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WO2025010160A1 - Procédés et compositions pour stabiliser des concatémères - Google Patents

Procédés et compositions pour stabiliser des concatémères Download PDF

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Publication number
WO2025010160A1
WO2025010160A1 PCT/US2024/035395 US2024035395W WO2025010160A1 WO 2025010160 A1 WO2025010160 A1 WO 2025010160A1 US 2024035395 W US2024035395 W US 2024035395W WO 2025010160 A1 WO2025010160 A1 WO 2025010160A1
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Prior art keywords
staple
region
sequence
nucleic acid
molecule
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English (en)
Inventor
Kurt PATTERSON
Min-Jui Richard Shen
Brittany Ann ROHRMAN
Andrew Sparks
Evan Hurowitz
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Pacific Biosciences of California Inc
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Pacific Biosciences of California Inc
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Publication of WO2025010160A1 publication Critical patent/WO2025010160A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present application generally relates to molecular biology and more specifically to compositions and methods for enhancing the stability of amplifying and sequencing nucleic acids, as well as their methods of use.
  • a method of forming stabilized concatemers includes: (a) providing a plurality of concatemers, wherein individual concatemers include multiple instances of a target sequence and multiple instances of at least one adapter sequence; (b) contacting the plurality of concatemers with staple molecules to form a plurality of stabilized concatemers; wherein individual staple molecules include at least a first region and a second region; and wherein the first region and the second region of staple molecules each hybridize to a different instance of the at least one adapter sequence.
  • Methods of the subject application may further include any of the below aspects:
  • step (a) includes rolling circle amplification (RCA) with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template includes the target sequence and the at least one adapter sequence.
  • RCA rolling circle amplification
  • A4. The method of aspect A3, wherein the linear nucleic acid template includes a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • A5. The method of aspect A4, wherein the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  • step (b) further includes hybridizing the staple molecules to the sense strand.
  • A29 The method of aspect A28, wherein the surface is a continuous surface.
  • A34 The method of aspect A33, wherein the 3’ end of the staple molecule hybridizes within 40 nucleotides from the 3’ end of the target sequence.
  • A35 The method of aspect Al, wherein at least one of the first region and the second region of the staple molecule includes a 3’ end of the staple molecule and hybridizes to a primer binding site of the at least one adapter sequence.
  • A36 The method of aspect Al , wherein at least one of the first region and second region of the staple molecule include a 3’ end that allows for ternary complex formation.
  • A40 The method of aspect A39, wherein the at least one adapter sequence within the sequence unit includes a first adapter sequence 3 ’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • A46 The method of aspect A45, wherein the 3’ end of the staple molecule includes a blocker that prevents nucleotide incorporation.
  • A48 The method of aspect A47, wherein the 3’ end of the staple molecule is capped with a moiety that prevents ternary complex formation.
  • A49 The method of aspect A47, wherein the 3’ end of the staple molecule includes a mismatch and a blocker, optionally wherein the blocker is a 3’ phosphate blocker.
  • A60 The method of aspect A59, wherein the dendrimer includes polyamidoamine (PAM AM).
  • PAM AM polyamidoamine
  • A64 The method of aspect A59, wherein neither the first region nor the second region of the staple molecule includes a 3’ end that allows for ternary complex formation or extension.
  • A65 The method of aspect A50, wherein the spacer is coupled to the 3’ end of the first region.
  • A68 The method of aspect Al, wherein a plurality of staple molecules are hybridized to the same concatemer.
  • A71 The method of aspect A70, wherein the polymer is a PAMAM denrimer.
  • A77 The method of any one of aspects A74 to A76, further including blocking the staple primer to prevent further sequencing from the staple primer.
  • A80 The method of aspect A79, further including sequencing a second portion of the concatemer from the another primer while the staple primer remains hybdridized to the concatemer.
  • A81 The method of any one of aspects A74 to A80, including at least 100 cycles of sequencing.
  • Compsoitions may generally include aspect Bl of a stabilized concatemer, including: (a) a concatemer including multiple instances of a target sequence and multiple instances of at least one adapter sequence; and (b) one or more staple molecules; wherein individual staple molecules include at least a first region and a second region; andwherein the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence.
  • compositions may futher include any of the below aspects:
  • composition of aspect Bl wherein the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template includes the target sequence and the at least one adapter sequence.
  • composition of aspect B2 wherein the circular nucleic acid template is a product of circularization of a linear nucleic acid template including the target sequence and at least one adapter sequence.
  • the linear nucleic acid template includes a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • BIO The composition of aspect B9, wherein the concatemer is attached to a surface subsequent to hybridizing the concatemer with one or more staple molecules.
  • Bl The composition of aspect BIO, wherein the surface is a structured surface.
  • composition of aspect B12, wherein the surface is a structured surface.
  • composition of aspect B2 wherein the concatemer is a sense strand, and the composition further includes a plurality of antisense strands produced from the amplification of the sense strand.
  • Bl 7 The composition of aspect Bl 6, wherein at least some of the one or more staple molecules are one or more staple primers used to amplify the sense strand to produce the plurality of antisense strands.
  • Bl 8. The method of aspect B2, wherein the one or more staple molecules hybridize to the concatemer during RCA.
  • Bl 9. The composition of aspect Bl, wherein individual instances of the at least one adapter sequence includes a primer binding site.
  • UMI unique molecular identifier
  • composition of aspect Bl, wherein instances of the target sequence include a sense sequence or an antisense sequence.
  • composition of aspect Bl, wherein at least one of the first region and the second region of the staple molecule includes a 3’ end of the staple molecule and hybridizes to a primer binding site of the at least one adapter sequence.
  • composition of aspect Bl, wherein neither the first region nor the second region of the staple molecule includes a 3’ end that allows for ternary complex formation or extension.
  • composition of aspect B39, wherein the at least one adapter sequence within the sequence unit includes a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • composition of aspect B40, wherein at least one of the first region and the second region of the staple molecule includes a 3’ end of the staple molecule and hybridizes to the 3 ’ adapter.
  • composition of aspect Bl wherein the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence that are spaced at least 100 nucleotides apart.
  • B45 The composition of aspect Bl, wherein the 3’ end of the staple molecule includes at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation.
  • composition of aspect B45, wherein the 3’ end of the staple molecule includes a blocker that prevents nucleotide incorporation.
  • composition of aspect B47, wherein the 3’ end of the staple molecule includes a mismatch and a blocker, optionally wherein the blocker is a 3’ phosphate blocker.
  • [140] B50 The composition of aspect Bl, wherein the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • composition of aspect B59, wherein neither the first region nor the second region of the staple molecule includes a 3’ end that allows for ternary complex formation or extension.
  • a method of sequencing may genererally include aspect Cl of A method of sequencing a target sequence, the method including: (i) providing the plurality of stabilized concatemers of any one of aspects B1-B68 or by any of the methods of A1-A68; and (ii) sequencing at least a first portion of the target sequence.
  • Methods of sequencing may further include any of the below aspects:
  • sequencing step (ii) includes sequencing by binding (SBB) using the staple molecule as a staple primer.
  • SBB includes cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes including the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • C28 The method of aspect Cl, wherein the average increase in FWHM of the stabilized concatemers after at least 100 sequencing cycles, as measured in at least one dimension, is reduced at least 5-fold by the staple molecules.
  • C29 The method of aspect Cl, wherein the stabilize concatemer is sequenced at a read length of greater than 150 cycles.
  • Kits may generally include aspect DI to a kit that includes the staple molecules of any one of aspects 1-166.
  • Kits may further include any of the below aspects:
  • kit of aspect DI further including one or more of the following: (i) one or more staple primers, (ii) one or more non-staple primers, (iii) one or more adapters, (iv) one or more polymerases, (v) one or more ligases, (vi) one or more splint oligonucleotides, (vii) a flow cell, (viii) a plurality of labeled nucleotides, (ix) a plurality of reversibly terminated nucleotides, (x) a capping moiety, or any combination thereof.
  • a kit including: (i) a first adapter, (ii) a second adapter, and (iii) a staple molecule, wherein the first and the second region of the staple molecule each hybridize to a different instance of te first and/or the second adapter.
  • kit of aspect El further including: (iv) reagents sufficient to form a stabilized concatemer from a target nucleic acid, wherein the stabilized concatemer includes the first adapter, the second adapter, and the staple molecule.
  • Fig. 1 shows a schematic of a nucleic acid cluster on a flow cell surface and a schematic of a plurality of staple molecules binding to two adapter sequences of a nucleic acid cluster.
  • FIG. 2 shows a schematic of staple molecules (with and without a polynucleotide spacer) binding to two separate instances of an “A” adapter sequence of a nucleic acid cluster.
  • Fig. 3 shows a schematic of staple molecules (with and without a polynucleotide spacer) that are mismatched and blocked at the 3’ end binding to two separate instances of an “P” adapter sequence of a nucleic acid cluster.
  • Fig. 4 shows a schematic of a set of two staple molecules, wherein a first region of the first and second staple molecules are designed to bind to each other, thereby forming a doublestranded region of DNA, and a second region of the first and second staple molecules are designed to bind to separate instances of an “A” adapter sequence of a nucleic acid cluster.
  • Fig. 5 shows a schematic of a set of two staple molecules, wherein a first region of the first and second staple molecules are designed to bind to each other, thereby forming a doublestranded region of DNA, wherein the double-stranded region is elongated by the inclusion of a polynucleotide spacer, and a second region of the first and second staple molecules are designed to bind to separate instances of an “A” adapter sequence of a nucleic acid cluster.
  • Fig. 6 shows a schematic of a plurality of staple molecules bound to a non-DNA linker and their interaction with multiple “P” adapter sequences of a nucleic acid cluster, and schematics of two exemplary non-DNA linkers.
  • Fig. 7 shows a schematic of a nucleic acid cluster and a schematic of a mixture of staple molecules (with and without polynucleotide spacers) binding to an “A” adapter sequence and a “P” adapter sequence of a nucleic acid cluster.
  • Fig. 8 shows exam images of a concatemer sequencing experiment.
  • the left column shows exam images after the first cycle of sequencing and the right column shows exam images after the last (177 th ) cycle of sequencing.
  • the top row shows exam images of concatemer stabilized with staple molecules (“+Staples”) and the bottom row shows exam images of concatemers that were not stabilized with staple molecules (“No Staples”).
  • Fig. 9 shows the results of a sequencing experiment wherein one array of concatemers included staple molecules (left column; “+Staples”) and the other array omitted staple molecules (right column; “SOP”).
  • the top row shows the 50 th percentile of raw intensity values for each exam (A-T-G-C) collected over 177 cycles of sequencing versus the observed background (or “off intensity”) averaged over the full lane of the flow cell (“ON/OFF P50”).
  • the middle row shows graphs of the average amount of fluorescence of a pixel cluster for each exam in the column direction (“column FWHM”).
  • the bottom row shows graphs of the average amount of fluorescence of a pixel cluster for each exam in the row direction (“row FWHM”).
  • Fig. 9 shows the results of a sequencing experiment wherein one array of concatemers included staple molecules (left column; “+Staples”) and the other array omitted staple molecules (right column; “SOP”).
  • the top row shows the 50 th percentile of raw intensity values
  • FIG. 10 shows four swaths of tiles collected during sequencing of concatemers stabilized with staple molecules (left column; “+Staples”) versus concatemers that were not stabilized (right column; “No Staples”).
  • the top row indicates the total number of individual concatemers sequenced in each tile.
  • the average read length (out of 177 cycles) of the concatemers within each tile for the 50 th percentile is shown in the middle row.
  • the average read length of the concatemers within each tile for the 25 th percentile is shown in the bottom row.
  • Fig. 11 shows the results of a single-end RCA clustering/sequencing experiment employing concatemers stabilized with staple molecules, where the stabilized concatemers were conjugated to different fluorophores. The 25 th percentile read lengths and 90% accurate O-score values are shown for the various fluorophore configurations.
  • Fig. 12 shows the results of a paired-end RCA clustering/sequencing experiment demonstrating the feasibility of reading dual-indices on paired-end concatemers stabilized with staple molecules.
  • Fig. 13 shows a schematic of buffer exchange during RCA.
  • Fig. 14 shows an embodiment of the paired-end sequencing workflow beginning at the end of the paired-end RCA clustering workflow.
  • Fig. 15 shows the clonality of stabilized concatemers at different seeding densities.
  • Fig. 16 shows the FWHM of stabilized concatemers compared to concatemers not stabilized with staple molecules, allowing the comparison of concatemer sizes.
  • nucleotide can be used to refer to a native nucleotide or analog thereof.
  • examples include, but are not limited to, nucleotide triphosphates (NTPs) such as ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs), or nonnatural analogs thereof such as dideoxyribonucleotide triphosphates (ddNTPs) or reversibly terminated nucleotide triphosphates (rtNTPs).
  • NTPs nucleotide triphosphates
  • rNTPs ribonucleotide triphosphates
  • dNTPs deoxyribonucleotide triphosphates
  • rtNTPs nonnatural analogs thereof such as dideoxyribonucleotide triphosphates (ddNTPs) or reversibly terminated nucleotide triphosphates (rtNTPs).
  • template means a nucleic acid, or portion thereof, having a sequence of nucleotide bases that act as a guide for producing a complementary copy of the sequence.
  • a template can be copied via extension of a primer that is hybridized at or adjacent to the template. Extension can be mediated by a polymerase or ligase.
  • a template can include, or can be, DNA, RNA, or analogs thereof.
  • a template can include a linear nucleic acid template, a circular nucleic acid template, or a linear nucleic acid template that is circularized.
  • the term “concatemer,” when used in reference to a nucleic acid molecule, means a continuous nucleic acid molecule that contains multiple copies of a common sequence linked in series, such as, for example, multiple copies of a target sequence and one or more adapter sequences linked in series.
  • the term “concatemer,” when used in reference to a nucleotide sequence means a continuous nucleotide sequence that contains multiple copies of a common sequence in series.
  • Each copy of the sequence can be referred to as a “sequence unit” of the concatemer.
  • a sequence unit can have a length of at least 10 bases, 50 bases, 100 bases, 250 bases, 500 bases or more.
  • a concatemer can include at least 2, 5, 10, 50, 100 or more sequence units.
  • a sequence unit can include subregions having any of a variety of functions such as an adapter sequence region, primer binding region, target sequence region, tag region, unique molecular identifier (UMI), or the like.
  • the term “common sequence” means a sequence of nucleotides that is the same for two or more nucleic acid molecules.
  • the sequence that is common to two or more nucleic acids can include all or part of the nucleic acids that are being compared.
  • the common sequence can have a length of at least 5, 10, 25, 50, 100, 250, 500, 1000 or more nucleotides. Alternatively, or additionally, the length can be at most 1000, 500, 250, 100, 50, 25, 10, or 5 nucleotides.
  • a population of nucleic acid molecules can include individual molecules that have a region of common sequence between the individuals (e.g., a ‘universal primer’ or “universal primer binding site’) and a region of variable sequence that differs from one individual to another (e.g., a ‘target region’).
  • a region of common sequence between the individuals e.g., a ‘universal primer’ or “universal primer binding site’
  • a region of variable sequence that differs from one individual to another e.g., a ‘target region’
  • sense and antisense are used herein to distinguish members of a pair of complementary nucleic acid molecules or sequences. The terms are intended as context specific identifiers. The terms are interchangeable in accordance with their use in the art of molecular biology. As such, a strand that is identified as a “sense strand” in one context can be referred to as an “antisense” strand in a second context. This is independent of how similar or different the first context is compared to the second context.
  • the term “circular,” when used in reference to a nucleic acid strand, means that the strand has no terminus (that is, the strand lacks a 3’ end and a 5’ end). Accordingly, the 3’ oxygen and the 5’ phosphate moi eties of every nucleotide monomer in a circular strand are covalently attached to an adjacent nucleotide monomer in the strand.
  • a circular DNA strand can serve as a template for producing a concatemeric amplicon via rolling circle amplification (RCA), wherein each sequence unit of the concatemeric amplicon is the reverse complement of the circular nucleic acid strand.
  • a circular nucleic acid can be double stranded.
  • One or both strands in a double stranded nucleic acid can lack a 3’ end and a 5’ end.
  • One strand in a double stranded nucleic acid can have a gap (absence of at least one nucleotide monomer relative to the other strand) or nick (absence of a phosphodiester bond between two nucleotide monomers), so long as the other strand is circular.
  • polymerase can be used to refer to a nucleic acid synthesizing enzyme, including but not limited to, DNA polymerase, RNA polymerase, reverse transcriptase, primase, and transferase.
  • the polymerase has one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization may occur.
  • the polymerase may catalyze the polymerization of nucleotides to the 3’ end of the first strand of the double stranded nucleic acid molecule.
  • a polymerase catalyzes the addition of a next correct nucleotide to the 3’ oxygen group of the first strand of the double stranded nucleic acid molecule via a phosphodi ester bond, thereby covalently incorporating the nucleotide to the first strand of the double stranded nucleic acid molecule.
  • a polymerase need not be capable of nucleotide incorporation under one or more conditions used in a method set forth herein.
  • a mutant polymerase may be capable of forming a ternary complex but incapable of catalyzing nucleotide incorporation.
  • a polymerase can have strand displacement activity, such as Phi29.
  • a polymerase can lack strand displacement activity.
  • a polymerase can have 5' 3' exonuclease activity.
  • a polymerase can lack 5' 3' exonuclease activity.
  • the term “adapter sequence” refers to a known synthetic sequence of nucleic acids. Adapter sequences can act as starting points for reading bases for a number of positions beyond each adapter sequence-target sequence junction, and optionally bases can be read in both directions from the adapter sequence. Adapter sequences can be engineered so as to comprise one or more of the following: 1) a length of about 10 to about 100 nucleotides, 2) features so as to be ligated to the 5’ end and/or the 3’ end of a target sequence, 3) different and distinct anchor binding sites at the 5’ end and/or the 3’ end of the adapter sequence for use in sequencing of adjacent target sequences, and 4) optionally one or more restriction sites.
  • the adapter sequences to which the first and second regions of the staple molecules hybridize can be identical (e g., the first and second regions of a staple molecule hybridize to different instances of the same adapter sequence in the concatemer), adapter sequences that are distinct from each other, or adapter sequences that are at least partially conserved in sequence.
  • the staple molecule comprises a primer sequence on it’s 3’ end that hybridizes to a primer binding site of the concatemer, and a tail sequence on its 5’ end that hybridizes to an adapter of the concatemer.
  • staple molecule may be referred to herein as a “staple primer”.
  • staple molecules can further comprise without limitation one or more of the following components: a reversible terminator moiety, a blocker or blocking moiety, a cap or capping moiety, a double-stranded nucleic acid region, optionally at least one nucleotide mismatch in the double-stranded portion, a 3’ phosphate blocker, a spacer between the first and second regions, or any combination thereof.
