Compositions and methods for pairwise sequencing

Abstract

The present disclosure provides compositions and methods for performing pairwise sequencing, as well as compositions and methods for generating concatemer template molecules for pairwise sequencing. Concatemers can be generated using a rolling circle amplification reaction, which is performed either on a support or in solution and then distributed on a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers that serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction uses detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., with nucleotide units), the signals emitted by the nucleotides or nucleotide units involved in the parallel sequencing reaction along the concatemer result in increased signal intensity for each concatemer.

Description

[Technical field]

[0001] Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents, and / or patent applications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 63 / 212,059, filed June 17, 2021, U.S. Provisional Patent Application No. 17 / 377,284, filed July 15, 2021, now issuing as U.S. Patent No. 11,220,707, U.S. Provisional Patent Application No. 17 / 377,285, filed July 15, 2021, now issuing as U.S. Patent No. 11,236,388, and U.S. Provisional Patent Application No. 17 / 377,285, filed July 15, 2021, now issuing as U.S. Patent No. 11,236,388. This application claims the benefit of U.S. Patent Application No. 17 / 377,279, filed on May 15, 2021, U.S. Patent Application No. 17 / 377,283, filed on July 15, 2021, U.S. Patent Application No. 17 / 521,239, filed on November 8, 2021, and U.S. Patent Application No. 17 / 554,396, filed on December 17, 2021, the contents of each of which are incorporated by reference herein in their entirety.

[0003] The present disclosure provides compositions and methods of using same for performing pairwise sequencing, as well as compositions and methods of using same for generating concatemeric template molecules for pairwise sequencing. [Background technology]

[0004] Polynucleotide sequencing technology has applications in biomedical research and medical environments. Improved methods of polynucleotide sequencing require enhanced surface chemistry, on-support polynucleotide amplification, and base calling. Currently, these factors pose barriers to existing sequencing technologies, which result in limited throughput and poor signal-to-noise ratios, and ultimately increased costs associated with polynucleotide sequencing.

[0005] There is a need for new polynucleotide sequencing methods with improved surface chemistry, on-support amplification, and base calling. The present disclosure provides methods and compositions for improving polynucleotide sequencing. Summary of the Invention

[0006] The present disclosure provides a method for pairwise sequencing comprising: a) providing a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each of the plurality of immobilized single-stranded nucleic acid concatemer template molecules comprising at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the concatemer template molecule, and each of the plurality of concatemer template molecules is immobilized to a first surface primer immobilized to a support, the immobilized first surface primer lacking the nucleotide having the cleavable moiety; b) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands, each of the immobilized concatemer template molecules having two or more extended forward sequencing primer strands hybridized thereto; and c) sequencing the plurality of immobilized concatemer template molecules. and replacing the retained, extended, forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained, immobilized, single-stranded nucleic acid concatemer template molecule by performing a primer extension reaction; d) removing the retained, immobilized, concatemer template molecule by creating an abasic site at a nucleotide(s) having a cleavable moiety in the immobilized, single-stranded concatemer template molecule and creating a gap at the abasic site to retain the plurality of forward extension strands and generate a plurality of gap-containing single-stranded nucleic acid concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized surface primers; and e) sequencing the plurality of retained forward extension strands, thereby generating a plurality of extended reverse sequencing primer strands, each of the retained forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto.

[0007] In some embodiments, each of the concatemeric template molecules in the plurality of concatemeric template molecules is covalently linked to an immobilized first surface primer. In some embodiments, each of the concatemeric template molecules in the plurality of concatemeric template molecules is hybridized to an immobilized first surface primer. In some embodiments, each immobilized concatemer template molecule in the plurality of concatemer template molecules comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence for a first immobilized surface primer, (iv) two or more copies of a universal binding sequence for a second immobilized surface primer, (v) two or more copies of a universal binding sequence for a first soluble amplification primer, (vi) two or more copies of a universal binding sequence for a second soluble amplification primer, (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0008] In some embodiments, the sequencing in step (b) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing in step (e) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions.

[0009] In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking a nucleotide having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in each concatemer template molecule is hybridized to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have a 3'OH extendable end. In some embodiments, the plurality of immobilized second surface primers have a 3'non-extendable end. In some embodiments, the 3'non-extendable end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

[0010] The disclosure also provides a method for pairwise sequencing comprising: a) providing a support having a plurality of first surface primers immobilized thereon, each of the first surface primers having a 3′ extendable end and lacking a nucleotide having a cleavable moiety; b) generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules by hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized first surface primers and performing a rolling circle amplification reaction with a plurality of strand displacement polymerases and a plurality of nucleotides including dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site, thereby generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules having at least one nucleotide having a cleavable moiety, each single-stranded nucleic acid concatemer template molecule being covalently linked to the immobilized first surface primer; and c) sequencing the plurality of immobilized concatemer template molecules. Thus, generating a plurality of extended forward sequencing primer strands, each immobilized concatemer template molecule having two or more extended forward sequencing primer strands hybridized thereto; d) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with the plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecules by performing a primer extension reaction; e) removing the retained immobilized concatemer template molecules by creating a non-basic site at the nucleotide(s) having a cleavable moiety in the immobilized single-stranded concatemer template molecule and creating a gap at the non-basic site to retain the plurality of forward extension strands and generate a plurality of gap-containing single-stranded nucleic acid concatemer template molecules while retaining the plurality of immobilized first surface primers; and f) sequencing the plurality of retained forward extension strands, therebygenerating a plurality of extended reverse sequencing primer strands, each of the forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto;

[0011] In some embodiments, each of the single stranded circular nucleic acid library molecules in the plurality of single stranded circular nucleic acid library molecules comprises a sequence of interest, and each individual library molecule further comprises any one of: (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer, (iii) a universal binding sequence for the first immobilized surface primer, (iv) a universal binding sequence for the second immobilized surface primer, (v) a universal binding sequence for the first soluble amplification primer, (vi) a universal binding sequence for the second soluble amplification primer, (vii) a universal binding sequence for a soluble compaction oligonucleotide, (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence, or any combination of two or more thereof.

[0012] In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule produced by a rolling circle amplification reaction comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer; (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer; (iii) two or more copies of a universal binding sequence for a first immobilized surface primer; (iv) two or more copies of a universal binding sequence for a second immobilized surface primer; (v) two or more copies of a universal binding sequence for a first soluble amplification primer; (vi) two or more copies of a universal binding sequence for a second soluble amplification primer; (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide; (viii) two or more copies of a sample barcode sequence; and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0013] In some embodiments, the sequencing in step (c) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing in step (f) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions.

[0014] In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking a nucleotide having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in each concatemer template molecule is hybridized to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have a 3'OH extendable end. In some embodiments, the plurality of immobilized second surface primers have a 3'non-extendable end. In some embodiments, the 3'non-extendable end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

[0015] The disclosure also provides a method for pairwise sequencing comprising: a) contacting a plurality of single-stranded circular nucleic acid library molecules in solution with a plurality of first soluble amplification primers, a plurality of strand-displacing polymerases, and a plurality of nucleotides including dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site, under conditions suitable for forming a plurality of library-primer duplexes and suitable for conducting a rolling circle amplification reaction, thereby generating a plurality of single-stranded nucleic acid concatemers having at least one nucleotide having a cleavable moiety; b) distributing the rolling circle amplification reaction onto a support having a plurality of first surface primers immobilized thereon under conditions suitable for hybridizing one or more portions of the individual single-stranded concatemers to one or more immobilized first surface primers, each of the first surface primers lacking the nucleotide having the cleavable moiety; and c) continuing the rolling circle amplification reaction on the support to generate a plurality of immobilized concatemers. d) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands, each immobilized concatemer template molecule having two or more extended forward sequencing primer strands hybridized thereto; e) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecules by performing a primer extension reaction; f) generating an abasic site in the immobilized single-stranded concatemer template molecule at a nucleotide(s) having a cleavable moiety and generating a gap at the abasic site to retain the plurality of forward extension strands and generate a plurality of gap-containing single-stranded nucleic acid concatemer template molecules while retaining the plurality of immobilized first surface primers.g) removing the retained immobilized concatemeric template molecules; and g) sequencing the plurality of retained forward extension strands, thereby generating a plurality of extended reverse sequencing primer strands, each of the forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto.

[0016] In some embodiments, each of the single stranded circular nucleic acid library molecules in the plurality of single stranded circular nucleic acid library molecules comprises a sequence of interest, and each individual library molecule further comprises any one of: (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer, (iii) a universal binding sequence for the first immobilized surface primer, (iv) a universal binding sequence for the second immobilized surface primer, (v) a universal binding sequence for the first soluble amplification primer, (vi) a universal binding sequence for the second soluble amplification primer, (vii) a universal binding sequence for a soluble compaction oligonucleotide, (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence, or any combination of two or more thereof.

[0017] In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule produced by a rolling circle amplification reaction comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer; (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer; (iii) two or more copies of a universal binding sequence for a first immobilized surface primer; (iv) two or more copies of a universal binding sequence for a second immobilized surface primer; (v) two or more copies of a universal binding sequence for a first soluble amplification primer; (vi) two or more copies of a universal binding sequence for a second soluble amplification primer; (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide; (viii) two or more copies of a sample barcode sequence; and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0018] In some embodiments, the sequencing in step (d) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing in step (g) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions.

[0019] In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking a nucleotide having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in each concatemer template molecule is hybridized to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have a 3'OH extendable end. In some embodiments, the plurality of immobilized second surface primers have a 3'non-extendable end. In some embodiments, the 3'non-extendable end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

[0020] The disclosure provides a method for pairwise sequencing comprising: a) providing a support having a plurality of first surface primers immobilized thereon, each first surface primer in the plurality of first surface primers comprising a first portion (SP1-A) and a second portion (SP1-B), each first surface primer comprising a 3' extendable end and lacking a nucleotide having a cleavable portion that can be cleaved to generate an abasic site in the first surface primer; and b) contacting the plurality of first surface primers with a plurality of single-stranded linear nucleic acid library molecules, each of the library molecules having at a 5' end a universal sequence (SP1-A') that binds to the first portion of the immobilized first surface primer and each of the library molecules having at a 3' end a universal sequence (SP1-B') that binds to the second portion of the immobilized first surface primer, wherein the contacting is performed under conditions suitable for hybridizing the individual library molecules to the immobilized first surface primers to form circularized library molecules, a) contacting the plurality of immobilized single-stranded nucleic acid concatemer template molecules with a plurality of strand-displacing polymerases and a plurality of nucleotides, the plurality of nucleotides including dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site, thereby generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules having at least one nucleotide having a cleavable moiety, each of the single-stranded nucleic acid concatemer template molecules being covalently linked to the immobilized first surface primer; and, e) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands.generating individual immobilized concatemer template molecules having two or more extended forward sequencing primer strands hybridized thereto; f) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with the plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecules by performing a primer extension reaction; g) generating an abasic site in the immobilized single-stranded concatemer template molecule at the nucleotide(s) having a cleavable moiety and forming a gap. removing the retained immobilized concatemer template molecules by generating gaps at the abasic sites to retain the plurality of forward extension strands and to generate a plurality of gap-containing single-stranded nucleic acid concatemer template molecules while retaining the plurality of immobilized first surface primers; and h) sequencing the plurality of retained forward extension strands, thereby generating a plurality of extended reverse sequencing primer strands, each of the forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto.

[0021] In some embodiments, an individual linear library molecule in the plurality of linear library molecules comprises a sequence of interest, and the library molecule further comprises any one of: (i) a universal binding sequence for the soluble forward sequencing primer, (ii) a universal binding sequence for the soluble reverse sequencing primer, (iii) a universal binding sequence for the first portion of the immobilized first surface primer (SP1-A), (iv) a universal binding sequence for the second portion of the immobilized first surface primer (SP1-B), (v) a universal binding sequence for the immobilized second surface primer, (vi) a universal binding sequence for the first soluble amplification primer, (vii) a universal binding sequence for the second soluble amplification primer, (viii) a universal binding sequence for the soluble compaction oligonucleotide, (ix) a sample barcode sequence, and / or (x) a unique molecular index sequence, or any combination of two or more thereof.

[0022] In some embodiments, each immobilized single stranded nucleic acid concatemer template molecule produced by a rolling circle amplification reaction comprises two or more copies of a sequence of interest, wherein each immobilized concatemer template molecule comprises: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer; (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer; (iii) two or more copies of a universal binding sequence for a first portion of the immobilized first surface primer (SP1-A); (iv) two or more copies of a universal binding sequence for a second portion of the immobilized first surface primer (SP1-B); (v) two or more copies of a universal binding sequence for the immobilized second surface primer, (vi) two or more copies of a universal binding sequence for the first soluble amplification primer, (vii) two or more copies of a universal binding sequence for the second soluble amplification primer, (viii) two or more copies of a universal binding sequence for the soluble compaction oligonucleotide, (ix) two or more copies of a sample barcode sequence, and / or (x) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0023] In some embodiments, the sequencing in step (e) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing in step (h) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions.

[0024] In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking a nucleotide having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in each concatemer template molecule is hybridized to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have a 3'OH extendable end. In some embodiments, the plurality of immobilized second surface primers have a 3'non-extendable end. In some embodiments, the 3'non-extendable end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

[0025] In some embodiments, closing the gaps in the circularized library molecules comprises performing a polymerase-catalyzed gap-filling reaction using the immobilized first surface primer as a template molecule and ligating the nicks to form covalently closed circular molecules, each of which is hybridized to the immobilized first surface primer. In some embodiments, closing the nicks in the circularized library molecules comprises performing a ligation reaction to form covalently closed circular molecules, each of which is hybridized to the immobilized first surface primer.

[0026] The present disclosure provides a method for pairwise sequencing comprising: a) providing a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each of the plurality of immobilized single-stranded nucleic acid concatemer template molecules lacking a cleavable moiety that can be cleaved to generate an abasic site in the concatemer template molecule, and each of the plurality of concatemer template molecules is immobilized to a first surface primer immobilized to a support, the immobilized first surface primer lacking a nucleotide having a cleavable moiety; b) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands, each of the immobilized concatemer template molecules having two or more extended forward sequencing primer strands hybridized thereto; and c) retaining the plurality of immobilized concatemer template molecules and subjecting the plurality of extended forward sequencing primer strands to a plurality of soluble amplification products. displacing the plurality of forward extension strands by performing a primer extension reaction with a primer and a plurality of strand displacing polymerases to generate a plurality of forward extension strands and a plurality of partially displaced forward extension strands hybridized to the immobilized concatemeric template molecules to form a plurality of immobilized amplicons, wherein the primer extension reaction generates a plurality of separated forward extension strands (e.g., not hybridized to the immobilized concatemeric template molecules); d) sequencing the plurality of immobilized partially displaced forward extension strands, thereby generating a first plurality of extended reverse sequencing primer strands, and sequencing the plurality of immobilized separated forward extension strands, thereby generating a second plurality of extended reverse sequencing primer strands, wherein each immobilized partially displaced forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto, and in each immobilized separated forward extension strand,and generating a nucleic acid sequence having two or more extended reverse sequencing primer strands hybridized thereto.

[0027] In some embodiments, each of the concatemeric template molecules in the plurality of concatemeric template molecules is covalently linked to an immobilized first surface primer. In some embodiments, each of the concatemeric template molecules in the plurality of concatemeric template molecules is hybridized to an immobilized first surface primer. In some embodiments, each immobilized concatemer template molecule in the plurality of concatemer template molecules comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence for a first immobilized surface primer, (iv) two or more copies of a universal binding sequence for a second immobilized surface primer, (v) two or more copies of a universal binding sequence for a first soluble amplification primer, (vi) two or more copies of a universal binding sequence for a second soluble amplification primer, (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0028] In some embodiments, the sequencing in step (b) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing in step (d) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized partially displaced forward extension strands and a plurality of immobilized separated extended forward sequencing primer strands and performing one or more sequencing reactions.

[0029] In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking a nucleotide having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in each concatemer template molecule is hybridized to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have a 3'OH extendable end. In some embodiments, the plurality of immobilized second surface primers have a 3'non-extendable end. In some embodiments, the 3'non-extendable end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

[0030] The disclosure also provides a method for pairwise sequencing comprising: a) providing a support having a plurality of first surface primers immobilized thereon, each of the first surface primers having a 3' extendable end and lacking a nucleotide having a cleavable moiety; b) generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules by hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized first surface primers and performing a rolling circle amplification reaction with a plurality of strand displacement polymerases and a plurality of nucleotides lacking a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site, thereby generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each single-stranded nucleic acid concatemer template molecule being covalently linked to the immobilized first surface primer; and c) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands, each of the individual immobilized single-stranded nucleic acid concatemer template molecules being covalently linked to the immobilized first surface primer. generating immobilized concatemeric template molecules having two or more extended forward sequencing primer strands hybridized thereto; d) retaining the plurality of immobilized concatemeric template molecules and displacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands by performing a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand displacing polymerases to generate a plurality of forward extension strands hybridized to the immobilized concatemeric template molecules and a plurality of partially displaced forward extension strands to form a plurality of immobilized amplicons, the primer extension reaction generating a plurality of separated forward extension strands (e.g., not hybridized to the immobilized concatemeric template molecules); e) sequencing the plurality of immobilized partially displaced forward extension strands, thereby generating a first plurality of extended reverse sequencing primer strands and sequencing the plurality of immobilized separated forward extension strands, therebygenerating a second plurality of extended reverse sequencing primer strands, each of the immobilized partially displaced forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto, and each of the immobilized separated forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto;

[0031] In some embodiments, each of the single stranded circular nucleic acid library molecules in the plurality of single stranded circular nucleic acid library molecules comprises a sequence of interest, and each individual library molecule further comprises any one of: (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer, (iii) a universal binding sequence for the first immobilized surface primer, (iv) a universal binding sequence for the second immobilized surface primer, (v) a universal binding sequence for the first soluble amplification primer, (vi) a universal binding sequence for the second soluble amplification primer, (vii) a universal binding sequence for a soluble compaction oligonucleotide, (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence, or any combination of two or more thereof.

[0032] In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule produced by a rolling circle amplification reaction comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer; (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer; (iii) two or more copies of a universal binding sequence for a first immobilized surface primer; (iv) two or more copies of a universal binding sequence for a second immobilized surface primer; (v) two or more copies of a universal binding sequence for a first soluble amplification primer; (vi) two or more copies of a universal binding sequence for a second soluble amplification primer; (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide; (viii) two or more copies of a sample barcode sequence; and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0033] In some embodiments, the sequencing in step (c) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing in step (e) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized partially displaced forward extension strands and a plurality of immobilized separated extended forward sequencing primer strands and performing one or more sequencing reactions.

[0034] In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking a nucleotide having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in each concatemer template molecule is hybridized to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have a 3'OH extendable end. In some embodiments, the plurality of immobilized second surface primers have a 3'non-extendable end. In some embodiments, the 3'non-extendable end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

[0035] The disclosure also provides a method for pairwise sequencing comprising: a) contacting a plurality of single-stranded circular nucleic acid library molecules in solution with a plurality of first soluble amplification primers, a plurality of strand-displacing polymerases, and a plurality of nucleotides lacking a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site, under conditions suitable for forming a plurality of library-primer duplexes and suitable for performing a rolling circle amplification reaction, thereby generating a plurality of single-stranded nucleic acid concatemers; b) distributing the rolling circle amplification reaction onto a support having a plurality of first surface primers immobilized thereon under conditions suitable for hybridizing one or more portions of the individual single-stranded concatemers to one or more immobilized first surface primers, each of the first surface primers lacking a nucleotide having a cleavable moiety; c) continuing the rolling circle amplification reaction on the support to generate a plurality of immobilized concatemer template molecules; and d) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of single-stranded nucleic acid concatemers. generating a plurality of extended forward sequencing primer strands, each immobilized concatemeric template molecule having two or more extended forward sequencing primer strands hybridized thereto; e) retaining the plurality of immobilized concatemeric template molecules and displacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands by performing a primer extension reaction with a plurality of second soluble amplification primers and a plurality of strand displacing polymerases to generate a plurality of forward extension strands hybridized to the immobilized concatemeric template molecules and a plurality of partially displaced forward extension strands to form a plurality of immobilized amplicons, the primer extension reaction generating a plurality of separated forward extension strands (e.g., not hybridized to the immobilized concatemeric template molecules); and f) sequencing the plurality of immobilized partially displaced forward extension strands, thereby generating a first plurality of extended reverse sequencing primer strands;and sequencing the plurality of immobilized separated forward extension strands, thereby generating a second plurality of extended reverse sequencing primer strands, each of the immobilized partially displaced forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto, each of the immobilized separated forward extension strands having two or more extended reverse sequencing primer strands hybridized thereto.

[0036] In some embodiments, each of the single stranded circular nucleic acid library molecules in the plurality of single stranded circular nucleic acid library molecules comprises a sequence of interest, and each individual library molecule further comprises any one of: (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer, (iii) a universal binding sequence for the first immobilized surface primer, (iv) a universal binding sequence for the second immobilized surface primer, (v) a universal binding sequence for the first soluble amplification primer, (vi) a universal binding sequence for the second soluble amplification primer, (vii) a universal binding sequence for a soluble compaction oligonucleotide, (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence, or any combination of two or more thereof.

[0037] In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule produced by a rolling circle amplification reaction comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer; (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer; (iii) two or more copies of a universal binding sequence for a first immobilized surface primer; (iv) two or more copies of a universal binding sequence for a second immobilized surface primer; (v) two or more copies of a universal binding sequence for a first soluble amplification primer; (vi) two or more copies of a universal binding sequence for a second soluble amplification primer; (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide; (viii) two or more copies of a sample barcode sequence; and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0038] In some embodiments, the sequencing in step (d) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatemeric template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing in step (f) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized partially displaced forward extension strands and a plurality of immobilized separated extended forward sequencing primer strands and performing one or more sequencing reactions.

