

29. The method of any one of claims 16-28, wherein a bead of said plurality of beads comprises at least 1000 single-stranded template nucleic acid molecules of said plurality of single-stranded template nucleic acid molecules.

30. The method of claim 29, wherein said at least 1000 single-stranded template nucleic acid molecules are substantially identical copies.

31. A method for sequencing data generation, comprising: (a) loading a plurality of beads comprising a plurality of single-stranded template nucleic acid molecules attached thereto, onto a substrate, wherein a plurality of first sequencing primers is hybridized to said plurality of single-stranded template nucleic acid molecules;

(b) generating a first set of sequencing data on said plurality’ of single-stranded template nucleic acid molecules by extending said plurality of first sequencing primers;

(c) on said substrate, denaturing extension products of said plurality of first sequencing primers from said plurality of single-stranded template nucleic acid molecules and re-hybridizing a plurality’ of second sequencing primers to said plurality of single-stranded template nucleic acid molecules; and

(d) generating a second set of sequencing data on said plurality of single-stranded template nucleic acid molecules by extending said plurality of second sequencing primers.

32. The method of claim 31 wherein (d) comprises performing sequencing-by-synthesis.

33. The method of any one of claims 31-32, wherein (d) comprises repeating a plurality of cycles of (i) extending said plurality’ of second sequencing primers using a plurality of nucleotides comprising labeled nucleotides in a flow, and (ii) detecting the presence or absence of a labeled nucleotide incorporated into the extending plurality of second sequencing primers to generate said sequencing data.

34. The method of claim 33, wherein said plurality of nucleotides are non-terminated.

35. The method of claim 33, wherein said plurality of nucleotides are reversibly terminated.

36. The method of any one of claims 33-35, wherein said plurality of nucleotides comprises a mixture of said labeled nucleotides and unlabeled nucleotides.

37. The method of any one of claims 33-36, wherein said plurality of nucleotides are nucleotides of a single base type.

38. The method of any one of claims 31-37, wherein said substrate is rotated prior to, during, or subsequent to said loading said plurality of beads onto said substrate.

39. The method of any one of claims 31-38. wherein said substrate is rotated prior to, during, or subsequent to said denaturing said extension products of said plurality of first sequencing primers from said plurality of single-stranded template nucleic acid molecules.

40. The method of any one of claims 31-39, wherein said substrate is rotated prior to, during, or subsequent to said re-hybridizing said plurality⁷ of second sequencing primers to said plurality of single-stranded template nucleic acid molecules.

41. The method of any one of claims 31-40, wherein said substrate is rotated prior to, during, or subsequent to said extending said plurality of second sequencing primers.

42. The method of any one of claims 31-41, wherein said denaturing in (c) comprises treating said extension products of said plurality of first sequencing primers hybridized to said plurality^ of single-stranded template nucleic acid molecules with sodium hydroxide (NaOH).

43. The method of any' one of claims 31-42, wherein said plurality of beads are loaded onto a plurality⁷ of individually addressable locations on said substrate.

44. The method of any one of claims 31-43, wherein a bead of said plurality' of beads comprises at least 1000 single-stranded template nucleic acid molecules of said plurality of single-stranded template nucleic acid molecules.

45. The method of claim 44, wherein said at least 1000 single-stranded template nucleic acid molecules are substantially identical copies.

46. A method for amplification, comprising:

(a) providing a first plurality of supports, each having coupled thereto a plurality of a first type of surface primers;

(b) hybridizing a template molecule to a support of the first plurality⁷ of supports;

(c) amplifying the template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the template molecule;

(d) contacting the first amplified support with a substrate, wherein the substrate comprises a second plurality of supports coupled thereto, wherein a support of the second plurality of supports comprises a plurality of a second type of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer; (e) hybridizing one or more copies of the template molecule to a support of the second plurality of supports;

(!) and amplifying the one or more copies of the template molecule to provide a second amplified support coupled to the substrate.

47. The method of claim 46, further comprising analyzing the second amplified support to determine a sequence of the template molecule.

48. The method of claim 46, wherein the second amplified support comprises a plurality of nucleic acid molecules having substantially 100% sequence identity.

49. The method of claim 46. wherein the plurality of nucleic acid molecules are copies of copies of the template molecule.

50. The method of claim 46, wherein the amplifying (f) is performed in the absence of solution primers.

51. A method for amplification, comprising:

(b) hybridizing a template molecule to a support of the first plurality of supports;

(c) contacting the first plurality of supports to a substrate, thereby coupling the support to the substrate;

(d) amplifying the template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the template molecule;

(e) contacting the substrate with a second plurality of supports comprising a plurality of a second type of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer;

(f) hybridizing one or more copies of the template molecule to a support of the second plurality of supports; and

(g) ampli Tying the one or more copies of the template molecule to provide a plurality of copies of the template molecule coupled to the first amplified support.