  • the spacer can be a polynucleotide sequence or a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker can be a branched poly electrolyte species, such as polyethylene glycol or a dendrimer, such as poly(amidoamine).
  • at least one of the first region and second region of the staple molecule may further comprise one or more of the following components: a 3’ end that allows for extension, a 3’ end that allows for ternary complex formation, a 3’ end that is reversibly terminated, or any combination thereof.
  • primer-template nucleic acid hybrid refers to a nucleic acid having a double stranded region such that one of the strands is a primer and the other strand is a template.
  • the two strands can be parts of a contiguous nucleic acid molecule (e g., a hairpin structure) or the two strands can be separable molecules that are not covalently attached to each other.
  • the term “cluster,” when used in reference to nucleic acids, refers to a population of nucleic acids that is attached to a solid support, for example, at a site in an array of sites on the solid support.
  • the term “clonal population” refers to a population of nucleic acids that is homogeneous with respect to a particular nucleic acid sequence. The homogenous sequence is typically at least 10 nucleotides long, but can be even longer including for example, at least 50, 100, 500, 1000 or 2500 nucleotides long.
  • a clonal population can be derived from a single template nucleic acid.
  • a clonal population can include at least 2, 10, 100, 1000 or more copies of a particular nucleic acid sequence.
  • branched polyelectrolytes include what are generally referred to as “dendrimers” that are repeatedly branched, roughly spherical three-dimensional molecules with nanometer-scale dimensions.
  • Dendrimer species can comprise controlled terminal surface chemistry with one or more functional groups that include, but are not limited to, amines, carboxyl, and hydroxyl groups.
  • Branched polyamine that comprises a protonated structure that interacts and forms complexes with the negatively charged backbone of DNA.
  • adaptor elements may still be employed with branched polyamines in the embodiments described herein for alternative purposes or to provide improved binding characteristics for the dendrimer species to the nucleic acid.
  • the branched poly electrolyte is a poly(amidoamine) dendrimer species (also referred to as PAMAM), for example, a G2 PAMAM dendrimer molecule with 16 branches having the amine (NH2) terminal surface chemistry.
  • PAMAM poly(amidoamine) dendrimer species
  • Non-limiting examples of branched polyelectrolyte also include G4 (64 branches with the amine terminal group) and G5 (128 branches with the amine terminal group) PAMAM dendrimer species.
  • blocking moiety when used in reference to a nucleotide, means a part of the nucleotide that inhibits or prevents the 3’ oxygen of the nucleotide from forming a covalent linkage to a next correct nucleotide during a nucleic acid polymerization reaction.
  • the blocking moiety of a “reversibly terminated” nucleotide can be removed from the nucleotide analog, or otherwise modified, to allow the 3 ’-oxygen of the nucleotide to covalently link to a next correct nucleotide.
  • Such a blocking moiety is referred to herein as a “reversible terminator moiety.”
  • reversible terminator moieties are set forth in U.S. Pat Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or 8,034,923; or PCT publications WO 91/06678 or WO 07/123744, each of which is incorporated herein by reference.
  • a nucleotide that has a blocking moiety or reversible terminator moiety can be at the 3’ end of a nucleic acid, such as a primer, or the nucleotide can be a monomer that is not covalently attached to a nucleic acid.
  • the term “capping moiety,” when used in reference to a nucleic acid, means a moiety that when present in a nucleic acid hinders or precludes the 3’ end of the nucleic acid from binding to a polymerase and next correct nucleotide to form a ternary complex.
  • Moieties that create a steric block to ternary complex formation are particularly useful and include, for example, a polymerization or ligation product that extends a primer to the end of a template to which the primer is hybridized.
  • Another example of a steric block is a mismatched nucleotide.
  • a capping moiety can have a positive or negative charge that hinders or prevents ternary complex formation.
  • the term “array” refers to a population of molecules that is attached to one or more solid support such that the molecules can be distinguished from each other.
  • An array can include different molecules that are each located at different addressable sites on a solid support.
  • An array can include separate solid supports each functioning as a site that bears a different molecule, wherein the different molecules can be identified according to the locations of the solid supports on a surface to which the solid supports are attached, or according to the locations of the solid supports in a liquid such as a fluid stream.
  • the molecules of the array can be, for example, nucleotides, nucleic acid primers, nucleic acid templates or nucleic acid enzymes such as polymerases, ligases, exonucleases, or combinations thereof.
  • the term “site,” when used in reference to an array, means a location in the array where a particular molecule is present.
  • a site can contain only a single molecule, or it can contain a population of several molecules of the same species (an ensemble of the molecules).
  • a site can include a population of molecules that are different species (e g., a population of ternary complexes having different template sequences).
  • Sites of an array are typically discrete. The discrete sites can be contiguous, or they can have interstitial spaces between each other.
  • An array useful herein can have, for example, sites that are separated by less than 100 microns, 50 microns, 10 microns, 5 microns, 1 micron, or 0.5 micron.
  • an array can have sites that are separated by greater than 0.5 micron, 1 micron, 5 microns, 10 microns, 50 microns or 100 microns.
  • the sites can each have an area of less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron or less.
  • a site may also be referred to as a “feature” of an array.
  • Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, cyclic olefins, polyimides etc ), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers.
  • plastics including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, cyclic olefins, polyimides etc
  • nylon ceramics
  • resins Zeonor
  • silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers
  • a “vessel” is a container that functions to isolate one chemical process (e.g., a binding event; an incorporation reaction; etc.) from another, or to provide a space in which a chemical process can take place.
  • Examples of vessels useful in connection with the disclosed technique include, but are not limited to, flow cells, wells of a multi-well plate; microscope slides; tubes (e.g., capillary tubes); droplets, vesicles, test tubes, trays, centrifuge tubes, features in an array, tubing, channels in a substrate etc.
  • cycle when used in reference to a sequencing procedure, refers to the portion of a sequencing run that is repeated to indicate the presence of a nucleotide. Typically, a cycle includes several steps such as steps for delivery of reagents, washing away unreacted reagents and detecting signals indicative of changes occurring in response to added reagents.
  • the term “deblock” means to remove or modify a reversible terminator moiety of a nucleotide to render the nucleotide extendable.
  • the nucleotide can be present at the 3’ end of a primer such that deblocking renders the primer extendable.
  • Exemplary deblocking reagents and methods are set forth in U.S. Pat Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or 8,034,923; or PCT publications WO 91/06678 or WO 07/123744, each of which is incorporated herein by reference.
  • exogenous when used in reference to a moiety of a molecule, means a chemical moiety that is not present in a natural analog of the molecule.
  • an exogenous label of a nucleotide is a label that is not present on a naturally occurring nucleotide.
  • an exogenous label that is present on a polymerase is not found on the polymerase in its native milieu.
  • extension when used in reference to a nucleic acid, means a process of adding at least one nucleotide to the 3’ end of the nucleic acid.
  • polymerase extension when used in reference to a nucleic acid, refers to a polymerase catalyzed process of adding at least one nucleotide to the 3’ end of the nucleic acid.
  • a nucleotide or oligonucleotide that is added to a nucleic acid by extension is said to be incorporated into the nucleic acid.
  • incorporating can be used to refer to the process of joining a nucleotide or oligonucleotide to the 3’ end of a nucleic acid by formation of a phosphodiester bond.
  • extendable when used in reference to a nucleotide, means that the nucleotide has an oxygen or hydroxyl moiety at the 3’ position, and is capable of forming a covalent linkage to a next correct nucleotide.
  • An extendable nucleotide can be at the 3’ position of a polymeric nucleic acid or it can be a monomeric nucleotide.
  • a nucleotide that is extendable will lack blocking moieties such as reversible terminator moieties.
  • immobilized when used in reference to a molecule, refers to direct or indirect, covalent, or non-covalent attachment of the molecule to a surface such as a surface of a solid support. In some configurations, covalent attachment may be preferred, but generally all that is required is that the molecules (e.g., nucleic acids) remain immobilized or attached to the surface under the conditions in which surface retention is intended.
  • label refers to a molecule, or moiety thereof, that provides a detectable characteristic.
  • the detectable characteristic can be, for example, an optical signal such as absorbance of radiation, fluorescence emission, luminescence emission, fluorescence lifetime, fluorescence polarization, or the like; Rayleigh and/or Mie scattering; binding affinity for a ligand or receptor; magnetic properties; electrical properties; charge; mass; radioactivity or the like.
  • Exemplary labels include, without limitation, a fluorophore, luminophore, chromophore, nanoparticle (e.g., gold, silver, carbon nanotubes), heavy atoms, radioactive isotope, mass label, charge label, spin label, receptor, ligand, or the like.
  • next correct nucleotide refers to the nucleotide or nucleotide type that will bind and/or incorporate at the 3’ end of a primer to complement a base in a template strand to which the primer is hybridized.
  • the base in the template strand is referred to as the “next base” and is immediately 5’ of the base in the template that is hybridized to the 3’ end of the primer.
  • the next correct nucleotide can be referred to as the “cognate” of the next base and vice versa.
  • Cognate nucleotides that interact with each other in a ternary complex or in a double stranded nucleic acid are said to “pair” with each other.
  • a nucleotide having a base that is not complementary to the next template base is referred to as an “incorrect”, “mismatch” or “non-cognate” nucleotide.
  • non-catalytic metal ion refers to a metal ion that, when in the presence of a polymerase enzyme, does not facilitate phosphodiester bond formation needed for chemical incorporation of a nucleotide into a primer.
  • a non-catalytic metal ion may interact with a polymerase, for example, via competitive binding compared to catalytic metal ions. Accordingly, a non-catalytic metal ion can act as an inhibitory metal ion.
  • a “divalent non- catalytic metal ion” is a non-catalytic metal ion having a valence of two.
  • divalent non-catalytic metal ions examples include, but are not limited to, Ca2+, Zn2+, Co2+, Ni2+, and Sr2+.
  • the trivalent Eu3+ and Tb3+ions are non-catalytic metal ions having a valence of three.
  • ternary complex refers to an intermolecular association between a polymerase, a double-stranded nucleic acid, and a nucleotide.
  • the polymerase facilitates interaction between a next correct nucleotide and a template strand of a primed nucleic acid.
  • a next correct nucleotide can interact with the template strand via Watson- Crick hydrogen bonding.
  • stabilized ternary complex means a ternary complex having promoted or prolonged existence or a ternary complex for which disruption has been inhibited. Generally, stabilization of the ternary complex prevents covalent incorporation of the nucleotide component of the ternary complex into the primed nucleic acid component of the ternary complex.
  • Staple molecules specifically interact and hybridize with concatemers, and this structural interaction serves to stabilize the concatemers, which further serves to ease downstream processes, including without limitation, loading of concatemers onto arrays and utilizing the stabilized concatemers in applications such as sequencing reactions.
  • concatemers are continuous nucleic acid molecule that contain multiple copies of a common sequence.
  • concatemers of use in the aspects and embodiments discussed herein contain repeating copies of a target sequence and one or more adapter sequences.
  • concatemers increase in volume, they can become increasingly difficult to load onto a surface such as an array, and often such concatemers may, in applications such as sequencing or other reactions that utilize signaling molecules, further exhibit a reduction in signal intensity.
  • These issues that can arise with concatemers are often referred to as the concatemers becoming destabilized or unstable. Additives used to compact or stabilize concatemers can mitigate these issues. However, many additives become gradually washed away from the concatemers during downstream processes such as sequencing reactions, because such additives often rely on non-specific interactions with the concatemers for their stabilizing influence.
  • staple molecules described herein are configured such that the sequences of the first and second region of the staple molecules hybridize to adapter sequences within the concatemer. As a result, regions of the concatemer are drawn toward each other in various directions, compacting and otherwise stabilizing the structure of the overall molecule.
  • the methods and compositions described herein increase the compactness of a concatemer by linking different parts of the concatemer sequence across the three-dimensional structure of that concatemer molecule using staple molecules.
  • the methods and compositions of the invention inhibit the molecular dispersion of concatemers during sequencing.
  • staple oligonuclotides i.e., staple molecules comprising an oligonucleotide sequence as described herein
  • staple oligonucleotides are bound, directly or indirectly, to two or more regions of a concatemer.
  • staple oligonucleotides can be hybridized directly to two or more adaptor regions, can be hybridized to each other, and/or can be bound to an intermediate.
  • one portion of a staple oligonucleotide (e.g., one end of the staple oligonucleotide) can hybridize to a single-stranded portion of the first adapter region while another portion of the staple oligonucleotide (e.g., its other end) can hybridize to a single-stranded portion of the second adapter region or to a single-stranded portion of another instance of the first adaptor region.
  • portions of staple oligonucleotides may hybridize to each other or to an intermediate.
  • portions of two or more staple oligonucleotides may each hybridize to a same single intermediate oligonucleotide or to different intermediate oligonucleotides presented by a particle. Such hybridization can bridge different fragments, keeping the fragments resulting from a given concatemer tightly clustered.
  • Staple oligonucleotides can be functionalized (e.g., at their 3’ and/or 5’ ends) for cross-linking to each other or thorough an intermediate.
  • biotinylated staple oligonucleotides can be cross-linked through streptavidin (e.g., through addition of streptavidin after hybridizing staple oligonucleotides to first and/or second adaptor regions).
  • staple oligonucleotides can be functionalized with a click chemistry group, such as a strain-promoted click chemistry group, may be cross-linked through a multivalent intermediate presenting multiple instances of the complementary click chemistry partner.
  • a click chemistry group such as a strain-promoted click chemistry group
  • One exemplary strain- promoted click chemistry partners include dibenzocyclooctyne (DBCO) and azide, and another is Trans-Cyclooctene (TCO) and tetrazine, and derivatives thereof.
  • DBCO- functionalized staple oligonucleotides could be cross linked by a dendrimer presenting a plurality of azides.
  • a single oligonucleotide can optionally serve as both a primer and a staple oligonucleotide.
  • the 5' end of the first sequencing primer can hybridize to the second adapter region while the 3' end hybridizes to the first adapter region (such that the first sequencing primer also serves as a staple oligonucleotide), and/or the 5' end of the sequencing primer can hybridize to the first adapter region while the 3' end hybridizes to the second adapter region (such that sequencing primer also serves as a staple oligonucleotide).
  • the portion of the adapter regions complementary to the sequencing and staple portions of such primers will generally not overlap each other so that the primers are not competing for their binding sites.
  • the present disclosure provides methods of forming stabilized concatemers.
  • concatemers often spread out, become less compact, or “streak”, resulting in a reduction in quality, intensity, and read length during sequencing.
  • the concatemers increase in volume and exhibit a reduction in signal intensity, they are said to destabilize or become unstable.
  • Additives used to compact or “stabilize” concatemers can mitigate these issues. However, these additives are gradually washed out of the concatemers during sequencing due to their non-specific interactions with the concatemers.
  • stabilized concatemers are concatemers that are resistant to molecular diffusion and spectral bleeding due to the hybridization of staple molecules with concatemers.
  • staple molecules specifically interact with concatemers, and are thus resistant to being washed out of concatemers during sequencing. Additionally, staple molecules facilitate a stabilization of concatemers by bridging segments of a concatemer, causing a reduction in the volume of a concatemer and an increase in signal intensity.
  • any aspects and embodiments of the methods of forming stabilized concatemers described herein can utilize any of the aspects and/or embodiments of the compositions described below and can further be used in any of the methods of use also described herein.
  • the method of making concatemers can include without limitation the following steps: 1) isolation and processing of nucleic acids, 2) attaching adapter sequence(s) to the nucleic acids and circularizing the template, 3) performing rolling circle amplification (RCA) to create a single stranded concatemer (a first strand or “sense strand”), and optionally 4) performing multiple displacement amplification (MDA) to create a plurality of seconds strands (“antisense strands”) of the first (“sense”) strand of the concatemer.
  • RCA rolling circle amplification
  • MDA multiple displacement amplification
  • Nucleic acids can be isolated using methods known in the art including, for example, those described in Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference.
  • Nucleic acids can be fragmented using methods known in the art including, for example, 1) physical methods, such as, for example, acoustic shearing, sonication, or hydrodynamic shearing, or 2) enzymatic methods, such as, for example, DNase I digestion, restriction endonucleases, or transposases, or 3) chemical fragmentation methods, such as, for example, chemical shearing using heat and divalent metal cation(s). Selection of nucleic acids based on ideal fragment length can be performed using methods known in the art including, for example, gel electrophoresis or bead-based size selection. The aforementioned steps lead to the formation of a linear nucleic acid template comprising a target sequence.
  • a splint oligonucleotide After ligation of at least one adapter sequences to the linear nucleic acid template, a splint oligonucleotide hybridizes to the 5’ and 3’ ends of the linear template to form a circular nucleic acid template comprising a target sequence and one or more adapter sequences.
  • a splint oligonucleotide need not be used to ligate the ends, for example, when using CircLigase TM (Epicenter, Madison WI) or other enzyme capable of splint-free ligation of nucleic acid ends.
  • CircLigase TM CircLigase TM (Epicenter, Madison WI) or other enzyme capable of splint-free ligation of nucleic acid ends.
  • An exonuclease can be used to remove remaining linear fragments.
  • RCA can be performed using methods known in the art including, for example, those described in Lizardi et al., Nat. Genet. 19:225-232 (1998).
  • the method involves a polymerase, such as, for example, 029 (phi29) DNA polymerase, extending a primer that is annealed to a circular nucleic acid template such that multiple laps of the polymerase around the circular template produces a concatemeric single stranded nucleic acid (a “sense strand”) that contains multiple tandem repeats, each of the repeats being complementary to the circular nucleic acid template.
  • a polymerase such as, for example, 029 (phi29) DNA polymerase
  • RCA can be performed initially in the presence of a low concentration of a polymer, such as dendrimers (e.g., polyamidoamine (PAMAM)), and subsequently in the presence of a polymer.
  • a polymer such as dendrimers (e.g., polyamidoamine (PAMAM)
  • PAMAM polyamidoamine
  • an RCA reaction is stopped by denaturing the polymerase, for example, by heating the sample at 60°C, 65°C, 70°C, 75°C, 80°C, or more.
  • an RCA reaction is stopped by removing one or more components of RCA, such as the polymerase, and dNTPs. Components of RCA can be removed by, for example, washing.
  • one or more antisense strands can be made by replicating the concatemeric sense strand, for example, using multiple displacement amplification (MDA).
  • MDA multiple displacement amplification
  • MDA can be performed using methods known in the art including, for example, those described in Lizardi et al., Nat. Genet. 19:225-232 (1998). Generally, primers are hybridized to one or more regions of the concatemeric single stranded nucleic acid, and a polymerase, such as, for example, 29 DNA polymerase, will extend the primers annealed to the concatemeric single stranded nucleic acid to produce a plurality of single stranded nucleic acids (a plurality of “antisense strands”).