[0039] In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking a nucleotide having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in each concatemer template molecule is hybridized to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have a 3'OH extendable end. In some embodiments, the plurality of immobilized second surface primers have a 3'non-extendable end. In some embodiments, the 3'non-extendable end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

[0040] The disclosure also provides a method for pairwise sequencing, comprising: a) providing a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each of the plurality of immobilized single-stranded nucleic acid concatemer template molecules comprising at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the concatemer template molecule, each of the plurality of concatemer template molecules being immobilized to a first surface primer immobilized to a support, the immobilized first surface primer comprising the nucleotide having the cleavable moiety, the support further comprising a plurality of immobilized second surface primers, the plurality of immobilized second surface primers lacking the nucleotide having the cleavable moiety and having an extendable terminal 3′ OH group, the immobilized concatemer template molecules comprising two or more copies of a universal binding sequence for the immobilized second surface primer (the support comprising an excess of immobilized first and second surface primers compared to the number of immobilized concatemer template molecules); and b) providing a plurality of immobilized single-stranded nucleic acid concatemer template molecules comprising at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the concatemer template molecule. a) sequencing a number of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers, thereby generating a plurality of extended forward sequencing primer strands, each of the immobilized concatemer template molecules having two or more extended forward sequencing primer strands hybridized thereto; c) removing the extended forward sequencing primer strands and retaining the immobilized concatemer template molecules; and d) generating a first plurality of immobilized forward extension strands by hybridizing at least a portion of each of the immobilized concatemer template molecules to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to a portion of the immobilized concatemer template molecules to generate a plurality of forward extension strands, each of the plurality of forward extension strands having a sequence complementary to at least a portion of the immobilized concatemer template molecules and covalently linked to the immobilized second surface primer.e) contacting the plurality of immobilized concatemeric template molecules and the plurality of immobilized forward extension strands with a relaxation solution comprising at least one chaotropic agent; f) dissociating at least a portion of the immobilized concatemeric template molecules from the immobilized second surface primers, retaining the immobilized forward extension strands, and rehybridizing at least a portion of the immobilized concatemeric template molecules to one of the immobilized second surface primers that is not covalently linked to the forward extension strands, wherein the dissociating and reassociating comprises a temperature ramp up, a temperature plateau, and a temperature ramp down, and washing the relaxation solution from the support; and g) contacting the rehybridized immobilized concatemeric template molecules with an amplification solution to perform a primer extension reaction from the second surface primers that have been rehybridized to a portion of the immobilized concatemeric template molecules to generate a plurality of newly synthesized forward extension strands. h) repeating steps (e)-(g) at least once; i) removing the retained immobilized concatemeric template molecule by creating a non-basic site at the nucleotide(s) having a cleavable moiety in the immobilized single-stranded concatemeric template molecule and the immobilized first surface primer, creating a gap at the non-basic site, thereby retaining the plurality of immobilized forward extension strands and retaining the plurality of immobilized second surface primers, while generating a plurality of gap-containing nucleic acid molecules; and j) sequencing the plurality of retained immobilized forward extension strands with a plurality of soluble reverse sequencing primers, thereby generating a plurality of extended reverse sequencing primer strands.

[0041] In some embodiments, each of the concatemeric template molecules in the plurality of concatemeric template molecules is covalently linked to an immobilized first surface primer. In some embodiments, each of the concatemeric template molecules in the plurality of concatemeric template molecules is hybridized to an immobilized first surface primer. In some embodiments, each immobilized concatemer template molecule in the plurality of concatemer template molecules comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence for a first immobilized surface primer, (iv) two or more copies of a universal binding sequence for a second immobilized surface primer, (v) two or more copies of a universal binding sequence for a first soluble amplification primer, (vi) two or more copies of a universal binding sequence for a second soluble amplification primer, (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0042] The disclosure also provides a method for pairwise sequencing comprising: a) providing a support having a plurality of first and second surface primers immobilized thereon, wherein the first surface primer has a cleavable moiety that can be cleaved to generate an abasic site, the second surface primer lacks a nucleotide having a cleavable moiety, and the second surface primer has an extendable terminal 3′ OH group; and b) generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules by hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized first surface primers and performing a rolling circle amplification reaction using a plurality of strand displacement polymerases and a plurality of nucleotides including dATP, dCTP, dGTP, dTTP, and a plurality of nucleotides having a cleavable moiety that can be cleaved to generate an abasic site, thereby generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules having at least one nucleotide having a cleavable moiety, wherein each single-stranded nucleic acid concatemer template molecule is hybridized to the plurality of immobilized first surface primers. a first surface primer covalently linked to the first immobilized concatemer template molecule; c) sequencing the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers, thereby generating a plurality of extended forward sequencing primer strands, each of the immobilized concatemer template molecules having two or more extended forward sequencing primer strands hybridized thereto; d) removing the extended forward sequencing primer strands and retaining the immobilized concatemer template molecules; and e) generating a first plurality of immobilized forward extension strands by hybridizing at least a portion of each of the immobilized concatemer template molecules to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to a portion of the immobilized concatemer template molecules to generate a plurality of forward extension strands, each of the immobilized concatemer template molecules having a sequence complementary to at least a portion of the immobilized concatemer template molecules;f) contacting the plurality of immobilized concatemer template molecules and the plurality of immobilized forward extension strands with a relaxation solution comprising at least one chaotropic agent; g) dissociating at least a portion of the immobilized concatemer template molecules from the immobilized second surface primers, retaining the immobilized forward extension strands, and rehybridizing at least a portion of the immobilized concatemer template molecules to one of the immobilized second surface primers that is not covalently linked to the forward extension strand, wherein the dissociating and reassociating comprises a temperature ramp up, a temperature plateau, and a temperature ramp down, and washing the relaxation solution from the support; and h) contacting the rehybridized immobilized concatemer template molecules with an amplification solution to effect a primer extension reaction from the second surface primers that have been rehybridized to a portion of the immobilized concatemer template molecules to produce a plurality of new concatemer template molecules. i) generating a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of the immobilized concatemer template molecule and covalently linked to an immobilized second surface primer; i) repeating steps (f)-(h) at least once; j) removing the retained immobilized concatemer template molecule by creating a non-basic site at the nucleotide(s) having a cleavable moiety in the immobilized single-stranded concatemer template molecule and the immobilized first surface primer, creating gaps at the non-basic site to generate a plurality of gap-containing nucleic acid molecules while retaining a plurality of immobilized forward extension strands and retaining a plurality of immobilized second surface primers; and k) sequencing the plurality of retained immobilized forward extension strands with a plurality of soluble reverse sequencing primers, thereby generating a plurality of extended reverse sequencing primer strands.

[0043] In some embodiments, each of the single stranded circular nucleic acid library molecules in the plurality of single stranded circular nucleic acid library molecules comprises a sequence of interest, and each individual library molecule further comprises any one of: (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer, (iii) a universal binding sequence for the first immobilized surface primer, (iv) a universal binding sequence for the second immobilized surface primer, (v) a universal binding sequence for the first soluble amplification primer, (vi) a universal binding sequence for the second soluble amplification primer, (vii) a universal binding sequence for a soluble compaction oligonucleotide, (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence, or any combination of two or more thereof.

[0044] In some embodiments, each immobilized concatemer template molecule in the plurality of concatemer template molecules comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence for a first immobilized surface primer, (iv) two or more copies of a universal binding sequence for a second immobilized surface primer, (v) two or more copies of a universal binding sequence for a first soluble amplification primer, (vi) two or more copies of a universal binding sequence for a second soluble amplification primer, (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0045] The disclosure also provides a method for pairwise sequencing comprising: a) contacting a plurality of single-stranded circular nucleic acid library molecules in solution with a plurality of first soluble amplification primers, a plurality of strand displacement polymerases, and a plurality of nucleotides including dATP, dCTP, dGTP, dTTP, and a plurality of nucleotides having a cleavable moiety that can be cleaved to generate an abasic site, under conditions suitable for forming a plurality of library-primer duplexes and suitable for performing a rolling circle amplification reaction, thereby generating a plurality of single-stranded nucleic acid concatemers having at least one nucleotide having a cleavable moiety; and b) distributing the rolling circle amplification reaction onto a support having a plurality of first surface primers immobilized thereon under conditions suitable for hybridizing one or more portions of the individual single-stranded concatemers to the one or more immobilized first surface primers, wherein the immobilized first surface primers comprise the nucleotide having the cleavable moiety, and the support further comprises a plurality of immobilized second surface primers, and the plurality of immobilized second surface primers comprise the nucleotide having the cleavable moiety. and having an extendable terminal 3' OH group; c) continuing the rolling circle amplification reaction on the support in the presence of a plurality of nucleotides, including a plurality of nucleotides having a cleavable moiety, to produce a plurality of immobilized concatemer template molecules; d) sequencing the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers, thereby producing a plurality of extended forward sequencing primer strands, each immobilized concatemer template molecule having two or more extended forward sequencing primer strands hybridized thereto; e) removing the extended forward sequencing primer strands and retaining the immobilized concatemer template molecules; f) hybridizing at least a portion of each immobilized concatemer template molecule to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to a portion of the immobilized concatemer template molecule,a) generating a first plurality of immobilized forward extension strands by generating a plurality of forward extension strands, the plurality of forward extension strands having sequences complementary to at least a portion of the immobilized concatemeric template molecules and covalently linked to immobilized second surface primers; g) contacting the plurality of immobilized concatemeric template molecules and the plurality of immobilized forward extension strands with a relaxation solution comprising at least one chaotropic agent; h) dissociating at least a portion of the immobilized concatemeric template molecules from the immobilized second surface primers, retaining the immobilized forward extension strands, and rehybridizing at least a portion of the immobilized concatemeric template molecules to one of the immobilized second surface primers that is not covalently linked to the forward extension strands, the dissociating and reassociating comprising a temperature ramp up, a temperature plateau, and a temperature ramp down, and washing the relaxation solution from the support; and i) contacting the rehybridized immobilized concatemeric template molecules with a relaxation solution comprising at least one chaotropic agent. contacting the immobilized concatemer template molecules with an amplification solution to perform a primer extension reaction from the second surface primer rehybridized to a portion of the immobilized concatemer template molecules to generate a plurality of newly synthesized forward extension strands, the plurality of newly synthesized forward extension strands having a sequence that is complementary to at least a portion of the immobilized concatemer template molecules and that are covalently linked to the immobilized second surface primer; j) repeating steps (g)-(i) at least once; k) removing the retained immobilized concatemer template molecules by creating a non-basic site at the nucleotide(s) having a cleavable moiety in the immobilized single-stranded concatemer template molecules and the immobilized first surface primer, and creating gaps at the non-basic site to retain the plurality of immobilized forward extension strands and generate a plurality of gap-containing nucleic acid molecules while retaining the plurality of immobilized second surface primers; l) separating the plurality of retained immobilized forward extension strands from the plurality of immobilized second surface primers.and sequencing with a plurality of soluble reverse sequencing primers, thereby generating a plurality of extended reverse sequencing primer strands.

[0046] In some embodiments, each of the single stranded circular nucleic acid library molecules in the plurality of single stranded circular nucleic acid library molecules comprises a sequence of interest, and each individual library molecule further comprises any one of: (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer, (iii) a universal binding sequence for the first immobilized surface primer, (iv) a universal binding sequence for the second immobilized surface primer, (v) a universal binding sequence for the first soluble amplification primer, (vi) a universal binding sequence for the second soluble amplification primer, (vii) a universal binding sequence for a soluble compaction oligonucleotide, (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence, or any combination of two or more thereof.

[0047] In some embodiments, each immobilized concatemer template molecule in the plurality of concatemer template molecules comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence for a first immobilized surface primer, (iv) two or more copies of a universal binding sequence for a second immobilized surface primer, (v) two or more copies of a universal binding sequence for a first soluble amplification primer, (vi) two or more copies of a universal binding sequence for a second soluble amplification primer, (vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0048] In any of the above or related embodiments, the support comprises a planar substrate, the planar substrate comprising glass, fused silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof.

[0049] In any of the above or related embodiments, the substrate comprises at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less, and at least one of the hydrophilic polymer coatings comprises a branched hydrophilic polymer having at least four branches.

[0050] In any of the above or related embodiments, the 5' ends of the plurality of first surface primers are immobilized to a support or to a coating on a support. In any of the above or related embodiments, the plurality of first surface primers comprises modified oligonucleotide molecules having 2-10 phosphorothioate bonds at their 5' ends to confer resistance to nuclease degradation.

[0051] In any of the above or related embodiments, the 5' ends of the plurality of second surface primers are immobilized to a support or to a coating on a support. In some embodiments, the plurality of second surface primers comprises modified oligonucleotide molecules having 2-10 phosphorothioate bonds at the 5' ends to confer resistance to nuclease degradation.

[0052] In any of the above or related embodiments, the immobilized concatemeric template molecule comprises at least one nucleotide having a cleavable moiety that comprises uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine.

[0053] In any of the above or related embodiments, the nucleotides having the cleavable moieties are located at randomly distributed positions on each immobilized concatemeric template molecule in the plurality of concatemeric template molecules.

[0054] In any of the above or related embodiments, 0.01-30% of the thymidine nucleotides in each immobilized concatemeric template molecule are replaced with uridine.In any of the above or related embodiments, 0.01-30% of the guanosine nucleotides in each immobilized concatemeric template molecule are replaced with 8-oxo-7,8-dihydroguanine or deoxyinosine.

[0055] In any of the above or related embodiments, the soluble forward sequencing primer comprises a 3'OH extendable end and lacks a nucleotide having a cleavable moiety. In any of the above or related embodiments, the soluble reverse sequencing primer comprises a 3'OH extendable end and lacks a nucleotide having a cleavable moiety.

[0056] In any of the above or related embodiments, the first soluble amplification primer comprises a 3'OH extendable end and lacks a nucleotide having a cleavable moiety. In any of the above or related embodiments, the second soluble amplification primer comprises a 3'OH extendable end and lacks a nucleotide having a cleavable moiety.

[0057] In any of the above or related embodiments, the forward sequencing step includes a) contacting a plurality of sequencing polymerases with (i) a plurality of immobilized concatemeric template molecules, and (ii) a plurality of soluble forward sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of multiplexed polymerases, each of the multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising immobilized concatemeric template molecules hybridized to a soluble forward sequencing primer; and b) contacting the multiplexed sequencing polymerases with a plurality of soluble forward sequencing primers. The method includes contacting a plurality of nucleotides with a nucleotide sequence under conditions suitable for binding of at least one nucleotide to a multiplexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog, at least one nucleotide analog being labeled with a fluorophore and having a removable chain-terminating moiety at a sugar 3' position; c) incorporating at least one nucleotide into the 3' end of the hybridized forward sequencing primer, thereby generating a plurality of nascent extended forward sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0058] In any of the above or related embodiments, the reverse sequencing step includes a) contacting a plurality of sequencing polymerases with (i) the plurality of retained forward extension strands, and (ii) a plurality of soluble reverse sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of multiplexed polymerases, each of the multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising the retained forward extension strand hybridized to a soluble reverse sequencing primer; and b) contacting the plurality of multiplexed sequencing polymerases with a plurality of nucleotides, contacting under conditions suitable for binding of at least one nucleotide to the multiplexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog, at least one nucleotide analog being labeled with a fluorophore and having a removable chain-terminating moiety at a sugar 3' position; c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer, thereby generating a plurality of nascent extended reverse sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0059] In some embodiments, the reverse sequencing of step (a) comprises hybridizing a plurality of soluble reverse sequencing primers to the plurality of retained forward extension strands in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0060] In some embodiments, the reverse sequencing step comprises a) contacting a plurality of sequencing polymerases with (i) the plurality of immobilized partially displaced forward extension strands, (ii) the plurality of immobilized separated extended forward sequencing primer strands, and (iii) a plurality of soluble reverse sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of multiplexed polymerases, each of the multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, and the nucleic acid duplex comprising a soluble reverse sequencing primer hybridized to the immobilized partially displaced forward extension strands or the immobilized separated extended forward sequencing primer strands. , b) contacting a plurality of multiplexed sequencing polymerases with a plurality of nucleotides under conditions suitable for binding of at least one nucleotide to the multiplexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog, at least one nucleotide analog being labeled with a fluorophore and having a removable chain-terminating moiety at a sugar 3' position; c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer, thereby generating a plurality of nascent extended reverse sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0061] In some embodiments, the reverse sequencing of step a) comprises hybridizing a plurality of soluble reverse sequencing primers to the plurality of retained forward extension strands in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0062] In any of the above or related embodiments, the forward sequencing and reverse sequencing steps include 1) performing a sequencing reaction at a position on the template molecule using a multivalent molecule that binds but does not incorporate, 2) performing a sequencing reaction at the same position on the template molecule using an incorporated nucleotide, and 3) repeating steps a) and b) at the next position on the template molecule.

[0063] In any of the above or related embodiments, the forward sequencing step and the reverse sequencing step include a) contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first multiplexed polymerases, each of the plurality of first multiplexed polymerases comprising a first sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising a nucleic acid template molecule hybridized to a sequencing primer, and wherein (1) the plurality of nucleic acid template molecules comprises a plurality of immobilized concatemeric template molecules and the plurality of soluble primers comprises a plurality of soluble forward sequencing primers, or (2) the plurality of nucleic acid template molecules comprises a plurality of retained forward extension strands and the plurality of soluble sequencing primers comprises a plurality of soluble reverse sequencing primers. a) contacting a plurality of first multiplex polymerases with a plurality of detectably labeled multivalent molecules under conditions suitable for binding complementary nucleotide units of the multivalent molecules to at least two of the plurality of first multiplex polymerases, thereby forming a plurality of multivalent multiplex polymerases, wherein the conditions inhibit incorporation of the complementary nucleotide units into the sequencing primers of the plurality of multivalent multiplex polymerases, and each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each of the nucleotide arms being attached to a nucleotide unit; b) detecting the plurality of multivalent multiplex polymerases; and c) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first multiplex polymerases in the plurality of multivalent multiplex polymerases, thereby determining the sequence of the nucleic acid template.

[0064] In some embodiments, the step of reverse sequencing comprises hybridizing a plurality of soluble reverse sequencing primers to the plurality of retained forward extension strands in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0065] In some embodiments, the method includes: e) dissociating the plurality of multivalent hybrid polymerases to remove the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; f) contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contacting is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second hybrid polymerases, each of the plurality of second hybrid polymerases comprising a second sequencing polymerase bound to the retained nucleic acid duplexes; and g) contacting the plurality of second hybrid polymerases with the plurality of nucleic acid duplexes. the second multiplexed polymerase of step (f), where the contacting is performed under conditions suitable for binding a complementary nucleotide from the plurality of nucleotides to at least two of the second multiplexed polymerases of step (f), thereby forming a plurality of nucleotide multiplexed polymerases, and the conditions are suitable for promoting incorporation of the bound complementary nucleotide into the sequencing primer of the nucleotide multiplexed polymerase; h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide multiplexed polymerase; and d) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide multiplexed polymerase.

[0066] In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising: a) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a nucleic acid template molecule, thereby forming a first binding complex, where a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and b) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule, thereby forming a second binding complex, where a second nucleotide unit of the second multivalent molecule binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecules form an avidity complex.

[0067] In some embodiments, (i) the first sequencing primer comprises a soluble forward sequencing primer and the nucleic acid template molecule comprises an immobilized concatemeric template molecule, (ii) the second sequencing primer comprises a soluble forward sequencing primer and the nucleic acid template molecule comprises the same immobilized concatemeric template molecule, and (iii) the first and second sequencing primers have the same sequence.

[0068] In some embodiments, (i) the first sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises a retained forward extension strand, (ii) the second sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises the same retained forward extension strand, and (iii) the first and second sequencing primers have the same sequence.

[0069] In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising: a) contacting a plurality of first sequencing polymerases and a plurality of second sequencing primers with different portions of the nucleic acid template molecule to form at least a first and a second multiplexed polymerase on the same nucleic acid template molecule; and b) contacting a plurality of multivalent molecules with at least a first and a second multiplexed polymerase on the same nucleic acid template molecule under conditions suitable for binding of a single multivalent molecule from the plurality of multivalent molecules to the first and the second multiplexed polymerases, wherein at least a first nucleotide unit of the single multivalent molecule is bound to the first multiplexed polymerase comprising the first sequencing primer hybridized to the first portion of the nucleic acid template molecule, thereby forming a first binding complex and forming a single multivalent a) contacting the first and second binding complexes bound to the same multivalent molecule to form an avidity complex; b) detecting the first and second binding complexes on the same nucleic acid template molecule; and c) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the nucleic acid template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the same nucleic acid template molecule.

[0070] In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, and the nucleic acid template molecule comprises an immobilized concatemeric template molecule, (ii) the plurality of second sequencing primers comprises a plurality of second soluble forward sequencing primers, and the nucleic acid template molecule comprises the same immobilized concatemeric template molecule, and (iii) the plurality of first and second sequencing primers have the same sequence.

[0071] In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecule comprises a retained forward extension strand, (ii) the plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, and the nucleic acid template molecule comprises the same retained forward extension strand, and (iii) the plurality of first and second sequencing primers have the same sequence.

[0072] In any of the above or related embodiments, the forward sequencing step and the reverse sequencing step include a) contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid template molecules, and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first multiplexed polymerases, each of the plurality of first multiplexed polymerases comprising a first sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising a nucleic acid template molecule hybridized to a soluble sequencing primer, and wherein (1) the plurality of nucleic acid template molecules comprise a plurality of immobilized concatemeric template molecules and the plurality of sequencing primers comprise a plurality of soluble forward sequencing primers, or (2) the plurality of nucleic acid template molecules comprise a plurality of immobilized partially displaced forward extension strands and the plurality of sequencing primers comprise a plurality of soluble reverse sequencing primers, or (3) the plurality of nucleic acid template molecules comprise a plurality of immobilized concatemeric template molecules. a) contacting the plurality of first multiplexed polymerases with a plurality of detectably labeled multivalent molecules under conditions suitable for binding complementary nucleotide units of the multivalent molecules to at least two of the plurality of first multiplexed polymerases, thereby forming a plurality of multivalent multiplexed polymerases, wherein the conditions inhibit incorporation of the complementary nucleotide units into the sequencing primers of the plurality of multivalent multiplexed polymerases, and each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each of the nucleotide arms being attached to a nucleotide unit; b) detecting the plurality of multivalent multiplexed polymerases; and c) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first multiplexed polymerases in the plurality of multivalent multiplexed polymerases, thereby determining the sequence of the nucleic acid template.

[0073] In any of the above or related embodiments, the reverse sequencing step comprises hybridizing a plurality of soluble reverse sequence primers to the plurality of immobilized partially displaced forward extension strands or the plurality of immobilized separated extended forward sequencing primer strands in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0074] In some embodiments, the method includes: e) dissociating the plurality of multivalent hybrid polymerases to remove the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; f) contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contacting is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second hybrid polymerases, each of the plurality of second hybrid polymerases comprising a second sequencing polymerase bound to the retained nucleic acid duplexes; and g) contacting the plurality of second hybrid polymerases with the plurality of nucleic acid duplexes. the second multiplexed polymerase of step (f) contacting the nucleic acid sequence of the nucleotide-complexed polymerase with a nucleotide sequence that is suitable for binding a complementary nucleotide from the plurality of nucleotides to at least two of the second multiplexed polymerases of step (f), thereby forming a plurality of nucleotide-complexed polymerases, and the conditions are suitable for promoting incorporation of the bound complementary nucleotide into the sequencing primer of the nucleotide-complexed polymerase; h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase; and i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.