52. The method of claim 51, further comprising analyzing the plurality of copies of the template molecule to determine a sequence of the template molecule.

53. The method of claim 51, wherein the plurality of copies of the template molecule have substantially 100% sequence identity.

54. The method of claim 51, wherein the amplifying (g) is performed in the absence of solution primers.

55. A method of amplification, comprising:

(b) hybridizing and ligating a double-stranded template molecule to a support of the first plurality of supports, wherein a first strand of the double stranded template molecule is ligated to the support and a second strand of the doublestranded template molecule is hybridized to a surface primer of the first type of surface primers;

(c) amplifying the double-stranded template molecule in the absence of solution primers, providing a first amplified support coupled to one or more of copies of the second strand of the template molecule;

(d) contacting the first amplified support with a substrate, wherein the substrate comprises a second plurality of supports coupled thereto, wherein a support of the second plurality' of supports comprises a plurality of a second ty pe of surface primers coupled thereto, wherein the second type of surface primer is different from the first type of surface primer;

(e) hybridizing one or more copies of the second strand of the double-stranded template molecule to a support of the second plurality of supports; and

(f) amplifying the one or more copies of the second strand of the double-stranded template molecule and the first strand of the template molecule to provide a second amplified support coupled to the substrate.

56. The method of claim 55, wherein the coupling (c) comprises covalent coupling.

57. The method of claim 55, wherein the second amplified support comprises copies of copies of the second strand of the double-stranded template molecule and copies of the first strand of the template molecule.

58. The method of claim 55. wherein the second amplified support comprises a plurality of nucleic acid molecules having substantially 100% sequence identity.

59. The method of claim 55, wherein at least one nucleic acid molecule does not have 100% sequence identity to the plurality' of nucleic acid molecules.

60. The method of claim 55, wherein the amplifying (f) is performed in the absence of solution primers.

61. The method of claim 55, further comprising analyzing the plurality of copies of the template molecule to determine a sequence of the template molecule.

62. A method for post-amplification enrichment, comprising:

(a) subsequent to amplification of a plurality of library molecules, receiving a mixture of positive supports and negative supports, wherein each of the positive supports comprises at least one template strand derived from the plurality' of library' molecules, and yvherein each of the negative supports does not comprise a template strand derived from the plurality' of library molecules;

(b) contacting the mixture with a plurality of desthiobiotinylated sequencing primers to generate a plurality of desthiobiotin-bound complexes, each desthiobiotin-bound complex comprising a positive support of the positive supports, wherein a desthiobiotinylated sequencing primer of the plurality of desthiobiotinylated sequencing primers is hybridized to a template strand of the at least one template strand in the positive support:

(c) isolating the desthiobiotin-bound complexes from the negative supports in the mixture by contacting the mixture yvith (i) a plurality of magnetic beads comprising streptavidin and (ii) a magnet, and eluting: and

(d) isolating the positive supports from the plurality of magnetic beads bycontacting the isolated desthiobiotin-bound complexes yvith (i) a plurality of biotin moieties and (ii) a magnet, and eluting, yvherein the plurality of biotin moieties binds to the plurality of magnetic beads to displace the desthiobiotinylated sequencing primer from the plurality of magnetic beads.

63. The method of claim 62, wherein the contacting in (b) comprises contacting the mixture yvith a primer mixture, the primer mixture comprising the plurality of desthiobiotinylated sequencing primers and a plurality of sequencing primers.

64. The method of claim 63. wherein less than 30% of the primer mixture is the plurality of desthiobiotinylated sequencing primers.

65. The method of claim 64, wherein less than 15% of the primer mixture is the plurality of desthiobiotinylated sequencing primers.

66. The method of any one of claims 62-64, wherein, prior to the contacting in (b), any double-stranded nucleic acid molecules on the positive supports are denatured to generate single-stranded positive supports.

67. The method of claim 66, wherein the double-stranded nucleic acid molecules are denatured via a denaturing agent, heating, or both.

68. The method of claim 67, wherein the denaturing agent comprises sodium hydroxide.

69. The method of any one of claims 62-68, further comprising loading the isolated positive supports onto a substrate and sequencing the at least one template strand on the substrate.

70. The method of claim 69. further comprising denaturing any desthiobiotinylated sequencing primers of the plurality of desthiobiotinylated sequencing primers bound to template strands on the positive supports prior to sequencing.

71. The method of claim 70, wherein the denaturing is performed on the substrate.

72. The method of claim 70, wherein the denaturing is performed prior to loading the isolated positive supports on the substrate.

