Single chain splint chain and method of use

Abstract

The present disclosure provides compositions comprising nucleic acid single-stranded splint chains, including kits, as well as methods of employing the single-stranded splint chains. The single-stranded splint strand can hybridize to a portion of a linear library molecule to form a cyclized library-splint complex having gaps, where the gaps can be linked to form a covalently closed cyclic molecule, which can be subjected to downstream amplification and sequencing workflows.

Description

[0001] Related applications

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 316,790, filed on March 4, 2022, and U.S. Patent Application No. 17 / 725,042, filed on April 20, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The contents of the electronic sequence listing (ELEM-002_001WO_SequenceListing_ST26.xml; size: 68,293 bytes; and creation date: March 3, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure provides compositions comprising single-stranded splint strands of nucleic acids and methods of using the single-stranded splint strands. The single-stranded splint strands can hybridize with portions of library molecules to form a library-splint complex with gaps, wherein the gaps can be ligated to form covalently closed circular molecules that undergo downstream amplification and sequencing workflows. Background Art

[0006] The present disclosure relates to preparing polynucleotides from covalently closed circular molecule libraries and sequencing them. Improvements in second-generation sequencing technology have greatly improved sequencing speed and data output, resulting in high sample throughput of current sequencing platforms. Effectively preparing closed circular library molecules with target sequences is very important for downstream amplification and sequencing workflows. Another aspect of improving sequencing throughput is to add unique index sequences to DNA fragments during library preparation, which allows a large number of libraries to be assembled and sequenced simultaneously during each sequencing run. Therefore, there is a need for an alternative method for generating and sequencing circular library molecules containing target sequences and unique index sequences that is compatible with downstream second-generation sequencing technology. Compositions, methods, and test kits that meet this need are provided herein. Summary of the Invention

[0007] The present disclosure provides a library-splint complex (300) comprising: (i) a single-stranded nucleic acid library molecule (100) comprising a sequence of interest (110) flanked on one side by at least a first left universal adaptor sequence (120) and on the other side by at least a first right universal adaptor sequence (130); and (ii) a single-stranded splint chain (200) comprising a first region (210) having a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and further comprising a second region (220) having a universal binding sequence for the first right universal adaptor sequence (130) of the library molecule, thereby looping the library molecule to produce a library-splint complex (300) having a gap between the 5' end of the library molecule and the 3' end of the library molecule.

[0008] In some embodiments, the nucleic acid library molecule (100) further comprises: a second left universal adapter sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises: a second right universal adapter sequence (150). In some embodiments, the nucleic acid library molecule (100) further comprises: a first left sample index sequence (160). In some embodiments, the nucleic acid library molecule (100) further comprises: a first right sample index sequence (170). In some embodiments, the nucleic acid library molecule (100) further comprises: a first left unique identification sequence (180). In some embodiments, the nucleic acid library molecule (100) further comprises: a first right unique identification sequence (190).

[0009] In some embodiments, the nucleic acid library molecule (100) further comprises any one or any combination of two or more of the following: (i) a second left universal adapter sequence (140); (ii) a second right universal adapter sequence (150); (iii) a first left index sequence (160); (iv) a first right index sequence (170); (v) a first left unique identification sequence (180); and / or (vi) a first right unique identification sequence (190).

[0010] In some embodiments, the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) include: (i) a universal binding sequence for a forward sequencing primer; (ii) a universal binding sequence for a reverse sequencing primer; (iii) a universal binding sequence for a first surface primer; (iv) a universal binding sequence for a second surface primer; (v) a universal binding sequence for a forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and / or (vii) a universal binding sequence for a compacted oligonucleotide.

[0011] In some embodiments, the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) include: (i) a universal binding sequence for a forward sequencing primer; (ii) a universal binding sequence for a reverse sequencing primer; (iii) a universal binding sequence for a first surface primer; (iv) a universal binding sequence for a second surface primer; (v) a universal binding sequence for a forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and / or (vii) a universal binding sequence for a compacted oligonucleotide.

[0012] The present disclosure provides a method for generating a plurality of covalently closed circular library molecules (400), comprising: a) providing a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule of the plurality of single-stranded nucleic acid library molecules comprises a target sequence (110) flanked on one side by at least a first left universal adapter sequence (120) and on the other side by at least a first right universal adapter sequence (130); b) providing a plurality of single-stranded splint chains (200), wherein each single-stranded splint chain (200) of the plurality of single-stranded splint chains comprises a first region (210) and a second region (220), wherein the first region is capable of hybridizing with at least the first left universal adapter sequence (120) of each library molecule and the second region is capable of hybridizing with at least the first right universal adapter sequence (130) of each library molecule. c) hybridizing a plurality of single-stranded splint chains (200) to a plurality of single-stranded nucleic acid library molecules (100) such that a first region of one of the single-stranded splint chains (210) anneals to at least a first left universal adaptor sequence (120) of the library molecule, and such that a second region of the single-stranded splint chain (220) anneals to at least a first right universal sequence (130) of the library molecule, thereby circularizing each library molecule to form a plurality of library-splint complexes (300) having a gap between the 5' and 3' ends of the library molecule, wherein the gap is enzymatically ligatable; and d) ligating the gaps in the plurality of library-splint complexes (300), thereby generating a plurality of covalently closed circular library molecules (400).

[0013] In some embodiments, the method further comprises: (e) distributing the plurality of covalently closed circular library molecules (400) onto a support having a plurality of surface primers immobilized on the support under conditions suitable for hybridizing each covalently closed circular library molecule (400) to each immobilized surface primer, thereby immobilizing the plurality of covalently closed circular library molecules (400) to the support.

[0014] In some embodiments, the method further comprises: (f) contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on a support using the plurality of surface primers as immobilized amplification primers and using the plurality of covalently closed circular library molecules (400) as template molecules, thereby producing a plurality of immobilized nucleic acid concatemer molecules.

[0015] In some embodiments, the method further comprises: sequencing the plurality of immobilized concatemer molecules. In some embodiments, sequencing comprises: a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of composite polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises the concatemer molecules hybridized to the soluble sequencing primers; b) contacting the plurality of composite sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the composite sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the 3' position of the sugar; c) incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0016] In some embodiments, sequencing comprises: a) contacting a plurality of immobilized concatamer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first complex polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises the concatamer molecules hybridized to the soluble sequencing primers; b) contacting the plurality of complex sequencing 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 complex polymerases to form a plurality of multivalent-complex polymerases, thereby forming a plurality of multivalent-complex polymerases, and wherein the conditions inhibit incorporation of complementary nucleotide units into sequencing primers of the plurality of multivalent-complex polymerases, wherein each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; c) detecting the plurality of multivalent-complex polymerases; and d) identifying nucleobases of the complementary nucleotide units bound to the plurality of first complex polymerases in the plurality of multivalent-complex polymerases, thereby determining the sequence of the nucleic acid template.

[0017] In some embodiments, sequencing further comprises: e) dissociating the plurality of multivalent-complex polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining a 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 complex polymerases each comprising a second sequencing polymerase bound to the retained nucleic acid duplexes; g) contacting the plurality of second complex polymerases with a plurality of nucleotides, wherein the contacting is performed under conditions suitable for binding complementary nucleotides in the plurality of nucleotides to at least two of the second complex polymerases of step (f), thereby forming a plurality of nucleotide-complex polymerases, and the conditions are suitable for promoting incorporation of the bound complementary nucleotides into sequencing primers of the nucleotide-complex polymerases.

[0018] In some embodiments, sequencing further comprises: h) detecting a complementary nucleotide incorporated into a sequencing primer of a nucleotide-complex polymerase. In some embodiments, sequencing further comprises: h) detecting a complementary nucleotide incorporated into a sequencing primer of a nucleotide-complex polymerase; and i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase.

[0019] The present disclosure provides a method for forming at least one affinity complex, comprising: a) combining a first universal nucleic acid primer, a first DNA polymerase, and a first multivalent molecule with a first portion of a concatamer molecule according to claim 16, thereby forming a first binding complex, wherein the first nucleotide unit of the first multivalent molecule is bound to the first DNA polymerase; and b) combining a second universal nucleic acid primer, a second DNA polymerase, and the first multivalent molecule with a second portion of the same concatamer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule is bound to the second DNA polymerase, wherein the first binding complex and the second binding complex of the same multivalent molecule form an affinity complex, wherein the first multivalent molecule comprises a core attached to a plurality of nucleotide arms and each nucleotide arm is attached to a nucleotide unit, and wherein the concatamer molecule comprises two or more tandem repeats of a target sequence (110) and a universal primer binding site that binds the first universal nucleic acid primer and the second universal nucleic acid primer.

[0020] The present disclosure provides a method for sequencing by forming at least one affinity complex, comprising: a) binding a first universal nucleic acid primer, a first DNA polymerase, and a first multivalent molecule to a first portion of a concatemer template molecule of claim 16 , thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first DNA polymerase; b) binding a second universal nucleic acid primer, a second DNA polymerase, and the first multivalent molecule to a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule is bound to the second DNA polymerase, wherein the first binding complex and the second binding complex comprise the same multivalent molecule, wherein the first binding complex comprises a plurality of nucleotides attached to the first multivalent molecule. The invention relates to a method for preparing a multimeric template molecule comprising: a) a first nucleotide unit having a core and each nucleotide arm being attached to a nucleotide unit, wherein the multimeric molecule comprises two or more tandem repeats of a sequence of interest (110) and universal primer binding sites that bind a first universal nucleic acid primer and a second universal nucleic acid primer, and wherein the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the first nucleotide unit and the second nucleotide unit bound in the first binding complex and the second binding complex; c) detecting the first binding complex and the second binding complex on the same multimeric template molecule, and d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the multimeric template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the multimeric template molecule.

[0021] In some embodiments, the nucleic acid library molecule (100) further comprises: a second left universal adapter sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises: a second right universal adapter sequence (150). In some embodiments, the nucleic acid library molecule (100) further comprises: a first left sample index sequence (160). In some embodiments, the nucleic acid library molecule (100) further comprises: a first right sample index sequence (170). In some embodiments, the nucleic acid library molecule (100) further comprises: a first left unique identification sequence (180). In some embodiments, the nucleic acid library molecule (100) further comprises: a first right unique identification sequence (190).

[0022] In some embodiments, the nucleic acid library molecule (100) further comprises any one or any combination of two or more of the following: (i) a second left universal adapter sequence (140); (ii) a second right universal adapter sequence (150); (iii) a first left index sequence (160); (iv) a first right index sequence (170); (v) a first left unique identification sequence (180); and / or (vi) a first right unique identification sequence (190).

[0023] In some embodiments, the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) include: (i) a universal binding sequence for a forward sequencing primer; (ii) a universal binding sequence for a reverse sequencing primer; (iii) a universal binding sequence for a first surface primer; (iv) a universal binding sequence for a second surface primer; (v) a universal binding sequence for a forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and / or (vii) a universal binding sequence for a compacted oligonucleotide.

[0024] In some embodiments, the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) include: (i) a universal binding sequence for a forward sequencing primer; (ii) a universal binding sequence for a reverse sequencing primer; (iii) a universal binding sequence for a first surface primer; (iv) a universal binding sequence for a second surface primer; (v) a universal binding sequence for a forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and / or (vii) a universal binding sequence for a compacted oligonucleotide.

[0025] The present disclosure provides a method for sequencing nucleic acid molecules, comprising: a) providing a plurality of nucleic acid template molecules immobilized at random positions on a support, wherein each immobilized template molecule comprises an insert sequence region and a sample index, wherein each sample index comprises a 3-mer random sequence joined to a universal sample index sequence that identifies a sample source of the insert sequence, wherein different immobilized template molecules have different 3-mer random sequences and the same universal sample index sequence, and wherein the immobilized template molecules have different insert sequences; b) performing three cycles of polymerase-mediated sequencing of the 3-mer random sequences of the plurality of immobilized template molecules using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C, and T / U; The method further comprises the steps of: detecting and imaging an optical color signal emitted from the detectably labeled nucleotide reagent bound to the immobilized template molecule to determine the sequence of the 3-mer random sequence in each of the plurality of immobilized template molecules; and detecting and imaging a balanced diversity of A, G, C, and T / U nucleobases across the plurality of immobilized template molecules in each of the first, second, and third sequencing cycles; and c) using the image obtained in step (b) to generate a map of the positions of the plurality of immobilized template molecules, wherein the sequence of the insert region is not used to generate the map. In some embodiments, the balanced diversity of step b) is 25-75% of each of the nucleobases A, G, C, and T / U detected and imaged in each of the first, second, and third sequencing cycles.

[0026] In some embodiments, the method further comprises: d) sequencing a universal sample index sequence of a plurality of fixed template molecules; e) sequencing an insert sequence region of a plurality of fixed template molecules; and f) assigning the insert sequence of a given template molecule obtained in step (b) to the universal sample index sequence from the same given template molecule obtained in step (a), thereby identifying the sample source of the given insert sequence.

[0027] In some embodiments, the plurality of nucleic acid template molecules further comprises a second sample index comprising a second universal sample index sequence that identifies a sample source of the insert sequence, and the second sample index lacks the random sequence.

[0028] In some embodiments, the method further comprises: g) sequencing the 3-mer random sequences of the plurality of fixed template molecules to obtain a balanced diversity of A, G, C and T / U nucleobases detected and imaged in each of the first sequencing cycle, the second sequencing cycle and the third sequencing cycle, thereby generating a map of the positions of the plurality of fixed template molecules; h) sequencing the first universal sample index sequence of the plurality of fixed template molecules; i) sequencing the second universal sample index sequence of the plurality of fixed template molecules; j) sequencing the insert sequence regions of the plurality of fixed template molecules; and k) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insert sequence.

[0029] The present disclosure provides a method for sequencing nucleic acid molecules, comprising: a) providing a plurality of nucleic acid template molecules fixed at random positions on a support, wherein each fixed template molecule comprises an insert sequence region and a sample index, wherein each sample index comprises a 3-mer random sequence joined to a universal sample index sequence that identifies a sample source of the insert sequence, wherein the universal sample index sequence comprises 3-20 nucleotides, wherein different fixed template molecules have different 3-mer random sequences and the same universal sample index sequence, and wherein the fixed template molecules have different insert sequences; (b) performing four cycles of polymerase-mediated sequencing of the 3-mer random sequences of the plurality of fixed template molecules and the first base position of the universal sample index sequence using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C, and T / U; c) generating a map of the positions of the plurality of immobilized template molecules using the images of the four cycles of polymerase-mediated sequencing reaction obtained in step (b), wherein the sequence of the insert region is not used to generate the map. In some embodiments, the balanced diversity of step (b) is 25-75% of the nucleobases A, G, C, and T / U detected and imaged in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle, and the fourth sequencing cycle.

[0030] In some embodiments, the method further comprises: a) sequencing the remaining base positions of the universal sample index sequence of the plurality of fixed template molecules; b) sequencing the insert sequence region of the plurality of fixed template molecules; and c) assigning the insert sequence of the given template molecule obtained in step (b) to the universal sample index sequence obtained from the same given template molecule in step (a), thereby identifying the sample source of the given insert sequence. In some embodiments, the plurality of nucleic acid template molecules further comprises a second sample index comprising a second universal sample index sequence that identifies the sample source of the insert sequence, and the second sample index lacks a random sequence.

[0031] In some embodiments, the method further includes: a) sequencing the first base positions of the 3-mer random sequences and universal sample index sequences of multiple fixed template molecules to obtain a balanced diversity of A, G, C and T / U nucleobases detected and imaged in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle and the fourth sequencing cycle, thereby generating a map of the positions of the multiple fixed template molecules; b) sequencing the remaining base positions of the first universal sample index sequence of the multiple fixed template molecules; c) sequencing the second universal sample index sequence of the multiple fixed template molecules; d) sequencing the insert sequence region of the multiple fixed template molecules; and e) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insert sequence.

[0032] In certain embodiments, the support comprises a glass or plastic substrate. In certain embodiments, the support is configured on a flow cell channel, a flow cell, or a capillary lumen. In certain embodiments, the support is passivated with at least one hydrophilic polymer coating having a water contact angle of not more than 45 degrees.

[0033] In some embodiments, at least one hydrophilic polymer coating comprises 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. In some embodiments, at least one hydrophilic polymer coating comprises a branched hydrophilic polymer molecule having at least four branches. In some embodiments, at least one hydrophilic polymer coating comprises a polymer molecule having a molecular weight of at least 1000 Daltons.

[0034] In some embodiments, the immobilized template molecules comprise a plurality of immobilized concatemer molecules having a sample index and tandem repeats of an insert sequence. In some embodiments, the immobilized template molecules comprise a plurality of different clustered template molecules having a copy of the insert sequence and a copy of the sample index, wherein the clustered template molecules are generated via bridge amplification.

[0035] In some embodiments, the density of immobilized nucleic acid template molecules at random locations on the support is 10 4 –10 8 Pieces / mm 2 In some embodiments, the sample source of the insert sequence is genomic DNA, double-stranded cDNA, or free circulating cellular DNA.

[0036] In some embodiments, detectably labeled nucleotide reagents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups, and a fluorophore. In some embodiments, detectably labeled nucleotide reagents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety with a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups, and a fluorophore.

[0037] In some embodiments, the detectably labeled nucleotide agent comprises a multivalent molecule, each multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (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, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

[0038] In some embodiments, the detectably labeled nucleotide reagent bound to the immobilized template molecule comprises each immobilized template molecule that hybridizes to a sequencing primer to form a duplex, and the duplex binds to a polymerase to form a composite polymerase, and the composite polymerase binds to the detectably labeled nucleotide reagent.

[0039] In some embodiments, the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into the hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups, and a fluorophore. In some embodiments, the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into the hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety having a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups, and a fluorophore.

[0040] In some embodiments, the composite polymerase binds to the detectably labeled nucleotide agent under conditions suitable for binding the detectably labeled nucleotide agent to the composite polymerase, and the conditions are suitable for inhibiting nucleotide incorporation, wherein the detectably labeled nucleotide agent comprises a multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (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, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

[0041] In some embodiments, the immobilized template molecule comprises an immobilized concatamer molecule that hybridizes to a plurality of sequencing primers to form at least a first duplex and a second duplex on the same concatamer molecule, wherein the first duplex binds to a first polymerase and the second duplex binds to a second polymerase to form a first composite polymerase and a second composite polymerase, and the method comprises: a) contacting a plurality of multivalent molecules with the first composite polymerase and the second composite polymerase on the same concatamer template molecule, wherein each multivalent molecule comprises (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (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, and the spacer is attached to the linker, and the linker is attached to the nucleotide unit, wherein the contacting is performed under conditions suitable for binding a single multivalent molecule of the plurality of multivalent molecules to the first composite polymerase and the second composite polymerase, wherein a first nucleotide unit of the single multivalent molecule binds to the first composite polymerase, the first composite polymerase comprising a nucleotide unit that binds to the concatamer template. The invention further comprises the steps of: a) detecting a first sequencing primer that hybridizes to a first portion of a multivalent template molecule, thereby forming a first binding complex, and wherein a second nucleotide unit of the single multivalent molecule binds to a second complex polymerase, the second complex polymerase comprising a second sequencing primer that hybridizes to a second portion of the multivalent template, thereby forming a second binding complex, and wherein the first binding complex and the second binding complex bound to the same multivalent molecule form an affinity complex, wherein the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the first nucleotide unit and the second nucleotide unit bound in the first binding complex and the second binding complex; b) detecting the first binding complex and the second binding complex on the same multivalent template molecule; c) imaging an optical color signal emitted from a detectably labeled multivalent molecule that forms the first binding complex and the second binding complex on the same multivalent template molecule; and d) recognizing the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the multivalent template molecule, and recognizing the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the multivalent template molecule.

[0042] The present disclosure provides a method for sequencing nucleic acid molecules, comprising: a) providing a first plurality of library molecules, each of the plurality of library molecules comprising (i) an insert sequence region derived from a first sample source, (ii) a first sample index having a 3-mer random sequence joined to a first universal sample index sequence, and (iii) a second sample index having a second universal sample index sequence, the second sample index lacking the random sequence, wherein the combination of the first universal sample index sequence and the second universal sample index sequence uniquely identifies the first sample source of the insert sequence, wherein different first library molecules have different 3-mer random sequences and have different insert sequences; b) providing a second plurality of library molecules, each of the plurality of library molecules comprising (i) an insert sequence region derived from a second sample source, (ii) a third sample index having a 3-mer random sequence joined to a third universal sample index sequence, and (iii) a fourth sample index having a fourth universal sample index sequence, the fourth sample index lacking the random sequence, wherein the combination of the third universal sample index sequence and the fourth universal sample index sequence uniquely identifies the second sample source of the insert sequence, wherein different second library molecules have different 3-mer random sequences and having different insert sequences; c) pooling a first plurality of library molecules and a second plurality of library molecules; d) distributing the pooled library molecules onto a support and performing an amplification reaction to produce a plurality of clonally amplified template molecules immobilized at random positions on the support; (e) performing three cycles of polymerase-mediated sequencing reactions on the 3-mer random sequences of the first sample index and the third sample index using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C, and T / U, wherein the nucleotide reagents comprise a different detectable color label corresponding to each different type of nucleobase, wherein the three cycles Sequencing the circles comprises detecting and imaging an optical color signal emitted from a detectably labeled nucleotide reagent bound to an immobilized amplification template molecule, thereby determining the sequence of the 3-mer random sequence in each of the plurality of immobilized template molecules, and wherein in each of the first sequencing cycle, the second sequencing cycle, and the third sequencing cycle, a balanced diversity of A, G, C, and T / U nucleobases is detected and imaged among the plurality of immobilized amplification template molecules; and f) using the image obtained in step (e) to generate a map of the positions of the plurality of immobilized template molecules, wherein the sequence of the insert region is not used to generate the map. In some embodiments, the balanced diversity of step (e) is 25-75% of each of the nucleobases A, G, C, and T / U detected and imaged in each of the first sequencing cycle, the second sequencing cycle, and the third sequencing cycle.

[0043] In some embodiments, the method further comprises: g) sequencing a first universal sample index sequence of a plurality of fixed template molecules; h) sequencing a second universal sample index sequence of a plurality of fixed template molecules; i) sequencing the insert sequence regions of the plurality of fixed template molecules derived from the first library molecule; and j) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence from the same given template molecule, thereby identifying the first sample source of the given insert sequence.

[0044] In some embodiments, the method further comprises: g) sequencing a third universal sample index sequence of a plurality of fixed template molecules; h) sequencing a fourth universal sample index sequence of a plurality of fixed template molecules; i) sequencing the insert sequence regions of a plurality of fixed template molecules derived from a second library molecule; and j) assigning the insert sequence of a given template molecule obtained in step (c) to the third universal sample index sequence and the fourth universal sample index sequence from the same given template molecule, thereby identifying the second sample source of the given insert sequence.

[0045] The present disclosure provides a method for sequencing a nucleic acid molecule, comprising: a) providing a first plurality of library molecules, each molecule of the plurality of library molecules comprising (i) an insert sequence region derived from a first sample source, (ii) a first sample index having a 3-mer random sequence joined to a first universal sample index sequence, and (iii) a second sample index having a second universal sample index sequence, the second sample index lacking the random sequence, wherein a combination of the first universal sample index sequence and the second universal sample index sequence uniquely identifies the first sample source of the insert sequence, wherein the first universal sample index sequence comprises 3-20 nucleotides, and wherein different first library molecules have different b) providing a second plurality of library molecules, each of the plurality of library molecules comprising (i) an insert sequence region derived from the second sample source, (ii) a third sample index having a 3-mer random sequence joined to a third universal sample index sequence, and (iii) a fourth sample index having a fourth universal sample index sequence, the fourth sample index lacking the random sequence, wherein the combination of the third universal sample index sequence and the fourth universal sample index sequence uniquely identifies the second sample source of the insert sequence, wherein the third universal sample index sequence comprises 3-20 nucleotides, and wherein different second library molecules have different 3-mer random sequences and having different insert sequences; c) pooling the first plurality of library molecules and the second plurality of library molecules; d) distributing the pooled library molecules onto a support and performing an amplification reaction to generate a plurality of clonally amplified template molecules fixed to random positions on the support; e) performing four cycles of polymerase-mediated sequencing reactions on the 3-mer random sequences of the first sample sequence and the third sample index using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C, and T / U, and sequencing the first base position of the first universal sample index sequence and the third universal sample index sequence. a detectable color label, wherein the three cycles of sequencing include detecting and imaging an optical color signal emitted from a detectably labeled nucleotide reagent bound to an immobilized amplification template molecule, thereby determining the sequence of the 3-mer random sequence in each of the plurality of immobilized template molecules, and wherein in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle, and the fourth sequencing cycle, a balanced diversity of A, G, C, and T / U nucleobases is detected and imaged among the plurality of immobilized amplification template molecules; and f) using the image obtained in step (e) to generate a map of the positions of the plurality of immobilized template molecules, wherein the sequence of the insert region is not used to generate the map.In some embodiments, the equilibrium diversity of step (e) is 25-75% of each of the nucleobases A, G, C, and T / U detected and imaged in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle, and the fourth sequencing cycle.

[0046] In some embodiments, the method further comprises: g) sequencing the remaining base positions of the first universal sample index sequence of the plurality of fixed template molecules; h) sequencing the second universal sample index sequence of the plurality of fixed template molecules; i) sequencing the insert sequence regions of the plurality of fixed template molecules derived from the first library molecule; and j) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence from the same given template molecule, thereby identifying the first sample source of the given insert sequence.

[0047] In some embodiments, the method further comprises: g) sequencing the remaining base positions of the third universal sample index sequence of the plurality of fixed template molecules; h) sequencing the fourth universal sample index sequence of the plurality of fixed template molecules; i) sequencing the insert sequence regions of the plurality of fixed template molecules derived from the second library molecule; and j) assigning the insert sequence of the given template molecule obtained in step (c) to the third universal sample index sequence and the fourth universal sample index sequence from the same given template molecule, thereby identifying the second sample source of the given insert sequence.