  • RCA and MDA methods can be carried out isothermally. Generally, the polymerase used for RCA or MDA is a strand displacing polymerase.
  • the RCA and/or MDA reaction can be carried out in the presence of deoxyribose adenosine triphosphate (dATP), deoxyribose thymidine triphosphate (dTTP), deoxyribose guanosine triphosphate (dGTP), and deoxyribose cytidine triphosphate (dCTP) (or analogues thereof).
  • dATP deoxyribose adenosine triphosphate
  • dTTP deoxyribose thymidine triphosphate
  • dGTP deoxyribose guanosine triphosphate
  • dCTP deoxyribose cytidine triphosphate
  • the antisense strands generated during an MDA reaction can include adenine, guanine, cytosine, and thymine bases.
  • the MDA reaction can be carried out in the presence of deoxyribose uridine triphosphate (dUTP) (or
  • the antisense strands generated can include uracil bases (indicated by “stars” in the antisense strand) in addition to adenine, guanine, cytosine, and thymine bases when the MDA reaction is carried out in the presence of dUTP in addition to dATP, dTTP, dGTP, and dCTP.
  • Antisense strands with uridine bases can be digested after the antisense strands are sequenced.
  • the MDA reaction can be carried out in the presence of deoxyribonucleotide triphosphate with a modified base or a non-canonical base such that the antisense strands generated include one or more bases that are modified or non-canonical.
  • modified bases or non-canonical bases can target the antisense strands for degradation, such as enzymatic digestion.
  • the MDA reaction can be carried out in the presence of modified or non-canonical deoxyribonucleotide triphosphate (e.g., deoxy pseudouridine triphosphate) such that the antisense strands generated include one or more nucleotides that are modified or non-canonical (e.g., deoxyribose pseudouridine monophosphate).
  • modified or non-canonical deoxyribonucleotide triphosphate e.g., deoxy pseudouridine triphosphate
  • the antisense strands generated include one or more nucleotides that are modified or non-canonical (e.g., deoxyribose pseudouridine monophosphate).
  • Whether a base in an antisense strand is a thymine or an uracil depends on the relative concentration of dTTP and dUTP in the MDA reaction.
  • the concentration of dUTP (or deoxyribonucleotide trisphosphate with a modified base or a non-canonical base, or deoxyribonucleotide trisphosphate that is modified or non-canonical) in an MDA reaction can be lower than the concentration of another deoxyribose nucleotide triphosphate in the MDA reaction such that the percentage of uracil bases (or modified bases or non-canonical bases or nucleotides that are modified or non-canonical) present in the antisense strand is low.
  • the uracil bases (or modified bases or non-canonical bases or nucleotides that are modified or non-canonical) can be randomly distributed and present at a low percentage such that two antisense strands (any two antisense strands) include uracil bases (or modified bases or non-canonical bases or nucleotides that are modified or non-canonical) at different positions.
  • the concentration of a deoxyribonucleotide triphosphate (e.g., dATP, dTTP, dGTP, or dCTP), or the concentration of all deoxyribonucleotide triphosphates, in a RCA or MDA reaction can be about, be at least, be at least about, be at most, or be at most about, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM,
  • the concentration of dUTP in an MDA reaction can be, be about, be at least, be at least about, be at most, or be at most about, 0.001 mM, 0.002 mM, 0.003 mM, 0.004 mM, 0.005 mM, 0.006 mM, 0.007 mM, 0.008 mM, 0.009 mM, 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10
  • the ratio of the concentration of dUTP (or deoxy ribonucleotide trisphosphate with a modified base or a non- canonical base, or deoxyribonucleotide trisphosphate that is modified or non-canonical) relative to the concentration of dTTP (or the concentration of another deoxyribonucleotide triphosphate or the total concentration of deoxyribonucleotide triphosphate other than dUTP or deoxyribonucleotide trisphosphate with a modified base or a non-canonical base or deoxyribonucleotide trisphosphate that is modified or non-canonical) can be, be about, be at least, be at least about, be at most, or be at most about, 1 : 100, 1 :99, 1 :98, 1 :97, 1 :96, 1 :95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1
  • the percentage of deoxyribonucleotide triphosphates in the MDA reaction that are dUTP can be, be about, be at least about, be at most, or be at most about, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%
  • the concentration of dUTP (or deoxyribonucleotide trisphosphate with a modified base or a non-canonical base, or deoxyribonucleotide trisphosphate that is modified or non-canonical) relative to another deoxyribose nucleotide triphosphate such as dTTP in an MDA reaction can be low such that the percentage of uracil bases (or modified bases or non-canonical bases or deoxyribonucleotides that are modified or non-canonical) present in the antisense strand is low.
  • the ratio of nucleotides with bases that are uracil (or modified bases or non-canonical bases or nucleotides that are modified or non-canonical) relative to the bases that are thymine (or another base, or all bases that are not uracil) can be, be about, be at least, be at least about, be at most, or be at most about, 1:10000, 1:9000, 1:8000, 1:7000, 1:6000, 1:5000, 1:6000, 1:5000, 1:4000, 1:3000, 1:2000, 1:1000, 1:900, 1:800, 1:700, 1:600, 1:500, 1:400, 1:300, 1:200, 1:100, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85, 1:84, 1:83, 1:82,
  • the 5’ and 3’ ends of the linear nucleic acid are ligated so as to circularize the linear nucleic acid.
  • the surface-bound oligo is then subjected to an extension reaction, so as to have added at its 3’ end multiple monomer units of the originally linear nucleic acid via RCA.
  • the result is a concatemer of multimers of the original linear nucleic acid being tethered to the surface.
  • a plurality of second strands can subsequently be synthesized using MDA.
  • a concatemer in solution-phase synthesis, can be completely synthesized in solution and then deposited onto a surface for sequencing, or a concatemer can be partially synthesized in solution and then deposited onto a surface to finalize synthesis of the concatemer for sequencing.
  • a concatemer can be completely synthesized in solution, stabilized with staple molecules in solution, and then deposited onto a surface for sequencing.
  • a concatemer can be completely synthesized in solution, deposited onto a surface, and then be stabilized with staple molecules prior to sequencing.
  • the circular template can be annealed to a surface-bound oligo, and RCA can be performed as described above, optionally followed by MDA.
  • the circular template can be contacted to a primer and RCA can be performed in solution.
  • MDA can be performed to produce a plurality of second strands.
  • the concatemer synthesized in solution can be deposited onto a surface for sequencing.
  • the concatemer can be stabilized using staple molecules and then deposited onto a surface for sequencing.
  • a method of forming stabilized concatemers comprising: (a) providing a plurality of concatemers, wherein individual concatemers comprise multiple instances of a target sequence and multiple instances of at least one adapter sequence; (b) contacting the plurality of concatemers with staple molecules to form a plurality of stabilized concatemers; wherein individual staple molecules comprise at least a first region and a second region; and wherein the first region and the second region of staple molecules each hybridize to a different instance of the at least one adapter sequence.
  • references to “a target sequence” of a concatemer can further encompass a sequence that is reverse and complementary to the target sequence, and that references to “at least one adapter sequence” can further encompass a sequence that is reverse and complementary to the at least one adapter sequence.
  • staple molecules comprise at least a first region and a second region, wherein the first region and the second region of staple molecules each hybridize to a different instance of the at least one adapter sequence of a concatemer.
  • the sequences of the first and second region can be identical, partially conserved, or completely distinct. Further, the sequences of the first and second region can be of the same length, or the first region can be greater than or equal to the length of the second region, or the second region can be greater than or equal to the length of the first region.
  • Staple molecules can further comprise one or more of the following components: a reversible terminator moiety, a blocker or blocking moiety, a cap or capping moiety, a double-stranded nucleic acid region, optionally at least one nucleotide mismatch in the double-stranded portion, a 3’ phosphate blocker, a spacer between the first and second regions, or any combination thereof.
  • the spacer can be a polynucleotide sequence or a non-nucleotide polymer linker.
  • the nonnucleotide polymer linker can be a branched polyelectrolyte species, such as polyethylene glycol or a dendrimer, such as poly(amidoamine).
  • One strand in a double-stranded nucleic acid can have a gap (absence of at least one nucleotide monomer relative to the other strand) or nick (absence of a phosphodiester bond between two nucleotide monomers), so long as the other strand is circular.
  • Any of a variety of polymerases can be used in a method or composition set forth herein. Non-limiting examples of polymerases that may be used include naturally occurring polymerases and modified version thereof, including, but not limited to, mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics, analogs, and the like.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the method further comprises circularizing a linear nucleic acid template comprising the target sequence and the at least one adapter sequence.
  • the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  • a “splint oligonucleotide” is an oligonucleotide that, when hybridized to other polynucleotides, such as, for example, a first adapter sequence, or a second adapter sequence, or a (nucleic acid) template, or a linear (nucleic acid) template, acts as a “splint” to position the polynucleotides next to one another so that they can be ligated together.
  • the splint oligonucleotide is DNA or RNA.
  • the splint oligonucleotide can include a nucleotide sequence that is partially complimentary to nucleotide sequences from two or more different oligonucleotides. In some embodiments, the splint oligonucleotide assists in ligating a “donor” oligonucleotide and an “acceptor” oligonucleotide. In general, an RNA ligase, a DNA ligase, or another other variety of ligase is used to ligate two nucleotide sequences together.
  • the splint oligonucleotide is between 10 and 50 oligonucleotides in length, e.g., between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20 nucleotides in length. In some embodiments, the splint oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and 35, 15 and 30, or 15 and 25 nucleotides in length.
  • a splint oligonucleotide need not be used to ligate the ends, for example, when using CircLigase TM (Epicenter, Madison WI) or other enzyme capable of splint-free ligation of nucleic acid ends.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the method further comprises circularizing a linear nucleic acid template comprising the target sequence and the at least one adapter sequence.
  • the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  • the splint oligonucleotide is the primer hybridized to the circularized nucleic acid.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the method further comprises circularizing a linear nucleic acid template comprising the target sequence and the at least one adapter sequence.
  • the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  • the splint oligonucleotide is removed prior to RCA.
  • the splint oligonucleotide can be removed during or subsequent to RCA.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is immobilized on a surface during RCA.
  • Suitable surfaces include, but are not limited to, a structured surface, a planar substrate, a hydrogel, a nanohole array, a microparticle, a nanoparticle, a flow cell surface, a surface of a solid support, or a surface of a solid support within a flow cell.
  • the surface can be planar or curved.
  • the solid support can be made from any of a variety of materials used for analytical biochemistry.
  • Suitable materials may include, for example, glass, polymeric materials, silicon, quartz (fused silica), borofloat glass, silica, silica- based materials, carbon, metals, an optical fiber or bundle of optical fibers, sapphire, or plastic materials.
  • the material can be selected based on properties desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will utilize radiation of that wavelength. Conversely, it may be desirable to select a material that does not pass radiation of a certain wavelength (e.g., being opaque, absorptive, or reflective). Wavelength regions that may be pass or not pass through a particular material include, for example, UV, VIS (e.g., red, yellow, green, or blue) or IR. Other properties of a material that can be exploited are inertness or reactivity to certain reagents used in a downstream process, such as those set forth herein, or ease of manipulation, or low cost of manufacture.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the method further comprises depositing the plurality of stabilized concatemers on a surface subsequent to contacting step (b).
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the method further comprises depositing the plurality of stabilized concatemers on a surface subsequent to contacting step (b). In even further embodiments, the surface is a structured surface.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the method further comprises depositing the plurality of concatemers on a surface prior to contacting step (b).
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the method further comprises depositing the plurality of concatemers on a surface prior to contacting step (b). In even further embodiments, the surface is a structured surface.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • RCA produces a sense strand
  • the method further comprises amplifying the sense strand to produce a plurality of antisense strands.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • RCA produces a sense strand
  • the method further comprises amplifying the sense strand to produce a plurality of antisense strands.
  • at least some of the staple molecules are hybridized to adapter sequences of the plurality of antisense strands.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • RCA produces a sense strand
  • the method further comprises amplifying the sense strand to produce a plurality of antisense strands.
  • step (b) further comprises hybridizing the staple molecules to the sense strand.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • RCA produces a sense strand
  • the method further comprises amplifying the sense strand to produce a plurality of antisense strands.
  • step (b) further comprises hybridizing the staple molecules to the sense strand.
  • at least some of the staple molecules is a staple primer used to amplify the sense strand to produce a plurality of antisense strands.
  • the providing a plurality of concatemers of step (a) comprises RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • contacting step (b) occurs during RCA step (a).
  • individual instances of the at least one adapter sequence comprises a primer binding site.
  • individual instances of the at least one adapter sequence comprises a primer binding site.
  • individual instances of the at least one adapter sequence further comprise a tag region, optionally wherein the tag region is a sample index.
  • individual instances of the at least one adapter sequence comprises a primer binding site. In further embodiments, individual instances of the at least one adapter sequence further comprise a splint binding site. [301] In some embodiments, individual instances of the at least one adapter sequence comprises a primer binding site. In further embodiments, individual instances of the at least one adapter sequence further comprise a variable region, optionally wherein the variable region is a unique molecular identifier (UMI).
  • UMIs are sequences of nucleotides applied to or identified in polynucleotides that may be used to distinguish individual nucleic acid molecules that are present in an initial reaction from one another. In some cases, the UMI may comprise from about 5 to about 20 nucleotides.
  • the UMI may comprise less than about 5 or more than 20 nucleotides.
  • the UMI may be a unique sequence that varies across individual nucleic acid molecules. In some cases, the UMI may be a random sequence. In some cases, the UMI may be a predetermined sequence. In a sequencing reaction, UMIs may be sequenced along with the nucleic acid molecules with which they are associated to determine whether the read sequences are those of one source nucleic acid molecule or another.
  • the term “UMI” is used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide comprising that sequence information. Additional examples of UMIs and uses thereof are provided in, e.g., US 2016/0319345 Al, which is incorporated herein by reference.
  • the individual concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the individual concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the first region and the second region of the staple molecule each hybridize to a different instance of the at least one adapter sequence on a different antisense strand.
  • the individual concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the sense strand does not comprise uracil and the plurality of antisense strands comprises uracil.
  • all instances of the target sequences within the individual concatemers are identical.
  • instances of the target sequence comprise a sense sequence or an antisense sequence.
  • the plurality of concatemers is provided immobilized to a surface in a flow cell.
  • the plurality of concatemers is provided immobilized to a surface in a flow cell.
  • the surface is a continuous surface.
  • the plurality of concatemers is provided immobilized to a surface in a flow cell. In further embodiments, the plurality of concatemers is immobilized to binding sites of a structured surface of the flow cell.
  • the plurality of concatemers is provided in solution in step (a) and deposited on the surface of a flow cell in step (b).
  • sequence of the first region and the second region are identical.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for extension. Extension can be mediated by a polymerase or ligase.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for extension.
  • the 3’ end of the staple molecules hybridizes within 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 10, 5 or fewer nucleotides from the 3’end of the target sequence.
  • the 3’ end of the staple molecule hybridizes within 40 nucleotides from the 3’ end of the target sequence.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for ternary complex formation.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for ternary complex formation.
  • the 3’ end that allows for ternary complex formation is reversibly terminated.
  • Reversible termination can be performed using any reversible terminator moiety.
  • Exemplary reversible terminator moi eties such as, for example, reversible terminator moieties in which the 3 ’-OH group is replaced by a 3’-ONH2 moiety, are set forth in U.S. Pat Nos.
  • neither the first region nor the second region of the staple molecule comprises a 3’ end that allows for ternary complex formation or extension.
  • the plurality of concatemers comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the plurality of concatemers comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the at least one adapter sequence within the sequence unit comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the plurality of concatemers comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the at least one adapter sequence within the sequence unit comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first region of the staple molecule hybridizes to the first adapter sequence and the second region of the staple molecule hybridizes to the second adapter sequence.
  • the plurality of concatemers comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the at least one adapter sequence within the sequence unit comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • at least one of the first region and the second region of the staple molecule comprises a 3’ end of the staple molecule and hybridizes to the 3’ adapter.
  • the length of the first region and the second region of the staple molecule are equal. Alternatively, the length of the first region may be greater than the length of the second region of the staple molecule. In other instances, the length of the second region may be greater than the length of the first region of the staple molecule. In some embodiments, the first region of the staple molecule is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides in length. In some embodiments, the second region of the staple molecule is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides in length.
  • the first region and the second region of the staple molecule are each at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides in length. In an exemplary embodiment, the first region and the second region of the staple molecule are each at least 10 nucleotides in length. [324] In some embodiments, the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence that are spaced at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 or more nucleotides apart. In an exemplary embodiment, the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence that are spaced at least 100 nucleotides apart.
  • the blocking moiety of a “reversibly terminated” nucleotide can be removed from the nucleotide analog, or otherwise modified, to allow the 3 ’-oxygen of the nucleotide to covalently link to a next correct nucleotide.
  • a blocking moiety is referred to herein as a “reversible terminator moiety.”
  • a blocking moiety need not hinder or preclude ternary complex formation at the 3’ end of a nucleic acid to which the blocking moiety is attached.
  • a cap can be any capping moiety.
  • a capping moiety can have a positive or negative charge that hinders or prevents ternary complex formation.
  • a capping moiety can include a ligand that binds a receptor to hinder or prevent ternary complex formation such as a biotin (or analog thereof) that binds to streptavidin (or an analog thereof), an epitope that binds to an antibody (or functional fragment thereof), a carbohydrate that binds to a lectin, or the like. Further examples of capping moieties are described in US Pat. App. Pub. No. 2020/0032322 Al or Turcatti et al. Nucl. Acids. Res. 36(4) e25 (2008), each of which is incorporated herein by reference.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation. [328] In some embodiments, the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation. In yet further embodiments, the 3’ end of the staple molecule is capped with a moiety that prevents ternary complex formation.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation. In yet further embodiments, the 3’ end of the staple molecule comprises a mismatch and a blocker, optionally wherein the blocker is a 3’ phosphate blocker.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • a spacer Any suitable spacer known in the arts can be used.
  • the spacer can comprise a polynucleotide sequence.
  • the spacer can comprise a non-nucleotide polymer linker, such as, for example, a branched polyelectrolyte species.
  • branched poly electrolyte species include polyethylene glycol (PEG) and dendrimers.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer. In further embodiments, at least some of the staple molecules do not comprise a spacer.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides in length.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer is of variable length across different staple molecules.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a nonnucleotide polymer linker.
  • the non-nucleotide polymer linker comprises polyethylene glycol (PEG).
  • the PEG can comprise a PEG with an average molecular weight of about 200 Daltons (e.g., PEG-200) to about 8000 Daltons (e.g., PEG-8000).