73. The method of any one of claims 69-72, further comprising hybridizing a plurality of sequencing primers to template strands of the at least one template strand prior to sequencing.

74. The method of claim 73, wherein the hybridizing is performed on the substrate.

75. The method of claim 73, wherein the hybridizing is performed prior to loading the isolated positive supports on the substrate.

76. The method of any one of claims 62-75, wherein the positive support comprises a plurality' of template strand having substantially 100% sequence identity.

77. The method of any one of claims 62-76, wherein two respective template strands on two different positive supports of the positives supports are derived from two different library molecules of the plurality of library molecules.

78. The method of any one of claims 62-77, wherein the positive support comprises a bead.

79. A method of amplification, comprising:

(a) providing a support comprising a first plurality of primers and a second plurality of primers, wherein a first primer of the first plurality of primers comprising a first sequence and is coupled directly to a primer attachment site of the support and wherein a second primer of the second plurality of primers comprises a second sequence and is coupled to a primer attachment site of the support via a tether;

(b) hybridizing a template molecule to the first primer and extending the first primer to generate a first extended strand coupled to the support; and

(c) hybridizing the second primer to the first extended strand and extending the second primer to generate a second extended strand couple to the support, wherein the second extended strand comprises a sequence capable of hybridizing to another primer of the first plurality of primers.

80. The method of claim 79, wherein the first and second sequences do not have sequence complementarity.

81. The method of claim 79, further comprising repeating (b) and (c) to generate an amplified support comprising a first plurality of molecules that are copies of the first extended strand, wherein each of the first plurality' of molecules is coupled to the amplified support via a first primer.

82. The method of claim 81, wherein the amplified support further comprises a second plurality of molecules that are copies of the second extended strand, wherein each of the second plurality of molecules is coupled to the amplified support via a second primer.

83. The method of claim 79, wherein the second primer comprises a cleavage site comprising one or more cleavable moieties.

84. The method of claim 83, further comprising cleaving the cleavage site and removing the second extended strand from the support.

85. The method of claim 79. wherein the support comprises a polymer mesh, wherein a polymer of the polymer mesh comprises a primer attachment site.

86. The method of claim 85, wherein the primer attachment site comprises a coupling moiety.

87. The method of claim 85 or claim 86, wherein the primer attachment site comprises one of a click chemistry pair.

88. The method of claim 87, wherein the primer attachment site comprises a cyclooctyne.

89. A method of post-amplification enrichment, comprising

(b) subjecting the amplified product mixture to conditions sufficient to break the emulsion, and providing one or more oligos to hybridize to one or more support-coupled primers in the amplification product mixture;

(c) providing a plurality of single stranded binding proteins to bind to the plurality of solution amplification primers, and removing the plurality of single stranded binding proteins; and

(d) isolating the plurality of positive supports from the plurality of negative supports.

90. The method of claim 89, wherein the conditions sufficient to break the emulsion in (b) comprising, heating, agitation, the application of electrostatic force, or a combination thereof.

91. The method of claim 89, wherein the one or more oligos hybridize to at least 25% of the support-coupled primers.

92. The method of claim 91. wherein at least one support-coupled primer is coupled to a positive support.

93. The method of claim 91, wherein at least one support-coupled primer is couple to a negative support.

94. The method of claim 89, wherein the plurality of single stranded binding proteins bind to at least 25% of the plurality of solution amplification primers.

95. The method of claim 89, wherein the emulsion amplification product mixture is a result of the amplification of a plurality of library⁷ molecules.

96. The method of claim 95, wherein each of the positive supports comprises at least one template strand derived from the plurality of library molecules, and wherein each of the negative supports does not comprise a template strand derived from the plurality of library⁷ molecules.

97. The method of claim 96, wherein the isolating (d) comprises:

(e) contacting the amplification product mixture with a plurality of desthiobiotinylated sequencing primers to generate a plurality of desthiobiotinbound complexes, each desthiobiotin-bound complex comprising a positive support of the positive supports, wherein a desthiobiotinylated sequencing primer of the plurality of desthiobiotinylated sequencing primers is hybridized to a template strand of the at least one template strand in the positive support;

(1) isolating the desthiobiotin-bound complexes from the negative supports in the amplification product mixture by contacting the amplification product mixture with (i) a plurality of magnetic beads comprising streptavidin and (ii) a magnet, and eluting; and

(g) isolating the positive supports from the plurality of magnetic beads bycontacting the isolated desthiobiotin-bound complexes with (i) a plurality of biotin moieties and (ii) a magnet, and eluting, wherein the plurality of biotin moieties binds to the plurality of magnetic beads to displace the desthiobiotinylated sequencing primer from the plurality of magnetic beads.