[0048] In some embodiments, the method further comprises: a) sequencing the remaining base positions of the universal sample index sequence of the plurality of fixed template molecules; b) sequencing the insert sequence region of the plurality of fixed template molecules; and c) assigning the insert sequence of the given template molecule obtained in step (b) to the universal sample index sequence obtained from the same given template molecule in step (a), thereby identifying the sample source of the given insert sequence. In some embodiments, the plurality of nucleic acid template molecules further comprises a second sample index comprising a second universal sample index sequence that identifies the sample source of the insert sequence, and the second sample index lacks a random sequence.

[0049] In some embodiments, the method further includes: a) sequencing the first base positions of the 3-mer random sequences and universal sample index sequences of multiple fixed template molecules to obtain a balanced diversity of A, G, C and T / U nucleobases detected and imaged in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle and the fourth sequencing cycle, thereby generating a map of the positions of the multiple fixed template molecules; b) sequencing the remaining base positions of the first universal sample index sequence of the multiple fixed template molecules; c) sequencing the second universal sample index sequence of the multiple fixed template molecules; d) sequencing the insert sequence region of the multiple fixed template molecules; and e) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insert sequence.

[0050] In certain embodiments, the support comprises a glass or plastic substrate. In certain embodiments, the support is configured on a flow cell channel, a flow cell, or a capillary lumen. In certain embodiments, the support is passivated with at least one hydrophilic polymer coating having a water contact angle of not more than 45 degrees.

[0051] In some embodiments, at least one hydrophilic polymer coating comprises 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. In some embodiments, at least one hydrophilic polymer coating comprises a branched hydrophilic polymer molecule having at least four branches. In some embodiments, at least one hydrophilic polymer coating comprises a polymer molecule having a molecular weight of at least 1000 Daltons.

[0052] In some embodiments, the immobilized template molecules comprise a plurality of immobilized concatemer molecules having a sample index and tandem repeats of an insert sequence. In some embodiments, the immobilized template molecules comprise a plurality of different clustered template molecules having a copy of the insert sequence and a copy of the sample index, wherein the clustered template molecules are generated via bridge amplification.

[0053] In some embodiments, the density of immobilized nucleic acid template molecules at random locations on the support is 10 4 –108 Pieces / mm 2 In some embodiments, the sample source of the insert sequence is genomic DNA, double-stranded cDNA, or free circulating cellular DNA.

[0054] In some embodiments, detectably labeled nucleotide reagents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups, and a fluorophore. In some embodiments, detectably labeled nucleotide reagents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety with a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups, and a fluorophore.

[0055] In some embodiments, the detectably labeled nucleotide agent comprises a multivalent molecule, each multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (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, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

[0056] In some embodiments, the detectably labeled nucleotide reagent bound to the immobilized template molecule comprises each immobilized template molecule that hybridizes to a sequencing primer to form a duplex, and the duplex binds to a polymerase to form a composite polymerase, and the composite polymerase binds to the detectably labeled nucleotide reagent.

[0057] In some embodiments, the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into the hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups, and a fluorophore. In some embodiments, the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into the hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety having a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups, and a fluorophore.

[0058] In some embodiments, the composite polymerase binds to the detectably labeled nucleotide agent under conditions suitable for binding the detectably labeled nucleotide agent to the composite polymerase, and the conditions are suitable for inhibiting nucleotide incorporation, wherein the detectably labeled nucleotide agent comprises a multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (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, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

[0059] In some embodiments, the immobilized template molecule comprises an immobilized concatamer molecule that hybridizes to a plurality of sequencing primers to form at least a first duplex and a second duplex on the same concatamer molecule, wherein the first duplex binds to a first polymerase and the second duplex binds to a second polymerase to form a first composite polymerase and a second composite polymerase, and the method comprises: a) contacting a plurality of multivalent molecules with the first composite polymerase and the second composite polymerase on the same concatamer template molecule, wherein each multivalent molecule comprises (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (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, and the spacer is attached to the linker, and the linker is attached to the nucleotide unit, wherein the contacting is performed under conditions suitable for binding a single multivalent molecule of the plurality of multivalent molecules to the first composite polymerase and the second composite polymerase, wherein a first nucleotide unit of the single multivalent molecule binds to the first composite polymerase, the first composite polymerase comprising a nucleotide unit that binds to the concatamer template. The invention further comprises the steps of: a) detecting a first sequencing primer that hybridizes to a first portion of a multivalent template molecule, thereby forming a first binding complex, and wherein a second nucleotide unit of the single multivalent molecule binds to a second complex polymerase, the second complex polymerase comprising a second sequencing primer that hybridizes to a second portion of the multivalent template, thereby forming a second binding complex, and wherein the first binding complex and the second binding complex bound to the same multivalent molecule form an affinity complex, wherein the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the first nucleotide unit and the second nucleotide unit bound in the first binding complex and the second binding complex; b) detecting the first binding complex and the second binding complex on the same multivalent template molecule; c) imaging an optical color signal emitted from a detectably labeled multivalent molecule that forms the first binding complex and the second binding complex on the same multivalent template molecule; and d) recognizing the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the multivalent template molecule, and recognizing the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the multivalent template molecule.

[0060] The present disclosure provides a method for generating a plurality of library-splint complexes (300), comprising: a) providing a plurality of single-stranded nucleic acid library molecules (100), wherein each of the plurality of single-stranded nucleic acid library molecules comprises regions arranged in 5' to 3' order: (i) a first left universal adapter sequence (120) having a binding sequence for a second surface primer; (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer; (iii) a target sequence (110); (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer; (v) a first right universal adapter sequence (130) having a binding sequence for a first surface primer; b) providing a plurality of single-stranded splint chains (200), wherein each of the plurality of single-stranded splint chains (200) comprises regions arranged in 5' to 3' order: (i) a first region (210) having a universal binding sequence; 0), which hybridizes to the first left universal adapter sequence (120) of the linear single-stranded library molecule (100), and (ii) a second region (220) having a universal binding sequence, which hybridizes to the first right universal adapter sequence (130) of the linear single-stranded library molecule (100); c) a first region (210) of the single-stranded splint strand (200) which is suitable for hybridizing to the first left universal adapter sequence (120) of the linear single-stranded library molecule (100) and suitable for Under conditions where the second region (220) of the chain splint chain (200) hybridizes with the first right universal adapter sequence (130) of the linear single-stranded library molecule (100), multiple single-stranded splint chains (200) are hybridized with multiple single-stranded nucleic acid library molecules (100), thereby causing each library molecule to be looped to form multiple library-splint complexes (300), each of which has a gap between the 5' and 3' ends of the library molecules, wherein the gap is enzymatically ligatable.

[0061] In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left index sequence (160) and / or a first right index sequence (170). In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left unique identification sequence (180) and / or a first right unique identification sequence (190).

[0062] In some embodiments, the method further comprises: d) ligating the gaps in each library-splint complex (300), thereby generating a plurality of covalently closed circular library molecules (400). In some embodiments, the method further comprises: e) contacting the plurality of covalently closed circular library molecules (400) with at least one exonuclease to remove the plurality of single-stranded splint strands (200) and retain the plurality of covalently closed circular library molecules (400).

[0063] In some embodiments, each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules comprises a first right universal adapter sequence (130) having a binding sequence for a first surface primer, and the method further comprises: f) distributing the plurality of covalently closed circular library molecules (400) onto a support having a plurality of first surface primers immobilized on the support under conditions suitable for hybridizing each covalently closed circular library molecule (400) to each immobilized second surface primer, thereby immobilizing the plurality of covalently closed circular library molecules (400) to the support.

[0064] In some embodiments, the method further comprises: g) contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on a support using the plurality of first surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (400) as template molecules, thereby producing a plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0065] In some embodiments, the plurality of fixed first surface capture primers on the support are located at predetermined or random positions on the support. In some embodiments, the plurality of fixed second surface capture primers on the support are in fluid communication with each other to allow a solution of a reagent (e.g., an enzyme, nucleotide, divalent cation, etc.) to flow onto the support so that the plurality of fixed first surface primers can react with the reagent in a massively parallel manner substantially simultaneously. In some embodiments, the density of the plurality of fixed nucleic acid concatemer molecules on the support is 10 4 –10 8 Pieces / mm 2 .

[0066] In some embodiments, the method further comprises: a) sequencing the plurality of immobilized concatemers, wherein sequencing comprises: b) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of composite polymerases, each composite polymerase comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to a soluble sequencing primer; c) contacting the plurality of composite sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the composite sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the 3' position of the sugar; d) incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; and e) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0067] In some embodiments, the plurality of nucleotides comprises a removable chain-terminating portion at the 3' sugar group, wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azido 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 wherein the removable chain-terminating portion can be cleaved with a chemical compound to produce an extendable 3'OH portion on the sugar group. In some embodiments, the plurality of nucleotides comprises a type of nucleotide selected from dATP, dGTP, dCTP, dTTP, and dUTP. 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, dTTP, and / or dUTP.

[0068] In some embodiments, the method further comprises: sequencing the plurality of immobilized concatemer molecules, wherein the sequencing comprises: a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble universal sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first composite polymerases, each first composite polymerase comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to the soluble sequencing primer; b) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble universal sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first composite polymerases, each first composite polymerase comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to the soluble sequencing primer; and b) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble universal sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first composite polymerases, each first composite polymerase comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to the soluble sequencing primer; The method of claim 1, wherein the plurality of multivalent-complex polymerases are contacted with a plurality of detectably labeled multivalent molecules under conditions that inhibit incorporation of complementary nucleotide units into sequencing primers of the plurality of multivalent-complex polymerases, wherein each multivalent molecule of the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; c) detecting the plurality of multivalent-complex polymerases; and d) identifying nucleobases of the complementary nucleotide units bound to the plurality of first complex polymerases of the plurality of multivalent-complex polymerases, thereby determining the sequence of the nucleic acid template.

[0069] In some embodiments, the method further comprises: e) dissociating the plurality of multivalent-complex polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining a 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 complex polymerases, each comprising a second sequencing polymerase bound to the retained nucleic acid duplexes; g) contacting the plurality of second complex polymerases with a plurality of nucleotides, the plurality of nucleotides comprising at least one nucleotide analog having a removable chain-terminating portion at the sugar 3' position, wherein the contacting is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second complex polymerases of step (f), thereby forming a plurality of nucleotide-complex polymerases, and the conditions are suitable for promoting incorporation of the bound complementary nucleotides into sequencing primers of the nucleotide-complex polymerases.

[0070] In some embodiments, the method further comprises: h) detecting a complementary nucleotide incorporated into a sequencing primer of a nucleotide-complex polymerase. In some embodiments, the method further comprises: h) detecting a complementary nucleotide incorporated into a sequencing primer of a nucleotide-complex polymerase; and i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase.

[0071] The present disclosure provides a method for sequencing by forming at least one affinity complex, comprising: a) binding a first universal sequencing primer, a first sequencing polymerase, and a first detectably labeled multivalent molecule to a first portion of a concatemer molecule of claim 16 , thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first sequencing polymerase; b) binding a second universal sequencing primer, a second sequencing polymerase, and a first detectably labeled multivalent molecule to a second portion of the same concatemer molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule is bound to the second sequencing polymerase; and c) binding a second universal sequencing primer, a second sequencing polymerase, and a first detectably labeled multivalent molecule to a second portion of the same concatemer molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule is bound to the second sequencing polymerase. The first binding complex and the second binding complex form an affinity complex, wherein the first detectably labeled multivalent molecule comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit, wherein the concatemer molecule comprises two or more tandem repeats of a sequence of interest (110) and a universal primer binding site that binds a first universal sequencing primer and a second universal sequencing primer, and wherein the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first nucleotide unit and the second nucleotide unit in the first binding complex and the second binding complex; and c) detecting the first binding complex and the second binding complex on the same concatemer molecule.

[0072] In some embodiments, the plurality of nucleotide arms attached to the core of each multivalent molecule have the same type of nucleotide unit, and wherein the type of nucleotide unit is 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 type having a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0073] In some embodiments, the plurality of nucleotides comprises a removable chain-terminating portion at the 3' sugar group, wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azido 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 wherein the removable chain-terminating portion can be cleaved with a chemical compound to produce an extendable 3'OH portion on the sugar group. In some embodiments, the plurality of nucleotides comprises a type of nucleotide selected from dATP, dGTP, dCTP, dTTP, and dUTP. 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, dTTP, and / or dUTP.

[0074] In some embodiments, the support comprises a glass or plastic substrate. In some embodiments, the support is passivated with at least one hydrophilic polymer coating having a water contact angle of no more than 45 degrees. In some embodiments, the at least one hydrophilic polymer coating comprises 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The novel features of the present invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description which sets forth exemplary embodiments in which the principles of the invention are utilized, and in the accompanying drawings which illustrate:

[0076] Figure 1Schematic diagram showing hybridization of an exemplary linear single-stranded library molecule (100) with a single-stranded splint molecule / strand (200), thereby looping the library molecule to form a gapped library-splint complex (300). The library molecule (100) comprises a target sequence (110) flanked on one side by a first left universal adapter sequence (120) and on the other side by a first right universal adapter sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule.

[0077] Figure 2 Schematic diagram showing hybridization of an exemplary linear single-stranded library molecule (100) with a single-stranded splint molecule / chain (200), thereby looping the library molecule to form a gapped library-splint complex (300). The library molecule (100) includes a target sequence (110) flanked on one side by a first left universal adapter sequence (120) and a second left universal adapter sequence (140), and on the other side by a second right universal adapter sequence (150) and a first right universal adapter sequence (130). The single-stranded splint chain (200) includes a first region (210) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule.

[0078] Figure 3 Schematic diagram showing hybridization of an exemplary linear single-stranded library molecule (100) with a single-stranded splint molecule / strand (200), thereby looping the library molecule to form a gapped library-splint complex (300). The exemplary library molecule (100) comprises: a first left universal adapter sequence (120); an optional first left unique identification sequence (180); a first left index sequence (160); a second left universal adapter sequence (140); a target sequence (110); a second right universal adapter sequence (150); a first right index sequence (170); and a first right universal adapter sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule.

[0079] Figure 4Schematic diagram showing hybridization of an exemplary linear single-stranded library molecule (100) with a single-stranded splint molecule / strand (200), thereby looping the library molecule to form a gapped library-splint complex (300). The exemplary library molecule (100) comprises: a first left universal adapter sequence (120); a first left index sequence (160); a second left universal adapter sequence (140); a target sequence (110); a second right universal adapter sequence (150); a first right index sequence (170); an optional first right unique identification sequence (190); and a first right universal adapter sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule.

[0080] Figure 5 Schematic diagram showing hybridization of an exemplary linear single-stranded library molecule (100) to a single-stranded splint molecule / strand (200), thereby circularizing the library molecule to form a gapped library-splint complex (300). The library molecule (100) comprises: a first left universal adapter sequence (120); a first left junction adapter sequence (125); a first left index sequence (160); a second left junction adapter sequence (165); a second left universal adapter sequence (140); a third left junction adapter sequence (145); a target sequence (110); a third right junction adapter sequence (155); a second right universal adapter sequence (150); a second right junction adapter sequence (175); a first right index sequence (170); a first right junction adapter sequence (135); an optional first right unique identification sequence (190); and a first right universal adapter sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. For simplicity, the library-splint complex (300) is not shown with any ligation adaptors. The skilled artisan will recognize that the library-splint complex (300) may comprise any one or any combination of two or more of the ligation adaptors present in the library molecule (100).

[0081] Figure 6Three schematic diagrams of exemplary covalently closed circular library molecules are shown, each hybridized to a single-stranded splint strand (200). The top schematic diagram (A) shows a covalently closed circular library molecule (400) having a target sequence (110), a first right universal adapter sequence (130), and a first left universal adapter sequence (120). The middle schematic diagram (B) shows a covalently closed circular library molecule (400) having a target sequence (110), a second right universal adapter sequence (150), a first right universal adapter sequence (130), a first left universal adapter sequence (120), and a second left universal adapter sequence (140). The bottom schematic (C) shows a covalently closed circular library molecule (400) having a sequence of interest (110), a second right universal adapter sequence (150), a first right index sequence (170), a first right universal adapter sequence (130), a first left universal adapter sequence (120), an optional first left unique identification sequence (180), a first left index sequence (160), and a second left universal adapter sequence (140).

[0082] Figure 7 Schematic diagram showing an exemplary library-splint complex undergoing a ligation reaction to close the gap to form a covalently closed circular library molecule (400), which is hybridized to a single-stranded splint strand (200), where the single-stranded splint strand (200) is used as an amplification primer for a rolling circle amplification reaction. The dotted line represents the nascent extension product.

[0083] Figure 8 The nucleotide sequence of an exemplary single-stranded splint molecule / chain (200) is shown. The exemplary single-stranded splint chain comprises a first region (210; SEQ ID NO: 193) and a second region (220; SEQ ID NO: 194).

[0084] Figures 9A-9F Table 1 (of 6) lists sequences of exemplary first left index sequences (160) and first right index sequences (170) having short random sequences (eg, NNN).

[0085] Figure 10 is a schematic diagram of exemplary Y-shaped adaptors, each of which has a double-stranded annealing region that includes blunt ends, or the ends may have 5' or 3' overhang regions. Figure 10 Also shown are exemplary Y-shaped adaptors with mismatch portions of varying lengths.

[0086] Figure 11 is a schematic diagram of an exemplary Y-shaped adaptor having a first strand comprising a binding sequence for a first sequencing primer and a binding sequence for a first surface primer. Figure 11Also shown is an exemplary Y-shaped adapter having a second strand comprising a binding sequence for a second sequencing primer and a binding sequence for a second surface primer. The first strand optionally includes a first sample index sequence. The second strand optionally includes a second sample index sequence.

[0087] Figure 12 Schematic diagram of a schematic diagram of an exemplary Y-shaped adaptor having a first strand comprising a binding sequence for a first sequencing primer and a binding sequence for a first surface primer. Figure 12 Also shown is an exemplary Y-shaped adapter having a second strand comprising a binding sequence for a second sequencing primer and a binding sequence for a second surface primer. The first strand optionally includes a first sample index sequence having a short random sequence (e.g., NNN). The second strand optionally includes a second sample index sequence having a short random sequence (e.g., NNN).

[0088] Figure 13 Schematic diagram illustrating an exemplary library preparation workflow in which nucleic acid fragments are linked to a Y-shaped adapter at both ends. The Y-shaped adapter comprises a first strand comprising a binding sequence for a first sequencing primer, a first sample index sequence, and a binding sequence for a first surface primer. The Y-shaped adapter comprises a second strand comprising a binding sequence for a second sequencing primer, a second sample index sequence, and a binding sequence for a second surface primer.

[0089] Figure 14 Schematic diagram illustrating an exemplary library preparation workflow in which nucleic acid fragments are linked to a Y-shaped adapter at both ends. The Y-shaped adapter includes a first strand comprising a binding sequence for a first sequencing primer, a first sample index sequence having a short random sequence (e.g., NNN), and a binding sequence for a first surface primer. The Y-shaped adapter includes a second strand comprising a binding sequence for a second sequencing primer, a second sample index sequence, and a binding sequence for a second surface primer.

[0090] Figure 15 Schematic diagram illustrating an exemplary library preparation workflow, in which nucleic acid fragments are connected to Y-shaped adapters at both ends to produce adapter-insert-adapter molecules. The Y-shaped adapter includes a first strand comprising a binding sequence for a first sequencing primer. The Y-shaped adapter includes a second strand comprising a binding sequence for a second sequencing primer. The adapter-insert-adapter molecule is subjected to a first primer extension reaction using a first tailed PCR primer carrying the binding sequence for the first surface primer to produce a first extension product. The first extension product is subjected to a second primer extension reaction using a second tailed PCR primer carrying the binding sequence for the second surface primer to produce a second extension product.

[0091] Figure 16Schematic diagram illustrating an exemplary library preparation workflow, wherein nucleic acid fragments are connected to Y-shaped adapters at both ends to produce adapter-insert-adapter molecules. The Y-shaped adapter includes a first chain comprising a binding sequence for a first sequencing primer. The Y-shaped adapter includes a second chain comprising a binding sequence for a second sequencing primer. A first tailed PCR primer pair, comprising the binding sequence for a first surface primer, a first sample index sequence, and the binding sequence for a first surface primer, is subjected to a first primer extension reaction to produce a first extension product. A second tailed PCR primer pair, comprising the binding sequence for a second surface primer, a second sample index sequence, and the binding sequence for a second surface primer, is subjected to a second primer extension reaction to produce a second extension product.

[0092] Figure 17 Schematic diagram showing an exemplary library preparation workflow, wherein the nucleic acid fragments are connected to a Y-shaped adapter at both ends to produce an adapter-insert-adapter molecule. The Y-shaped adapter includes a first chain comprising a binding sequence for a first sequencing primer. The Y-shaped adapter includes a second chain comprising a binding sequence for a second sequencing primer. Using a first tailed PCR primer carrying the binding sequence of a first surface primer, a first sample index sequence with a short random sequence (e.g., NNN), and a binding sequence for a first surface primer, the adapter-insert-adapter molecule is subjected to a first primer extension reaction to produce a first extension product. Using a second tailed PCR primer carrying the binding sequence of a second surface primer, a second sample index sequence, and a binding sequence for a second surface primer, the first extension product is subjected to a second primer extension reaction to produce a second extension product.

[0093] Figure 18 is a schematic diagram of an exemplary low binding support comprising a glass substrate and alternating hydrophilic coatings covalently or non-covalently adhered to the glass, and which further comprises chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides). In alternative embodiments, the support can be made of any material, such as glass, plastic, or polymeric material.

[0094] Figure 19 are schematic diagrams of various exemplary configurations of multivalent molecules. Left (Class I): Schematic diagrams of multivalent molecules having a "starburst" or "helter-skelter" configuration. Center (Class II): Schematic diagrams of multivalent molecules having a dendrimer configuration. Right (Class III): Schematic diagrams of various multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. The nucleotide unit is designated as 'N', biotin is designated as 'B', and streptavidin is designated as 'SA'.

[0095] Figure 20 is a schematic diagram of an exemplary multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0096] Figure 21 is a schematic diagram of an exemplary multivalent molecule comprising a dendritic core attached to multiple nucleotide arms.

[0097] Figure 22 Shown is a schematic diagram of an exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, wherein the nucleotide arms comprise biotin, a spacer, a linker, and nucleotide units.

[0098] Figure 23 is a schematic diagram of an exemplary nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit.

[0099] Figure 24 Shown are the chemical structures of exemplary spacers (top) and various exemplary linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker (bottom).

[0100] Figure 25 The chemical structures of various exemplary linkers are shown, including Linkers 1-9.

[0101] Figure 26 The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0102] Figure 27 The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0103] Figure 28 The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0104] Figure 29 The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0105] Figure 30 The chemical structure of an exemplary biotinylated nucleotide arm is shown. In this example, the nucleotide unit is linked to the linker via a propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base.

[0106] Figure 31 1 is a graph showing the nucleotide base diversity of the right sample index sequence (170) including the universal right sample index and the 3-mer random sequence (NNN). The graph shows that the nucleotide diversity of the 3-mer random sequence (NNN) for A and T base recognition is approximately 30%, and the nucleotide diversity for C and G base recognition is approximately 20%.

[0107] Figure 32 is a graph showing the nucleotide base diversity of the left sample index sequence (160) lacking the 3-mer random sequence (NNN). The graph shows that the nucleotide diversity of A and T base recognition is approximately 40%, the nucleotide diversity of C base recognition is approximately 15%, and the nucleotide diversity of G base recognition is approximately 5%. DETAILED DESCRIPTION

[0108] Throughout this application, various publications, patents and / or patent applications are cited. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference into this application in their entirety in order to more fully describe the state of the art to which this disclosure pertains.

[0109] definition:

[0110] 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.

[0111] Unless otherwise defined, the technology and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art, unless otherwise defined. Generally speaking, the terms related to the technology of molecular biology as described herein, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production and hybridization are well-known in the art and commonly used those. Technology and procedures as described herein are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, referring to Sambrook et al., Molecular Cloning:A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature utilized in conjunction with laboratory procedures as described herein and technology is well-known in the art and commonly used those.

[0112] Unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as any word used in the singular, include plural referents unless expressly and unequivocally limited to one referent.

[0113] It will be understood that use of alternative terms (eg, "or") is taken to mean one or both or any combination of the alternatives.

[0114] As used herein, the term "and / or" should be taken to mean a specific disclosure of each of the specified features or components with or without the other. For example, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include: "A and B"; "A or B"; "A" (only A); and "B" (only B). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" herein is intended to cover 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" (only A); "B" (only B); and "C" (only C).

[0115] As used herein and in the appended claims, the terms "comprises," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be non-limiting, such that the listing of one or more items in a list is not exclusive of other items that can be substituted or added to the listed items. It should be understood that whenever aspects are described herein with the language "comprising," similar aspects described with "consisting of" and / or "consisting essentially of" are also otherwise provided.

[0116] As used herein, the terms "about" and "approximately" refer to values ​​or compositions within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" or "approximately" may mean within one or more standard deviations. Alternatively, "about" or "approximately" may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. In addition, particularly for biological systems or processes, these terms may mean a value of up to an order of magnitude or up to 5 times. When a specific value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that specific value or composition. In addition, where a range and / or sub-range of values ​​is provided, the range and / or sub-range may include the endpoints of the range and / or sub-range.

