Methods, compositions, and kits for improving efficiency of seeding flow cells with polynucleotides

Abstract

The present disclosure relates to methods, compositions, and kits for improving the efficiency of seeding a flow cell with polynucleotides and their applications, including for sequencing.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 143,680, filed January 29, 2021, the disclosure of which is incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present disclosure relates to methods, compositions, and kits for improving the efficiency of seeding a flow cell with polynucleotides and their applications, including for sequencing. [Background technology]

[0003] DNA sequencing is undergoing rapid change, and capillary sequencing is no longer the technology of choice for most ultra-high throughput applications. A new generation of instruments utilizing primed synthesis within a flow cell to simultaneously obtain the sequence of millions of different DNA templates has transformed the field.

[0004] Next-generation sequencing (NGS) platforms typically utilize nucleic acid fragment libraries. Nucleic acid fragment libraries can be prepared using PCR-based methods or transposon-based methods. In the latter case, free transposon ends and modified transposases are used to form transposome complexes. The transposome complexes are then used to fragment and tag the nucleic acids to generate sequencer-ready libraries. Transposome complexes can be formed in solution or attached to a solid support such as beads. To use transposome complexes bound to a solid support (e.g., transposome beads) in a flow cell, the nucleic acid components of the transposome complexes must be released and then seeded onto the flow cell surface. Typically, the means to do so is by the use of heat or a competitive binding agent (e.g., biotin) used in excess. However, heat leads to increased diffusion in the flow cell, which disrupts the spatial colocalization of the formed clusters. Summary of the Invention

[0005] The present disclosure provides compositions, methods, and kits that address the problem of low seeding efficiency of flow cells. In particular, the compositions, methods, and kits of the present disclosure provide for the release of amplified nucleic acids from beads to seed flow cells by using exclusion amplification (ExAmp) to copy translocated polynucleotides on beads using primers in solution ("solution primers"). More specifically, strand invasion by the use of solution primers results in displacement of the copied strands into solution, thereby effectively releasing them from the beads and facilitating seeding of the flow cells. In additional embodiments, the beads may further comprise attached amplification primers to enhance amplification and displacement of amplified nucleic acids from the beads. In other embodiments, the seeding efficiency of flow cells may be improved or further improved by the use of on-flow cell linear amplification and shallow liquid denaturation / seeding workflows.

[0006] In certain embodiments, the present disclosure provides a method for seeding a flow cell with polynucleotides released from beads, comprising: (i) annealing a first primer to a polynucleotide strand bound to a bead, wherein the 5' end of the polynucleotide strand is attached to a first attachment agent; (ii) annealing a polynucleotide strand comprising an adaptor comprising a sequence complementary to a sequence of a first primer, the first primer being in solution, (iii) performing primer invasion of the double stranded polynucleotide product by using an additional amount of the first primer and a recombinase, (iv) displacing and releasing a strand of the double stranded polynucleotide product of step (ii) from the bead by extending from an additional first primer in step (iii) using a polymerase, (v) seeding a flow cell by attaching the strand released in step (iv) to a surface of the flow cell using a second attachment agent, and optionally repeating steps (iii)-(v) multiple times. In a further embodiment, the beads are generally spherical or generally ovoid in shape.In another embodiment, the beads are about 500 nm, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3 μM, 3.1 μM, 3.2μM, 3.3μM, 3.4μM, 3.5μM, 3.6μM, 3.7μM, 3.8μM, 3.9μM, 4μM, 4.1μM, 4.2μM, 4.3μM, 4.4μM M, 4.5 μM, 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM, 5.0 μM, 5.1 μM, 5.2 μM, 5.3 μM, 5.4 μM, 5.5 μM, 5.6 μM, 5.7μM, 5.8μM, 5.9μM, 6μM, 6.1μM, 6.2μM, 6.3μM, 6.4μM, 6.5μM, 6.6μM, 6.7μM, 6.8μM, 6.9μM M, 7μM, 7.1μM, 7.2μM, 7.3μM, 7.4μM, 7.5μM, 7.6μM, 7.7μM, 7.8μM, 7.9μM, 8μM, 8.1μM, 8.2μM M, 8.3 μM, 8.4 μM, 8.5 μM, 8.6 μM, 8.7 μM, 8.8 μM, 8.9 μM, 9.0 μM, 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM, 9.5 μM, 9.6 μM, 9.7 μM, 9.8 μM, 9.9 μM, 10 μM, or a diameter ranging between or including any two of the aforesaid distances. In yet another embodiment, the beads have a diameter of about 500 nm, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, or a range including or between any two of the aforementioned distances. In certain embodiments, the beads are composed of a polymeric material, a silica material, a zirconia material, or a superparamagnetic material. In further embodiments, the beads are composed of a superparamagnetic material.In still further embodiments, the beads are functionalized with a coating or layer selected from biotin, streptavidin, amine groups, carboxyl groups, epoxy groups, tosyl groups, antibodies or antigens, polyhistidine tags, or carriers comprising metal ions, and receptors or ligands. In certain embodiments, the beads are not immobilized on one or more surfaces of the flow cell.

[0007] In an alternative embodiment, the beads are immobilized on one or more surfaces of the flow cell using an attachment agent. In another embodiment, the attachment agent is a biotin / streptavidin bond or base pairing between two complementary sequences. In yet another embodiment, the polynucleotide strands bound to the beads comprise genomic DNA. In a further embodiment, the polynucleotide strands bound to the beads comprise a long template DNA. In yet a further embodiment, the polynucleotide strands bound to the beads comprise DNA from a subject's sample. In certain embodiments, the subject's sample is urine, blood, saliva, tissue, serum, plasma, sputum, bile, feces, hair, skin, and / or primary cell samples. In another embodiment, the polynucleotide strands are tagmented and attached to the beads by the use of transposomes linked to the beads. In yet another embodiment, the transposomes linked to the beads comprise transposase Tn5, or a mutant or variant thereof. In further embodiments, the polynucleotide strand comprises a first portion of the sequence bound to the bead by a first attachment agent and a second portion of the sequence tagmented and ligated to the first portion of the sequence by the use of a transposome linked to the bead and a ligase. In yet further embodiments, the transposome linked to the bead comprises a transposase Tn5, or a mutant or variant thereof. In another embodiment, the first portion of the sequence comprises one or more barcode or index sequences, one or more universal primer sequences, and / or a mosaic end sequence. In yet another embodiment, the first portion of the sequence comprises one or more barcode or index sequences, one or more universal primer sequences, and a mosaic end sequence, the mosaic end sequence being located at the 3' end of the first portion of the sequence. In certain embodiments, the adaptor comprises a first sequence that is complementary to the first portion of the sequence of the polynucleotide strand and can hybridize with the first portion, and the adaptor comprises a second sequence that is not complementary to the first portion of the sequence of the polynucleotide strand and cannot hybridize with the first portion.In another embodiment, the second sequence of the adaptor comprises a sequence that is complementary to the sequence of the first primer. In yet another embodiment, the second portion of the sequence of the polynucleotide strand comprises gDNA. In certain embodiments, the second portion of the sequence of the polynucleotide strand comprises a long template DNA. In a further embodiment, the second portion of the sequence of the polynucleotide strand comprises DNA from a sample of the subject.

[0008] In still further embodiments, the subject sample is urine, blood, saliva, tissue, serum, plasma, sputum, bile, feces, hair (or hair), skin, and / or primary cell sample. In certain embodiments, the first and second attachment agents are selected from biotin / streptavidin binding, covalent binding, base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier that includes a metal ion, and a receptor that binds to a ligand. In another embodiment, the first attachment agent is biotin / streptavidin or avidin binding and the second attachment agent is base pairing between two complementary sequences. In yet another embodiment, the polynucleotide chain comprises a biotin residue at the 5' end and the bead comprises a streptavidin residue. In a further embodiment, the first primer has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In a still further embodiment, the first primer has the sequence of SEQ ID NO: 2. In certain embodiments, a DNA polymerase is used to extend the sequence from the first primer. In further embodiments, the method provides a linear copy of the polynucleotide strand. In certain embodiments, the flow cell is a patterned flow cell. In further embodiments, the strand released from the bead is bound by a capture agent on the flow cell surface. In still further embodiments, the capture agent is located on an array of features of the patterned flow cell, and the capture agent is not located on the interstitial area between the features. In another embodiment, the array of features of the patterned flow cell is a well. In yet another embodiment, the capture agent is a capture nucleic acid attached to the surface of the flow cell, and the strand comprises a sequence that can be released from the bead and hybridize with a primer attached to the surface of the flow cell. In a further embodiment, the capture nucleic acid comprises a universal capture sequence. In still further embodiments, the universal capture sequence comprises P5 5'-AATGATACGGCGACCACCGA-3' ((SEQ ID NO: 1)) or P7 (5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 2)), or a fragment thereof.In alternative embodiments, the universal capture sequence comprises anti-P5: 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 3) or anti-P7: 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 4), or a fragment thereof. In further embodiments, the capture nucleic acid further comprises a sequencing primer binding site (SBS), a target specific capture region, a restriction site, and / or a linker region. In still further embodiments, the SBS is SBS3: 5'-ACACTCTTTCCCTACACGACGCTCTTCCG ATCT-3' (SEQ ID NO: 5) or SBS8: 5'-CGGTCTCGGCATTCCTGCTGAACCGC TCTTCCGATCT-3' (SEQ ID NO: 6). In certain embodiments, the method further comprises performing an on-flow cell linear amplification reaction to make copies of the polynucleotides seeded on the flow cell surface, denaturing the copied polynucleotides and seeding them on the flow cell surface. In a further embodiment, the on-flow cell linear amplification reaction comprises a low temperature AMS1 first extension reaction. In yet a further embodiment, the copied polynucleotides are denatured by use of a hybridization buffer comprising a chemical denaturant. In another embodiment, the chemical denaturant is formamide. In yet another embodiment, the mixture comprises HT1. In a further embodiment, the hybridization buffer comprising a chemical denaturant shallowly coats the surface of the patterned flow cell such that the array features on the patterned flow cell surface are in fluidic contact with each other. In a still further embodiment, the method further comprises forming clusters on the surface of the patterned flow cell by using bridge amplification to make multiple copies of each seeded polynucleotide. In yet another embodiment, the method further comprises sequencing by synthesis the clusters generated on the surface of the patterned flow cell.

