Compositions and methods for capturing and amplifying target polynucleotides using modified capture primers - Patents.com

Abstract

A composition for capturing a target polynucleotide on a surface of a substrate is provided. The composition may include a plurality of capture primers bound to a surface of the substrate, the plurality of capture primers comprising modified nucleic acids, and a plurality of orthogonal capture primers bound to a surface of the substrate, the plurality of capture primers comprising modified nucleic acids. The modified nucleic acid of the capture primers may include locked nucleic acid (LNA), peptide nucleic acid (PNA), or super T. The modified nucleic acid of the orthogonal capture primers may include locked nucleic acid (LNA), peptide nucleic acid (PNA), or super T.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 128,675, entitled "Compositions and Methods for Capturing and Amplifying Target Polynucleotides Using Modified Capture Primers," filed December 21, 2020, the entire contents of which are incorporated by reference herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy is named IP-2054-PCT_SL.txt and is 4,174 bytes in size.

[0003] This application relates to cluster amplification. [Background technology]

[0004] Cluster amplification is an approach for amplifying polynucleotides, for example, intended for use in gene sequencing. Target polynucleotides are captured by primers (e.g., P5 and P7 capture primers) bound to the substrate surface in a flow cell, forming "seeds" at random locations on the surface. Amplification cycles are performed to form clusters on the surface around each seed. The clusters contain copies of the seed polynucleotide and complementary copies (which may be referred to together as "amplicons"). For example, FIG. 1 shows a schematic of polynucleotide amplification on a substrate. Capture and amplification of a single seed 111 on a substrate region 101 can result in monoclonal clusters 121 that can substantially fill the substrate region with amplicons of the seed 111 and can be easily used for sequencing-by-synthesis, or SBS (the dashed and dotted circles are intended to represent the expansion of the clusters over time). In some situations, the substrate is patterned to define regions bound to different clusters, such as wells that can be filled with the respective clusters. Summary of the Invention

[0005] Examples provided herein relate to the use of modified capture primers to capture and amplify target polynucleotides. Compositions and methods for performing such capture and amplification are disclosed.

[0006] In some examples, a composition for capturing a target polynucleotide on a surface of a substrate is provided herein. The composition may include a plurality of capture primers bound to the surface of the substrate. Each capture primer may include a modified nucleic acid. The composition may also include a plurality of orthogonal capture primers bound to the surface of the substrate. Each orthogonal capture primer may include a modified nucleic acid.

[0007] In some examples, the modified nucleic acid of the capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T. In some examples, the capture primer further comprises a deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and the DNA are distributed between the 5' end and the 3' end of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the capture primer and the DNA is located at the 3' end of the capture primer.

[0008] In some examples, the modified nucleic acid of the orthogonal capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T. In some examples, the orthogonal capture primer further comprises a deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and the DNA are distributed between the 5' end and the 3' end of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the orthogonal capture primer and the DNA is located at the 3' end of the orthogonal capture primer.

[0009] In some examples, the composition further comprises a first target polynucleotide, each of which comprises a first adaptor that is complementary to the capture primer and a second adaptor that is complementary to the orthogonal capture primer. The composition may further comprise a second target polynucleotide that is complementary to each of the first target polynucleotides.

[0010] In some examples, a first adaptor of some of the first target polynucleotides hybridizes to each of the capture primers to form a first duplex, and a second adaptor of some of the first target polynucleotides hybridizes to each of the orthogonal capture primers to form a second duplex. In some examples, the first and second duplexes each have a melting temperature (Tm) greater than the Tm of a third duplex formed by hybridization of the second target polynucleotide to each of the first target polynucleotides that are complementary to the second target polynucleotide. In some examples, the first and second duplexes each have a Tm that is about 80°C to about 110°C. In some examples, the first and second duplexes each have a Tm that is about 85°C to about 105°C. In some examples, the first and second duplexes each have a Tm that is about 90°C to about 100°C.

[0011] In some instances, substantially none of the second target polynucleotides hybridize in solution to any of the first target polynucleotides.

[0012] In some examples, the composition further comprises about 1% to about 100% formamide (% v / v), or about 5% to about 80% formamide (% v / v).

[0013] In some examples, the composition further comprises about 100 to about 800 mM Na+, or about 200 to about 800 mM Na+.

[0014] In some examples, the capture primer is a modified P5 capture primer and the orthogonal capture primer is a modified P7 capture primer.

[0015] In some examples, each of the capture primers comprises from about 5 to about 20 modified nucleic acids and each of the orthogonal capture primers comprises from about 5 to about 20 modified nucleic acids.

[0016] In some examples, each of the capture primers comprises at least about 9 modified nucleic acids, each of the orthogonal capture primers comprises at least about 9 modified nucleic acids, or each of the capture primers comprises at least about 12 modified nucleic acids, each of the orthogonal capture primers comprises at least about 12 modified nucleic acids, or each of the capture primers comprises at least about 15 modified nucleic acids, and each of the orthogonal capture primers comprises at least about 15 modified nucleic acids.

[0017] In some examples, a method for capturing and amplifying a target polynucleotide on a surface of a substrate is provided herein. The method may include contacting a composition with a fluid. The composition may include a plurality of capture primers bound to a surface of a substrate, each of the capture primers comprising a modified nucleic acid, and a plurality of orthogonal capture primers bound to a surface of the substrate, each of the orthogonal capture primers comprising a modified nucleic acid. The fluid may include a first target polynucleotide, each of the first target polynucleotides comprising a first adaptor that is complementary to the capture primer and a second adaptor that is complementary to the orthogonal capture primer, and a second target polynucleotide that is complementary to each of the first target polynucleotides. The method may include hybridizing a first adaptor of some of the first target polynucleotides to each of the capture primers to form a first duplex while inhibiting hybridization of a second target polynucleotide in the fluid to the first target polynucleotide, hybridizing a second adaptor of some of the first target polynucleotides to each of the orthogonal capture primers to form a second duplex, and then amplifying the first target polynucleotides, where amplifying includes generating an amplicon for each of the first target polynucleotides.

[0018] In some examples, the modified nucleic acid of the capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T. In some examples, the capture primer further comprises a deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and the DNA are distributed between the 5' end and the 3' end of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the capture primer and the DNA is located at the 3' end of the capture primer.

[0019] In some examples, the modified nucleic acid of the orthogonal capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T. In some examples, the orthogonal capture primer further comprises a deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and the DNA are distributed between the 5' end and the 3' end of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the orthogonal capture primer and the DNA is located at the 3' end of the orthogonal capture primer.

[0020] In some examples, the first and second duplexes each have a melting temperature (Tm) greater than the Tm of a third duplex formed by hybridization of the second target polynucleotide to each of the first target polynucleotides that are complementary to the second target polynucleotide. In some examples, the first and second duplexes each have a Tm that is about 80°C to about 110°C. In some examples, the first and second duplexes each have a Tm that is about 85°C to about 105°C. In some examples, the first and second duplexes each have a Tm that is about 90°C to about 100°C.

[0021] In some instances, substantially none of the second target polynucleotides hybridize in solution to any of the first target polynucleotides.

[0022] In some examples, hybridization is carried out in about 1% to about 100% formamide (% v / v), or about 5% to about 80% formamide (% v / v).

[0023] In some examples, hybridization is carried out at about 100 to about 800 mM Na+, or about 200 to about 800 mM Na+.

[0024] In some examples, the capture primer is a modified P5 capture primer and the orthogonal capture primer is a modified P7 capture primer.

[0025] In some examples, each of the capture primers comprises about 5 to about 20 modified nucleic acids, and each of the orthogonal capture primers comprises about 5 to about 20 modified nucleic acids. In some examples, each of the capture primers comprises at least about 9 modified nucleic acids, and each of the orthogonal capture primers comprises at least about 9 modified nucleic acids, or each of the capture primers comprises at least about 12 modified nucleic acids, and each of the orthogonal capture primers comprises at least about 12 modified nucleic acids, or each of the capture primers comprises at least about 15 modified nucleic acids, and each of the orthogonal capture primers comprises at least about 15 modified nucleic acids.

[0026] It should be understood that any corresponding features / embodiments of each of the aspects of the present disclosure as described herein may be implemented together in any suitable combination, and that any feature / embodiment from any one or more of these aspects may be implemented with any of the features of the other aspects as described herein in any suitable combination to obtain advantages as described herein. [Brief description of the drawings]

[0027] [Figure 1] 1 shows a schematic diagram of amplification of a polynucleotide on a substrate. [Figure 2A] 1 illustrates generally the compositions and operations in a process flow for capturing a polynucleotide on a substrate using a capture primer as known in the art. [Figure 2B] 1 illustrates generally the compositions and operations in a process flow for capturing a polynucleotide on a substrate using a capture primer as known in the art. [Figure 2C] 1 illustrates generally the compositions and operations in a process flow for capturing a polynucleotide on a substrate using a capture primer as known in the art. [Figure 3A] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 3B] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 3C] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 3D] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 3E] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 3F] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 3G] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 3H] 1A-1C are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for capture and amplification of polynucleotides on a substrate using capture primers of the present invention. [Figure 4] 1 shows a schematic of an exemplary duplex between a polynucleotide and one of the capture primers of the invention, according to some examples. [Figure 5A]1 is a plot showing exemplary effects of conditions on capture of polynucleotides by capture primers of the invention. [Figure 5B] 1 is a plot showing exemplary effects of conditions on capture of polynucleotides by capture primers of the invention. [Figure 5C] 1 is a plot showing exemplary effects of conditions on capture of polynucleotides by capture primers of the invention. [Figure 6] 1 shows an exemplary flow chart of operations in a method for capturing and amplifying a polynucleotide using a capture primer of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Examples provided herein relate to capturing and amplifying polynucleotides using modified capture primers. Compositions and methods for performing such capture and amplification are disclosed.