[0117] As used herein, the terms "peptide," "polypeptide," and "protein," and other related terms, are used interchangeably and refer to polymers of amino acids and are not limited to any particular length. Polypeptides may include natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not undergone post-translational modifications, such as proteolytic cleavage, cleavage due to ribosome skipping, hydroxylation, methylation, lipidation, acetylation, sumoylation, ubiquitination, glycosylation, phosphorylation, and / or disulfide bond formation. These terms encompass natural and artificial proteins, protein fragments, and polypeptide analogs (such as mutants, variants, chimeric proteins, and fusion proteins) of protein sequences, as well as proteins that have been post-translationally or otherwise covalently or non-covalently modified.

[0118] The term "cell biological sample" refers to a section of a single cell, a plurality of cells, a tissue, an organ, an organism or any of these cell biological samples. A cell biological sample can be extracted from an organism (e.g., a biopsy) or obtained from a cell culture grown in a liquid or a culture dish. A cell biological sample comprises a fresh, frozen, freshly frozen or archived sample (e.g., formalin-fixed paraffin-embedded; FFPE). A cell biological sample can be embedded in wax, a resin, an epoxy resin or agar. A cell biological sample can be fixed in, for example, any one of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton or glutaraldehyde or any combination of two or more thereof. A cell biological sample can be sectioned or unsectioned. A cell biological sample can be stained, decolorized or unstained.

[0119] The nucleic acid of interest can be extracted from a cell or cell biological sample using any of a variety of techniques known to those skilled in the art. For example, a typical DNA extraction procedure involves: (i) collecting a cell sample or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane (i.e., cell lysis) to release the 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 the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during cell membrane lysis. A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolating genomic DNA from human samples) and the DNAeasy kit (for isolating genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the DNAeasy kit (for isolating genomic DNA from animal or plant samples) from Promega (Madison, WI). and ReliaPrep TM Series of kits.

[0120] As used herein, the term "polymerase" and its variants include enzymes comprising a domain that binds nucleotides (or nucleosides), wherein 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 analogs thereof) into nucleic acid chains. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, the polymerase is included in one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase includes other enzymatic activities, such as, for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand displacement activity. Polymerases may include, but are not limited to, naturally occurring polymerases and any of their subunits 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 comprising a nucleotide binding domain. In certain embodiments, polymerases may be isolated from cells or generated using recombinant DNA technology or chemical synthesis methods. In certain embodiments, polymerases may be expressed in prokaryotes, eukaryotes, viruses or phage organisms. In certain embodiments, polymerases may be post-translationally modified proteins or fragments thereof. Polymerases may be derived from prokaryotes, eukaryotes, viruses or phages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0121] The term "strand displacement" refers to the ability of a polymerase to partially separate a double-stranded nucleic acid chain and synthesize a new chain in a template-based manner. The strand displacement polymerase displaces the complementary strand from the template strand and catalyzes the synthesis of a new chain. The strand displacement polymerase includes mesophilic and thermophilic polymerases. The strand displacement polymerase includes wild-type enzymes and variants (including exonuclease-minus mutants, mutant versions, chimeric enzymes, and truncated enzymes). Examples of strand displacement polymerases include: phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment (exo-) of Bsu DNA polymerase, Bca DNA polymerase (exo-), the Klenow fragment of Escherichia coli (E.coli) DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV virus reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (eg, MagniPhi from Expedeon), or a variant EquiPhi29 DNA polymerase (eg, from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (eg, from 4basebio).

[0122] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," and other related terms are used interchangeably and refer to polymers of nucleotides and are not limited to any specific 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 generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, wherein the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside bonds, such as phosphodiester bonds. Nucleic acids include non-natural internucleoside bonds, including phosphorothioate, thiol phosphorothioate, or peptide nucleic acid (PNA) bonds. In some embodiments, nucleic acids comprise a type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0123] As used herein, the terms "operably linked" and "operably joined" or related terms refer to the juxtaposition of components. The juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the bond linking the two components together comprises a phosphodiester bond. The first and second nucleic acid components can be linked together, wherein the first nucleic acid component can confer a function to the second nucleic acid component. For example, the link between the primer binding sequence and the target sequence forms a nucleic acid library molecule having a portion that can bind to the primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a target nucleic acid sequence) can be linked to a vector, wherein the link allows the transgene sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgene expression. 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 expression of the level, timing, and / or position of the transgene.

[0124] The terms "linked," "joined," "attached," "attached," and variations thereof encompass any type of fusion, bonding, adhesion, or association between any combination of compounds or molecules that is sufficiently stable to withstand use in a particular procedure. The procedure may include, but is not limited to, nucleotide binding; nucleotide incorporation; deblocking (e.g., removal of a chain terminating moiety); washing; removal; flow; detection; imaging and / or identification. For example, such bonds may include: covalent bonds, ionic bonds, hydrogen bonds, dipole-dipole bonds, hydrophilic bonds, hydrophobic bonds, or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, and the like. In some embodiments, such bonds occur within a molecule, such as linking the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonds may occur between a combination of different molecules, or between a molecule and a non-molecule, including, but not limited to, bonds between a nucleic acid molecule and a solid surface; bonds between a protein and a detectable reporter moiety; bonds between a nucleotide and a detectable reporter moiety; and the like. Some examples of bonds can be found in, for example, Hermanson, G., “Bioconjugate Techniques”, 2nd ed. (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).

[0125] The term "primer" and related terms used herein refer to oligonucleotides that can hybridize with DNA and / or RNA polynucleotide templates to form duplex molecules. Primers can be single-stranded or have single-stranded and double-stranded portions along their entire length. Primers include natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers can have any length, but generally range from 4 to 50 nucleotides. Typical primers include 5' ends and 3' ends. The 3' end of a primer can include a 3'OH portion that serves as a nucleotide polymerization initiation site in a primer extension reaction catalyzed by a polymerase. Alternatively, the 3' end of a primer can lack a 3'OH portion or can include an end 3' blocking group that inhibits the nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide along the length of the primer or more than one nucleotide can be labeled with a detectable reporter gene portion. Primers can be in solution (e.g., soluble primers) or can be fixed to a support (e.g., capture primers).

[0126] The terms "template nucleic acid," "template polynucleotide," "target nucleic acid," "target polynucleotide," "template strand," and other variations refer to a nucleic acid strand used as a base nucleic acid molecule in any amplification and / or sequencing method described herein. A template nucleic acid can be single-stranded or double-stranded, or a template nucleic acid can have single-stranded or double-stranded portions. Template nucleic acids can be obtained from naturally occurring sources, recombinant forms, or chemically synthesized to include any type of nucleic acid analog. Template nucleic acids can be linear, concatemeric, circular, or in other forms.

[0127] The term "adapter" and related terms refer to oligonucleotides that can be operably linked (attached) to a target polynucleotide, wherein the adaptor pair conferring function to the co-joined adaptor-target molecule. Adaptors include DNA, RNA, chimeric DNA / RNA or their analogs. Adaptors may include at least one ribonucleoside residue. Adaptors may be single-stranded, double-stranded or have single-stranded and / or double-stranded portions. Adaptors may be configured as linear, stem-loop, hairpin or Y-shaped forms. Adaptors may be of any length, including 4 to 100 nucleotides or longer. Adaptors may have blunt ends, overhanging ends or a combination of the two. Overhanging ends include 5' overhangs and 3' overhangs. The 5' end of a single-stranded adaptor or one chain of a double-stranded adaptor may have a 5' phosphate group or lack a 5' phosphate group. Adaptors may include a 5' tail (e.g., a tailed adaptor) that is not hybridized to a target polynucleotide, or adaptors may be tailless. At least a portion of an adaptor comprises a known and predetermined sequence. The adapter may comprise 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 comprise a random sequence or a degenerate sequence. The adapter may comprise at least one inosine residue. The adapter may comprise at least one phosphorothioate, phosphorothiol, and / or phosphoramidite bond. The adapter may comprise at least one barcode sequence that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. The adapter may comprise at least one unique identification sequence (e.g., a molecular identifier) ​​that can be used to uniquely identify the nucleic acid molecule to which the adapter is attached. In some embodiments, the unique identification sequence comprises 2-12 or more nucleotides with a known sequence. For example, the unique identification sequence comprises a known random sequence, wherein the nucleotide at each position is randomly selected from a nucleotide having a base A, G, C, T, or U. The adapter may comprise at least one restriction enzyme recognition sequence, including 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.

[0128] The term "universal sequence" and related terms refer to a sequence common to two or more polynucleotide molecules in a nucleic acid molecule. For example, an adaptor with a universal sequence can be operably joined to multiple polynucleotides such that the co-joined population of molecules carries the same universal adaptor sequence. Examples of universal adaptor sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0129] When used to refer to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization" or other related terms refer to the hydrogen bonding between two different nucleic acids to form a duplex 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 duplex region. Hybridization can include Watson-Crick or Hoogstein bonding to form a duplex double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid or a single nucleic acid can be completely complementary or partially complementary. The complementary nucleic acid chains do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing or can be other forms of base pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides.

[0130] When used in reference to nucleic acids, the terms "extend," "extending," "extension," and other variants 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 end of a nucleic acid chain, resulting in extension of the nucleic acid chain. Nucleotide incorporation can be performed using natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, including primer extension catalyzed by DNA polymerase or RNA polymerase.

[0131] The term "nucleotide" and related terms refer to molecules comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and at least one phosphate group. Regular or non-regular nucleotides are consistent with the use of the term. In certain embodiments, nucleotides comprise monophosphate, diphosphate or triphosphate, or corresponding phosphate analogs. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be unlabeled or labeled with a detectable reporter moiety.

[0132] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings, which are commonly found in nucleic acids, including naturally occurring, substituted, modified or engineered variants or analogs thereof. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with appropriate complementary bases. Exemplary bases include, but are not limited to, purines and pyrimidines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethyleneadenine, N 6 -Δ 2 -Isopentenyl adenine (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-deazapurines 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; muscimol; inosines; hydroxymethylcytosines; 5-methylcytosines; bases (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", pages 385-394, CRC Press, Boca Raton, Fla.

[0133] Nucleotides (and nucleosides) generally contain a sugar moiety, such as 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 other sugar moieties (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'-mercaptocarboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptocarboxyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0134] In certain embodiments, the nucleotide comprises a chain with one, two or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In certain embodiments, the nucleotide is an analog with a phosphorus chain, wherein the phosphorus atom is linked together with an intervening O, S, NH, methylene or ethylene. In certain embodiments, the phosphorus atom in the chain comprises a substituted side group, including O, S or BH3. In certain embodiments, the chain comprises a phosphate group substituted with an analog, including phosphoramide, phosphorothioate, phosphorodithioate and O-methylphosphoramidite groups.

[0135] The term "rolling circle amplification" generally refers to an amplification method that utilizes a circularized nucleic acid template molecule containing a target sequence of interest, an amplification primer binding sequence, and optionally one or more adapter sequences, such as a sequencing primer binding sequence and / or a sample index sequence. The rolling circle amplification reaction can be performed under isothermal amplification conditions and includes a circularized nucleic acid template molecule, an amplification primer, a strand-displacing polymerase, and a plurality of nucleotides to generate concatemers containing tandem repeats of the circular template molecule and any adapter sequences present in the original circularized nucleic acid template molecule. The concatemers can self-collapse to form nucleic acid nanospheres. The shape and size of the nanospheres can be further compressed by including a pair of inverted repeats in the circular template molecule or by performing the rolling circle amplification reaction with one or more compacting oligonucleotides. One advantage of using rolling circle amplification to generate clonal amplicons for sequencing workflows is that duplicate copies of the target sequence in the nanosphere can be sequenced simultaneously to increase signal strength. In some embodiments, the rolling circle amplification reaction can be performed in the presence of multiple compactions having at least four consecutive guanines. The rolling circle amplification reaction generates concatemers containing duplicate copies of the universal binding sequence for the compacting oligonucleotides. At least one compacted oligonucleotide can form a guanine quadruplex and hybridize to a universal binding sequence for the compacted oligonucleotide, and the resulting concatemer can fold to form an intramolecular G-quadruplex structure. The concatemer can self-collapse to form a compact nanosphere. The formation of guanine quadruplexes and G-quadruplexes in the nanosphere can increase the stability of the nanosphere to maintain its compact size and shape, so that it can withstand the repeated flow of reagents used to perform any sequencing workflow described herein.

[0136] The terms "amplify," "amplifying," "amplification," and other related terms when used in reference to nucleic acids include generating multiple copies of an original polynucleotide template molecule, wherein the copies comprise a sequence complementary to the template sequence, and / or the copies comprise a sequence identical to the template sequence. In some embodiments, the copies comprise a sequence substantially identical to the template sequence, and / or a sequence substantially identical to a sequence complementary to the template sequence.

[0137] The term "reporter moiety," "reporter moieties," or related terms refers to a compound that generates or causes a detectable signal. A reporter moiety is sometimes referred to as a "label." Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. The reporter moiety generates a detectable signal caused by a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity, or a neighboring event). A neighboring event includes two reporter moieties that are close to each other, or associate with each other, or are bound to each other. It is well known to those skilled in the art that the reporter moiety is selected so that each absorbs excitation radiation and / or fluoresces at a wavelength that can be distinguished from other reporter moieties, to allow monitoring of the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties with spectrally distinct emission curves or with minimal overlapping spectral emission curves can be selected. The reporter moiety can be linked (eg, operably linked) to a nucleotide, nucleoside, nucleic acid, enzyme (eg, a polymerase or reverse transcriptase), or support (eg, a surface).

[0138] Reporter moieties (or labels) include fluorescent labels or fluorophores. Exemplary fluorescent moieties that can be used as fluorescent labels or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthol fluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamido fluorescein, maleimide fluorescein, SAMSA-fluorescein, thiosemicarbazide fluorescein, hydrazine carbonate methylthioacetamido fluorescein, 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 hydrazide, 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 / 550C3, BODIPY 530 / 550C3-SE, BODIPY 530 / 550C3 hydrazide, BODIPY 493 / 503C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FLIA, BODIPY 530 / 551IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl triazoide, 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 cyanine dyes, benzindolium cyanine dyes, pyridinium cyanine dyes, thiazolium cyanine dyes, quinolinium cyanine dyes, imidazolium cyanine dyes, Cy 3. Cy5, lanthanide chelates and derivatives, such as 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, diphenylethylene, DEG dyes, NR dyes, near-infrared dyes, and other dyes known in the art, such as Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th edition;Those described in Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Edition, Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof;Cyanine dyes can exist in sulfonated or non-sulfonated form and consist of two indolenine, benzindolium, pyridinium, thiazolium and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which may comprise 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 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 oxopyrrolidin-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 may include 1-(6-((2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-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). )penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium or 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-sulfonindol-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) and Cy7 (which may contain 1-(5-carboxypentyl) )-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium or 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 digit identifies the number of carbon atoms between the two indolenine groups. Cy2 is an oxazole derivative rather than an indolenine, and the benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0139] In some embodiments, the reporter moiety can be a FRET pair, so that multiple classifications can be performed in a single excitation and imaging step. As used herein, FRET can include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0140] As used herein, the term "support" refers to a substrate designed for the deposition of biological molecules or biological samples for measurement and / or analysis. Examples of biological molecules to be deposited on a support include nucleic acids (e.g., DNA, RNA), polypeptides, carbohydrates, lipids, single cells or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

[0141] In some embodiments, the support is solid, semi-solid, or a combination thereof. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosities. 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, comprising a capillary or the inner surface of a capillary.

[0142] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can have a regular or irregular texture, including bumps, etchings, holes, three-dimensional scaffolds, or any combination thereof.

[0143] In some embodiments, the support comprises microbeads having any shape, including spheres, hemispheres, cylinders, barrels, rings, disks, rods, cones, triangles, cubes, polygons, tubes, or threads.

[0144] The support may be made of any material, including but not limited to glass, fused silica, silicon, polymers (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.

[0145] The present disclosure provides multiple (for example, two or more) nucleic acid template molecules that are fixed to an upholder.In certain embodiments, the multiple nucleic acid template molecules fixed have the same sequence or have different sequences.In certain embodiments, each nucleic acid template molecule in a plurality of nucleic acid template molecules is fixed to different sites on the upholder.In certain embodiments, two or more each nucleic acid template molecules in a plurality of nucleic acid templates are fixed to the site on the upholder.

[0146] The term "array" refers to a support comprising a plurality of sites located at predetermined positions on the support to form an array of sites. The sites can be discrete and separated by gaps. In some embodiments, the predetermined sites on the support can 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 straight lines, hexagonal patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, and the like. The spacing between different pairs of sites may be the same or different. In some embodiments, the support comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 sites or more sites, wherein the sites are located at predetermined positions on the support. In some embodiments, the plurality of predetermined sites (e.g., 10 2 –10 15 In some embodiments, the nucleic acid template molecules are immobilized at a plurality of predetermined sites by hybridizing with the immobilized surface capture primers, or the nucleic acid template molecules are covalently attached to the surface capture primers. In some embodiments, the nucleic acid template molecules are immobilized at a plurality of predetermined sites, for example, at 10 2 –10 15 In some embodiments, the fixed nucleic acid template molecules are clonally amplified to generate fixed nucleic acid clusters at multiple predetermined sites. In some embodiments, each fixed nucleic acid cluster comprises a linear cluster, or comprises a single-stranded or double-stranded concatemer.

[0147] In some embodiments, a support comprising a plurality of sites located at random positions on the support is referred to herein as a support having randomly positioned sites thereon. The positions of the randomly positioned sites on the support are not predetermined. The plurality of randomly positioned sites are arranged in a disordered and / or unpredictable manner on the support. In some embodiments, the support comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 sites or more sites, wherein the sites are randomly positioned on the support. In some embodiments, the plurality of randomly positioned sites (e.g., 10 2 –10 15 In some embodiments, the nucleic acid template molecules are immobilized at a plurality of randomly positioned sites by hybridization with an immobilized surface capture primer, or the nucleic acid template molecules are covalently attached to the surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly positioned sites, for example, at 10 2 –10 15 In certain embodiments, the nucleic acid templates are cloned and amplified to generate fixed nucleic acid clusters at multiple randomly positioned sites. In certain embodiments, each fixed nucleic acid cluster comprises a linear cluster, or comprises a single-stranded or double-stranded concatemer.

[0148] In some embodiments, a plurality of fixed surface capture primers on a support (e.g., located at predetermined or random positions on the support) are in fluid communication with each other to allow a solution of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, etc.) to flow onto the support so that the plurality of fixed surface capture primers on the support can react with the reagents in a massively parallel manner substantially simultaneously. In some embodiments, the fluid communication of the plurality of fixed surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) on the plurality of fixed surface capture primers substantially simultaneously.

[0149] In some embodiments, the multiple fixed nucleic acid clusters on the support are in fluid communication with each other to allow a solution of a reagent (e.g., an enzyme, a nucleotide, a divalent cation, etc.) to flow onto the support so that the multiple fixed nucleic acid clusters on the support can react with the reagent in a large-scale parallel manner substantially simultaneously. In some embodiments, the fluid communication of the multiple fixed nucleic acid clusters can be used to perform nucleotide binding assays and / or perform nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​the multiple fixed nucleic acid clusters substantially simultaneously, and optionally perform detection and imaging of large-scale parallel sequencing.

[0150] In certain embodiments, term " fixed " and related terms refer to nucleic acid molecules attached to support or the coating on support or embedded in the matrix formed by coating on support by covalent bond or non-covalent interaction, wherein nucleic acid molecules include the extension product of surface capture primer, nucleic acid template molecule and capture primer.The extension product of capture primer includes nucleic acid concatemer (for example, nucleic acid cluster).Nucleic acid molecules can be fixed on the predetermined or random position on support.Nucleic acid molecules can be fixed on support on passivated coating or within predetermined or random position.

[0151] In some embodiments, the term "immobilized" and related terms refer to an enzyme (e.g., a polymerase) that is attached to a support, or to a coating on a support, or embedded in a substrate formed by a coating on a support, by covalent bonds or non-covalent interactions. The enzyme can be immobilized at a predetermined or random position on the support. The enzyme can be immobilized at a predetermined or random position on or within a passivated coating on a support.

[0152] In some embodiments, one or more nucleic acid template molecules are fixed on a support, for example, fixed at a site on a support. In some embodiments, one or more nucleic acid template molecules are clonally amplified. In some embodiments, one or more nucleic acid template molecules are clonally amplified from a support (for example, in a solution), then deposited onto a support and fixed on a support. In some embodiments, the clonal amplification reaction of one or more nucleic acid template molecules is carried out on a support, resulting in immobilization on a support. In some embodiments, one or more nucleic acid template molecules are clonally amplified (for example, in a solution or on a support) using a nucleic acid amplification reaction, including any one or any combination of the following: 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), loop-to-loop amplification, helicase-dependent amplification, recombinase-dependent amplification and / or single-stranded binding (SSB) protein-dependent amplification.

[0153] The term "surface primer" and related terms refer to single-stranded oligonucleotides that are fixed to a support and comprise a sequence that can hybridize with at least a portion of a nucleic acid template molecule. Surface capture primers can be used for fixing template molecules to a support via hybridization. Surface capture primers can be fixed to a support in a manner that resists primer removal during flow, washing, suction, and changes in temperature, pH, salt, chemicals, and / or enzyme conditions. Typically, but not necessarily, the 5' end of the surface capture primer can be fixed to a support or a coating on a support (or embedded in a coating on a support). Alternatively, the inside or 3' end of the surface capture primer can be fixed to a support.

[0154] The sequence of the surface capture primer can be fully or partially complementary to at least a portion of the nucleic acid template molecule along its length. The support can include a plurality of fixed surface capture primers with the same sequence or with two or more different sequences. The surface capture primer can be any length, for example 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, or longer lengths.

[0155] The surface capture primer can have a terminal 3' nucleotide, and this terminal 3' nucleotide has a sugar 3' OH part, which can be extended for nucleotide polymerization (for example, polymerization catalyzed by polymerase). The surface capture primer can have a terminal 3' nucleotide, and this terminal 3' nucleotide has a 3' sugar position connected to the chain termination part that inhibits nucleotide polymerization. Deblocking agents can be used to remove (for example, to block) 3' chain termination part, so that 3' end is converted into an extendable 3' OH end. The example of chain termination part includes: alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azido group, amino group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group or silyl group. Azide type chain termination part includes azido, azido and azidomethyl group. Examples of deblocking agents include phosphine compounds such as tris(2-carboxyethyl)phosphine (TCEP) and bissulfotriphenylphosphine (BS-TPP) for chain-terminating groups azido, azido and azidomethyl groups. Examples of deblocking agents include tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) for chain-terminating groups alkyl, alkenyl, alkynyl and allyl. Examples of deblocking agents include Pd / C for chain-terminating groups aryl and benzyl. Examples of deblocking agents include phosphine, β-mercaptoethanol or dithiothreitol (DTT) for chain-terminating groups amine, amide, ketone, isocyanate, phosphate, thio and disulfide. Examples of deblocking agents include potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine and Zn in acetic acid (AcOH) for carbonate chain-terminating groups. Examples of deblocking agents include tetrabutylammonium fluoride, pyridine-HF, and ammonium fluoride and triethylamine trihydrofluoride for the chain-terminating groups urea and silyl.

[0156] The term "sequencing" and related terms refer to methods for obtaining nucleotide sequence information from nucleic acid molecules, typically by determining the identity of at least some nucleotides (including their nucleobase components) within the nucleic acid molecule. In some embodiments, the sequence information for a given region of a nucleic acid molecule includes identifying each nucleotide in the region being sequenced. In some embodiments, the sequencing information only identifies some nucleotides in the region, while the identities of some nucleotides remain undetermined or incorrectly determined. Any suitable method for sequencing can be used. In exemplary embodiments, sequencing can include label-free or ion-based sequencing methods. In some embodiments, sequencing can include labeled or dye-containing nucleotides or fluorescence-based nucleotide sequencing methods. In some embodiments, sequencing can include polymerase-based cloning sequencing or bridge sequencing methods. In some embodiments, sequencing uses a polymerase and a multivalent molecule to generate at least one affinity complex, wherein each multivalent molecule comprises a plurality of nucleotide units tethered to a core. In some embodiments, sequencing uses a polymerase and free nucleotides to perform sequencing-by-synthesis. In some embodiments, sequencing uses a ligase and multiple sequence-specific oligonucleotides to perform sequencing-by-ligation. DETAILED DESCRIPTION

[0158] Introduction: Cyclization using a single-strand splint chain

[0159] The present disclosure provides compositions comprising single-stranded nucleic acid splint chains, including kits, and methods employing the single-stranded splint chains.

[0160] The single-stranded splint chain (200) can be used in a one-pot multienzyme reaction to circularize linear library molecules (e.g., see Figure 1-Figure 5 ). The single-stranded splint chain (200) includes a first region (210) that hybridizes to a sequence on one end of a linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. The two regions of the single-stranded splint chain (e.g., (210) and (220)) are designed to increase the efficiency of circularization of linear nucleic acid molecules. The single-stranded splint chains and methods using them have the advantage of providing efficient circularization with as little as 0.25 pmol of library molecules.

[0161] The methods described herein can be performed manually or for automation because annealing and multi-enzyme reactions can be performed in a single reaction vessel (one pot) by combining some enzymatic reactions (e.g., phosphorylation and ligation) and by adding subsequent enzymes (e.g., exonucleases), without the need for intermediate alcohol precipitation or organic extraction.

[0162] Single chain splint chain

[0163] The present disclosure provides a nucleic acid single-stranded splint chain (200) comprising a first region (210) that hybridizes to a sequence on one end of a linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. The two regions of the single-stranded splint chain (e.g., (210) and (220)) are designed to hybridize to a universal adapter sequence at the end of a single-stranded linear library molecule (100) having a target sequence (110). For example, the first region (210) of the single-stranded splint chain hybridizes to one end of the library molecule, and the second region (220) of the single-stranded splint chain hybridizes to the other end of the library molecule, thereby looping the library molecule to produce a library-splint complex (300) that includes a gap (e.g., see Figure 1-Figure 5 The gaps can be enzymatically ligated to produce a covalently closed circular molecule (400) in which the ends of the library molecules are covalently joined.