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a non- nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • Species of dendrimer that can be used in the methods, compositions and systems disclosed herein includes, but is not limited to a branched polyamine that comprises a protonated structure that interacts and forms complexes with the negatively charged backbone of DNA.
  • Dendrimer species can comprise controlled terminal surface chemistry with one or more functional groups that include, but are not limited to, amines, carboxyl, and hydroxyl groups.
  • the branched polyelectrolyte is a poly(amidoamine) dendrimer species (also referred to as PAMAM), for example, a G2 PAM AM dendrimer molecule with 16 branches having the amine (NH2) terminal surface chemistry.
  • PAMAM poly(amidoamine) dendrimer species
  • Non-limiting examples of branched polyelectrolyte also include G4 (64 branches with the amine terminal group) and G5 (128 branches with the amine terminal group) PAMAM dendrimer species.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a nonnucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the dendrimer comprises poly amidoamine (PAMAM). Any species of PAMAM described above, both those specifically stated and those that are implied by the totality of the disclosure, can be used.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a non- nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the staple molecules hybridize to 3, 4, 5, 6, 7, 8, 9, or 10 or more instances of the at least one adapter.
  • the staple molecules hybridize to 3 or more instances of the at least one adapter.
  • the staple molecule hybridizes to 10 or more instances of the at least one adapter sequence.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a non- nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the staple molecules hybridize to 3 or more instances of the at least one adapter.
  • a majority of the staple molecules hybridize to adapter sequences flanking at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 or more different target sequences.
  • a majority of the staple molecules hybridize to adapter sequences flanking at least 100 different target sequences.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a non- nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • neither the first region nor the second region of the staple molecule comprises a 3’ end that allows for ternary complex formation or extension.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the spacer is coupled to the 5’ end of the second region.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the spacer is coupled to a 3’ end of the second region and wherein the staple molecule cannot act as a primer.
  • a plurality of staple molecules is hybridized to the same concatemer.
  • concatemers often have a structure that tends to spread or smear during sequencing and as a result exhibit a reduction in the quality, intensity, and read length during sequencing of concatemers. Therefore, stabilization of concatemers can be measured using various metrics related to 1) changes in the volume or size of a concatemer, 2) signal intensity and resolution from the concatemer during sequencing, and 3) changes in read length during sequencing of concatemers.
  • individual fields of view are collected and are referred to as tiles.
  • a single row of tiles collected across the lane of a flow cell is referred to as a swatch. Metrics related to stabilization are collected during the sequencing reaction.
  • raw intensity values for each nucleotide exam are collected for all cycles of the sequencing run and is referred to as “ON intensity”. Further, raw intensity values for the observed background, or “OFF intensity”, is collected. The 50 th percentile of raw intensity values for each exam versus the observed background is averaged over the full lane of the flow cell (“ON/OFF intensity”). As a concatemer becomes diffuse, molecular dispersion results in spectral bleeding that interferes with the discrimination between intensity values of the concatemer from the intensity of the background.
  • Sequencing read lengths can be highly varied based on the method of sequencing employed, as well as various other considerations, such as those described above, and others known in the arts. As concatemers become destabilized, the length of a sequencing run should be reduced to maintain the integrity of the sequencing reads. However, this results in a shorter read length that may not capture at least some of variant sequences present. With respect to read length, stabilization can thus be measured by changes in the length of the sequencing run, that is to say, the number of cycles in the sequencing reaction, or by changes in the read length produced.
  • the concatemer may be formed by rolling circle amplification (RCA), such as in solution before deposition on a solid surface or from a primer bound to a solid surface.
  • the primer is a splint primer upon which a template comprising the target and adaptor sequences is circularized prior to RCA.
  • a compaction additive such as PAMAM may be added or increased in concentration.
  • compositions described herein can be used in any of the aspects and/or embodiments of the methods of forming stabilized concatemers described above or in the methods of use described below.
  • Nucleic acids that are used in a method or composition herein can be deoxyribonucleic acids (DNA), such as, for example, genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA), or the like. Additionally, nucleic acids used in a method or composition herein can also be ribonucleic acids (RNA), such as, for example, mRNA, ribosomal RNA, tRNA, or the like. Further, nucleic acids used in a method or composition herein can also be nucleic acid analogs. For example, a nucleic acid analog can be used as a template for an amplification or sequencing process set forth herein.
  • DNA deoxyribonucleic acids
  • cDNA complementary DNA
  • nucleic acids used in a method or composition herein can also be ribonucleic acids (RNA), such as, for example, mRNA, ribosomal RNA, tRNA, or the like.
  • RNA ribonucleic acids
  • Nucleic acids used herein for example, as a template to produce a concatemer or as a target for sequencing, can be derived from a biological source, synthetic source, or amplification product.
  • Primers used herein can include, or can be, DNA, RNA, or analogs thereof.
  • a nucleic acid can be obtained from a preparative method such as genome, transcriptome or other nucleic acid isolation, genome fragmentation, gene cloning and/or amplification.
  • One or more nucleic acids can be obtained from an amplification technique such as polymerase chain reaction (PCR), emulsion PCR, random prime amplification, RCA, MDA, or the like.
  • PCR polymerase chain reaction
  • emulsion PCR random prime amplification
  • RCA and MDA can be particularly useful for producing concatemeric products.
  • Exemplary methods for isolating, amplifying and fragmenting nucleic acids to produce templates for analysis on an array are set forth in US Pat. Nos. 6,355,431 or 9,045,796, each of which is incorporated herein by reference.
  • Amplification can also be carried out using a method set forth in Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference.
  • a nucleic acid template containing a target sequence subject to the methods described herein can be derived or generated from a sample.
  • the sample can include one or more organisms.
  • the nucleic acid template may be obtained or derived from the sample without performing polymerase chain reaction.
  • the nucleic acid template may be obtained or derived from the sample by performing a few cycles of polymerase chain reaction, such as at most one cycle, two cycles, three cycles, four cycles, five cycles, six cycles, seven cycles, eight cycles, nine cycles, or ten cycles or more than ten cycles.
  • Different lengths of nucleic acid templates (or target sequences) are contemplated herein.
  • a nucleic acid can be at least, be at least about, be at most, or be at most about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,
  • Exemplary organisms from which nucleic acids can be derived include, for example, a mammal, such as, for example, a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate, human or non-human primate; a plant, such as, for example, Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola, or soybean; an algae, such as, for example, Chlamydomonas reinhardtii; a nematode, such as, for example, Caenorhabditis elegans; an insect, such as, for example, Drosophila melanogaster, mosquito, fruit fly, or honey bee; an arachnid, such as, for example, a spider; a fish, such as, for example, a zebrafish; a reptile; an amphibian, such as, for example, a mammal,
  • Nucleic acids can also be derived from a prokaryote such as a bacterium, such as, for example, Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an archaea; a virus, such as, for example, Hepatitis C virus or human immunodeficiency virus; or a viroid. Nucleic acids can be derived from a homogeneous culture or population of the above organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
  • a primer refers to a nucleic acid having a sequence that binds to a nucleic acid at or near a template sequence.
  • the primer binds in a configuration that allows replication of the template, for example, via polymerase extension of the primer.
  • staple molecules are configured such that the first region and/or second region of a staple molecule anneals to one or more adapter sequences and then is extended into the target sequence by a polymerase.
  • the staple molecules are said to be or act as “staple primers”.
  • a staple molecule and an “additional primer” are used in one or more of the steps of a given method or composition.
  • an “additional primer” may refer to a primer, as previously defined above, or a separate staple molecule configured to be a staple primer. To delineate embodiments where the additional primer is not a staple primer, the additional primer may be referred to as a “non-staple primer”.
  • primers can have an identical length. In other embodiments, primers can have different lengths.
  • the length of a primer (or two of more primers of a type or population, or each primer of a type or population) can be, be about, be at least, be at least about, be at most, be at most about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
  • the length of a primer (or two of more primers of a type or population, or each primer of a type or population) can be, be about, be at least, be at least about, be at most, be at most about, 100 A, 110 A, 120 A, 130 A, 140 A, 150 A, 160 A, 170 A, 180 A, 190 A, 200 A, 300 A, 400 A, 500 A, 600 A, 700 A, 800 A, 900 A, 1000 A, 0.2 pm, 0.3 pm, 0.4 pm, 0.5 pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, or a number or a range between any two of these values.
  • the ratio of the length of a primer of one type or population (e.g., a capture primer) and the length of a primer of the same type or population (e.g., a capture primer), or the ratio of the length of a primer of one type or population (e.g., a capture primer) and the length of a primer of another type or population (e.g., an amplification primer), can vary.
  • the ratio of the lengths of two primers of one type or population, or the ratio of the length of two primers of different types or populations can be, be about, be at least, be at least about, be at most, or be at most about, 1:100, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85, 1:84, 1:83, 1:82, 1:81, 1:80, 1:79, 1:78, 1:77, 1:76, 1:75, 1:74, 1:73, 1:72, 1:71, 1:70, 1:69, 1:68, 1:67,
  • polymerases can be used in the methods set forth herein, for example, to replicate a nucleic acid template, form a concatemer, form a nucleic acid cluster, form a stabilized concatemer, and the like.
  • Polymerases that may be used include naturally occurring polymerases and modified variations thereof, including, but not limited to, mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics, and analogs.
  • Naturally occurring polymerases and modified variations thereof are not limited to polymerases that have the ability to catalyze a polymerization reaction.
  • the naturally occurring and/or modified variations thereof have the ability to catalyze a polymerization reaction in at least one condition that is not used during formation or examination of a stabilized ternary complex.
  • the naturally occurring and/or modified variations that participate in stabilized ternary complexes have modified properties, for example, enhanced binding affinity to nucleic acids, reduced binding affinity to nucleic acids, enhanced binding affinity to nucleotides, reduced binding affinity to nucleotides, enhanced specificity for next correct nucleotides, reduced specificity for next correct nucleotides, reduced catalysis rates, catalytic inactivity etc.
  • Mutant polymerases include, for example, polymerases wherein one or more amino acids are replaced with other amino acids, or insertions or deletions of one or more amino acids.
  • Exemplary polymerase mutants that can be used to form a stabilized ternary complex include, for example, those set forth in US Pat. App. Ser. No. 15/866,353, published as US Pat. App. Pub.
  • a polymerase can be useful, for example, in a template amplification process, primer modification process such as a primer extension step or primer capping step, examination step or combination thereof.
  • the different activities can follow from differences in the structure (e.g., via natural activities, mutations, or chemical modifications). Nevertheless, polymerase can be obtained from a variety of known sources and applied in accordance with the teachings set forth herein and recognized activities of polymerases.
  • Useful DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases and phage DNA polymerases.
  • Bacterial DNA polymerases include E. coli DNA polymerases I, II and III, IV and V, the KI enow fragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase and Sulfolobus solfataricus (Sso) DNA polymerase.
  • Eukaryotic DNA polymerases include DNA polymerases a, b, g, d, €, h, z, 1, s, m, and k, as well as the Revl polymerase (terminal deoxycytidyl transferase) and terminal deoxynucleotidyl transferase (TdT).
  • Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases, PZA DNA polymerase, phi- 15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase, and T4 polymerase.
  • thermostable and/or thermophilic DNA polymerases such as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavus (TH) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp.
  • GB-D polymerase Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (IDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp.
  • Tma Thermotoga maritima
  • Bst Bacillus stearothermophilus
  • KOD Pyrococcus Kodakaraensis
  • Pfx DNA polymerase Pfx DNA polymerase
  • Thermococcus sp. JDF-3 (IDF-3) DNA polymerase Thermococcus
  • RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and Kll polymerase; Eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and Archaea RNA polymerase.
  • viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and Kll polymerase
  • Eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V
  • Archaea RNA polymerase such as Archaea RNA polymerase.
  • a polymerase having an intrinsic 3'-5' proofreading exonuclease activity can be useful for some embodiments.
  • Polymerases that substantially lack 3'-5' proofreading exonuclease activity are also useful in some embodiments, for example, in some sequencing embodiments. Absence of exonuclease activity can be a wild type characteristic or a characteristic imparted by a variant or engineered polymerase structure.
  • exo minus Klenow fragment is a mutated version of Klenow fragment that lacks 3'-5' proofreading exonuclease activity. Klenow fragment and its exo minus variant can be useful in a method or composition set forth herein.
  • a concatemer (or “a concatemeric strand of nucleic acid molecules” or “a concatemer strand” or other derivative linguistic phrasings) comprises multiple instances of a target sequence and multiple instances of at least one adapter sequence.
  • references to “a target sequence” of a concatemer can further encompass a sequence that is reverse and complementary to the target sequence, and that references to “at least one adapter sequence” can further encompass a sequence that is reverse and complementary to the at least one adapter sequence.
  • a concatemer can be made prior to, or as part of, a method set forth herein.
  • a concatemer can include multiple copies of a sequence unit (such as, for example, a sequence unit comprising a target sequence and at least one adapter sequence) linked in series.
  • a concatemeric strand of nucleic acid molecules can include at least 2, 10, 25, 100 or more sequence units.
  • the number of sequence units in a concatemer strand can be, for example, at most 100, 25, 10 or 2 sequence units.
  • the number of sequence units in a concatemer that is produced by RCA will be a function of the number of times a polymerase completes a lap around a circular template during replication.
  • the content of each sequence unit that is produced by RCA will be the reverse complement of the content of the circular template that was replicated.
  • a circular template can comprise two adapter sequences, and a target sequence, wherein the first adapter sequence is 3’ to the target sequence and the second adapter sequence is 5’ to the target sequence.
  • adapter sequences are known synthetic sequence of nucleic acids interspersed at regular intervals. Adapter sequences can act as starting points for reading bases for a number of positions beyond each adapter sequence-target sequence junction, and optionally bases can be read in both directions from the adapter sequence.
  • the adapter sequences can have any of a variety of functions including, but not limited to, providing a binding site that complements a capture probe (e.g. a capture probe attached to a solid support), providing a primer binding site for replicating the circular template, providing a primer binding site for replicating a complement of the circular template, providing a tag that is associated with the target region (e.g.
  • Adapter sequences can be engineered so as to comprise one or more of the following: 1) a length of about 10 to about 100 nucleotides, 2) features so as to be ligated to the 5’ end and/or the 3 ’ end of a target sequence, 3) different and distinct anchor binding sites at the 5’ end and/or the 3’ end of the adapter sequence for use in sequencing of adjacent target sequences, and 4) optionally one or more restriction sites.
  • the adapter sequences or portions thereof can be common to a population of circular templates or to a population of concatemers. Whether or not the adapter sequences have common sequences, the target regions in the population of circular templates or in the population of concatemers can have different sequences.
  • sequence units between two or more concatemers, or between two or more circular templates can have common sequence regions (e.g., universal primer binding sites or universal capture probe binding sites) and/or the sequence units can have regions of differing sequence (e.g., different target sequences).
  • the length of a sequence unit in a concatemer or the length of a circular template can be selected to suit a particular application of the methods set forth herein. For example, the length can be at least about 50, 100, 250, 500, 1000, 1 x 10 4 , 1 x IO 3 or more nucleotides.
  • a nucleic acid cluster can contain one or more strands of a nucleic acid concatemer.
  • a cluster can contain only a single strand of the nucleic acid concatemer.
  • the single concatemer strand can be produced by an RCA reaction.
  • a nucleic acid cluster can contain a first strand (e.g., a sense strand) that is a concatemer along with one or more second strands (e.g., an antisense strand) that are complimentary to the first strand.
  • the one or more second strands can be produced, for example, by multiple displacement amplification (MDA) performed on a concatemeric template.
  • MDA multiple displacement amplification
  • a nucleic acid cluster can be made prior to, or as part of, a method set forth herein.
  • a cluster can include one or more concatemeric strands. In some configurations, a cluster contains no more than one concatemeric strand. Alternatively, a cluster can include a plurality of concatemer strands, for example, including at least 2, 4, 10, 50, 100 or more sense strands of a concatemer. Alternatively, or additionally, the number of concatemer strands in a cluster can be, for example, including at most 100, 50, 10, 4, 2 or 1 sense strands of the concatemer.
  • the concatemer strands in a particular cluster can have the same target sequence, for example, being sense strands of the same concatemer.
  • a cluster can have a plurality of different concatemer strands, such a cluster being non-clonal.
  • a cluster that contains at least one sense strand of a concatemer can further contain at least one antisense strand of the concatemer.
  • a cluster that contains one or more sense strands of a concatemer can further contain a plurality of antisense strands.
  • the plurality of antisense strands can include at least 2, 4, 10, 50, 100 or more antisense strands of a particular concatemer.
  • the number of antisense strands in a cluster can be, for example, at most 100, 50, 10, 4, 2 or 1 antisense strands of a particular concatemer.
  • the number of antisense strands can be, be about, be at least, be at least about, be at most, or be at most about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, or a number or a range between any two of these values.
  • a cluster can contain a single sense concatemer strand (no more than one sense strand) and a single antisense concatemer strand (no more than one antisense strand).
  • a cluster can contain at least one sense strand of a concatemer and multiple antisense strands of the concatemer.
  • the number of antisense strands in a cluster can outnumber the sense strands in the cluster.
  • the number of sense strands in a cluster can outnumber the antisense strands in the cluster. Note that the antisense strand of the concatemer need not be the same length as the sense strand.
  • the antisense strand can have more sequence units than the sense strand, or the antisense strand can have fewer sequence units than the sense strand.
  • the number of sequence units in an antisense strand can fall in a range set forth herein for sense strands of a concatemer.
  • An antisense strand of a concatemer need not have more than one sequence unit. Indeed, an antisense strand need not have a complete sequence unit.
  • a cluster that contains at least one sense strand of a concatemer can further contain at least one antisense strand of the concatemer hybridized to the sense strand via Watson-Crick base pairing.
  • the sense strand of a particular concatemer can hybridize to at least 2, 4, 10, 50, 100 or more antisense strands of the particular concatemer.
  • the sense strand of a particular concatemer can be hybridize to, for example, at most 100, 50, 10, 4, 2 or 1 antisense strands of the particular concatemer.
  • a staple molecule comprises at least a first region and a second region, wherein the first region and the second region of the staple molecule each hybridize to a different instance of at least one adapter sequence of a concatemer.
  • references to “a target sequence” of a concatemer can further encompass a sequence that is reverse and complementary to the target sequence
  • references to “at least one adapter sequence” can further encompass a sequence that is reverse and complementary to the at least one adapter sequence.
  • the first region and/or the second region of a staple molecule will hybridize to at least one adapter sequence and/or a sequence that is reverse and complementary to the at least one adapter sequence.
  • sequence of the first region and the second region are identical. In other instances, the sequence of the first and second regions are partially conserved, and in others the sequences are discrete.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for extension.
  • the 3’ end of the staple molecules hybridizes within 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 10, 5 or fewer nucleotides from the 3’end of the target sequence.