[0009] In certain embodiments, a method for seeding a flow cell with polynucleotides released from beads comprises: (i) providing beads comprising a polynucleotide strand bound to the beads, wherein a 5' end of the polynucleotide strand is bound to the beads using a first attachment agent, the polynucleotide strand comprises an adaptor comprising a sequence that is complementary to a sequence of the first primer, the beads also comprising an attached first amplification primer, the first amplification primer being attached to the beads by a third attachment agent, the first amplification primer having a sequence that is different from and not complementary to the sequence of the first primer, the polynucleotide strand comprising a sequence that is complementary to the first amplification primer; and (ii) annealing the first primer to the polynucleotide strand bound to the beads, wherein the first primer is annealed to the bead by a third attachment agent, the first amplification primer being ... (iii) extending from the first primer using a polymerase to form a double-stranded polynucleotide product; (iv) performing primer invasion of the double-stranded polynucleotide product by using an additional amount of the first primer and / or first amplification primer and a recombinase; (v) displacing and releasing a strand of the double-stranded polynucleotide product of step (iii) from the bead by extending from the additional first primer and / or first amplification primer in step (iv) using a polymerase; (vi) seeding a flow cell by attaching the strand released in step (v) to the surface of the flow cell using a second attachment agent; and optionally repeating steps (ii)-(vi) multiple times.In another embodiment, the disclosure provides a method for seeding a flow cell with polynucleotides released from beads, the method comprising: (i) providing beads comprising a polynucleotide strand bound to the beads, wherein a 5' end of the polynucleotide strand is bound to the beads using a biotin / streptavidin bond, the beads also comprising a bound second amplification primer, the second amplification primer being bound to the beads by a third attachment agent, the second amplification primer having a sequence different from and not complementary to the sequence of the second primer, the polynucleotide strand comprising a sequence complementary to the second amplification primer, (ii) annealing the second amplification primer to the polynucleotide strand bound to the beads, (iii) extending from the second amplification primer using a polymerase to form a double stranded polynucleotide product, and then The method includes step (a) or steps (b) and (b'): (a) introducing beads into a flow cell and releasing the double-stranded polynucleotide product from the beads as a single-stranded polynucleotide product using heat and excess biotin, or (b) performing primer invasion of the double-stranded polynucleotide product by using a second primer and a recombinase, the second primer being in solution, (b') displacing and releasing a strand of the double-stranded polynucleotide product from the beads by extending from the second primer with a polymerase, (iv) seeding the single-stranded polynucleotide product released in step (a) or the strand of the double-stranded polynucleotide product released from the beads in step (b') onto the surface of the flow cell using a second attachment agent, and optionally repeating steps (ii) to (iv) multiple times. In a further embodiment, the beads are immobilized on the surface of the flow cell using an attachment agent. In still further embodiments, the attachment agent is selected from a biotin / streptavidin bond, a covalent bond, base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier that includes a metal ion, and a receptor that binds to a ligand.In still further embodiments, the attachment agent is a biotin / streptavidin bond or base pairing between two complementary sequences. In alternative embodiments, the beads are not immobilized to the surface of the flow cell using an attachment agent. In certain embodiments, the flow cell is a patterned flow cell. In further embodiments, the strands released from the beads are bound by a capture agent on the flow cell surface. In still further embodiments, the capture agent is located on an array of features of the patterned flow cell, and the capture agent is not located on the interstitial region between the features. In another embodiment, the array of features of the patterned flow cell is a well. In yet another embodiment, the capture agent is a capture nucleic acid attached to the surface of the flow cell, and the strands include a sequence that can be released from the beads and hybridize to a primer attached to the surface of the flow cell. In a further embodiment, the capture nucleic acid includes a universal capture sequence. In yet further embodiments, the universal capture sequence comprises P5 5'-AATGATACGGCGACCACCGA-3' ((SEQ ID NO:1)) or P7 (5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO:2)), or a fragment thereof. In alternative embodiments, the universal capture sequence comprises anti-P5: 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO:3) or anti-P7: 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO:4)), or a fragment thereof. In further embodiments, the capture nucleic acid further comprises a sequencing primer binding site (SBS), a target specific capture region, a restriction site, and / or a linker region. In still further embodiments, the SBS is SBS3: 5'-ACACTCTTTCCCTACACGACGCTCTTCCG ATCT-3' (SEQ ID NO:5) or SBS8: 5'-CGGTCTCGGCATTCCTGCTGAACCGC TCTTCCGATCT-3' (SEQ ID NO:6). In certain embodiments, the method further includes performing an on-flow cell linear amplification reaction to make copies of the polynucleotides seeded on the flow cell surface, and denaturing the copied polynucleotides and seeding them on the flow cell surface.In a further embodiment, the on-flow cell linear amplification reaction comprises a low-temperature AMS1 first extension reaction. In yet a further embodiment, the copied polynucleotides are denatured by the use of a hybridization buffer comprising a chemical denaturant. In another embodiment, the chemical denaturant is formamide. In yet another embodiment, the hybridization buffer comprises HT1. In a further embodiment, the hybridization buffer comprising a chemical denaturant shallowly coats the surface of the patterned flow cell such that the array features on the patterned flow cell surface are in fluidic contact with each other. In still a further embodiment, the method further comprises forming clusters on the surface of the patterned flow cell by using bridge amplification to make multiple copies of each seeded polynucleotide. In yet another embodiment, the method further comprises sequencing by synthesis the clusters generated on the surface of the patterned flow cell.

[0010] In certain embodiments, the disclosure provides a method for seeding a flow cell with polynucleotides released from beads, comprising: (i) providing beads comprising a polynucleotide strand bound to the beads, wherein a 5' end of the polynucleotide strand is bound to the beads using a biotin / streptavidin bond, the beads also comprising first and second amplification primers bound thereto, the first and second amplification primers being bound to the beads by an attachment agent, the first amplification primer having a sequence that is at least 99% identical to a sequence of the first primer, the second amplification primer having a sequence that is at least 99% identical to a sequence of the second primer, the polynucleotide strand comprising a sequence that is complementary to the first amplification primer and a sequence that is complementary to the second amplification primer; (ii) annealing the first and second amplification primers to the polynucleotide strand bound to the beads; and (iii) extending from the first and second amplification primers using a polymerase to produce a double-stranded polynucleotide product. and then performing either step (A) or steps (B) and (B'): (A) introducing the beads into a flow cell and releasing the double-stranded polynucleotide products from the beads as single-stranded polynucleotide products using heat and excess biotin, or (B) performing primer invasion of the double-stranded polynucleotide products by using a first primer and / or a second primer and a recombinase, where the first and second primers are in solution, (B') displacing and releasing the strands of the double-stranded polynucleotide products from the beads by extending from the first and / or second primers with a polymerase; (iv) seeding the single-stranded polynucleotide products released in step (A) or the strands of the double-stranded polynucleotide products released from the beads in step (B') onto a surface of a flow cell using an attachment agent, and optionally repeating steps (ii)-(iv) multiple times. In a further embodiment, the beads are immobilized on the surface of the flow cell using an adhesive.In still further embodiments, the attachment agent is selected from biotin / streptavidin binding, a covalent bond, a base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier that contains a metal ion, and a receptor that binds to a ligand. In still further embodiments, the attachment agent is a biotin / streptavidin binding or a base pairing between two complementary sequences. In alternative embodiments, the beads are not immobilized to the surface of the flow cell using an attachment agent. In certain embodiments, the flow cell is a patterned flow cell. In further embodiments, the strands released from the beads are bound by a capture agent on the flow cell surface. In still further embodiments, the capture agent is located on an array of features of the patterned flow cell, and the capture agent is not located on the interstitial area between the features. In another embodiment, the array of features of the patterned flow cell is a well. In yet another embodiment, the capture agent is a capture nucleic acid attached to the surface of the flow cell, and the strands include a sequence that can be released from the beads and hybridized to a primer attached to the surface of the flow cell. In a further embodiment, the capture nucleic acid includes a universal capture sequence. In yet further embodiments, the universal capture sequence comprises P5 5'-AATGATACGGCGACCACCGA-3' ((SEQ ID NO:1)) or P7 (5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO:2)), or a fragment thereof. In alternative embodiments, the universal capture sequence comprises anti-P5: 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO:3) or anti-P7: 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO:4)), or a fragment thereof. In further embodiments, the capture nucleic acid further comprises a sequencing primer binding site (SBS), a target specific capture region, a restriction site, and / or a linker region. In still further embodiments, the SBS is SBS3: 5'-ACACTCTTTCCCTACACGACGCTCTTCCG ATCT-3' (SEQ ID NO:5) or SBS8: 5'-CGGTCTCGGCATTCCTGCTGAACCGC TCTTCCGATCT-3' (SEQ ID NO:6).In certain embodiments, the method further comprises performing an on-flow cell linear amplification reaction to generate copies of the seeded polynucleotides on the flow cell surface, and denaturing and seeding the copied polynucleotides on the flow cell surface. In further embodiments, the on-flow cell linear amplification reaction comprises a low temperature AMS1 first extension reaction. In still further embodiments, the copied polynucleotides are denatured by use of a hybridization buffer comprising a chemical denaturant. In another embodiment, the chemical denaturant is formamide. In yet another embodiment, the hybridization buffer comprises HT1. In still further embodiments, the hybridization buffer comprising a chemical denaturant shallowly coats the surface of the patterned flow cell such that the array features on the patterned flow cell surface are in fluidic contact with each other. In still further embodiments, the method further comprises forming clusters on the surface of the patterned flow cell by using bridge amplification to generate multiple copies of each seeded polynucleotide. In yet another embodiment, the method further comprises sequencing by synthesis the clusters generated on the surface of the patterned flow cell.

[0011] In certain embodiments, the present disclosure also provides a method for increasing the seeding efficiency of polynucleotides on the surface of a flow cell, comprising: performing an on-flow cell linear amplification reaction to generate copies of the seeded polynucleotides on the flow cell surface; and denaturing and seeding the copied polynucleotides on the flow cell surface. In a further embodiment, the on-flow cell linear amplification reaction comprises a low-temperature AMS1 first extension reaction. In still a further embodiment, the copied polynucleotides are denatured by use of a hybridization solution comprising a chemical denaturant. In another embodiment, the chemical denaturant is formamide. In yet another embodiment, the hybridization solution comprises HT1. In a further embodiment, the mixture shallowly coats the surface of the patterned flow cell such that the array features on the patterned flow cell surface are in fluid contact with each other. In still a further embodiment, the method further comprises forming clusters on the surface of the patterned flow cell by using bridge amplification to generate multiple copies of each seeded polynucleotide. In another embodiment, the method further comprises sequencing by synthesis the clusters generated on the surface of the patterned flow cell.

[0012] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the following description. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0013] [Figure 1]Provided is an overview of the steps constituting an exemplary method of the present disclosure for enhancing or improving the efficiency of seeding a flow cell with transposomal beads. As shown, step 1 provides for priming off the 3' end of the Y-shaped adaptor on the bead with a primer such as P7 or P5 in solution. Step 2 provides for extension from the bound primer to create a double-stranded nucleic acid product by using exclusion amplification. Then, in step 3, as an extension product is formed using exclusion amplification, this product displaces the previously copied strand in solution (step 4a). The released strand is then seeded onto the surface of a flow cell for analysis and application (step 4b). [Diagram 2] An embodiment of the disclosed method is provided in which the beads contain an amplification primer (eg, P5) that has a different sequence than a primer in solution (eg, P7). [Diagram 3] An embodiment of the disclosed method is provided in which the beads contain an amplification primer (eg, P7) that has the same or nearly the same sequence as a primer (eg, P7) in solution. [Figure 4] An embodiment of the disclosed method is provided in which a bridge amplification approach is used to amplify the library on beads and seed the amplification products onto the surface of a flow cell. [Diagram 5] We demonstrate that when two or more DNA library molecules are seeded into the same well of a patterned flow cell, either one dominant cluster (blue) emerges or two (or more) equivalent polyclonal clusters (blue + pink) emerge. [Figure 6] The workflow for on-flow cell linear amplification is shown. (A) Polynucleotides of a DNA library are seeded into the wells of a flow cell. The workflow for on-flow cell linear amplification is shown. (B) A first extension using Illumina™ AMS1 results in double-stranded DNA (one copied anchor strand with the original strand). (C) After shallow liquid denaturation / seeding, the strands leave the original well and are seeded close to the well. (D) After bridge amplification, there is a higher probability of collecting sequencing information from the DNA library. [Figure 7A] Results of linear amplification on the wells of a flow cell are presented. (A) Clusters formed using the standard seeding protocol. [Figure 7B] (B) Results of linear amplification on the wells of a flow cell. [Figure 8] Photographs of the bottom wells of patterned flow cells are provided to assess whether the methods disclosed herein can be used to seed a flow cell with a minimal amount of DNA. Top photo: Photograph of the bottom of a flow cell loaded with a standard amount of DNA (300 pM). Middle photo: Photograph of the bottom of a flow cell loaded with 10 pM of DNA. Bottom photo: Photograph of the bottom of a flow cell loaded with 10 pM of DNA and P7 oligonucleotide and ExAmp. The photographs show that when using the amplification process described herein, the wells of the patterned flow cell can be fully occupied with a small amount of DNA at the start (i.e., 10 pM). [Figure 9] To assess whether the amount of amplification could be controlled during the loading process, a photograph of the bottom well of a patterned flow cell is provided. As shown in the photograph, the amount of amplification exhibited a dose-dependent relationship with the amount of P7 oligo used. [Figure 10] 1 provides photographs of the bottom and top of wells from a patterned flow cell using the disclosed method to release DNA from beads using P7 oligos and seed the wells of the flow cell. As shown in the top two photographs, both the top and bottom of the wells were seeded using the disclosed method. In stark contrast, the top of the wells was not seeded when beads only were used (bottom right photograph), while the bottom of the wells was minimally seeded when beads only were used (bottom left photograph).