[0029] The modified capture primers provided herein can hybridize more strongly with target polynucleotides than previously known capture primers, thereby increasing the efficiency with which target polynucleotides are captured on substrates. Illustratively, due to the stronger binding, clusters can be prepared on substrates using lower concentrations of target polynucleotides compared to previously known capture primers. Under conditions in which polynucleotide capture and amplification using previously known primers is performed, for example in the manner described further below with reference to Figures 2A-2C, hybridization and solution-based annealing at the surface act as competing processes, resulting in some polynucleotides being unavailable for capture or amplification. In comparison, in the method of the present invention described with reference to Figures 3A-3H, 4, 5A-5C, and 6, the capture primers of the present invention allow for the method to be performed under conditions that reduce or inhibit solution-based annealing as a competing process, thereby substantially increasing the availability of polynucleotides for capture and amplification, and allowing the use of higher concentrations of target polynucleotides to prepare clusters on substrates compared to previously known capture primers, and reducing the overall seeding time.

[0030] We first provide a brief explanation of some of the terms used herein, followed by a description of some exemplary compositions and exemplary methods for capturing and amplifying polynucleotides using the present capture primers.

[0031] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting. Furthermore, the use of the term "having" as well as other forms such as "have", "has" and "had" is not limiting. As used herein, in transitional phrases or claim texts, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrase "having at least" or "including at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may include additional features or components.

[0032] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to account for small variations due to processing variations and the like. For example, they can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0033] As used herein, "hybridize" refers to the non-covalent binding of a first polynucleotide to a second polynucleotide along the length of the polynucleotides to form a double stranded "duplex." For example, two DNA polynucleotide strands can associate through complementary base pairing. The strength of association between a first and a second polynucleotide increases with the complementarity between the nucleotide sequences in the polynucleotides. The strength of hybridization between polynucleotides can be characterized by the melting temperature (Tm) at which 50% of the polynucleotide strands of the duplex separate. Polynucleotides that are "partially" hybridized to each other mean that they have sequences that are complementary to each other, but such sequences hybridize to each other along only a portion of their length to form a partial duplex. Polynucleotides that are "unable" to hybridize include those that are physically separated from each other with an insufficient number of bases that can contact each other to hybridize to each other.

[0034] As used herein, the term "nucleotide" is intended to mean a molecule that includes a sugar moiety, a backbone component that includes at least one phosphate group, and, in some instances, a nucleobase. A nucleotide that lacks a nucleobase may be referred to as "abasic." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine triphosphate (DTPA ... Deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0035] As used herein, the term "nucleotide" is also intended to encompass any "nucleotide analogs", i.e., to refer to a type of nucleotide that includes a modified nucleobase, sugar moiety, and / or backbone moiety (such as a phosphate or amide) compared to a naturally occurring nucleotide. Nucleotide analogs may also be referred to as "modified nucleic acids". Exemplary modified nucleobases include inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-aminoguanine, 2 ... Examples of such nucleotide analogs include azothymine, 5-uracil, 4-thiouracil, 8-halogen adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halogen substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. As is known in the art, certain nucleotide analogs, such as adenosine 5'-phosphosulfate, cannot be incorporated into polynucleotides. The backbone moiety of a nucleotide can include any suitable number of phosphates, such as 3, 4, 5, 6, or more than 6 phosphates. Nucleotide analogs also include locked nucleic acid (LNA), peptide nucleic acid (PNA), and 5-hydroxybutynyl-2'-deoxyuridine ("Super T"). LNA contains an RNA-like backbone in which the ribose moiety contains an additional bridge connecting the 2' oxygen and the 4' carbon. PNA contains a backbone that contains N-(2-aminoethyl)-glycine units linked by peptide bonds and nucleobases attached to the backbone via methylene bridges and carbonyl groups.Super-T contains a 5-hydroxybutynyl-2'-deoxyuridine nucleobase. Exemplary structures of LNA, PNA, and Super-T are shown below.

[0036] [ka]

[0037] As used herein, the term "polynucleotide" refers to a molecule that comprises a sequence of nucleotides linked together. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogs, such as locked nucleic acid (LNA) and peptide nucleic acid (PNA). A polynucleotide can include a single-stranded sequence of nucleotides, such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides, such as double-stranded DNA, or a mixture of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA and products of PCR and amplification. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. A polynucleotide can include non-naturally occurring DNA, such as enantiomeric DNA, LNA, or PNA. The exact sequence of nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: genes or gene fragments (e.g., probes, primers, expressed sequence tags (ESTs), or serial analysis of gene expression (SAGE) tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers or amplified copies of any of the foregoing. Polynucleotides may have "chimeric" structures that include adjacent sections of different types of polynucleotides, such as adjacent sections of PNA and RNA, DNA and RNA, DNA and PNA.

[0038] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles a polynucleotide by polymerizing nucleotides into a polynucleotide. A polymerase can bind to a primed single-stranded target polynucleotide and grow it by successively adding nucleotides to the primer to form a "complementary copy" polynucleotide having a sequence complementary to that of the target polynucleotide. Another polymerase, or the same polymerase, can then form a copy of the target nucleotide by forming a complementary copy of the complementary copy polynucleotide. Any such copy may be referred to herein as an "amplicon." A DNA polymerase can bind to a target polynucleotide and then move downstream while growing the target polynucleotide by successively adding nucleotides to the free hydroxyl group at the 3' end of the polynucleotide chain (amplicon growth). A DNA polymerase can synthesize a complementary DNA molecule from a DNA template, and an RNA polymerase can synthesize an RNA molecule from a DNA template (transcription). Polymerases may use short RNA or DNA strands (primers) to initiate strand growth. Some polymerases may displace the strand upstream of the site where they add a base to the strand. Such polymerases may also be said to be strand displacing, that is, to have the activity of removing a complementary strand from the template strand that is read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, the large fragment of Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase, or sequencing grade T7 exo-polymerase. Some polymerases cleave the strand in front of them and effectively replace it with the growing strand (5' exonuclease activity). Some polymerases have the activity of degrading the strand behind them (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0039] As used herein, the term "primer" is defined as a polynucleotide to which a nucleotide can be added via a free 3'OH group. A primer may include a 3' block that prevents polymerization until the block is removed. A primer may include a modification at the 5' end to allow a coupling reaction or to allow the primer to be attached to another moiety. A primer may include one or more moieties, such as 8-oxo-G, that can be cleaved under appropriate conditions, such as UV light, chemicals, enzymes, etc. The length of a primer may be any suitable number of bases long and may include a suitable combination of natural and / or non-natural nucleotides. A target polynucleotide may include an "adapter" that hybridizes to (has a sequence complementary to) the primer and can be amplified to generate a complementary copy polynucleotide by adding a nucleotide to the free 3'OH group of the primer. A "capture primer" is intended to mean a primer that can bind to a substrate and hybridize to a first adapter of a target polynucleotide, and an "orthogonal capture primer" is intended to mean a primer that can bind to a substrate and hybridize to a second adapter of that target polynucleotide. The first adaptor may have a sequence complementary to that of the capture primer, and the second adaptor may have a sequence complementary to that of the orthogonal capture primer. The capture primer and the orthogonal capture primer may have independent sequences that are different from each other. Additionally, the capture primer and the orthogonal capture primer may differ from each other in at least one other characteristic. For example, the capture primer and the orthogonal capture primer may have different lengths from each other, either the capture primer or the orthogonal capture primer may include a non-nucleic acid portion (such as a blocking group or a removal portion) that the other of the capture primer or the orthogonal capture primer does not have, or may include any suitable combination of such characteristics. A "modified capture primer" is a capture primer or an orthogonal capture primer that includes multiple nucleic acid analogs, such as LNA, PNA, or super T. The modified capture primer may further include multiple naturally occurring nucleic acids, such as, but not limited to, DNA.

[0040] As used herein, the term "substrate" refers to a material used as a carrier for the compositions described herein. Exemplary substrate materials may include glass, silica, plastic, quartz, metal, metal oxide, organosilicate (e.g., polyhedral organosilsesquioxane (POSS)), polyacrylate, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of a POSS may be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some examples, substrates used in this application include silica-based substrates such as glass, condensed silica, or other silica-containing materials. In some examples, substrates may include silicon, silicon nitride, or hydrogenated silicones. In some examples, substrates used in this application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Exemplary plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material or a combination thereof. In certain examples, the substrate has at least one surface including glass or a silicon-based polymer. In some examples, the substrate may include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface including a metal oxide. In one example, the surface includes tantalum oxide or tin oxide. Acrylamide, enone, or acrylate may also be utilized as a substrate material or component. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramic, polyimide, quartz, resin, polymer, quartz, resin, polymer, and copolymer. In some examples, the substrate and / or substrate surface may be or include quartz. In some other examples, the substrate and / or substrate surface may be or include a semiconductor such as GaAs or ITO.The foregoing list is intended to be illustrative, but not limiting, of the present application. The substrate may comprise a single material or multiple different materials. The substrate may be a composite or laminate. In some examples, the substrate comprises an organosilicate material. The substrate may be flat, round, spherical, rod-shaped, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0041] In some examples, the surface is a patterned surface. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more regions can be features in which one or more capture primers are present. The features can be separated by gap regions in which no capture primers are present. In some examples, the pattern can be an xy format of features in rows and columns. In some examples, the pattern can be a repeating sequence of features and / or gap regions. In some examples, the pattern can be a random sequence of features and / or gap regions. In some examples, the substrate includes an array of wells (recesses) in a surface. The wells can be provided with substantially vertical sidewalls. The wells can be manufactured as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As is understood in the art, the techniques used depend on the composition and shape of the array substrate.