  • the 3’ end of the staple molecule hybridizes within 40 nucleotides from the 3’ end of the target sequence.
  • at least one of the first region and the second region of the staple molecule comprises a 3’ end of the staple molecule and hybridizes to a primer binding site of the at least one adapter sequence.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for ternary complex formation.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for ternary complex formation.
  • the 3’ end that allows for ternary complex formation is reversibly terminated.
  • Reversible termination can be performed using any reversible terminator moiety.
  • Exemplary reversible terminator moieties such as, for example, reversible terminator moieties in which the 3 ’-OH group is replaced by a 3’-ONH2 moiety, are set forth in U.S. Pat Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or 8,034,923; or PCT publications WO 91/06678 or WO 07/123744, each of which is incorporated herein by reference.
  • neither the first region nor the second region of the staple molecule comprises a 3’ end that allows for ternary complex formation or extension.
  • the concatemer comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the at least one adapter sequence within the sequence unit comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • at least one of the first region and the second region of the staple molecule comprises a 3’ end of the staple molecule and hybridizes to the 3’ adapter.
  • the length of the first region and the second region of the staple molecule are equal. Alternatively, the length of the first region may be greater than the length of the second region of the staple molecule. In other instances, the length of the second region may be greater than the length of the first region of the staple molecule. In some embodiments, the first region of the staple molecule is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length. In some embodiments, the second region of the staple molecule is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length.
  • the first region and the second region of the staple molecule are each at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length. In an exemplary embodiment, the first region and the second region of the staple molecule are each at least 10 nucleotides in length.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence that are spaced at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more nucleotides apart.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence that are spaced at least 100 nucleotides apart.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation.
  • a blocker can be any blocking moiety.
  • a blocking moiety refers to a part of a nucleotide that inhibits or prevents the 3’ oxygen of the nucleotide from forming a covalent linkage to a next correct nucleotide during a nucleic acid polymerization reaction.
  • the blocking moiety of a “reversibly terminated” nucleotide can be removed from the nucleotide analog, or otherwise modified, to allow the 3 ’-oxygen of the nucleotide to covalently link to a next correct nucleotide.
  • a blocking moiety is referred to herein as a “reversible terminator moiety.”
  • a blocking moiety need not hinder or preclude ternary complex formation at the 3’ end of a nucleic acid to which the blocking moiety is attached.
  • a cap can be any capping moiety.
  • a capping moiety can have a positive or negative charge that hinders or prevents ternary complex formation.
  • a capping moiety can include a ligand that binds a receptor to hinder or prevent ternary complex formation such as a biotin (or analog thereof) that binds to streptavidin (or an analog thereof), an epitope that binds to an antibody (or functional fragment thereof), a carbohydrate that binds to a lectin, or the like. Further examples of capping moieties are described in US Pat. App. Pub. No. 2020/0032322 Al or Turcatti et al. Nucl. Acids. Res. 36(4) e25 (2008), each of which is incorporated herein by reference.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation. In yet further embodiments, the 3’ end of the staple molecule is capped with a moiety that prevents ternary complex formation.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation. In yet further embodiments, the 3’ end of the staple molecule comprises a mismatch and a blocker, optionally wherein the blocker is a 3’ phosphate blocker.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • a spacer Any suitable spacer known in the arts can be used.
  • the spacer can comprise a polynucleotide sequence.
  • the spacer can comprise a non-nucleotide polymer linker, such as, for example, a branched poly electrolyte species.
  • branched poly electrolyte species include polyethylene glycol (PEG) and dendrimers.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer. In further embodiments, at least some of the one or more staple molecules do not comprise a spacer.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence is variable in length.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence comprises a double-stranded DNA sequence.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence comprises a double-stranded DNA sequence.
  • the first region and the second region each comprise a 3’ end that is hybridized to the concatemer.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is of variable length across different staple molecules.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises polyethylene glycol (PEG).
  • the PEG can comprise a PEG with an average molecular weight of about 200 Daltons (e.g., PEG-200) to about 8000 Daltons (e.g., PEG-8000).
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • Species of dendrimer that can be used in the methods, compositions and systems disclosed herein includes, but is not limited to a branched polyamine that comprises a protonated structure that interacts and forms complexes with the negatively charged backbone of DNA.
  • Dendrimer species can comprise controlled terminal surface chemistry with one or more functional groups that include, but are not limited to, amines, carboxyl, and hydroxyl groups.
  • the branched polyelectrolyte is a poly (amidoamine) dendrimer species (also referred to as PAMAM), for example, a G2 PAMAM dendrimer molecule with 16 branches having the amine (NH2) terminal surface chemistry.
  • PAMAM poly (amidoamine) dendrimer species
  • Nonlimiting examples of branched polyelectrolyte also include G4 (64 branches with the amine terminal group) and G5 (128 branches with the amine terminal group) PAMAM dendrimer species.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the dendrimer comprises polyamidoamine (PAMAM). Any species of PAMAM described above, both those specifically stated and those that are implied by the totality of the disclosure, can be used.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a non- nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the staple molecules hybridize to 3, 4, 5, 6, 7, 8, 9, 10 or more instances of the at least one adapter.
  • the staple molecules hybridize to 3 or more instances of the at least one adapter.
  • the staple molecule hybridizes to 10 or more instances of the at least one adapter sequence.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a non- nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the staple molecules hybridize to 3 or more instances of the at least one adapter.
  • a majority of the staple molecules hybridize to adapter sequences flanking at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more different target sequences.
  • a majority of the staple molecules hybridize to adapter sequences flanking at least 100 different target sequences.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • neither the first region nor the second region of the staple molecule comprises a 3’ end that allows for ternary complex formation or extension.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the spacer is coupled to the 5’ end of the second region.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the spacer is coupled to a 3’ end of the second region and wherein the staple molecule cannot act as a primer.
  • a plurality of staple molecules is hybridized to the same concatemer.
  • a concatemer, a nucleic acid cluster, and/or a nucleic acid can be attached to a surface of a solid support.
  • the solid support can be made from any of a variety of materials used for analytical biochemistry. Suitable materials may include, for example, glass, polymeric materials, silicon, quartz (fused silica), Borofloat glass, silica, silica-based materials, carbon, metals, an optical fiber or bundle of optical fibers, sapphire, or plastic materials.
  • the material can be selected based on properties desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will utilize radiation of that wavelength.
  • Wavelength regions that may be pass or not pass through a particular material include, for example, UV, VIS (e.g., red, yellow, green, or blue) or IR.
  • UV, VIS e.g., red, yellow, green, or blue
  • IR IR
  • composition of a bead can vary, depending for example, on the format, chemistry and/or method of attachment to be used.
  • exemplary bead compositions include solid supports, optionally including chemical functionalities that are used in protein and nucleic acid capture methods.
  • compositions include, for example, plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as SepharoseTM, cellulose, nylon, crosslinked micelles and TeflonTM, as well as other materials set forth in “Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind., which is incorporated herein by reference.
  • a particle such as a bead or microsphere
  • a particle can be symmetrically shaped (e.g., spherical or cylindrical) or irregularly shaped (e.g., controlled pore glass).
  • particles can be porous, thus increasing the surface area available for capture of ternary complexes or components thereof. Exemplary sizes for beads used herein can range from nanometers to millimeters or from about 10 nm to about 1 mm.
  • Exemplary bead-based arrays that can be used include, without limitation, a BeadChipTM Array available from Illumina, Inc. (San Diego, CA) or arrays such as those described in U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCT Publication No. WO 00/63437, each of which is incorporated herein by reference.
  • Beads can be located at discrete locations, such as wells, on a solid-phase support, whereby each location accommodates a single bead.
  • discrete locations where beads reside can each include a plurality of beads as described, for example, in U.S. Pat. App. Pub. Nos. 2004/0263923 Al, 2004/0233485 Al, 2004/0132205 Al, or 2004/0125424 Al, each of which is incorporated herein by reference.
  • beads can be arrayed or otherwise spatially distinguished.
  • Exemplary bead-based arrays that can be used include, without limitation, a BeadChipTM Array available from Illumina, Inc. (San Diego, CA) or arrays such as those described in U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCT Publication No. WO 00/63437, each of which is incorporated herein by reference.
  • Beads can be located at discrete locations, such as wells, on a solid-phase support, whereby each location accommodates a single bead.
  • discrete locations where beads reside can each include a plurality of beads as described, for example, in U.S. Pat. App. Pub. Nos. 2004/0263923 Al, 2004/0233485 Al, 2004/0132205 Al, or 2004/0125424 Al, each of which is incorporated herein by reference.
  • the methods can be carried out in a multiplex format whereby multiple different types of nucleic acids are detected in parallel.
  • Other types of arrays can be used instead of bead arrays, including, for example, those set forth in further detail below.
  • parallel processing can provide cost savings, time savings, and uniformity of conditions.
  • An array or method of the present disclosure can be configured to include at least 2, 10, 100, l x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 9 , or more different nucleic acids.
  • an array or method of the present disclosure can be configured to include at most 1 x 10 9 , 1 x 10 6 , 1 x 10 5 , 1 x 10 4 , l x 10 3 , 100, 10, 2 or fewer, different nucleic acids.
  • the nucleic acids can be attached to different sites of an array. As such, the number of sites in an array can be in a range exemplified here for different nucleic acids.
  • various reagents or products set forth herein e.g., primer-template nucleic acid hybrids or stabilized ternary complexes
  • arrays made by photolithographic synthesis of nucleic acids for example, an Affymetrix GeneChipTM array.
  • a spotted array can also be used to attach presynthesized nucleic acids to array sites according to some embodiments.
  • An exemplary spotted array is a CodeLinkTM Array available from Amersham Biosciences.
  • Another array that is useful is one that is manufactured using inkjet printing methods such as SurePrintTM Technology available from Agilent Technologies.
  • the methods used for attaching nucleic acid probes to these arrays can be modified to attach nucleic acid primers for use in amplifying (e g., via RCA) and/or sequencing target nucleic acids to which the primers hybridize.
  • arrays include those that are used in nucleic acid sequencing applications.
  • methods and compositions that are used to attach amplicons of genomic fragments (often referred to as clusters) to form arrays can be particularly useful. Examples are described in Bentley et al, Nature 456:53-59 (2008), PCT Pub. Nos. WO 91/06678; WO 04/018497 or WO 07/123744; U.S. Pat. Nos. 7,057,026; 7,211,414; 7,315,019; 7,329,492 or 7,405,281; or U.S. Pat. App. Pub. No. 2008/0108082 Al, each of which is incorporated herein by reference.
  • a nucleic acid, a concatemer, and/or a nucleic acid cluster can be attached to a solid support (e.g., a site of an array) via covalent or non-covalent bonds.
  • a solid support can be covalently or non-covalently attached at or near the 5’ end of a concatemeric nucleic acid. This configuration can result, for example, when the concatemer has been produced by RCA performed by extending a primer that is attached to the solid support at or near its 5’ end.
  • Attachment of a nucleic acid to a solid support can be mediated by any of a variety of surface chemistries such as reaction of a carboxylate moiety or succinimidyl ester moiety on the solid support with an amine-modified nucleic acid, reaction of an alkylating reagent (e.g.
  • iodoacetamide or maleimide on the solid support with a thiol-modified nucleic acid, reaction of an epoxysilane or isothiocyanate modified solid support with an amine-modified nucleic acid, reaction of an aminophenyl or aminopropyl modified solid support with a succinylated nucleic acid, reaction of an aldehyde or epoxide modified solid support with a hydrazide-modified nucleic acid or reaction of a thiol modified solid support with a thiol modified nucleic acid.
  • the members of the preceding reactive pairs can be switched with regard to being present on the solid support or the nucleic acid.
  • Click chemistry can be useful for attaching nucleic acids to solid supports. Exemplary reagents and methods for click chemistry are set forth in U.S. Pat. Nos. 6,737,236; 7,375,234; 7,427,678 and 7,763,736, each of which is incorporated herein by reference.
  • the solid support can include two (or more, such as three, four, five, six, seven, eight, nine, ten, or more) types or populations of primers.
  • the two or more types of primers can be, for example, as multiple capture primers and multiple amplification primers.
  • the types of primers can include multiple capture primers and multiple sequencing primers for sequencing, for example, the first strands generated by extending the capture primers).
  • the density of one type or population of primers (e.g., capture primers) on the solid support can be higher than the density of another type or population of primers (e.g., amplification primers) on the solid support.
  • the density of one type or population of primers on the solid support can be the same as the density of another type or population of primers on the solid support.
  • the density of a type or population of primers (or all primers) can very.
  • the density of a type or population of primers (or all primers) on a solid support can be, be about, be at least, be at least about, be at most, or be at most about, 1 x IO 10 , 2 x IO 10 , 3 x IO 10 , 4 x IO 10 , 5 x IO 10 , 6 x IO 10 , 7 x IO 10 , 8 x IO 10 , 9 x IO 10 , 1 x 10 11 , 2 x 10 11 , 3 x 10 11 , 4 x 10 11 , 5 x 10 11 , 6 x 10 11 ,
  • the separation distance or average separation distance between two adjacent primers of the same type or population (or of two different types or populations) can be, be about, be at least, be at least about, be at most, or be at most about, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42
  • ratios of the number of one type or population of primers and the numbers of another type or population of primers are contemplated by the present disclosure.
  • the ratio of the number of one type or population of primers and the numbers of another type or population of primers can be, be about, be at least, be at least about, be at most, or be at most about, 1 : 100, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85, 1:84, 1:83,
  • the average distance between locations on the solid support two neighboring or closest capture primers are attached to can be greater than (or less than or equal to) the length of one of the two capture primers, lengths of the two capture primers, an average length of the two capture primers, or the total length of the two capture primers (or 0.1 x, 0.2 x, 0.3 x, 0.4 x, 0.5 x, 0.6 x, 0.7 x, 0.8 x, 0.9 x, of the length).
  • An average distance between locations on the solid support two neighboring or closest amplification primers of the plurality of amplification primers are attached to can be greater than (or less than, or equal to) the length of one of the two amplification primers, lengths of the two amplification primers, an average length of the two amplification primers, or the total length of the two capture primers (or 0.1 x, 0.2 x, 0.3 x, 0.4 x, 0.5 x, 0.6 x, 0.7 x, 08 x, 0.9 x, 1.0 x, 1.1 x, 1.2 x, 1.3 x, 1.4 x, 1.5 x, 1.6 x, 1.7 x, 1.8 x, 1.9 x, 2 x, 3 x, 4 x, 5 x, 6 x, 7 x, 8 x, 9 x, 10 x of any of the lengths).
  • a flow cell allows for convenient fluidic manipulation by passing solutions into and out of a fluidic chamber that contacts support-bound analyte(s).
  • the flow cell also provides for detection of the fluidically manipulated components.
  • a detector can be positioned to detect signals from the solid support, such as signals from a label that is recruited to the solid support during a sequencing process.
  • Exemplary flow cells that can be used are described, for example, in US Pat. App. Pub. No. 2010/0111768 Al, WO 05/065814 or US Pat. App. Pub. No. 2012/0270305 Al, each of which is incorporated herein by reference.
  • a nucleic acid, a concatemer, and/or a nucleic acid cluster can be attached to a surface of the flow cell and/or to a solid support in a flow cell.
  • the concatemer and/or nucleic acid cluster can comprise a first strand (sense strand) and/or a plurality of second strands (antisense strands).
  • the nucleic acid, the concatemer, and/or the nucleic acid cluster is attached to the surface of the flow cell and/or the solid support in the flow cell by a covalent attachment.
  • the first strand is covalently attached to a surface of a flow cell and/or a solid support in the flow cell, and the plurality of second strands is not covalently attached.
  • the plurality of second strands can be retained to the flow cell surface or solid support in the flow cell due to Watson- Crick base pairing to the first strand.
  • a stabilized concatemer composition comprising: (a) a concatemer comprising multiple instances of a target sequence and multiple instances of at least one adapter sequence; and (b) one or more staple molecules; wherein individual staple molecules comprise at least a first region and a second region; and wherein the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence.
  • references to “a target sequence” of a concatemer can further encompass a sequence that is reverse and complementary to the target sequence
  • references to “at least one adapter sequence” can further encompass a sequence that is reverse and complementary to the at least one adapter sequence.
  • the first region and/or the second region of a staple molecule will hybridize to at least one adapter sequence and/or a sequence that is reverse and complementary to the at least one adapter sequence.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • a circular nucleic acid template (such as, for example, a circular nucleic acid template comprising a target sequence and at least one adapter sequence) can be single-stranded or double-stranded. One or both strands in a double-stranded nucleic acid can lack a 3’ end and a 5’ end.
  • One strand in a double-stranded nucleic acid can have a gap (absence of at least one nucleotide monomer relative to the other strand) or nick (absence of a phosphodiester bond between two nucleotide monomers), so long as the other strand is circular.
  • Any of a variety of polymerases can be used in a method or composition set forth herein. Non-limiting examples of polymerases that may be used include naturally occurring polymerases and modified version thereof, including, but not limited to, mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics, analogs, and the like.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the circular nucleic acid template is a product of circularization of a linear nucleic acid template comprising the target sequence and at least one adapter sequence. Circularization of a linear nucleic acid template can be performed using any method known in the art. In some embodiments, circularization of a linear nucleic acid template is performed using a ligase. Circularization of a linear nucleic acid template can be performed using any suitable ligase, such as, for example, T4 DNA ligase.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the circular nucleic acid template is a product of circularization of a linear nucleic acid template comprising the target sequence and at least one adapter sequence.
  • the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the circular nucleic acid template is a product of circularization of a linear nucleic acid template comprising the target sequence and at least one adapter sequence.
  • the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  • a “splint oligonucleotide” is an oligonucleotide that, when hybridized to other polynucleotides, such as, for example, a first adapter sequence, or a second adapter sequence, or a (nucleic acid) template, or a linear (nucleic acid) template, acts as a “splint” to position the polynucleotides next to one another so that they can be ligated together.
  • the splint oligonucleotide is DNA or RNA.
  • the splint oligonucleotide can include a nucleotide sequence that is partially complimentary to nucleotide sequences from two or more different oligonucleotides.
  • the splint oli onucleotide assists in ligating a “donor” oligonucleotide and an “acceptor” oligonucleotide.
  • an RNA ligase, a DNA ligase, or another other variety of ligase is used to ligate two nucleotide sequences together.
  • the splint oligonucleotide is between 10 and 50 oligonucleotides in length, e.g., between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20 nucleotides in length. In some embodiments, the splint oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and 35, 15 and 30, or 15 and 25 nucleotides in length.
  • a splint oligonucleotide need not be used to ligate the ends, for example, when using CircLigase TM (Epicenter, Madison WI) or other enzyme capable of splint-free ligation of nucleic acid ends.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the circular nucleic acid template is a product of circularization of a linear nucleic acid template comprising the target sequence and at least one adapter sequence.