[0075] In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising: a) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a nucleic acid template molecule, thereby forming a first binding complex, where a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and b) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule, thereby forming a second binding complex, where a second nucleotide unit of the second multivalent molecule binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecules form an avidity complex.

[0076] In some embodiments, (i) the first sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises an immobilized concatemer template molecule, (ii) the second sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises the same immobilized concatemer template molecule, and (iii) the first and second sequencing primers have the same sequence. In some embodiments, (i) the first sequencing primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises an immobilized partially displaced forward extension strand, (ii) the second sequencing primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises the same immobilized partially displaced forward extension strand, and (iii) the first and second sequencing primers have the same sequence. In some embodiments, (i) the first sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises an immobilized, separate, extended forward sequencing primer strand, (ii) the second sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises the same immobilized, separate, extended forward sequencing primer strand, and (iii) the first and second sequencing primers have the same sequence.

[0077] In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising: a) contacting a plurality of first sequencing polymerases and a plurality of second sequencing primers with different portions of the nucleic acid template molecule to form at least a first and a second multiplexed polymerase on the same nucleic acid template molecule; and b) contacting a plurality of multivalent molecules with at least a first and a second multiplexed polymerase on the same nucleic acid template molecule under conditions suitable for binding of a single multivalent molecule from the plurality of multivalent molecules to the first and the second multiplexed polymerases, wherein at least a first nucleotide unit of the single multivalent molecule is bound to the first multiplexed polymerase comprising the first sequencing primer hybridized to the first portion of the nucleic acid template molecule, thereby forming a first binding complex and forming a single multivalent a) contacting the first and second binding complexes bound to the same multivalent molecule to form an avidity complex; b) detecting the first and second binding complexes on the same nucleic acid template molecule; and c) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the nucleic acid template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the same nucleic acid template molecule.

[0078] In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, the nucleic acid template molecule comprises an immobilized concatemer template molecule, (ii) the plurality of second sequencing primers comprises a plurality of second soluble forward sequencing primers, the nucleic acid template molecule comprises the same immobilized concatemer template molecule, and (iii) the plurality of first and second sequencing primers have the same sequence. In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, the nucleic acid template molecule comprises an immobilized partially displaced forward extension strand, (ii) the plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, the nucleic acid template molecule comprises the same immobilized partially displaced forward extension strand, and (iii) the plurality of first and second sequencing primers have the same sequence. In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecule comprises an immobilized, separated, extended forward sequencing primer strand, (ii) the plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, and the nucleic acid template molecule comprises the same immobilized, separated, extended forward sequencing primer strand, and (iii) the plurality of first and second sequencing primers have the same sequence.

[0079] In any of the above or related embodiments, each nucleotide in the plurality of nucleotides comprises an aromatic base, a 5-carbon sugar, and one to ten phosphate groups, and the aromatic base of the nucleotide comprises adenine, guanine, cytosine, thymine, or uracil. In some embodiments, the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, and dTTP. In some embodiments, the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, and / or dTTP. In some embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a fluorescently labeled nucleotide. In some embodiments, at least one of the nucleotides in the plurality of nucleotides lacks a fluorophore label.

[0080] In any of the above or related embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a chain terminating moiety attached to the 3'-OH sugar position via a cleavable moiety, wherein the chain terminating moiety comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0081] In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl, and allyl are cleavable / removable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (i) the chain terminating moieties aryl and benzyl are cleavable / removable with H2 (ii) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, and disulfide are cleavable / removable with thiol reagents including beta-mercaptoethanol or dithiothritol (DTT); and (iii) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, and disulfide are cleavable / removable with phosphine reagents including tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxypropyl)phosphine (THPP). (iv) amine, amide, keto, isocyanate, phosphate, thio, disulfide chain terminating moieties are cleavable / removable with 4-dimethylaminopyridine (4-DMAP); (v) carbonate chain terminating moieties are cleavable / removable with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn(AcOH) in acetic acid; and (vi) urea and silyl chain terminating moieties are cleavable with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0082] In some embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a chain terminating moiety attached to the 3'-OH sugar position via a cleavable moiety, the chain terminating moiety comprising a 3'O-azido or 3'O-azidomethyl group. In some embodiments, (i) the chain terminating 3'O-azido and 3'O-azidomethyl groups are cleavable / removable with a phosphine compound including a derivatized trialkylphosphine moiety, a derivatized triarylphosphine moiety, tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxypropyl)phosphine (THPP), and (ii) the chain terminating 3'O-azido and 3'O-azidomethyl groups are cleavable / removable with 4-dimethylaminopyridine (4-DMAP).

[0083] In any of the above or related embodiments, each multivalent molecule in the plurality of multivalent molecules comprises (a) a core and (b) a plurality of nucleotide arms, the plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core attachment moieties, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0084] In some embodiments, the core comprises an avidin-type moiety and the core attachment moiety comprises biotin. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligoethylene glycol chain having 2-6 subunits. In some embodiments, the linker further comprises an aromatic moiety. In some embodiments, the nucleotide unit comprises an aromatic base, a 5-carbon sugar, and 1-10 phosphate groups. In some embodiments, the linker is attached to the nucleotide unit via the base.

[0085] In some embodiments, the nucleotide arms attached to the core have the same type of nucleotide unit, the type of nucleotide unit being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the plurality of multivalent molecules comprises one type of multivalent molecule, each multivalent molecule in the plurality of multivalent molecules having the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each of the two or more types of multivalent molecules having a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0086] In some embodiments, the plurality of multivalent molecules are fluorescently labeled multivalent molecules, in some embodiments (i) the core of each fluorescently labeled multivalent molecule is attached to a fluorophore that corresponds to the nucleotide unit attached to the nucleotide arm, (ii) at least one of the nucleotide arms comprises a linker attached to a fluorophore that corresponds to the nucleotide unit attached to the nucleotide arm, and / or (iii) at least one of the nucleotide arms comprises a nucleotide unit attached to a fluorophore that corresponds to the nucleotide unit attached to the nucleotide arm.

[0087] In some embodiments, a plurality of the multivalent molecules lacks a fluorophore.

[0088] In some embodiments, at least one of the multivalent molecules in the plurality of multivalent molecules comprises a nucleotide unit having a chain terminating moiety attached to the 3'-OH sugar position via a cleavable moiety, wherein the chain terminating moiety comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0089] In some embodiments, (i) the chain terminating moieties alkyl, alkenyl, alkynyl, and allyl are cleavable / removable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (ii) the chain terminating moieties aryl and benzyl are cleavable / removable with H2 (iii) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, and disulfide are cleavable / removable with thiol reagents including beta-mercaptoethanol or dithiothritol (DTT); and (iv) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, and disulfide are cleavable / removable with phosphine reagents including tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxypropyl)phosphine (THPP). (v) amine, amide, keto, isocyanate, phosphate, thio, disulfide chain terminating moieties are cleavable / removable with 4-dimethylaminopyridine (4-DMAP), (vi) carbonate chain terminating moieties are cleavable / removable with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn(AcOH) in acetic acid, and (vii) urea and silyl chain terminating moieties are cleavable with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0090] In some embodiments, at least one of the multivalent molecules in the plurality of multivalent molecules comprises a nucleotide unit having a chain terminating moiety attached to its 3'-OH sugar position via a cleavable moiety, wherein the chain terminating moiety comprises a 3'O-azido or 3'O-azidomethyl group.

[0091] In some embodiments, (i) the chain terminating moieties 3'O-azido and 3'O-azidomethyl groups are cleavable / removable with a phosphine compound including a derivatized trialkylphosphine moiety, a derivatized triarylphosphine moiety, tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxypropyl)phosphine (THPP), and (ii) the chain terminating moieties 3'O-azido and 3'O-azidomethyl are cleavable / removable with 4-dimethylaminopyridine (4-DMAP).

[0092] In some embodiments, the plurality of sequencing polymerases in step (a) comprises a recombinant wild-type DNA polymerase and the plurality of nucleotides in step (b) comprises fluorescently labeled nucleotides having a removable chain-terminating moiety at the 3' sugar position.

[0093] In some embodiments, the plurality of sequencing polymerases in step (a) comprises a mutant DNA polymerase and the plurality of nucleotides in step (b) comprises fluorescently labeled nucleotides having a removable chain-terminating moiety at the 3' sugar position.

[0094] In some embodiments, the plurality of first sequencing polymerases in step (a) comprises a recombinant wild-type DNA polymerase. In some embodiments, the plurality of first sequencing polymerases in step (a) comprises a mutant DNA polymerase.

[0095] In some embodiments, the plurality of second sequencing polymerases in step (f) comprises a recombinant wild-type DNA polymerase and the plurality of nucleotides in step (b) comprises fluorescently labeled nucleotides having a removable chain-terminating moiety at the 3' sugar position.

[0096] In some embodiments, the plurality of second sequencing polymerases in step (f) comprises a mutant DNA polymerase and the plurality of nucleotides in step (b) comprises fluorescently labeled nucleotides having a removable chain-terminating moiety at the 3' sugar position.

[0097] In any of the above or related embodiments, replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecule by performing a primer extension reaction comprises: (i) contacting at least one extended forward sequencing primer strand with a plurality of strand displacement polymerases and a plurality of nucleotides, in the absence of a soluble amplification primer, under conditions suitable for performing a strand displacement primer extension reaction using the at least one extended forward sequencing primer strand to initiate a primer extension reaction, thereby generating a forward extension strand covalently linked to the extended forward sequencing primer strand, wherein the forward extension strand is hybridized to the immobilized concatemer template molecule.

[0098] In any of the above or related embodiments, replacing the plurality of extended forward sequencing primer strands with the plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecule by performing a primer extension reaction comprises removing the plurality of extended forward sequencing primer strands by: (i) contacting the plurality of extended forward sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease; (ii) contacting the plurality of extended forward sequencing primer strands with a denaturing reagent comprising any combination of formamide, acetonitrile, guanidinium chloride, and / or a pH buffer; or (iii) contacting the plurality of extended forward sequencing primer strands with 100% formamide.

[0099] In any of the above or related embodiments, replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecule by performing a primer extension reaction includes (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecule, and (ii) reacting the plurality of retained immobilized concatemer molecules with a second plurality of soluble forward sequencing primers, a plurality of nucleotides, and a plurality of primer extension polymerases and a plurality of soluble phosphatase. and contacting the word sequencing primer with the plurality of retained immobilized concatemer template molecules under conditions suitable for hybridizing the word sequencing primer to the plurality of retained immobilized concatemer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension strands, wherein the plurality of nucleotides include dATP, dGTP, dCTP, and dTTP, but lack dUTP, and are resistant to uridine-containing template strands in the plurality of primer extension polymerases, and wherein the soluble sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized concatemer molecules.

[0100] In some embodiments, the contacting comprises contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble forward sequencing primers in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0101] In any of the above or related embodiments, replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecules by performing a primer extension reaction can include (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules, and (ii) reacting the plurality of retained immobilized concatemer molecules with a plurality of soluble amplification primers, a plurality of nucleotides, and a plurality of primer extension polymerases, and hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatemer template molecules. and contacting the immobilized concatemer molecules with the soluble amplification primers under conditions suitable for hybridizing the soluble amplification primers to the soluble amplification primer binding sequences in the retained immobilized concatemer molecules and suitable for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension strands, wherein the plurality of nucleotides include dATP, dGTP, dCTP, and dTTP, but lack dUTP, and are resistant to uridine-containing template strands in the plurality of primer extension polymerases, and the soluble sequencing primers hybridize to the forward sequencing primer binding sequences in the retained immobilized concatemer molecules.

[0102] In some embodiments, the contacting comprises contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble amplification primers in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0103] In some embodiments, the method further comprises contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble compaction oligonucleotides.

[0104] In any of the above or related embodiments, displacing the plurality of extended forward sequencing primer strands comprises (i) contacting at least one extended forward sequencing primer strand with a plurality of strand displacement polymerases and a plurality of nucleotides, in the absence of soluble amplification primers, under conditions suitable for conducting a strand displacement primer extension reaction using the at least one extended forward sequencing primer strand to initiate a primer extension reaction, thereby generating a plurality of forward extension strands, a plurality of partially displaced extended forward sequencing strands, and a plurality of separated extended forward sequencing primer strands.

[0105] In any of the above or related embodiments, replacing the plurality of extended forward sequencing primer strands comprises removing the plurality of extended forward sequencing primer strands by: (i) contacting the plurality of extended forward sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease; (ii) contacting the plurality of extended forward sequencing primer strands with a denaturing reagent comprising any combination of formamide, acetonitrile, guanidinium chloride, and / or a pH buffer; or (iii) contacting the plurality of extended forward sequencing primer strands with 100% formamide.

[0106] In any of the above or related embodiments, displacing the plurality of extended forward sequencing primer strands includes (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemeric template molecules; and (ii) contacting the plurality of retained immobilized concatemeric molecules with a second plurality of soluble forward sequencing primers, a plurality of nucleotides, and a plurality of strand displacing polymerases under conditions suitable for hybridizing the plurality of soluble forward sequencing primers to the plurality of retained immobilized concatemeric template molecules and suitable for conducting a polymerase-catalyzed strand displacement reaction, thereby displacing the immobilized concatemeric template molecules. and generating a plurality of forward extension strands and a plurality of partially displaced extended forward sequencing strands hybridized to the retained immobilized concatemer template molecules to form a plurality of immobilized amplicons, wherein the primer extension reaction generates a plurality of separated extended forward sequencing primer strands (e.g., not hybridized to the immobilized concatemer template molecules), wherein the plurality of nucleotides include dATP, dGTP, dCTP, and dTTP, but lack dUTP, and the soluble forward sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized concatemer molecule.

[0107] In some embodiments, the contacting comprises contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble forward sequencing primers in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0108] In any of the above or related embodiments, displacing the plurality of extended forward sequencing primer strands includes (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemeric template molecule; and (ii) contacting the plurality of retained immobilized concatemeric molecules with a plurality of soluble amplification primers, a plurality of nucleotides, and a plurality of strand displacing polymerases under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatemeric template molecules and suitable for performing a polymerase-catalyzed strand displacement reaction, thereby displacing the immobilized concatemeric template molecules. and generating a plurality of forward extension strands and a plurality of partially displaced extended forward sequencing strands hybridized to the concatemer template molecule to form a plurality of immobilized amplicons, wherein the primer extension reaction generates a plurality of separated extended forward sequencing primer strands (e.g., not hybridized to the immobilized concatemer template molecule), wherein the plurality of nucleotides include dATP, dGTP, dCTP, and dTTP, but lack dUTP, and the soluble amplification primer hybridizes to a soluble amplification primer binding sequence in the retained immobilized concatemer molecule.

[0109] In some embodiments, the contacting comprises contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble amplification primers in the presence of a high efficiency hybridization buffer, the high efficiency hybridization buffer comprising (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer, (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer, (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5, and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer.

[0110] In any of the above or related embodiments, at least one of the retained immobilized concatemer template molecules comprises one or more nucleotides having a cleavable moiety, the cleavable moiety comprising uridine, or 8-oxo-7,8-dihydroguanine, or deoxyinosine. In any of the above or related embodiments, the retained immobilized concatemer template molecule comprises one or more uridines, and generating an abasic site at the uridine comprises contacting the retained immobilized concatemer template molecule with uracil DNA glycosylase (UDG). In any of the above or related embodiments, the retained immobilized concatemer template molecule comprises one or more 8-oxoG, and generating an abasic site at the 8-oxoG comprises contacting the retained immobilized concatemer template molecule with Fpg enzyme (formamidopyrimidine DNA glycosylase). In any of the above or related embodiments, the retained immobilized concatemeric template molecule includes one or more deoxyinosines, and generating an abasic site in the deoxyinosine includes contacting the retained immobilized concatemeric template molecule with an AlkA glycosylase enzyme.

[0111] In any of the above or related embodiments, the method further includes generating gaps at the abasic sites to generate at least one gap-containing concatemeric template molecule, where the generating includes contacting the retained, immobilized template molecule containing the one or more abasic sites with endonuclease IV, an AP lyase (e.g., an apurinic DNA lyase or an apyrimidinic DNA lyase), an FPG glycosylase / AP lyase, and / or an endo VIII glycosylase / AP lyase.

[0112] In any of the above or related embodiments, the immobilized concatemeric template molecules comprise 0.1-30% uridine and the plurality of wild-type sequencing polymerases provide an error rate of at least 0.1-fold incorporation of dUTP compared to the error rate of incorporation of dTTP. In any of the above or related embodiments, the immobilized concatemeric template molecules comprise 0.1-30% uridine and the plurality of mutant sequencing polymerases provide an error rate of at least 0.1-fold incorporation of dUTP compared to the error rate of incorporation of dTTP. In any of the above or related embodiments, the immobilized concatemeric template molecules comprise 0.1-30% uridine and the plurality of wild-type sequencing polymerases provide an error rate of at least 0.1-fold incorporation of dUTP compared to the error rate of incorporation of dTTP. In any of the above or related embodiments, the immobilized concatemeric template molecules comprise 0.1-30% uridines, and the plurality of mutant sequencing polymerases provide an error rate for incorporating dUTP that is at least 0.1-fold greater than the error rate for incorporating dTTP.

[0113] In any of the above or related embodiments, the ratio of the first base fluorescent signal of R2 (e.g., reverse sequencing) to the first base fluorescent signal of R1 (e.g., forward sequencing) is at least 0.7 for sequencing using 1, 2, 3, or 4 dye colors.

[0114] In any of the above or related embodiments, the rolling circle amplification step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating immobilized concatemeric template molecules having a more compact size and / or shape compared to a rolling circle amplification reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0115] In any of the above or related embodiments, the primer extension reaction of a step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating a plurality of forward extension strands having a more compact size and / or shape compared to a primer extension reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0116] In any of the above or related embodiments, the rolling circle amplification step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating concatemeric molecules having a more compact size and / or shape compared to a rolling circle amplification reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0117] In any of the above or related embodiments, the primer extension reaction step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating a plurality of forward extension strands having a more compact size and / or shape compared to a primer extension reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0118] In any of the above or related embodiments, the rolling circle amplification step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating immobilized concatemeric template molecules having a more compact size and / or shape compared to a rolling circle amplification reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0119] In any of the above or related embodiments, the primer extension reaction step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating a plurality of forward extension strands having a more compact size and / or shape compared to a primer extension reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0120] In any of the above or related embodiments, the primer extension reaction step includes a plurality of compaction oligonucleotides and / or hexamine, thereby generating a plurality of primer extension products having a more compact size and / or shape compared to a primer extension reaction in the absence of the compaction oligonucleotides and / or hexamine, the plurality of primer extension products including a plurality of forward extension strands, a plurality of partially displaced extended forward sequencing strands, and a plurality of separated extended forward sequencing primer strands.

[0121] In any of the above or related embodiments, the rolling circle amplification step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating immobilized concatemeric template molecules having a more compact size and / or shape compared to a rolling circle amplification reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0122] In any of the above or related embodiments, the primer extension reaction step includes a plurality of compaction oligonucleotides and / or hexamine, thereby generating a plurality of primer extension products having a more compact size and / or shape compared to a primer extension reaction in the absence of the compaction oligonucleotides and / or hexamine, the plurality of primer extension products including a plurality of forward extension strands, a plurality of partially displaced extended forward sequencing strands, and a plurality of separated extended forward sequencing primer strands.

[0123] In any of the above or related embodiments, the rolling circle amplification step includes a plurality of compaction oligonucleotides and / or hexamines, thereby generating a plurality of concatemeric molecules having a more compact size and / or shape compared to a rolling circle amplification reaction in the absence of the compaction oligonucleotides and / or hexamines.

[0124] In any of the above or related embodiments, the primer extension reaction step includes a plurality of compaction oligonucleotides and / or hexamine, thereby generating a plurality of primer extension products having a more compact size and / or shape compared to a primer extension reaction in the absence of the compaction oligonucleotides and / or hexamine, the plurality of primer extension products including a plurality of forward extension strands, a plurality of partially displaced extended forward sequencing strands, and a plurality of separated extended forward sequencing primer strands.

[0125] In any of the above or related embodiments, the plurality of immobilized concatemer template molecules or the plurality of immobilized concatemer molecules have a FWHM (full width at half maximum) of about 5 μm or less. In any of the above or related embodiments, the plurality of forward extension strands have a FWHM (full width at half maximum) of about 5 μm or less. In any of the above or related embodiments, the plurality of primer extension products have a FWHM (full width at half maximum) of about 5 μm or less.