[0042] The features within the patterned surface of the substrate may include wells in which a covalently bonded gel, such as poly(N-(5-azidoacetamylpentyl)acrylamide-co-acrylamide) (PAZAM), is patterned and arrayed on glass, silicon, plastic, or other suitable material. This process creates a gel pad used for sequencing, which may be stable over many cycles of sequencing operations. Covalently bonding the polymer to the wells may be useful to maintain the gel in the structured features throughout the life of the structured substrate during various applications. However, in many instances, the gel does not need to be covalently bonded to the wells. For example, in some conditions, silane-free acrylamide (SFA) that is not covalently bonded to any part of the structured substrate may be used as the gel material.

[0043] In certain other embodiments, a structured substrate can be created by patterning a suitable material with wells (e.g., microwells or nanowells), coating the patterned material with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as the azido form of SFA (azido-SFA)), and polishing the gel-coated material, for example by chemical or mechanical polishing, thereby retaining the gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions of the surface of the structured substrate between the wells. Primers can then be bound to the gel material. A solution of target polynucleotides (e.g., a fragmented human genome or a portion thereof) can then be contacted with the polished substrate such that individual target nucleotides are seeded into individual wells through interaction with primers attached to the gel material, but the target polynucleotides do not occupy the interstitial regions due to the absence or inactivity of the gel material. Amplification of the target polynucleotides can be confined within the wells due to the absence or inactivity of the gel in the interstitial regions, preventing outward migration of clusters. The process is easily manufacturable, scalable, and utilizes conventional micro- or nano-fabrication methods.

[0044] Patterned substrates may include, for example, wells etched into a slide or chip. The pattern of wells etched and geometric shapes may be of a variety of different shapes and sizes, and such features may be physically or functionally separable from one another. Particularly useful substrates with such structural features include patterned substrates that allow selection of the size of solid particles, such as microspheres. An exemplary patterned substrate with these characteristics is the etched substrate used in conjunction with BEAD array technology (Illumina, Inc., San Diego, Calif.).

[0045] In some examples, the substrates described herein form at least a part of a flow cell, or are located within or attached to a flow cell. A flow cell may include a flow chamber divided into multiple lanes or multiple sectors. Examples of flow cells and substrates for the manufacture of flow cells that may be used in the methods and compositions described herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, Calif.).

[0046] As used herein, the term "directly", when used in reference to a layer covering the surface of a substrate, is intended to mean that the layer covers the surface of the substrate without a significant intermediate layer, such as, for example, an adhesive layer or a polymer layer. A layer that directly covers a surface can be attached to this surface through any chemical or physical interaction, such as covalent or non-covalent bonds.

[0047] As used herein, the term "immobilized" when used in reference to a polynucleotide is intended to mean direct or indirect attachment to a substrate via a covalent or non-covalent bond. In certain instances, a covalent bond may be used, or any other suitable bond may be used where the polynucleotide remains stationary or attached to the substrate under the conditions for which the substrate is intended to be used, such as for amplification or sequencing of the polynucleotide. A polynucleotide used as a capture primer or as a target polynucleotide may be immobilized such that the 3' end is available for enzymatic extension and at least a portion of the sequence can hybridize to a complementary sequence. Immobilization may occur via hybridization to a surface-attached oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide may be in a 3' to 5' orientation. Alternatively, immobilization may occur by means other than base-pairing hybridization, such as a covalent bond. Illustratively, a chemical functional group may be incorporated at the 5' end of one of the capture primers of the invention via a chemical linker. The chemical functional groups on the capture primer can be attached to the substrate surface via any suitable combination of one or more non-covalent interactions (e.g., electrostatic, metal-ligand binding, hybridization, etc.) or covalent interactions (e.g., copper click chemistry, copper-free click chemistry, etc.).

[0048] As used herein, the term "array" refers to a collection of substrate regions that can be distinguished from one another according to their relative positions. Different molecules (such as polynucleotides) in different regions of an array can be distinguished from one another according to the position of the regions in the array. An individual region of an array can contain one or more molecules of a particular type. For example, a substrate region can contain a single target polynucleotide with a particular sequence, or a substrate region can contain several polynucleotides with the same sequence (or its complementary sequence). Regions of an array can contain different features from one another on the same substrate. Exemplary features include, but are not limited to, wells in the substrate, beads (or other particles) in or on the substrate, protrusions from the substrate, ridges on the substrate, or channels in the substrate. Regions of an array can each comprise different regions on different substrates. Different molecules attached to separate substrates can be identified according to the position of the substrate on the surface to which the substrates are associated, or according to the position of the substrate in a liquid or gel. Exemplary arrays in which separate substrates are disposed on a surface include, but are not limited to, those with beads in wells.

[0049] As used herein, the term "plurality" is intended to mean a population of two or more distinct members. A plurality can range in size from small, medium, large, to very large. A small size plurality can range, for example, from a few members to tens of members. A medium size plurality can range, for example, from tens of members to about 100 members or hundreds of members. A large plurality can range, for example, from about hundreds of members to about 1000 members, thousands of members, and tens of thousands of members. A very large plurality can range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, a plurality can range in size from 2 to 100 million or more, as well as all sizes measured as the number of members between and above the exemplary ranges above. Exemplary polynucleotide complexes include, for example, about 1×10 5 That's it, 5 x 10 5 or more, or 1×10 6A population of 1 or more distinct polynucleotides is included. Thus, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the plurality can be set, for example, by the theoretical diversity of polynucleotide sequences in a sample.

[0050] As used herein, the term "double-stranded," when used in reference to a polynucleotide, is intended to mean that all or substantially all of the nucleotides in a polynucleotide are hydrogen bonded to their respective nucleotides in a complementary polynucleotide. A "partially" double-stranded polynucleotide has at least about 10%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of its nucleotides, but fewer than all of the nucleotides, are hydrogen bonded to nucleotides in a complementary polynucleotide.

[0051] As used herein, the term "single-stranded" when used in reference to a polynucleotide means that none of the nucleotides in a polynucleotide are hydrogen bonded to their respective nucleotides in a complementary polynucleotide. A polynucleotide that is "not capable of hybridizing" to another polynucleotide may be single-stranded.

[0052] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the object of an analysis or action, and may be referred to as a "library polynucleotide," "template polynucleotide," or "library template." The analysis or action may include subjecting the polynucleotide to capture, amplification, sequencing, and / or other procedures. The target polynucleotide may include a nucleotide sequence that is added to the target sequence to be analyzed. For example, the target polynucleotide may include one or more adapters, such as adapters that function as primer binding sites, the flanking portions of which are the target polynucleotide sequence to be analyzed. The target polynucleotide hybridized to the capture primer may include nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide, such that not all of the target polynucleotide is suitable for extension. In certain examples, multiple target polynucleotides may have first and second adapters that are the same as each other, although they may have different sequences from each other. Two adapters that may flank a particular target polynucleotide sequence may have the same sequence as each other, or complementary sequences to each other, or the two adapters may have different sequences. Thus, a species in a plurality of target polynucleotides may include, for example, a region of known sequence flanked by regions of unknown sequence that are evaluated by sequencing (e.g., SBS). In some examples, the target polynucleotide carries an adapter at a single end, and such an adapter may be located at either the 3' or 5' end of the target polynucleotide. The target polynucleotide may be used without an adapter, in which case the primer binding sequence may directly use a sequence present in the target polynucleotide.

[0053] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in the terminology is not intended to indicate any particular difference in size, sequence, or other properties, unless otherwise specified. For clarity of explanation, when describing a particular method or composition that includes several polynucleotide species, different terms may be used to distinguish one species of polynucleotide from another species.

[0054] As used herein, the term "amplicon" when used in reference to a nucleic acid refers to a product of the replication of a nucleic acid, which product has a nucleotide sequence that is substantially the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. "Amplification" and "amplifying" refer to the process of creating an amplicon of a polynucleotide. A first amplicon of a target polynucleotide can typically be a complementary copy. Further amplicons are copies made from the target polynucleotide or the first amplicon after the generation of the first amplicon. Subsequent amplicons can have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It will be understood that a small number of mutations of a polynucleotide (e.g., due to amplification artifacts) may occur when generating an amplicon of that polynucleotide.

[0055] A substrate region that contains substantially only amplicons of a given polynucleotide may be referred to as "monoclonal," while a substrate region that contains amplicons of polynucleotides having sequences that differ from one another may be referred to as "polyclonal." A polyclonal region of a substrate may contain different subregions that are each monoclonal. Each such monoclonal region may correspond to a "cluster" generated from a "seed," whether within a larger polyclonal region or by itself. A "seed" may refer to a single target polynucleotide, while a "cluster" may refer to a collection of amplicons of that target polynucleotide.

[0056] Compositions and methods for capturing and amplifying polynucleotides Examples provided herein relate to compositions and methods for capturing and amplifying target polynucleotides using modified capture primers that can be used under solution-based conditions that reduce or inhibit annealing while allowing the modified capture primer to capture target polynucleotides from solution. In comparison, previously known capture primers can be used under conditions where solution-based annealing competes with capture of target polynucleotides from solution, such competition reducing the availability of target polynucleotides for capture.