  • the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  • the splint oligonucleotide is the primer hybridized to the circularized nucleic acid.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the circular nucleic acid template is a product of circularization of a linear nucleic acid template comprising the target sequence and at least one adapter sequence.
  • the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  • the splint oligonucleotide is removed prior to RCA.
  • the splint oligonucleotide can be removed during or subsequent to RCA.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is immobilized on a surface during RCA.
  • Suitable surfaces include, but are not limited to, a structured surface, a planar substrate, a hydrogel, a nanohole array, a microparticle, a nanoparticle, a flow cell surface, a surface of a solid support, or a surface of a solid support within a flow cell.
  • the surface can be planar or curved.
  • the solid support can be made from any of a variety of materials used for analytical biochemistry.
  • Suitable materials may include, for example, glass, polymeric materials, silicon, quartz (fused silica), borofloat glass, silica, silica-based materials, carbon, metals, an optical fiber or bundle of optical fibers, sapphire, or plastic materials.
  • the material can be selected based on properties desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will utilize radiation of that wavelength. Conversely, it may be desirable to select a material that does not pass radiation of a certain wavelength (e.g., being opaque, absorptive, or reflective). Wavelength regions that may be pass or not pass through a particular material include, for example, UV, VIS (e.g., red, yellow, green, or blue) or IR. Other properties of a material that can be exploited are inertness or reactivity to certain reagents used in a downstream process, such as those set forth herein, or ease of manipulation, or low cost of manufacture.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the concatemer is attached to a surface subsequent to hybridizing the concatemer with one or more staple molecules.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the concatemer is attached to a surface subsequent to hybridizing the concatemer with one or more staple molecules. In even further embodiments, the surface is a structured surface.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the concatemer is attached to a surface prior to hybridizing the concatemer with one or more staple molecules.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the primer is in solution during RCA.
  • the concatemer is attached to a surface prior to hybridizing the concatemer with one or more staple molecules. In even further embodiments, the surface is a structured surface.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the concatemer is a sense strand, and the composition further comprises a plurality of antisense strands produced from the amplification of the sense strand.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the concatemer is a sense strand, and the composition further comprises a plurality of antisense strands produced from the amplification of the sense strand.
  • at least some of the one or more staple molecules are hybridized to adapter sequences of the plurality of antisense strands.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the concatemer is a sense strand, and the composition further comprises a plurality of antisense strands produced from the amplification of the sense strand.
  • the one or more staple molecules are hybridized to the sense strand.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the concatemer is a sense strand, and the composition further comprises a plurality of antisense strands produced from the amplification of the sense strand.
  • the one or more staple molecules are hybridized to the sense strand.
  • at least some of the one or more staple molecules are one or more staple primers used to amplify the sense strand to produce the plurality of antisense strands.
  • the concatemer is a product of RCA with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  • the one or more staple molecules hybridize to the concatemer during RCA.
  • individual instances of the at least one adapter sequence comprises a primer binding site.
  • individual instances of the at least one adapter sequence comprises a primer binding site.
  • individual instances of the at least one adapter sequence further comprise a tag region, optionally wherein the tag region is a sample index.
  • individual instances of the at least one adapter sequence comprises a primer binding site. In further embodiments, individual instances of the at least one adapter sequence further comprise a splint binding site.
  • individual instances of the at least one adapter sequence comprises a primer binding site.
  • individual instances of the at least one adapter sequence further comprise a variable region, optionally wherein the variable region is a unique molecular identifier (UMI).
  • UMIs are sequences of nucleotides applied to or identified in polynucleotides that may be used to distinguish individual nucleic acid molecules that are present in an initial reaction from one another.
  • the UMI may comprise from about 5 to about 20 nucleotides.
  • the UMI may comprise less than about 5 or more than 20 nucleotides.
  • the UMI may be a unique sequence that varies across individual nucleic acid molecules.
  • the UMI may be a random sequence. In some cases, the UMI may be a predetermined sequence. In a sequencing reaction, UMIs may be sequenced along with the nucleic acid molecules with which they are associated to determine whether the read sequences are those of one source nucleic acid molecule or another.
  • the term “UMI” is used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide comprising that sequence information. Additional examples of UMIs and uses thereof are provided in, e.g., US 2016/0319345 Al, which is incorporated herein by reference.
  • the concatemer comprises a sense strand hybridized to a plurality of antisense strands.
  • the concatemer comprises a sense strand hybridized to a plurality of antisense strands.
  • the first region and the second region of the staple molecule each hybridize to a different instance of the at least one adapter sequence on a different antisense strand.
  • the concatemer comprises a sense strand hybridized to a plurality of antisense strands.
  • the sense strand does not comprise uracil and the plurality of antisense strands comprises uracil.
  • instances of the target sequence comprise a sense sequence or an antisense sequence.
  • the concatemer is provided immobilized to a surface in a flow cell.
  • the concatemer is provided immobilized to a surface in a flow cell. In further embodiments, the concatemer is immobilized to binding sites of a structured surface of the flow cell.
  • a staple molecule comprises at least a first region and a second region, wherein the first region and the second region of the staple molecule each hybridize to a different instance of at least one adapter sequence of a concatemer.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for extension. Extension can be mediated by a polymerase or ligase.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for extension.
  • the 3’ end of the staple molecules hybridizes within 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 10, 5 or fewer nucleotides from the 3’end of the target sequence.
  • the 3’ end of the staple molecule hybridizes within 40 nucleotides from the 3’ end of the target sequence.
  • At least one of the first region and the second region of the staple molecule comprises a 3’ end of the staple molecule and hybridizes to a primer binding site of the at least one adapter sequence.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for ternary complex formation.
  • At least one of the first region and second region of the staple molecule comprise a 3’ end that allows for ternary complex formation.
  • the 3’ end that allows for ternary complex formation is reversibly terminated.
  • Reversible termination can be performed using any reversible terminator moiety.
  • Exemplary reversible terminator moi eties such as, for example, reversible terminator moieties in which the 3 ’-OH group is replaced by a 3’-ONH2 moiety, are set forth in U.S. Pat Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or 8,034,923; or PCT publications WO 91/06678 or WO 07/123744, each of which is incorporated herein by reference.
  • neither the first region nor the second region of the staple molecule comprises a 3’ end that allows for ternary complex formation or extension.
  • the concatemer comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the concatemer comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the at least one adapter sequence within the sequence unit comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the concatemer comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the at least one adapter sequence within the sequence unit comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • the first region of the staple molecule hybridizes to the first adapter sequence and the second region of the staple molecule hybridizes to the second adapter sequence.
  • the concatemer comprises a repeating sequence unit comprising the target sequence and the at least one adapter sequence.
  • the at least one adapter sequence within the sequence unit comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  • at least one of the first region and the second region of the staple molecule comprises a 3’ end of the staple molecule and hybridizes to the 3’ adapter.
  • the first region and the second region of the staple molecule are each at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length. In an exemplary embodiment, the first region and the second region of the staple molecule are each at least 10 nucleotides in length.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence that are spaced at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more nucleotides apart.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence that are spaced at least 100 nucleotides apart.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation.
  • a blocker can be any blocking moiety.
  • a blocking moiety refers to a part of a nucleotide that inhibits or prevents the 3’ oxygen of the nucleotide from forming a covalent linkage to a next correct nucleotide during a nucleic acid polymerization reaction.
  • the blocking moiety of a “reversibly terminated” nucleotide can be removed from the nucleotide analog, or otherwise modified, to allow the 3 ’-oxygen of the nucleotide to covalently link to a next correct nucleotide.
  • a blocking moiety is referred to herein as a “reversible terminator moiety.”
  • a blocking moiety need not hinder or preclude ternary complex formation at the 3’ end of a nucleic acid to which the blocking moiety is attached.
  • a cap can be any capping moiety.
  • a capping moiety can have a positive or negative charge that hinders or prevents ternary complex formation.
  • a capping moiety can include a ligand that binds a receptor to hinder or prevent ternary complex formation such as a biotin (or analog thereof) that binds to streptavidin (or an analog thereof), an epitope that binds to an antibody (or functional fragment thereof), a carbohydrate that binds to a lectin, or the like. Further examples of capping moieties are described in US Pat. App. Pub. No. 2020/0032322 Al or Turcatti et al. Nucl. Acids. Res. 36(4) e25 (2008), each of which is incorporated herein by reference.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule comprises a blocker that prevents nucleotide incorporation.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation. In yet further embodiments, the 3’ end of the staple molecule is capped with a moiety that prevents ternary complex formation.
  • the 3’ end of the staple molecule comprises at least one mismatch, a blocker that prevents nucleotide incorporation, or a cap that prevents ternary complex formation. In further embodiments, the 3’ end of the staple molecule prevents ternary complex formation. In yet further embodiments, the 3’ end of the staple molecule comprises a mismatch and a blocker, optionally wherein the blocker is a 3’ phosphate blocker.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • a spacer Any suitable spacer known in the arts can be used.
  • the spacer can comprise a polynucleotide sequence.
  • the spacer can comprise a non -nucleotide polymer linker, such as, for example, a branched poly electrolyte species.
  • branched poly electrolyte species include polyethylene glycol (PEG) and dendrimers.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer. In further embodiments, at least some of the one or more staple molecules do not comprise a spacer.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence is variable in length.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence comprises a double-stranded DNA sequence.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a polynucleotide sequence.
  • the polynucleotide sequence comprises a double-stranded DNA sequence.
  • the first region and the second region each comprise a 3’ end that is hybridized to the concatemer.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is of variable length across different staple molecules.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises polyethylene glycol (PEG).
  • the PEG can comprise a PEG with an average molecular weight of about 200 Daltons (e.g., PEG-200) to about 8000 Daltons (e.g., PEG-8000).
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • Species of dendrimer that can be used in the methods, compositions and systems disclosed herein includes, but is not limited to a branched polyamine that comprises a protonated structure that interacts and forms complexes with the negatively charged backbone of DNA.
  • Dendrimer species can comprise controlled terminal surface chemistry with one or more functional groups that include, but are not limited to, amines, carboxyl, and hydroxyl groups.
  • the branched polyelectrolyte is a poly(amidoamine) dendrimer species (also referred to as PAMAM), for example, a G2 PAMAM dendrimer molecule with 16 branches having the amine (NH2) terminal surface chemistry.
  • PAMAM poly(amidoamine) dendrimer species
  • Non- limiting examples of branched poly electrolyte also include G4 (64 branches with the amine terminal group) and G5 (128 branches with the amine terminal group) PAMAM dendrimer species.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the dendrimer comprises polyamidoamine (PAMAM). Any species of PAMAM described above, both those specifically stated and those that are implied by the totality of the disclosure, can be used.
  • the first region and the second region of at least some of the staple molecules are separated by a spacer.
  • the spacer comprises a non- nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • the staple molecules hybridize to 3 or more instances of the at least one adapter.
  • a majority of the staple molecules hybridize to adapter sequences flanking at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more different target sequences.
  • a majority of the staple molecules hybridize to adapter sequences flanking at least 100 different target sequences.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer comprises a non-nucleotide polymer linker.
  • the non-nucleotide polymer linker comprises a dendrimer.
  • neither the first region nor the second region of the staple molecule comprises a 3’ end that allows for ternary complex formation or extension.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the spacer is coupled to the 5’ end of the second region.
  • the first region and the second region of at least some of the one or more staple molecules are separated by a spacer.
  • the spacer is coupled to the 3’ end of the first region.
  • the spacer is coupled to a 3’ end of the second region and wherein the staple molecule cannot act as a primer.
  • a plurality of staple molecules is hybridized to the same concatemer.
  • kits comprising: the staple molecules of any one of the first aspect and embodiments, second aspect and embodiments, or fifth aspects and embodiments.
  • the kit further comprises one or more of the following: (i) one or more staple primers, (ii) one or more non-staple primers, (iii) one or more adapter sequences, (iv) one or more polymerases, (v) one or more ligases, (vi) one or more splint oligonucleotides, (vii) a flow cell, (viii) a plurality of labeled nucleotides, (ix) a plurality of reversibly terminated nucleotides, (x) a capping moiety, or any combination thereof.
  • kits comprising: (i) a first adapter sequence, (ii) a second adapter sequence, and (iii) a staple molecule, wherein the first and the second region of the staple molecule each hybridize to a different instance of the first and/or the second adapter sequence.
  • the kit further comprises (iv) reagents sufficient to form a stabilized concatemer from a target nucleic acid, wherein the stabilized concatemer comprises the first adapter, the second adapter, and the staple molecule.
  • reagents sufficient to form a stabilized concatemer from a target nucleic acid, wherein the stabilized concatemer comprises the first adapter, the second adapter, and the staple molecule.
  • a variety of methods have been developed for determining the sequences of portions of a nucleic acid template, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules.
  • the result of any sequencing methodology is the production of a set of sequence reads (or other derivative linguistic phrasings). Therefore, any method of sequencing can also be referred to as a method for producing a set of sequence reads. It is contemplated that all current sequencing methodologies, as well as future sequencing methodologies, can be improved using the aspects and embodiments of the compositions of stabilized concatemers and the aspects and embodiments of the methods of making stabilized concatemers described above.
  • methods of sequencing can be stratified based on 1) the type of nucleic acid molecule utilized as a template (DNA-sequencing or RNA-sequencing), 2) the number of ends of a template that are sequenced (single-end sequencing or paired-end sequencing), and 3) the read length produced from the sequencing method (short-read sequencing or long-read sequencing).
  • methods of sequencing can be stratified at a high-level based on the sequencing “generation”.
  • DNA sequencing and RNA sequencing methodologies primarily differ based on the type of nucleic acid molecule utilized as the template or starting material in a given workflow.
  • DNA sequencing is the process of determining the nucleotide order of a given DNA fragment.
  • RNA sequencing is the process of determining the nucleotide order of a given RNA fragment. RNA is less stable than DNA and is more prone to nuclease attack experimentally. Therefore, methods of RNA sequencing commonly reverse transcribe RNA from a sample to generate complementary DNA (cDNA) fragments and then subject the cDNA to a given sequencing methodology to determine its sequence.
  • cDNA complementary DNA
  • single-send sequencing methods are configured to determine the sequence at one end of a nucleic acid template
  • paired-end sequencing methods are configured to determine the sequences at opposite ends of a nucleic acid template.
  • sequence of a first end is determined by extending a first primer along the first strand of a nucleic acid template.
  • sequence of the second end can be determined by extending a second primer along the second strand of the nucleic acid template.
  • the first and second primer are extended in opposite directions and towards each other. Because of this orientation, inter alia, some paired-end sequencing methods require each of the strands to be sequenced in the absence of the other strand. Accordingly, some paired-end methods typically require steps of removing strands or synthesizing strands between two sequencing reads. Other methods of paired-end sequencing allow for the sequencing of a first strand (sense strand) of a target nucleic acid in the presence of the second strand (antisense strand) of the target nucleic acid.
  • High-throughput sequencing refers to “short-read” and “long-read” sequencing methods (as well as exome sequencing, genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interaction (ChlP-sequencing), and characterization of the epigenome).
  • RNA-Seq transcriptome profiling
  • CholP-sequencing DNA-protein interaction
  • Next-generation sequencing also referred to as second-generation sequencing, massive parallel sequencing, or massively parallel sequencing, is any of several high-throughput approaches for sequencing nucleic acid molecules using the concept of massively parallel processing.
  • Next-generation sequencing methods are well-known in the art and primarily comprises short-read sequencing methods.
  • Non-limiting examples of second-generation sequencing include: 454 pyrosequencing, sequencing-by-synthesis (SBS), sequencing-by- ligation, sequencing-by-hybridization, sequencing-by-binding (SBB), Illumina dye sequencing, Solexa sequencing, ion semiconductor sequencing, ABI SOLiD sequencing, sequencing using combinatorial probe anchor ligation (cP AL sequencing), RNA-Seq, and the like.
  • Third-generation sequencing Long-read sequencing, also known as third-generation sequencing, is a class of sequencing methods currently under active development. A few third-generation sequencing methods are well-known in the art despite the relatively nascent state of the field. Non-limiting examples of third generation sequencing include: Pacific Biosciences’ single molecule real-time (SMRT) sequencing, Oxford Nanopore Technologies’ nanopore sequencing, Helicos’ single molecule fluorescent sequencing, and the like. 7. Sequencing-by-Binding:
  • SBB Sequence-by-binding
  • SBB has been described in U.S. Patent No. 10,443,098, U.S. Patent No. 10,246,744, and U.S. Patent Application Publication No. 2018/0044727 Al; the content of each of which is incorporated herein by reference in its entirety.
  • the polymerase undergoes conformational transitions between open and closed conformations during discrete steps of the reaction.
  • the polymerase binds to a primed template nucleic acid to form a binary complex, also referred to herein as the pre-insertion conformation.
  • an incoming nucleotide is bound and the polymerase fingers close, forming a pre-chemistry conformation comprising the polymerase, primed template nucleic acid and nucleotide; wherein the bound nucleotide has not been incorporated.
  • This step also referred to herein as an examination step, may be followed by a chemical incorporation step wherein a phosphodiester bond is formed with concomitant pyrophosphate cleavage from the nucleotide (nucleotide incorporation).
  • the polymerase, primed template nucleic acid and newly incorporated nucleotide produce a post-chemistry, pre-translation conformation.
  • both the pre-chemistry conformation and the pre-translocation conformation comprise a polymerase, primed template nucleic acid and nucleotide, wherein the polymerase is in a closed state
  • either conformation may be referred to herein as a ternary complex.
  • the polymerase configuration and/or interaction with a nucleic acid may be monitored during an examination step to identify the next correct base in the nucleic acid sequence.
  • reaction conditions can be changed to disengage the polymerase from the primed template nucleic acid and changed again to remove from the local environment any reagents that inhibit polymerase binding.
  • the SBB procedure includes an “examination” step that identifies the next template base, and optionally an “incorporation” step that adds one or more complementary nucleotides to the 3 ’-end of the primer component of the primed template nucleic acid. Identity of the next correct nucleotide to be added is determined either without, or before chemical linkage of that nucleotide to the 3 ’-end of the primer through a covalent bond.
  • the examination step can involve providing a primed template nucleic acid to be used in the procedure and contacting the primed template nucleic acid with a polymerase enzyme (e.g., a DNA polymerase) and one or more test nucleotides being investigated as the possible next correct nucleotide. Further, there is a step that involves monitoring or measuring the interaction between the polymerase and the primed template nucleic acid in the presence of the test nucleotides.