[0126] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0127] [Figure 1] FIG. 1 is a schematic diagram showing an exemplary single-stranded nucleic acid concatemer template molecule immobilized on an immobilized first surface primer. The immobilized concatemer template molecule comprises at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the immobilized concatemer template molecule. In some embodiments, the immobilized concatemer template molecule can be generated by performing a rolling circle amplification reaction on the support. The various primer binding sequence arrangements are for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. FIGS. 2-12 show the workflow for pairwise sequencing the immobilized concatemer template molecule shown in FIG. 1. [Diagram 2]Schematic diagram showing an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in FIG. 1. The forward sequencing reaction can be carried out with multiple soluble forward sequencing primers to generate multiple extended forward sequencing primer strands. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Diagram 3] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer, thereby generating a forward extended strand. [Figure 4] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer, thereby generating a forward extended strand. [Diagram 5] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer, thereby generating a forward extended strand. [Figure 6] 1 is a schematic diagram showing an exemplary method for generating an abasic site at a nucleotide having a cleavable moiety in an immobilized single-stranded concatemer template molecule, generating a gap at the abasic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. 3 or 4. [Figure 7] FIG. 7 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemer template molecule shown in FIG. 6. [Figure 8]5 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable moiety in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. [Figure 9] FIG. 9 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemer template molecule shown in FIG. 8. [Figure 10] Schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 7. The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to or covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strands are not immobilized to a support. For simplicity, FIGS. 1-10 show exemplary immobilized concatemer molecules with one copy of the sequence of interest and various universal primer binding sites. It will be understood by those skilled in the art that the immobilized concatemer molecules can include two or more tandem copies containing the sequence of interest and various universal primer binding sites. [Figure 11]10 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 9. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to or covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strands are not immobilized to a support. For simplicity, FIGS. 1-11 show exemplary immobilized concatemer molecules with one copy of the sequence of interest and various universal primer binding sites. It will be understood by those skilled in the art that the immobilized concatemer molecules can include two or more tandem copies containing the sequence of interest and various universal primer binding sites. [Figure 12] 1 is a schematic diagram showing an exemplary support on which a first and a second surface primer are immobilized. A portion of the immobilized concatemer template molecule shown in FIG. 1 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Figure 13]FIG. 1 is a schematic diagram showing an exemplary single-stranded nucleic acid concatemer template molecule immobilized on an immobilized first surface primer. The immobilized concatemer template molecule comprises at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the immobilized concatemer template molecule. In some embodiments, the immobilized concatemer template molecule can be generated by performing a rolling circle amplification reaction in solution and distributing the rolling circle amplification reaction on a support. The various primer binding sequence arrangements are for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. FIGS. 14-25 show the workflow for pairwise sequencing the immobilized concatemer template molecule shown in FIG. 13. [Figure 14] 14 is a schematic diagram showing an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 13. The forward sequencing reaction can be carried out with multiple soluble forward sequencing primers. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Figure 15] FIG. 1 is a schematic showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer. [Figure 16] FIG. 1 is a schematic diagram showing an exemplary method for replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer. [Figure 17] FIG. 1 is a schematic diagram showing an exemplary method for replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer. [Figure 18]1 is a schematic diagram showing an exemplary method for generating an abasic site at a nucleotide having a cleavable moiety in an immobilized single-stranded concatemer template molecule, generating a gap at the abasic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. 15 or 16. [Figure 19] FIG. 19 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemer template molecule shown in FIG. 18. [Figure 20] 17 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable moiety in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. [Figure 21] FIG. 21 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemer template molecule shown in FIG. 20. [Figure 22]22 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 19. The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers. The retained forward extension strand shown in FIG. 22 is a concatemer molecule that can contain two or more tandem copies of the sequence of interest and various primer binding sites. Such a concatemer molecule can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strand is not hybridized to the first surface primer or is not covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strand is not immobilized to a support. For simplicity, FIGS. 13-23 show an exemplary immobilized concatemer molecule with one copy of the sequence of interest and various universal primer binding sites. It will be understood by those skilled in the art that the immobilized concatemer molecule can contain two or more tandem copies containing the sequence of interest and various universal primer binding sites. [Figure 23]23 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 21. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The retained forward extension strand shown in FIG. 23 is a concatemer molecule that contains two or more tandem copies of a sequence of interest and various primer binding sites. Such a concatemer molecule can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strand is not hybridized to the first surface primer or is not covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strand is not immobilized to a support. For simplicity, FIGS. 13-23 show an exemplary immobilized concatemer molecule with two tandem copies containing a sequence of interest and various universal primer binding sites. One of skill in the art will appreciate that the immobilized concatemeric molecules can contain three or more tandem copies containing the sequence of interest and various universal primer binding sites. [Figure 24] 13 is a schematic diagram showing an exemplary support on which a first and a second surface primer are immobilized. A portion of the immobilized concatemer template molecule shown in FIG. 13 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Diagram 25] FIG. 1 is a schematic diagram showing an exemplary support having a first surface primer immobilized thereon, which in some embodiments can be used to perform an on-support pairwise sequencing workflow. [Figure 26]25A-25C are schematic diagrams showing an exemplary on-support rolling circle amplification reaction using a nucleic acid circular library molecule, an immobilized first surface primer as shown in FIG. 25, and a mixture of nucleotides including a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site. The rolling circle amplification reaction generates immobilized concatemer template molecules having at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the immobilized single-stranded nucleic acid concatemer template molecule. The arrangement of the various primer binding sequences in the nucleic acid circular library molecule is for illustrative purposes. It will be understood by those skilled in the art that many other arrangements are possible. Figures 26-37 show the workflow for pairwise sequencing the immobilized concatemer template molecules as shown in FIG. 26. [Figure 27] 27 is a schematic diagram showing an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 26. The forward sequencing reaction can be carried out with a plurality of soluble forward sequencing primers to generate a plurality of extended forward sequencing primer strands. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Figure 28] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer, thereby generating a forward extended strand. [Figure 29] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer, thereby generating a forward extended strand. [Diagram 30]FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer, thereby generating a forward extended strand. [Diagram 31] 2 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable moiety in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. 28 or 29. [Diagram 32] FIG. 32 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemer template molecule shown in FIG. 31. [Diagram 33] 30 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable portion in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. [Diagram 34] FIG. 34 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemer template molecule shown in FIG. 33. [Diagram 35]3 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 32. The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to or covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strands are not immobilized to a support. For simplicity, FIGS. 26-36 show exemplary immobilized concatemer molecules with one copy of the sequence of interest and various universal primer binding sites. It will be understood by those skilled in the art that the immobilized concatemer molecules can include two or more tandem copies containing the sequence of interest and various universal primer binding sites. [Diagram 36] 35 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 34. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to or covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strands are not immobilized to a support. For simplicity, FIGS. 26-36 show exemplary immobilized concatemer molecules with one copy of the sequence of interest and various universal primer binding sites. It will be understood by those skilled in the art that the immobilized concatemer molecules can include two or more tandem copies containing the sequence of interest and various universal primer binding sites. [Figure 37]26 is a schematic diagram showing an exemplary support on which a first and a second surface primer are immobilized. A portion of the immobilized concatemer template molecule shown in FIG. 26 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Figure 38] 3 is a schematic diagram showing an exemplary rolling circle amplification reaction in solution using a nucleic acid circular library molecule, a soluble first amplification primer, and a mixture of nucleotides including a nucleotide having a cleavable portion that can be cleaved to generate an abasic site. The rolling circle amplification reaction generates concatemer molecules in solution having at least one nucleotide having a cleavable portion that can be cleaved to generate an abasic site in a single stranded nucleic acid concatemer molecule. The arrangement of the various primer binding sequences in the nucleic acid circular library molecule is for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. Figures 38-52 show the workflow for pairwise sequencing the concatemer molecules shown in Figure 38. [Figure 39] FIG. 39 is a schematic diagram showing an exemplary method that includes distributing the rolling circle amplification reaction shown in FIG. 38 onto a support having a first surface primer immobilized thereon. The concatemer molecules are allowed to hybridize to the immobilized first surface primer. [Diagram 40] FIG. 1 is a schematic showing an exemplary method illustrating the rolling circle amplification reaction being allowed to continue on a support, thereby generating immobilized concatemeric template molecules that include at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the immobilized concatemeric template molecule. [Diagram 41]FIG. 41 is a schematic diagram showing an exemplary immobilized concatemeric template molecule produced by the method shown in FIG. 40. [Diagram 42] 41 shows a schematic diagram of an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 41. The forward sequencing reaction can be carried out with multiple soluble forward sequencing primers. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Diagram 43] FIG. 1 is a schematic showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer. [Diagram 44] FIG. 1 is a schematic diagram showing an exemplary method for replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer. [Diagram 45] FIG. 1 is a schematic diagram showing an exemplary method for replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer. [Diagram 46] 4 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable moiety in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. 43 or 44. [Figure 47] FIG. 47 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemeric template molecule shown in FIG. 46. [Figure 48]45 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable portion in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. [Figure 49] FIG. 49 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemer template molecule shown in FIG. 48. [Figure 50] 47. A schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 47. The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers. The retained forward extension strand shown in FIG. 50 is a concatemer molecule that can contain two or more tandem copies of the sequence of interest and various primer binding sites. Such a concatemer molecule can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to the first surface primer or are not covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strands are not immobilized to a support. For simplicity, FIGS. 41-50 show exemplary immobilized concatemer molecules with one copy of the sequence of interest and various universal primer binding sites. It will be understood by those skilled in the art that the immobilized concatemer molecules can contain two or more tandem copies containing the sequence of interest and various universal primer binding sites. [Figure 51]Schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in FIG. 49. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The retained forward extension strand shown in FIG. 51 is a concatemer molecule that contains two or more tandem copies of a sequence of interest and various primer binding sites. Such a concatemer molecule can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strand is not hybridized to or covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strand is not immobilized to a support. For simplicity, FIGS. 41-51 show an exemplary immobilized concatemer molecule with two tandem copies containing a sequence of interest and various universal primer binding sites. One of skill in the art will appreciate that the immobilized concatemeric molecules can contain three or more tandem copies containing the sequence of interest and various universal primer binding sites. [Figure 52] 41 is a schematic diagram showing an exemplary support on which a first and a second surface primer are immobilized. A portion of the immobilized concatemer template molecule shown in FIG. 41 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Diagram 53]Schematic diagram showing a linear single-stranded library molecule (top left schematic) hybridizing to a double-stranded splint molecule (bottom left schematic) to generate a circular library molecule (right schematic) with two gaps. The splint molecule comprises a first splint strand (long strand) hybridized to a second splint strand (short strand). The first splint strand comprises a left sequence that hybridizes to a sequence on one end of the linear single-stranded library molecule and a right sequence that hybridizes to a sequence on the other end of the linear single-stranded library molecule. An inner portion of the first splint strand hybridizes to the second splint strand. [Figure 54] 54 is a schematic diagram showing the circular library molecule shown in Figure 53 (left schematic), which is subjected to a ligation reaction to generate a single-stranded covalently closed circular molecule hybridized to the first splint strand (middle schematic), which is subjected to an RCA reaction in which the 3' end of the first splint strand is used to prime a rolling circle amplification reaction (right schematic). [Figure 55] 55-72 are schematic diagrams showing exemplary supports with a first surface primer immobilized thereon, in some embodiments, the supports can be used to perform on-support ligation reactions for pairwise sequencing workflows. Figures 55-72 show on-support ligation and pairwise sequencing workflows. [Figure 56] Schematic diagram showing an exemplary single-stranded linear library molecule comprising various universal adaptor sequences for a sequence of interest and a primer binding site. The arrangement of the various universal adaptor sequences in this diagram is for illustrative purposes. One of skill in the art will appreciate that many other arrangements and combinations of universal adaptor sequences are possible. [Figure 57] FIG. 1 is a schematic diagram showing an exemplary single-stranded linear library molecule hybridizing to an immobilized first surface primer to form a circularized library molecule with an asymmetrically positioned gap or nick. [Figure 58](Left) A schematic diagram showing an exemplary single-stranded linear library molecule hybridized to an immobilized first surface primer to form a circularized library molecule with an asymmetrically located gap or nick. Figure 58 (right) is a schematic diagram showing an exemplary single-stranded linear library molecule hybridized to an immobilized first surface primer to form a circularized library molecule with a symmetrically located gap or nick. The schematic diagrams shown in Figures 57 and 58 represent several embodiments of circularized library molecules comprising single-stranded linear library molecules hybridized to an immobilized first surface primer. [Figure 59] FIG. 1 is a schematic diagram showing exemplary covalently closed circular library molecules generated by covalently closing a gap or nick. [Figure 60] (Left) Schematic diagram showing exemplary covalently closed circular library molecules generated by covalently closing a gap or nick. Figure 60 (right) Schematic diagram showing exemplary covalently closed circular library molecules generated by covalently closing a gap or nick. The schematics shown in Figures 57 and 58 depict several embodiments of covalently closed circular library molecules hybridized to an immobilized first surface primer. [Figure 61] 56 is a schematic diagram showing an exemplary on-support rolling circle amplification reaction using covalently closed circular library molecules, the immobilized first surface primer shown in Figure 55, and a mixture of nucleotides including nucleotides having a cleavable moiety that can be cleaved to generate an abasic site. The rolling circle amplification reaction generates immobilized concatemeric template molecules having at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the immobilized single stranded nucleic acid concatemeric template molecule. [Figure 62]61 shows a schematic diagram of an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 61. The forward sequencing reaction can be carried out with a plurality of soluble forward sequencing primers to generate a plurality of extended forward sequencing primer strands. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Figure 63] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer, thereby generating a forward extended strand. [Figure 64] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer, thereby generating a forward extended strand. [Figure 65] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer, thereby generating a forward extended strand. [Figure 66] 6 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable moiety in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. 63 or 64. [Figure 67] FIG. 67 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemeric template molecule shown in FIG. [Figure 68]65 is a schematic diagram showing an exemplary method for generating a non-basic site at a nucleotide having a cleavable portion in an immobilized single-stranded concatemer template molecule, generating a gap at the non-basic site to retain a plurality of forward extension strands, and generating a plurality of gap-containing concatemer template molecules while retaining a plurality of immobilized first surface primers. The forward extension strands can be generated by the method shown in FIG. [Figure 69] FIG. 69 is a schematic diagram showing an exemplary retained forward extension strand following removal of the gap-containing concatemeric template molecule shown in FIG. 68. [Figure 70] 68 is a schematic diagram showing an exemplary reverse sequencing reaction carried out on the retained forward extension strand shown in Figure 67. The reverse sequencing reaction can be carried out with multiple soluble reverse sequencing primers. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to the first surface primer or are not covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strands are not immobilized on a support. [Figure 71] 69 shows a schematic diagram of an exemplary reverse sequencing reaction carried out on the retained forward extension strand shown in Figure 69. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to the first surface primer or covalently linked to the first surface primer. Thus, the extended reverse sequencing primer strands are not immobilized to a support. [Figure 72]61 is a schematic diagram showing an exemplary support on which a first and a second surface primer are immobilized. A portion of the immobilized concatemer template molecule shown in FIG. 61 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Figure 73] 7 is a schematic diagram showing an exemplary single-stranded nucleic acid concatemer template molecule immobilized on an immobilized first surface primer. In some embodiments, the immobilized concatemer template molecule can be generated by performing a rolling circle amplification reaction on a support. The various primer binding sequence arrangements are for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. Figures 73-79 show the workflow for pairwise sequencing the immobilized concatemer template molecule shown in Figure 73. [Figure 74] 73 shows a schematic diagram of an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 73. The forward sequencing reaction can be carried out with multiple soluble forward sequencing primers to generate multiple extended forward sequencing primer strands. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Figure 75] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer and a strand displacing polymerase in the presence of a compaction oligonucleotide, thereby generating a forward extension strand hybridized to an immobilized concatemeric template molecule and a partially displaced forward extension strand, thereby forming an immobilized amplicon. [Figure 76] FIG. 76 is a schematic diagram showing a continuation of the exemplary strand displacement method shown in FIG. 75, where the polymerase-catalyzed strand displacement reaction produces a forward extension strand hybridized to the immobilized concatemeric template molecule and a partially displaced forward extension strand, as well as a separated forward extension strand that is not hybridized to the immobilized concatemeric template molecule. [Figure 77] FIG. 1 is a schematic diagram showing an exemplary hybridization complex comprising a forward extension strand and a partially substituted forward extension strand hybridized to an immobilized concatemeric template molecule, and an immobilized separated forward extension strand hybridized to the partially substituted forward extension strand. [Figure 78] 78 is a schematic diagram showing an exemplary reverse sequencing reaction carried out in the hybridization complex shown in FIG. 77. The reverse sequencing reaction can be carried out with multiple soluble reverse sequencing primers in the partially displaced forward extension strand and the immobilized separated forward extension strand. The reverse sequencing reaction produces an extended reverse sequencing primer strand. For simplicity, FIG. 78 shows one copy of the extended reverse sequencing primer strand in the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand in the immobilized separated forward extension strand. It will be understood by those skilled in the art that the partially displaced forward extension strand and the immobilized separated forward extension strand can include two or more extended reverse sequencing primer strands hybridized thereon. [Figure 79]73 is a schematic diagram showing an exemplary support on which a first and a second surface primer are immobilized. A portion of the immobilized concatemer template molecule shown in FIG. 73 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Figure 80] FIG. 8 is a schematic diagram showing an exemplary single-stranded nucleic acid concatemer template molecule immobilized on an immobilized first surface primer. In some embodiments, the immobilized concatemer template molecule can be generated by performing a rolling circle amplification reaction in solution and distributing the rolling circle amplification reaction on a support. The various primer binding sequence arrangements are for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. FIG. 80-FIG. 86 show the workflow for pairwise sequencing the immobilized concatemer template molecule shown in FIG. 80. [Figure 81] 80 shows a schematic diagram of an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 80. The forward sequencing reaction can be carried out with multiple soluble forward sequencing primers. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Figure 82] FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer and a strand displacing polymerase in the presence of a compaction oligonucleotide, thereby generating a forward extension strand hybridized to an immobilized concatemeric template molecule and a partially displaced forward extension strand, thereby forming an immobilized amplicon. [Figure 83] FIG. 83 is a schematic diagram showing a continuation of the exemplary strand displacement method shown in FIG. 82, in which the polymerase-catalyzed strand displacement reaction produces a forward extension strand hybridized to the immobilized concatemeric template molecule and a partially displaced forward extension strand, as well as a separated forward extension strand that is not hybridized to the immobilized concatemeric template molecule. [Figure 84] FIG. 1 is a schematic diagram showing an exemplary hybridization complex comprising a forward extension strand and a partially substituted forward extension strand hybridized to an immobilized concatemeric template molecule, and an immobilized separated forward extension strand hybridized to the partially substituted forward extension strand. [Figure 85] 85 is a schematic diagram showing an exemplary reverse sequencing reaction carried out in the hybridization complex shown in FIG. 84. The reverse sequencing reaction can be carried out with multiple soluble reverse sequencing primers in the partially displaced forward extension strand and the immobilized separated forward extension strand. The reverse sequencing reaction produces an extended reverse sequencing primer strand. For simplicity, FIG. 85 shows one copy of the extended reverse sequencing primer strand in the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand in the immobilized separated forward extension strand. It will be understood by those skilled in the art that the partially displaced forward extension strand and the immobilized separated forward extension strand can include two or more extended reverse sequencing primer strands hybridized thereon. [Figure 86]80 is a schematic diagram showing an exemplary support on which a first and a second surface primer are immobilized. A portion of the immobilized concatemer template molecule shown in FIG. 80 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Figure 87] FIG. 1 is a schematic diagram showing an exemplary support having a first surface primer immobilized thereon, which in some embodiments can be used to perform an on-support pairwise sequencing workflow. [Figure 88] 87A-87C are schematic diagrams showing an exemplary on-support rolling circle amplification reaction using a nucleic acid circular library molecule and an immobilized first surface primer as shown in FIG. 87. The rolling circle amplification reaction produces immobilized single-stranded nucleic acid concatemer template molecules. The arrangement of the various primer binding sequences in the nucleic acid circular library molecule is for illustrative purposes. One of skill in the art will appreciate that many other arrangements are possible. FIGS. 87-94 show the workflow for pairwise sequencing the immobilized concatemer template molecules shown in FIG. 87. [Figure 89] FIG. 1 is a schematic diagram showing an exemplary single-stranded nucleic acid concatemer template molecule immobilized to an immobilized first surface primer. [Figure 90] 89 shows a schematic diagram of an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 89. The forward sequencing reaction can be carried out with multiple soluble forward sequencing primers to generate multiple extended forward sequencing primer strands. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Figure 91]FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer and a strand displacing polymerase in the presence of a compaction oligonucleotide, thereby generating a forward extension strand hybridized to an immobilized concatemeric template molecule and a partially displaced forward extension strand, thereby forming an immobilized amplicon. [Figure 92] FIG. 92 is a schematic diagram showing a continuation of the exemplary strand displacement method shown in FIG. 91, where the polymerase-catalyzed strand displacement reaction produces a forward extension strand hybridized to the immobilized concatemeric template molecule and a partially displaced forward extension strand, as well as a separated forward extension strand that is not hybridized to the immobilized concatemeric template molecule. [Figure 93] FIG. 1 is a schematic diagram showing an exemplary hybridization complex comprising a forward extension strand and a partially substituted forward extension strand hybridized to an immobilized concatemeric template molecule, and an immobilized separated forward extension strand hybridized to the partially substituted forward extension strand. [Figure 94] 94 is a schematic diagram showing an exemplary reverse sequencing reaction carried out in the hybridization complex shown in FIG. 93. The reverse sequencing reaction can be carried out with multiple soluble reverse sequencing primers in the partially displaced forward extension strand and the immobilized separated forward extension strand. The reverse sequencing reaction produces an extended reverse sequencing primer strand. For simplicity, FIG. 94 shows one copy of the extended reverse sequencing primer strand in the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand in the immobilized separated forward extension strand. It will be understood by those skilled in the art that the partially displaced forward extension strand and the immobilized separated forward extension strand can include two or more extended reverse sequencing primer strands hybridized thereon. [Figure 95] 95-103 are schematic diagrams showing an exemplary in-solution rolling circle amplification reaction using a nucleic acid circular library molecule, a soluble first amplification primer, and a mixture of nucleotides. The rolling circle amplification reaction produces single-stranded nucleic acid concatemer molecules in solution. The arrangement of the various primer binding sequences in the nucleic acid circular library molecule is for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. Figures 95-103 show the workflow for pairwise sequencing the concatemer molecules shown in Figure 96. [Figure 96] 96 is a schematic diagram showing an exemplary method that includes distributing the rolling circle amplification reaction shown in Figure 95 onto a support having a first surface primer immobilized thereon. The concatemer molecules are allowed to hybridize to the immobilized first surface primer. [Figure 97] FIG. 1 is a schematic diagram illustrating an exemplary method showing a rolling circle amplification reaction continuing on a support, thereby generating immobilized concatemeric template molecules. [Figure 98] FIG. 1 is a schematic diagram showing an exemplary single-stranded nucleic acid concatemer template molecule immobilized to an immobilized first surface primer. [Figure 99] 99 is a schematic diagram showing an exemplary forward sequencing reaction carried out on the immobilized concatemer template molecule shown in Figure 98. The forward sequencing reaction can be carried out with multiple soluble forward sequencing primers. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon. [Figure 100]FIG. 1 is a schematic diagram showing an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer and a strand displacing polymerase in the presence of a compaction oligonucleotide, thereby generating a forward extension strand hybridized to an immobilized concatemeric template molecule and a partially displaced forward extension strand, thereby forming an immobilized amplicon. [Figure 101] FIG. 101 is a schematic diagram showing a continuation of the exemplary strand displacement method shown in FIG. 100, in which the polymerase-catalyzed strand displacement reaction produces a forward extension strand hybridized to the immobilized concatemeric template molecule and a partially displaced forward extension strand, as well as a separated forward extension strand that is not hybridized to the immobilized concatemeric template molecule. [Figure 102] FIG. 1 is a schematic diagram showing an exemplary hybridization complex comprising a forward extension strand and a partially substituted forward extension strand hybridized to an immobilized concatemeric template molecule, and an immobilized separated forward extension strand hybridized to the partially substituted forward extension strand. [Figure 103] 103 is a schematic diagram showing an exemplary reverse sequencing reaction carried out in the hybridization complex shown in FIG. 102. The reverse sequencing reaction can be carried out with multiple soluble reverse sequencing primers in the partially displaced forward extension strand and the immobilized separated forward extension strand. The reverse sequencing reaction produces an extended reverse sequencing primer strand. For simplicity, FIG. 103 shows one copy of the extended reverse sequencing primer strand in the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand in the immobilized separated forward extension strand. It will be understood by those skilled in the art that the partially displaced forward extension strand and the immobilized separated forward extension strand can include two or more extended reverse sequencing primer strands hybridized thereon. [Figure 104] Schematic diagrams of various exemplary configurations of multivalent molecules. Left: Schematic diagram of a multivalent molecule with a starburst or helter-skelter configuration. Center: Schematic diagram of a multivalent molecule with a dendrimer configuration. Right: Schematic diagram of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS bearing biotin and dNTPs. Nucleotide units are represented as "N", biotin is represented as "B", and streptavidin is represented as "SA". [Figure 105] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a generic core attached to multiple nucleotide arms. [Figure 106] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms. [Figure 107] 1 shows a schematic diagram of an exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, the nucleotide arms comprising biotin, a spacer, a linker, and a nucleotide unit. [Figure 108] FIG. 1 is a schematic diagram of an exemplary nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit. [Fig. 109] The chemical structures of exemplary spacers are shown, as well as various exemplary linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker. [Figure 110] 1 shows the chemical structures of various exemplary linkers, including linkers 1-9. [Figure 111] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 112] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 113] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Fig. 114]The chemical structure of an exemplary nucleotide arm is shown. In this example, the nucleotide unit is connected to the linker via a propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base. This nucleotide arm shows an exemplary biotinylated nucleotide arm. [Figure 115] 1 is an illustrative schematic diagram of one embodiment of a low-binding support comprising a glass substrate and alternating layers of a hydrophilic coating, the alternating layers of the hydrophilic coating being covalently or non-covalently attached to the glass and further comprising chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides and circularized oligonucleotides). In alternative embodiments, the support can be made of any material, such as glass, plastic, or polymeric materials. [Fig. 116A] FIG. 1 is a schematic diagram of a G-quadruplex (eg, a G-quadruplex). [Fig. 116B] FIG. 1 is a schematic diagram of an intramolecular G-quadruplex structure. [Figure 117] 1 is a schematic diagram of an exemplary single cycle showing a run in a nucleic acid relaxation buffer with temperature ramp up and ramp down, a wash step, and a run in a flexing amplification buffer containing a strand-displacing DNA polymerase with temperature ramp up and MDA incubation and ramp down. One or more cycles of run in a flexing amplification buffer containing a strand-displacing DNA polymerase with temperature ramp up and MDA amplification and ramp down can be performed. [Fig. 118] (Left) Graph showing error rate from R1 sequencing reads of template molecules with various levels of uracil. Figure 118 (right) Graph showing phasing rate from R1 sequencing reads of template molecules with various levels of uracil. The data shows that sequencing template molecules with lower levels of incorporated uracil result in lower error and phasing rates. The level of uracil in the template molecule also affects the intensity ratio of R2 / R1 reads. [Figure 119] 1 is a graph showing the increased signal intensity ratio for R2 / R1 sequencing reads when the sequencing workflow uses a cleavage reagent that contains a compound that reduces photodamage to nucleic acids. Lanes 1, 3, 5, and 7 show the R2 / R1 signal intensity using different cleavage reagent formulations that do not have a compound that reduces photodamage. Lanes 2, 4, 6, and 8 show the R2 / R1 signal intensity using the corresponding cleavage reagent formulations that contain a compound that reduces photodamage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0128] definition The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. Generally, the terms related to molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization techniques described herein are well known and commonly used in the art. The techniques and procedures described herein are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with the experimental procedures and techniques described herein, as well as the experimental procedures and techniques, are well known and commonly used in the art.