[0164] The terminal sequences of the linear nucleic acid library molecules each comprise at least a first universal adapter sequence and a second universal adapter sequence. In some embodiments, the first universal adapter sequence and the second universal adapter sequence of the linear library molecule each comprise binding sequences for a first capture primer and a second capture primer immobilized on a support.

[0165] In some embodiments, the first region (210) of the single-stranded splint strand comprises a first universal adapter sequence comprising a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0166] In some embodiments, the second region (220) of the single-stranded splint strand comprises a second universal adapter sequence comprising a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0167] In some embodiments, the single-stranded splint chain (200) comprises one or more phosphorothioate bonds at its 5' and / or 3' end to confer resistance to nucleic acid exonucleases. In some embodiments, the single-stranded splint chain (200) comprises one or more phosphorothioate bonds at an internal position to confer resistance to nucleic acid endonucleases. In some embodiments, the single-stranded splint chain (200) comprises one or more 2'-O-methylcytosine bases at its 5' and / or 3' end or at an internal position. In some embodiments, the 5' end of the single-stranded splint chain (200) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the single-stranded splint chain (200) comprises a terminal 3'OH group or a terminal 3' blocking group.

[0168] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adapter sequence (120) of a library molecule, wherein the first region (210) comprises the sequence

[0169] 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193) (e.g., Figure 7 A- Figure 7 C).

[0170] In some embodiments, the first left universal adaptor sequence (120) in the library molecule comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199).

[0171] In some embodiments, the second region of the single-stranded splint strand (220) comprises the universal binding sequence of the first right universal adapter sequence (130) of the library molecule, wherein the second region (220) comprises the sequence

[0172] 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194) (e.g., Figure 7 A- Figure 7 C).

[0173] In some embodiments, the first right universal adaptor sequence (130) in the library molecule comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200).

[0174] In some embodiments, the single-stranded splint chain (200) comprises the sequence

[0175] 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195) (e.g., Figure 7 A- Figure 7 C).

[0176] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adapter sequence (120) of a library molecule, wherein the first region (210) comprises the sequence

[0177] 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0178] In some embodiments, the second region of the single-stranded splint strand (220) comprises the universal binding sequence of the first right universal adapter sequence (130) of the library molecule, wherein the second region (220) comprises the sequence

[0179] 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0180] In some embodiments, the single-stranded splint chain (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0181] Library-splint complex

[0182] The present disclosure provides a library-splint complex (300) comprising: (i) a single-stranded nucleic acid library molecule (100) comprising a sequence of interest (110) flanked on one side by at least a first left universal adapter sequence (120) and on the other side by at least a first right universal adapter sequence (130); and (ii) a single-stranded splint chain (200) comprising a first region (210) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. In the library-splint complex (300), the first region (210) of the single-stranded splint chain hybridizes to at least the first left universal adapter sequence (120) of the library molecule, and the second region (220) of the single-stranded splint chain hybridizes to at least the first right universal adapter sequence (130) of the library molecule, thereby circularizing the library molecule to produce the library-splint complex (300) (see, e.g., Figure 1-Figure 5 ).

[0183] In the library-splint complex (300), the first region (210) of the single-stranded splint strand comprises a first universal adaptor sequence that can hybridize to a first universal binding sequence at one end of a linear nucleic acid library molecule (e.g., see Figure 1-Figure 5). In some embodiments, the first region (210) of the single-stranded splint chain includes a first universal adapter sequence, which includes a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, and a universal binding sequence for a compaction oligonucleotide. In some embodiments, the length of the single-stranded splint chain (200) may be 20-150 nucleotides, or a length of 60-100 nucleotides, a length of 70-90 nucleotides, or a length of 60-80 nucleotides. In some embodiments, the single-stranded splint chain (200) includes one or more phosphorothioate bonds at the 5' and / or 3' end to confer nuclease resistance. In some embodiments, the single-stranded splint chain (200) includes one or more phosphorothioate bonds at an internal position to confer nuclease resistance. In some embodiments, the single-stranded splint chain (200) includes one or more 2'-O-methylcytosine bases at the 5' and / or 3' end or at an internal position. In some embodiments, the 5' end of the single-stranded splint chain (200) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the single-stranded splint chain (200) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0184] The second region (220) of the single-stranded splint strand comprises a second universal adaptor sequence that can hybridize to a second universal binding sequence at the other end of the linear nucleic acid library molecule (e.g., see Figure 1-Figure 5 In some embodiments, the second region (220) of the single-stranded splint strand comprises a second universal adapter sequence comprising a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0185] In the library-splint complex (300), the first region (210) of the single-stranded splint strand hybridizes to at least the first left universal adapter sequence (120) of the library molecule, and the second region (220) of the single-stranded splint strand hybridizes to at least the first right universal adapter sequence (130) of the library molecule, thereby circularizing the library molecule to produce the library-splint complex (300). The library-splint complex (300) comprises a gap between the 5' end of the library molecule and the 3' end of the library molecule (e.g., see Figure 1-Figure 5 ). In some embodiments, the gap is enzymatically ligatable.

[0186] In the library-splint complex (300), the first region of the single-stranded splint strand (210) can hybridize with the sense strand or antisense strand of a double-stranded nucleic acid library molecule. In the library-splint complex (300), the second region of the single-stranded splint strand (220) can hybridize with the sense strand or antisense strand of a double-stranded nucleic acid library molecule. The double-stranded nucleic acid library molecule can be denatured to produce single-stranded sense and antisense library strands. The single-stranded library molecule (100) can be the sense strand or the antisense strand.

[0187] In the library-splint complex (300), the first region (210) of the single-stranded splint strand does not hybridize to the target sequence (110), and the second region (220) of the single-stranded splint strand does not hybridize to the target sequence (110).

[0188] In some embodiments, the 5' end of the single-stranded library molecule (100) in the library-splint complex (300) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the single-stranded library molecule comprises a terminal 3' OH group or a terminal 3' blocking group.

[0189] In some embodiments, the nucleic acid library molecule (100) further comprises a second left universal adapter sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises a second right universal adapter sequence (150). An exemplary library-splint complex (300) is shown in Figure 1-Figure 5 In some embodiments, the nucleic acid library molecule (100) may further comprise an additional left universal adapter sequence and / or a right universal adapter sequence.

[0190] In some embodiments, the nucleic acid library molecule (100) further comprises a first left index sequence (160). In some embodiments, the nucleic acid library molecule (100) further comprises a first right index sequence (170). The length of the first right index sequence (170) can be 3-20 nucleotides. In some embodiments, the first left index sequence (160) includes a sample index sequence. The length of the first left index sequence (160) can be 3-20 nucleotides. In some embodiments, the first right index sequence (170) includes another sample index sequence. The sequences of the left sample index sequence and the right sample index sequence (e.g., (160) and (170)) can be the same or different from each other. The sample index sequence can be used to distinguish target sequences obtained from different sample sources in multiplexed assays. An exemplary library-splint complex (300) is shown in Figure 1-Figure 5 middle.

[0191] A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Figures 9A-9FThe first right index sequence (170) may include a short random sequence (eg, NNN) or lack a short random sequence. The length of the short random sequence may be 3-20 nucleotides.

[0192] In some embodiments, the nucleic acid library molecule (100) further comprises at least one junction adapter sequence positioned between any of the universal adapter sequences described herein (e.g., see Figure 5). For example, the first left junction adapter sequence (125) may be located between the first left universal adapter sequence (120) and the first left index sequence (160). The second left junction adapter sequence (165) may be located between the first left index sequence (160) and the second left universal adapter sequence (140). The third junction adapter sequence (145) may be located between the second left universal adapter sequence (140) and the target sequence (110). The first right junction adapter sequence (135) may be located between the first right universal sequence (130) and the first right index sequence (170). The second right junction adapter sequence (175) may be located between the first right index sequence (170) and the second right universal adapter sequence (150). The third right junction adapter sequence (155) may be located between the second right universal adapter sequence (150) and the target sequence (110). Any junction adapter sequence comprises any sequence and may be 3-60 nucleotides in length. Any junction adapter sequence comprises a universal sequence or a unique sequence. Any ligation adapter sequence includes a random sequence (e.g., NNN) of 3-20 nucleotides. Any ligation adapter sequence includes a binding sequence for an amplification primer, a sequencing primer, or a compacting oligonucleotide. Any ligation adapter sequence includes a binding sequence for a fixed surface primer (e.g., a capture primer). Any ligation adapter sequence includes a sample index sequence. Any ligation adapter sequence includes a unique identification sequence. Any ligation adapter sequence, particularly a ligation adapter sequence (145) includes a Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 209). Any ligation adapter sequence, particularly a ligation adapter sequence (155) includes a Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 210). The Tn5 transposon end sequence can be introduced into the library molecule (100) via a transposase-mediated reaction under conditions suitable for forming a transposon synaptic complex, the reaction comprising contacting double-stranded input DNA (e.g., genomic DNA) with a Tn-5 type transposase and a double-stranded oligonucleotide comprising a Tn transposon end sequence (SEQ ID NO: 209) linked to a universal adapter sequence or a sample index sequence. In the double-stranded oligonucleotide, the Tn transposon end sequence (SEQ ID NO: 209) can be located 5' or 3' relative to the universal adapter sequence or the sample index sequence.

[0193] A multiplex workflow is initiated by preparing a library with a sample index using one or two index sequences (e.g., a left index sequence and / or a right index sequence). A separate library with a sample index can be prepared using a first left index sequence (160) and / or a first right index sequence (170) using input nucleic acids isolated from different sources. Libraries with sample indexes can be pooled together to produce a multiple library mixture, and the pooled libraries can be looped, amplified, and / or sequenced. The sequence of the insertion region, together with the first left index sequence (160) and / or the first right index sequence (170), can be used to identify the source of the input nucleic acid. In some embodiments, any number of libraries with sample indexes can be pooled together, for example, 2-10, or 10-50, or 50-100, or 100-200, or more than 200 libraries with sample indexes can be pooled. Exemplary nucleic acid sources include naturally occurring, recombinant, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or entire organisms. Exemplary nucleic acid sources include fresh, frozen, fresh frozen or archived sources (e.g., formalin fixed paraffin embedded; FFPE). The skilled artisan will recognize that nucleic acids can be isolated from many other sources. Nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0194] In some embodiments, the nucleic acid library molecule (100) further comprises: an optional first left unique identification sequence (180). In some embodiments, the nucleic acid library molecule (100) further comprises an optional first right unique identification sequence (190). In some embodiments, the first left unique identification sequence (180) and the first right unique identification sequence (190) each comprise a sequence that is used to uniquely identify each target sequence (e.g., an insertion sequence) to which a unique adapter is attached among a population of other target molecular sequences. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular labeling. In some embodiments, the unique identification sequence (180) and / or (190) comprises 2-12 or more nucleotides with a known sequence. For example, the unique identification sequence comprises a known random sequence, wherein the nucleotide at each position is randomly selected from nucleotides having the bases A, G, C, T, or U. The unique identification sequence (180) and / or (190) can be used for a molecular labeling procedure. An exemplary library-splint complex (300) is shown in Figure 1-Figure 5 middle.

[0195] In some embodiments, the nucleic acid library molecule (100) comprises any one or any combination of two or more of the following: a first left universal adapter sequence (120); a second left universal adapter sequence (140); a first left index sequence (160); a first left unique identification sequence (180); a first right universal adapter sequence (130); a second right universal adapter sequence (150); a first right index sequence (170); and / or a first right unique identification sequence (190). Figure 1-Figure 5 Some exemplary library-splint complexes (300) are shown.

[0196] In some embodiments, the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) include: a universal binding sequence for a forward sequencing primer or a reverse sequencing primer; a universal binding sequence for a first surface primer or a second surface primer; a universal binding sequence for a forward amplification primer or a reverse amplification primer; and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) may further include additional left universal adapter sequences.

[0197] In some embodiments, the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) include: a universal binding sequence for a forward sequencing primer or a reverse sequencing primer; a universal binding sequence for a first surface primer or a second surface primer; a universal binding sequence for a forward amplification primer or a reverse amplification primer; and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) may further include additional right universal adapter sequences.

[0198] In some embodiments, an exemplary library-splint complex (300) comprises: (a) a single-stranded nucleic acid library molecule (100); and (b) a single-stranded splint strand (200).

[0199] In an exemplary library-splint complex (300), a single-stranded nucleic acid library molecule (100) comprises components arranged in 5' to 3' order: (i) a first left universal adapter sequence (120) having a binding sequence for a second surface primer; (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer; (iii) a target sequence (110); (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer; and (v) a first right universal adapter sequence (130) having a binding sequence for a first surface primer.

[0200] In the exemplary library-splint complex (300), the single-stranded splint chain (200) includes components arranged in 5' to 3' order: a first region (210); and a second region (220).

[0201] In an exemplary library-splint complex (300), a portion of a single-stranded splint strand (200) hybridizes with a portion of a library molecule (100), thereby looping the library molecule to produce a library-splint complex (300) such that a first region (210) of the single-stranded splint strand hybridizes with a binding sequence (120) for a first surface primer, and a second region (220) of the single-stranded splint strand hybridizes with a binding sequence (130) for a second surface primer. The library-splint complex (300) comprises a gap between the 5' end of the library molecule and the 3' end of the library molecule, wherein the gap is enzymatically ligatable. An exemplary library-splint complex (300) is shown in Figure 1-Figure 5 middle.

[0202] In the exemplary library-splint complex (300), the first region (210) of the single-stranded splint strand does not hybridize to the sequence of interest (110), and the second region (220) of the single-stranded splint strand does not hybridize to the sequence of interest (110).

[0203] In some embodiments, any library-splint complex (300) described herein comprises a plurality of library-splint complexes (300), wherein the sequence of interest (110) of each library-splint complex in the plurality of library-splint complexes comprises the same sequence of interest or a different sequence of interest.

[0204] In some embodiments, the first left universal adaptor sequence (120) in the library molecule comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199).

[0205] In some embodiments, the first left universal adaptor sequence (120) in the library molecule comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203).

[0206] In some embodiments, the second left universal adaptor sequence (140) in the library molecules comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 201).

[0207] In some embodiments, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 205).

[0208] In some embodiments, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 206).

[0209] In some embodiments, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 202).

[0210] In some embodiments, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 207).

[0211] In some embodiments, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 208).

[0212] In some embodiments, the first right universal adaptor sequence (130) in the library molecule comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200).

[0213] In some embodiments, the first right universal adaptor sequence (130) in the library molecule comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 204).

[0214] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adaptor sequence (120) of the library molecule, wherein the first region (210) comprises the following sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0215] In some embodiments, the second region of the single-stranded splint strand (220) comprises a universal binding sequence for the first right universal adaptor sequence (130) of the library molecule, wherein the second region (220) comprises the following sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0216] In some embodiments, the single-stranded splint chain (200) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195). See, for example, Figure 7 A- Figure 7 C.

[0217] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adapter sequence (120) of the library molecule, wherein the first region (210) comprises the following sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0218] In some embodiments, in the method, the second region of the single-stranded splint strand (220) comprises a universal binding sequence for the first right universal adaptor sequence (130) of the library molecule, wherein the second region (220) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0219] In some embodiments, in the method, the single-stranded splint chain (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0220] The present disclosure provides a reaction mixture comprising a plurality of any library-splint complex (300) described herein. In some embodiments, the reaction mixture comprises a plurality of any library-splint complex (300) described herein and T4 polynucleotide kinase. In some embodiments, the reaction mixture comprises a plurality of any library-splint complex (300) described herein and a ligase. In some embodiments, the reaction mixture comprises a plurality of any library-splint complex (300) described herein and a T4 polynucleotide kinase and a ligase. In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0221] Covalently closed ring molecules

[0222] The present disclosure provides a covalently closed circular library molecule (400) comprising: a sequence of interest (110), at least a first left universal adapter sequence (120), and at least a first right universal adapter sequence (130). An exemplary covalently closed circular library molecule is shown in Figure 6 A- Figure 6In C. In some embodiments, the covalently closed circular library molecule (400) further comprises a second left universal adapter sequence (140). In some embodiments, the covalently closed circular molecule (400) further comprises a second right universal adapter sequence (150). In some embodiments, the covalently closed circular molecule (400) further comprises additional left and / or right universal adapter sequences.

[0223] In some embodiments, the covalently closed circular molecule (400) further comprises a first left index sequence (160) and / or a first right index sequence (170). The length of the first left index sequence (160) can be 3-20 nucleotides. The length of the first right index sequence (170) can be 3-20 nucleotides. The sample index sequence can be used to distinguish target sequences obtained from different sample sources in multiplexed assays. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Figures 9A-9F The first right index sequence (170) and / or the first left index sequence (160) may include a short random sequence (e.g., NNN) or lack a short random sequence. The length of the short random sequence may be 3-20 nucleotides.

[0224] A multiplex workflow is initiated by preparing a library with a sample index using one or two index sequences (e.g., a left index sequence and / or a right index sequence). A separate library with a sample index can be prepared using a first left index sequence (160) and / or a first right index sequence (170) using input nucleic acids isolated from different sources. Libraries with sample indexes can be pooled together to produce a multiple library mixture, and the pooled libraries can be looped, amplified, and / or sequenced. The sequence of the insertion region, together with the first left index sequence (160) and / or the first right index sequence (170), can be used to identify the source of the input nucleic acid. In some embodiments, any number of libraries with sample indexes can be pooled together, for example, 2-10, or 10-50, or 50-100, or 100-200, or more than 200 libraries with sample indexes can be pooled. Exemplary nucleic acid sources include naturally occurring, recombinant, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or entire organisms. Exemplary nucleic acid sources include fresh, frozen, fresh frozen or archived sources (e.g., formalin fixed paraffin embedded; FFPE). The skilled artisan will recognize that nucleic acids can be isolated from many other sources. Nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0225] In some embodiments, the covalently closed circular molecule (400) further comprises an optional first left unique identification sequence (180) and / or an optional first right unique identification sequence (190). In some embodiments, the first left unique identification sequence (180) and the first right unique identification sequence (190) each comprise a sequence that uniquely identifies each target sequence (e.g., insertion sequence) to which a unique adapter is attached, among a population of other target molecular sequences (e.g., molecular tags).

[0226] In some embodiments, the covalently closed circular molecule (400) includes any one or any combination of two or more of: a first left universal adapter sequence (120); a second left universal adapter sequence (140); a first left index sequence (160); a first left unique identification sequence (180); a first right universal adapter sequence (130); a second right universal adapter sequence (150); a first right index sequence (170); and / or a first right unique identification sequence (190). In some embodiments, the first left index sequence (160) includes a sample index sequence. In some embodiments, the first right index sequence (170) includes another sample index sequence. The sequences of the right sample index sequence and the left sample index sequence (e.g., (170) and (160)) can be the same or different from each other. The sample index sequence can be used to distinguish target sequences obtained from different sample sources in multiplexed assays. In some embodiments, the first left unique identification sequence (180) and the first right unique identification sequence (190) each comprise a sequence that is used to uniquely identify each target sequence (e.g., an insertion sequence) to which a unique adapter is attached among a population of other target molecular sequences. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular labeling. In some embodiments, the unique identification sequence comprises 2-12 or more nucleotides with a known sequence. For example, the unique identification sequence (180) and / or (190) comprises a known random sequence, wherein the nucleotide at each position in the unique identification sequence is randomly selected from a group having a nucleobase A, G, C, T, or U. The unique identification sequence (180) and / or (190) can be used for a molecular labeling procedure. Exemplary covalently closed circular library molecules are shown in Figure 6 A- Figure 6 C.

[0227] In some embodiments, in the covalently closed circular molecule (400), the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) comprise: a universal binding sequence for a forward sequencing primer or a reverse sequencing primer; a universal binding sequence for a first surface primer or a second surface primer; a universal binding sequence for a forward amplification primer or a reverse amplification primer; and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the covalently closed circular molecule (400) may further comprise an additional left universal adapter sequence.

[0228] In some embodiments, in the covalently closed circular molecule (400), the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) include: a universal binding sequence for a forward sequencing primer or a reverse sequencing primer; a universal binding sequence for a first surface primer or a second surface primer; a universal binding sequence for a forward amplification primer or a reverse amplification primer; and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the covalently closed circular molecule (400) may further include additional right universal adapter sequences.

[0229] In some embodiments, the covalently closed circular molecule (400) further comprises at least one ligation adapter sequence positioned between any of the universal adapter sequences described herein (e.g., see Figure 5). For example, the first left junction adapter sequence (125) may be located between the first left universal adapter sequence (120) and the first left index sequence (160). The second left junction adapter sequence (165) may be located between the first left index sequence (160) and the second left universal adapter sequence (140). The third junction adapter sequence (145) may be located between the second left universal adapter sequence (140) and the target sequence (110). The first right junction adapter sequence (135) may be located between the first right universal sequence (130) and the first right index sequence (170). The second right junction adapter sequence (175) may be located between the first right index sequence (170) and the second right universal adapter sequence (150). The third right junction adapter sequence (155) may be located between the second right universal adapter sequence (150) and the target sequence (110). Any junction adapter sequence comprises any sequence and may be 3-60 nucleotides in length. Any junction adapter sequence comprises a universal sequence or a unique sequence. Any ligation adapter sequence includes a random sequence (e.g., NNN) of 3-20 nucleotides. Any ligation adapter sequence includes a binding sequence for an amplification primer, a sequencing primer, or a compacting oligonucleotide. Any ligation adapter sequence includes a binding sequence for a fixed surface primer (e.g., a capture primer). Any ligation adapter sequence includes a sample index sequence. Any ligation adapter sequence includes a unique identification sequence. Any ligation adapter sequence, particularly a ligation adapter sequence (145) includes a Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 209). Any ligation adapter sequence, particularly a ligation adapter sequence (155) includes a Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 210). The Tn5 transposon end sequence can be introduced into the library molecule (100) via a transposase-mediated reaction under conditions suitable for forming a transposon synaptic complex, the reaction comprising contacting double-stranded input DNA (e.g., genomic DNA) with a Tn-5 type transposase and a double-stranded oligonucleotide comprising a Tn transposon end sequence (SEQ ID NO: 209) linked to a universal adapter sequence or a sample index sequence. In the double-stranded oligonucleotide, the Tn transposon end sequence (SEQ ID NO: 209) can be located 5' or 3' relative to the universal adapter sequence or the sample index sequence.

[0230] In some embodiments, an exemplary covalently closed circular molecule (400) comprises: (i) a first left universal adapter sequence (120) having a binding sequence for a second surface primer (120); (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer; (iii) a target sequence (110); (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer; (v) a first right universal adapter sequence (130) having a binding sequence for a first surface primer (130); and (vi) optionally, the covalently closed circular molecule (400) hybridizes to a single-stranded splint strand (200) (e.g., see Figure 6 A- Figure 6 C).

[0231] In the exemplary covalently closed circular molecule (400), the first and second regions (210 and 220, respectively) of the single-stranded splint strand do not hybridize to the sequence of interest (110).

[0232] In some embodiments, any covalently closed circular molecule (400) described herein comprises a plurality of covalently closed circular molecules (400), wherein the target sequence (110) of each covalently closed circular molecule (400) in the plurality of covalently closed circular molecules comprises the same target sequence or a different target sequence.

[0233] In some embodiments, the first left universal adaptor sequence (120) in the covalently closed circular molecule comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199).

[0234] In some embodiments, the first left universal adaptor sequence (120) in the covalently closed circular molecule comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203).

[0235] In some embodiments, the second left universal adaptor sequence (140) in the covalently closed circular molecule comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 201).

[0236] In some embodiments, the second left universal adaptor sequence (140) in the covalently closed circular molecule comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 205).

[0237] In some embodiments, the second left universal adaptor sequence (140) in the covalently closed circular molecule comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 206).

[0238] In some embodiments, the second right universal adaptor sequence (150) in the covalently closed circular molecule comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 202).

[0239] In some embodiments, the second right universal adaptor sequence (150) in the covalently closed circular molecule comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 207).

[0240] In some embodiments, the second right universal adaptor sequence (150) in the covalently closed circular molecule comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 208).

[0241] In some embodiments, the first right universal adaptor sequence (130) in the covalently closed circular molecule comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200).

[0242] In some embodiments, the first right universal adaptor sequence (130) in the covalently closed circular molecule comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 204).

[0243] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adaptor sequence (120) of the library molecule, wherein the first region (210) comprises the following sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0244] In some embodiments, the second region of the single-stranded splint strand (220) comprises a universal binding sequence for the first right universal adaptor sequence (130) of the library molecule, wherein the second region (220) comprises the following sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0245] In some embodiments, the single-stranded splint chain (200) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195). See, for example, Figure 7 A- Figure 7 C.

[0246] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adapter sequence (120) of the library molecule, wherein the first region (210) comprises the following sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0247] In some embodiments, in the method, the second region of the single-stranded splint strand (220) comprises a universal binding sequence for the first right universal adaptor sequence (130) of the library molecule, wherein the second region (220) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0248] In some embodiments, in the method, the single-stranded splint chain (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0249] The present disclosure provides a reaction mixture comprising a plurality of any covalently closed circular molecules (400) described herein and at least one exonuclease. In some embodiments, the exonuclease comprises any one of Exonuclease I, Thermostable Exonuclease I, and / or T7 Exonuclease, or any combination of two or more thereof.

[0250] Kit including single-stranded splint chain

[0251] The present disclosure provides at least one kit for looping linear nucleic acid library molecules to form multiple library-splint complexes (300), each library-splint complex having a gap. In some embodiments, the kit can be used to loop single-stranded nucleic acid library molecules having a target sequence (110), which is flanked by at least a first left universal adapter sequence (120) on one side and at least a first right universal adapter sequence (130) on the other side. In some embodiments, the looped library molecules can be converted into covalently closed circular molecules, which can undergo rolling circle amplification (RCA) reactions to generate nucleic acid concatemers. The concatemers can be fixed to a support. The fixed concatemers can undergo massively parallel sequencing reactions.