  • a polymerase enzyme e.g., a DNA polymerase
  • An examination step typically includes the following substeps: (1) providing a primed template nucleic acid (i.e., a template nucleic acid molecule hybridized with a primer that optionally may be blocked from extension at its 3 ’-end); (2) contacting the primed template nucleic acid with a reaction mixture that includes a polymerase and at least one nucleotide; (3) monitoring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of the nucleotide(s) and without chemical incorporation of any nucleotide into the primed template nucleic acid; and (4) determining from the monitored interaction the identity of the next base in the template nucleic acid (i.e., the next correct nucleotide).
  • a primed template nucleic acid i.e., a template nucleic acid molecule hybridized with a primer that optionally may be blocked from extension at its 3 ’-end
  • a reaction mixture that includes a polymerase and at least one nucleotide
  • the examination step may be controlled so that nucleotide incorporation is either attenuated or accomplished. If nucleotide incorporation is attenuated, a separate incorporation step may be performed. The separate incorporation step may be accomplished without the need for monitoring, as the base has already been identified during the examination step. If nucleotide incorporation proceeds during examination, subsequent nucleotide incorporation may be attenuated by a stabilizer that traps the polymerase on the nucleic acid after incorporation. A reversibly terminated nucleotide may be used in the incorporation step to prevent the addition of more than one nucleotide during a single cycle.
  • the SBB method allows for controlled determination of a template nucleic acid base without the need for labeled nucleotides, as the interaction between the polymerase and template nucleic acid can be monitored without a label on the nucleotide.
  • the controlled nucleotide incorporation can also provide accurate sequence information of repetitive and homopolymeric regions without necessitating use of a labeled nucleotide.
  • template nucleic acid molecules may be sequenced under examination conditions which do not require attachment of template nucleic acid or polymerase to a solid-phase support.
  • primed template nucleic acids to be sequenced are attached to a solid support, such as an interior surface of a flow cell.
  • the examination step may be controlled, in part, by providing reaction conditions to prevent chemical incorporation of a nucleotide, while allowing determination of the identity of the next correct base on the primed template nucleic acid molecule.
  • reaction conditions may be referred to as examination reaction conditions.
  • Examination typically involves detecting polymerase interaction with a template nucleic acid. Detection may include optical, electrical, thermal, acoustic, chemical, and mechanical means. Generally, the examination step involves binding a polymerase to the polymerization initiation site of a primed template nucleic acid in a reaction mixture comprising one or more nucleotides and monitoring the interaction. The examination step of the sequencing reaction may be repeated 1, 2, 3, 4 or more times prior to the incorporation step. The examination and incorporation steps may be repeated until the desired sequence of the template nucleic acid is obtained.
  • SBB involves contacting of the primed template nucleic acid molecule with a reaction mixture that includes a polymerase and one or more nucleotide molecules preferably occurs under conditions that stabilize formation of the ternary complex, and that destabilize formation of binary complexes.
  • the formation of the ternary complex or the stabilized ternary complex can be employed to ensure that only one nucleotide is added to the template nucleic acid primer per cycle of sequencing, wherein the added nucleotide is sequestered within the ternary complex.
  • the controlled incorporation of a single nucleotide per sequencing cycle enhances sequencing accuracy for nucleic acid regions comprising homopolymer repeats.
  • a reaction mixture used in the examination step can include 1, 2, 3, or 4 types of nucleotide molecules.
  • the nucleotides can be selected from dATP, dTTP (or dUTP), dCTP, and dGTP.
  • the reaction mixture can comprise one or more triphosphate nucleotides and one or more diphosphate nucleotides.
  • a ternary complex can form between the primed template nucleic acid, the polymerase, and any one of the four nucleotide molecules so that four types of ternary complexes may be formed.
  • Monitoring or measuring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of a nucleotide molecule may be accomplished in many different ways. For example, monitoring can include measuring association kinetics for the interaction between the primed template nucleic acid, the polymerase, and any one of the four nucleotide molecules. Monitoring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of a nucleotide molecule can include measuring equilibrium binding constants between the polymerase and primed template nucleic acid molecule (i.e., equilibrium binding constants of polymerase to the template nucleic acid in the presence of any one or the four nucleotides).
  • the monitoring includes measuring the equilibrium binding constant of the polymerase to the primed template nucleic acid in the presence of any one of the four nucleotides.
  • Monitoring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of a nucleotide molecule includes measuring dissociation kinetics of the polymerase from the primed template nucleic acid in the presence of any one of the four nucleotides.
  • the monitoring step can include monitoring the steady state interaction of the polymerase with the primed template nucleic acid molecule in the presence of the first nucleotide molecule, without chemical incorporation of the first nucleotide molecule into the primer of the primed template nucleic acid molecule.
  • the monitoring can include monitoring dissociation of the polymerase with the primed template nucleic acid molecule in the presence of the first nucleotide molecule, without chemical incorporation of the first nucleotide molecule into the primer of the primed template nucleic acid molecule.
  • the monitoring can include monitoring association of the polymerase with the primed template nucleic acid molecule in the presence of the first nucleotide molecule, without chemical incorporation of the first nucleotide molecule into the primer of the primed template nucleic acid molecule.
  • the test nucleotides in these procedures may be native nucleotides (i.e., unlabeled), labeled nucleotides (e.g., fluorescently labeled nucleotides), or nucleotide analogs (e.g., nucleotides modified to include reversible terminator moieties).
  • a chemical block on the 3’ nucleotide of the primer of the primed template nucleic acid molecule e.g., a reversible terminator moiety on the base or sugar of the nucleotide
  • a chemical block on the 3’ nucleotide of the primer of the primed template nucleic acid molecule e.g., a reversible terminator moiety on the base or sugar of the nucleotide
  • the absence of a catalytic metal ion in the reaction mixture or the absence of a catalytic metal ion in the active site of the polymerase prevents the chemical incorporation of the nucleotide into the primer of the primed template nucleic acid.
  • the identity of the next correct base or nucleotide can be determined by monitoring the presence, formation, and/or dissociation of the ternary complex.
  • the identity of the next base may be determined without chemically incorporating the next correct nucleotide to the 3 ’-end of the primer.
  • the identity of the next base can be determined by monitoring the affinity of the polymerase to the primed nucleic acid template in the presence of added nucleotides.
  • SBB can include an incorporation step.
  • the incorporation step involves chemically incorporating one or more nucleotides at the 3 ’-end of a primer bound to a template nucleic acid.
  • the incorporation reaction may be facilitated by an incorporation reaction mixture.
  • the incorporation reaction mixture can have a different composition of nucleotides than the examination reaction.
  • the examination reaction can include one type of nucleotide and the incorporation reaction can include another type of nucleotide.
  • the examination reaction comprises one type of nucleotide and the incorporation reaction comprises four types of nucleotides, or vice versa.
  • the examination reaction mixture can be altered or replaced by the incorporation reaction mixture.
  • Nucleotides present in the reaction mixture but not sequestered in a ternary complex may cause multiple nucleotide insertions.
  • a wash step can be employed prior to the chemical incorporation step to ensure only the nucleotide sequestered within a trapped ternary complex is available for incorporation during the incorporation step.
  • the trapped polymerase complex may be a ternary complex, a stabilized ternary complex or ternary complex involving the polymerase, primed template nucleic acid and next correct nucleotide.
  • SBS Sequencing-by-synthesis
  • SBS generally involves the enzymatic extension of a nascent primer through the iterative addition of nucleotides against a template strand to which the primer is hybridized.
  • SBS has been described, for example, in US Pat. Nos. 5,302,509 and 6,828,100, and US Pat. App. Pub. No. 2009/0247414 Al; the content of each is incorporated herein by reference in its entirety.
  • SBS differs from SBB, above, in that labeled nucleotides are incorporated into the extending strand, assayed and then the label is removed or deactivated, and the 3’ block removed, to iteratively sequence a template.
  • a labeled base is not incorporated into an extending strand. Rather, ternary complex formation is assayed, usually for the presence of a labeled base but sometimes for the presence of a labeled polymerase or other feature, after which point the complex is disassembled and a 3’ blocked, unlabeled base is used to extend the primer strand.
  • SBS can be initiated by contacting target nucleic acids, attached to sites in a flow cell, with one or more labeled nucleotides, DNA polymerase, etc. Those sites where a primer is extended using the target nucleic acid as template will incorporate a labeled nucleotide that can be detected. Detection can include scanning using an apparatus or method set forth herein.
  • the labeled nucleotides can further include a reversible termination property that terminates further primer extension once a nucleotide has been added to a primer.
  • a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety.
  • a deblocking reagent can be delivered to the vessel (before or after detection occurs). Washes can be carried out between the various delivery steps. The cycle can be performed n times to extend the primer by n nucleotides, thereby detecting a sequence of length n.
  • One or more reagents used in an SBS process can optionally be delivered via a mixed-phase fluid (e.g., a fluid foam, fluid slurry or fluid emulsion), contacted with a mixed-phase fluid, and/or removed by a mixed-phase fluid.
  • a mixed-phase fluid e.g., a fluid foam, fluid slurry or fluid emulsion
  • a mixed-phase fluid can be removed from a flow cell for detection during an SBS process.
  • the average full width at half maximum (FWHM) of the stabilized concatemers does not increase by more than 5% after at least 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 150, 175, or 200 sequencing cycles. In an exemplary embodiment, the average FWHM of the stabilized concatemers, as measured across at least one dimension, does not increase by more than 5% after at least 100 sequencing cycles.
  • the average increase in FWHM of the stabilized concatemers after at least 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 150, 175, or 200 sequencing cycles, as measured in at least one dimension, is reduced at least 2-fold, 3-fold, 4-fold, or 5-fold by the staple molecules.
  • the average increase in FWHM of the stabilized concatemers after 100 sequencing cycles, as measured in at least one dimension, is reduced by at least 5-fold by the staple molecules.
  • the stabilized concatemer is sequenced at a read length of greater than 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 cycles. In an exemplary embodiment, the stabilized concatemer is sequenced at a read length of greater than 150 cycles. 3. Improved Intensity and Resolution of Signals:
  • the stabilized concatemer is sequenced at a read length of greater than 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 cycles.
  • an average signal obtained from the stabilized concatemers in the final cycle of sequencing is 10%, 20%, 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% greater with the staple molecule compared to an average signal obtained from concatemers that are not stabilized by staple molecules.
  • the stabilized concatemer is sequenced at a read length of greater than 150 cycles and an average signal obtained from the stabilized concatemers in the final cycle of sequencing is 50% greater with the staple molecule compared to an average signal obtained from concatemers that are not stabilized by staple molecules.
  • the read length may be defined by a signal to noise ratio, below which bases are no longer called.
  • the read lenth may be a cycle at which signal to noise drops below 2, 3, or 4.
  • the read length may be defined by an accuracy below which bases are no longer called.
  • the read length may be a cycle at which base call accuracy drops below 99%, 99.5%, 99.9%, 99.95%, or 99.99%.
  • a variety of methods have been developed for determining the sequences of different portions of a nucleic acid template. In some cases, these methods are configured to determine the sequence at one end of a nucleic acid template and are thus referred to as “single-end” sequencing. While any single-end sequencing methodology can be utilized in the aspects and embodiments described above and below, exemplary workflows for the synthesis of concatemers in need of single-end sequencing are provided, such as those illustrated in Fig. 13, to facilitate a further understanding of the disclosure herein.
  • the single-end clustering workflow can begin with the synthesis of a concatemer from a linear nucleic acid template comprising a target sequence.
  • the concatemer can be synthesized in solution in a vessel or attached to a surface, as described above.
  • the linear nucleic acid template is prepared from the extraction of nucleic acids from a sample, followed by fragmentation and size selection. Methods for the isolation, fragmentation, and size capture of nucleic acids are described above.
  • One or more adapter sequences are linked to the linear nucleic acid template, as described above.
  • the linear nucleic acid template now comprising a target sequence and one or more adapter sequences, can be annealed to a guide oligonucleotide having regions complementary to the one or more adapters of the linear nucleic acid.
  • the guide oligo is bound to a surface
  • the guide oligo functions as a splint (or “splint oligonucleotide”), causing a conformational change to the linear nucleic acid such that the 5’ and 3’ ends are brought together in close proximity.
  • the 5’ and 3’ ends of the linear nucleic acid template are ligated so as to circularize the template.
  • the two ends can be ligated while hybridized to a splint oligonucleotide in solution or on a surface (such as, for example, the surface of a solid support, or a surface of a flow cell, or a surface of a solid support in a flow cell).
  • a surface such as, for example, the surface of a solid support, or a surface of a flow cell, or a surface of a solid support in a flow cell.
  • the linear nucleic acid template can be circularized without a splint oligonucleotide using CircLigaseTM (Epicenter, Madison WI) or other enzyme capable of splint free ligation of nucleic acid ends.
  • the guide oligo is then subjected to an extension reaction, so as to have added at its 3’ end multiple monomer units of the originally linear nucleic acid via RCA.
  • an initial seeding/extension phase is initiated, allowing nascent concatemers to form and begin amplifying.
  • This phase can be carried out in the presence of a first reaction mix comprising: a non-catalytic metal ion, a dendrimer, such as, for example, PAMAM, a polymerase, and a plurality of dNTPs.
  • a buffer exchange is performed, as shown in Fig. 15. Subsequently, synthesis of the concatemer continues during a second extension phase.
  • the second extension phase is carried out in the presence of a second reaction mix comprising a higher concentration of a dendrimer and a non-catalytic metal ion, but with no additional polymerase added to the mixture.
  • the result is a concatemer of multimers of the previously linear nucleic acid.
  • the concatemer can be tethered to a surface via the guide oligo, or the concatemer can be attached to a surface after synthesis in solution.
  • the RCA reaction may be terminated via heat inactivation of the polymerase, or through washing, alone or in combination with chemical inactivation.
  • the RCA reaction is stopped by denaturing the polymerase, for example, by heating the sample at 60°C, 65°C, 70°C, 75°C, 80°C, or more.
  • an RCA reaction is stopped by removing one or more components of RCA, such as the polymerase, and dNTPs. Components of RCA can be removed by, for example, washing.
  • the concatemer can be stabilized by providing staple molecules before, during, or after inactivation of the polymerase, as described further in below.
  • the concatemer can be subjected to any number of singleend sequencing methodologies or the concatemer can be provided for a paired-end clustering workflow, as described below.
  • paired-end sequencing A variety of methods have been developed for determining the sequences of different portions of a nucleic acid template. In some cases, these methods are configured to determine the sequences at opposite ends of a nucleic acid template and are thus referred to as “paired-end” sequencing. Some methods for the paired-end sequencing of a nucleic acid template require the degradation of at least one strand of a concatemer. In other configurations, paired-end sequencing does not require the degradation of at least one strand of a concatemer. While any paired-end sequencing methodology can be utilized in the aspects and embodiments described above and below, exemplary workflows for the paired-end clustering and sequencing of concatemers are provided, such as those illustrated in Fig. 14, to facilitate a further understanding of the disclosure herein.
  • the exemplary paired-end clustering/ sequencing workflow can begin at any of the steps of the single-end clustering workflow described above.
  • the description of the paired-end clustering/ sequencing workflow will begin with a first strand of a concatemer having been synthesized/provided and the RCA reaction having been terminated.
  • the second reaction mixture is removed by washing and replaced with an appropriate buffer/reaction mixture.
  • a plurality of primers is hybridized to the first strand of the concatemer and a plurality of second strands of the concatemer is generated by MDA.
  • the 3’ ends of the second strands of the concatemer are blocked or capped to prevent further amplification during sequencing.
  • Additional primers are hybridized to the plurality of second strands and sequencing is performed on the plurality of second strands.
  • the additional primers can comprise non-staple primers and/or staple molecules configured to act as staple primers.
  • the plurality of second strands is removed after sequencing the plurality of second strands and the first strand of the concatemer is hybridized to an additional primer to allow a sequencing reaction to occur.
  • the additional primers can comprise non-staple primers and/or staple molecules configured to act as staple primers.
  • a method of sequencing comprising: (i) providing the plurality of stabilized concatemers of any one of the second aspect and/or embodiments of the second aspect or by any of the methods of the first aspect and/or embodiments of the first aspect; and (ii) sequencing at least a first portion of the target sequence.
  • the sequencing step (ii) uses reversibly terminated nucleotides.
  • the staple molecules comprise staple primers in sequencing step (ii).
  • the staple molecules comprise staple primers in sequencing step (ii).
  • the staple primers are blocked or capped, and an additional primer is hybridized to the concatemer followed by sequencing from the additional primer.
  • the additional primer can be a non-staple primer.
  • the additional primer can be a staple molecule that is a staple primer.
  • the sequencing step (ii) comprises sequencing by synthesis (SBS).
  • the sequencing step (ii) comprises sequencing by binding (SBB)
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • SBB comprises cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes comprising the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • the examination step can involve providing a primed template nucleic acid to be used in the procedure and contacting the primed template nucleic acid with a polymerase enzyme (e.g., a DNA polymerase) and one or more test nucleotides being investigated as the possible next correct nucleotide. Further, there is a step that involves monitoring or measuring the interaction between the polymerase and the primed template nucleic acid in the presence of the test nucleotides.
  • a polymerase enzyme e.g., a DNA polymerase
  • An examination step typically includes the following substeps: (1) providing a primed template nucleic acid (i.e., a template nucleic acid molecule hybridized with a primer that optionally may be blocked from extension at its 3 ’-end); (2) contacting the primed template nucleic acid with a reaction mixture that includes a polymerase and at least one nucleotide; (3) monitoring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of the nucleotide(s) and without chemical incorporation of any nucleotide into the primed template nucleic acid; and (4) determining from the monitored interaction the identity of the next base in the template nucleic acid (i.e., the next correct nucleotide).
  • a primed template nucleic acid i.e., a template nucleic acid molecule hybridized with a primer that optionally may be blocked from extension at its 3 ’-end
  • a reaction mixture that includes a polymerase and at least one nucleotide
  • the examination step may be controlled so that nucleotide incorporation is either attenuated or accomplished. If nucleotide incorporation is attenuated, a separate incorporation step may be performed. The separate incorporation step may be accomplished without the need for monitoring, as the base has already been identified during the examination step. If nucleotide incorporation proceeds during examination, subsequent nucleotide incorporation may be attenuated by a stabilizer that traps the polymerase on the nucleic acid after incorporation. A reversibly terminated nucleotide may be used in the incorporation step to prevent the addition of more than one nucleotide during a single cycle.
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • SBB comprises cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes comprising the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • the stabilized ternary complex comprises at least one of a labeled nucleotide and a labeled polymerase. [552] Any suitable label can be used.
  • Non-limiting examples of labels that may be used in certain embodiments include: Acridine Orange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Allophycocyanin (APC), AMCA/AMCA-X, 7-Aminoactinomycin D (7-AAD), 7-Amino-4-m ethylcoumarin, 6- Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAGTM CBQCA, ATTO-TAGTM FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue Fluor
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • SBB comprises cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes comprising the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • the cyclical steps of SBB are repeated at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more times. In an exemplary embodiment, the cyclical steps of SBB are repeated 50 or more times.