[0130] Unless otherwise required by context herein, singular terms include the plural and plural terms include the singular. Use of the singular forms "a," "an," and "the," as well as the singular form of any word, includes plural references unless expressly and unambiguously limited to a reference to one.

[0131] The use of alternative terms (eg, "or") is understood to mean either one or both of the alternatives, or any combination thereof.

[0132] As used herein, the term "and / or" should be understood to mean a specific disclosure of each of the specified features or components with or without the other. For example, when used herein in phrases such as "A and / or B", the term "and / or" is intended to include "A and B", "A or B", "A" (A alone), and "B" (B alone). In a similar manner, when used in phrases such as "A, B, and / or C", the term "and / or" is intended to include each of the following aspects: "A, B, and C", "A, B, or C", "A or C", "A or B", "B or C", "A and B", "B and C", "A and C", "A" (alone), "B" (alone), and "C" (alone).

[0133] As used in this specification and the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be open-ended so that one or more items in a list do not exclude other items that may be substituted for or added to the listed items. Wherever an embodiment is described herein with the term "comprising," it is understood that alternatively similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0134] As used herein, the term "about" or "approximately" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, the acceptable error range depending in part on how the value or composition is measured or determined, i.e., on the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) or more, depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be considered to be within an acceptable error range for this particular value or composition. Also, when a range and / or subrange of values ​​is provided, the range and / or subrange can include the endpoints of the range and / or subrange.

[0135] The term "biological sample" refers to a single cell, multiple cells, tissue, organ, organism, or section of any of these biological samples. Biological samples may be extracted from an organism (e.g., biopsy) or obtained from cell cultures growing in liquid or in culture dishes. Biological samples include fresh samples, frozen samples, fresh frozen samples, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) samples. Biological samples may be embedded in wax, resin, epoxy, or agar. Biological samples may be fixed, for example, in any one of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton®, or glutaraldehyde, or any combination of two or more thereof. Biological samples may or may not be sectioned. Biological samples may be stained, destained, or unstained.

[0136] Nucleic acids of interest can be extracted from biological samples using any of several techniques known to those of skill in the art. For example, a typical DNA extraction procedure includes (i) collecting a cell or tissue sample from which DNA is to be extracted, (ii) disrupting cell membranes (i.e., lysing cells) to release DNA and other cytoplasmic components, (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA, and (iv) purifying DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during cell membrane lysis. A variety of suitable commercially available nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolation of genomic DNA from human samples) and the DNAeasy kit (for isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI).

[0137] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms, are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acid (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside linkages, e.g., phosphodiester linkages. Nucleic acids can lack phosphate groups. Nucleic acids include non-natural internucleoside linkages, where the non-natural internucleoside linkages include phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0138] The terms "universal sequence," "universal adapter sequence," and related terms refer to a sequence in a nucleic acid molecule that is common between two or more polynucleotide molecules. For example, an adapter with the same universal sequence can be ligated to multiple polynucleotides, such that a population of co-ligated molecules will possess the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or support-immobilized capture primers).

[0139] As used herein, the terms "operably linked" and "operably linked" or related terms refer to the juxtaposition of components. The juxtaposed components may be covalently linked together. For example, two nucleic acid components may be enzymatically ligated together, and the bond linking the two components together comprises a phosphodiester bond. A first and a second nucleic acid component may be linked together, and the first nucleic acid component may confer a function to the second nucleic acid component. For example, the bond between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion capable of binding to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) may be ligated into a vector, and the bond allows for expression or function of the transgene sequence contained within the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, etc. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of expression of the transgene.

[0140] The terms "bound," "linked," "attached," "added," and variations thereof include any type of fusion, binding, adhesion, or association between any combination of compounds or molecules that have sufficient stability to withstand use in a particular procedure. Procedures can include, but are not limited to, nucleotide binding, nucleotide incorporation, deblocking (e.g., removal of chain terminating moieties), washing, removal, flow, detection, imaging, and / or identification. Such binding can include, for example, covalent bonds, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonds, bonds or associations involving van der Waals forces, and mechanical bonds. In some embodiments, such binding occurs intramolecularly, for example, by joining the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such binding can occur between different molecular combinations or between molecules and non-molecules, including, but not limited to, binding between a nucleic acid molecule and a solid surface, binding between a protein and a detectable reporter moiety, and binding between a nucleotide and a detectable reporter moiety. Some examples of conjugation can be found, for example, in Hermanson, G., "Bioconjugate Techniques", Second Edition (2008), Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998), Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998).

[0141] The term "adapter" and related terms refer to an oligonucleotide that can be operatively attached (appended) to a target polynucleotide, where the adapter confers a function to the co-ligated adapter-target molecule. Adapters include DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adapters can include at least one ribonucleoside residue. Adapters can be single-stranded or double-stranded, or can have single-stranded and / or double-stranded portions. Adapters can be configured to be linear, stem-loop, hairpin, or Y-shaped in form. Adapters can be of any length, including from 4 to 100 or more nucleotides. Adapters can have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' overhangs and 3' overhanging ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, can have a 5' phosphate group or can lack a 5' phosphate group. The adapter may include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tailed adapter), or the adapter may be tailless. The adapter may include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter may include a random or degenerate sequence. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, phosphorothiolate, and / or phosphoroamidate bond. The adapter may include a barcode sequence, which may be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. The adapter may include a unique identification sequence (e.g., a unique molecular index, UMI, or unique molecular tag), which may be used to uniquely identify the nucleic acid molecule to which the adapter is attached.In some embodiments, the unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false positive variant calls, and / or increase the sensitivity of variant detection. The adapter can include at least one restriction enzyme recognition sequence, where the at least one restriction enzyme recognition sequence includes any one or any combination of two or more selected from the group consisting of type I, type II, type III, type IV, type Hs, or type IIB.

[0142] The terms "nucleic acid template," "template polynucleotide," "nucleic acid target," "target polynucleotide," "template strand," and other variations refer to a nucleic acid strand that serves as a base nucleic acid molecule for any of the analytical methods described herein (e.g., primer extension, amplification, and / or sequencing). The template nucleic acid may be single-stranded or double-stranded, or the template nucleic acid may have single-stranded or double-stranded portions. The template nucleic acid may be obtained from a naturally occurring source or recombinant form, or may be chemically synthesized to include any type of nucleic acid analog. The template nucleic acid may be linear, circular, or in other forms. The template nucleic acid may include an insertion region having an insert sequence, also known as the sequence of interest. The template nucleic acid may also include at least one adaptor sequence. The template nucleic acid may be a concatemer having two or tandem copies of the sequence of interest and at least one adaptor sequence. The insertion region may be isolated in any form, including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, total genomic DNA obtained from fresh frozen paraffin embedded tissue, needle biopsies, circulating tumor cells, cell-free circulating DNA, or any type of nucleic acid library. The insertion region may be isolated from any source, including organisms such as prokaryotes, eukaryotes (e.g., human, plant, and animal), fungi, viral cells, tissues, normal or diseased cells or tissues, bodily fluids including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, semen, environmental samples, culture samples, or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The insertion region can be isolated from any organ, including the head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testes, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and compositional analysis.

[0143] As used herein, the term "polymerase" and variations thereof include enzymes that include a nucleotide (or nucleoside) binding domain, where the polymerase can form a complex with a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities, including, but not limited to, base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. The polymerase can be any enzyme that can catalyze the polymerization of nucleotides (including their analogs) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, the polymerase includes one or more active sites, where nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, the polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand displacement activity. Polymerases can include, but are not limited to, naturally occurring polymerases and any subunits thereof and truncations, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes that contain a nucleotide binding domain. In some embodiments, the polymerase may be isolated from a cell or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, the polymerase may be expressed in a prokaryotic, eukaryotic, viral, or phage organism. In some embodiments, the polymerase can be a post-translationally modified protein or fragment thereof. The polymerase can be derived from a prokaryotic, eukaryotic, viral, or phage organism.Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0144] The term "strand displacement" refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acid and synthesize a new strand in a template-based manner. Strand displacement polymerases displace the complementary strand from the template strand and catalyze new strand synthesis. Strand displacement polymerases include mesophilic and thermophilic polymerases. Strand displacement polymerases include wild-type enzymes and variants including exonuclease minus mutants, mutated versions, chimeric enzymes, and truncated enzymes. Examples of strand displacement polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0145] As used herein, the term "DNA primase-polymerase" and related terms refer to an enzyme that has DNA polymerase and RNA primase activity. DNA primase-polymerase enzymes can use deoxyribonucleotide triphosphates to synthesize DNA primers on single-stranded DNA templates in a template sequence-dependent manner, and can extend the primer strand in the presence of catalytic divalent cations (e.g., magnesium and / or manganese) via nucleotide polymerization (e.g., primer extension). DNA primase-polymerases include enzymes that are members of DnaG-like primases (e.g., bacteria) and AEP-like primases (archaea and eukaryotes). An exemplary DNA primase-polymerase enzyme is Tth PrimPol from Thermus thermophilus HB27.

[0146] As used herein, the term "fidelity" refers to the accuracy of DNA polymerization by a template-dependent DNA polymerase. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporating an incorrect nucleotide, i.e., a nucleotide that is not complementary to the template nucleotide). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3' to 5' exonuclease activity of the DNA polymerase.

[0147] As used herein, the term "binding complex" refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, where the nucleic acid duplex includes a nucleic acid template molecule hybridized to a nucleic acid primer. In the binding complex, the free nucleotide or nucleotide unit may or may not be bound to the 3' end of the nucleic acid primer at a position opposite the complementary nucleotide in the nucleic acid template molecule. A "ternary complex" is an example of a binding complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, where the free nucleotide or nucleotide unit is bound to the 3' end of the nucleic acid primer (as part of the nucleic acid duplex) at a position opposite the complementary nucleotide in the nucleic acid template molecule.

[0148] The term "duration" and related terms refer to the length of time during which a binding complex remains stable without any of the components dissociating, the components of the binding complex including the nucleic acid template and nucleic acid primer, the polymerase, the nucleotide unit of the multivalent molecule, or the free (e.g., unconjugated) nucleotide. The nucleotide unit or free nucleotide may be complementary or non-complementary to the nucleotide residue in the template molecule. The nucleotide unit or free nucleotide may be bound to the 3' end of the nucleic acid primer at a position opposite the complementary nucleotide residue in the nucleic acid template molecule. The duration indicates the stability of the binding complex and the strength of the binding interaction. The duration can be measured by observing the onset and / or duration of the binding complex, for example, by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide, or a labeling reagent including one or more nucleotides, can be present in the binding complex, thus allowing the signal from the label to be detected during the duration of the binding complex. One exemplary label is a fluorescent label. The bound complex (e.g., ternary complex) remains stable until it is subjected to conditions that cause dissociation of interactions between the polymerase, the template molecule, the primer, and / or any of the nucleotide units or nucleotides. For example, dissociation conditions include contacting the bound complex with any one of detergent, EDTA, and / or water, or any combination thereof.

[0149] The term "primer" and related terms as used herein refers to an oligonucleotide capable of hybridizing to a DNA and / or RNA polynucleotide template to form a duplex molecule. A primer comprises natural nucleotides and / or nucleotide analogs. A primer can be a recombinant nucleic acid molecule. A primer can be of any length, but typically ranges from 4 to 50 nucleotides. A typical primer comprises a 5' end and a 3' end. The 3' end of the primer can comprise a 3'OH moiety that functions as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of the primer can lack a 3'OH moiety or can comprise a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one or more nucleotides along the length of the primer can be labeled with a detectable reporter moiety. The primer can be in solution (e.g., a soluble primer) or immobilized on a support (e.g., a capture primer).

[0150] When used in reference to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization", or other related terms, refers to hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule with a double-stranded region. Hybridization can include Watson-Crick or Hoogstein binding to form a double-stranded double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, can be fully complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other over their entire length. Complementary base pairing can be standard AT or CG base pairing, or other forms of base pairing interactions. Double-stranded nucleic acids can include mismatched base pairing nucleotides.

[0151] When used in reference to nucleic acids, the terms "extend", "extending", "extension" and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides into the terminal 3'OH terminus of a nucleic acid strand (e.g., a nucleic acid primer), resulting in an extension of the nucleic acid strand (e.g., an extended primer). Nucleotide incorporation can be performed with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method of extending a nucleic acid molecule can be used, with suitable methods including primer extension catalyzed by DNA polymerase or RNA polymerase.

[0152] In some embodiments, any of the amplification primer sequence, sequencing primer sequence, capture primer sequence (capture oligonucleotide), target capture sequence, circularization anchor sequence, sample barcode sequence, spatial barcode sequence, or anchor region sequence can be about 3-50 nucleotides in length, or about 5-40 nucleotides in length, or about 5-25 nucleotides in length.

[0153] The term "nucleotide" and related terms refer to a molecule that includes an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or nonstandard nucleotides are consistent with the use of this term. In some embodiments, the phosphate includes a monophosphate, diphosphate, or triphosphate, or the corresponding phosphate analogs. The term "nucleoside" refers to a molecule that includes an aromatic base and a sugar. Nucleotides and nucleosides can be unlabeled or labeled with a detectable reporter moiety.

[0154] Nucleotides (and nucleosides) typically contain a heterocyclic base that contains a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, which are commonly found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants, or analogs thereof. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases are purines and pyrimidines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N 6 -Δ 2 -Isopentenyladenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -Methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine, and O 6 -methylguanine; 7-deaza-purines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indoles, such as nitroindole and 4-methylindole; pyrroles, such as nitropyrrole; nebularine; inosine; hydroxymethylcytosine; 5-methycytosine; base (Y); and methylated, glycosylated, and acylated base moieties, etc. Additional exemplary bases can be found in Fasman, 1989, in "Practical Handbook of Biochemistry and Molecular Biology", pp. 385-394, CRC Press, Boca Raton, Fla.

[0155] Nucleotides (and nucleosides) typically include a sugar moiety, e.g., a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48), an acyclic moiety (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez, et al., 1997 BIOORGANIC & MEDICINAL Chemistry Letters vol. 7:3013-3016), and another sugar moiety (Joeng, et al., 1993 J. Med. Chem. 36:2627-2638; Kim, et al., 1993 J. Med. Chem. 36:30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). Sugar moieties include ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic, or other modified sugars.

[0156] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain are O, S, or BH. 3 In some embodiments, the tethers include phosphate groups replaced with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methyl phosphoramidate groups.

[0157] The terms "reporter moiety", "reporter moieties" or related terms refer to a compound that generates or can be caused to generate a detectable signal. Reporter moieties are often referred to as "labels". Any suitable reporter moiety can be used, suitable reporter moieties include luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. Reporter moieties generate a detectable signal that results from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity, or a proximity event). A proximity event involves two reporter moieties coming into close proximity to each other, associating with each other, or binding to each other. It is well known to those skilled in the art to select reporter moieties such that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties, allowing the presence of different reporter moieties in the same or different reactions to be monitored. Two or more different reporter moieties may be selected that have spectrally distinct emission profiles or that have minimal overlapping spectral emission profiles. The reporter moiety may be bound (e.g., operably bound) to a nucleotide, a nucleoside, a nucleic acid, an enzyme (e.g., a polymerase or a reverse transcriptase), or a support (e.g., a surface).

[0158] The reporter moiety (or label) comprises a fluorescent label or a fluorophore. Exemplary fluorescent moieties that can function as fluorescent labels or fluorophores include fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine and rhodamine derivatives, such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, Coumarin and Coumarin derivatives such as AMCA, AMCA-NHS, AMCA-Sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives, such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanines and derivatives, such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5,Lanthanide chelates and derivatives, such as, but not limited to, BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbenes, DEG dyes, NR dyes, near infrared dyes, and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition, Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes can exist in either sulfonated or non-sulfonated form and consist of two indolenine, benzoindolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, which is 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium, or can include 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which can include1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-yne 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl) Cy5 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), and Cy6 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for "cyanine" and the first number identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0159] In some embodiments, the reporter moieties can be FRET pairs, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET can include excitation exchange (Förster) transfer, or electron exchange (Dexter) transfer.

[0160] As used herein, the term "support" refers to a substrate designed for the deposition of biomolecules or biological samples for assay and / or analysis. Examples of biomolecules deposited on a support include nucleic acids (e.g., DNA, RNA), polypeptides, sugars, lipids, single cells, or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, stool, sweat, tears, and fluids from tissues or organs.

[0161] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0162] In some embodiments, the surface of the support can be substantially smooth, hi some embodiments, the support can be textured, either regularly or irregularly, and includes bumps, etchings, pores, three-dimensional scaffolds, or any combination thereof.

[0163] In some embodiments, the support comprises a bead having any shape, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disk-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0164] The support may be made of any material, including, but not limited to, glass, fused silica, silicon, polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0165] The support can have a plurality (e.g., two or more) of nucleic acid templates immobilized thereon. The plurality of immobilized nucleic acid templates have the same sequence or different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at different sites on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at sites on the support.

[0166] The term "array" refers to a support that includes a plurality of sites located at predetermined locations on the support to form an array of sites. The sites may be dispersed and separated by interstitial regions. In some embodiments, the predetermined sites on the support may be arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, the plurality of predetermined sites are arranged in an organized manner on the support. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, including linear, hexagonal, lattice, patterns with reflection symmetry, or patterns with rotational symmetry, etc. The pitch between different pairs of sites may be the same or may vary. In some embodiments, the support is at least 10 2 at least 10 sites 3 at least 10 sites 4 at least 10 sites5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 sites, or at least 10 15 In some embodiments, the substrate comprises a plurality of predetermined sites (e.g., 10 or more sites), the sites being located at predetermined locations on the substrate. 2 ~10 15 At least one of the plurality of predefined sites (e.g., 10 or more sites) is immobilized with the nucleic acid template, forming a nucleic acid template array. In some embodiments, the nucleic acid template is immobilized at a plurality of predefined sites by hybridization to the immobilized surface capture primer, or the nucleic acid template is covalently attached to the surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of predefined sites, e.g., 10 or more sites. 2 ~10 15 A nucleic acid template that is immobilized at more than one site. In some embodiments, the immobilized nucleic acid template is clonally amplified to generate immobilized nucleic acid polonies at a plurality of predetermined sites. In some embodiments, each immobilized nucleic acid polony comprises a single-stranded or double-stranded concatemer.

[0167] In some embodiments, a support comprising a plurality of sites located at random locations on the support is referred to herein as a support having randomly located sites thereon. The locations of the randomly located sites on the support are not predetermined locations. The plurality of randomly located sites are arranged on the support in a random and / or unpredictable manner. In some embodiments, the support comprises at least 10 2 at least 10 sites 3at least 10 sites 4 at least 10 sites 5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 sites, or at least 10 15 In some embodiments, the substrate comprises a plurality of randomly located sites (e.g., 10 or more sites), the sites being randomly located on the substrate. 2 ~10 15 10 or more sites) are immobilized with the nucleic acid template to form a nucleic acid template immobilized support. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites by hybridization to an immobilized surface capture primer, or the nucleic acid template is covalently attached to the surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites, e.g., 10 2 ~10 15 A nucleic acid template immobilized at one or more sites. In some embodiments, the immobilized nucleic acid template is clonally amplified to generate immobilized nucleic acid polonies at multiple randomly located sites. In some embodiments, each immobilized nucleic acid polony comprises a single-stranded or double-stranded concatemer.

[0168] When used for low-binding surface coatings, one or more layers of the multi-layer surface coating may comprise a branched polymer or may be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinyl pyridine), branched poly(vinyl pyrrolidone) (branched PVP), branched), poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxylethyl methacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched poly-lysine, branched poly-glucoside, and dextran.

[0169] In some embodiments, branched polymers used to make one or more layers of any of the multilayer surfaces disclosed herein may contain at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, or at least 40 branches.

[0170] Linear, branched, or hyperbranched polymers used to make one or more layers of any of the multilayer surfaces disclosed herein may have a molecular weight of at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 daltons.