[0252] The present disclosure provides a kit comprising a single-stranded nucleic acid splint chain (200), each of which comprises a first region (210) and a second region (220). The first region (210) comprises a first universal adapter sequence that can hybridize with a first universal binding sequence at one end of a linear nucleic acid library molecule. The second region (220) comprises a second universal adapter sequence that can hybridize with a second universal binding sequence at the other end of a linear nucleic acid library molecule. In some embodiments, the first region (210) of the single-stranded splint chain comprises a first universal adapter sequence that comprises a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compacted oligonucleotide. In some embodiments, the second region (220) of the single-stranded splint chain includes a second universal adapter sequence, which includes a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the length of the single-stranded splint chain (200) may be 20-150 nucleotides, or a length of 60-100 nucleotides, a length of 70-90 nucleotides, or a length of 60-80 nucleotides. In some embodiments, the single-stranded splint chain (200) includes one or more thiophosphate bonds at the 5' and / or 3' end to confer nuclease resistance. In some embodiments, the single-stranded splint chain (200) includes one or more thiophosphate bonds at an internal position to confer nuclease resistance. In some embodiments, the single-stranded splint chain (200) includes one or more 2'-O-methylcytosine bases at the 5' and / or 3' end or at an internal position. In some embodiments, the 5' end of the single-stranded splint chain (200) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the single-stranded splint chain (200) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0253] In some embodiments, the kit includes a single-stranded splint chain (200) having a first region (210) comprising the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0254] In some embodiments, the kit includes a single-stranded splint chain (200) having a second region (220) comprising the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0255] In some embodiments, the kit comprises a single-stranded splint chain (200) comprising the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195).

[0256] In some embodiments, the kit further comprises an adapter having a left universal adapter sequence (120) for preparing a plurality of library molecules, wherein the library molecules will include the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199) that can bind to a surface primer. In some embodiments, the adapter having a left universal adapter sequence (120) further comprises a sample index sequence (160). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter. For example, see Figure 12 A. Figure 12 B and Figure 12 C.

[0257] In some embodiments, the kit further comprises an adapter having a right universal adapter sequence (130) for preparing a plurality of library molecules, wherein the library molecules will include the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200) that can bind to a surface primer. In some embodiments, the adapter having the right universal adapter sequence (130) further comprises a sample index sequence (170). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter. For example, see Figure 12 A. Figure 12 B and Figure 12 C.

[0258] In some embodiments, the kit further comprises an adapter having a left universal adapter sequence (140) for preparing a plurality of library molecules, wherein the library molecules will comprise the following sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 201), which can bind to a sequencing primer. In some embodiments, the adapter having a left universal adapter sequence (140) further comprises a sample index sequence (160). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter. For example, see Figure 12 A. Figure 12 B and Figure 12 C.

[0259] In some embodiments, the kit further comprises an adapter having a right universal adapter sequence (150) for preparing a plurality of library molecules, wherein the library molecules will comprise the following sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 202), which can bind to a sequencing primer. In some embodiments, the adapter having the right universal adapter sequence (150) further comprises a sample index sequence (170). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter. For example, see Figure 12 A. Figure 12 B and Figure 12 C.

[0260] In some embodiments, the kit includes a single-stranded splint chain (200) having a first region (210) comprising the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0261] In some embodiments, the kit includes a single-stranded splint chain (200) having a second region (220) comprising the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0262] In some embodiments, the kit includes a single-stranded splint chain (200) comprising the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0263] In some embodiments, the kit further comprises an adapter having a left universal adapter sequence (120) for preparing a plurality of library molecules, wherein the library molecules will include the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203) that can bind to a surface primer. In some embodiments, the adapter having the left universal adapter sequence (120) further comprises a sample index sequence (160). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter.

[0264] In some embodiments, the kit further comprises an adapter having a right universal adapter sequence (130) for preparing a plurality of library molecules, wherein the library molecules will include the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 204) that can bind to a surface primer. In some embodiments, the adapter having the right universal adapter sequence (130) further comprises a sample index sequence (170). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter.

[0265] In some embodiments, the kit further comprises an adapter having a left universal adapter sequence (140) for preparing a plurality of library molecules, wherein the library molecules will comprise the following sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 205), which can bind to a sequencing primer. In some embodiments, the adapter having the left universal adapter sequence (140) further comprises a sample index sequence (160). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter.

[0266] In some embodiments, the kit further comprises an adapter having a right universal adapter sequence (150) for preparing a plurality of library molecules, wherein the library molecules will comprise the following sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 207), which can bind to a sequencing primer. In some embodiments, the adapter having the right universal adapter sequence (150) further comprises a sample index sequence (170). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter.

[0267] In some embodiments, the kit further comprises an adapter having a left universal adapter sequence (140) for preparing a plurality of library molecules, wherein the library molecules will comprise the following sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 206), which can bind to a sequencing primer. In some embodiments, the adapter having the left universal adapter sequence (140) further comprises a sample index sequence (160). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter.

[0268] In some embodiments, the kit further comprises an adapter having a right universal adapter sequence (150) for preparing a plurality of library molecules, wherein the library molecules will comprise the following sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 208), which can bind to a sequencing primer. In some embodiments, the adapter having the right universal adapter sequence (150) further comprises a sample index sequence (170). The adapter can be a single-stranded adapter (e.g., a PCR primer), a double-stranded adapter, a bubble adapter, or a Y-shaped adapter.

[0269] In some embodiments, the test kit further comprises a plurality of sample indexes, comprising a plurality of first left indexes (160) and / or a plurality of first right index sequences (170). The length of the first left index sequence (160) may be 3-20 nucleotides. The length of the first right index sequence (170) may be 3-20 nucleotides. In some embodiments, the test kit may comprise a separate container or a well of a multi-well plate (e.g., a 96-well plate) that holds a single first left index (160) or a single first right index (170). In some embodiments, the test kit may comprise a separate container or a well of a multi-well plate (e.g., a 96-well plate) that holds a pair of single first left indexes (160) and a single first right index (170). In some embodiments, the test kit comprises a first left index (160) and / or a plurality of first right index sequences (170) located in a multi-well plate (e.g., a 96-well plate). A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Figures 9A-9F The first left index sequence (160) may include a short random sequence (e.g., NNN) or lack a short random sequence. The first right index sequence (170) may include a short random sequence (e.g., NNN) or lack a short random sequence. The length of the short random sequence may be 3-20 nucleotides.

[0270] In some embodiments, the kit includes a plurality of tailed PCR primers, the plurality of tailed PCR primers including a first left index sequence (160). The tailed PCR primers may include: (i) a first left universal adapter sequence (120) having a binding sequence for a second surface primer; (ii) a first left index sequence (160); (iii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer or a second sequencing primer. The first left index sequence (160) may also include a random sequence (e.g., NNN). See, e.g., Figure 17 .

[0271] In some embodiments, the kit includes a plurality of tailed PCR primers, the plurality of tailed PCR primers including a first right index sequence (170). The tailed PCR primers may include: (i) a first right universal adapter sequence (130) having a binding sequence for a first surface primer; (ii) a first right index sequence (170); and (iii) a second right universal adapter sequence (150) having a binding sequence for a first sequencing primer or a second sequencing primer. The first right index sequence (170) may also include a random sequence (e.g., NNN). See, e.g., Figure 17 .

[0272] In some embodiments, the kit comprises a nucleic acid single-stranded splint strand (200) and further comprises T4 polynucleotide kinase. In some embodiments, the kit further comprises a ligase, wherein the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. In some embodiments, the kit further comprises at least one endonuclease, which comprises any one of exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease, or any combination of two or more thereof.

[0273] In some embodiments, the kit includes at least one buffer for hybridizing a plurality of single-stranded splint chains (200) and a plurality of nucleic acid library molecules (100). In some embodiments, the kit includes a buffer for performing multiple enzymatic reactions in a single reaction vessel, including (i) phosphorylating the 5' end of the single-stranded splint chain (200), (ii) ligating a gap in the library-splint complex (300), and / or (iii) exonuclease digestion of the single-stranded splint chain (200) from the covalently closed circular molecule (400). Alternatively, the kit includes two or more separate buffers, wherein a first buffer can be used to perform the phosphorylation reaction, a second buffer can be used to perform the ligation reaction, and a third buffer can be used to perform the exonuclease digestion reaction.

[0274] In some embodiments, the kit includes one or more containers containing any of the single-stranded splint chains described herein (200). The kit may further include one or more containers containing T4 polynucleotide kinase, at least one ligase, and / or at least one exonuclease. The kit may include any of these components in any combination and may be included in a single container, or may be included in separate containers, or any combination thereof.

[0275] The kit may include instructions for using the kit to perform a reaction to circularize linear nucleic acid library molecules to form a plurality of library-splint complexes (300), each library-splint complex having a gap. The kit may include instructions for using the kit to generate covalently closed circular library molecules (400) by ligating the gaps in the library-splint complexes (300).

[0276] Kit includes adapters and tailed PCR primers for library preparation

[0277] The present disclosure provides a library preparation kit for preparing a plurality of linear library molecules by ligating both ends of a fragmented target nucleic acid (e.g., an insertion region (110)) to Y-shaped adapters to generate a plurality of adapter-insert-adapter constructs, and appending additional adapter sequences to the adapter-insert-adapter constructs (e.g., Figure 17 Any single-chain splint molecule described herein can be used (e.g., Figure 3 and Figure 4 ) The resulting linear library molecules are circularized to form covalently closed circular molecules.

[0278] In some embodiments, the library preparation kit includes a Y-shaped adapter. The Y-shaped adapter comprises a first nucleic acid chain and a second nucleic acid chain, wherein a portion of the two chains are fully complementary to each other and anneal together, and the other portion of the two chains are not complementary to each other and mismatched. In some embodiments, the connecting end of the Y-shaped adapter comprises an annealing portion that forms a blunt end or an overhanging end (e.g., a 5' or 3' overhanging end). In some embodiments, in the library preparation kit, the first chain of the Y-shaped adapter comprises an oligonucleotide having the sequence 5'-TGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 211). In some embodiments, in the library preparation kit, the second chain of the Y-shaped adapter comprises an oligonucleotide having the following sequence: 5'-CGTGCTGGATTGGCTCACCAGACACCTTCC GACAT-3' (SEQ ID NO: 212)

[0279] In some embodiments, the library preparation kit comprises a first tailed PCR primer comprising the sequence 5'-GATCAGGTGAGGCTGCGACGACTNNNNNNNNN NNNAGTTGACAAGCGGTAGCCTGCACACCTTCCGACAT-3' (SEQ ID NO: 213), wherein the 12-base N sequence represents the right sample index sequence (170). In some embodiments, the first nine 'N's in the 12-base N sequence comprise the universal sample index sequence for the right sample index. In some embodiments, the last three "NNN"s (underlined) in the 12-base N sequence comprise a 3-mer random sequence designed to provide nucleotide diversity and color balance in each sequencing cycle.

[0280] In some embodiments, the library preparation kit comprises a second tailed PCR primer comprising the sequence 5'-CATGTAATGCACGTACTTTCAGGGTNNNNNNNNNN

[0281] CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 214), wherein the 10-base N sequence represents the left sample index sequence (160). In some embodiments, the 10-base N sequence comprises a universal sample index sequence for the left sample index.

[0282] Methods for forming multiple library-splint complexes

[0283] The present disclosure provides a method for forming a plurality of library-splint complexes (300), comprising: (a) providing a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule of the plurality of single-stranded nucleic acid library molecules comprises a sequence of interest (110) flanked on one side by at least a first left universal adapter sequence (120) and on another side by at least a first right universal adapter sequence (130) (e.g., see Figure 1-Figure 5 ).

[0284] The method for forming a plurality of library-splint complexes (300) further comprises step (b): providing a plurality of single-stranded splint chains (200), wherein each single-stranded splint chain (200) in the plurality of single-stranded splint chains comprises a first region (210) capable of hybridizing to at least a first left universal adapter sequence (120) of each library molecule and a second region (220) capable of hybridizing to at least a first right universal adapter sequence (130) of each library molecule. An exemplary single-stranded splint chain (200) is shown in FIG. Figure 1-Figure 5 In some embodiments, the single-stranded splint chain (200) can be 20-150 nucleotides in length, or 60-100 nucleotides in length, 70-90 nucleotides in length, or 60-80 nucleotides in length.

[0285] The method of forming a plurality of library-splint complexes (300) further comprises the step (c): hybridizing a plurality of single-stranded splint chains (200) with a plurality of single-stranded nucleic acid library molecules (100). The hybridization is performed under conditions suitable for hybridizing each library molecule with each single-stranded splint chain, such that a first region of one of the single-stranded splint chains (210) anneals to at least a first left universal adapter sequence (120) of the library molecule, and a second region of the single-stranded splint chain (220) anneals to at least a first right universal sequence (130) of the library molecule, thereby looping each library molecule to form a plurality of library-splint complexes (300). In some embodiments, the library-splint complex (300) comprises a gap between the 5' and 3' ends of the library molecule (e.g., Figure 1-Figure 5 ). In some embodiments, the gap is enzymatically ligatable.

[0286] In some embodiments, in the method of forming a plurality of library-splint complexes (300), the first region of the single-stranded splint strand (210) comprises a first universal adapter sequence that can hybridize to a first universal binding sequence at one end of the linear nucleic acid library molecule (120). In some embodiments, the first region of the single-stranded splint strand (210) comprises a first universal adapter sequence that comprises a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0287] In some embodiments, in the method for forming a plurality of library-splint complexes (300), the second region (220) of the single-stranded splint strand comprises a second universal adapter sequence that can hybridize to a second universal binding sequence at the other end of the linear nucleic acid library molecule. In some embodiments, the second region (220) of the single-stranded splint strand comprises a second universal adapter sequence that comprises a universal binding sequence for a forward sequencing primer or a reverse sequencing primer, a universal binding sequence for a first surface primer or a second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0288] In some embodiments, in a method of forming a plurality of library-splint complexes (300), the single-stranded splint chain (200) comprises one or more phosphorothioate bonds at its 5' and / or 3' end to confer resistance to exonucleases. In some embodiments, the single-stranded splint chain (200) comprises one or more phosphorothioate bonds at an internal position to confer resistance to endonucleases. In some embodiments, the single-stranded splint chain (200) comprises one or more 2'-O-methylcytosine bases at its 5' and / or 3' end or at an internal position. In some embodiments, the 5' end of the single-stranded splint chain (200) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the single-stranded splint chain (200) comprises a terminal 3'OH group or a terminal 3' blocking group.

[0289] In some embodiments, in a method of forming a plurality of library-splint complexes (300), a first region of a single-stranded splint strand (210) can hybridize with a sense strand or an antisense strand of a double-stranded nucleic acid library molecule. In the library-splint complex (300), a second region of a single-stranded splint strand (220) can hybridize with a sense strand or an antisense strand of a double-stranded nucleic acid library molecule. The double-stranded nucleic acid library molecule can be denatured to produce single-stranded sense and antisense library strands. The double-stranded nucleic acid library molecule can be denatured to produce the single-stranded nucleic acid library molecule (100) of step (a).

[0290] In some embodiments, in the method for forming a plurality of library-splint complexes (300), the first and second regions (210 and 220, respectively) of the single-stranded splint strand do not hybridize to the sequence of interest (110).

[0291] In some embodiments, in the method of forming a plurality of library-splint complexes (300), the nucleic acid library molecule (100) further comprises a second left universal adapter sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises a second right universal adapter sequence (150). In some embodiments, the nucleic acid library molecule (100) may further comprise additional left universal adapter sequences and / or right universal adapter sequences.

[0292] In some embodiments, in a method of forming multiple library-splint complexes (300), the nucleic acid library molecule (100) further comprises a first left index sequence (160) and / or a first right index sequence (170). In some embodiments, the first left index sequence (160) comprises a sample index sequence. The length of the first left index sequence (160) can be 3-20 nucleotides. In some embodiments, the first right index sequence (170) comprises another sample index sequence. The length of the first right index sequence (170) can be 3-20 nucleotides. The sequences of the left sample index sequence and the right sample index sequence (e.g., (160) and (170)) can be the same or different from each other. The sample index sequences can be used to distinguish target sequences obtained from different sample sources in multiple assays. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Figures 9A-9F The first left index sequence (160) may include a short random sequence (e.g., NNN) or lack a short random sequence. The first right index sequence (170) may include a short random sequence (e.g., NNN) or lack a short random sequence. The length of the short random sequence (e.g., NNN) may be 3-20 nucleotides.

[0293] A multiplex workflow is initiated by preparing a library with a sample index using one or two index sequences (e.g., a left index sequence and / or a right index sequence). A separate library with a sample index can be prepared using a first left index sequence (160) and / or a first right index sequence (170) using input nucleic acids isolated from different sources. Libraries with sample indexes can be pooled together to produce a multiple library mixture, and the pooled libraries can be looped, amplified, and / or sequenced. The sequence of the insertion region, together with the first left index sequence (160) and / or the first right index sequence (170), can be used to identify the source of the input nucleic acid. In some embodiments, any number of libraries with sample indexes can be pooled together, for example, 2-10, or 10-50, or 50-100, or 100-200, or more than 200 libraries with sample indexes can be pooled. Exemplary nucleic acid sources include naturally occurring, recombinant, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or entire organisms. Exemplary nucleic acid sources include fresh, frozen, fresh frozen or archived sources (e.g., formalin fixed paraffin embedded; FFPE). The skilled artisan will recognize that nucleic acids can be isolated from many other sources. Nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0294] In some embodiments, in a method for forming a plurality of library-splint complexes (300), the nucleic acid library molecule (100) further comprises: an optional first left unique identification sequence (180) and / or an optional first right unique identification sequence (190). In some embodiments, the first left unique identification sequence (180) and the first right unique identification sequence (190) each comprise a sequence that uniquely identifies each target sequence (e.g., an insertion sequence) to which a unique adapter is attached, among a population of other target molecular sequences. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular labeling. In some embodiments, the unique identification sequence comprises 2-12 or more nucleotides having a known sequence. For example, the unique identification sequence comprises a known random sequence, wherein the nucleotide at each position is randomly selected from nucleotides having the bases A, G, C, T, or U. The unique identification sequences (180) and / or (190) can be used for a molecular labeling procedure.

[0295] In some embodiments, in a method for forming multiple library-splint complexes (300), the nucleic acid library molecule (100) comprises any one or any combination of two or more of the following in any order: a first left universal adapter sequence (120); a second left universal adapter sequence (140); a first left index sequence (160); a first left unique identification sequence (180); a first right universal adapter sequence (130); a second right universal adapter sequence (150); a first right index sequence (170); and / or a first right unique identification sequence (190).

[0296] In some embodiments, in a method for forming a plurality of library-splint complexes (300), the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) include: a universal binding sequence for a forward sequencing primer or a reverse sequencing primer; a universal binding sequence for a first surface primer or a second surface primer; a universal binding sequence for a forward amplification primer or a reverse amplification primer; and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) may further include additional left universal adapter sequences.

[0297] In some embodiments, in a method for forming a plurality of library-splint complexes (300), the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) include: a universal binding sequence for a forward sequencing primer or a reverse sequencing primer; a universal binding sequence for a first surface primer or a second surface primer; a universal binding sequence for a forward amplification primer or a reverse amplification primer; and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) may further include additional right universal adapter sequences.

[0298] In some embodiments, in the method for forming a plurality of library-splint complexes (300), the nucleic acid library molecule (100) further comprises at least one junction adapter sequence positioned between any of the universal adapter sequences described herein (e.g., see Figure 5). For example, the first left junction adapter sequence (125) may be located between the first left universal adapter sequence (120) and the first left index sequence (160). The second left junction adapter sequence (165) may be located between the first left index sequence (160) and the second left universal adapter sequence (140). The third junction adapter sequence (145) may be located between the second left universal adapter sequence (140) and the target sequence (110). The first right junction adapter sequence (135) may be located between the first right universal sequence (130) and the first right index sequence (170). The second right junction adapter sequence (175) may be located between the first right index sequence (170) and the second right universal adapter sequence (150). The third right junction adapter sequence (155) may be located between the second right universal adapter sequence (150) and the target sequence (110). Any junction adapter sequence comprises any sequence and may be 3-60 nucleotides in length. Any junction adapter sequence comprises a universal sequence or a unique sequence. Any ligation adapter sequence includes a random sequence (e.g., NNN) of 3-20 nucleotides. Any ligation adapter sequence includes a binding sequence for an amplification primer, a sequencing primer, or a compacting oligonucleotide. Any ligation adapter sequence includes a binding sequence for a fixed surface primer (e.g., a capture primer). Any ligation adapter sequence includes a sample index sequence. Any ligation adapter sequence includes a unique identification sequence. Any ligation adapter sequence, particularly a ligation adapter sequence (145) includes a Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 209). Any ligation adapter sequence, particularly a ligation adapter sequence (155) includes a Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 210). The Tn5 transposon end sequence can be introduced into the library molecule (100) via a transposase-mediated reaction under conditions suitable for forming a transposon synaptic complex, the reaction comprising contacting double-stranded input DNA (e.g., genomic DNA) with a Tn-5 type transposase and a double-stranded oligonucleotide comprising a Tn transposon end sequence (SEQ ID NO: 209) linked to a universal adapter sequence or a sample index sequence. In the double-stranded oligonucleotide, the Tn transposon end sequence (SEQ ID NO: 209) can be located 5' or 3' relative to the universal adapter sequence or the sample index sequence.

[0299] The present disclosure provides a method for forming a plurality of library-splint complexes (300), comprising: (a) providing a plurality of single-stranded splint chains (200), wherein each single-stranded splint chain (200) comprises regions arranged in 5' to 3' order: (i) a first region (210) having a universal binding sequence that hybridizes to a sequence on one end of a linear single-stranded library molecule (e.g., 120), and (ii) a second region (220) having a universal binding sequence that hybridizes to a sequence on the other end of the linear single-stranded library molecule (e.g., 130). The method for forming a plurality of library-splint complexes (300) further comprises the step (b): hybridizing a plurality of single-stranded splint strands (200) to a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule comprises regions arranged in 5' to 3' order: (i) a first left universal adapter sequence (120) having a binding sequence for the second surface primer; (ii) a second left universal adapter sequence (140) having a binding sequence for the first sequencing primer or the second sequencing primer; (iii) a target sequence (110); (iv) a second right universal adapter sequence (150) having a binding sequence for the second sequencing primer or the first sequencing primer; and (v) ) a first right universal adapter sequence (130) having a binding sequence for a first surface primer, wherein hybridization is performed under conditions suitable for hybridization of the single-stranded splint strand (200) to the library molecule (100), thereby circularizing the library molecule to produce a library-splint complex (300), such that the first region (210) of the single-stranded splint strand hybridizes to the binding sequence for the second surface primer (120), and such that the second region (220) of the single-stranded splint strand hybridizes to the binding sequence for the first surface primer (130), wherein the library-splint complex (300) comprises a gap between the terminal 5' end and the 3' end of the library molecule, and wherein the gap is enzymatically ligatable (e.g., see Figure 2 In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left index sequence (160) and / or a first right index sequence (170) (e.g., see Figure 3 and Figure 4 ). In some embodiments, in a plurality of given library-splint complexes (300), the sequences of the first left index (160) and the first right index (170) are the same or different from each other. The length of the first left index sequence (160) can be 3-20 nucleotides. The length of the first right index sequence (170) can be 3-20 nucleotides. The first left index sequence (160) and / or the first right index sequence (170) can include a short random sequence (e.g., NNN). The length of the short random sequence can be 3-20 nucleotides. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Figures 9A-9FIn some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprise a first left unique identification sequence (180) and / or a first right unique identification sequence (190) that can be used for molecular labeling (e.g., see Figure 3 and Figure 4 ).

[0300] In some embodiments, in the method, the first left universal adaptor sequence (120) in the library molecule comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199).

[0301] In some embodiments, in the method, the first left universal adaptor sequence (120) in the library molecule comprises the sequence 5'-AATGATACG GCGACCACCGA-3' (SEQ ID NO: 203).

[0302] In some embodiments, in the method, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 201).

[0303] In some embodiments, in the method, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 205).

[0304] In some embodiments, in the method, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 206).

[0305] In some embodiments, in the method, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 202).

[0306] In some embodiments, in the method, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 207).

[0307] In some embodiments, in the method, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 208).

[0308] In some embodiments, in the method, the first right universal adaptor sequence (130) in the library molecule comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200).

[0309] In some embodiments, in the method, the first right universal adaptor sequence (130) in the library molecule comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 204).

[0310] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adapter sequence (120) of the library molecule, wherein the first region (210) comprises the following sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0311] In some embodiments, in the method, the second region of the single-stranded splint strand (220) comprises a universal binding sequence of the first right universal adaptor sequence (130) of the library molecule, wherein the second region (220) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0312] In some embodiments, in this method, the single-stranded splint chain (200) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195). See, for example, Figure 7 A- Figure 7 C.

[0313] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for a first left universal adapter sequence (120) of the library molecule, wherein the first region (210) comprises the following sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0314] In some embodiments, in the method, the second region of the single-stranded splint strand (220) comprises a universal binding sequence for the first right universal adaptor sequence (130) of the library molecule, wherein the second region (220) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0315] In some embodiments, in the method, the single-stranded splint chain (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0316] In some embodiments, any method for forming a plurality of library-splint complexes (300) described herein may further include at least one enzymatic reaction, including a phosphorylation reaction, a ligation reaction, and / or a nuclease exonuclease reaction. The enzymatic reactions can be performed sequentially or substantially simultaneously. The enzymatic reactions can be performed in a single reaction vessel. Alternatively, a first enzymatic reaction can be performed in a first reaction vessel, then transferred to a second reaction vessel for performing a second enzymatic reaction, and then transferred to a third reaction vessel for performing a third enzymatic reaction.