[0057] For example, Figures 2A-2C show schematic compositions and operations in a process flow for capturing polynucleotides on a substrate using a capture primer known in the art. The composition 2000 shown in Figure 2A includes a substrate 200 including a capture primer 231 and an orthogonal capture primer 232 bound thereto, and a fluid (solution) containing a target polynucleotide, e.g., a polynucleotide intended for capture, amplification, and sequencing using sequencing-by-synthesis (SBS). The target polynucleotide may include or be provided in the form of a duplex (illustratively, duplex D1, D2, and D3) between target polynucleotides having complementary sequences to each other. For example, duplex D1 may include a first target polynucleotide comprising sequence 211, a first adaptor 221, and a second adaptor 222, and a second target nucleotide that is complementary to the first target polynucleotide, e.g., comprises complementary sequence 211', a first complementary adaptor 221', and a second complementary adaptor 222'. Similarly, duplex D2 may include a first target polynucleotide comprising sequence 212, a first adaptor 221, and a second adaptor 222, and a second target nucleotide that is complementary to the first target polynucleotide, e.g., comprises complementary sequence 212', a first complementary adaptor 221', and a second complementary adaptor 222'. For example, duplex D3 may include a first target polynucleotide comprising sequence 213, a first adaptor 221, and a second adaptor 222, and a second target polynucleotide that is complementary to the first target polynucleotide, e.g., comprises complementary sequence 212', a first complementary adaptor 221', and a second complementary adaptor 222'. Sequences 211, 212, and 213 may be different from one another, and it may be desirable to determine their sequences using SBS. First adaptor 221 may be complementary to capture primer 231 such that they can hybridize under suitable conditions, and second adaptor 222 may be complementary to orthogonal capture primer 232 such that they can hybridize under appropriate conditions.Although not specifically shown, each capture primer 231 or each orthogonal capture primer 232 can include a cleavable moiety, such as 8-oxo-G.

[0058] The capture primer 231 can be, for example, a P5 capture primer, and the orthogonal capture primer 232 can be, for example, a P7 capture primer. A P5 capture primer commercially available from Illumina, Inc. (San Diego, Calif.) has the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 1). A P7 capture primer commercially available from Illumina, Inc. has the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 2). The first adaptor 221 can be, for example, a complementary P5 adaptor (cP5), and the second adaptor 222 can be, for example, a complementary P7 adaptor (cP7). A complementary P5 adaptor commercially available from Illumina, Inc. (San Diego, Calif.) has the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 3). A complementary P7 adaptor, commercially available from Illumina, Inc. (San Diego, Calif.), can have the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO:4).

[0059] Prior to attempting to capture the target polynucleotide on substrate 200 for subsequent amplification and sequencing, duplexes D1, D2, and D3 are melted in a manner as shown in Figure 2B to yield a single-stranded target polynucleotide having a first adaptor 221 available for hybridization to a capture primer 231 and a second adaptor 222 available for hybridization to an orthogonal capture primer 232. Such melting may be performed, for example, by altering the temperature and / or composition of the solution in which the duplexes D1, D2, and D3 are placed. For example, duplexes D1, D2, and D3 may be exposed to a sufficient amount of formamide solution, such as about 1% to 100% formamide (% v / v), or about 5% to about 80% formamide (% v / v), or about 10% to about 50% formamide (% v / v), or about 1% to about 20% formamide (% v / v), to dissociate the duplexes at the current solution temperature. Additionally or alternatively, the temperature of the solution may be raised above the melting temperature (Tm) of the duplexes in the current solution composition. It will be understood that the particular Tm of the duplex may depend on the composition of the solution (e.g., salt (Na+) concentration and formamide concentration, if present), as well as the length and sequence of the polynucleotides in the duplex.

[0060] As described with reference to FIG. 2B, after the duplexes D1, D2, and D3 are melted, the first adaptor 221 may be available to hybridize to the capture primer 231 and the second adaptor 222 may be available to hybridize to the orthogonal capture primer 232. However, the solution and temperature conditions that caused such melting may also inhibit the first adaptor 221 from hybridizing to the capture primer 231 and inhibit the second adaptor 222 from hybridizing to the orthogonal capture primer 232. To facilitate such hybridization and thus the capture of the target polynucleotide on the substrate 200, the temperature and / or composition of the solution into which the target polynucleotide is again placed can be altered. For example, a sufficient amount of formamide may be removed from the solution at the current solution temperature. Additionally or alternatively, the temperature of the solution may be reduced below the melting temperature (Tm) of the duplex between (a) the first adaptor 221 and the capture primer 231, and (b) the second adaptor 222 and the orthogonal capture primer 232 in the current solution composition.

[0061] In the non-limiting example shown in Figure 2C, this further change in conditions, e.g., solution temperature and / or composition, facilitates hybridization between second adaptor 222 attached to sequence 211 and one of capture primers 232, and between first adaptor 221 attached to sequence 213 and one of capture primers 231. However, this further change in conditions also promotes annealing between target polynucleotides in solution. For example, first and second target polynucleotides, each of which includes sequences complementary to one another, may reanneal to one another along their lengths, such as, illustratively, sequences 212 and 212' reannealing along their lengths to reform duplex D2. Additionally, first and second target polynucleotides that include sequences that are not complementary to one another may partially anneal to one another, illustratively at adapters 221 and 221' and adapters 222 and 222', such as non-complementary sequences 214 and 211' that anneal substantially only at their adapters to form duplex D4 in the manner shown in Figure 2C. Target polynucleotides that anneal to form duplexes in solution, illustratively target polynucleotides that include sequences 212 and 214 in the non-limiting example shown in Figure 2C, are unavailable to hybridize with capture primer 231 or orthogonal capture primer 232, and therefore cannot be captured, much less amplified or sequenced.

[0062] In comparison, the modified capture primers of the present invention can hybridize to the adaptors of the target polynucleotides under conditions in which annealing does not substantially occur in the solution, such as, for example, salt concentrations, formamide content, and / or temperatures in which annealing does not substantially occur in the solution. Thus, the target polynucleotides may not substantially anneal in such a solution and may form double strands and thus remain available to hybridize with the capture primers present, which may then be amplified and sequenced or otherwise used as desired. Additionally, the modified capture primers of the present invention can hybridize to the adaptors of the target polynucleotides under conditions that inhibit any non-specific or non-productive hybridization of the target polynucleotides to the capture primers or adaptors in the solution, such that it can be expected that substantially all hybridization events are productive and result in double strands that can be sequenced.

[0063] 3A-3H are schematic diagrams illustrating exemplary compositions and operations in an exemplary process flow for the capture and amplification of polynucleotides on a substrate using the capture primers of the present invention. The composition 3000 shown in FIG. 3A includes a substrate 300 including a capture primer 331 and an orthogonal capture primer 332 bound thereto, and a fluid (solution) containing a target polynucleotide, e.g., a polynucleotide intended for capture, amplification, and sequencing using sequencing by synthesis (SBS). The target polynucleotide may include or be provided in the form of a duplex (illustratively, duplexes D5, D6, and D7) between target polynucleotides having sequences complementary to each other. For example, in a manner similar to that described with reference to Figure 2A, duplex D5 may comprise a first target polynucleotide comprising sequence 311, a first adaptor 321, and a second adaptor 322, and a second target nucleotide that is complementary to the first target polynucleotide, e.g., comprises complementary sequence 311', a first complementary adaptor 321', and a second complementary adaptor 322'. For example, duplex D6 may comprise a first target polynucleotide comprising sequence 312, a first adaptor 321, and a second adaptor 322, and a second target nucleotide that is complementary to the first target polynucleotide, e.g., comprises complementary sequence 312', a first complementary adaptor 321', and a second complementary adaptor 322'. For example, duplex D7 may include a first target polynucleotide comprising sequence 313, a first adaptor 321, and a second adaptor 322, and a second target polynucleotide that is complementary to the first target polynucleotide, e.g., comprises complementary sequence 312', a first complementary adaptor 321', and a second complementary adaptor 322'. Sequences 311, 312, and 313 may be different from one another, and it may be desirable to determine their sequences using SBS. First adaptor 321 may be complementary to capture primer 331 such that it can hybridize under suitable conditions, and second adaptor 322 may be complementary to modified orthogonal capture primer 332 such that it can hybridize under appropriate conditions.

[0064] Each of the capture primers 331 may include multiple nucleic acids that increase the Tm of the duplex between the capture primer 331 and the first adaptor 321 compared to the Tm of the duplex between the first adaptor 321 and the complementary first adaptor 321'. For example, the modified nucleic acid of the capture primer 331 may include a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T, each of which may be expected to increase the Tm of the duplex between the capture primer 331 and the first adaptor 321 compared to the Tm of the duplex between the first adaptor 321 and the complementary first adaptor 321'. The capture primer 331 may further include DNA. The modified nucleic acid and DNA may be distributed between the 5' and 3' ends of the capture primer. For example, the sequence of the capture primer 331 may include one or more DNA molecules, followed by one or more modified nucleic acids, followed by one or more DNA molecules, followed by one or more modified nucleic acids, etc. Alternatively, the modified nucleic acid can be placed at the 5' end of the capture primer and the DNA can be placed at the 3' end of the capture primer, for example, in a manner as described below with reference to Figure 4. Although not specifically shown, each capture primer 331 can also include a cleavable moiety, such as 8-oxo-G.