[0171] In some embodiments, for example, when at least one layer of a multilayer surface comprises a branched polymer, the number of covalent bonds between the branched polymer molecules of the deposited layer and the molecules of the previous layer can range from about 1 covalent bond per molecule to about 32 covalent bonds per molecule. In some embodiments, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, or at least 32 covalent bonds per molecule.

[0172] Any reactive functional groups remaining after coupling of a material layer to a surface can optionally be blocked by coupling small inert molecules using high yield coupling chemistry. For example, if amine coupling chemistry is used to attach a new material layer to the previous one, any remaining amine groups can then be acetylated or inactivated by coupling with a small amino acid such as glycine.

[0173] The number of layers of low non-specific binding material, e.g., hydrophilic polymeric material, deposited on the surface can range from 1 to about 10. In some embodiments, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some embodiments, the number of layers can be up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1. Any of the lower and upper limits described in this paragraph can be combined to form ranges included within the present disclosure, e.g., in some embodiments, the number of layers can range from about 2 to about 4. In some embodiments, all of the layers can include the same material. In some embodiments, each layer can include a different material. In some embodiments, multiple layers can include multiple materials. In some embodiments, at least one layer can include a branched polymer. In some embodiments, all of the layers can include a branched polymer.

[0174] One or more layers of low non-specific binding material may be deposited on and / or conjugated to the substrate surface, in some cases, using a polar protic solvent, a polar or polar aprotic solvent, a non-polar solvent, or any combination thereof. In some embodiments, the solvent used for layer deposition and / or coupling may include an alcohol (e.g., methanol, ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile, dimethylsulfoxide (DMSO), dimethylformamide (DMF), etc.), water, an aqueous buffer solution (e.g., phosphate buffer, phosphate buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any combination thereof. In some embodiments, the organic component of the solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, with the balance being water or an aqueous buffer solution. In some embodiments, the aqueous component of the solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, with the balance being organic solvent. The pH of the solvent mixture used can be less than 6, about 6, 6.5, 7, 7.5, 8, 8.5, 9, or greater than pH 9.

[0175] The term "branched polymer" and related terms refer to a polymer having multiple functional groups that facilitate conjugation to biologically active molecules such as nucleotides, where the functional groups may be on the side chains of the polymer or may be directly attached to the central core or central backbone of the polymer. A branched polymer can have a linear backbone with one or more functional groups that dissociate from the backbone for conjugation. A branched polymer can also be a polymer with one or more side chains, where the side chains have sites that are suitable for conjugation. Examples of functional groups include, but are not limited to, hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activated sulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

[0176] The term "immobilized" and related terms, when used in reference to immobilized nucleic acids, refers to nucleic acid molecules that are attached to a support via covalent or non-covalent interactions, or to a coating on a support, or embedded within a matrix formed by a coating on a support, the nucleic acid molecules comprising a surface capture primer, a nucleic acid template molecule, and an extension product of the capture primer. The extension product of the capture primer comprises a nucleic acid concatemer capable of forming a nucleic acid polony.

[0177] In some embodiments, one or more nucleic acid templates are immobilized on a support, e.g., immobilized at a site on a support. In some embodiments, one or more nucleic acid templates are clonally amplified. In some embodiments, one or more nucleic acid templates are clonally amplified off the support (e.g., in solution), then deposited on the support and immobilized on the support. In some embodiments, a clonal amplification reaction of one or more nucleic acid templates is performed on the support, resulting in immobilization on the support. In some embodiments, one or more nucleic acid templates are clonally amplified (e.g., in solution or on the support) using a nucleic acid amplification reaction, the nucleic acid amplification reaction comprising any one of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-stranded binding (SSB) protein-dependent amplification, or any combination thereof.

[0178] The terms "surface primer", "surface capture primer" and related terms refer to a single-stranded oligonucleotide that is immobilized to a support and comprises a sequence that can hybridize to at least a portion of a nucleic acid template molecule. Surface primers can be used to immobilize template molecules to a support via hybridization. Surface primers can be immobilized to a support in a manner that resists primer removal during flow, washing, aspiration, and changes in temperature, pH, salt, chemical, and / or enzyme conditions. Typically, but not necessarily, the 5' end of the surface primer can be immobilized to the support. Alternatively, an inner portion or the 3' end of the surface primer can be immobilized to the support.

[0179] Surface primers include DNA, RNA, or analogs thereof. Surface primers can include combinations of DNA and RNA. The sequences of the surface primers can be fully or partially complementary along their length to at least a portion of the nucleic acid template molecule (e.g., a linear or circular template molecule). The support can include a plurality of immobilized surface primers having the same sequence or two or more different sequences. Surface primers can be any length, for example, 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides long, or longer.

[0180] A surface primer can include a terminal 3' nucleotide with a sugar 3' OH moiety that is extendable for nucleotide polymerization (e.g., polymerase-catalyzed polymerization). A surface primer can include a terminal 3' nucleotide with a moiety that blocks polymerase-catalyzed extension. A surface primer can include a terminal 3' nucleotide with a 3' sugar position attached to a chain-terminating moiety that inhibits nucleotide polymerization. A 3' chain-terminating moiety can be removed (e.g., deblocked) using a deblocking agent to convert the 3' end to an extendable 3' OH end. Examples of chain-terminating moieties include alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl groups. Azide-type chain-terminating moieties, including azide, azido, and azidomethyl groups. Examples of deblocking agents include phosphine compounds such as tris(2-carboxyethyl)phosphine (TCEP) and bis-sulfotriphenylphosphine (BS-TPP) for the chain terminating groups azide, azido, and azidomethyl. Examples of deblocking agents include tetrakis(triphenylphosphine)palladium(0) (Pd(PPh) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ) for the chain terminating groups alkyl, alkenyl, alkynyl, and allyl. 3 ) 4 ) for aryl and benzyl chain terminating groups. Examples of deblocking agents include Pd / C. Examples of deblocking agents include phosphines, beta mercaptoethanol, or dithiothreitol (DTT) for amine, amide, keto, isocyanate, phosphate, thio, and disulfide chain terminating groups. Examples of deblocking agents include potassium carbonate (K 2 CO 3 ) in MeOH for carbonate chain terminating groups. 2 CO 3), triethylamine in pyridine, or Zn(AcOH) in acetic acid. Examples of deblocking agents include tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, and triethylamine trihydrofluoride for the chain terminating groups urea and silyl.

[0181] In some embodiments, the multiple immobilized surface capture primers on the support are in fluid communication with each other, allowing solutions of reagents (e.g., linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and reagents, etc.) to flow over the support, allowing the multiple immobilized surface capture primers on the support to react essentially simultaneously with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the multiple immobilized surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) essentially simultaneously on the multiple immobilized surface capture primers.

[0182] In some embodiments, the multiple immobilized single-stranded nucleic acid concatemer template molecules on the support are in fluid communication with each other, allowing solutions of reagents (e.g., soluble primers, enzymes, nucleotides, divalent cations, buffers, and reagents, etc.) to flow over the support, allowing the multiple immobilized concatemer template molecules on the support to essentially simultaneously react with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the multiple immobilized single-stranded nucleic acid concatemer template molecules can be used to essentially simultaneously perform nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​the multiple immobilized single-stranded nucleic acid concatemer template molecules, and optionally, to perform detection and imaging for massively parallel sequencing.

[0183] The terms "amplify," "amplifying," "amplification," and other related terms, when used in reference to nucleic acids, include producing multiple copies of an original polynucleotide template molecule, where the copies contain a sequence that is complementary to the template sequence or where the copies contain a sequence that is the same as the template sequence. In some embodiments, the copies contain a sequence that is substantially identical to the template sequence or a sequence that is substantially identical to the sequence that is complementary to the template sequence.

[0184] The present disclosure provides a variety of pH buffering agents, the full names of which are listed herein. The term "Tris" refers to the pH buffer tris(hydroxymethyl)-aminomethane. The term "TrisHCl" refers to the pH buffer tris(hydroxymethyl)-aminomethane hydrochloride. The term "tricine" refers to the pH buffer N-[tris(hydroxymethyl)methyl]glycine. The term "bicine" refers to the pH buffer N,N-bis(2-hydroxyethyl)glycine. The term "bis-trispropane" refers to the pH buffer 1,3 bis[tris(hydroxymethyl)methylamino]propane. The term "HEPES" refers to the pH buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The term "MES" refers to the pH buffer 2-(N-morpholino)ethanesulfonic acid. The term "MOPS" refers to the pH buffer 3-(N-morpholino)propanesulfonic acid. The term "MOPSO" refers to 3-(N-morpholino)-2-hydroxypropanesulfonic acid, a pH buffer. The term "BES" refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, a pH buffer. The term "TES" refers to 2-[(2-hydroxy-1,1 bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid), a pH buffer. The term "CAPS" refers to 3-(cyclohexylamino)-1-propanesulfonic acid, a pH buffer. The term "TAPS" refers to N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, a pH buffer. The term "TAPSO" refers to N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid, a pH buffer. The term "ACES" refers to N-(2-acetamido)-2-aminoethanesulfonic acid, a pH buffer. The term "PIPES" refers to piperazine-1,4-bis(2-ethanesulfonic acid), a pH buffering agent.

[0185] Introduction The present disclosure provides compositions and methods of using same for performing pairwise sequencing, as well as compositions and methods of using same for generating concatemeric template molecules for pairwise sequencing.

[0186] Pairwise sequencing includes obtaining a first sequencing read of a first region of a first nucleic acid strand (e.g., sense strand) and obtaining a second sequencing read of a second region of a second nucleic acid strand that is complementary to the first strand (e.g., antisense strand), where the first and second strands correspond to two complementary strands of the same double-stranded template molecule. The first sequencing read of the first sequenced region and the second sequencing read of the second sequenced region can have overlapping sequences that correspond to complementary sequences from the first and second strands of the double-stranded template molecule. The first and second sequencing reads can be aligned such that the overlapping sequencing reads can provide sequence information of the paired regions in the original double-stranded nucleic acid source (e.g., paired regions in a genome), and the accuracy of the sequence information can be confirmed with a high level of confidence from the first and second sequencing reads. The first sequencing read of the first sequenced region and the second sequencing read of the second sequenced region do not necessarily have to have overlapping sequences, in which case the sequence information of the paired regions in the original double-stranded nucleic acid source may not be confirmed with a high level of confidence. The first and second sequencing reads may start at one end of their respective template molecules or at an internal position.

[0187] The compositions and methods for pairwise sequencing described herein provide several advantages that improve the quality of sequencing data, including increased signal strength that improves base calling accuracy.Pairwise sequencing methods also save time by eliminating the need to prepare separate nucleic acid libraries, each corresponding to the sense and antisense strands of a double-stranded template molecule with a sequence of interest.In addition, pairwise sequencing methods generate and sequence the sense and antisense strands that are directly present on the support / substrate that is used to carry out the sequencing reaction.

[0188] The present disclosure provides a pairwise sequencing method that uses a support having a plurality of surface primers immobilized thereon. The immobilized surface primers are in fluid communication with each other, allowing various solutions, such as linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and reagents, to flow over the support, whereby the plurality of immobilized surface primers (and products generated from the immobilized surface primers) react with the solutions in a massively parallel manner.

[0189] The present disclosure provides a pairwise sequencing method, comprising the steps of: (a) providing a plurality of single-stranded nucleic acid concatemer template molecules immobilized on a support; (b) sequencing the plurality of immobilized concatemer template molecules with a first plurality of sequencing polymerases, a plurality of soluble forward sequencing primers, and a first plurality of multivalent molecules, thereby generating a plurality of extended forward sequencing primer strands; (c) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized concatemer template molecules by performing a primer extension reaction; (d) removing the retained immobilized concatemer template molecules while retaining the plurality of forward extension strands; and (e) sequencing the plurality of retained forward extension strands with a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers, and a second plurality of multivalent molecules. In some embodiments, each concatemer template molecule in the plurality of concatemer template molecules is immobilized to a surface primer, and the surface primer is immobilized to a support. In some embodiments, each concatemer template molecule is covalently linked to a surface primer, or each concatemer template molecule is hybridized to a surface primer. In some embodiments, the immobilized surface primer comprises or lacks a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the surface primer. In some embodiments, the plurality of concatemer template molecules comprises at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the concatemer template molecule. In some embodiments, the plurality of concatemer template molecules lacks a nucleotide having a cleavable moiety.Exemplary nucleotides having a cleavable moiety (eg, in a surface primer or a concatemer template molecule) include uridine, 8-oxo-7,8-dihydroguanine, and deoxyinosine.

[0190] In some embodiments, the pairwise sequencing method includes a rolling circle amplification reaction, which is carried out on a support by distributing a plurality of single-stranded circular library molecules on a support on which a plurality of surface primers are immobilized. Each surface primer is designed to capture a single circular library molecule via hybridization. The rolling circle amplification reaction can be carried out on a support. In some embodiments, for an on-support RCA reaction, a solution of single-stranded circular library molecules is flowed onto the support, whereby each circular molecule is captured via hybridization to each surface primer. Each circular library molecule includes at least a sequence of interest and a universal surface primer binding site, and optionally includes a universal sequencing primer binding site, a universal amplification primer binding site, an additional surface primer binding site, and a sample barcode and / or a molecular index. A single immobilized surface primer captures a single circular library molecule, and the rolling circle amplification reaction generates single-stranded linear concatemers covalently bound to the immobilized surface primer by using the terminal 3' end of the surface primer as a primer extension initiation site. Thus, each concatemer molecule is immobilized on the support as a concatemer covalently bound to an immobilized surface primer. A single-stranded concatemer contains multiple tandem copies of the sequence of interest and the universal sequencing primer binding site. A single surface primer captures a single circular library molecule, generating a single concatemer molecule.

[0191] In some embodiments, the pairwise sequencing method includes a rolling circle amplification reaction, which is carried out in solution to generate a plurality of concatemers distributed on a support on which a plurality of surface primers are immobilized. Each surface primer is designed to capture a single concatemer with a complementary sequence of the circular library molecule through hybridization. The rolling circle amplification reaction can continue on the support. In some embodiments, for an in-solution RCA reaction, a plurality of single-stranded circular library molecules are subjected to a rolling circle amplification reaction in a reactor. Each circular library molecule includes at least a sequence of interest and a universal surface primer binding site, and optionally includes a universal sequencing primer binding site, a universal amplification primer binding site, an additional surface primer binding site, and a sample barcode and / or a molecular index. The RCA reaction can be carried out for a very short period of time or for a longer period of time, thereby generating a plurality of concatemers hybridized to each circular library molecule, which are then distributed on a support on which a plurality of surface primers are immobilized. A solution of concatemer molecules is flowed onto the support, whereby individual concatemer molecules are captured via hybridization to individual surface primers. Each individual concatemer molecule contains at least a sequence of interest, a universal surface primer binding site(s), a universal sequencing primer binding site, and optionally a sample barcode and / or a molecular index. A single immobilized surface primer captures a single concatemer molecule, and the rolling circle amplification reaction (now on the support) continues, thereby extending the single-stranded concatemers hybridized to the immobilized surface primers. Thus, each individual concatemer molecule is immobilized on the support as a concatemer hybridized to the immobilized surface primer. The single-stranded concatemers contain multiple tandem copies of the sequence of interest and the universal sequencing primer binding site.A single surface primer captures a single concatemer molecule, generating a single extended concatemer molecule.

[0192] Rolling circle amplification reactions, performed either in solution or on a support, generate concatemers immobilized on a support. Immobilized concatemers offer several advantages over non-concatemeric molecules. The number of tandem copies in a concatemer is adjustable by controlling the time, temperature, and concentration of the reagents of the rolling circle amplification reaction in solution or on a support. Concatemers can self-disintegrate into compact nucleic acid nanoballs. Including one or more compaction oligonucleotides in the RCA reaction can further compact the size and / or shape of the nanoballs. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers that serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction uses detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., with nucleotide units), the signals emitted by the nucleotides or nucleotide units involved in the parallel sequencing reaction along the concatemer result in increased signal intensity for each concatemer. Multiple portions of a given concatemer can be sequenced simultaneously. Furthermore, multiple binding complexes can be formed along a particular concatemer molecule, each binding complex containing a sequencing polymerase bound to a multivalent molecule, and the multiple binding complexes remain stable without dissociation, resulting in increased duration, which increases signal intensity and reduces imaging time.

[0193] The level of sequencing accuracy can be further improved by obtaining partially or completely overlapping sequencing reads from both the sense and antisense strands and aligning the sequencing reads to provide overlapping sequencing data.

[0194] Thus, the pairwise sequencing compositions and methods described herein provide improved sequencing data quality in a massively parallel manner.

[0195] Methods for pairwise sequencing - generation of abasic sites The present disclosure provides a method for pairwise sequencing, comprising the step (a): providing a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each of the plurality of immobilized single-stranded nucleic acid concatemer template molecules comprising at least one nucleotide having a cleavable moiety, each of the plurality of concatemer template molecules being immobilized to a first surface primer immobilized to a support, the immobilized first surface primer lacking the nucleotide having the cleavable moiety. In some embodiments, the support comprises a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support comprises a plurality of first and second surface primers.

[0196] In some embodiments, each immobilized concatemer template molecule is covalently linked to an immobilized surface primer (e.g., the first immobilized surface primer) (Figure 1). In an alternative embodiment, each immobilized concatemer template molecule is hybridized to an immobilized surface primer (e.g., the first immobilized surface primer) (Figure 13).

[0197] In some embodiments, each concatemer template molecule in the plurality of concatemer template molecules comprises two or more copies of a sequence of interest, and each immobilized concatemer template molecule further comprises any one of: (i) two or more copies of a universal binding sequence for the soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence for the soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence for the first immobilized surface primer, (iv) two or more copies of a universal binding sequence for the second immobilized surface primer, (v) two or more copies of a universal binding sequence for the first soluble amplification primer, (vi) two or more copies of a universal binding sequence for the second soluble amplification primer, (vii) two or more copies of a universal binding sequence for the soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0198] In some embodiments, the universal binding sequence for the forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, the universal binding sequence for the reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence for the immobilized first surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, the universal binding sequence (or its complementary sequence) for a soluble compaction oligonucleotide can hybridize to at least a portion of the soluble compaction oligonucleotide.

[0199] In some embodiments, the cleavable moieties in the immobilized concatemer template molecule of step (a) can be converted to abasic sites in the immobilized concatemer template molecule. In some embodiments, the cleavable moieties in the immobilized concatemer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8-oxoG), or deoxyinosine. In the concatemer template molecule, uridine can be converted to abasic sites using uracil DNA glycosylase (UDG), 8-oxoG can be converted to abasic sites using FPG glycosylase, and deoxyinosine can be converted to abasic sites using AlkA glycosylase. In some embodiments, the immobilized concatemer template molecule includes 1-20, 20-40, 40-60, 60-80, 80-100, or more nucleotides with cleavable moieties. In some embodiments, about 0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or higher percent of the dTTP in the immobilized concatemeric template molecules are replaced with nucleotides having a cleavable moiety. In some embodiments, the nucleotides having a cleavable moiety are distributed at random positions along each immobilized concatemeric template molecule. In some embodiments, the nucleotides having a cleavable moiety are distributed at different positions in different immobilized concatemeric template molecules.

[0200] In some embodiments, the immobilized first surface primer comprises a single stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The immobilized first surface primer may be immobilized to a support or may be immobilized to a coating on a support. The immobilized first surface primer may be embedded or attached (coupled) to a coating on a support. In some embodiments, the 5' end of the immobilized first surface primer is immobilized to a support or immobilized to a coating on a support. Alternatively, the inner portion or 3' end of the immobilized first surface primer may be immobilized to a support or may be immobilized to a coating on a support. The support comprises a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer can be any length, for example, 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides long, or longer. In some embodiments, the 3' end of the immobilized first surface primer comprises an extendable 3'OH moiety. In some embodiments, the 3' end of the immobilized first surface primer comprises a 3' non-extendable portion.

[0201] In some embodiments, the plurality of immobilized first surface primers comprises at least one phosphorothioate diester bond at their 5' ends, which can make the first surface primers resistant to exonuclease degradation. In some embodiments, the plurality of immobilized first surface primers comprises 2-5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprises at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which can make the first surface primers resistant to exonuclease degradation.

[0202] In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA), where the at least one LNA comprises a methylene bridge bond between the 2' oxygen and the 4' carbon of the pentose ring. The immobilized first surface primer comprising at least one LNA can be resistant to nuclease digestion and can exhibit an increased melting temperature when hybridized to the forward extension strand.

[0203] In some embodiments, the immobilized concatemeric template molecule further comprises two or more copies of a universal binding sequence (or its complementary sequence) for the immobilized second surface primer having a different sequence from the first immobilized surface primer. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The immobilized second surface primer may be immobilized to the support or immobilized to a coating on the support. The immobilized second surface primer may be embedded or attached (coupled) to a coating on the support. In some embodiments, the 5' end of the immobilized second surface primer is immobilized to the support or immobilized to a coating on the support. Alternatively, the inner portion or the 3' end of the immobilized second surface primer may be immobilized to the support or immobilized to a coating on the support. The support comprises a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer can be of any length, for example, 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides long, or longer.

[0204] In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3'OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extendable moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a moiety that blocks primer extension (e.g., a non-extendable terminal 3' end), such as a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group. The immobilized second surface primer is not extendable in a primer extension reaction. The immobilized second surface primer lacks a nucleotide with a cleavable moiety.

[0205] In some embodiments, the plurality of immobilized second surface primers comprises at least one phosphorothioate diester bond at their 5' ends, which can make the second surface primers resistant to exonuclease degradation. In some embodiments, the plurality of immobilized second surface primers comprises 2-5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprises at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which can make the second surface primers resistant to exonuclease degradation.

[0206] In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule is linked or immobilized to an immobilized first surface primer, and at least a portion of each concatemer template molecule is hybridized to an immobilized second surface primer, which functions to hold a portion of the immobilized concatemer template molecule down to the support (see Figures 12 and 24).

[0207] In some embodiments, the support is 1 mm 2About 10 per 2 ~10 15 In some embodiments, the support comprises 1 mm 2 About 10 per 2 ~10 15 In some embodiments, the support comprises 1 mm 2 About 10 per 2 ~10 15 The immobilized surface primer comprises an immobilized first surface primer and an immobilized second surface primer.

[0208] The immobilized surface primers (e.g., a first and a second surface primer) are in fluid communication with one another, allowing various solutions, such as linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and reagents, to flow over the support, such that the multiple immobilized surface primers (and primer extension products generated from the immobilized surface primers) react with the solutions in a massively parallel manner.