[0317] In some embodiments, any of the methods for forming a plurality of library-splint complexes (300) described herein further comprises performing separate and sequential phosphorylation and ligation reactions, the phosphorylation and ligation reactions being performed in separate reaction vessels. In some embodiments, the method for forming a plurality of library-splint complexes (300) further comprises step (c1): contacting the plurality of single-stranded splint chains (200) and the plurality of single-stranded nucleic acid library molecules (100) with T4 polynucleotide kinase in a first reaction vessel under conditions suitable for phosphorylating the 5' ends of the plurality of single-stranded splint chains (200) and / or the plurality of single-stranded nucleic acid library molecules (100); and transferring the phosphorylation reaction to a second reaction vessel. In some embodiments, the method for forming a plurality of library-splint complexes (300) further comprises step (d1): contacting the plurality of phosphorylated single-stranded splint strands (200) and the plurality of phosphorylated single-stranded nucleic acid library molecules (100) with a ligase in a second reaction vessel under conditions suitable for enzymatically ligating the gaps, thereby generating a plurality of covalently closed circular library molecules (400) each hybridized to the single-stranded splint strands (200). In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0318] In some embodiments, any of the methods for forming a plurality of library-splint complexes (300) described herein further comprises performing sequential phosphorylation and ligation reactions, the reactions being performed sequentially in the same reaction vessel. In some embodiments, the method for forming a plurality of library-splint complexes (300) further comprises step (c2): contacting the plurality of single-stranded splint strands (200) and the plurality of single-stranded nucleic acid library molecules (100) with T4 polynucleotide kinase in a first reaction vessel under conditions suitable for phosphorylating the 5' ends of the plurality of single-stranded splint strands (200) and the plurality of single-stranded nucleic acid library molecules (100). In some embodiments, the method for forming a plurality of library-splint complexes (300) further comprises step (d2): contacting the phosphorylated single-stranded splint strands (200) and the phosphorylated single-stranded nucleic acid library molecules (100) with a ligase in the same first reaction vessel under conditions suitable for enzymatically ligating the nicks, thereby generating a plurality of covalently closed circular library molecules (400) each hybridized to the single-stranded splint strands (200). In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0319] In some embodiments, any of the methods for forming a plurality of library-splint complexes (300) described herein further comprises performing substantially simultaneous phosphorylation and ligation reactions, the reactions being performed together in the same reaction vessel. In some embodiments, the method for forming a plurality of library-splint complexes (300) further comprises step (c3): contacting the plurality of single-stranded splint strands (200) and the plurality of single-stranded nucleic acid library molecules (100) with (i) T4 polynucleotide kinase and (ii) ligase in a first reaction vessel under conditions suitable for phosphorylating the 5' ends of the plurality of single-stranded splint strands (200) and the plurality of single-stranded nucleic acid library molecules (100), and the conditions being suitable for enzymatic ligation of the gaps, thereby producing a plurality of covalently closed circular library molecules (400) each hybridized to the single-stranded splint strands (200). In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0320] In some embodiments, any of the methods for forming a plurality of library-splint complexes (300) described herein further comprises the optional step of enzymatically removing a plurality of single-stranded splint chains (200) from a plurality of covalently closed circular library molecules (400), comprising the step of contacting the plurality of covalently closed circular library molecules (400) with at least one exonuclease to remove the plurality of single-stranded splint chains (200) and retain the plurality of covalently closed circular library molecules (400). In some embodiments, the exonuclease reaction can be performed in the same reaction buffer used to perform the phosphorylation and / or ligation reactions, or in different reaction buffers. In some embodiments, the exonuclease reaction can be performed in a third reaction vessel after the phosphorylation reaction (step c1, see above) is performed in a first reaction vessel and the ligation reaction (step d1, see above) is performed in a second reaction vessel. In some embodiments, the exonuclease reaction can be performed in a first reaction vessel after the phosphorylation reaction (step c2, see above) is performed in a first reaction vessel and the sequential ligation reaction (step d2, see above) is performed in the first reaction vessel. In some embodiments, the exonuclease reaction can be performed in the first reaction vessel after the substantially simultaneous phosphorylation and ligation reactions (step c3, see above) are performed in the first reaction vessel. In some embodiments, the at least one exonuclease comprises any combination of two or more of Exonuclease I, Thermolabile Exonuclease I, and / or T7 Exonuclease.

[0321] Rolling circle amplification method

[0322] The present disclosure provides a method for performing a rolling circle amplification reaction on a covalently closed circular library molecule (400). The rolling circle amplification reaction can be performed after phosphorylation and ligation, or after ligation. In some embodiments, the rolling circle amplification reaction can be performed on a covalently closed circular library molecule (400) hybridized with a single-stranded splint chain (200). In some embodiments, for example, the rolling circle amplification reaction can be performed after a nuclease exonuclease reaction, at which point the covalently closed circular library molecule (400) no longer hybridizes with the single-stranded splint chain (200). In some embodiments, the covalently closed circular library molecule (400) can be distributed on a support and then undergo a rolling circle amplification reaction. In some embodiments, the covalently closed circular library molecule (400) can undergo a rolling circle amplification reaction in a solution and then be distributed on a support. In some embodiments, the rolling circle amplification reaction can use the retained single-stranded splint chain (200) as an amplification primer, or the single-stranded splint chain (200) can be removed (e.g., via exonuclease digestion) and replaced with a soluble amplification primer.

[0323] Rolling circle amplification on support

[0324] In some embodiments, a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules lacking hybridized single-stranded splint chains (200), and wherein each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules comprises a universal binding sequence for a first surface primer, the method comprising step (a): distributing the plurality of covalently closed circular library molecules (400) onto a support having a plurality of first surface primers fixed thereto under conditions suitable for hybridizing each covalently closed circular library molecule (400) to each fixed first surface primer, thereby fixing the plurality of covalently closed circular library molecules (400).

[0325] In some embodiments, in the method of performing a rolling circle amplification reaction, the plurality of first surface primers immobilized on the support comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0326] In some embodiments, in the method of performing a rolling circle amplification reaction, the plurality of first surface primers immobilized on the support comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0327] A single first surface primer can hybridize to a covalently closed circular library molecule (400) having the universal binding sequence of the first surface primer.

[0328] In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (b): contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on a support using a plurality of first surface primers as immobilized amplification primers and a plurality of covalently closed circular library molecules (400) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules immobilized on the first surface primers. In some embodiments, the plurality of nucleotides comprises any combination of two or more of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, each immobilized concatemer is covalently attached to each first surface primer. In some embodiments, each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules comprises a universal binding sequence for the first surface primer and the second surface primer (e.g., (130) and (120), respectively), such that the rolling circle amplification reaction generates concatemer molecules having multiple tandem copies of the universal binding sequence for the first surface primer and the second surface primer. In certain embodiments, the support further comprises a plurality of second surface primers. In certain embodiments, the second surface primers for fixing are used for fixing at least a portion of the concatemer molecule to the support. In certain embodiments, the second surface primers for fixing have non-extendable 3 ' ends and cannot be used for amplification. In certain embodiments, the concatemer can be subjected to sequencing reaction.

[0329] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199 or its complement).

[0330] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203 or its complement).

[0331] A single second surface primer can hybridize with a portion of a concatemer molecule having a universal binding sequence of the second surface primer. In certain embodiments, the fixed second surface primer is used to fix at least a portion of the concatemer molecule to a support. In certain embodiments, the fixed second surface primer has a non-extendable 3' end and cannot be used for amplification. In certain embodiments, the fixed concatemer can be subjected to a sequencing reaction.

[0332] In some embodiments, in methods for performing rolling circle amplification reactions, a plurality of covalently closed circular library molecules (400) can be dispensed onto a support that is coated with one or more compounds to create a passivation layer on the support (e.g., Figure 18). In some embodiments, the passivation layer forms a porous or semi-porous layer. In some embodiments, one or more types of surface primers, concatemer template molecules and / or polymerases can be attached to the passivation layer to be fixed to the support. In some embodiments, the support comprises a low non-specific binding surface that can improve the nucleic acid hybridization and amplification performance on the support. In general, the support may comprise one or more layers of covalently or non-covalently attached low binding chemical modification layers, such as silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to fix multiple nucleic acid concatemer molecules to the support. In some embodiments, the support may comprise a functionalized polymer coating that is covalently bound to at least a portion of the support via chemical groups on the support, primers grafted to the functionalized polymer coating, and a water-soluble protective coating on the primers and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of a hydrophilic polymer coating and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise a branched PEG having at least 4 branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity as measured by a water contact angle, wherein the water contact angle does not exceed 45 degrees. In some embodiments, the density of the covalently closed circular library molecules (400) immobilized to the support or to the coating on the support is about 10 2 -10 6 Pieces / mm 2 , or about 10 6 -10 9 Pieces / mm 2 , or about 10 9 -10 12 Pieces / mm 2 In some embodiments, a plurality of covalently closed circular library molecules (400) are immobilized to a support or to a coating on a support at predetermined sites on the support (or a coating on a support); or to a coating on a support (or a coating on a support) at random sites.

[0333] In some embodiments, in a method of performing a rolling circle amplification reaction, the dispensing of step (a) (e.g., dispensing a plurality of covalently closed circular library molecules (400) onto a support) can be performed in the presence of a high efficiency hybridization buffer comprising: (i) a first polar aprotic solvent having a dielectric constant of no greater than 40 and having a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of no greater than 115 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 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (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. In some embodiments, the high-efficiency hybridization buffer further comprises betaine. In-solution rolling circle amplification using soluble amplification primers

[0334] In some embodiments, a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules (400) lacking hybridized single-stranded splint chains (200), wherein each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules comprises a universal binding sequence for a forward amplification primer and a universal binding sequence for a first surface primer, the method comprising: (a) hybridizing the plurality of covalently closed circular library molecules and the plurality of soluble forward amplification primers in a solution; and (b) performing a first rolling circle amplification reaction by contacting the plurality of covalently closed circular library molecules (400) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction in a solution using the plurality of forward amplification primers and using the plurality of covalently closed circular library molecules (400) as template molecules, thereby producing a plurality of nucleic acid concatemer molecules having portions that are still hybridized to their covalently closed circular library molecules (400). In some embodiments, the method for performing a rolling circle amplification reaction further comprises the step (c): distributing the plurality of concatemer molecules onto a support having a plurality of first surface primers fixed thereon under conditions suitable for hybridizing at least a portion of the concatemer molecules with a plurality of fixed first surface primers, thereby immobilizing the plurality of concatemer molecules. The plurality of fixed concatemer molecules remain hybridized to their covalently closed circular library molecules (400). In some embodiments, the method for performing a rolling circle amplification reaction further comprises the step (d): contacting the fixed plurality of concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a second rolling circle amplification reaction on the support using a plurality of covalently closed circular library molecules (400) as template molecules, thereby extending the plurality of fixed nucleic acid concatemer molecules. In some embodiments, the first rolling circle amplification reaction and / or the second rolling circle amplification reaction can be performed with a plurality of nucleotides comprising any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, each fixed concatemer hybridizes to each first surface primer. In some embodiments, each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules comprises a universal binding sequence for a first surface primer and a second surface primer (e.g., (130) and (120), respectively), such that an in-solution rolling circle amplification reaction produces a concatemer molecule having multiple tandem copies of the universal binding sequence for the first surface primer and the second surface primer. In some embodiments, the support further comprises a plurality of second surface primers. In some embodiments, the fixed second surface primer is used to fix at least a portion of the concatemer molecule to the support. In some embodiments, the fixed second surface primer has a non-extendable 3' end and cannot be used for amplification. In some embodiments, the fixed concatemer can be subjected to a sequencing reaction.

[0335] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support include the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194). A single first surface primer can hybridize to a covalently closed circular library molecule (400) having a universal binding sequence of the first surface primer.

[0336] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support include the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197). A single first surface primer can hybridize to a covalently closed circular library molecule (400) having a universal binding sequence of the first surface primer.

[0337] In some embodiments, the plurality of second surface primers immobilized on the support comprise the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199 or its complement). A single second surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence of the second surface primer.

[0338] In some embodiments, the plurality of second surface primers immobilized on the support include the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203 or its complement). A single second surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence of the second surface primer.

[0339] In certain embodiments, the fixed second surface primer is used to fix at least a portion of the concatemer molecule to a support. In certain embodiments, the fixed second surface primer has a non-extendable 3' end and cannot be used for amplification. In certain embodiments, the fixed concatemer can be subjected to a sequencing reaction.

[0340] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support that is coated with one or more compounds to produce a passivation layer on the support (e.g., Figure 18). In some embodiments, the passivation layer forms a porous or semi-porous layer. In some embodiments, one or more types of surface primers, concatemer template molecules and / or polymerases can be attached to the passivation layer to be fixed to the support. In some embodiments, the support comprises a low non-specific binding surface that can improve the nucleic acid hybridization and amplification performance on the support. In general, the support may comprise one or more layers of covalently or non-covalently attached low binding chemical modification layers, such as silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to fix multiple nucleic acid concatemer molecules to the support. In some embodiments, the support may comprise a functionalized polymer coating that is covalently bound to at least a portion of the support via chemical groups on the support, primers grafted to the functionalized polymer coating, and a water-soluble protective coating on the primers and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of a hydrophilic polymer coating and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise a branched PEG having at least 4 branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity as measured by a water contact angle, wherein the water contact angle does not exceed 45 degrees. In some embodiments, the density of concatemer molecules immobilized to the support or to the coating on the support is about 10 2 -10 6 / mm 2 , or about 10 6 -10 9 / mm 2 , or about 10 9 -10 12 / mm 2 In some embodiments, a plurality of concatemer molecules are immobilized to the support or to the coating on the support at predetermined sites on the support (or the coating on the support); or immobilized to the coating on the support at random sites on the support (or the coating on the support).

[0341] In some embodiments, the partitioning of step (c) can be performed in the presence of a high efficiency hybridization buffer comprising: (i) a first polar aprotic solvent having a dielectric constant of no greater than 40 and having a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of no greater than 115 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 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high efficiency hybridization buffer comprises: (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. In some embodiments, the high efficiency hybridization buffer further comprises betaine. In-solution rolling circle amplification using single-stranded splint chains

[0342] In some embodiments, a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules hybridized to a single-stranded splint chain (200), wherein each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules comprises a universal binding sequence for a first surface primer, the method comprising: (a) contacting the plurality of covalently closed circular library molecules (400) hybridized to the single-stranded splint chain (200) with a plurality of strand displacement polymerases and a plurality of nucleotides in a solution under conditions suitable for performing a first rolling circle amplification reaction using the single-stranded splint chain (200) as an amplification primer, thereby generating a plurality of concatemer molecules (e.g., see Figure 6 A- Figure 6 C).

[0343] In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (b): distributing the plurality of concatemer molecules hybridized with the covalently closed circular library molecules (400) onto a support having the plurality of first surface primers immobilized thereon, under conditions suitable for hybridizing at least a portion of the concatemer to the plurality of immobilized first surface primers, thereby immobilizing the plurality of concatemer molecules. The plurality of immobilized concatemer molecules remain hybridized with the covalently closed circular library molecules (400).

[0344] In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (c): contacting the plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a second rolling circle amplification reaction on a support using a plurality of covalently closed circular library molecules (400) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules.

[0345] In some embodiments, the first rolling circle amplification reaction and / or the second rolling circle amplification reaction can be performed with multiple nucleotides in any combination of two or more of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, each fixed concatemer hybridizes with each first surface primer. In some embodiments, each covalently closed circular library molecule (400) in the multiple covalently closed circular library molecules comprises a universal binding sequence of a first surface primer and a second surface primer (e.g., (130) and (120) respectively), such that the rolling circle amplification reaction in solution produces a concatemer molecule having multiple tandem copies of the universal binding sequence for the first surface primer and the second surface primer. In some embodiments, the support further comprises multiple second surface primers. In some embodiments, the fixed second surface primer is used to fix at least a portion of the concatemer molecule to the support. In some embodiments, the fixed second surface primer has an unextendable 3' end and cannot be used for amplification. In some embodiments, the fixed concatemer can be subjected to sequencing reaction.

[0346] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support include the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194). A single first surface primer can hybridize to a covalently closed circular library molecule (400) having a universal binding sequence of the first surface primer.

[0347] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support include the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197). A single first surface primer can hybridize to a covalently closed circular library molecule (400) having a universal binding sequence of the first surface primer.

[0348] In some embodiments, the plurality of second surface primers immobilized on the support comprise the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199 or its complement). A single second surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence of the second surface primer.

[0349] In some embodiments, the plurality of second surface primers immobilized on the support include the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203 or its complement). A single second surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence of the second surface primer.

[0350] In certain embodiments, the fixed second surface primer is used to fix at least a portion of the concatemer molecule to a support. In certain embodiments, the fixed second surface primer has a non-extendable 3' end and cannot be used for amplification. In certain embodiments, the fixed concatemer can be subjected to a sequencing reaction.

[0351] In some embodiments, the plurality of concatemer molecules of step (b) can be dispensed onto a support that is coated with one or more compounds to produce a passivation layer on the support (e.g., Figure 18 ). In some embodiments, the passivation layer forms a porous or semi-porous layer. In some embodiments, surface primers, concatemer template molecules and / or polymerases can be attached to the passivation layer to be fixed to the support. In some embodiments, the support comprises a low non-specific binding surface that can improve the nucleic acid hybridization and amplification performance on the support. In general, the support may comprise one or more layers of covalently or non-covalently attached low binding chemical modification layers, such as silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to fix multiple nucleic acid concatemer molecules to the support. In some embodiments, the support may comprise a functionalized polymer coating that is covalently bound to at least a portion of the support via chemical groups on the support, primers grafted to the functionalized polymer coating, and a water-soluble protective coating on the primers and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of a hydrophilic polymer coating and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise a branched PEG having at least 4 branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity as measured by a water contact angle, wherein the water contact angle does not exceed 45 degrees. In some embodiments, the density of concatemer molecules immobilized to the support or to the coating on the support is about 10 2 -10 6 / mm 2 , or about 10 6 -10 9 / mm 2 , or about 10 9 -10 12 / mm2 In some embodiments, a plurality of concatemer molecules are immobilized to the support or to the coating on the support at predetermined sites on the support (or the coating on the support); or immobilized to the coating on the support at random sites on the support (or the coating on the support).

[0352] In some embodiments, the partitioning of step (b) can be performed in the presence of a high efficiency hybridization buffer comprising: (i) a first polar aprotic solvent having a dielectric constant of no greater than 40 and having a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of no greater than 115 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 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (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. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0353] Methods used for sequencing

[0354] The present disclosure provides methods for sequencing any fixed concatemer molecules described herein. Any method for performing rolling circle amplification reactions described herein can be used to produce multiple concatemer molecules fixed to a support, and the fixed concatemer molecules can be subjected to sequencing reactions. In some embodiments, the sequencing reaction uses detectably labeled nucleotide analogs. In some embodiments, the sequencing reaction uses a two-stage sequencing reaction, including combining a detectably labeled multivalent molecule and incorporating a nucleotide analog. In some embodiments, the sequencing reaction uses unlabeled nucleotide analogs. The terms concatemer molecule and template molecule are used interchangeably.

[0355] In some embodiments, any rolling circle amplification reaction described herein (e.g., RCA performed on a support or in solution) can be used to generate immobilized concatemers, each concatemer containing a tandem repeat unit of a target sequence and any adapter sequence present in the covalently closed circular library molecule (400). For example, the tandem repeat unit comprises: (i) a first left universal adapter sequence (120) having a binding sequence for a second surface primer, (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer, (iii) a target sequence (110), (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer, (v) a first right universal adapter sequence (130) having a binding sequence for a first surface primer, and (vii) a first left index sequence (160) and / or a first right index sequence (170) (see, e.g., Figure 3-Figure 4 In some embodiments, the tandem repeat unit further comprises a first left unique identification sequence (180) and / or a first right unique identification sequence (190).

[0356] Fixed concatemers can collapse into compact nucleic acid nanoballs by themselves. During the RCA reaction, one or more compacting oligonucleotides are included to further compact the size and / or shape of the nanoballs. Given the increase in the number of tandem repeat units in a concatemer, the number of sites for hybridizing with multiple sequencing primers (e.g., sequencing primers with universal sequences) around the concatemer is increased, and the sequencing primers are used as multiple starting sites for the sequencing reaction catalyzed by polymerase. When sequencing reaction adopts detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., with nucleotide units), the signal emitted by the nucleotides or nucleotide units participating in the parallel sequencing reactions along the concatemer produces an increased signal intensity for each concatemer. A plurality of parts of a given concatemer can be sequenced simultaneously. In addition, a plurality of binding complexes can be formed along a specific concatemer molecule, and each binding complex comprises a sequencing polymerase that is bonded to a multivalent molecule, wherein the plurality of binding complexes remain stable and do not dissociate, resulting in an increased residence time, which increases signal intensity and reduces imaging time.

[0357] Methods for sequencing using nucleotide analogs

[0358] The present disclosure provides a method for sequencing any immobilized concatemer molecule described herein, the method comprising the step (a): contacting a sequencing polymerase with (i) a nucleic acid concatemer molecule and (ii) a nucleic acid sequencing primer, wherein the contacting is performed under conditions suitable for binding the sequencing polymerase to the nucleic acid concatemer molecule hybridized to the nucleic acid primer, wherein the nucleic acid concatemer molecule hybridized to the nucleotide primer forms a nucleic acid duplex. In some embodiments, the sequencing polymerase comprises a recombinant mutant sequencing polymerase that can bind and incorporate nucleotide analogs. In some embodiments, the sequencing primer comprises a 3' extendable end.

[0359] In some embodiments, in a method for sequencing a concatemer molecule, a sequencing primer comprises a 3' extendable end or a 3' non-extendable end. In some embodiments, a plurality of nucleic acid concatemer molecules comprise amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, a plurality of nucleic acid concatemer molecules comprise a copy of a target sequence of interest. In some embodiments, a plurality of nucleic acid molecules comprise two or more tandem copies of a target sequence of interest (e.g., concatemers). In some embodiments, the nucleic acid concatemer molecules in a plurality of nucleic acid concatemer molecules comprise the same target sequence of interest or different target sequences of interest. In some embodiments, a plurality of nucleic acid concatemer molecules and / or a plurality of nucleic acid primers are in solution or fixed to a support. In some embodiments, when a plurality of nucleic acid concatemer molecules and / or a plurality of nucleic acid primers are fixed to a support, binding to a first sequencing polymerase generates a plurality of fixed first composite polymerases. In some embodiments, a plurality of nucleic acid concatemer molecules and / or nucleic acid primers are fixed to 10 on a support. 2 –10 15 In some embodiments, the combination of multiple concatemer molecules and nucleic acid primers with multiple first sequencing polymerases generates 10 2 –10 15 In some embodiments, the plurality of immobilized first composite polymerases on the support are immobilized at predetermined or random sites on the support. In some embodiments, the plurality of immobilized first composite polymerases are in fluid communication with each other to allow a solution of reagents (e.g., an enzyme comprising a sequencing polymerase, a multivalent molecule, nucleotides, and / or divalent cations) to flow onto the support, such that the plurality of immobilized composite polymerases on the support react with the reagent solution in a massively parallel manner.

[0360] In some embodiments, the method for sequencing further comprises step (b): contacting the sequencing polymerase with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to a sequencing polymerase (which binds to a nucleic acid duplex) and suitable for polymerase-catalyzed incorporation of nucleotides. In some embodiments, the sequencing polymerase is contacted with a plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises at least one nucleotide analog having a chain termination moiety at the sugar 2' or 3' position. In some embodiments, the chain termination moiety is removable from the sugar 2' or 3' position to convert the chain termination moiety into an OH or H group. In some embodiments, the plurality of nucleotides comprises at least one nucleotide lacking a chain termination moiety. In some embodiments, at least one nucleotide is labeled with a detectable reporter moiety (e.g., a fluorophore).

[0361] In some embodiments, the method for sequencing further comprises step (c): incorporating at least one nucleotide into the 3' end of the extendable primer under conditions suitable for incorporating at least one nucleotide. In some embodiments, the conditions suitable for binding the nucleotide to the polymerase and for incorporating the nucleotide may be the same or different. In some embodiments, the conditions suitable for incorporating the nucleotide comprise adding at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the at least one nucleotide binds to the sequencing polymerase and is incorporated into the 3' end of the extendable primer. In some embodiments, incorporating the nucleotide into the 3' end of the primer in step (c) comprises a primer extension reaction.

[0362] In certain embodiments, the method for sequencing further comprises step (d): repeating at least one nucleotide incorporating into the 3' end of the extendable primer in steps (b) and (c) at least once. In certain embodiments, a plurality of nucleotides include a plurality of nucleotides labeled with a detectable reporter moiety. The detectable reporter moiety includes a fluorophore. In certain embodiments, the fluorophore is attached to a nucleotide base. In certain embodiments, the fluorophore is attached to a nucleotide base with a joint that can be cleaved / removed from the base. In certain embodiments, at least one of the plurality of nucleotides is not labeled with a detectable reporter moiety. In certain embodiments, the specific detectable reporter moiety (e.g., fluorophore) attached to the nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to allow detection and identification of the nucleotide base. In certain embodiments, the method further comprises: detecting the at least one incorporated nucleotide at step (c) and / or (d). In certain embodiments, the method further comprises: identifying the at least one incorporated nucleotide at step (c) and / or (d). In some embodiments, the sequence of a concatemer of nucleic acid molecules can be determined by detecting and identifying nucleotides bound to a sequencing polymerase, thereby determining the sequence of the concatemer molecule. In some embodiments, the sequence of a concatemer of nucleic acid molecules can be determined by detecting and identifying nucleotides incorporated into the 3' end of a primer, thereby determining the sequence of the concatemer molecule.