[0065] Similarly, each of the orthogonal capture primers 332 may include a plurality of modified nucleic acids that increase the Tm of the duplex between the orthogonal capture primer 332 and the second adaptor 322 compared to the Tm of the duplex between the second adaptor 322 and the complementary second adaptor 322'. For example, the modified nucleic acids of the orthogonal capture primer 332 may include a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T, each of which may be expected to increase the Tm of the duplex between the orthogonal capture primer 332 and the second adaptor 322 compared to the Tm of the duplex between the second adaptor 322 and the complementary second adaptor 322'. The orthogonal capture primer 332 may further include a deoxyribonucleic acid (DNA). The modified nucleic acid and DNA may be distributed between the 5' and 3' ends of the orthogonal capture primer. For example, the sequence of the orthogonal capture primer 332 may include one or more DNA molecules, followed by one or more modified nucleic acids, followed by one or more DNA molecules, followed by one or more modified nucleic acids, etc. Alternatively, the modified nucleic acid can be placed at the 5' end of the capture primer and the DNA can be placed at the 3' end of the capture primer, for example, in a manner as described below with reference to Figure 4. Although not specifically shown, each of the orthogonal capture primers 332 can also include a cleavable moiety, such as 8-oxo-G.

[0066] It should be noted that the modified nucleic acid of capture primer 331 can be, but is not necessarily, the same type of modified nucleic acid as orthogonal capture primer 332. Illustratively, capture primer 331 and orthogonal capture primer 332 can comprise LNA, capture primer 331 and orthogonal capture primer 332 can comprise PNA, or capture primer 331 and orthogonal capture primer 332 can comprise super-T. Alternatively, capture primer 331 can comprise LNA while orthogonal capture primer 332 can comprise PNA or super-T, capture primer 331 can comprise PNA while orthogonal capture primer 332 can comprise LNA or super-T, or capture primer 331 can comprise super-T while orthogonal capture primer 332 can comprise LNA or PNA. In yet other examples, the orthogonal capture primer 332 may comprise an LNA while the capture primer 331 may comprise a PNA or a super-T; the orthogonal capture primer 332 may comprise a PNA while the capture primer 331 may comprise an LNA or a super-T; or the orthogonal capture primer 332 may comprise a super-T while the capture primer 331 may comprise an LNA or a PNA.

[0067] LNA is very similar to DNA, with the only difference being the linker connecting the 2' and 4' carbons. PNA is structured by carboxylic acid and amino groups, similar to polypeptides. SuperT contains a modified thymidine base with a butyne group. In the case of LNA and SuperT base modifications, the increased rigidity of the molecular structure and stronger base stacking may increase the binding energy of the duplex interaction (321-331 or 322-332) compared to the DNA / DNA duplex (321-321' or 322-322'). Meanwhile, the melting point of the duplex containing uncharged PNA (321-331 or 322-332) may be increased compared to the DNA / DNA duplex (321-321' or 322-322') due to the reduced electrostatic repulsion. When the capture primer sequences are modified to include such modified nucleic acids, the melting temperature between the surface / solution duplex (321-331 or 322-332) can be significantly higher than the melting temperature of the solution / solution DNA duplex (321-321' or 322-322'). This allows the target polynucleotides 311, 312, 313 to be seeded under a set of conditions (e.g., salt concentration, formamide concentration, and temperature) that do not allow reannealing of adapters in solution. Thus, the target polynucleotides 311, 312, 313 can be seeded without substantial loss from adapter reannealing, as described with reference to FIG. 2C.

[0068] Prior to attempting to capture the target polynucleotide on substrate 300 for subsequent amplification and sequencing, double stranded D5, D6, and D7 are melted in a manner as shown in Figure 3B to yield a single stranded target polynucleotide having a first adaptor 321 available for hybridization to a capture primer 331 and a second adaptor 322 available for hybridization to an orthogonal capture primer 332. In a manner similar to that described with reference to Figure 2B, such melting may be performed, for example, by altering the temperature and / or composition of the solution in which the double stranded D5, D6, and D7 are disposed. For example, duplexes D5, D6, and D7 may be exposed to a sufficient amount of formamide in solution to dissociate the duplexes at the current solution temperature, e.g., about 1% to 100% formamide (v / v), or about 5% to about 80% formamide (% v / v), or about 10% to about 50% formamide (% v / v), or about 1% to about 20% formamide (% v / v). Additionally or alternatively, the temperature of the solution may be raised above the melting temperature (Tm) of the duplexes in the current solution composition. It will be understood that the particular Tm of the duplex may depend on the composition of the solution (e.g., salt (Na+) concentration and formamide concentration, if present), as well as the length and sequence of the polynucleotides in the duplex.

[0069] As described with reference to FIG. 3B, after the duplexes D5, D6, and D7 are melted, the first adaptor 321 may hybridize to the capture primer 331 and the second adaptor 322 may hybridize to the orthogonal capture primer 332 under the same conditions used to melt the duplexes D5, D6, and D7 in the manner shown in FIG. 3C. That is, the solution and temperature conditions that caused such melting may also not inhibit the first adaptor 321 from hybridizing to capture primer 331, nor may the second adaptor 322 from hybridizing to the orthogonal capture primer 332. Instead, such conditions may inhibit any annealing between the adaptor 321 and the complementary adaptor 321′, and any re-annealing between the adaptor 322 and the complementary adaptor 322′ may inhibit the formation of any duplex as described with reference to FIG. 2C. Thus, each of the target polynucleotides may remain available for capture and may be captured on the substrate without necessarily changing the conditions used to melt the duplexes D5, D6, and D7, e.g., without necessarily reducing the concentration of formaldehyde, e.g., without necessarily reducing the concentration of formamide.

[0070] In a non-limiting example shown in Figure 3C, under conditions that inhibit annealing between target polynucleotides in solution, first adaptors 321 of some of the target polynucleotides hybridize to each of the capture primers 331 to form a first duplex, and second adaptors 322 of some of the target polynucleotides hybridize to each of the orthogonal capture primers 332 to form a second duplex. Each of the first and second duplexes can have a higher melting temperature (Tm) than the Tm of a third duplex formed by hybridization of the second target polynucleotide to each of the first target polynucleotides to which it is complementary. Illustratively, a first adaptor 321 attached to sequence 312 hybridizes to one of the capture primers 331 to form duplex D8, a second adaptor 322 attached to sequence 311 hybridizes to one of the orthogonal capture primers 332 to form duplex D9, and a second adaptor 322 attached to sequence 313 hybridizes to one of the orthogonal capture primers 332 to form duplex D10.

[0071] The Tm of the duplex between the first adaptor 321 and the capture primer 331 may be greater than the Tm of the duplex between the first adaptor 321 and the complementary first adaptor 321', e.g., may be at least about 5° C., at least about 10° C., at least about 15° C., at least about 20° C., or at least about 25° C. greater than the Tm of the duplex between the first adaptor 321 and the complementary first adaptor 321'. Similarly, the Tm of the duplex between the second adaptor 322 and the orthogonal capture primer 332 may be greater than the Tm of the duplex between the second adaptor 322 and the complementary second adaptor 322', e.g., may be at least about 5° C., at least about 10° C., at least about 15° C., at least about 20° C., or at least about 25° C. greater than the Tm of the duplex between the first adaptor 321 and the complementary first adaptor 321'. Thus, the target polynucleotide may be captured onto the substrate 300 preferably at a temperature below the Tm of the duplex between the first adaptor 321 and the capture primer 331, below the Tm of the duplex between the second adaptor 322 and the orthogonal capture primer 332, which is above the Tm of the duplex between the first adaptor 321 and the complementary first adaptor 321', and above the Tm of the duplex between the second adaptor 322 and the complementary second adaptor 322'.

[0072] Illustratively, the Tm of duplexes D8, D9, and D10 shown in FIG. 3C can exceed the Tm of any of the duplexes D1, D2, D3, and D4 described with reference to FIG. 2A and FIG. 2C. For example, duplexes D8, D9, and D10 can each have a melting temperature (Tm) of about 80° C. to about 110° C., e.g., about 85° C. to about 105° C., or about 90° C. to about 100° C. In comparison, the Tm of duplexes D1, D2, D3, and D4 can be less than about 80° C., e.g., less than about 75° C., less than about 70° C., less than about 65° C., or less than about 60° C. As a result of the significantly different Tm of the duplex between the adaptor and the surface primer compared to the Tm of the duplex between the adaptors in solution, substantially none of the second target polynucleotides hybridize in solution to any of the first target polynucleotides. Thus, duplexes D8, D9, and D10 can be readily formed in certain conditions where duplexes D1, D2, D3, and D4 cannot be formed. The duplexes can be formed on the surface of the substrate 300 at different locations, for example according to a Poisson distribution. Although not specifically shown, it will be understood that the substrate 300 can be patterned, for example, to define different regions in which duplexes can each be formed, and in which clusters can then be formed using amplification. Additionally, while the examples described with reference to Figures 3A-3H are shown to suggest the use of a flat substrate, it will be apparent that more complex substrates can be used, such as wells, each of which can be seeded with a target polynucleotide and in which substantially monoclonal clusters can be formed.