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and implementations of the disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a flow cell" includes a plurality of such flow cells, reference to "the DNA library" includes references to one or more DNA libraries, and so forth.

[0016] Also, unless otherwise stated, the use of "or" means "and / or." Similarly, "comprise," "comprises," "comprising," "include," "includes," "including," "have," "haves," and "having" are interchangeable and are not intended to be limiting.

[0017] It is further understood that where the descriptions of various embodiments use the term "comprising," those of ordinary skill in the art will understand that in some specific instances, an embodiment can be alternatively described using the phrase "consisting essentially of" or "consisting of."

[0018] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions of the present disclosure, exemplary methods, devices, and materials are described herein.

[0019] The term "amplify" or "amplification" herein is intended to mean the process of increasing the number of template polynucleotide sequences by generating copies of the template. The amplification process can be either linear or exponential. In exponential amplification, the number of copies made from the template polynucleotide sequence increases at an exponential rate. For example, in an ideal exponential amplification reaction of 30 rounds, one copy of the template DNA is multiplied by 2 copies. 30 or 1,073,741,824 copies. However, bridge amplification as described herein typically does not occur under ideal conditions, and a 30-cycle "exponential" reaction may only produce hundreds to thousands of copies of the original template, primarily due to the limited local concentration of surface-bound primers and competition with template rehybridization. In linear amplification, the number of copies made from a template polynucleotide sequence increases at a linear rate. For example, in an ideal 4-hour amplification reaction with a copy rate of 2000 copies per minute, each copy of template DNA produces 480,000 copies. In certain embodiments, the compositions, methods, and kits of the present disclosure provide a linear amplification reaction for releasing polynucleotides from beads.

[0020] The term "capture agent" refers to an agent present on the surface of a flow cell that can bind to a target biomolecule, typically a nucleic acid-based biomolecule, such that the target biomolecule is immobilized on the surface of the flow cell. In particular, a "capture agent" is specific to the target biomolecule and does not bind to non-target biomolecules or other compounds. Exemplary capture agents include receptors and / or ligands with their respective binding partners bound to the target nucleic acid, examples of which are described herein above. Particularly useful capture agents are capture nucleic acids that are complementary to the sequence of one or more target nucleic acids. For example, the capture nucleic acid present on the surface of the flow cell can have a universal capture sequence that is complementary to a universal sequence present in the target nucleic acid. In some embodiments, the capture nucleic acid can also function as a primer for amplification of the target nucleic acid (whether or not it also contains a universal sequence). The universal capture sequence can include, for example, a region that has the sequence of a universal Illumina® capture primer or a region that specifically hybridizes to a universal Illumina® capture primer. Universal Illumina® capture primers include, for example, P5 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 1) or P7 (5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 2)), or fragments thereof. The region that specifically hybridizes to the universal Illumina® capture primer can include, for example, Illumina® capture primer P5 ("anti-P5": 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 3) or P7 ("anti-P7": 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 4)), or a fragment thereof. Other capture nucleic acids can include a universal capture sequence and a target-specific capture region. The capture nucleic acid can further include a sequencing primer binding site (SBS), a target-specific capture region, a predetermined cleavage site such as a restriction site, and a linker region, e.g., a linker region separating two or more restriction sites. Some capture primers can include, for example, a universal capture region and an SBS.An SBS can include, for example, a region having the sequence of an Illumina® sequencing primer or a fragment thereof, or a region that specifically hybridizes to an Illumina® sequencing primer or a fragment thereof. Illumina® sequencing primers include, for example, SBS3 (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 5)) or SBS8 (5'-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3' (SEQ ID NO: 6)). A region that specifically hybridizes to an Illumina® sequencing primer or a fragment thereof can include, for example, the reverse complement of Illumina® sequencing primer SBS3 ("anti-SBS3": 5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3' (SEQ ID NO: 7)) or SBS8 ("anti-SBS8": 5'-AGATCGGAAGAGCGGTTCAGCAGGAATG CCGAGACCG-3' (SEQ ID NO: 8)), or a fragment thereof. A capture nucleic acid can have any combination of regions, for example, any combination of Illumina® P5, P7, SBS3, or SBS8 primer regions or fragments thereof, including combinations such as P5-SBS3 and P7-SBS8 or fragments thereof.

[0021] The capture nucleic acid may be blocked at the 3' end (3'-blocked) or may not be blocked at the 3' end (3'-unblocked). A capture nucleic acid with a blocked 3' end may be, for example, a 3'-phosphate end. Some capture nucleic acids with a blocked 3' end can be deblocked. Deblocking can occur in an enzymatic or chemical reaction. An enzymatic reaction can be mediated, for example, by a kinase or a phosphatase. For example, a 3'-phosphate terminated primer can be deblocked by a kinase (e.g., T4 kinase).

[0022] The capture nucleic acid may include a predetermined (non-random) cleavage site. Possible predetermined cleavage sites are disclosed, for example, in U.S. Pat. No. 8,715,966 (B2). Cleavage at a predetermined site may occur as non-enzymatic cleavage, such as enzymatic or chemical cleavage. Enzymatic cleavage at a predetermined site, such as a restriction site, may be mediated, for example, by a restriction enzyme, such as a restriction endonuclease. In some embodiments, the predetermined cleavage site in the primer may include a uracil base. Cleavage may occur by treating the uracil-containing primer with uracil DNA glycosylase to form a basic site in the primer, followed by treatment with an endonuclease, heat, or alkali to cleave the primer at the basic site. In some aspects, the predetermined cleavage site includes a diol linker, which can be cleaved by treatment with periodate. In some embodiments, the predetermined cleavage site includes 8-oxo-guanine.

[0023] The predetermined cleavage site may comprise an enzyme restriction site. Any restriction enzyme or any enzyme restriction site known to those skilled in the art may be used in the methods or compositions provided herein. For example, the restriction endonuclease may be a type I enzyme (EC 3.1.21.3), a type II enzyme (EC 3.1.21.4), a type III enzyme (EC 3.1.21.5), or a type IV enzyme (EC 3.1.21.5). Restriction endonucleases can include, for example, but are not limited to, Alu I, Ava I, Bam HI, Bg1 II, Eco P15 I, Eco RI, Eco RII, Eco RV, Hae III, Hga I, Hha I, Hind III, Hinf I, Hpa I, Kpn I, Mbo I, Not I, Pst I, Pvu II, Sac I, Sal I, Sap I, Sau 3A, Sca I, Sma I, Spe I, Sph I, Sst I, Stu I, Taq I, Xba I, or Xma I. Restriction endonucleases can be recombinant restriction enzymes. Recombinant restriction enzymes can include, but are not limited to, fusion proteins that include a natural or engineered DNA binding domain (e.g., zinc finger domain, TAL effector domain) and a nuclease domain (e.g., the cleavage domain of the type IIS restriction enzyme FokI).

[0024] In certain embodiments, a capture agent such as a capture nucleic acid can be attached to a specific feature arranged on the surface of a patterned flow cell. For example, the capture agent can be attached to the surface of an array feature (e.g., a well) on a patterned flow cell. Alternatively, the capture agent is bound to the surface of the flow cell in a random manner (i.e., a non-patterned flow cell). Attachment can be via an intermediate structure such as a bead, particle, or gel. Attachment of a capture nucleic acid to an array feature of a patterned flow cell by using a gel is described in WO2008 / 093098, which is incorporated herein by reference. Exemplary gels that can be used in the methods and devices described herein include, but are not limited to, those with colloidal structures, such as polymer mesh structures such as agarose, gelatin, or those with cross-linked polymer structures, such as polyacrylamide, SFA (see, e.g., U.S. Patent Application Publication No. 2011 / 0059865(A1), which is incorporated herein by reference), or PAZAM (see, e.g., U.S. Patent Application Publication No. 2014 / 0079923(A1), U.S. Patent No. 9,815,916(B2), and U.S. Patent No. 10,208,142(B2), the disclosures of which are incorporated herein by reference). With respect to PAZAM, capture agents can be attached to residual azides on the PAZAM surface by using click chemistry.

[0025] As used herein, the term "clonal population" refers to a population of nucleic acids that are homogeneous for a particular nucleotide sequence. The homogeneous sequence may be at least 10 nucleotides in length, or may be longer, e.g., at least 50, 100, 250, 500, or 1000 nucleotides in length. A clonal population may be derived from a single target or template nucleic acid. Essentially, all nucleic acids in a clonal population have the same nucleotide sequence. It will be understood that minor mutations (e.g., due to amplification artifacts) may occur without departing from clonality.

[0026] The terms "denaturing" and "denaturation" are broad terms that primarily refer to the physical separation of interacting DNA bases within a single-stranded polynucleotide sequence and its complement, a Watson-Crick DNA duplex. These terms also refer to the physical separation of both of these strands. In the broadest sense, these terms refer to a process that creates a condition that allows annealing of another primer oligonucleotide or polynucleotide sequence to one or both of the strands of the duplex.

[0027] As used herein, the term "flow cell" is intended to mean a chamber having a surface across which one or more fluidic reagents can flow and to which nucleic acids can be bound and seeded on the surface of the flow cell. Generally, a flow cell has an inlet and an outlet opening to facilitate fluid flow. Examples of flow cells and associated fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Patent No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211,414, U.S. Patent No. 7,315,019, U.S. Patent No. 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0028] In certain embodiments, the flow cell comprises a solid support having a surface to which nucleic acids can be bound. In some aspects, the surface contains a lawn of capture agents that can bind to polynucleotides released from the beads. In some examples, the surface is a patterned surface. A "patterned surface" can refer to an arrangement (such as an array) of different regions (such as amplification sites) in or on the exposed surface of a solid support. For example, one or more of the regions can be features in which one or more capture agents are present. The features can be separated by interstitial regions in which no capture agents are present. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some examples, the pattern can be a random arrangement of features and / or interstitial regions. In some embodiments, the surface is a patterned surface containing an array of wells with capture agents that bind to the released polynucleotides from the beads, with interstitial regions between the wells that lack capture agents. In some cases, the substrates are exemplified in these references for applications using beads in the wells. The patterned flow cells can be used with the compositions, methods, and kits of the present disclosure.

[0029] A patterned flow cell includes an array of features, such as microwells or nanowells, on a glass, silicon, plastic, or other suitable solid support. The array of features can include a capture agent, which in one embodiment can include a capture agent, such as poly(N-(5-azidoacetamidylpentyl)acrylamide, PAZAM, which is covalently attached to a gel on the surface of the well. See, e.g., U.S. Patent Application Publication No. 2013 / 184796, WO 2016 / 066586, and WO 2015 / 002813, each of which is incorporated by reference in its entirety. The lifetime of the flow cell can be extended by covalently attaching a polymer to the features on the flow cell surface. However, in many instances, the gel does not need to be covalently attached to the features on the flow cell surface. For example, silane free acrylamide (PAZAM) can be used. acrylamide, SFA, see, for example, U.S. Patent No. 8,563,477, which is incorporated herein by reference in its entirety, can be used as the gel material. SFA is not covalently bound to the features on the flow cell surface. Examples of flow cells with patterned surfaces that can be used in the methods and compositions described herein are described in U.S. Patent Nos. 8,778,848, 8,778,849, and 9,079,148, and U.S. Patent Application Publication No. 2014 / 0243224, each of which is incorporated herein by reference in its entirety. An array of features on the surface of a flow cell can be used to capture single nucleic acid template molecules to seed the subsequent formation of homogenous colonies, for example, via bridge amplification. Such patterned arrays are particularly useful for nucleic acid sequencing applications.