[0209] In some embodiments, the pairwise sequencing method further comprises step (b): sequencing a plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands. The sequencing step (b) comprises contacting a plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers under conditions suitable for hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites / sequences of the immobilized concatemer template molecules, and performing a forward sequencing reaction using one or more types of sequencing polymerase, a plurality of nucleotides and / or multivalent molecules, and the hybridized first forward sequencing primer. The forward sequencing reaction can generate a plurality of extended forward sequencing primer strands. In some embodiments, each immobilized concatemer template molecule has multiple copies of the forward sequencing primer binding site, and each forward sequencing primer binding site can hybridize to the first forward sequencing primer. Each forward sequencing primer binding site in a given immobilized concatemer template molecule can be hybridized to a forward sequencing primer and subjected to a sequencing reaction. Each immobilized concatemer template molecule can be subjected to two or more sequence reactions, each sequencing reaction starting from the first forward sequencing primer hybridized to the forward sequencing primer binding site (see, for example, Figures 2 and 14). In some embodiments, the soluble forward sequencing primer comprises a 3'OH extendable end. In some embodiments, the soluble forward sequencing primer comprises a 3' blocking portion that can be removed to generate a 3'OH extendable end.In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. In some embodiments, the sequencing reaction comprises a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, the multivalent molecules being labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and / or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in FIG. 108 and exemplary multivalent molecules are shown in FIGS. 104-107.

[0210] In some embodiments, the pairwise sequencing method further comprises step (c): retaining a plurality of immobilized concatemer template molecules and replacing a plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized single-stranded nucleic acid concatemer template molecules. The plurality of extended forward sequencing primer strands can be removed and replaced with a plurality of forward extension strands by performing a primer extension reaction (see Figures 3-5 and Figures 15-17).

[0211] In some embodiments, step (c) comprises contacting at least one extended forward sequencing primer strand with a plurality of strand displacement polymerases and a plurality of nucleotides in the absence of soluble amplification primers under conditions suitable for performing a strand displacement primer extension reaction using at least one extended forward sequencing primer strand to initiate a primer extension reaction, thereby generating a forward extension strand covalently linked to the extended forward sequencing primer strand, the forward extension strand hybridizing to the immobilized concatemer template molecule. For example, one of the extended forward sequencing primer strands can function as a primer for the strand displacement polymerase. The strand displacement polymerase can extend the extended forward sequencing primer strand and displace the downstream extended forward sequencing primer strand while synthesizing an extended strand that displaces the downstream extended forward sequencing primer strand (Figures 3 and 15). The newly extended strand is covalently linked to the extended forward sequencing primer strand, and the immobilized concatemeric template molecule is retained.

[0212] The primer extension reaction can optionally include a plurality of compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III) to generate forward extension strands. The individual forward extension strands can collapse into nanoballs having a more compact size and / or shape compared to nanoballs generated from a primer extension reaction performed without compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III). Including compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III) in the primer extension reaction can improve the FWHM (full width at half maximum) of the spot image of the nanoballs. The spot image can be represented as a Gaussian spot, and the size can be measured as the FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanoball spot can be about 10 μm or less.

[0213] Examples of strand displacement polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0214] In some embodiments, step (c) comprises: (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules; and (ii) contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble forward sequencing primers (e.g., a second plurality of soluble forward sequencing primers), a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer-extending polymerases under conditions suitable for hybridizing the plurality of soluble forward sequencing primers to the plurality of retained immobilized concatemer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extended strands, wherein the soluble sequencing primers hybridize to the forward sequencing primer binding sequences in the retained immobilized concatemer molecules (FIGS. 4 and 16). The primer extension reaction can optionally include a plurality of compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III) to generate forward extension strands. The individual forward extension strands can collapse into nanoballs having a more compact size and / or shape compared to nanoballs generated from a primer extension reaction performed without compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III). Including compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III) in the primer extension reaction can improve the FWHM (full width at half maximum) of the spot image of the nanoballs. The spot image can be represented as a Gaussian spot, and the size can be measured as the FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanoball spot can be about 10 μm or less.

[0215] In some embodiments, in step (c), the conditions suitable for hybridizing the plurality of soluble forward sequencing primers to the plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include hybridizing the retained immobilized concatemer template molecules with the soluble primers in the presence of a primer extension polymerase, a plurality of nucleotides, and a high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant that is 40 or less and having a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant that is 115 or less and present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high efficiency hybridization buffer further comprises betaine.

[0216] In some embodiments, step (c) comprises: (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules; and (ii) contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer extension polymerases under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatemer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension strands, in which the soluble amplification primers hybridize to the soluble amplification primer binding sequences in the retained immobilized concatemer molecules (FIGS. 5 and 17). The primer extension reaction can optionally include a plurality of compaction oligonucleotides and / or hexamine (e.g., cobalt hexamine III) to generate the forward extension strands. Individual forward extension strands can collapse into nanoballs with a more compact size and / or shape compared to nanoballs generated from a primer extension reaction carried out without compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III). Including compaction oligonucleotides and / or hexamines (e.g., cobalt hexamine III) in the primer extension reaction can improve the FWHM (full width at half maximum) of the nanoball spot image. The spot image can be represented as a Gaussian spot, and the size can be measured as the FWHM. Smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanoball spot can be about 10 μm or less.

[0217] In some embodiments, in step (c), the conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include hybridizing the retained immobilized concatemer template molecules with the soluble primers in the presence of a primer extension polymerase, a plurality of nucleotides, and a high efficiency hybridization buffer. In some embodiments, the high efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant that is 40 or less and having a polarity index of 4 to 9, (ii) a second polar aprotic solvent having a dielectric constant that is 115 or less and present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids, (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8, and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high efficiency hybridization buffer further comprises betaine.

[0218] In some embodiments, in step (c), the multiple extended forward sequencing primer strands can be removed using an enzyme or chemical reagent. For example, the multiple extended forward sequencing primer strands can be enzymatically degraded using a 5' to 3' double-stranded DNA exonuclease, which includes T7 exonuclease (e.g., from New England Biolabs, catalog number M0263S). In some embodiments, the multiple extended forward sequencing primer strands can be removed at a temperature that favors nucleic acid denaturation.

[0219] In some embodiments, in step (c), a denaturing reagent may be used to remove multiple extended forward sequencing primer strands, the denaturing reagent comprising any one of the following compounds, such as formamide, acetonitrile, guanidinium chloride, and / or a pH buffer (such as, for example, Tris-HCl, MES, HEPES, or MOPS), or any combination thereof. Optionally, the denaturing reagent may further comprise PEG.

[0220] In some embodiments, in step (c), the multiple extended forward sequencing primer strands may be removed using elevated temperatures (e.g., heat) with or without a nucleic acid denaturing reagent. The multiple extended forward sequencing primer strands may be subjected to a temperature of about 45-50°C, or about 50-60°C, or about 60-70°C, or about 70-80°C, or about 80-90°C, or about 90-95°C, or higher.

[0221] In some embodiments, in step (c), the multiple extended forward sequencing primer strands may be removed using 100% formamide at a temperature of about 65° C. for about 3 minutes and washing with about 50 mM NaCl or a reagent containing an equivalent ionic strength and having a pH of about 6.5-8.5.

[0222] In some embodiments, the primer extension polymerase of step (c) comprises a high-fidelity polymerase. In some embodiments, the primer extension polymerase of step (c) comprises a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include Q5U Hot Start high fidelity DNA polymerase (e.g., catalog number M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., a mixture of Taq and Deep Vent DNA polymerase, catalog number M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., catalog number M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., catalog number M0490S from New England Biolabs), Bst DNA polymerase (e.g., large fragment, catalog number M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog number M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., catalog number M0269S from New England Biolabs), E. coli DNA polymerase (e.g., catalog number M0209S from New England Biolabs), Therminator DNA polymerase (e.g., catalog number M0261S from New England Biolabs), Vent DNA polymerase, and Deep Vent DNA polymerase.

[0223] The pairwise method described herein can provide increased accuracy in downstream sequencing reaction because step (c) replaces the extended forward sequencing primer strand generated in step (b) with a forward extension strand with reduced base errors. The extended forward sequencing primer strand may or may not contain nucleotides that are generated in step (b) and mis-incorporated due to polymerase-catalyzed mismatched bases. When step (c) is performed with high fidelity DNA polymerase, the resulting forward extension strand may have reduced base errors compared to the extended forward sequencing primer strand. The forward extension strand is used as a nucleic acid template for downstream sequencing step (see, for example, step (e) below). Thus, step (c) can increase the sequencing accuracy of downstream step (e), and thus increase the overall sequencing accuracy of pairwise sequencing workflow.

[0224] In some embodiments, the pairwise sequencing method further comprises step (d): removing the retained immobilized concatemer template molecules by creating an abasic site in the immobilized single-stranded concatemer template molecule at the nucleotide(s) having the cleavable moiety and creating a gap at the abasic site to retain the plurality of forward extension strands and generate a plurality of gap-containing single-stranded nucleic acid concatemer template molecules while retaining the plurality of immobilized surface primers (FIGS. 6 and 18).

[0225] Abasic sites are generated on the retained concatemer template strand containing a nucleotide with a cleavable moiety. In some embodiments, the cleavable moiety in the retained concatemer template molecule comprises a uridine, 8-oxo-7,8-dihydroguanine (e.g., 8 oxoG), or deoxyinosine. Abasic sites can be removed to generate a plurality of single-stranded nucleic acid template molecules with gaps while retaining a plurality of forward extension strands. Abasic sites can be generated by contacting the immobilized concatemer template molecule with an enzyme that removes a nucleobase in a nucleotide with a cleavable moiety. Uracil in the retained concatemer template strand can be converted to an abasic site using uracil DNA glycosylase (UDG). 8 oxoG in the retained concatemer template strand can be converted to an abasic site using FPG glycosylase. Deoxyinosine in the retained concatemer template strand can be converted to an abasic site using AlkA glycosylase.

[0226] In some embodiments, in step (d), the gaps can be generated by contacting the abasic sites in the immobilized concatemer template molecule with an enzyme or mixture of enzymes that have lyase activity that breaks the phosphodiester backbone at the 5' and 3' sides of the abasic site to release an abasic deoxyribose and generate the gap (FIGS. 6 and 18). The abasic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase. In some embodiments, generating the abasic sites and removing the abasic sites to generate the gaps can be accomplished using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (Uracil-Specific Excision Reagent Enzyme, from New England Biolabs) or thermolabile USER (also from New England Biolabs).

[0227] In some embodiments, in step (d), the plurality of gap-containing template molecules may be removed using enzymes, chemicals, and / or heat. After the gap removal procedure, the plurality of retained forward extension strands (see, e.g., Figures 7 and 9, and Figures 19 and 21) are hybridized to the retained immobilized surface primers.

[0228] For example, the plurality of gap-containing template molecules can be enzymatically degraded using a 5' to 3' double-stranded DNA exonuclease, including T7 exonuclease (e.g., from New England Biolabs, catalog number M0263S). When a 5' to 3' double-stranded DNA exonuclease is used to remove the gap-containing template molecule, the plurality of soluble amplification primers in step (c) can include at least one phosphorothioate diester bond at their 5' ends, which can make the soluble amplification primers resistant to exonuclease degradation. In some embodiments, the plurality of soluble amplification primers in step (c) include 2 to 5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of soluble amplification primers in step (c) comprises at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which can render the forward sequence primer resistant to exonuclease degradation.

[0229] In some embodiments, the plurality of gap-containing template molecules can be removed using a chemical reagent that favors nucleic acid denaturation. The denaturing reagent can include any one of the following compounds, such as formamide, acetonitrile, guanidinium chloride, and / or a buffer (such as Tris-HCl, MES, or HEPES), or any combination thereof.

[0230] In some embodiments, the plurality of gap-containing template molecules may be removed using elevated temperatures (e.g., heat) with or without a nucleic acid denaturing reagent. The gap-containing template molecules may be subjected to a temperature of about 45-50° C., or about 50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C., or about 90-95° C., or higher.

[0231] In some embodiments, the gap-containing template molecules may be removed using 100% formamide at a temperature of about 65° C. for about 3 minutes and washing with about 50 mM NaCl or a reagent containing an equivalent ionic strength and having a pH of about 6.5-8.5.

[0232] In some embodiments, the pairwise sequencing method further comprises step (e): sequencing the plurality of retained forward extension strands, thereby generating a plurality of extended reverse sequencing primer strands. In some embodiments, the sequencing of step (e) comprises contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers under conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strands, and by carrying out a sequencing reaction using the hybridized reverse sequencing primers, the forward sequencing reaction generating a plurality of extended reverse sequencing primer strands (FIGS. 10 and 11, and FIG. 22 and 23). The extended reverse sequencing primer strands are hybridized to the retained forward extension strands. The retained forward extension strands are hybridized to the first surface primer. The extended reverse sequencing primer strand is not hybridized to or covalently linked to the first surface primer, and therefore is not immobilized to a support.

[0233] For simplicity, Figures 7 and 9 show exemplary retained forward extension strands, each of which has one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will understand that the retained forward extension strand can contain two or more tandem copies containing the sequence of interest and various universal primer binding sites. Thus, the reverse sequencing reaction can generate multiple extended reverse sequencing primer strands hybridized to the same retained forward extension strand.

[0234] In some embodiments, in step (e), the conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand include contacting the plurality of soluble reverse sequencing primers and the retained forward extension strand with a high efficiency hybridization buffer. In some embodiments, the high efficiency hybridization buffer includes (i) a first polar aprotic solvent having a dielectric constant that is 40 or less and having a polarity index of 4 to 9, (ii) a second polar aprotic solvent having a dielectric constant that is 115 or less and present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids, (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8, and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high efficiency hybridization buffer further comprises betaine.

[0235] In an alternative embodiment, the sequencing in step (e) comprises performing a sequencing reaction using the immobilized surface primers as sequencing primers to generate multiple reverse sequencing strands.

[0236] In some embodiments, the reverse sequencing reaction of step (e) comprises contacting a plurality of soluble reverse sequencing primers with the reverse sequencing primer binding sequences of the retained forward extension strands, one or more types of sequencing polymerase, and a plurality of nucleotides or a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primers comprise a 3'OH extendable end. In some embodiments, the soluble reverse sequencing primers comprise a 3' blocking moiety that can be removed to generate a 3'OH extendable end. In some embodiments, the soluble reverse sequencing primers lack a nucleotide with a cleavable moiety. Sequencing reactions using nucleotides and / or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate a plurality of extended reverse sequencing primer strands. In some embodiments, each retained forward extension strand has multiple copies of the reverse sequencing primer binding sequence / site, and each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Each reverse sequencing primer binding site in a given retained forward extension strand can be hybridized to a reverse sequencing primer and subjected to a sequencing reaction. Thus, each retained forward extension strand can be subjected to two or more sequence reactions, each sequencing reaction starting from a reverse sequencing primer hybridized to a reverse sequencing primer binding site (see, for example, Figures 10 and 11 and Figures 22 and 23). In some embodiments, the sequencing reaction comprises a plurality of nucleotides (or their analogs) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, the multivalent molecules being labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

[0237] In some embodiments, at least one wash step may be performed after any of steps (a)-(e). The wash step may be performed with a wash buffer comprising a pH buffer, a metal chelator, a salt, and a detergent.

[0238] In some embodiments, the pH buffering compound in the wash buffer comprises any one of Tris, Tris-HCl, Tricine, Bicine, Bis-Tris Propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (also known as 2-aminomethanol; MEA), citrate compounds, citrate mixtures, NaOH, and / or KOH, or any combination of two or more thereof. In some embodiments, the pH buffering agent can be present in the wash buffer at a concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM. In some embodiments, the pH of the pH buffering agent present in any of the reagents described herein can be adjusted to a pH of about 4-9, or a pH of about 5-9, or a pH of about 5-8.

[0239] In some embodiments, the metal chelator in the wash buffer comprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), DPTA (diethylenetriaminepentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium citrate. In some embodiments, the wash buffer comprises a chelator at a concentration of about 0.01-50 mM, or about 0.1-20 mM, or about 0.2-10 mM.

[0240] In some embodiments, the salt in the wash buffer is NaCl, KCl, NH 2 SO 4In some embodiments, the detergent comprises an ionic detergent, such as SDS (sodium dodecyl sulfate). The wash buffer can include a monovalent salt at a concentration of about 25-500 mM, or about 50-250 mM, or about 100-200 mM.

[0241] In some embodiments, the detergent in the wash buffer comprises a non-ionic detergent, such as Triton® X-100, Tween® 20, Tween® 80, or Nonidet P-40. In some embodiments, the detergent comprises a zwitterionic detergent, such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfate (DetX). In some embodiments, the detergent comprises LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is present in the wash buffer at a concentration of about 0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%, or about 0.2-0.25%.

[0242] On-support RCA and pairwise sequencing - generation of abasic sites The present disclosure provides a pairwise sequencing method comprising step (a): providing a support having a plurality of surface primers (e.g., a plurality of first surface primers) immobilized thereon, each of the surface primers having a 3'OH extendable end and lacking a nucleotide having a cleavable moiety (FIG. 25). For example, the surface primers lack uridine, 8-oxo-7,8-dihydroguanine (e.g., 8 oxoG), and deoxyinosine. In some embodiments, the support comprises a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support comprises a plurality of first and second surface primers.

[0243] In some embodiments, the immobilized first surface primers comprise single-stranded oligonucleotides comprising DNA, RNA, or a combination of DNA and RNA. The first surface primers comprise sequences along their length that are fully or partially complementary to at least a portion of the nucleic acid library molecules (e.g., linear or circular library molecules). The first surface primers can comprise a terminal 3' nucleotide having a sugar 3' OH moiety that is extendable for nucleotide polymerization (e.g., polymerase-catalyzed polymerization).

[0244] The immobilized first surface primer may be immobilized to the support or to a coating on the support. The immobilized first surface primer may be embedded or attached (coupled) to a coating on the support. In some embodiments, the 5' end of the immobilized first surface primer is immobilized to the support or to a coating on the support. Alternatively, the inner portion or the 3' end of the immobilized first surface primer may be immobilized to the support or to a coating on the support. The support comprises multiple immobilized first surface primers having the same sequence. The immobilized first surface primer can be any length, for example, 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides long, or longer.

[0245] In some embodiments, the plurality of immobilized first surface primers comprises at least one phosphorothioate diester bond at their 5' ends, which can make the first surface primers resistant to exonuclease degradation. In some embodiments, the plurality of immobilized first surface primers comprises 2-5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprises at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which can make the first surface primers resistant to exonuclease degradation.

[0246] In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA), where the at least one LNA comprises a methylene bridge bond between the 2' oxygen and the 4' carbon of the pentose ring. The immobilized first surface primer comprising at least one LNA can be resistant to nuclease digestion and can exhibit an increased melting temperature when hybridized to the forward extension strand.

[0247] In some embodiments, the support further comprises a plurality of second surface primers immobilized thereon (FIG. 37). The second surface primers have a different sequence than the first immobilized surface primers. The immobilized second surface primers of step (a) comprise single-stranded oligonucleotides comprising DNA, RNA, or a combination of DNA and RNA. The second surface primers comprise sequences along their length that are fully complementary or partially complementary to at least a portion of the immobilized single-stranded concatemer template molecules. The immobilized second surface primers may be immobilized to the support or immobilized to a coating on the support. The immobilized second surface primers may be embedded or attached (coupled) to a coating on the support. In some embodiments, the 5' end of the immobilized second surface primer is immobilized to the support or immobilized to a coating on the support. Alternatively, the inner portion or the 3' end of the immobilized second surface primer may be immobilized to the support or immobilized to a coating on the support. The support comprises a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer can be any length, for example, 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides long, or longer. In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3'OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extendable moiety. The 3' end of the immobilized second surface primer comprises a moiety that blocks primer extension, for example, a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group. The immobilized second surface primer is not extendable in a primer extension reaction. The immobilized second surface primer lacks a nucleotide with a cleavable moiety.

[0248] In some embodiments, the plurality of immobilized second surface primers comprises at least one phosphorothioate diester bond at their 5' ends, which can make the second surface primers resistant to exonuclease degradation. In some embodiments, the plurality of immobilized second surface primers comprises 2-5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprises at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which can make the second surface primers resistant to exonuclease degradation.

[0249] In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule is covalently linked to an immobilized first surface primer, and at least one portion of each concatemer template molecule is hybridized to an immobilized second surface primer (Figure 37). The immobilized second surface primer functions to hold a portion of the immobilized concatemer template molecule down to the support. The immobilized concatemer template molecule has two or more copies of a universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. In some embodiments, the second surface primers include a terminal 3' blocking group that makes them non-extendable. In some embodiments, the second surface primers have a terminal 3' extendable end.

[0250] In some embodiments, the support is 1 mm 2 About 10 per 2 ~10 15 In some embodiments, the support comprises 1 mm2 About 10 per 2 ~10 15 In some embodiments, the support comprises 1 mm 2 About 10 per 2 ~10 15 The immobilized surface primer comprises an immobilized first surface primer and an immobilized second surface primer.

[0251] The immobilized surface primers (e.g., a first and a second surface primer) are in fluid communication with one another, allowing various solutions, such as linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and reagents, to flow over the support, such that the multiple immobilized surface primers (and primer extension products generated from the immobilized surface primers) react with the solutions in a massively parallel manner.

[0252] In some embodiments, the pairwise sequencing method further comprises step (b): hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers, and performing a rolling circle amplification reaction using a plurality of strand-displacing polymerases and a plurality of nucleotides including dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety, thereby generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each of which is linked (covalently linked) to an immobilized first surface primer (e.g., the immobilized first surface primer) (Figure 26). In some embodiments, the rolling circle amplification reaction can be performed in the presence or absence of a plurality of compaction oligonucleotides.

[0253] In some embodiments, the single-stranded circular nucleic acid library molecules comprise covalently closed circular molecules. In some embodiments, the single-stranded circular nucleic acid library molecules can be removed from the concatemer template molecules by at least one washing step, the at least one washing step being performed under conditions suitable for retaining the single-stranded nucleic acid concatemer template molecules, each concatemer template molecule being operably linked to the immobilized first surface primer.

[0254] In some embodiments, each of the single-stranded circular nucleic acid library molecules in the plurality of single-stranded circular nucleic acid library molecules comprises a sequence of interest, and each immobilized concatemeric template molecule further comprises any one of: (i) a universal binding sequence for a soluble forward sequencing primer (or a complementary sequence thereof), (ii) a universal binding sequence for a soluble reverse sequencing primer (or a complementary sequence thereof), (iii) a universal binding sequence for a first immobilized surface primer (or a complementary sequence thereof), (iv) a universal binding sequence for a second immobilized surface primer (or a complementary sequence thereof), (v) a universal binding sequence for a first soluble amplification primer (or a complementary sequence thereof), (vi) a universal binding sequence for a second soluble amplification primer (or a complementary sequence thereof), (vii) a universal binding sequence for a soluble compaction oligonucleotide (or a complementary sequence thereof), (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence, or any combination of two or more thereof.