[0073] As shown in FIG. 3D, after the initial hybridization described with reference to FIG. 3C, each of the target polynucleotides 311, 312, 313 can be amplified to form respective amplicons 311', 312', and 313', respectively. Following such amplification, the target polynucleotides 311, 312, 313 can be dehydrogenated in a manner as shown in FIG. 3E, while the amplicons 311', 312', and 313' remain covalently attached to the substrate 300. Note that such dehydrogenation does not necessarily have to be performed. For example, instead of dehydrogenating the target polynucleotides 311, 312, 313, such polynucleotides can remain hybridized to the substrate and can be further amplified using a strand invasion process, which may be referred to as ExAmp, as known in the art.

[0074] As shown in FIG. 3F, after the initial amplification described with reference to FIGS. 3D-3E, the resulting amplicon may bend so that it may hybridize to other capture primers or orthogonal capture primers on the substrate 300. For example, the complementary first adaptor 321' of amplicon 311' may hybridize to one of the capture primers 331, the complementary second adaptor 322' of amplicon 312' may hybridize to one of the orthogonal capture primers 332, and the complementary first adaptor 321' of amplicon 313' may hybridize to one of the capture primers 331. The duplexes between the amplicon adaptors and the respective capture or orthogonal capture primers may have similar or the same Tm as duplexes D8, D9, and D10, and thus may remain hybridized to facilitate further amplification of the target polynucleotide.

[0075] FIG. 3G shows the composition of FIG. 3F after another amplification run. As can be seen, the composition includes an additional amplicon 311 of amplicon 311', an additional amplicon 312 of amplicon 312', and an additional amplicon 313 of amplicon 313'. Such additional amplicons can hybridize to the amplicons generated from them. The solution can be increased to dehydrogenate the adapters of the amplicons from the capture primer or orthogonal capture primer in a manner as shown in FIG. 3H. The amplification run can be repeated any suitable number of times to generate additional amplicons of amplicons 311, 311', 312, 312', 313, and 313'. The amplification run can be formed any suitable number of times, for example, with the amplicons of target polynucleotides 311, 312, or 313 substantially occupying the respective substrate regions (not specifically shown) in substantially monoclonal clusters. For example, the amplicons within each of the substrate regions may comprise at least about 60% of the amplicons of one selected target polynucleotide, or at least about 70% of the amplicons of one selected target polynucleotide, or at least about 80% of the amplicons of one selected target polynucleotide, or at least about 90% of the amplicons of one selected target polynucleotide, or at least about 95% of the amplicons of one selected target polynucleotide, or at least about 98% of the amplicons of one selected target polynucleotide, or at least about 99% of the amplicons of one selected target polynucleotide, or 100% of the amplicons of one selected target polynucleotide.

[0076] As noted above, in some examples, certain capture primers and orthogonal capture primers may include non-nucleotide moieties. Such non-nucleotide moieties include, but are not limited to, removal moieties, whereby a portion of the capture primer may be selectively removed. The removal moieties may be located at any suitable position along the length of any suitable primer, and may be, but are not necessarily, the same type of removal moiety as each other. Following a desired number of further amplification operations, such as those described with reference to Figures 3E-3H, a portion of the capture primer 331 or orthogonal capture primer 332 may be removed by reacting an appropriate enzyme or reagent with the removal moiety.

[0077] The capture primers and orthogonal capture primers of the present invention may include any suitable number, type, and arrangement of modified nucleic acids that sufficiently increase the Tm of the duplex between the capture primer or orthogonal capture primer and the adaptor of the target polynucleotide. FIG. 4 shows a schematic of an exemplary duplex between a polynucleotide and one of the capture primers of the present invention according to some examples. In the non-limiting example shown in FIG. 4, a modified nucleic acid 441 (such as a PNA or LNA) is placed at the 5' end of the capture primer 331, and a DNA 442 is placed at the 3' end of the capture primer. An optional T spacer 443 is placed between the DNA 442 and the surface of the substrate 400 (e.g., a flow cell). The modified nucleic acid 441 and the DNA 442 together may be considered to provide a "chimeric" grafted primer structure. The target polynucleotide may include a library template 311 attached to an adaptor 321 that includes a first subsequence 341 that is complementary to the sequence of the modified nucleic acid 441, and a second subsequence 342 that is complementary to the sequence of the DNA 442. Alternatively, modified nucleic acids (such as PNA, LNA, or Super T) and DNA are distributed between the 5' and 3' ends of the capture primer in a manner as further described above. The orthogonal capture primer 332 may be configured similarly to the capture primer 331 shown in Figure 4, but may include, for example, a chimeric grafted primer structure having a different sequence. Alternatively, modified nucleic acids (such as PNA, LNA, or Super T) and DNA may be distributed between the 5' and 3' ends of the orthogonal capture primer in a manner as further described above.

[0078] In some examples, each of the capture primers 331 may contain about 5 to about 20 modified nucleic acids, and each of the orthogonal capture primers 332 may contain about 5 to about 20 modified nucleic acids. However, it is understood that the number of modified nucleic acids may be optimally adjusted to obtain an appropriate Tm under conditions that allow duplex formation on the substrate surface while suppressing duplex formation in solution, and that each of the capture primers and the orthogonal capture primers need not contain the same number, type, or distribution of modified nucleic acids. Illustratively, each of the capture primers may contain at least about 9 modified nucleic acids, each of the orthogonal capture primers may contain at least about 9 modified nucleic acids, or each of the capture primers may contain at least about 12 modified nucleic acids, each of the orthogonal capture primers may contain at least about 12 modified nucleic acids, or each of the capture primers may contain at least about 15 modified nucleic acids, and each of the orthogonal capture primers may contain at least about 15 modified nucleic acids.

[0079] It should be noted that although the capture primer 331 and the orthogonal capture primer 332 contain modified nucleic acids, they may have similar or identical sequences to the capture primers 231 and 232 if appropriate. That is, the capture primer 331 may be a modified P5 capture primer, and the orthogonal capture primer is a modified P7 capture primer. For example, the capture primer 331 may be or include a P5 primer having a sequence provided elsewhere herein, where at least some of the P5 DNA bases are replaced with their PNA, LNA, or super T analogs. Additionally or alternatively, the orthogonal capture primer 332 may be or include a P7 primer having a sequence provided elsewhere herein, where at least some of the P7 DNA bases are replaced with their PNA, LNA, or super T analogs. Non-limiting examples of chimeric P5 and P7 sequences containing LNA of various lengths, and the calculated Tm of duplexes between the chimeric P5 or P7 sequences and their respective adaptors cP5 or cP7 (750 mM Na+) are shown in Table 1 below, where bold indicates LNA and non-bold indicates DNA. Commercially available P5 and P7 sequences, and the calculated Tm of duplexes between the P5 or P7 sequences and their respective adaptors cP5 or cP7 are shown for reference.

[0080] [Table 1]

[0081] From the examples provided in Table 1, it can be seen that for a P5 sequence, replacing 15 DNA bases with LNA can increase the Tm of the duplex between the sequence and the cP5 adaptor by about 21° C., replacing 12 DNA bases with LNA can increase the Tm of the duplex between the sequence and the cP5 adaptor by about 16° C., and replacing 9 DNA bases with LNA can increase the Tm of the duplex between the sequence and the cP5 adaptor by about 11° C. Additionally, from the examples provided in Table 1, it can be seen that for a P7 sequence, replacing 15 DNA bases with LNA can increase the Tm of the duplex between the sequence and the cP7 adaptor by about 21° C., replacing 12 DNA bases with LNA can increase the Tm of the duplex between the sequence and the cP7 adaptor by about 17° C., and replacing 9 DNA bases with LNA can increase the Tm of the duplex between the sequence and the cP7 adaptor by about 12° C. It will be understood that the number of modified nucleic acids in any sequence provided herein to provide any suitable Tm for use in a capture primer or orthogonal capture primer as provided herein.

[0082] One consideration for implementing the modified capture primers of the present invention is that such primers are compatible with suitable enzymes, such as recombinases, single-stranded binding proteins, and polymerases, so that they can be used for cluster generation and sequencing. Chimeric structures including PNA or LNA, as described with reference to Figure 4, are expected to be compatible with such enzymes in a manner similar to that described in the following references: Levin et al., "Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers," Nucleic Acids Research 34(20):e142, 11 pages (2006), and Misra et al., "Polyamide nucleic acid-DNA chimera lacking the phosphate backbone are novel primers for polymerase reaction catalyzed by DNA polymerases," Biochemistry 37:1917-1925 (1998), the contents of which are incorporated herein by reference in their entirety. Further information on LNA can be found in Braasch et al., "Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA," Chemistry & Biology 8:1-7 (2001), the entire contents of which are incorporated herein by reference. Providing DNA at the 3' end of a chimeric structure as described with reference to Figure 4 can help maintain expected enzymatic activity by placing unmodified DNA bases at positions where the enzyme may interact, while providing modified nucleic acid at the 5' end of the chimeric structure to improve the strength of binding to the adapter.

[0083] Additionally, as described elsewhere herein and as will be appreciated by those of skill in the art, the Tm of any given duplex may vary based on the composition of the solution in which the duplex is placed. For example, adding formaldehyde to a solution may decrease the Tm of a given duplex, while adding salt to a solution may increase the Tm of a given duplex. Thus, the exemplary Tms (and differences between Tms) provided herein are purely exemplary and may vary based on the particular composition of the solution in which the duplex is placed. Figures 5A-5C are plots showing exemplary effects of conditions on capture of polynucleotides by capture primers of the invention. In the non-limiting example shown in Figure 5A, the percent double stranded DNA (dsDNA) is shown as a function of temperature in a solution containing 0% v / v formamide and 750 mM Na+ for a duplex formed by hybridization between cP7 and a P7 standard library capture primer (curve 501) and for a duplex formed by hybridization between cP7 and an LNA modified P7 capture primer (curve 502). The LNA modified P7 capture primer has the sequence CAAGCAGAAGACGGCATAC(8-oxoG)AGAT (SEQ ID NO: 13) (bold in the sequence indicates LNA) and was modeled to have a Tm of 20°C when duplexed with cP7.