[0030] The array of features on the flow cell surface can have, for example, at least about 10, e.g., at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 1,000,000, or at least about 5,000,000 or more features / cm. 2 The material may have any of a variety of densities, such as:

[0031] The array of features (e.g., wells) on the surface of the patterned flow cell may appear as a grid of spots or patches. The features may be located in a repeating pattern or in an irregular, non-repeating pattern. Particularly useful patterns are hexagonal patterns, rectilinear patterns, grid patterns, patterns with mirror symmetry, patterns with rotational symmetry, and the like. Asymmetric patterns may also be useful. The pitch may be the same between different pairs of nearest neighbor features, or the pitch may vary between different pairs of nearest neighbor features. In certain embodiments, the features (e.g., wells) of the patterned flow cell array are each about 100 nm 2 , 250 nm 2 , 500 nm 2 , 1 μm 2 , 2.5μm 2 , 5 μm 2 , 10μm 2 , 100μm 2 , or 500 μm 2 Alternatively or additionally, the features (e.g., wells) of the array can each have an area of ​​about 1 mm 2 , 500μm 2 , 100μm 2 , 25μm 2 , 10μm 2 , 5 μm 2 , 1 μm 2 , 500 nm 2 , or 100 nm 2 In practice, the region can have a size within a range between upper and lower limits selected from those exemplified above.

[0032] The arrays of features (e.g., wells) on the patterned flow cell surface may be distinct and separated by interstitial regions. The size of the features and / or the spacing between regions may vary such that the arrays may be high density, medium density, or low density. High density arrays are characterized by having features separated by less than about 15 μm. Medium density arrays have features separated by about 15-30 μm, while low density arrays have regions separated by more than 30 μm. Arrays may have regions separated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. Interstitial regions having a substantially low amount or concentration of capture agent compared to the features of the array are advantageous. Interstitial regions lacking capture agent are particularly advantageous. For example, the relatively low amount or absence of capture agent in the interstitial regions favors localization of target nucleic acids, and subsequently generated clusters, to the desired features. In certain embodiments, the features may be concave features (e.g., wells) within the surface, and the features may contain gel material. The gel-containing features (e.g., PAZAM) can be separated from one another by interstitial regions on the surface where gel is substantially absent or, if present, where the gel is substantially unable to support localization of nucleic acids. Methods and compositions for making and using substrates with gel-containing features, such as wells.

[0033] As used herein, the term "flow cell surface" refers to a portion of a flow cell that is accessible for contacting a reagent, bead, or analyte. The flow cell surface may be substantially flat or planar. Alternatively, the flow cell surface may be rounded or contoured. Exemplary contours that may be included on the flow cell surface are wells, depressions, posts, ridges, channels, and the like. Flow cell surfaces include glass, e.g., modified or functionalized glass; plastic, e.g., acrylic, polystyrene, or copolymers of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or Teflon™; polysaccharides or cross-linked polysaccharides, e.g., agarose or sepharose; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metals; inorganic glasses; fiber optic bundles, or various other polymers. A single material or a mixture of several different materials can be used to form the surface of the flow cell. Examples of flow cells and associated fluidic systems and detection platforms that can readily be used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Pat. No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Pat. No. 7,329,492, U.S. Pat. No. 7,211,414, U.S. Pat. No. 7,315,019, U.S. Pat. No. 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0034] The term "isothermal" as used herein refers to a process in which the temperature of a system or device remains constant, i.e., ΔT = 0. This occurs when the system / device is in contact with an external thermal reservoir (e.g., a heater, heat bath, thermoelectric controller (TEC), etc.) and actions or changes occur within the system / device at a rate that allows the system / device to continually adjust to the temperature of the reservoir through heat exchange.

[0035] As used herein, the term "polymerase" is intended to be consistent with its use in the art and includes, for example, an enzyme that uses a nucleic acid as a template strand to produce a complementary copy of a nucleic acid molecule. Typically, a DNA polymerase binds to the template strand and then moves downstream along the template strand, sequentially adding nucleotides to the free hydroxyl group at the 3' end of the growing nucleic acid strand. DNA polymerases typically synthesize complementary DNA molecules from a DNA template, and RNA polymerases typically synthesize RNA molecules from a DNA template (transcription). Polymerases can use short RNA or DNA strands (called primers) to initiate strand growth. Some polymerases can displace the strand upstream of the site where they add a base to the strand. Such polymerases can also be said to be strand displacing, which is said to have the activity of removing the 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 their preceding strand and effectively replace it with the growing strand (5' exonuclease activity). Some polymerases have the activity to degrade their trailing strand (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0036] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA); however, where appropriate, one of skill in the art will recognize that the systems and devices herein can also be utilized with ribonucleic acid (RNA). These terms should be understood to include, as equivalents, analogs of either DNA or RNA made from nucleotide analogs. As used herein, the term also encompasses cDNA, which is the complementary or copy DNA generated from an RNA template, for example, by the action of reverse transcriptase.

[0037] A "primer oligonucleotide" or "primer" is an oligonucleotide sequence capable of specifically annealing to a single-stranded polynucleotide sequence to be amplified under conditions encountered in the primer annealing step of each cycle of an isothermal amplification reaction. In general, an amplification reaction requires at least two amplification primers, often designated as "forward" and "reverse" primers. In certain embodiments, the forward and reverse primers can be identical. A primer oligonucleotide can include a "template-specific portion," which is a sequence of nucleotides capable of annealing to a primer binding sequence in a single-stranded polynucleotide molecule to be amplified during the annealing step (or its complement when the template is considered single stranded). A primer binding sequence is generally a known sequence and is therefore specifically complementary to a sequence within known sequence I and known sequence II of a single-stranded polynucleotide molecule. The length of the primer binding sequence need not be the same as the length of known sequence I or II, but can be shorter, for example, 16-50 nucleotides long, 16-40 nucleotides long, or 20-30 nucleotides long. The optimal length of the primer oligonucleotide depends on many factors, but it is generally the case that the primer is sufficiently long (complex) so that annealing to sequences other than the primer binding sequence is highly unlikely.

[0038] The polynucleotide molecules to be amplified are typically in single-stranded form, such as ssDNA or RNA, or in double-stranded DNA (dsDNA) form (e.g., genomic DNA fragments, PCR, and amplification products, etc.). Thus, single-stranded polynucleotides can be the sense or antisense strand of a polynucleotide duplex. Methods for preparing single-stranded polynucleotide molecules suitable for use in the disclosed systems / devices are known in the art, e.g., treatment with hydroxide followed by dilution, which can utilize standard techniques. The exact sequence of the primary polynucleotide molecule is generally not critical to the present disclosure and can be known or unknown. Single-stranded polynucleotide molecules can represent genomic DNA molecules (e.g., human genomic DNA), including both intron and exon sequences (coding sequences), as well as non-coding regulatory sequences such as promoter and enhancer sequences. In certain embodiments, the polynucleotide molecules to be amplified are DNA libraries. In further embodiments, the DNA libraries are generated using library preparation kits. In still further embodiments, the library preparation kit is from Illumina, Inc. (e.g., AmpliSeq™ kits, COVIDSeq™ kits, Illumina DNA prep kits, Illumina RNA prep kits, Nextera™ kits, SureCell WTA™ kits, TruSeq™ kits, and TruSight™ kits).

[0039] As used herein, the term "recombinase" is intended to be consistent with its use in the art and includes, for example, RecA protein, T4 uvsX protein, any homologous protein or protein complex from any phylum, or functional variants thereof. Eukaryotic RecA homologs are generally named Rad51 after the first member of this group to be identified. Other non-homologous recombinases (e.g., RecT or RecO) can be utilized in place of RecA.

[0040] As used herein, the term "single-stranded binding protein" is intended to refer to any protein that has the function of binding to single-stranded nucleic acid, for example, to prevent premature annealing, to protect single-stranded nucleic acid from nuclease digestion, to remove secondary structure from nucleic acid, or to facilitate nucleic acid replication. The term is intended to include, but is not necessarily limited to, proteins formally identified as single-stranded binding proteins by the Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). Exemplary single-stranded binding proteins include, but are not limited to, E. coli SSB, T4 gp32, T7 gene 2.5 SSB, phage phi 29 SSB, any homologous protein or protein complex from any phylum, or functional variants thereof.

[0041] As used herein, "solid phase amplification" refers to a nucleic acid amplification reaction carried out on the surface of a channel of a flow cell such that, upon formation, all or a portion of the amplification product is immobilized on a solid support.

[0042] Library preparation is a critical first step for all next-generation sequencing (NGS) applications and generally employs several common steps. In vitro transposition has been used to generate sequencer-ready libraries from genomic DNA (gDNA) with dramatic time savings and reduced input requirements over previous methods. Transposome-based library construction improves the DNA library preparation process by eliminating the need for a separate DNA fragmentation step and removing the requirement for ligation between DNA fragments. Fragmentation efficiency is highly dependent on the enzyme-to-DNA ratio. Thus, variable DNA input amounts will produce inconsistencies in the fragment size distribution of the library. This introduces variability in the library preparation process that can have downstream effects on sequencing coverage.

[0043] Bead-linked transposome technology has been used to prepare libraries for whole genome sequencing technologies (e.g., Illumina™ DNA prep) as well as targeted sequencing applications (e.g., Illumina™ DNA prep for enrichment). This methodology utilizes transposomes of known concentration directly conjugated to beads to bind a fixed amount of DNA. It therefore offers broad applicability and supports a broad spectrum of DNA input ranges, as well as integrated extraction of blood and saliva samples to generate normalized libraries for sequencing to facilitate a quantification-free workflow. Libraries generated outside the standard parameters of the workflow highlight novel applications for whole genome sequencing, including human genome assembly and variant calling from less than 1 ng of DNA input, customization of insert size, and preparation of libraries from short fragments and heavily degraded samples. For large and complex genomes, coverage across the genome, including challenging regions, was improved compared to other library preparation methods. Libraries were successfully generated from amplicons of various sizes (50 bp to 11 kb), although reduced efficiency was observed for amplicons smaller than 250 bp. On-bead tagmentation chemistry is also applicable to targeted sequencing, supporting a wide range of DNA input amounts, various sample types, and a wide range of applications including fixed panels, custom panels, and whole-exome sequencing. Thus, bead-linked transposomes can be effectively used for whole genome sequencing as well as targeted resequencing. The source of DNA can be from the environment, from a subject's sample, etc. DNA can be extracted from virtually any part of the subject's body. For example, input DNA can be extracted from a variety of sample types, including but not limited to urine, blood, saliva, tissue, serum, plasma, sputum, bile, feces, hair (or hair), skin, and / or primary cell samples.

[0044] The embodiments provided herein include capturing polynucleotides released from the bead-linked transposomes disclosed herein on the surface of a flow cell. The bead-linked transposomes are loaded into a flow cell, and the methods of the present disclosure are used to release the polynucleotides from the bead-linked transposomes and seed the surface of the flow cell.