[0255] In some embodiments, the universal binding sequence for the forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, the universal binding sequence for the reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence for the immobilized first surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, the universal binding sequence (or its complementary sequence) for a soluble compaction oligonucleotide can hybridize to at least a portion of the soluble compaction oligonucleotide.

[0256] In some embodiments, the rolling circle amplification reaction of step (b) generates a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each of the plurality of immobilized single-stranded nucleic acid concatemer template molecules comprising concatemers having at least one nucleotide having a cleavable moiety and two or more copies of a sequence of interest, wherein the immobilized concatemer template molecules contain (i) two or more copies of a universal binding sequence (or a complementary sequence thereof) for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence (or a complementary sequence thereof) for a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) for an immobilized first surface primer, and (iv) a universal binding sequence (or a complementary sequence thereof) for an immobilized second surface primer. (iv) two or more copies of a universal binding sequence (or its complementary sequence) for the immobilized second surface primer, (v) two or more copies of a universal binding sequence (or its complementary sequence) for the first soluble amplification primer, (vi) two or more copies of a universal binding sequence (or its complementary sequence) for the second soluble amplification primer, (vii) two or more copies of a universal binding sequence (or its complementary sequence) for the soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence, or any combination of two or more thereof.

[0257] In some embodiments, the plurality of immobilized single-stranded nucleic acid concatemer template molecules generated by the rolling circle amplification reaction of step (b) further comprises tw...

Claims

1. A method for paired-end sequencing, comprising: a) providing a plurality of immobilized single-stranded nucleic acid concatemer template molecules, each of said plurality of immobilized single-stranded nucleic acid concatemer template molecules comprising at least one nucleotide having a cleavable moiety that can be cleaved to generate a non-basic site in an individual concatemer template molecule, wherein individual concatemer template molecules in said plurality of concatemer template molecules comprise two or more copies of a target sequence, as well as binding sequences for a forward sequencing primer, a reverse sequencing primer, and a surface primer, and are covalently linked or hybridized to a surface primer immobilized on a support, said immobilized surface primer lacking a nucleotide having a cleavable moiety, said providing; b) sequencing said plurality of immobilized concatemer template molecules with a first plurality of sequencing polymerases, a plurality of soluble forward sequencing primers, and a first plurality of multivalent molecules, thereby generating a plurality of extended forward sequencing primer strands, wherein individual immobilized concatemer template molecules have two or more extended forward sequencing primer strands hybridized thereon, said generating; c) retaining said plurality of immobilized concatemer template molecules, thereby generating retained immobilized concatemer template molecules, and replacing said plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to said retained immobilized concatemer template molecules by performing a primer extension reaction; d) generating a non-basic site in said immobilized concatemer template molecule at said nucleotide(s) having a cleavable moiety, generating a gap at said non-basic site, and removing said retained immobilized concatemer template molecules by generating a plurality of gap-containing concatemer template molecules while retaining said plurality of forward extension strands and said plurality of immobilized surface primers; e) sequencing the plurality of retained forward extension strands with a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers, and a second plurality of multivalent molecules, thereby generating a plurality of extended reverse sequencing primer strands, wherein each retained forward extension strand has two or more extended reverse sequencing primer strands hybridized thereon; wherein the individual multivalent molecules in the first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) each comprise (i) a core and (ii) a plurality of nucleotide arms, the plurality of nucleotide arms comprising a core attachment portion, a spacer, a linker, and a nucleotide unit; the core is attached to the plurality of nucleotide arms via their core attachment portions, the spacer is attached to the linker, and the linker is attached to the nucleotide unit. **Claim 2**: a) the nucleotide unit of the individual multivalent molecule of step (b) binds to a first polymerase bound to a nucleic acid duplex comprising an immobilized concatemer template molecule hybridized to a forward sequencing primer; b) the nucleotide unit of the individual multivalent molecule of step (e) binds to a second polymerase bound to a nucleic acid duplex comprising a retained forward extension strand hybridized to a reverse sequencing primer; c) the core comprises streptavidin and the core attachment portion comprises biotin; d) in the spacer, comprising a polyethylene glycol (PEG) moiety, the linker comprising an aliphatic chain having 2 to 6 subunits, or an oligoethylene glycol chain having 2 to 6 subunits; or e) the plurality of nucleotide arms attached to the core have the same type of nucleotide unit, the type of nucleotide unit being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP; The method according to claim 1. **Claim 3** a) The first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) comprise one type of multivalent molecule, and each multivalent molecule in the plurality of multivalent molecules has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, or, b) The first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) comprise a mixture of any combination of two or more types of multivalent molecules, and each of the two or more types of multivalent molecules has a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The method according to claim 1. **Claim 4** At least one multivalent molecule in the first plurality of multivalent molecules of step (b) is labeled with a fluorophore, and at least one multivalent molecule in the second plurality of multivalent molecules of step (e) is labeled with a fluorophore. The method according to claim 1. **Claim 5** The nucleotide(s) having the cleavable moiety comprises uridine, and generating the non-basic site with the nucleotide(s) having the cleavable moiety in step (d) comprises contacting the immobilized concatemer template molecule with uracil DNA glycosylase (UDG), or, Generating the plurality of gap-containing single-stranded nucleic acid concatemer template molecules in step (d) comprises contacting the retained immobilized template molecule containing one or more non-basic sites with endonuclease IV, AP lyase, FPG glycosylase / AP lyase, and / or endoVIII glycosylase / AP lyase, wherein the AP lyase comprises depurinated site DNA lyase or depyrimidinated site DNA lyase. The method according to claim 1. **Claim 6** The support comprises at least one hydrophilic polymer coating layer and a plurality of surface primers immobilized on the at least one hydrophilic polymer coating layer, and the at least one hydrophilic polymer coating layer has a water contact angle of 45 degrees or less. The method according to claim 1. **Claim 7** The at least one hydrophilic polymer coating layer contains a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran, The at least one hydrophilic polymer coating layer contains polyethylene glycol (PEG), The at least one hydrophilic polymer coating layer contains polymer molecules having a molecular weight of at least 1000 Daltons, The at least one hydrophilic polymer coating layer contains branched polymer molecules having 4 to 8 branches, or The method according to claim 6, wherein the support comprises one hydrophilic polymer coating layer and a plurality of surface primers at a surface density of at least 1000 / μm ２ ​ Claim 8 The support is (i) a first coating layer comprising a first monolayer of hydrophilic polymer molecules tethered to the support; a second coating layer comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer; and a third coating layer comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first layer, the second layer, or the third layer are the first layer, the second layer, or the third layer comprising a branched polymer layer; or (ii) the surface primer is immobilized on the hydrophilic polymer molecules of the second monolayer or the third monolayer, and the surface primer is distributed at a plurality of depths throughout the second layer or the third layer; (iii) one or more of the at least one hydrophilic polymer coating layer contains a plurality of surface primers at a surface density of at least 1000 / μm ２ ; or, (iv) One or more of the at least one hydrophilic polymer coating layer contains a plurality of surface primers at a surface density of 1000 to 15,000 per 1 μm ２ inclusive. The method according to claim 7, comprising Claim 9 When the fluorescence image of the support is obtained by contacting the support with a fluorescently labeled nucleotide (Cy3-labeled nucleotide) and acquiring the fluorescence image under no signal saturation conditions using an inverted fluorescence microscope and a camera while the support is immersed in a buffer solution, the hydrophilic polymer coating layer on the support exhibits a contrast-to-noise (CNR) ratio of at least 20; The support comprises glass or plastic; or The method according to claim 8, wherein the support is configured on a flow cell or inside a capillary lumen.

10. At least one of the immobilized concatemer template molecules among the plurality of concatemer template molecules includes a uridine-containing concatemer template molecule in which up to 30% of thymidine is replaced by uridine; and, Step c) includes removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules, and performing a primer extension reaction with a plurality of soluble extension primers, a plurality of nucleotides, and a primer extension polymerase, thereby generating a plurality of forward extension strands hybridized to the retained immobilized concatemer template molecules; The method according to claim 1.

11. (a) At least one of the immobilized concatemer template molecules among the plurality of immobilized concatemer template molecules lacks uridine, (b) The immobilized concatemer template molecule includes two or more copies of a target sequence and two or more copies of a universal binding sequence for a soluble amplification primer, and the plurality of soluble extension primers include a plurality of soluble amplification primers that hybridize to the universal binding sequence for the soluble amplification primer, or, (c) The immobilized concatemer template molecule includes two or more copies of a target sequence and two or more copies of a universal binding sequence for a soluble forward sequencing primer, and the plurality of soluble extension primers include a plurality of soluble forward sequencing primers that hybridize to the universal binding sequence for the soluble forward sequencing primer, The method according to claim 10.

12. The nucleotide units of the first plurality of multivalent molecules in step (b) contain a removable chain termination moiety at the 3'-sugar group, the nucleotide units of the second plurality of multivalent molecules in step (e) contain a removable chain termination moiety at the 3'-sugar group, the removable chain termination moiety contains an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, and the removable chain termination moiety is cleavable with a chemical compound so as to generate an extendable 3'-OH moiety on the sugar group; or, the sequencing in step (b) is a1) contacting the plurality of first sequencing polymerases with (i) the plurality of immobilized concatemer template molecules or the plurality of retained forward extension strands, and (ii) the plurality of soluble sequencing primers including a plurality of soluble forward sequencing primers or a plurality of soluble reverse sequencing primers, the contacting being performed under conditions suitable for forming a plurality of first composite polymerases, each of the plurality of first composite polymerases including a first sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex including a nucleic acid template molecule hybridized to a soluble sequencing primer, the contacting; b1) contacting the plurality of first composite polymerases with a plurality of fluorophore-labeled multivalent molecules to form a plurality of multivalent composite polymerases, the contacting being performed under conditions suitable for binding complementary nucleotide units of the multivalent molecules to at least two of the plurality of first composite polymerases, thereby forming a plurality of multivalent composite polymerases, the conditions inhibiting the incorporation of the complementary nucleotide units into the hybridized sequencing primers of the plurality of multivalent composite polymerases, the forming; c1) detecting the plurality of multivalent composite polymerases; d1) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first composite polymerases in the plurality of multivalent composite polymerases, thereby determining the sequence of the nucleic acid template; The method according to claim 1. **Claim 13** e1) dissociating the plurality of multivalent composite polymerases by removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; f1) contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, the contacting being carried out under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second composite polymerases, each of the plurality of second composite polymerases comprising a second sequencing polymerase bound to a retained nucleic acid duplex; and g1) contacting the plurality of second composite polymerases with a plurality of nucleotides, the contacting being carried out under conditions suitable for binding complementary nucleotides to at least two of the second composite polymerases of step (f), thereby forming a plurality of nucleotide composite polymerases, the conditions being suitable for promoting the incorporation of the bound complementary nucleotides into the hybridized sequencing primers of the nucleotide composite polymerases, thereby generating a plurality of extended sequencing primer strands, the plurality of extended sequencing primer strands comprising a plurality of extended forward sequencing primer strands or a plurality of extended reverse sequencing primer strands. The method according to claim 12, further comprising the contacting. **Claim 14** Generating the non-basic site of step (d) at the uridine of the immobilized concatemer template molecule comprises contacting the immobilized concatemer template molecule with uracil DNA glycosylase (UDG), or Generating the plurality of gap-containing concatenated template molecules of step (d) comprises contacting the retained and immobilized template molecule containing one or more non-basic sites with endonuclease IV, AP lyase, FPG glycosylase / AP lyase, and / or endoVIII glycosylase / AP lyase, wherein the AP lyase comprises a depurinated site DNA lyase or a depyrimidinated site DNA lyase, the method of claim 10.

15. The support is a) a first coating layer comprising a first monolayer of hydrophilic polymer molecules tethered to the support, b) a second coating layer comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer, and c) a third coating layer comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first layer, the second layer, or the third layer comprise a branched polymer layer, the method of claim 1.

16. The surface primer is immobilized on the hydrophilic polymer molecules of the second monolayer or the third monolayer, and the surface primer is distributed at a plurality of depths throughout the second layer or the third layer, the method of claim 15.

17. The cleavable moiety comprises uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine, the method of claim 1.

18. A method for paired-end sequencing comprising a) providing a support having a plurality of surface primers immobilized thereon, wherein the surface primers comprise a 3'-extendable end and lack a nucleotide having a cleavable moiety that can be cleaved to generate a non-basic site at the surface primer, providing b) Hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized surface primers and performing a rolling circle amplification reaction using a plurality of strand-displacing polymerases and a plurality of nucleotides, thereby generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules, wherein each single-stranded nucleic acid concatemer template molecule comprises two or more copies of the target sequence, as well as binding sequences for a forward sequencing primer, a reverse sequencing primer, and a surface primer, and is covalently linked to the immobilized surface primer, and the plurality of nucleotides lack nucleotides having a cleavable moiety that can be cleaved to generate a non-basic site in the concatemer template molecule; generating; c) Sequencing the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers, thereby generating a plurality of extended forward sequencing primer strands, wherein each immobilized concatemer template molecule has two or more extended forward sequencing primer strands hybridized thereon; generating; d) Holding the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands by performing a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand-displacing polymerases, thereby generating a plurality of forward extension strands and a plurality of partially replaced forward extension strands, wherein the forward extension strands and the partially replaced forward extension strands are hybridized to the immobilized concatemer template molecules, forming a plurality of immobilized amplicons; generating; e) sequencing the plurality of immobilized, partially substituted forward extension strands with a plurality of soluble reverse sequencing primers, thereby generating a plurality of extended reverse sequencing primer strands, wherein each immobilized, partially substituted forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto, and wherein the immobilized, partially substituted forward extension strand remains hybridized to the retained immobilized concatemer template molecule during the sequencing of step (e); including, method. **Claim 19** a) each of the single-stranded circular nucleic acid library molecules in the plurality of single-stranded circular nucleic acid library molecules contains a target sequence, and each individual library molecule (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer, (iii) a universal binding sequence for an immobilized surface primer, (iv) a universal binding sequence for a first soluble amplification primer, (v) a universal binding sequence for a second soluble amplification primer, (vi) a universal binding sequence for a soluble compaction oligonucleotide, (vii) a sample barcode sequence, and / or (viii) any one of the unique molecular index sequences, or any combination of two or more of them; or, b) each individual immobilized single-stranded nucleic acid concatemer template molecule generated by the rolling circle amplification reaction contains two or more copies of the target sequence, and each individual immobilized concatemer template molecule (i) two or more copies of the universal binding sequence for a soluble forward sequencing primer, (ii) two or more copies of the universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of the universal binding sequence for an immobilized surface primer, (iv) two or more copies of the universal binding sequence for a first soluble amplification primer, (v) two or more copies of the universal binding sequence for a second soluble amplification primer, (vi) two or more copies of the universal binding sequence for the soluble compacted oligonucleotide, (vii) two or more copies of the sample barcode sequence, and / or (viii) any one of two or more copies of the unique molecular index sequence, or any combination of two or more of them, The method according to claim 18.

20. The support includes at least one hydrophilic polymer coating layer and a plurality of surface primers immobilized on the at least one hydrophilic polymer coating layer, and the at least one hydrophilic polymer coating layer has a water contact angle of 45 degrees or less. When the fluorescence image of the support is obtained by contacting the support with a fluorescently labeled nucleotide (Cy3-labeled nucleotide) and acquiring the fluorescence image under no signal saturation conditions using an inverted fluorescence microscope and a camera while the support is immersed in a buffer solution, the hydrophilic coating layer on the support exhibits a contrast-to-noise (CNR) ratio of at least 20. The method according to claim 18.

21. Replacing the plurality of extended forward sequencing primer strands in step (d) (i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules; (ii) contacting the plurality of retained immobilized concatemer molecules with the plurality of soluble amplification primers, a plurality of nucleotides, and a plurality of strand displacement polymerases under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatemer template molecules and for performing a polymerase-catalyzed strand displacement reaction, thereby generating a plurality of forward extension strands hybridized to the immobilized concatemer template molecules and a plurality of partially displaced extended forward sequencing strands to form a plurality of immobilized amplicons. wherein the strand displacement polymerase includes phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), E. coli DNA polymerase Klenow fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV virus reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase, The method according to claim 18.

22. wherein the forward sequencing in step (c) a1) contacting a plurality of sequencing polymerases and a plurality of the soluble forward sequencing primers with a plurality of immobilized concatemer template molecules, the contacting being performed under conditions suitable for forming a plurality of complex polymerases, each of the plurality of complex polymerases including a sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex including an immobilized concatemer template molecule hybridized to a soluble forward sequencing primer; b1) contacting the plurality of complex sequencing polymerases with a plurality of nucleotides and at least one nucleotide analog under conditions suitable for binding the at least one nucleotide analog to the complex sequencing polymerases, the plurality of nucleotides including at least one nucleotide analog, the at least one nucleotide analog being labeled with a fluorophore and having a removable chain terminating moiety at the 3'-position of the sugar; c1) incorporating at least one nucleotide into the 3'-end of the hybridized forward sequencing primer, thereby generating a plurality of nascent extended forward sequencing primers; d1) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide; In at least one of the nucleotides among the plurality of nucleotides in step (b1), the nucleotide contains a removable chain termination moiety attached to the 3'-carbon position of the sugar group, and the removable chain termination moiety is an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, and the removable chain termination moiety is cleavable with a chemical compound so as to generate an extendable 3'-OH moiety on the sugar group. The method according to claim 18.

23. The reverse sequencing in step (e) is a2) contacting a plurality of sequencing polymerases and a plurality of the soluble reverse sequencing primers with the plurality of immobilized partially substituted forward extension strands, the contacting being performed under conditions suitable for forming a plurality of complex polymerases, each of the plurality of complex polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising a soluble reverse sequencing primer hybridized to the immobilized partially substituted forward extension strand; contacting; b2) contacting the plurality of complex sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the complex sequencing polymerase, the plurality of nucleotides including at least one nucleotide analog, the at least one nucleotide analog being labeled with a fluorophore and having a removable chain termination moiety at the sugar 3'-position; contacting; c2) incorporating at least one nucleotide into the 3'-end of the hybridized reverse sequencing primer, thereby generating a plurality of nascent extended reverse sequencing primers; d2) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide. In the plurality of nucleotides in step (b2), at least one of the nucleotides includes a removable chain termination moiety attached to the 3'-carbon position of the sugar group, and the removable chain termination moiety is an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, and the removable chain termination moiety is cleavable with a chemical compound so as to generate an extendable 3'-OH moiety on the sugar group. The method according to claim 18.

24. The forward sequencing in step (c) and the reverse sequencing in step (e) are a3) contacting a plurality of first sequencing polymerases and a plurality of soluble sequencing primers with a plurality of nucleic acid template molecules, the contacting being performed under conditions suitable for forming a plurality of first complex polymerases, each of the plurality of first complex polymerases including a first sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex including the nucleic acid template molecule hybridized to the soluble sequencing primer, (1) the plurality of nucleic acid template molecules including a plurality of the immobilized concatemer template molecules, and the plurality of sequencing primers including a plurality of the soluble forward sequencing primers, or (2) the plurality of nucleic acid template molecules including a plurality of immobilized partially substituted forward extension strands, and the plurality of sequencing primers including a plurality of the soluble reverse sequencing primers, the contacting; b3) contacting the plurality of first composite polymerases with a plurality of detectably labeled multivalent molecules to form a plurality of multivalent composite polymerases, wherein each individual multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each of the nucleotide arms being attached to a nucleotide unit, and the contacting is performed under conditions suitable for binding complementary nucleotide units of the multivalent molecules to at least two of the plurality of first composite polymerases, thereby forming a plurality of multivalent composite polymerases, the conditions inhibiting the incorporation of the complementary nucleotide units into the sequencing primers of the plurality of multivalent composite polymerases, and forming; c3) detecting the plurality of multivalent composite polymerases; d3) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first composite polymerases in the plurality of multivalent composite polymerases, thereby determining the sequence of the nucleic acid template, the method according to claim 18, comprising:

25. e3) dissociating the plurality of multivalent composite polymerases, removing the plurality of first sequencing polymerases and their attached multivalent molecules, and retaining the plurality of nucleic acid duplexes; f3) contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, the contacting being performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second composite polymerases; g3) further comprising contacting the plurality of second composite polymerases with a plurality of nucleotides and complementary nucleotides from the plurality of nucleotides under conditions suitable for binding the complementary nucleotides to at least two of the second composite polymerases of step (f3), thereby forming a plurality of nucleotide composite polymerases, the conditions being suitable for promoting the incorporation of the bound complementary nucleotides into the sequencing primers of the nucleotide composite polymerases, the method according to claim 24.

26. h3) detecting the complementary nucleotides incorporated within the sequencing primer of the nucleotide complex polymerase; i3) identifying the nucleobases of the complementary nucleotides incorporated within the sequencing primer of the nucleotide complex polymerase; The method according to claim 25, further comprising. **Claim 27** The method further comprising forming at least one avidity complex in step (b3), the method comprising: a4) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a nucleic acid template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; forming; b4) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the second multivalent molecule binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex; The method according to claim 24, comprising. **Claim 28** (i) the first sequencing primer comprises a soluble forward sequencing primer and the nucleic acid template molecule comprises an immobilized concatemer template molecule, (ii) the second sequencing primer comprises a soluble forward sequencing primer and the nucleic acid template molecule comprises the same immobilized concatemer template molecule, and (iii) the first and second sequencing primers have the same sequence; or, The method according to claim 27, wherein (i) the first sequencing primer comprises a soluble reverse sequencing primer, and the nucleic acid template molecule comprises an immobilized partially substituted forward extension strand; (ii) the second sequencing primer comprises a soluble reverse sequencing primer, and the nucleic acid template molecule comprises the same immobilized partially substituted forward extension strand; and (iii) the first and second sequencing primers have the same sequence.

29. The method according to claim 24, wherein each of the plurality of multivalent molecules in the plurality of multivalent molecules comprises (a) a core and (b) a plurality of nucleotide arms, and the plurality of nucleotide arms comprise (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, the core is attached to the plurality of nucleotide arms via the core attachment portion, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

30. (i) the core comprises streptavidin and the core attachment portion comprises biotin; (ii) the linker comprises an aliphatic chain having 2 to 6 subunits or an oligoethylene glycol chain having 2 to 6 subunits; and (iii) the nucleotide unit comprises an aromatic base, a pentose sugar, and one to ten phosphate groups; or the plurality of nucleotide arms attached to the core have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP; or the plurality of multivalent molecules comprise one type of multivalent molecule, and each multivalent molecule in the plurality of multivalent molecules has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP; or the plurality of multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules, and each of the two or more types of multivalent molecules has a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The method according to claim 29.

31. The method according to claim 24, wherein the plurality of detectably labeled multivalent molecules comprise fluorescently labeled multivalent molecules.

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