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • SBB comprises cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes comprising the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • the method further comprises capping the staple primer.
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • SBB comprises cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes comprising the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • the method further comprises capping the staple primer.
  • the method further comprises hybridizing a non-staple primer to the stabilized concatemer and sequencing a second portion of the target sequence in a SBB process.
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • SBB comprises cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes comprising the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • the method further comprises capping the staple primer.
  • the method further comprises hybridizing a non-staple primer to the stabilized concatemer and sequencing a second portion of the target sequence in a SBB process.
  • the second portion of the target sequence (a) overlaps with at least part of the first portion, (b) is upstream of the first portion, or (c) is downstream of the first portion.
  • the sequencing step (ii) comprises SBB using the staple molecule as a staple primer.
  • SBB comprises cyclical steps of (A) extension: adding a reversibly terminated nucleotide to the staple primer, (B) examination: forming and detecting stabilized ternary complexes comprising the staple primer, and (C) activation: cleaving the reversible terminator from the staple primer.
  • the method further comprises capping the staple primer.
  • the method further comprises hybridizing a non-staple primer to the stabilized concatemer and sequencing a second portion of the target sequence in a SBB process. In a particular embodiment of the even further embodiments, sequencing the second portion of the target sequence is repeated a plurality of times.
  • individual stabilized concatemers of the plurality of stabilized concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • individual stabilized concatemers of the plurality of stabilized concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence on a different antisense strand of the plurality of antisense strands.
  • the method further comprises sequencing at least some of the plurality of antisense strands of the plurality of stabilized concatemers.
  • the method further comprises degrading the plurality of antisense strands of the plurality of stabilized concatemers, thereby releasing the staple molecules, optionally wherein degrading comprises digesting uracil nucleotides of the antisense strands.
  • individual stabilized concatemers of the plurality of stabilized concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence on a different antisense strand of the plurality of antisense strands.
  • the method further comprises sequencing at least some of the plurality of antisense strands of the plurality of stabilized concatemers.
  • the method further comprises degrading the plurality of antisense strands of the plurality of stabilized concatemers, thereby releasing the staple molecules, optionally wherein degrading comprises digesting uracil nucleotides of the antisense strands.
  • the degrading comprises digesting uracil with uracil-DNA glycosylase.
  • the method further comprises degrading the plurality of antisense strands of the plurality of stabilized concatemers, thereby releasing the staple molecules, optionally wherein degrading comprises digesting uracil nucleotides of the antisense strands.
  • the method further comprises contacting the plurality of concatemers with staple molecules that each hybridize to a different instance of the at least one adapter sequence on the sense strand.
  • individual stabilized concatemers of the plurality of stabilized concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence on a different antisense strand of the plurality of antisense strands.
  • the method further comprises sequencing at least some of the plurality of antisense strands of the plurality of stabilized concatemers.
  • the method further comprises degrading the plurality of antisense strands of the plurality of stabilized concatemers, thereby releasing the staple molecules, optionally wherein degrading comprises digesting uracil nucleotides of the antisense strands.
  • the method further comprises contacting the plurality of concatemers with staple molecules that each hybridize to a different instance of the at least one adapter sequence on the sense strand.
  • the method further comprises sequencing the sense strand of individual concatemers.
  • individual stabilized concatemers of the plurality of stabilized concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the first region and the second region of a staple molecule each hybridize to a different instance of the at least one adapter sequence on a different antisense strand of the plurality of antisense strands.
  • the method further comprises sequencing at least some of the plurality of antisense strands of the plurality of stabilized concatemers.
  • the method further comprises degrading the plurality of antisense strands of the plurality of stabilized concatemers, thereby releasing the staple molecules, optionally wherein degrading comprises digesting uracil nucleotides of the antisense strands.
  • the method further comprises contacting the plurality of concatemers with staple molecules that each hybridize to a different instance of the at least one adapter sequence on the sense strand.
  • the method further comprises sequencing the sense strand of individual concatemers. In a particular embodiment that is even more specific, the sequencing of the sense strand is initiated from the staple molecules hybridized to the sense strand.
  • individual stabilized concatemers of the plurality of stabilized concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the staple molecule is hybridized to the sense strand.
  • individual stabilized concatemers of the plurality of stabilized concatemers comprise a sense strand hybridized to a plurality of antisense strands.
  • the staple molecule is hybridized to the sense strand.
  • the stabilized concatemer comprises a plurality of staple molecules hybridized to the sense strand, and wherein the plurality of staple molecules is extended to form the plurality of antisense strands.
  • the average full width at half maximum (FWHM) of the stabilized concatemers does not increase by more than 5% after at least 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 150, 175, or 200 sequencing cycles. In an exemplary embodiment, the average FWHM of the stabilized concatemers, as measured across at least one dimension, does not increase by more than 5% after at least 100 sequencing cycles.
  • the average increase in FWHM of the stabilized concatemers after at least 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 150, 175, or 200 sequencing cycles, as measured in at least one dimension is reduced at least 2-fold, 3-fold, 4-fold, or 5-fold by the staple molecules.
  • the average increase in FWHM of the stabilized concatemers after 100 sequencing cycles, as measured in at least one dimension is reduced by at least 5-fold by the staple molecules.
  • the stabilized concatemer is sequenced at a read length of greater than 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 cycles. In an exemplary embodiment, the stabilized concatemer is sequenced at a read length of greater than 150 cycles.
  • the stabilized concatemer is sequenced at a read length of greater than 20, 30, 40, 50, 60, 70, 80, 80, 100, 125, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 cycles.
  • an average signal obtained from the stabilized concatemers in the final cycle of sequencing is 10%, 20%, 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% greater per pixel with the staple molecule compared to an average signal obtained from concatemers that are not stabilized by staple molecules.
  • One of the difficulties plaguing various sequencing methodologies relates to the efficient, high density loading of analyte molecules of interest, such as, for example, a concatemer or a stabilized concatemer, onto arrays.
  • Methods for loading analyte molecules of interest onto arrays are described in, for example, U.S. Pat Nos. 8,906,831 and 10,300,452, each of which is incorporated herein by reference in its entirety for all purposes and in particular for all teachings related to methods of loading analytes onto arrays.
  • a concatemer can be attached to a surface of a solid support.
  • the solid support can be made from any of a variety of materials used for analytical biochemistry, A particularly useful solid support is a particle, such as a bead or microsphere.
  • Populations of beads can be used for attachment of populations of nucleic acids.
  • the composition of a bead can vary, depending for example, on the format, chemistry and/or method of attachment to be used.
  • the geometry of a particle, such as a bead or microsphere also can correspond to a wide variety of different forms and shapes.
  • beads can be arrayed or otherwise spatially distinguished.
  • Exemplary bead-based arrays that can be used include, without limitation, a BeadChipTM Array available from Illumina, Inc. (San Diego, CA) or arrays such as those described in U.S. Pat. Nos.
  • Beads can be located at discrete locations, such as wells, on a solid-phase support, whereby each location accommodates a single bead.
  • discrete locations where beads reside can each include a plurality of beads as described, for example, in U.S. Pat. App. Pub. Nos. 2004/0263923 Al, 2004/0233485 Al, 2004/0132205 Al, or 2004/0125424 Al, each of which is incorporated herein by reference.
  • the methods can be carried out in a multiplex format whereby multiple different types of nucleic acids are detected in parallel.
  • Other types of arrays can be used instead of bead arrays, including, for example, those set forth in further detail above.
  • parallel processing can provide cost savings, time savings, and uniformity of conditions.
  • An array or method of the present disclosure can be configured to include at least 2, 10, 100, 1 x I O 3 , 1 x I O 4 , 1 x I O 3 , 1 x 10 6 , 1 x 10 9 , or more different nucleic acids.
  • an array or method of the present disclosure can be configured to include at most l x 10 9 , 1 x 10 6 , 1 x IO 3 , 1 x 10 4 , l x 10 3 , 100, 10, 2 or fewer, different nucleic acids.
  • the nucleic acids can be attached to different sites of an array. As such, the number of sites in an array can be in a range exemplified here for different nucleic acids.
  • various reagents or products set forth herein e.g., primer-template nucleic acid hybrids or stabilized ternary complexes
  • the density of a type or population of primers (or all primers) on an array can be, be about, be at least, be at least about, be at most, or be at most about, 1 x 10 10 , 2 x 10 10 , 3 x 10 10 , 4 x 10 10 , 5 x 10 10 , 6 x 10 10 , 7 x 10 10 , 8 x 10 10 , 9 x 10 10 , 1 x 10 11 , 2 x 10 11 , 3 x 10 11 , 4 x 10 11 , 5 x 10 11 , 6 x 10 11 , 7 x 10 11 , 8 x 10 11 , 9 x 10 11 , 1 x 10 12 , 2 x 10 12 , 3 x 10 12 , 4 x 10 12 , 5 x 10 12 , 6 x 10 12 , 7 x 10 12 , 8 x 10 12 , 9 x 10 12 , 1 x 10 13 , 2 x 10 13 , 3 x 10 12
  • the separation distance or average separation distance between two adjacent primers of the same type or population (or of two different types or populations) can be, be about, be at least, be at least about, be at most, or be at most about, 10 nm, 1 1 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42
  • ratios of the number of one type or population of primers and the numbers of another type or population of primers are contemplated by the present disclosure.
  • the ratio of the number of one type or population of primers and the numbers of another type or population of primers can be, be about, be at least, be at least about, be at most, or be at most about, 1 : 100, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85, 1:84, 1:83,
  • the average distance between locations on the solid support two neighboring or closest capture primers are attached to can be greater than (or less than or equal to) the length of one of the two capture primers, lengths of the two capture primers, an average length of the two capture primers, or the total length of the two capture primers (or 0.1 x, 0.2 x, 0.3 x, 0.4 x, 0.5 x, 0.6 x, 0.7 x, 0.8 x, 0.9 x, of the length).
  • An average distance between locations on the solid support two neighboring or closest amplification primers of the plurality of amplification primers are attached to can be greater than (or less than, or equal to) the length of one of the two amplification primers, lengths of the two amplification primers, an average length of the two amplification primers, or the total length of the two capture primers (or 0.1 x, 0.2 x, 0.3 x, 0.4 x, 0.5 x, 0.6 x, 0.7 x, 08 x, 0.9 x, 1.0 x, 1.1 x, 1.2 x, 1.3 x, 1.4 x, 1.5 x, 1.6 x, 1.7 x, 1.8 x, 1.9 x, 2 x, 3 x, 4 x, 5 x, 6 x, 7 x, 8 x, 9 x, 10 x of any of the lengths).
  • concatemers are synthesized via solid-phase synthesis.
  • solid-phase synthesis of concatemers results in the formation of concatemers that are tethered to a surface, such as, for example, a surface of an array.
  • Solid-phase synthesis of concatemers can be performed according to any of the configurations described above.
  • concatemers can be synthesized in solution, stabilized using staple molecules in solution, and then deposited onto a surface.
  • the embodiments characterized by stabilization of a concatemer prior to deposition onto a surface are prone to aggregation of concatemers.
  • these embodiments can further comprise providing a crowding agent, such as, for example, PEG, to inhibit the formation of aggregates of (stabilized) concatemers. Any suitable crowding agent known in the arts can be utilized.
  • the stabilized concatemers synthesized via in solution synthesis can be deposited onto an array according to any of the configurations described above.
  • concatemers are synthesized in solution, deposited onto a surface, and then stabilized using staple molecules.
  • Embodiments characterized by stabilization of a concatemer subsequent to deposition onto a surface are less prone to aggregation of concatemers.
  • these embodiments can also further comprise providing a crowding agent, such as, for example, PEG, to inhibit the formation of aggregates of (stabilized) concatemers. Any suitable crowding agent known in the arts can be utilized.
  • the concatemers synthesized via in solution synthesis can be deposited onto an array according to any of the configurations described above.
  • FIG. 1 shows a schematic of a concatemer on a flow cell surface and a magnified section of the concatemer illustrating stabilization of the concatemer using various staple molecules binding to different instances of adapter sequences of the concatemer.
  • staple molecules can be designed such that the first and second regions of a staple molecule hybridize to different instances of an adapter sequence of a concatemer.
  • staple molecules can further comprise a polynucleotide spacer of variable length between the first and second regions of a staple molecule.
  • staple molecules were configured to hybridize to separate instances of an “A” adapter sequence of a concatemer. Neither the first region nor the second region of the staple molecules comprised a mismatched and blocked 3’ end, thereby allowing the staple molecules to act as staple primers to facilitate sequencing of a first portion of the stabilized concatemer.
  • an additional primer to facilitate sequencing of at least part of the stabilized concatemer.
  • the additional primer can be a non-staple primer, or it can be an additional staple molecule configured to act as a staple primer.
  • staple molecules were configured to hybridize separate instances of a “P” adapter sequence.
  • the second region of the staple molecules were designed to further comprise a mismatched and blocked 3’ end, preventing the staple molecules from acting as staple primers.
  • an additional primer would be necessary to facilitate sequencing of the stabilized concatemer.
  • the additional primer can be a non-staple primer, or it can be a staple molecule configured to act as a staple primer. 3. Sequencing two sections concurrently with a set of staple primers:
  • staple molecules can be configured to facilitate concurrent sequencing of multiple instances of a portion of a stabilized concatemer.
  • a first staple molecule and a second staple molecule were utilized.
  • the first region of the first staple molecule was configured to have a sequence that was reverse and complementary to the first region of the second staple molecule, thereby allowing the formation of a doublestranded DNA (dsDNA) bridge between the first and second staple molecules.
  • the second region of the first and second staple molecules were configured to hybridize separate instances of an “A” adapter sequence of the concatemer.
  • the staple molecules can act as staple primers to facilitate sequencing of multiple instances of a portion of the stabilized concatemer.
  • the dsDNA bridge can be extended by incorporating a polynucleotide spacer into the staple primers.
  • the sequence of the polynucleotide spacer of the first staple molecule and the sequence of the polynucleotide spacer of the second staple molecule should be reverse and complementary to each other.
  • staple molecules can be configured such that the first region and second region are separated by a non-nucleotide polymer linker, such as, for example, polyethylene glycol (PEG) or polyamidoamine (PAMAM).
  • a non-nucleotide polymer linker such as, for example, polyethylene glycol (PEG) or polyamidoamine (PAMAM).
  • the second region of staple molecules comprising a non-nucleotide polymer linker were configured to hybridize to a plurality of “P” adapter sequences.
  • the second regions of the staple molecules comprise a mismatched and blocked 3’ end.
  • sequencing of the stabilized concatemer may require an additional primer or sequencing can be initiated by a second region of the staple molecule that does not comprise a mismatched and blocked 3’ end.
  • an additional primer may still be utilized to sequence portions of the stabilized concatemer.
  • staple molecules can be designed such that the first and second regions of a staple molecule hybridize to distinct adapter sequences of a concatemer or to adapter sequences that are partially overlapping.
  • a mixture of five separate staple molecules were configured such that the first portion of the staple molecules will bind to partially conserved portions of the “P” adapter sequence and the second portion of the staple molecules will bind to partially conserved portions of the “A” adapter sequence.
  • Figures 8-10 show the results of a concatemer sequencing experiment in which staple molecules were included in an array compared to an array where the staple molecules were omitted.
  • Figure 8 shows exam images taken at the beginning and end of sequencing demonstrating prolonged cluster stability with staple molecules. After 177 cycles of sequencing, clusters with staple molecules that bridge concatemers together (top row) outperform clusters without staple molecules (bottom row). The yellow circle in the bottom right panel highlights areas where there is considerably more streaking in the clusters without staple molecules. Conversely, when staple primers are used, the clusters maintain their structure with minimal to no evidence of streaking and are overall brighter at the end of the run (noted in the yellow circle in the top right panel). As shown in Fig. 9, sequencing data confirms clusters with staple molecules (+Staples column) improve stability during sequencing compared to a standard workflow (SOP column) omitting staple molecules.
  • SOP column standard workflow
  • the top row includes the 50 th percentile of raw intensity values for each exam (A-T-G-C) collected over 177 cycles of sequencing (“ON intensity”) versus the observed background (“OFF intensity”) averaged over the full lane of the flow cell.
  • ON intensity the average intensity
  • OFF intensity the observed background
  • the black circle highlights improved separation between ON/OFF intensity, which allows for longer read lengths through additional cycles of sequencing, in the clusters with staple molecules.
  • FWHM refers to an individual cluster’s normal distribution of fluorescent signal across some number of pixels.
  • the graph shows the average amount of fluorescence of a pixel cluster for each exam in the column direction (middle row) and in the row direction (bottom row) over the course of a sequencing run. Clusters that change shape/ size can increase the FWHM in one or both directions during a run.
  • the clusters containing staple molecules produce little change in FWHM during sequencing, whereas the standard workflow omitting staple molecules show an increase in FWHM, suggesting that the clusters are streaking or otherwise losing their shape due to the omission of staple molecules.
  • Individual fields of view are collected during sequencing and are referred to as tiles, as shown in Fig. 10.
  • a single row of tiles collected across the lane of a flow cell is referred to as a swath. In this run, four swaths of tiles were sequenced per lane.
  • the top panel indicates the total number of individual clusters sequenced in each tile.
  • the average read length of the clusters (out of 177 cycles) within each tile for the 50 th (middle row) and 25 th (bottom row) percentile is increased in the clusters containing staple molecules compared to the clusters where the staple molecules were omitted. Further, the clusters containing staple molecules demonstrate less of a gradient in read length performance, demonstrating an improved sequencing performance compared clusters omitting staple molecules.
  • Staple molecules improve the performance and read-length during single-end sequencing:
  • Figure 11 shows the results of an experiment where concatemers were stabilized using staple molecules and subsequently subjected to the single-end sequencing workflow described above using various fluorophore-labeled nucleotides. Following stabilization with staple molecules and optimization of the conjugated fluorophore, read lengths of 200 base pairs were obtained. I).
  • Example 4 Paired-end Sequencing
  • Figure 12 shows the results of a separate experiment where concatemers comprising a sense strand and a plurality of antisense strands were stabilized using staple molecules and subsequently subjected to the paired-end sequencing workflow described above. The results of this experiment demonstrate the feasibility of sequencing both strands of a concatemer following stabilization with staple molecules.

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Abstract

La présente invention concerne des procédés, des compositions et des kits permettant de constituer des concatémères stabilisés au moyen de molécules agrafe. De plus, cette invention concerne des procédés d'utilisation de concatémères stabilisés pour améliorer les résultats d'applications en aval, y compris des applications de séquençage.
PCT/US2024/035395 2023-07-06 2024-06-25 Procédés et compositions pour stabiliser des concatémères Pending WO2025010160A1 (fr)

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