[0045] In some examples, the bead-linked transposome or the polynucleotide released from the bead-linked transposome is captured on the surface of the flow cell by interaction of the capture agent on the surface of the flow cell with the released polynucleotide or transposome bead. Examples of capture agents for immobilizing transposome beads on the surface of the flow cell in the disclosed methods include, but are not limited to, at least a portion of the nucleic acid linked to the adaptor on the bead or a portion of the nucleic acid released from the transposome bead; or a member of a receptor-ligand binding pair (e.g., avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.) that can bind to the target nucleic acid (or the linking moiety attached thereto); or a capture nucleic acid that is complementary to a chemical reagent that can form a covalent bond with the target nucleic acid (or the linking moiety attached thereto).

[0046] In some examples, the capture agent is a first member of a specific binding pair located on a flow cell and binds to a second member of the specific binding pair located on a transposomal bead or to a polynucleotide released from the bead. For example, the flow cell is functionalized with a first member of a specific binding pair and the polynucleotide released from the bead comprises a second member of the specific binding pair.

[0047] In some instances, the capture agents are present at discrete locations on the flow cell, e.g., in instances where the flow cell is a patterned flow cell containing an array of wells, the capture agents can be present in the wells of the patterned flow cell.

[0048] In addition, the flow cell may contain bead trapping structures such as pillars / posts, weirs, cup-like structures, etc. to immobilize / trap beads at defined locations within the flow cell. These trapping structures can be fabricated by etching into glass or silicone, or by photolithography of photopatternable materials.

[0049] In certain embodiments, the present disclosure describes a method for enhancing or improving the efficiency of seeding a flow cell with bead-linked transposomes, comprising the step of transposing a nucleic acid onto a transposomal bead. After transposition, both 5' ends of the transposed strand are linked to the bead-linked transposome via a strong biotin / streptavidin bond. This has previously required the use of excess biotin and heat to release the molecules from the beads. Heat also leads to increased diffusion in the flow cell, which destroys the spatial co-location of the formed clusters. The present disclosure provides an improved or enhanced method for improving nucleic acid release and flow cell seeding that does not require the use of heat or excess biotin.

[0050] In another embodiment, the present disclosure describes a method for enhancing or improving the efficiency of seeding a flow cell with bead-linked transposomes, comprising releasing nucleic acids from transposome beads and seeding the nucleic acids onto one or more surfaces of a flow cell such that closely linked nucleic acids are spatially co-localized on selected locations on the surface of the flow cell. The spatial co-localization of nucleic acids on the flow cell surface acts as a form of indexing, thereby allowing sequencing reads to be linked together into contigs.

[0051] In certain embodiments, the disclosed method is advantageously PCR-free. In another embodiment, the disclosed method exhibits high efficiency of release / seeding such that almost all transposed molecules are captured on one or more surfaces of the flow cell. Typically, the nucleic acid molecules are released from the beads by the use of heat / excess biotin. However, one drawback of using the above techniques is that the patterned flow cells require 2-3 or more molecules to efficiently seed the nanowells, whereas the use of standard methodologies is less efficient and results in reduced capture efficiency of transposed molecules and therefore more "holes" in coverage for each contig. Compared to standard techniques, the disclosed method provides improved or enhanced release and seeding efficiency of flow cells with polynucleotides.

[0052] FIG. 1 provides an exemplary embodiment of the disclosed method used to enhance or improve the efficiency of seeding a flow cell with bead-linked transposomes. As shown, the method uses a solution primer (e.g., P7) to prime off the 3' end of a Y-shaped adaptor on a bead (step 1). Exclusion amplification is used to extend the primer, creating the dsDNA structure seen in step 2. Further strand invasion then occurs via more P7 primers and recombinase (step 3). As these are extended by polymerase, the products displace the previously copied strand into solution (step 4a), which is then available for seeding and amplification onto a surface (step 4b). Incubating the P7 / exclusion amplification mix with the beads performs additional rounds of extension, primer invasion and release, resulting in linear copying of the strand from the bead. The result overcomes the need for multiple strands to seed each nanowell of a patterned flow cell, which is also less susceptible to the biases of typical two-primer solution PCR (thus preserving the good GC bias of PCR-free libraries).

[0053] A further embodiment of the present disclosure is shown in FIG. 2, where an amplification primer different from the solution primer is included on the bead. For example, the bead can include an amplification primer such as P5. Thus, in step 3 of FIG. 1, in addition to the solution primer (e.g., P7) that binds to the adapter, there will be an additional primer invasion at the bead surface by the amplification primer (e.g., P5). As a result, more copies are made from each strand, which is closer to exponential amplification rather than linear amplification. Although the use of amplification primers can introduce PCR-type bias, this method is a surface-based amplification rather than bulk solution-based PCR, i.e., there is less competition for reagents. For example, cluster-based surface amplification has less bias than nanolibrary amplification. This concept is illustrated in FIG. 2.

[0054] An alternative embodiment of the disclosed method is shown in FIG. 3. In this embodiment, the beads contain the same amplification primer as the solution primer (e.g., both are P7). Exclusion amplification (e.g., ExAMP) is then used for localized linear amplification onto the amplification primer surrounding each translocation event. The beads are then introduced into a flow cell and either (a) uses a heat / excess biotin means to release the amplified polynucleotide from the streptavidin / biotin bond, or (b) uses an exclusion amplification (e.g., ExAmp) and a second solution primer means to release the amplified polynucleotide via invasion / displacement. The latter means further provides amplification across the surface of the bead.

[0055] A further alternative embodiment of the disclosed method is that the beads contain two or more different types of amplification primers (e.g., P5 and P7). In this embodiment, after transposition, surface-based clustering of the library can be performed along with amplification. The beads are then introduced into a flow cell and either (a) use heat / excess biotin means to release the amplified polynucleotides from the streptavidin / biotin bond, or (b) use exclusion amplification (i.e., ExAmp) and solution primer means to release the amplified polynucleotides via invasion / displacement. For the latter means, solution primers that correlate to one or the other amplification primers can be used, or solution primers that correlate to multiple types of amplification primers are used. The amplification primers on the beads should be inactive against transposition or rendered inactive by the use of single-stranded binding proteins, etc.

[0056] 4 provides an embodiment of the disclosed method in which a bridge amplification approach is used to amplify the library on beads and seed the amplification products into nanowells. In this approach, soluble primers are first hybridized to the library and extended using an amplification mixture (e.g., AMS). The library is then denatured from the beads by flowing in a 40% formamide / HT1 mixture and then increasing the temperature to >80°C. The denatured library strands are seeded by lowering the temperature to 20°C. Another round of extension and denaturation is then performed by exchanging reagents.

[0057] The compositions, methods, and kits disclosed herein relate to the efficient release of polynucleotides from beads seeded and amplified on the surface of a flow cell to provide readable clusters for maximum molecular coverage of polynucleotides.As illustrated in Figure 5, when two or more DNA library molecules are seeded on the same well of a patterned flow cell, it will either end up with only one dominant cluster, or two or more equivalent clusters resulting in polyclonality.In both cases, there is a loss of valuable information from polynucleotides that did not become thermal clusters.

[0058] To provide even more extensive seeding of patterned flow cells, the present disclosure provides an on-flow cell linear amplification / shallow denaturation process that provides more efficient and extensive seeding within the cluster cloud region. As illustrated in Figures 6A-6D, after seeding polynucleotides on the surface of the flow cell, a low-temperature first extension with Illumina™ AMS1 is used to copy the polynucleotides. A "shallow liquid" denaturation and seeding step is then used to re-seed the copied polynucleotides near the wells. The reagents used for single-pot denaturation / seeding are a mixture of LDR (formamide) and HT1. A bridge amplification step is then used. Using the aforementioned process, sequencing information from all polynucleotides released from the beads can be realized.

[0059] In certain embodiments, the problem of low seeding efficiency from bead-linked transposomes on patterned flow cells is solved by using exclusion amplification (e.g., ExAmp) with solution primers to copy the transposition templates on the beads. Subsequent strand invasion by more solution primers then results in displacement of the copied strands into solution, thereby effectively releasing them from the beads and allowing them to seed onto the surface of the flow cell in close proximity to the beads. Further embodiments of this method may include bead-bound amplification primers to enhance amplification and displacement of the bead-bound templates.

[0060] The methods described herein can be used to generate clusters of various densities on the flow cell surface. For example, the flow cell surface may have a cluster density (K / mm) of about 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1050, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2500, 3000, 4000, 5000, or a range of densities including or between any two of the foregoing values. 2 The density of the clusters is related to the number of beads used and the size of the beads.

[0061] Nucleic acids can be attached to beads by single-point covalent attachment to the surface at or near the 5' end of the nucleic acid or at or near the 3' end of the nucleic acid. In embodiments where the nucleic acid functions as an amplification primer, the attachment is configured to free the template-specific portion of the primer to anneal to its cognate template and free the 3' hydroxyl group for primer extension. Any suitable covalent attachment agent known in the art may be used for this purpose. The attachment chemistry selected will depend on the nature of the bead surface and any derivatization or functionalization applied to it. Examples of attachment agents may include, but are not limited to, biotin / streptavidin bonds, covalent bonds, base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier that contains a metal ion, and a receptor that binds to a ligand. The amplification primers attached to the beads may include moieties that may be non-nucleotide chemical modifications to facilitate attachment. In certain embodiments, the amplification primers may include a biotin residue at the 5' end that can form a bond with streptavidin or avidin on the surface of the bead. In another embodiment, the amplification primer may contain a sulfur-containing nucleophile, such as a phosphorothioate or thiophosphate, located at the 5' end. In the case of polyacrylamide-based beads, this nucleophile binds to bromoacetamide groups present on the surface of the beads. Attachment may also occur via ligand-receptor interactions as described elsewhere herein.

[0062] Beads for use with the patterned flow cells disclosed herein are comprised of polymeric materials (e.g., polystyrene or polystyrene divinylbenzene, silica materials, zirconia materials, and / or superparamagnetic materials, etc.) that have been "functionalized" by application of a layer or coating of material that contains reactive groups that allow for the covalent attachment of nucleic acids or other biomolecules. Examples of such coatings include, but are not limited to, streptavidin, amine groups, carboxyl groups, epoxy groups, tosyl groups, or ligands (e.g., receptors, receptor ligands, etc.). In such embodiments, polynucleotides may be covalently attached directly to reactive groups (e.g., tosyl groups) in a layer or coating on the beads, although the coating or layer itself may be non-covalently attached to the beads. In further embodiments, beads disclosed herein may have several layers, which may have different compositions. Beads disclosed herein are generally spherical, but may also be ovoid. Beads may include a generally hydrophilic surface or a generally hydrophobic surface.Beads are approximately 500 nm, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3 μM, 3.1 μM, 3.2 μM, 3.3 μM. M, 3.4μM, 3.5μM, 3.6μM, 3.7μM, 3.8μM, 3.9μM, 4μM, 4.1μM, 4.2μM, 4.3μM, 4.4μM, 4.5μM, 4. 6μM, 4.7μM, 4.8μM, 4.9μM, 5.0μM, 5.1μM, 5.2μM, 5.3μM, 5.4μM, 5.5μM, 5.6μM, 5.7μM, 5.8μM M, 5.9μM, 6μM, 6.1μM, 6.2μM, 6.3μM, 6.4μM, 6.5μM, 6.6μM, 6.7μM, 6.8μM, 6.9μM, 7μM, 7.1μM M, 7.2μM, 7.3μM, 7.4μM, 7.5μM, 7.6μM, 7.7μM, 7.8μM, 7.9μM, 8μM, 8.1μM, 8.2μM, 8.3μM, 8. The beads have a diameter of 4 μM, 8.5 μM, 8.6 μM, 8.7 μM, 8.8 μM, 8.9 μM, 9.0 μM, 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM, 9.5 μM, 9.6 μM, 9.7 μM, 9.8 μM, 9.9 μM, 10 μM, or a range including or between any two of the aforementioned distances (including fractional increments thereof). The beads typically contain multiple bound polynucleotides that may or may not have the same sequence. The number of bound polynucleotides depends largely on the size of the beads and the number of available reactive groups on the bead surface. The beads can be composed of a variety of materials, including controlled pore glass beads, paramagnetic beads, polystyrene, triazole, sepharose beads, nanocrystals, and others known in the art, for example, as described in the Microsphere Detection Guide from Bangs Laboratories, Fishers Ind. The beads can be made from biological or non-biological materials. Magnetic beads are particularly useful due to the ease of manipulation of the magnetic beads using magnets in the various steps of the methods described herein.