[0084] In FIG. 5A, it can be seen that a temperature of about 77° C. corresponds to the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer (curve 501), and a temperature of about 91° C. corresponds to the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer (curve 502). At temperatures above about 85° C., curve 501 shows that the percentage of dsDNA is small (e.g., less than 1%) relative to the duplex formed by hybridization between cP7 and the P7 standard library capture primer. Additionally, at temperatures below about 90° C., curve 501 shows that the percentage of dsDNA is about 100% relative to the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer. Thus, from FIG. 5A, it can be seen that at temperatures of about 85° C. to 90° C., the hybridization between cP7 and the P7 standard library primer can be substantially completely inhibited, while the hybridization between cP7 and the LNA-modified P7 capture primer forms a very stable duplex. Thus, within this temperature range, the template polynucleotide can be captured from the solution with high efficiency and substantially free of competition from the solution-based annealing process as described with reference to FIG. 2C. It will be understood that the specific temperature range suitable for carrying out such an operation can vary depending on the specific composition of the solution. Any other capture primer and adapter sequence can be expected to show similar temperature dependence of duplex formation.

[0085] For example, the non-limiting example shown in Figure 5B shows the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer (curve 503) and the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer (curve 504) as a function of salt concentration (% v / v) of 750 mM Na+. As in Figure 5A, it can be seen that at 0% v / v formamide, a temperature of about 77°C corresponds to the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer (curve 503) and a temperature of about 91°C corresponds to the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer (curve 504). As the concentration of formamide increases, the Tm of both the duplex formed by hybridization between cP7 and the P7 standard library capture primer and the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases. For example, at a concentration of about 5% v / v formamide, the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer decreases to about 69° C., and the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases to about 88° C. At a concentration of about 10% v / v formamide, the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer decreases to about 66° C., and the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases to about 85° C. At a concentration of approximately 15% v / v formamide, the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer decreases to approximately 63°C, and the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases to approximately 82°C.At a concentration of approximately 20% v / v formamide, the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer decreases to approximately 58°C, and the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases to approximately 79°C.

[0086] Thus, from FIG. 5B, it can be seen that at any given concentration of formamide (e.g., about 1% to about 20% formamide (% v / v), or about 5% to about 20% formamide (% v / v)), there exists a temperature range in which hybridization between cP7 and the P7 standard library capture primer can be substantially completely inhibited, while hybridization of cP7 and the LNA modified P7 capture primer can form a very stable duplex. Thus, within this temperature range, the template polynucleotide can be captured from solution with high efficiency and substantially free of competition from the solution-based annealing process as described with reference to FIG. 2C. Any other capture primer and adapter sequence can be expected to show a similar formamide concentration dependence of duplex formation.

[0087] As another example, a non-limiting example shown in FIG. 5C shows the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer (curve 505) and the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer (curve 506) as a function of salt concentration (mM Na+). At 750 mM Na+, it can be seen that a temperature of about 72° C. corresponds to the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer (curve 505) and a temperature of about 89° C. corresponds to the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer (curve 506). As the concentration of salt decreases, the Tm decreases for both the duplex formed by hybridization between cP7 and the P7 standard library capture primer and the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer. For example, at a concentration of about 500 mM Na+, the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer decreases to about 70° C., and the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases to about 87° C. At a concentration of about 100 mM Na+, the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer decreases to about 55° C., and the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases to about 77° C. At a concentration of about 0 mM Na+, the Tm of the duplex formed by hybridization between cP7 and the P7 standard library capture primer decreases to about 42° C., and the Tm of the duplex formed by hybridization between cP7 and the LNA modified P7 capture primer decreases to about 58° C.

[0088] Thus, from FIG. 5C, it can be seen that at any given concentration of salt (e.g., at concentrations of about 100 to about 800 mM Na+, or 200 to about 800 mM Na+), there exists a temperature range in which hybridization between cP7 and the P7 standard library capture primer can be substantially completely inhibited, while hybridization of cP7 and the LNA modified P7 capture primer can form a very stable duplex. Thus, within this temperature range, the template polynucleotide can be captured from solution with high efficiency and substantially free of competition from the solution-based annealing process as described with reference to FIG. 2C. Any other capture primer and adapter sequence can be expected to show a similar salt concentration dependence of duplex formation.

[0089] It will be understood that the exemplary compositions described herein may be used in any suitable method for capturing and amplifying polynucleotides. For example, Figure 6 shows an exemplary flow of operations in a method 600 for capturing and amplifying polynucleotides using the present modified primers. Method 600 may be performed using composition 3000 described with reference to Figures 3A-3H, although method 600 may also be performed using any other suitable composition.

[0090] 6, a method 600 can include providing a composition including (a) a plurality of capture primers bound to a surface of a substrate, each of the capture primers comprising a modified nucleic acid, and (b) a plurality of orthogonal capture primers bound to a surface of the substrate, each of the capture primers comprising a modified nucleic acid (operation 610). The composition can be similar to composition 3000 described with reference to FIG. 3A, and can include, for example, capture primer 331 and orthogonal capture primer 332 bound to substrate 300.

[0091] The method 600 shown in Figure 6 may further include providing a fluid including (a) a first target polynucleotide, each of which includes a first adaptor that is complementary to a capture primer and a second adaptor that is complementary to an orthogonal capture primer, and (b) a second target polynucleotide that is complementary to each of the first target polynucleotides. The fluid may be similarly configured as described with reference to Figure 3A and may include, for example, target polynucleotides 311, 312, 313, each of which is bound to a first adaptor 321 and a second adaptor 322, and may include complementary target polynucleotides 311', 312', 313', each of which is bound to a complementary first adaptor 321' and a complementary second adaptor 322'.

[0092] 6 may further include contacting the composition with a fluid (operation 630). For example, the composition provided in operation 610 may be contacted with the fluid provided in operation 620. Illustratively, the composition may be provided to a flow cell and the fluid may flow to contact the composition within the flow cell.

[0093] The method 600 shown in FIG. 6 may further include (a) hybridizing a first adaptor of some of the first target polynucleotides to each of the capture primers to form a first duplex, while inhibiting hybridization of the second target polynucleotide to the first target polynucleotide (operation 640), and (b) hybridizing a second adaptor of some of the first target polynucleotides to each of the orthogonal capture primers to form a second duplex. For example, in a manner as described with reference to FIG. 3B-3C, hybridization of the first adaptor to capture primers and hybridization of the second adaptor to the orthogonal capture primer may be performed under conditions that inhibit annealing of the first and second target polynucleotides to each other. Exemplary conditions are described elsewhere herein. It should be noted that the same set of conditions may be used to cause dissociation of the first and second polynucleotides from each other and promote hybridization of the adaptor of the first polynucleotide to the capture primer of the present invention. In comparison, the use of conventional capture primers may require two conditions: a first condition that causes the first polynucleotide and the second polynucleotide to dissociate from each other, followed by a second condition that promotes hybridization of the adaptor of the first polynucleotide to the conventional capture primer.

[0094] The method 600 shown in Figure 6 may further include amplifying the first target polynucleotide, which includes generating an amplicon of each of the first target polynucleotides (operation 650). Non-limiting examples of how such amplicons may be generated are provided with reference to Figures 3D-3H.

[0095] Thus, while previously known capture primers may be used under conditions where non-productive rehybridization complexes may form in solution, the modified capture primers of the present invention (including orthogonal capture primers) overcome this drawback by using sequences that contain modified nucleic acids that confer stronger binding (and higher Tm) of the surface / solution duplex compared to the solution / solution duplex. The difference in Tm between the surface / solution and solution / solution duplexes may be achieved by using modified capture primer sequences that may include, for example, LNA, PNA, super T, or any combination thereof. By incorporating base modifications into the capture primer, there exists a temperature regime where the adaptor of the target polynucleotide can hybridize to the surface but cannot hybridize to other adaptors in solution.

[0096] Thus, the modified capture primers of the invention provide one or more of the following advantages: A) Library capture is more efficient: Where appropriate, lower concentrations of target polynucleotide input can be used. B) Post-denaturation of surface capture "on-flow cell" may be performed at elevated temperatures, thereby reducing or eliminating the need to integrate cooling to reach temperatures that promote hybridization in a timely manner; and / or C) Flow cells requiring large volumes of libraries can utilize multiple fluid pushes for seeding, for example, greatly increasing the turnaround time (TAT) from the start of sample preparation to obtaining sequencing data. With the higher seeding efficiency provided by the present capture primers, it is possible to use higher concentrations of target polynucleotides with fewer fluid pushes. This can save time during seeding.