[0363] In some embodiments, in a method for sequencing, a plurality of sequencing polymerases bound to a nucleic acid duplex comprises a plurality of composite polymerases having at least a first composite polymerase and a second composite polymerase, wherein (a) the first composite polymerase comprises a first sequencing polymerase bound to a first nucleic acid duplex, wherein the first nucleic acid duplex comprises a first nucleic acid template sequence hybridized to a first nucleic acid primer, (b) the second composite polymerase comprises a second sequencing polymerase bound to a second nucleic acid duplex, wherein the second nucleic acid duplex comprises a second nucleic acid template sequence hybridized to a second nucleic acid primer, (c) the first nucleic acid template sequence and the second nucleic acid template sequence comprise the same or different sequences, (d) the first nucleic acid concatemer and the second nucleic acid concatemer are clonally amplified, (e) the first primer and the second primer comprise an extendable 3' end or a non-extendable 3' end, and (f) the plurality of composite polymerases are immobilized to a support. In some embodiments, the plurality of composite polymerases immobilized to the support are immobilized at a density of about 10 2 –10 15 Complex polymerase / mm 2 .

[0364] Two-stage method for sequencing nucleic acids

[0365] The present disclosure provides a two-stage method for sequencing any immobilized concatemer molecule described herein. In some embodiments, the first stage generally comprises combining the multivalent molecule with a complex polymerase to form a multivalent-complex polymerase, and detecting the multivalent-complex polymerase.

[0366] In some embodiments, the first stage comprises step (a): contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid concatemer molecules and (ii) a plurality of nucleic acid primers, wherein the contacting is performed under conditions suitable for binding the plurality of first sequencing polymerases to the plurality of nucleic acid concatemer molecules and the plurality of nucleic acid primers, thereby forming a plurality of first composite polymerases, each first composite polymerase comprising a first sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid concatemer molecule hybridized to a nucleic acid primer. In some embodiments, the first polymerase comprises a recombinant mutant sequencing polymerase. In some embodiments, the sequencing primer comprises a 3' extendable end.

[0367] In some embodiments, in a method for sequencing a concatemer molecule, a sequencing primer comprises a 3' extendable end or a 3' non-extendable end. In some embodiments, a plurality of nucleic acid concatemer molecules comprise amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, a plurality of nucleic acid concatemer molecules comprise a copy of a target sequence of interest. In some embodiments, a plurality of nucleic acid molecules comprise two or more tandem copies of a target sequence of interest (e.g., concatemers). In some embodiments, the nucleic acid concatemer molecules in a plurality of nucleic acid concatemer molecules comprise the same target sequence of interest or different target sequences of interest. In some embodiments, a plurality of nucleic acid concatemer molecules and / or a plurality of nucleic acid primers are in solution or fixed to a support. In some embodiments, when a plurality of nucleic acid concatemer molecules and / or a plurality of nucleic acid primers are fixed to a support, binding to a first sequencing polymerase generates a plurality of fixed first composite polymerases. In some embodiments, a plurality of nucleic acid concatemer molecules and / or nucleic acid primers are fixed to 10 on a support. 2 –10 15 In some embodiments, the combination of multiple concatemer molecules and nucleic acid primers with multiple first sequencing polymerases generates 10 2 –10 15 In some embodiments, the plurality of immobilized first composite polymerases on the support are immobilized at predetermined or random sites on the support. In some embodiments, the plurality of immobilized first composite polymerases are in fluid communication with each other to allow a solution of reagents (e.g., an enzyme comprising a sequencing polymerase, a multivalent molecule, nucleotides, and / or divalent cations) to flow onto the support, such that the plurality of immobilized composite polymerases on the support react with the reagent solution in a massively parallel manner.

[0368] In some embodiments, the method for sequencing further comprises step (b): contacting the plurality of first complex polymerases with a plurality of multivalent molecules to form a plurality of multivalent-complex polymerases (e.g., binding complexes). In some embodiments, each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide (e.g., a nucleotide unit) (e.g., Figures 19-23 In some embodiments, the contacting of step (b) is performed under conditions suitable for binding the complementary nucleotide units of the multivalent molecule to at least two of the plurality of first complex polymerases, thereby forming a plurality of multivalent complex polymerases. In some embodiments, the conditions are suitable for inhibiting polymerase-catalyzed incorporation of the complementary nucleotide units into primers of the plurality of multivalent-complex polymerases. In some embodiments, the plurality of multivalent molecules comprises at least one having a plurality of nucleotide arms (e.g., Figures 19-23 ), each nucleotide arm is attached with a nucleotide analog (e.g., a nucleotide analog unit), wherein the nucleotide analog includes a chain termination portion located at the 2' and / or 3' position of the sugar. In some embodiments, the multiple multivalent molecules include at least one multivalent molecule comprising a plurality of nucleotide arms, each nucleotide arm being attached with a nucleotide unit lacking a chain termination portion. In some embodiments, at least one multivalent molecule in the multiple multivalent molecules is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the contacting of step (b) is performed in the presence of at least one non-catalytic cation comprising strontium, barium, and / or calcium.

[0369] In some embodiments, the method for sequencing further comprises step (c): detecting a plurality of multivalent-complex polymerases. In some embodiments, the detection comprises detecting a multivalent molecule bound to the complex polymerase, wherein the complementary nucleotide units of the multivalent molecule bind to the primer, but the incorporation of the complementary nucleotide units is inhibited. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety to allow detection. In some embodiments, the labeled multivalent molecule comprises a fluorophore attached to the core, linker, and / or nucleotide units of the multivalent molecule.

[0370] In some embodiments, the sequencing method further comprises step (d): identifying the nucleobases of the complementary nucleotide units bound to the plurality of first composite polymerases, thereby determining the sequence of the concatemer molecule. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety corresponding to a specific nucleotide unit attached to the nucleotide arms to allow identification of the complementary nucleotide units (e.g., the nucleotide bases adenine, guanine, cytosine, thymine, or uracil) bound to the plurality of first composite polymerases.

[0371] In some embodiments, the second stage of the two-stage sequencing method generally comprises nucleotide incorporation. In some embodiments, the method for sequencing further comprises step (e): dissociating the plurality of multivalent-complex polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, while retaining the plurality of nucleic acid duplexes.

[0372] In some embodiments, the method for sequencing further comprises step (f): contacting the plurality of retained nucleic acid duplexes from 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 composite polymerases each comprising a second sequencing polymerase bound to the retained nucleic acid duplexes. In some embodiments, the second sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[0373] In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is 100% identical to the amino acid sequence of the plurality of second sequencing polymerases of step (f). In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is different from the amino acid sequence of the plurality of second sequencing polymerases of step (f).

[0374] In some embodiments, the method for sequencing further comprises step (g): contacting a plurality of second composite polymerases with a plurality of nucleotides, wherein the contact is carried out under conditions suitable for combining complementary nucleotides from a plurality of nucleotides with at least two of the composite polymerases, thereby forming a plurality of nucleotide-composite polymerases. In some embodiments, the contact of step (g) is carried out under conditions suitable for promoting the incorporation of the complementary nucleotides of the combination into the primer of the nucleotide composite-polymerase through polymerase catalysis, thereby forming a plurality of nucleotide-composite polymerases. In some embodiments, the incorporation of nucleotides into the 3' end of the primer in step (g) comprises a primer extension reaction. In some embodiments, the contact of step (g) is carried out in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises natural nucleotides (e.g., non-analog nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides comprises removable or non-removable 2' and / or 3' chain termination portions. In some embodiments, the plurality of nucleotides comprises a plurality of unlabeled or detectable reporter moiety-labeled nucleotides. The detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker that is cleavable / removable or non-removable from the base. In some embodiments, at least one of the plurality of nucleotides is not labeled with a detectable reporter moiety. In some embodiments, the specific detectable reporter moiety (e.g., a fluorophore) attached to the nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of the nucleotide base.

[0375] In some embodiments, the method for sequencing further comprises step (h): detecting complementary nucleotides incorporated into the primer of the nucleotide-complex polymerase. In some embodiments, the plurality of nucleotides are labeled with a detectable reporter moiety to allow detection. In some embodiments, in the method for sequencing concatemer molecules, the detection of step (h) is omitted.

[0376] In some embodiments, the method for sequencing further comprises step (i): identifying the base of a complementary nucleotide incorporated into a primer of a nucleotide-complex polymerase. In some embodiments, the complementary nucleotide identified in step (i) can be used to confirm the identity of the complementary nucleotide of the multivalent molecule bound to the plurality of first complex polymerases in step (d). In some embodiments, the identification of step (i) can be used to determine the sequence of the nucleic acid concatemer molecule. In some embodiments, in the method for sequencing concatemer molecules, the identification of step (i) is omitted.

[0377] In some embodiments, the method for sequencing further comprises step (j): removing the chain terminating moiety from the incorporated nucleotides when step (g) is performed by contacting a plurality of second complex polymerases with a plurality of nucleotides comprising at least one nucleotide having a 2' and / or 3' chain terminating moiety.

[0378] In some embodiments, the method for sequencing further comprises step (k): repeating steps (a)-(g) and (j) at least once. In some embodiments, the method for sequencing further comprises step (k): repeating steps (a)-(j) at least once. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying the multivalent molecule that binds to the sequencing polymerase but is not incorporated into the 3' end of the primer in steps (c) and (d). In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined (or confirmed) by detecting and identifying the nucleotide that is incorporated into the 3' end of the primer in steps (h) and (i).

[0379] In some embodiments, in any method for sequencing a nucleic acid molecule, a plurality of first complex polymerases are bound to a plurality of multivalent molecules to form at least one affinity complex, the method comprising the steps of: (a) binding a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein the first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and (b) binding a second nucleic acid primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, wherein the first binding complex and the second binding complex of the same multivalent molecule form an affinity complex. In some embodiments, the first sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild-type or mutant polymerase described herein. The concatemer template molecule comprises a tandem repeat sequence of a target sequence and at least one universal sequencing primer binding site. The first nucleic acid primer and the second nucleic acid primer can bind to the sequencing primer binding site along the concatemer template molecule. Exemplary multivalent molecules are shown in Figures 19-23 middle.

[0380] In some embodiments, in any method for sequencing a nucleic acid molecule, wherein the method comprises binding a plurality of first composite polymerases to a plurality of multivalent molecules to form at least one affinity complex, the method comprises the steps of: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers with different portions of a concatemer nucleic acid concatemer molecule to form at least a first and a second composite polymerase on the same concatemer template molecule; and (b) contacting a plurality of detectably labeled multivalent molecules with at least the first composite polymerase and the second composite polymerase on the same concatemer template molecule under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first composite polymerase and the second composite polymerase, wherein at least a first nucleotide unit of the single multivalent molecule binds to the first composite polymerase that includes a first primer that hybridizes to a first portion of the concatemer template molecule, thereby forming a first binding complex (e.g., a first primer that hybridizes to a first portion of the concatemer template molecule). a ternary complex), and wherein at least a second nucleotide unit of the single multivalent molecule is bound to a second complex polymerase, the second complex polymerase comprising a second primer that hybridizes to a second portion of the concatemer template molecule, thereby forming a second binding complex (e.g., a second ternary complex), wherein the contacting is performed under conditions suitable for inhibiting the polymerase-catalyzed incorporation of the bound first and second nucleotide units into the first and second binding complexes, and wherein the first and second binding complexes bound to the same multivalent molecule form an affinity complex; and (c) detecting the first and second binding complexes on the same concatemer template molecule, and (d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule. In some embodiments, the plurality of sequencing polymerases comprises any wild-type or mutant sequencing polymerase described herein. The concatemer template molecule comprises a tandem repeat sequence of a sequence of interest and at least one universal sequencing primer binding site. A plurality of nucleic acid primers can bind to the sequencing primer binding sites along the concatemer template molecule. Exemplary multivalent molecules are shown in Figures 19-23 middle.

[0381] Combined sequencing

[0382] The present disclosure provides methods for sequencing any of the immobilized concatemer molecules described herein, wherein the sequencing method comprises a sequencing by binding (SBB) procedure employing unlabeled chain-terminating nucleotides. In some embodiments, a sequencing by binding (SBB) method comprises the steps of (a) sequentially contacting a primed template nucleic acid with at least two separate mixtures under conditions that stabilize a ternary complex, wherein the at least two separate mixtures each comprise a polymerase and nucleotides, whereby the sequential contacting results in contacting the primed template nucleic acid with nucleotides of base types homologous to a first, second, and third base types in the template under conditions that stabilize the ternary complex; (b) examining the at least two separate mixtures to determine whether a ternary complex is formed; and (c) identifying the next correct nucleotide of the primed template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of the first base type, the second base type, or the third base type, and wherein if a ternary complex is not present in step (b), the next correct nucleotide is inferred to be a nucleotide homolog of the fourth base type; (d) adding the next correct nucleotide to a primer of the primed template nucleic acid after step (b), thereby generating an extended primer; and (e) repeating steps (a) to (d) at least once for the primed template nucleic acid comprising the extended primer. Exemplary combined sequencing methods are described in US Pat. Nos. 10,246,744 and 10,731,141 (the contents of which are incorporated herein by reference in their entireties).

[0383] Multiple workflows

[0384] The present disclosure provides a multiplex workflow that generally includes preparing a population of separate sample-indexed covalently closed circular library molecules using a single-stranded splint chain (200) and a sample-indexed nucleic acid library carrying one or two index sequences (e.g., sequences of a left (160) and / or right (170) sample index). The first left index sequence (160) and the first right index sequence (170) are known sequences. Separate sample-indexed libraries can be prepared from input nucleic acids isolated from different sources, where the sample index sequences are used to distinguish between the different sources. The pooling step can be performed after generating the sample-indexed covalently closed circular library molecules or before generating the sample-indexed library-splint complex. The pooled molecules can undergo downstream multiplex amplification and / or multiplex sequencing reactions.

[0385] Traditional pooling workflow for multiplexing

[0386] In some embodiments, a method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources comprises: (a) providing two or more populations of single-stranded nucleic acid library molecules (100), each population of library molecules (100) contained in a separate compartment, wherein the nucleic acid library molecules in a given population comprise: (i) a first left universal adapter sequence (120) having a binding sequence for a second surface primer, (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer, (iii) a sequence of interest (110) isolated from a sample source, (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer, (v) a first right universal adapter sequence (130) having a binding sequence for a first surface primer, and (vii) a first left index sequence (160) and / or a first right index sequence (170), wherein the left (160) and / or right (170) indexes, alone or in combination, identify the sample source from which the sequence of interest (110) was isolated (e.g., see Figure 3 and Figure 4 In some embodiments, the single-stranded nucleic acid library molecule (100) further comprises a first left unique identification sequence (180) and / or a first right unique identification sequence (190).

[0387] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (b): providing a plurality of single-stranded splint chains (200), wherein each single-stranded splint chain (200) comprises a first region (210) and a second region (220), wherein the first region (210) can hybridize with the first left universal adapter sequence (120) of the single-stranded library molecule, and wherein the second region (220) can hybridize with the first right universal adapter sequence (130) of the single-stranded library molecule.

[0388] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (c): in a separate compartment, contacting a population of single-stranded nucleic acid library molecules (100) with a quota of multiple single-stranded splint chains (200), wherein the contacting is performed under conditions suitable for hybridizing a first region (210) of the single-stranded splint chain with a first left universal adapter sequence (120) of the single-stranded library molecule, and the conditions are suitable for hybridizing a second region (220) of the single-stranded splint chain with a first right universal adapter sequence (130) of the single-stranded library molecule, thereby circularizing the library molecules to produce a population (300) of library-splint complexes having a gap between the 5' end of the library molecule and the 3' end of the library molecule (see, for example, Figure 1-Figure 5 ). In some embodiments, the gap is enzymatically ligatable.

[0389] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from a plurality of sample sources further comprises the step (d) of contacting the population of library-splint complexes (300) with a ligase in a separate compartment under conditions suitable for enzymatic ligation of the gaps, thereby generating a population of covalently closed circular library molecules (400) each hybridized to a single-stranded splint strand (200) (e.g., see Figure 6 A- Figure 6 C).

[0390] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (e): pooling populations of covalently closed circular library molecules (400) from separate compartments to generate a multiplex mixture of covalently closed circular library molecules (400) comprising the multiplex mixture of target sequences isolated from multiple sample sources.

[0391] In some embodiments, the sequence of interest can be separated from two or more different sample sources (e.g., 2-10, or 10-50, or 50-100, or 100-250, or more than 250 different sample sources). Exemplary nucleic acid sources include naturally occurring, recombinant, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or entire organisms. Exemplary nucleic acid sources include fresh, frozen, freshly frozen, or archived sources (e.g., formalin-fixed paraffin-embedded; FFPE). Technicians will recognize that nucleic acids can be separated from many other sources. The sequence of interest in a given population has the same or different sequences.

[0392] In some embodiments, the number of populations of single-stranded nucleic acid library molecules (100) in step (a) can be 2-10, or 10-50, or 50-100, or 100-250, or more than 250 different populations of single-stranded nucleic acid library molecules (100). In some embodiments, any number of different populations of covalently closed circular library molecules (400) can be pooled together in step (e), for example, 2-10, or 10-50, or 50-100, or 100-200, or more than 200 different populations of covalently closed circular library molecules (400) can be pooled together.

[0393] The skilled artisan will recognize that any number of separate compartments (e.g., 2-10, or 10-50, or 50-100, or 100-250, or more separate compartments) (e.g., a multi-well plate, such as, for example, a 96-well plate) may be used in step (a).

[0394] In some embodiments, the 3' end of the single-stranded splint chain (200) hybridized to the covalently closed circular library molecule (400) of step (d) or (e) comprises an extendable 3' OH end, which can serve as an initiation point for a primer extension reaction (eg, a rolling circle amplification reaction).

[0395] In some embodiments, in step (d) or (e), the population of covalently closed circular library molecules (400) hybridized to the single-stranded splint strands (200) can be optionally reacted with at least one exonuclease to remove the plurality of single-stranded splint strands (200) and retain the plurality of covalently closed circular library molecules (400). In some embodiments, the at least one exonuclease comprises Exonuclease I, Thermolabile Exonuclease I, and / or T7 Exonuclease.

[0396] In some embodiments, the single-stranded nucleic acid library molecule (100) of step (a) further comprises any one or any combination of two or more of the following: a universal binding sequence for a forward amplification primer; a universal binding sequence for a reverse amplification primer; and / or a universal binding sequence for a compacted oligonucleotide.

[0397] Non-traditional pooled workflows for multiplexing

[0398] In some embodiments, a method for preparing a multiplex mixture of target sequences isolated from multiple sample sources comprises: (a) providing two or more populations of single-stranded nucleic acid library molecules (100), each population of library molecules (100), each population of single-stranded nucleic acid library molecules contained in a separate compartment, wherein the nucleic acid library molecules of a given population comprise: (i) a first left universal adapter sequence (120) having a binding sequence for a second surface primer, (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer, (iii) a target sequence (110) isolated from a sample source, (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer, (v) a first right universal adapter sequence (130) having a binding sequence for a first surface primer, and (vii) a first left index sequence (160) and / or a first right index sequence (170), wherein the left index (160) and / or right (170) index sequences individually or in combination identify the sample source from which the target sequence (110) was isolated; (e.g., see Figure 3 and Figure 4 In some embodiments, the single-stranded nucleic acid library molecule (100) further comprises a first left unique identification sequence (180) and / or a first right unique identification sequence (190).

[0399] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (b): pooling two or more populations of single-stranded nucleic acid library molecules (100) into the same compartment.

[0400] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (c): adding a plurality of single-stranded splint chains (200) to the compartment of step (b), wherein each single-stranded splint chain (200) comprises a first region (210) and a second region (220), wherein the first region (210) can hybridize with the first left universal adapter sequence (120) of the single-stranded library molecule, and wherein the second region (220) can hybridize with the first right universal adapter sequence (130) of the single-stranded library molecule.

[0401] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (d): incubating a population of single-stranded nucleic acid library molecules (100) and a plurality of single-stranded splint chains (200) under conditions suitable for hybridizing a first region (210) of the single-stranded splint chain to a first left universal adapter sequence (120) of the single-stranded library molecule, and the conditions are suitable for hybridizing a second region (220) of the single-stranded splint chain to a first right universal adapter sequence (130) of the single-stranded library molecule, thereby circularizing the library molecules to produce a population of library-splint complexes (300) having a gap between the 5' end of the library molecule and the 3' end of the library molecule (e.g., see Figure 1-Figure 5 ). In some embodiments, the gap is enzymatically ligatable.

[0402] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from a plurality of sample sources further comprises step (e): contacting the population of library-splint complexes (300) with a ligase in the compartment of step (d) under conditions suitable for enzymatic ligation of the gaps, thereby generating a population of covalently closed circular library molecules (400) each hybridized to a single-stranded splint strand (200), thereby generating a multiplex mixture of covalently closed circular library molecules (400) comprising the multiplex mixture of target sequences isolated from a plurality of sample sources (e.g., see Figure 6 A- Figure 6 C).

[0403] In some embodiments, the sequence of interest can be separated from two or more different sample sources (e.g., 2-10, or 10-50, or 50-100, or 100-250, or more than 250 different sample sources). Exemplary nucleic acid sources include naturally occurring, recombinant, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or entire organisms. Exemplary nucleic acid sources include fresh, frozen, freshly frozen, or archived sources (e.g., formalin-fixed paraffin-embedded; FFPE). Technicians will recognize that nucleic acids can be separated from many other sources. The sequence of interest in a given population has the same or different sequences.

[0404] In some embodiments, the number of populations of single-stranded nucleic acid library molecules (100) in step (a) can be 2-10, or 10-50, or 50-100, or 100-250, or more than 250 different populations of single-stranded nucleic acid library molecules (100). In some embodiments, any number of different populations of single-stranded nucleic acid library molecules can be pooled together in step (b), for example, 2-10, or 10-50, or 50-100, or 100-200, or more than 200 different populations of covalently closed circular library molecules (400) can be pooled together.

[0405] The skilled artisan will recognize that any number of separate compartments (e.g., 2-10, or 10-50, or 50-100, or 100-250, or more separate compartments) (e.g., a multi-well plate, such as, for example, a 96-well plate) may be used in step (a).

[0406] In some embodiments, the 3' end of the single-stranded splint strand (200) hybridized to the covalently closed circular library molecule (400) of step (e) comprises an extendable 3' OH end, which can serve as an initiation point for a primer extension reaction (eg, a rolling circle amplification reaction).

[0407] In some embodiments, in step (e), the population of covalently closed circular library molecules (400) hybridized to the single-stranded splint strands (200) can optionally be reacted with at least one exonuclease to remove the plurality of single-stranded splint strands (200) and retain the plurality of covalently closed circular library molecules (400). In some embodiments, the at least one exonuclease comprises Exonuclease I, Thermolabile Exonuclease I, and / or T7 Exonuclease.

[0408] In some embodiments, the single-stranded nucleic acid library molecule (100) of step (a) further comprises any one or any combination of two or more of the following: a universal binding sequence for a forward amplification primer; a universal binding sequence for a reverse amplification primer; and / or a universal binding sequence for a compacted oligonucleotide.

[0409] Multiplex rolling circle amplification reaction on support

[0410] In some embodiments, any multivalent mixture of covalently closed circular library molecules (400) described herein can be dispensed onto a support, wherein the method comprises: dispensing a plurality of covalently closed circular library molecules (400) onto a support having a plurality of first surface primers immobilized thereon under conditions suitable for hybridizing each covalently closed circular library molecule (400) to each immobilized first surface primer, thereby immobilizing the plurality of covalently closed circular library molecules (400); and contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on the support using the plurality of first surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (400) as template molecules, thereby producing a plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, the population of covalently closed circular library molecules remains hybridized to the single-stranded splint strands (200) and partitioned onto the support. In some embodiments, the single-stranded splint strands (200) have been removed from the population of covalently closed circular library molecules (400) using at least one exonuclease under conditions suitable for removing a plurality of single-stranded splint strands (200) while retaining a population of covalently closed circular library molecules (400).

[0411] In-solution multiplex rolling circle amplification reactions using soluble amplification primers immobilized on a support

[0412] In some embodiments, any multivalent mixture of covalently closed circular library molecules (400) described herein can be subjected to an in-solution rolling circle amplification reaction, the method comprising: contacting a population of covalently closed circular library molecules (400) hybridized to single-stranded splint strands (200) with at least one exonuclease under conditions suitable for removing a plurality of single-stranded splint strands (200) and retaining a population of covalently closed circular library molecules (400); and contacting the retained population of covalently closed circular library molecules (400) with a plurality of solubl...

Claims

1. A library-splint complex (300), comprising: (i) a single-stranded nucleic acid library molecule (100) comprising a sequence of interest (110) flanked on one side by at least a first left universal adaptor sequence (120) and on another side by at least a first right universal adaptor sequence (130); and (ii) a single-stranded splint chain (200), comprising a first region (210) having a universal binding sequence for the first left universal adapter sequence (120) of the library molecule, and further comprising a second region (220) having a universal binding sequence for the first right universal adapter sequence (130) of the library molecule, thereby circularizing the library molecule to produce a library-splint complex (300), wherein the library-splint complex has a gap between the 5' end of the library molecule and the 3' end of the library molecule.

2. The library-splint complex (300) according to claim 1, wherein the nucleic acid library molecule (100) further comprises: a second left universal adapter sequence (140).

3. The library-splint complex (300) according to claim 1, wherein the nucleic acid library molecule (100) further comprises: a second right universal adapter sequence (150).