[0063] The beads disclosed herein typically include bead-linked transposomes. Bead-linked transposomes can perform on-bead tagmentation on a provided DNA template (e.g., genomic DNA, long template DNA, etc.). Transposomes can be directly attached to beads using attachment agents such as biotin / streptavidin binding, covalent bonds, base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier that contains a metal ion, and a receptor that binds to a ligand. Alternatively, transposomes may be indirectly attached to beads by using a linker. Examples of linkers may include nucleotides, polymers, peptides, etc. In certain embodiments, transposomes are indirectly linked to beads via oligonucleotide linkers. In such cases, the oligonucleotide linkers may include terminal sequences (e.g., mosaic terminal sequences) that are recognized and bound by the transposomes. The transposase linked to the beads can be transposase Tn5, Sleeping Beauty transposase, Tn7 transposon, or mutations in each of the foregoing to improve activity and / or stability. In certain embodiments, the transposome linked to the beads comprises transposase Tn5, or a mutant or variant thereof. The oligonucleotide linker linking the transposome to the beads can be attached to the beads using an attachment agent, such as a biotin / streptavidin bond, a covalent bond, base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier that contains a metal ion, and a receptor that binds to a ligand. In certain embodiments, the oligonucleotide linker is attached to the beads via a biotin / streptavidin or avidin bond. The oligonucleotide linker may further comprise one or more barcode sequences, primer sequences, sequencing primer binding sites, and / or restriction enzyme recognition sites.

[0064] The clusters generated on the surface of the flow cell by releasing the polynucleotides from the immobilized beads can be of a size similar to or smaller than the area of ​​the surface occupied by the beads from which the clusters are generated, and the clusters are typically of similar size and intensity to each other. The uniform size, uniform intensity, and lack of overlapping can provide a preferred cluster density per unit area higher than that obtained from other random deposition methods, because these other methods often result in surfaces with overlapping clusters and different sizes and intensities. Images of dense, non-overlapping clusters with uniform size and intensity are typically easier to analyze than images in which a subset of clusters overlap each other.

[0065] In certain embodiments, the polynucleotide bound to the beads comprises genomic DNA. In further embodiments, the polynucleotide strand bound to the beads comprises a long template DNA. In yet further embodiments, The polynucleotide strands bound to the beads contain DNA from a sample of the subject. Examples of samples obtained from a subject include, but are not limited to, urine, blood, saliva, tissue, serum, plasma, sputum, bile, feces, hair (or hair), skin, and / or primary cell samples. The polynucleotides can be bound to the beads using an attachment agent. Examples of attachment agents include, but are not limited to, biotin / streptavidin binding, covalent binding, base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier containing a metal ion, and a receptor that binds to a ligand.

[0066] When strands are released from beads by using the methods disclosed herein, they bind to the capture agent on the surface of the flow cell closest to the bead, and the proximity of the beads to each other in the layer determines the proximity of the polynucleotides captured on the flow cell surface. The use of beads to seed a non-patterned flow cell provides an indirect "patterned array" based on the size of the beads used and their packing density on the surface of the flow cell. Alternatively, patterned flow cells can be used with the methods disclosed herein. After strands of polynucleotides are captured on the flow cell surface (flow cell seeding), cycles of extension with polymerase and dNTPs to copy the strands, followed by denaturation provide the desired array of attached nucleic acids in single-stranded form, which can then be subjected to cycles of isothermal amplification. Alternatively, the seeding efficiency of the flow cell can be further improved by performing an on-flow cell linear amplification of the seeded polynucleotides prior to isothermal amplification (e.g., bridge amplification), followed by the low-volume denaturation and seeding methods disclosed herein. In such an embodiment, various surfaces of the flow cell features contain loans of single-stranded primer sequences, allowing "bridge amplification" to occur. In bridge amplification, when the surface is exposed to conditions suitable for hybridization, single-stranded nucleic acid molecules to be amplified form bridges such that adapter sequences on their free ends hybridize with their complementary single-stranded primer sequences bound to the surface of the flow cell. Nucleotides and DNA polymerase are then transported to the flow cell to create complementary strands of the nucleic acid to be amplified. The created double-stranded sequences are then denatured by flowing in a denaturing reagent, and the process begins again, thus creating clusters of amplified nucleic acids without changing the temperature of the system during the amplification cycle. In a typical embodiment, the majority of the clusters are monoclonal and result from the amplification of a single original nucleic acid sequence.

[0067] Generally, the primer oligonucleotides used to create DNA clusters are single-stranded polynucleotides. They may also contain a mixture of natural and non-natural bases, as well as natural and non-natural backbone linkages, provided that any non-natural modifications do not preclude their function as primers (i.e., their ability to anneal to a template polynucleotide strand during the conditions of an amplification reaction and act as a starting point for the synthesis of a new polynucleotide strand complementary to the template strand). One of the primers may contain a modification that removes (cleaves) the primer from the surface, allowing the formation of a single-stranded cluster. Such linearized clusters may undergo hybridization with additional primer strands, allowing a sequencing reaction to occur. Extension products can then be generated by performing cycles of isothermal amplification on the covalently bound single-stranded polynucleotide molecules, such that each colony contains multiple copies of the original immobilized single-stranded polynucleotide molecule (and its complementary sequence). One cycle of amplification consists of the steps of hybridization, extension, and denaturation. Such steps are generally comparable in terms of reagent components (e.g., buffers, etc.) to conventional nucleic acid amplification procedures, such as PCR. Suitable reagents for amplifying nucleic acids (e.g., hybridizing, extending, etc.) are well known in the art. Exemplary reagents are described in more detail below.

[0068] Thus, a neutralization / hybridization buffer can be applied to the single-stranded polynucleotide molecule and a plurality of primer oligonucleotides such that the unbound ends of the surface-bound single-stranded polynucleotide molecules hybridize to the surface-bound primer oligonucleotides to form complexes (the primer oligonucleotides hybridize to and are complementary to a region or template-specific portion of the single-stranded polynucleotide molecule). This process creates a "bridge" structure. See also WO 0246456, U.S. Patent Application Serial No. 60 / 783,618, WO 9844151, and WO 0018957 for further discussion of bridge amplification.

[0069] Suitable neutralization / hybridization buffers are well known in the art (see Sambrooke et al., Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press, NY; Current Protocols, eds. Ausubelet et al.) as well as the schematic section describing amplification below. Suitable buffers may contain additives such as betaine or organic solvents to normalize the melting temperatures of different template sequences, and detergents. An exemplary hybridization buffer contains 2M betaine, 20 mM Tris, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton, 1.3% DMSO, pH 8.8.

[0070] An extension reaction is then carried out by applying to the bridge complex an extension solution containing an enzyme having polymerase activity and dNTPs. The primer oligonucleotide of the complex is extended by sequentially adding nucleotides to generate an extension product complementary to the single-stranded polynucleotide molecule. Suitable extension buffers / solutions are well known in the art (see, for example, Sambrooke et al., Molecular Cloning, A Laboratory Manual, 3 rd Ed., Cold Spring Harbor Laboratory Press, NY; Current Protocols, eds. Ausubel et al.) and the Examples below.

[0071] Examples of enzymes with polymerase activity that may be used in the disclosed system / device include DNA polymerases (Klenow fragment, T4 DNA polymerase) and thermostable DNA polymerases from various thermotolerant bacteria (such as Taq, VENT, Pfu, Bst, and Tfl DNA polymerases), as well as their genetically modified derivatives (such as TaqGold, VENT exo, Pfu exo, etc.). It should be understood that because the amplification reaction performed on the flow cell is isothermal, additional and / or alternative DNA polymerases may be used compared to the polymerase for thermocycling amplification, and in most embodiments, there is no specific requirement that the polymerase be thermostable. Also, enzymes with strand displacement activity, such as Bst polymerase, show superior performance in growth effect clusters for sequencing, although any DNA polymerase may be used.

[0072] The nucleoside triphosphate molecules used to create DNA clusters are typically deoxyribonucleotide triphosphates, such as dATP, dTTP, dCTP, dGTP. The nucleoside triphosphate molecules can be naturally occurring or non-naturally occurring.

[0073] After the hybridization and extension steps, the support and attached nucleic acid are subjected to denaturing conditions. Suitable denaturing buffers are well known in the art (see, for example, Sambrooke et al., Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press, NY; Current Protocols, eds. Ausubelet et al.). The disclosed system / device produces isothermal nucleic acid amplification, and thus the nucleic acid strands herein are not denatured by temperature increase or manipulation, but rather by other methods (e.g., chemical, physical, etc.). As an example, it is known that changes in pH and low ionic strength solutions can denature nucleic acids at substantially isothermal temperatures. Formamide and urea form new hydrogen bonds with the bases of the nucleic acid that break the hydrogen bonds that result in Watson-Crick base pairing. These result in single-stranded nucleic acid molecules. Alternatively, the strands can be separated by treatment with solutions of low salt and high pH (>12) or by using chaotropic salts (e.g., guanidinium hydrochloride). In certain embodiments, sodium hydroxide (NaOH) solution is used at a concentration of about 0.25 M to about 0.1 M. In alternative embodiments, 95% formamide in water, or 100% formamide is used. Such formamide embodiments show additional advantages since hydroxide treatment can damage the surface, resulting in lower intensity clusters in some cases. Such denaturing reagents, like other reagents used, are passed through the flow channel.

[0074] After denaturation, there are two immobilized nucleic acids, the first being the original immobilized single-stranded polynucleotide molecule and the second being its complement that extends from one of the immobilized primer oligonucleotides. Both the original immobilized single-stranded polynucleotide molecule and the immobilized extended primer oligonucleotide (complement) can then initiate a further round of amplification by subjecting the support to further cycles of hybridization, extension, and denaturation. Such further rounds of amplification result in a nucleic acid colony or "cluster" that contains multiple immobilized copies of the single-stranded polynucleotide sequence and its complementary sequence. The initial immobilization of the single-stranded polynucleotide molecule means that the single-stranded polynucleotide molecule can only hybridize with the primer oligonucleotide located at a distance within the entire length of the single-stranded polynucleotide molecule. Thus, the boundaries of the formed nucleic acid colony or cluster are limited to the relatively localized area where the original single-stranded polynucleotide molecule was immobilized. The terms "cluster" and "colony" are used interchangeably herein and refer to a distinct site on a solid support that is composed of multiple identical immobilized nucleic acid strands and multiple identical immobilized complementary nucleic acid strands. The term "clustered array" or "cluster array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood as requiring an ordered arrangement of the clusters.

[0075] In a typical embodiment, the nucleic acid to be amplified is bound to a bead and immobilized on the surface of a flow cell. Bead-linked transposomes can also be immobilized on the surface of a flow cell. The term "immobilized" as used herein is intended to encompass direct or indirect, covalent or non-covalent attachment, unless otherwise indicated explicitly or by context. In certain embodiments of the invention, covalent attachment is typical, but generally, all that is required is that the molecule (e.g., nucleic acid) remains immobilized or attached to the support under conditions for which the support is intended to be used, e.g., in amplification applications. Immobilized nucleic acid molecules for amplification can be obtained by directly attaching a suitably modified nucleic acid molecule (either single-stranded or double-stranded) to a suitable reactive surface, or by hybridization to a surface-immobilized primer, followed by copying the hybridized strand by extension cycles with polymerase and dNTPs. The extended strands or chemically attached double strands can then be subjected to denaturing conditions to remove the beads and generate the desired immobilized single-stranded nucleic acid molecules, which can then be subjected to cycles of isothermal amplification by the instrumentation described herein. The initial step of hybridizing DNA from solution onto the flow cell can be performed at a higher temperature than the subsequent amplification reaction, which can then be performed at a substantially isothermal temperature. The hybridization step can also be performed at the amplification temperature, except that the input nucleic acid strands are provided to the flow cell surface in single-stranded form attached to a bead.