[0097] It will be understood that the compositions and methods of the present invention are not limited to use with the particular procedures described above. For example, while Figures 3A-3H may be considered procedures consistent with "bridge amplification" or "surface-bound polymerase chain reaction," it will be understood that the compositions and methods of the present invention may be readily adapted for use with other amplification modalities. One such amplification modality is "exclusion amplification" or ExAmp. Exclusion amplification methods may allow for the amplification of a single target polynucleotide per substrate region and the generation of a substantially monoclonal population of amplicons in the substrate region. For example, the rate of amplification of a first captured target polynucleotide in a substrate region may be rapid relative to the slower transport rate and capture of target polynucleotides in the substrate region. Thus, a first target polynucleotide captured in a substrate region may be rapidly amplified and occupy the entire substrate region, thus inhibiting capture of additional target polynucleotides in the same substrate region. Alternatively, if the second target polynucleotide binds to the same substrate region after the first polynucleotide, the relatively rapid amplification of the first polynucleotide can result in sufficient substrate region occupation to result in a signal that is strong enough to perform sequencing by synthesis.The use of exclusion amplification can also result in a super-Poisson distribution of monoclonal substrate regions.That is, the fraction of substrate regions in the array that are substantially monoclonal can exceed the fraction predicted by Poisson distribution.

[0098] Increasing the super-Poisson distribution of useful clusters is useful because more substantially monoclonal substrate regions may result in higher quality signals and therefore improved SBS. However, the seeding of target polynucleotides into substrate regions may follow a spatial Poisson distribution, and the tradeoff for increasing the number of occupied substrate regions is increasing the number of polyclonal substrate regions. One way to obtain a higher super-Poisson distribution is for the seeding to occur quickly, followed by a delay between seeded target polynucleotides. The delay, called "kinetic delay," is believed to occur through biochemical reaction kinetics, thus allowing some seeded target polynucleotides to start earlier than other seeded targets. Exclusion amplification methods work by using a recombinase to promote the infiltration of primers (e.g., primers bound to substrate regions) into double-stranded DNA (e.g., target polynucleotides) when the recombinase mediates a sequence match. The compositions and methods of the present invention can be adapted for use with a recombinase to promote the infiltration of the capture primers and orthogonal capture primers of the present invention into target polynucleotides when the recombinase mediates a sequence match. Indeed, the compositions and methods of the present invention may be adapted for use with any surface-based polynucleotide amplification method, such as thermal PCR, chemically denaturing PCR, and enzyme-mediated methods, which may be referred to as recombinase polymerase amplification (RPA) or ExAmp.

[0099] Further comments While various illustrative embodiments have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the invention, and it is intended in the appended claims to cover all such changes and modifications that fall within the true spirit and scope of the invention.

[0100] It should be understood that any corresponding features / embodiments of each of the aspects of the present disclosure as described herein may be implemented together in any suitable combination, and that any feature / embodiment from any one or more of these aspects may be implemented with any of the features of the other aspect(s) as described herein in any suitable combination to obtain advantages as described herein.

Claims

1. 1. A composition for capturing a target polynucleotide on a surface of a substrate, comprising: a plurality of capture primers attached to the surface of the substrate, each of the capture primers comprising a modified nucleic acid; a plurality of orthogonal capture primers attached to the surface of the substrate, each of the orthogonal capture primers comprising a modified nucleic acid; a first target polynucleotide, each of which comprises a first adaptor that is complementary to the capture primer and a second adaptor that is complementary to the orthogonal capture primer; a second target polynucleotide complementary to each of the first target polynucleotides, the first adaptors of some of the first target polynucleotides hybridize to each of the capture primers to form a first duplex, and the second adaptors of some of the first target polynucleotides hybridize to each of the orthogonal capture primers to form a second duplex; The composition, wherein the first and second duplexes each have a melting temperature (Tm) greater than the Tm of a third duplex that may be formed by hybridization of the second target polynucleotide to each of the first target polynucleotides that are complementary to the second target polynucleotide.

2. 2. The composition of claim 1, wherein the modified nucleic acid of the capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T.

3. the capture primer further comprises deoxyribonucleic acid (DNA); Optionally, (i) the modified nucleic acid and the DNA are distributed between the 5' and 3' ends of the capture primer; or (ii) The composition of claim 1 or 2, wherein the modified nucleic acid is positioned at the 5' end of the capture primer and the DNA is positioned at the 3' end of the capture primer.

4. 4. The composition of any one of claims 1 to 3, wherein the modified nucleic acid of the orthogonal capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T.

5. The orthogonal capture primer further comprises deoxyribonucleic acid (DNA), and optionally (i) the modified nucleic acid and the DNA are distributed between the 5' and 3' ends of the capture primer; or 5. The composition of claim 4, wherein (ii) the modified nucleic acid is positioned at the 5' end of the orthogonal capture primer and the DNA is positioned at the 3' end of the orthogonal capture primer. (i) the first and second duplexes each have a melting temperature (Tm) that is between 80°C and 110°C; or (ii) the first and second duplexes each have a melting temperature (Tm) that is between 85° C. and 105° C.; or (iii) the first and second duplexes each have a melting temperature (Tm) that is between 90° C. and 100° C. The composition according to any one of claims 1 to 5.

7. 7. The composition of any one of claims 1 to 6, wherein substantially none of the second target polynucleotides hybridize in solution to any of the first target polynucleotides. (i) 1% to 100% formamide (% v / v), optionally 5% to 80% formamide (% v / v), and / or 8. The composition of any one of claims 1 to 7, further comprising (ii) 100-800 mM Na+, optionally 200-800 mM Na+.

9. 9. The composition of any one of claims 1 to 8, wherein the capture primer is a modified P5 capture primer and the orthogonal capture primer is a modified P7 capture primer. (a) each of the capture primers comprises 5 to 20 of the modified nucleic acids, and each of the orthogonal capture primers comprises 5 to 20 of the modified nucleic acids; and / or (b)(i) each of the capture primers comprises at least 9 of the modified nucleic acids and each of the orthogonal capture primers comprises at least 9 of the modified nucleic acids; or (ii) each of the capture primers comprises at least 12 of the modified nucleic acids and each of the orthogonal capture primers comprises at least 12 of the modified nucleic acids; or (iii) the composition of any one of claims 1 to 9, wherein each of the capture primers comprises at least 15 of the modified nucleic acids, and each of the orthogonal capture primers comprises at least 15 of the modified nucleic acids.

11. 1. A method for capturing and amplifying a target polynucleotide on a surface of a substrate, comprising: contacting a composition with a fluid, said composition comprising: a plurality of capture primers attached to the surface of the substrate, each of the capture primers comprising a modified nucleic acid; a plurality of orthogonal capture primers attached to the surface of the substrate, each of the orthogonal capture primers comprising a modified nucleic acid; The fluid is a first target polynucleotide, each of which comprises a first adaptor that is complementary to the capture primer and a second adaptor that is complementary to the orthogonal capture primer; a second target polynucleotide that is complementary to each of said first target polynucleotides; Meanwhile, inhibiting hybridization of the second target polynucleotide to the first target polynucleotide in the fluid; hybridizing the first adaptors of some of the first target polynucleotides to each of the capture primers to form a first duplex; hybridizing the second adaptors of some of the first target polynucleotides to each of the orthogonal capture primers to form second duplexes; and then amplifying the first target polynucleotide, wherein the amplifying comprises generating an amplicon for each of the first target polynucleotides; wherein the first and second duplexes each have a melting temperature (Tm) greater than a melting temperature (Tm) of a third duplex that may be formed by hybridization of the second target polynucleotide to the respective one of the first target polynucleotides that is complementary to the second target polynucleotide.

12. 12. The method of claim 11 , wherein the modified nucleic acid of the capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T.

13. the capture primer further comprises deoxyribonucleic acid (DNA); Optionally, (i) the modified nucleic acid and the DNA are distributed between the 5' and 3' ends of the capture primer; or 13. The method of claim 11 or 12, wherein (ii) the modified nucleic acid is positioned at the 5' end of the capture primer and the DNA is positioned at the 3' end of the capture primer.

14. 14. The method of any one of claims 11 to 13, wherein the modified nucleic acid of the orthogonal capture primer comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a super-T.

15. the orthogonal capture primer further comprises deoxyribonucleic acid (DNA); Optionally, (i) the modified nucleic acid and the DNA are distributed between the 5' and 3' ends of the capture primer; or 15. The method of claim 14, wherein (ii) the modified nucleic acid is positioned at the 5' end of the orthogonal capture primer and the DNA is positioned at the 3' end of the orthogonal capture primer. (i) the first and second duplexes each have a melting temperature (Tm) that is between 80°C and 110°C; and / or (ii) the first and second duplexes each have a melting temperature (Tm) that is between 85° C. and 105° C.; and / or (iii) the first and second duplexes each have a melting temperature (Tm) that is between 90° C. and 100° C.; and / or (iv) substantially none of the second target polynucleotides hybridize in solution to any of the first target polynucleotides; and / or (v) the hybridization is carried out in 1% to 100% formamide (% v / v), optionally the hybridization is carried out in 5% to 80% formamide (% v / v); and / or (vi) the hybridization is carried out at 100-800 mM Na+, optionally the hybridization is carried out at 200-800 mM Na+, and / or (vii) The method of any one of claims 11 to 15, wherein the capture primer is a modified P5 capture primer and the orthogonal capture primer is a modified P7 capture primer. (a) each of the capture primers comprises 5 to 20 of the modified nucleic acids, and each of the orthogonal capture primers comprises 5 to 20 of the modified nucleic acids; and / or (b)(i) each of the capture primers comprises at least 9 of the modified nucleic acids and each of the orthogonal capture primers comprises at least 9 of the modified nucleic acids; or (ii) each of the capture primers comprises at least 12 of the modified nucleic acids and each of the orthogonal capture primers comprises at least 12 of the modified nucleic acids; or (iii) the method of any one of claims 11 to 16, wherein each of the capture primers comprises at least 15 of the modified nucleic acids, and each of the orthogonal capture primers comprises at least 15 of the modified nucleic acids.

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