[0076] Some embodiments of preparing a template nucleic acid may include fragmenting a target nucleic acid. In some embodiments, barcoded or indexed adaptors are attached to the fragmented target nucleic acid (e.g., a DNA library). The adaptors may be attached using any number of methods known in the art, such as ligation (enzymatic or chemical), tagmentation, polymerase extension, etc. In some embodiments, insertion of a transposome containing a non-contiguous transposon sequence may result in fragmentation of the target nucleic acid. In some embodiments including loop transposomes, a target nucleic acid containing a transposon sequence may be fragmented at the fragmentation site of the transposon sequence. Further examples of methods useful for fragmenting target nucleic acids useful for embodiments provided herein can be found, for example, in U.S. Patent Application Publication No. 2012 / 0208705, U.S. Patent Application Publication No. 2012 / 0208724, and International Patent Application Publication No. 2012 / 061832, each of which is incorporated herein by reference in its entirety. In a further embodiment, the tagmented sequences are amplified using a cycle-limited PCR reaction comprising primers having sequences complementary to the adapter sequences, the sequences of SEQ ID NO:1 and SEQ ID NO:2, and optionally a barcode or index sequence. In a further embodiment, the sequences amplified using the cycle-limited PCR reaction are from / in a DNA library. In yet a further embodiment, the sequences amplified using the cycle-limited PCR reaction are denatured to single-stranded DNA and selectively bound by magnetic beads comprising a biotinylated capture probe that recognizes a specific sequence and streptavidin that interacts with the biotinylated capture probe. In a further embodiment, the magnetic beads comprising the biotinylated capture probe hybridized to the sequences amplified by cycle-limited PCR are immobilized on one or more surfaces of a flow cell using the methods of the present disclosure.

[0077] In some embodiments, linear amplification on beads and / or amplification of polynucleotides seeded on the flow cell surface can be performed using kinetic exclusion amplification (KEA), also referred to as exclusion amplification (ExAmp). Kinetic exclusion amplification reactions include components that promote the formation of selected amplification products (e.g., strands released from the beads). One example of an ExAmp component is a recombinase. Recombinases can promote selected amplification product formation by allowing repeated invasion / extension. More specifically, recombinases can promote invasion of target nucleic acid by polymerase, which uses the target nucleic acid as a template for amplification product formation, and extension of primers by polymerase (primer invasion). This process can occur more quickly than standard PCR, since no denaturation cycles (e.g., by heating or chemical denaturation) are required. Thus, recombinase-promoted amplification can be performed isothermally. In general, it is desirable to include ATP, or other nucleotides (or possibly non-hydrolyzable analogs thereof) in the recombinase-promoted amplification reagents to promote amplification. A mixture of recombinase and single stranded binding (SSB) protein is particularly useful since SSB can further enhance amplification. Exemplary formulations for recombinase-facilitated amplification include those sold as TwistAmp kits by TwistDx (Cambridge, UK). Useful components and reaction conditions for recombinase-facilitated amplification reagents are described in U.S. Patent No. 5,223,414 and U.S. Patent No. 7,399,590.

[0078] Helicase is another example of a component of a dynamic exclusion amplification reaction that promotes selected amplification product formation and, in some cases, increases the rate of amplification product formation. Because no denaturation cycles (e.g., by heating or chemical denaturation) are required, the process can occur more quickly than standard PCR. Thus, helicase-promoted amplification can be performed isothermally. A mixture of helicase and single-stranded binding (SSB) protein is particularly useful, as SSB can further promote amplification. Exemplary formulations for helicase-promoted amplification include those commercially available as IsoAmp kits from Biohelix (Beverly, MA). Further examples of useful formulations that include helicase proteins are described in U.S. Pat. Nos. 7,399,590 and 7,829,284. Origin binding protein is yet another example of a component that can be included in an ExAmp reaction to promote the formation of amplified linear products. The beads can be removed from the flow cell before or after the ExAmp-driven polynucleotide release step.

[0079] A variety of flow cell devices can be used to perform the methods of the present disclosure, including flow cell devices from Illumina, Inc. (e.g., HiSeq, NovaSeq, MiSeq, and NextSeq devices), F. Hoffmann-La Roche Ltd. (e.g., GS FLX and GS Junior devices), and Life Sciences (e.g., SOLiD / Ion Torrent devices). In certain embodiments, the flow cell device used to perform the methods of the present disclosure is an Illumina Inc. flow cell device.

[0080] In some embodiments, an exclusion amplification reaction can be used to amplify polynucleotides seeded on a flow cell surface. The exclusion amplification reaction proceeds until a sufficient number of amplicons are generated to fill the capacity of each amplification site (e.g., the wells of a patterned flow cell). Thus, filling an already seeded site to capacity inhibits a target nucleic acid from landing and amplifying at that site, thereby generating a clonal population of amplicons at that site. In some embodiments, apparent clonality can be achieved even if the amplification site is not filled to capacity before a second target nucleic acid arrives at the site. Under some conditions, amplification of a first target nucleic acid can proceed to a point where a sufficient number of copies are made to effectively exceed or overwhelm the generation of copies from a second target nucleic acid transported to the site. For example, in an embodiment using a bridge amplification process on a circular feature less than 500 nm in diameter, it was determined that after 14 cycles of exponential amplification for a first target nucleic acid, contamination from a second target nucleic acid at the same site generates an insufficient number of contaminating amplicons to adversely affect sequence synthesis analysis on an Illumina sequencing platform.

[0081] In some embodiments, dynamic exclusion may occur when a process occurs at a sufficiently fast rate to effectively exclude another event or process from occurring. Take as an example the creation of a nucleic acid array on a flow cell surface, where sites on the flow cell are randomly seeded with target nucleic acid from beads using the method of the present disclosure, and copies of the target nucleic acid are generated in an amplification process to fill each of the seeded sites to capacity. According to the dynamic exclusion method of the present disclosure, the seeding and amplification processes can proceed simultaneously under conditions where the amplification rate exceeds the seeding rate. Thus, the relatively fast rate at which copies are made at a site seeded by a first target nucleic acid effectively excludes a second nucleic acid from seeding that site for amplification. The binding equilibrium exclusion amplification method can be carried out as described in detail in the disclosure of US Patent Application Publication No. 2013 / 0338042.

[0082] Dynamic exclusion may take advantage of the relatively slow rate of initiating amplification (e.g., the slow rate of making a first copy of a target nucleic acid) versus the relatively fast rate of making subsequent copies of the target nucleic acid (or the first copy of a target nucleic acid). In the example of the previous paragraph, dynamic exclusion occurs due to a relatively slow rate of target nucleic acid seeding (e.g., relatively slow diffusion or transport) versus the relatively fast rate at which amplification occurs to fill a site with copies of a nucleic acid species. In another exemplary embodiment, dynamic exclusion may occur due to a delay in the formation of the first copy of a target nucleic acid seeded at a site (e.g., a delay or slow activation) versus the relatively fast rate at which subsequent copies are made to fill a site. In this example, individual sites or wells may be seeded with several different target nucleic acids (e.g., several target nucleic acids may be present at each site prior to amplification). However, the first copy formation of any given target nucleic acid may be activated randomly, which results in a relatively slow average rate of first copy formation compared to the rate at which subsequent copies are generated. In this case, individual sites may be seeded with several different target nucleic acids, but dynamic exclusion allows amplification of only one of those target nucleic acids. More specifically, when a first target nucleic acid is activated for amplification, the site or well is rapidly filled with copies of it, thereby preventing copies of a second target nucleic acid from being made at that site.

[0083] In some patterned flow cells, the patterned flow cells include wells having size dimensions (e.g., diameters) selected in a range that favors dynamic exclusion amplification (KEA) and formation of monoclonal populations of target-specific polynucleotides in the wells. Dynamic exclusion amplification of nucleic acid libraries is described, for example, in U.S. Patent Publication No. 2013 / 0338042. For example, well sizes (e.g., diameters) can vary between about 30 nm and about 1 μm, between about 50 nm and about 800 nm, between about 70 nm and about 600 nm, or between 100 nm and about 400 nm. In some embodiments, the wells have a diameter of about 400 nm. In some embodiments, the wells have a diameter of less than about 1 μm. Exemplary patterned flow cells include the Illumina® HiSeq-X10 patterned flow cell.

[0084] In further embodiments, the present disclosure provides a method to increase the seeding efficiency of patterned flow cells by performing on-flow cell linear amplification to copy polynucleotides captured on the flow cell surface, followed by "shallow liquid" denaturation and seeding to re-seed the polynucleotide copies into nearby regions. Examples of denaturants that may be used in such processes include, but are not limited to, the use of heat, sodium hydroxide treatment, high salt concentrations, formamide, guanidine, sodium salicylate, dimethyl sulfoxide, propylene glycol, and urea.

[0085] For the on-flow cell linear amplification step, any number of amplification techniques may be used, including ExAmp, as previously discussed herein. Alternatively, AMS1 first extension may be used to generate double-stranded DNA (one copied anchor strand with the original strand) on the flow cell surface. For the "shallow layer" denaturation step, a hybridization buffer containing a chemical denaturant (e.g., formamide), such as HT1 from Illumina™, may be used. Hybridization buffers generally contain a buffered salt solution, such as 5% SSPE or 5xSSC, and a small amount of a non-ionic detergent (e.g., 0.1% Tween or 0.1% sodium dodecyl sulfate). Hybridization buffers may contain a chemical denaturant, such as formamide or guanidine, in a percentage of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or any two of the aforementioned percentages or in a range between them. The amount of hybridization used should be sufficient to fluidly connect the wells of the patterned flow cell. After reseeding of the polynucleotides, bridge amplification can then be used to form clusters on the surface of the patterned flow cell for further processing, such as sequencing.

[0086] Kits and articles of manufacture are also described herein for use in the flow cell applications described herein. Such kits may include carriers, packages, or containers that are compartmentalized to receive one or more containers, such as vials, tubes, and the like, each of which contains one of the separate elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic.

[0087] For example, a container can contain one or more bead-linked transposomes and reagents (e.g., soluble primers, ExAmp, buffers, etc.) used to carry out the methods described herein. The container optionally has a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally contain a reagent along with an identifying description or label or instructions for its use in the methods described herein.

[0088] The kit typically includes one or more additional containers, each having one or more of a variety of materials (including additional reagents and / or devices, optionally in concentrated form) that are desirable from a commercial and user perspective for use of the bead-linked transposomes described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes, carriers, packages, containers, vials, and / or tube labels listing contents and / or instructions for use, and inserts containing instructions for use. A set of instructions for use is also typically included.

[0089] A label may be on or associated with a container. A label may be on a container when letters, numbers, or other symbols forming the label are attached, molded, or etched into the container itself, and a label may be associated with a container when the label is present, for example, as a package insert, in a receptacle or carrier that also holds the container. A label may be used to indicate that the contents are to be used in a particular flow cell application. A label may also indicate instructions for using the contents, such as the methods described herein. EXAMPLES

[0090] Evaluation of the performance of the on-flow cell linearized amplification method. To check the performance of the on-flow cell linearized amplification method, the workflow was tested on a Miseq flow cell to plot out all seeding / replated DNA library molecules. Figure 7A is the cluster cloud from the standard workflow (no shallow layer denaturation / replated), and Figure 7B is the cluster from the same batch of beads with shallow layer liquid denaturation / replated. The clusters in Figure 7B are clearly more than those in Figure 7A, indicating that the on-flow cell linear amplification workflow allows the original DNA library molecules to be successfully re-seeded to maximize the sequencing information.

[0091] Checking the number of replicate copies to confirm the performance of on-flow cell linear amplification: The generation of long DNA fragment islands from cluster clouds was also evaluated using the on-flow cell linear amplification workflow. Up to four replicate copies (both strands of the Y-adapter library are doubled by shallow layer reseeding) were observed, whereas only up to two replicate copies (both strands of the Y-adapter library) were observed with the standard workflow.

[0092] A number of embodiments of the present disclosure have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A method for seeding a flow cell with polynucleotides released from beads, comprising: (i) annealing a first primer to a polynucleotide strand bound to a bead, wherein the bead is not immobilized on one or more surfaces of the flow cell, a 5' end of the polynucleotide strand is bound to the bead using a first attachment agent, the polynucleotide strand comprises an adapter that includes a sequence that is complementary to a sequence of the first primer, and the first primer is in solution; (ii) extending the first primer using a polymerase to form a double-stranded polynucleotide product; (iii) performing primer invasion of the double stranded polynucleotide product by using additional amounts of the first primer and a recombinase; (iv) displacing and releasing a strand of the double stranded polynucleotide product of step (ii) from the bead by extending from the additional first primer in step (iii) using a polymerase; (v) seeding a flow cell by attaching the strands released in step (iv) to a surface of the flow cell using a second attachment agent; and optionally repeating steps (iii)-(v) multiple times. (i) the beads are generally spherical or generally ovoid in shape; and / or (ii) the beads have a diameter of about 500 nm, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, or a range including or between any two of the aforesaid distances; and / or (iii) the beads are composed of a polymeric material, a silica material, a zirconia material, or a superparamagnetic material; and / or (iv) the beads are functionalized with a coating or layer selected from biotin, streptavidin, amine groups, carboxyl groups, epoxy groups, tosyl groups, antibodies or antigens, polyhistidine tags, or carriers comprising metal ions, and receptors or ligands; and / or (v) the polynucleotide strand bound to the bead comprises genomic DNA; and / or (vi) the polynucleotide strand bound to the bead comprises a long template DNA; and / or (vii) the polynucleotide strands bound to the beads comprise DNA from a sample of interest; Optionally, the subject sample is a urine, blood, saliva, tissue, serum, plasma, sputum, bile, faeces, hair, skin, and / or primary cell sample; and / or (viii) the polynucleotide strand is tagmented and bound to the bead by the use of a transposome linked to the bead, and optionally The method of claim 1 , wherein the transposome linked to the bead comprises transposase Tn5, or a mutant or variant thereof.

3. 3. The method of claim 1 or 2, wherein the polynucleotide strand comprises a first portion of sequence bound to a bead by a first attachment agent and a second portion of sequence tagmented and ligated to the first portion of sequence by use of a transposome and a ligase linked to the bead. (i) the transposome linked to the bead comprises transposase Tn5, or a mutant or variant thereof; and / or (ii) the first portion of the sequence comprises one or more barcode or index sequences, one or more universal primer sequences, and / or a mosaic end sequence, and optionally the first portion of the sequence comprises one or more barcode or index sequences, one or more universal primer sequences, and a mosaic end sequence, the mosaic end sequence being located at the 3' end of the first portion of the sequence; and / or (iii) the adaptor comprises a first sequence that is complementary to and capable of hybridizing to the first portion of the sequence of the polynucleotide strand, and the adaptor comprises a second sequence that is not complementary to and capable of hybridizing to the first portion of the sequence of the polynucleotide strand, and optionally the second sequence of the adapter comprises a sequence that is complementary to the sequence of the first primer; and / or (iv) the second portion of the sequence of the polynucleotide strand comprises gDNA; and / or (v) the second portion of the sequence of the polynucleotide strand comprises a long template DNA; and / or (vi) the second portion of the sequence of the polynucleotide strand comprises DNA from a sample of a subject, and optionally 4. The method of claim 3, wherein the subject sample is a urine, blood, saliva, tissue, serum, plasma, sputum, bile, feces, hair, skin, and / or primary cell sample.

5. the first and second attachment agents are selected from a biotin / streptavidin bond, a covalent bond, base pairing between two complementary sequences, an antibody that binds to an antigen, an antibody that binds to another antibody, a polyhistidine tag that binds to a carrier that includes a metal ion, and a receptor that binds to a ligand, and optionally (i) the first attachment agent is a biotin / streptavidin or avidin bond and the second attachment agent is base pairing between two complementary sequences; or (ii) The method of any one of claims 1 to 4, wherein the first attachment agent is a biotin / streptavidin or avidin bond and the second attachment agent is base pairing between two complementary sequences, the polynucleotide strand comprises a biotin residue at the 5' end and the bead comprises a streptavidin residue. (i) the first primer has the sequence of SEQ ID NO:1 or SEQ ID NO:2, optionally the first primer has the sequence of SEQ ID NO:2, and / or (ii) extending the sequence from the first primer using a DNA polymerase; and / or (iii) The method of any one of claims 1 to 52, wherein the method provides a linear copy of the polynucleotide strand.

7. 1. A method for seeding a flow cell with polynucleotides released from beads, comprising: (i) providing beads comprising a polynucleotide strand bound to the beads, the beads not immobilized on one or more surfaces of the flow cell, a 5' end of the polynucleotide strand bound to the beads using a first attachment agent, the polynucleotide strand comprising an adaptor comprising a sequence that is complementary to a sequence of the first primer, the beads also comprising an attached first amplification primer, the first amplification primer bound to the beads by a third attachment agent, the first amplification primer having a sequence that is different from and not complementary to the sequence of the first primer, the polynucleotide strand comprising a sequence that is complementary to the first amplification primer; (ii) annealing the first primer to the polynucleotide strand bound to the bead, the first primer being in solution; (iii) extending the first primer using a polymerase to form a double-stranded polynucleotide product; (iv) performing primer invasion of the double stranded polynucleotide product by using an additional amount of the first primer and / or the first amplification primer and a recombinase; (v) displacing and releasing a strand of the double stranded polynucleotide product of step (iii) from the bead by extending from the additional first primer and / or the first amplification primer of step (iv) with a polymerase; (vi) seeding a flow cell by attaching the strands released in step (v) to a surface of the flow cell using a second attachment agent; and optionally repeating steps (ii)-(vi) multiple times.

8. 1. A method for seeding a flow cell with polynucleotides released from beads, comprising: (i) providing beads comprising a polynucleotide strand bound to the beads, the beads being not immobilized on one or more surfaces of the flow cell, a 5' end of the polynucleotide strand being bound to the beads using a biotin / streptavidin bond, the beads also comprising a bound second amplification primer, the second amplification primer being bound to the beads by a third attachment agent, the second amplification primer having a sequence different from and not complementary to the sequence of the second primer, the polynucleotide strand comprising a sequence complementary to the second amplification primer, and a sequence complementary to the second primer; (ii) annealing the second amplification primer to the polynucleotide strand bound to the beads; (iii) extending the second amplification primer using a polymerase to form a double-stranded polynucleotide product; Then, either step (a) or steps (b) and (b'): (a) introducing the beads into a flow cell and releasing the double-stranded polynucleotide products from the beads as single-stranded polynucleotide products using heat and excess biotin; or (b) performing primer invasion of the double stranded polynucleotide product by using the second primer and a recombinase, wherein the second primer is in solution; (b') displacing and releasing a strand of the double stranded polynucleotide product from the bead by extending from the second primer with a polymerase; (iv) seeding the single stranded polynucleotide product released in step (a) or the double stranded polynucleotide product released from the beads in step (b') onto the surface of a flow cell using a second attachment agent; and and optionally repeating steps (ii)-(iv) multiple times.

9. 1. A method for seeding a flow cell with polynucleotides released from beads, comprising: (i) providing beads comprising a polynucleotide strand bound to the beads, the beads not immobilized on one or more surfaces of the flow cell, a 5' end of the polynucleotide strand bound to the beads using a biotin / streptavidin bond, the beads also comprising first and second amplification primers bound thereto, the first and second amplification primers being bound to the beads by an attachment agent, the first amplification primer having a sequence that is at least 99% identical to a sequence of the first primer, the second amplification primer having a sequence that is at least 99% identical to a sequence of the second primer, the polynucleotide strand comprising a sequence that is complementary to the first amplification primer and a sequence that is complementary to the second amplification primer; (ii) annealing the first and second amplification primers to the polynucleotide strands bound to the beads; (iii) extending the first and second amplification primers using a polymerase to form a cluster of double-stranded polynucleotide products; Then, either step (A) or steps (B) and (B'): (A) introducing the beads into a flow cell and releasing the double-stranded polynucleotide products from the beads as single-stranded polynucleotide products using heat and excess biotin; or (B) performing primer invasion of the double-stranded polynucleotide product by using the first primer and / or the second primer and a recombinase, wherein the first and second primers are in solution; (B') displacing and releasing a strand of the double stranded polynucleotide product from the bead by extending from the first and / or second primer with a polymerase; (iv) seeding the strands of the single stranded polynucleotide product released in step (A) or the double stranded polynucleotide product released from the beads in step (B') onto the surface of a flow cell using an attachment agent; and and optionally repeating steps (ii)-(iv) multiple times.

10. The method of any one of claims 1 to 9, wherein the flow cell is a patterned flow cell.

11. The method of any one of claims 1 to 10, wherein the strands released from the beads are bound by a capture agent on the flow cell surface.

12. The method of claim 11 , wherein the capture agent is located on an array of features of a patterned flow cell, and the capture agent is not located on interstitial areas between the features.

13. 13. The method of claim 11 or 12, wherein the capture agent is a capture nucleic acid bound to the surface of the flow cell, and the strand released from the bead comprises a sequence that can hybridize to the primer bound to the surface of the flow cell.

14. The method of claim 13 , wherein the capture nucleic acid comprises a universal capture sequence.

15. (i) the universal capture sequence comprises: (a) P5 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO:1) or P7 (5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO:2)), or a fragment thereof; or (b) anti-P5: 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 3) or anti-P7: 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 4), or a fragment thereof; and / or (ii) the capture nucleic acid further comprises a sequencing primer binding site (SBS), a target specific capture region, a restriction site, and / or a linker region, and optionally The method of claim 14, wherein the SBS is SBS3: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 5) or SBS8: 5'-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3' (SEQ ID NO: 6).

16. The method further comprising: performing an on-flow cell linear amplification reaction to generate copies of the polynucleotides seeded on the flow cell surface; The method of any one of claims 1 to 15, further comprising denaturing the copied polynucleotides and seeding them onto a flow cell surface.

17. 1. A method for increasing the seeding efficiency of polynucleotides on a surface of a flow cell, comprising: performing an on-flow cell linear amplification reaction to generate copies of the polynucleotides seeded on the flow cell surface; denaturing the copied polynucleotides and seeding the flow cell surface.

18. The method of claim 17, wherein the copied polynucleotide is denatured by use of a hybridization solution containing a chemical denaturant, and optionally, the chemical denaturant is formamide; and / or (ii) the mixture is shallowly coated over the surface of the patterned flow cell such that the array features on the patterned flow cell surface are in fluid contact with one another; and / or (iii) the process comprises forming clusters on the surface of the patterned flow cell by using bridge amplification to generate multiple copies of each seeded polynucleotide, and optionally The method further comprising:

18. The method of claim 16 or 17, further comprising sequencing by synthesis the clusters generated on the surface of the patterned flow cell.

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