4. The library-splint complex (300) according to claim 1, wherein the nucleic acid library molecule (100) further comprises: a first left sample index sequence (160).

5. The library-splint complex (300) according to claim 1, wherein the nucleic acid library molecule (100) further comprises: a first right sample index sequence (170).

6. The library-splint complex (300) according to claim 1, wherein the nucleic acid library molecule (100) further comprises: a first left unique identification sequence (180).

7. The library-splint complex (300) according to claim 1, wherein the nucleic acid library molecule (100) further comprises: a first unique identification sequence (190).

8. The library-splint complex (300) according to claim 1, wherein the nucleic acid library molecule (100) further comprises any one of the following or any combination of two or more thereof: (i) a second left universal adaptor sequence (140); (ii) a second right universal adaptor sequence (150); (iii) a first left index sequence (160); (iv) a first right index sequence (170); (v) a first left unique identification sequence (180); and / or (vi) First right unique identification sequence (190).

9. The library-splint complex (300) according to claim 8, wherein the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) comprises: (i) universal binding sequence for forward sequencing primer; (ii) universal binding sequence for reverse sequencing primer; (iii) a universal binding sequence for the first surface primer; (iv) a universal binding sequence for a second surface primer; (v) universal binding sequence for forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and / or (vii) Universal binding sequences for compacted oligonucleotides.

10. The library-splint complex (300) of claim 8, wherein the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) comprises: (i) universal binding sequence for forward sequencing primer; (ii) universal binding sequence for reverse sequencing primer; (iii) a universal binding sequence for the first surface primer; (iv) a universal binding sequence for a second surface primer; (v) universal binding sequence for forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and / or (vii) Universal binding sequences for compacted oligonucleotides.

11. A method for producing a plurality of covalently closed circular library molecules (400), comprising: a) providing a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule of the plurality of single-stranded nucleic acid library molecules comprises a sequence of interest (110) flanked on one side by at least a first left universal adapter sequence (120) and on another side by at least a first right universal adapter sequence (130); b) providing a plurality of single-stranded splint chains (200), wherein each single-stranded splint chain (200) in the plurality of single-stranded splint chains comprises a first region (210) and a second region (220), wherein the first region is capable of hybridizing with the at least first left universal adapter sequence (120) of each library molecule, and the second region is capable of hybridizing with the at least first right universal adapter sequence (130) of each library molecule; c) hybridizing the plurality of single-stranded splint chains (200) with a plurality of single-stranded nucleic acid library molecules (100), such that the first region of one of the single-stranded splint chains (210) anneals to the at least first left universal adaptor sequence (120) of the library molecule, and such that the second region of the single-stranded splint chain (220) anneals to the at least first right universal sequence (130) of the library molecule, thereby circularizing each library molecule to form a plurality of library-splint complexes (300), wherein the library-splint complexes have a gap between the 5' and 3' ends of the library molecules, wherein the gap is enzymatically ligatable; and d) ligating the gaps in the plurality of library-splint complexes (300), thereby generating a plurality of covalently closed circular library molecules (400).

12. The method according to claim 11, further comprising: (e) distributing the plurality of covalently closed circular library molecules (400) onto a support having a plurality of surface primers immobilized on the support under conditions suitable for hybridizing each covalently closed circular library molecule (400) to each immobilized surface primer, thereby immobilizing the plurality of covalently closed circular library molecules (400) to the support.

13. The method according to claim 12, further comprising: (f) contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on the support using the plurality of surface primers as immobilized amplification primers and using the plurality of covalently closed circular library molecules (400) as template molecules, thereby producing a plurality of immobilized nucleic acid concatemer molecules.

14. The method according to claim 13, further comprising: The plurality of immobilized nucleic acid concatemer molecules are sequenced.

15. The method of claim 14, wherein the sequencing comprises: a) contacting a plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of composite polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises concatemer molecules hybridized to the soluble sequencing primers; b) contacting the plurality of composite sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the composite sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the 3' position of the sugar; c) incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; as well as d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

16. The method of claim 14, wherein the sequencing comprises: a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first composite polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises concatemer molecules hybridized to the soluble sequencing primers; b) contacting the plurality of composite sequencing polymerases with a plurality of detectably labeled multivalent molecules under conditions suitable for combining the complementary nucleotide units of the multivalent molecules with at least two of the plurality of first composite polymerases to form a plurality of multivalent-complex polymerases, thereby forming a plurality of multivalent-complex polymerases, and wherein the conditions inhibit incorporation of the complementary nucleotide units into sequencing primers of the plurality of multivalent-complex polymerases, wherein each multivalent molecule of the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; c) detecting the plurality of multivalent-complex polymerases; as well as d) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first complex polymerases among the plurality of multivalent-complex polymerases, thereby determining the sequence of the nucleic acid template.

17. The method according to claim 16, further comprising: e) dissociating the plurality of multivalent-complex polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining a 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 composite polymerases each comprising a second sequencing polymerase bound to the retained nucleic acid duplexes; as well as g) contacting the plurality of second composite polymerases with a plurality of nucleotides, wherein the contacting is carried out under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second composite polymerases of step (f), thereby forming a plurality of nucleotide-composite polymerases, and the conditions are suitable for promoting the incorporation of the bound complementary nucleotides into the sequencing primers of the nucleotide-composite polymerases.

18. The method according to claim 17, further comprising: h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase.

19. The method of claim 17, further comprising: h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase; as well as i) recognizing the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase.

20. A method of forming at least one affinity complex, comprising: a) combining a first universal nucleic acid primer, a first DNA polymerase and a first multivalent molecule with a first portion of the concatemer molecule according to claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first DNA polymerase; and b) combining a second universal nucleic acid primer, a second DNA polymerase, and the first multivalent molecule with a second portion of the same concatameric template molecule to form a second binding complex, wherein the second nucleotide unit of the first multivalent molecule binds to the second DNA polymerase, wherein the first binding complex and the second binding complex of the same multivalent molecule form an affinity complex, wherein the first multivalent molecule comprises a core attached to a plurality of nucleotide arms and each nucleotide arm is attached to a nucleotide unit, and wherein the concatameric molecule comprises two or more tandem repeats of a target sequence (110) and a universal primer binding site that binds the first universal nucleic acid primer and the second universal nucleic acid primer.

21. A method for sequencing by forming at least one affinity complex, comprising: a) combining a first universal nucleic acid primer, a first DNA polymerase and a first multivalent molecule with a first portion of the concatemer molecule according to claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first DNA polymerase; b) combining a second universal nucleic acid primer, a second DNA polymerase, and the first multivalent molecule with a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule binds to the second DNA polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex, wherein the first multivalent molecule comprises a core attached to a plurality of nucleotide arms and each nucleotide arm is attached to a nucleotide unit, wherein the concatemer molecule comprises two or more tandem repeats of a sequence of interest (110) and a universal primer binding site that binds the first universal nucleic acid primer and the second universal nucleic acid primer, and wherein the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first nucleotide unit and the second nucleotide unit in the first binding complex and the second binding complex; c) detecting the first binding complex and the second binding complex on the same concatemeric template molecule, and d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule.

22. The method according to claim 11, wherein the nucleic acid library molecule (100) further comprises: a second left universal adaptor sequence (140).

23. The method according to claim 11, wherein the nucleic acid library molecule (100) further comprises: a second right universal adaptor sequence (150).

24. The method according to claim 11, wherein the nucleic acid library molecule (100) further comprises: a first left sample index sequence (160).

25. The method according to claim 11, wherein the nucleic acid library molecule (100) further comprises: a first right sample index sequence (170).

26. The method according to claim 11, wherein the nucleic acid library molecule (100) further comprises: a first left unique identification sequence (180).

27. The method according to claim 11, wherein the nucleic acid library molecule (100) further comprises: a first unique identification sequence (190).

28. The method according to claim 11, wherein the nucleic acid library molecule (100) further comprises any one of the following or any combination of two or more thereof: (vii) a second left universal adaptor sequence (140); (viii) a second right universal adaptor sequence (150); (ix) a first left index sequence (160); (x) a first right index sequence (170); (xi) a first left unique identification sequence (180); and / or (xii) First right unique identification sequence (190).

29. The method of claim 28, wherein the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) comprises: (viii) universal binding sequence for forward sequencing primer; (ix) universal binding sequence for reverse sequencing primer; (x) a universal binding sequence for the first surface primer; (xi) a universal binding sequence for a second surface primer; (xii) a universal binding sequence for a forward amplification primer; (xiii) a universal binding sequence for a reverse amplification primer; and / or (xiv) Universal binding sequences for compacted oligonucleotides.

30. The method of claim 28, wherein the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) comprises: (viii) universal binding sequence for forward sequencing primer; (ix) universal binding sequence for reverse sequencing primer; (x) a universal binding sequence for the first surface primer; (xi) a universal binding sequence for a second surface primer; (xii) a universal binding sequence for a forward amplification primer; (xiii) a universal binding sequence for a reverse amplification primer; and / or (xiv) Universal binding sequences for compacted oligonucleotides.

31. A method for sequencing a nucleic acid molecule, comprising: a) providing a plurality of nucleic acid template molecules immobilized at random positions on a support, wherein each immobilized template molecule comprises an insert sequence region and a sample index, wherein each sample index comprises a 3-mer random sequence joined to a universal sample index sequence that identifies a sample source of the insert sequence, wherein different immobilized template molecules have different 3-mer random sequences and the same universal sample index sequence, and wherein the immobilized template molecules have different insert sequences; b) performing three cycles of polymerase-mediated sequencing reaction on the 3-mer random sequence of a plurality of fixed template molecules using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C and T / U, wherein the nucleotide reagents comprise a different detectable color label corresponding to each different type of nucleobase, wherein the three cycles of sequencing comprise detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to the fixed template molecules, thereby determining the sequence of the 3-mer random sequence in each of the plurality of fixed template molecules, and wherein in each of the first sequencing cycle, the second sequencing cycle and the third sequencing cycle, a balanced diversity of A, G, C and T / U nucleobases is detected and imaged among the plurality of fixed template molecules; and c) using the image obtained in step (b) to generate a map of the positions of the plurality of immobilized template molecules, wherein the sequence of the inserted region is not used to generate the map.

32. The method of claim 31 , wherein the balanced diversity of step (b) is 25-75% of each of the nucleobases A, G, C, and T / U detected and imaged in each of the first sequencing cycle, the second sequencing cycle, and the third sequencing cycle.

33. The method of claim 31, further comprising: d) sequencing the universal sample index sequence of the plurality of fixed template molecules; e) sequencing the insert sequence regions of the plurality of fixed template molecules; as well as f) assigning the insert sequence of the given template molecule obtained in step (b) to the universal sample index sequence from the same given template molecule obtained in step (a), thereby identifying the sample source of the given insert sequence.

34. The method of claim 31, wherein the plurality of nucleic acid template molecules further comprises a second sample index, the second sample index comprising a second universal sample index sequence identifying the sample source of the insert sequence, and the second sample index lacks a random sequence.

35. The method of claim 34, further comprising: g) sequencing the 3-mer random sequences of the plurality of immobilized template molecules to obtain a balanced diversity of A, G, C, and T / U nucleobases detected and imaged in each of the first sequencing cycle, the second sequencing cycle, and the third sequencing cycle, thereby generating a map of the positions of the plurality of immobilized template molecules; h) sequencing the first universal sample index sequence of the plurality of fixed template molecules; i) sequencing the second universal sample index sequence of the plurality of fixed template molecules; j) sequencing the insert sequence regions of the plurality of fixed template molecules; as well as k) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insert sequence.

36. A method for sequencing a nucleic acid molecule, comprising: a) providing a plurality of nucleic acid template molecules immobilized at random positions on a support, wherein each immobilized template molecule comprises an insert sequence region and a sample index, wherein each sample index comprises a 3-mer random sequence joined to a universal sample index sequence that identifies a sample source of the insert sequence, wherein the universal sample index sequence comprises 3-20 nucleotides, wherein different immobilized template molecules have different 3-mer random sequences and the same universal sample index sequence, and wherein the immobilized template molecules have different insert sequences; b) performing four cycles of polymerase-mediated sequencing reaction on the first base position of the 3-mer random sequence and the universal sample index sequence of the plurality of fixed template molecules using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C and T / U, wherein the nucleotide reagents comprise different detectable color labels corresponding to each different type of nucleobase, wherein the four cycles of sequencing include detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to the fixed template molecules, thereby determining the first base position of the universal sample index sequence and the sequence of the 3-mer random sequence in each of the plurality of fixed template molecules, and wherein in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle and the fourth sequencing cycle, a balanced diversity of A, G, C and T / U nucleobases is detected and imaged among the plurality of fixed template molecules; and c) using the images of the four cycles of polymerase-mediated sequencing reaction obtained in step (b) to generate a map of the positions of the plurality of immobilized template molecules, wherein the sequence of the insert region is not used to generate the map.

37. A method according to claim 36, wherein the balanced diversity of step (b) is 25-75% of each of the nucleobases A, G, C and T / U detected and imaged in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle and the fourth sequencing cycle.

38. The method of claim 36, further comprising: a) sequencing the remaining base positions of the universal sample index sequence of the plurality of fixed template molecules; b) sequencing the insert sequence regions of the plurality of fixed template molecules; as well as c) assigning the insert sequence of the given template molecule obtained in step (b) to the universal sample index sequence from the same given template molecule obtained in step (a), thereby identifying the sample source of the given insert sequence.

39. The method of claim 36, wherein the plurality of nucleic acid template molecules further comprises a second sample index comprising a second universal sample index sequence identifying the sample source of the insert sequence, and the second sample index lacks a random sequence.

40. The method of claim 39, further comprising: a) sequencing the 3-mer random sequences of the plurality of fixed template molecules and the first base positions of the universal sample index sequence to obtain a balanced diversity of A, G, C and T / U nucleobases detected and imaged in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle and the fourth sequencing cycle, thereby generating a map of the positions of the plurality of fixed template molecules; b) sequencing the remaining base positions of the first universal sample index sequence of the plurality of fixed template molecules; c) sequencing the second universal sample index sequence of the plurality of fixed template molecules; d) sequencing the insert sequence regions of the plurality of fixed template molecules; as well as e) assigning the insertion sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insertion sequence.

41. The method of any one of claims 30 to 40, wherein the support comprises a glass or plastic substrate.

42. The method of any one of claims 30 to 40, wherein the support is disposed on a flow cell channel, a flow cell, or a capillary lumen.

43. The method of any one of claims 30 to 40, wherein the support is passivated with at least one hydrophilic polymer coating having a water contact angle of no greater than 45 degrees.

44. The method of claim 43, wherein the at least one hydrophilic polymer coating comprises 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.

45. The method of claim 43, wherein the at least one hydrophilic polymer coating comprises branched hydrophilic polymer molecules having at least four branches.

46. ​​The method of claim 43, wherein the at least one hydrophilic polymer coating comprises polymer molecules having a molecular weight of at least 1000 Daltons.

47. The method of any one of claims 30 to 40, wherein the immobilized template molecules comprise a plurality of immobilized concatemer molecules having tandem repeats of the one sample index and the insert sequence.

48. The method of any one of claims 30 to 40, wherein the fixed template molecules comprise a plurality of different clustered template molecules having one copy of the insert sequence and one copy of the one sample index, wherein the clustered template molecules are generated via bridge amplification.

49. The method according to any one of claims 30 to 40, wherein the density of the immobilized nucleic acid template molecules at random positions on the support is 10 4 -10 8 Pieces / mm 2 .

50. The method of any one of claims 30 to 40, wherein the sample source of the insert sequence is genomic DNA, double-stranded cDNA, or cell-free circulating DNA.

51. The method of any one of claims 30 to 40, wherein the detectably labeled nucleotide reagents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups, and a fluorophore.

52. The method of any one of claims 30 to 40, wherein the detectably labeled nucleotide reagents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety having a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups, and a fluorophore.

53. A method according to any one of claims 30 to 40, wherein the detectably labeled nucleotide agent comprises a multivalent molecule, each multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

54. A method according to any one of claims 30 to 40, wherein the detectably labeled nucleotide reagent bound to the fixed template molecule comprises individual fixed template molecules that hybridize with a sequencing primer to form a duplex, and the duplex is bound to a polymerase to form a composite polymerase, and the composite polymerase is bound to a detectably labeled nucleotide reagent.

55. A method according to claim 54, wherein the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into the hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups and a fluorophore.

56. A method according to claim 54, wherein the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into the hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety with a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups and a fluorophore.

57. The method of claim 54, wherein the composite polymerase binds to the detectably labeled nucleotide agent under conditions suitable for binding the detectably labeled nucleotide agent to the composite polymerase, and the conditions are suitable for inhibiting nucleotide incorporation, wherein the detectably labeled nucleotide agent comprises a multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

58. The method of claim 54, wherein the immobilized template molecules comprise immobilized concatemer molecules that hybridize to a plurality of sequencing primers to form at least a first duplex and a second duplex on the same concatemer molecule, wherein the first and second duplexes bind to a first polymerase and the second duplexes bind to a second polymerase to form a first composite polymerase and a second composite polymerase, and wherein the method comprises: a) contacting a plurality of multivalent molecules with the first composite polymerase and the second composite polymerase on the same concatameric template molecule, wherein each multivalent molecule comprises (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, and the spacer is attached to the linker, and the linker is attached to the nucleotide unit, wherein the contacting is performed under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first composite polymerase and the second composite polymerase, wherein a first nucleotide unit of the single multivalent molecule binds to the first composite polymerase comprising the first sequencing primer hybridized to a first portion of the concatameric template molecule, thereby forming a first bound complex, and wherein a second nucleotide unit of the single multivalent molecule binds to the second composite polymerase comprising the second sequencing primer hybridized to a second portion of the concatameric template molecule, thereby forming a second bound complex, and wherein said first binding complex and said second binding complex bound to the same multivalent molecule form an affinity complex, wherein said contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first nucleotide unit and the second nucleotide unit in said first binding complex and said second binding complex; b) detecting the first binding complex and the second binding complex on the same concatemeric template molecule; c) imaging the optical color signal emitted from the detectably labeled multivalent molecule that forms the first binding complex and the second binding complex on the same concatemer template molecule; as well as d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule.

59. A method for sequencing a nucleic acid molecule, comprising: a) providing a first plurality of library molecules, each of the plurality of library molecules comprising (i) an insert sequence region derived from a first sample source, (ii) a first sample index having a 3-mer random sequence joined to a first universal sample index sequence, and (iii) a second sample index having a second universal sample index sequence, the second sample index lacking the random sequence, wherein a combination of the first universal sample index sequence and the second universal sample index sequence uniquely identifies the first sample source of the insert sequence, wherein different first library molecules have different 3-mer random sequences and have different insert sequences; b) providing a second plurality of library molecules, each of the plurality of library molecules comprising (i) an insert sequence region derived from a second sample source, (ii) a third sample index having a 3-mer random sequence joined to a third universal sample index sequence, and (iii) a fourth sample index having a fourth universal sample index sequence, the fourth sample index lacking a random sequence, wherein a combination of the third universal sample index sequence and the fourth universal sample index sequence uniquely identifies the second sample source of the insert sequence, wherein different second library molecules have different 3-mer random sequences and have different insert sequences; c) pooling said first plurality of library molecules and said second plurality of library molecules; d) distributing the pooled library molecules onto a support and performing an amplification reaction to generate a plurality of clonally amplified template molecules immobilized at random locations on the support; e) performing three cycles of polymerase-mediated sequencing reaction on the 3-mer random sequence of the first sample index and the third sample index using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C and T / U, wherein the nucleotide reagents comprise a different detectable color label corresponding to each different type of nucleobase, wherein the three cycles of sequencing comprise detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to fixed amplification template molecules, thereby determining the sequence of the 3-mer random sequence in each of the plurality of fixed template molecules, and wherein in each of the first sequencing cycle, the second sequencing cycle and the third sequencing cycle, a balanced diversity of A, G, C and T / U nucleobases is detected and imaged among the plurality of fixed amplification template molecules; and f) generating a map of the positions of the plurality of immobilized template molecules using the image obtained in step (e), wherein the sequence of the inserted region is not used to generate the map.

60. The method of claim 1, wherein the balanced diversity of step (e) is 25-75% of each of the nucleobases A, G, C, and T / U detected and imaged in each of the first sequencing cycle, the second sequencing cycle, and the third sequencing cycle.

61. The method of claim 59, further comprising: g) sequencing the first universal sample index sequence of the plurality of fixed template molecules; h) sequencing the second universal sample index sequence of the plurality of fixed template molecules; i) sequencing the insert sequence regions of the plurality of immobilized template molecules derived from the first library molecules; as well as j) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence derived from the same given template molecule, thereby identifying the first sample source of the given insert sequence.

62. The method of claim 59, further comprising: g) sequencing the third universal sample index sequence of the plurality of fixed template molecules; h) sequencing the fourth universal sample index sequence of the plurality of immobilized template molecules; i) sequencing the insert sequence regions of the plurality of immobilized template molecules derived from the second library molecules; as well as j) assigning the insert sequence of the given template molecule obtained in step (c) to the third universal sample index sequence and the fourth universal sample index sequence derived from the same given template molecule, thereby identifying the second sample source of the given insert sequence.

63. A method for sequencing a nucleic acid molecule, comprising: a) providing a first plurality of library molecules, each of the plurality of library molecules comprising (i) an insert sequence region derived from a first sample source, (ii) a first sample index having a 3-mer random sequence joined to a first universal sample index sequence, and (iii) a second sample index having a second universal sample index sequence, the second sample index lacking the random sequence, wherein a combination of the first universal sample index sequence and the second universal sample index sequence uniquely identifies the first sample source of the insert sequence, wherein the first universal sample index sequence comprises 3-20 nucleotides, wherein different first library molecules have different 3-mer random sequences and have different insert sequences; b) providing a second plurality of library molecules, each of the plurality of library molecules comprising (i) an insert sequence region derived from a second sample source, (ii) a third sample index having a 3-mer random sequence joined to a third universal sample index sequence, and (iii) a fourth sample index having a fourth universal sample index sequence, the fourth sample index lacking the random sequence, wherein a combination of the third universal sample index sequence and the fourth universal sample index sequence uniquely identifies the second sample source of the insert sequence, wherein the third universal sample index sequence comprises 3-20 nucleotides, wherein different second library molecules have different 3-mer random sequences and have different insert sequences; c) pooling said first plurality of library molecules and said second plurality of library molecules; d) distributing the pooled library molecules onto a support and performing an amplification reaction to generate a plurality of clonally amplified template molecules immobilized at random locations on the support; e) performing four cycles of polymerase-mediated sequencing reaction on the 3-mer random sequence of the first sample sequence and the third sample index using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C and T / U, and sequencing the first base position of the first universal sample index sequence and the third universal sample index sequence, wherein the nucleotide reagents comprise a different detectable color label corresponding to each different type of nucleobase, wherein the three cycles of sequencing include detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to fixed amplification template molecules, thereby determining the sequence of the 3-mer random sequence in each of the plurality of fixed template molecules, and wherein in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle and the fourth sequencing cycle, a balanced diversity of A, G, C and T / U nucleobases is detected and imaged among the plurality of fixed amplification template molecules; and f) generating a map of the positions of the plurality of immobilized template molecules using the image obtained in step (e), wherein The sequence of the inserted regions is not used to generate the map.

64. The method of claim 63, wherein the balanced diversity of step (e) is 25-75% of each of the nucleobases A, G, C, and T / U detected and imaged in each of the first sequencing cycle, the second sequencing cycle, the third sequencing cycle, and the fourth sequencing cycle.

65. The method of claim 63, further comprising: g) sequencing the remaining base positions of the first universal sample index sequence of the plurality of fixed template molecules; h) sequencing the second universal sample index sequence of the plurality of fixed template molecules; i) sequencing the insert sequence regions of the plurality of immobilized template molecules derived from the first library molecules; as well as j) assigning the insert sequence of the given template molecule obtained in step (c) to the first universal sample index sequence and the second universal sample index sequence derived from the same given template molecule, thereby identifying the first sample source of the given insert sequence.

66. The method of claim 59, further comprising: g) sequencing the remaining base positions of the third universal sample index sequence of the plurality of fixed template molecules; h) sequencing the fourth universal sample index sequence of the plurality of immobilized template molecules; i) sequencing the insert sequence regions of the plurality of immobilized template molecules derived from the second library molecules; as well as j) assigning the insert sequence of the given template molecule obtained in step (c) to the third universal sample index sequence and the fourth universal sample index sequence derived from the same given template molecule, thereby identifying the second sample source of the given insert sequence.

67. The method of any one of claims 59 to 66, wherein the support comprises a glass or plastic substrate.

68. The method of any one of claims 59 to 66, wherein the support is disposed on a flow cell channel, a flow cell, or a capillary lumen.

69. The method of any one of claims 59 to 66, wherein the support is passivated with at least one hydrophilic polymer coating having a water contact angle of no greater than 45 degrees.

70. The method of claim 69, wherein the at least one hydrophilic polymer coating comprises 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.

71. The method of claim 69, wherein the at least one hydrophilic polymer coating comprises branched hydrophilic polymer molecules having at least four branches.

72. The method of claim 69, wherein the at least one hydrophilic polymer coating comprises polymer molecules having a molecular weight of at least 1000 Daltons.

73. The method of any one of claims 59 to 66, wherein the fixed template molecules comprise a plurality of fixed concatemer molecules having tandem repeats of the one sample index and the insert sequence.

74. The method of any one of claims 59 to 66, wherein the fixed template molecules comprise a plurality of different clustered template molecules having one copy of the insert sequence and one copy of the one sample index, wherein the clustered template molecules are generated via bridge amplification.

75. The method of any one of claims 59 to 66, wherein the density of the immobilized nucleic acid template molecules at random positions on the support is 10 4 -10 8 Pieces / mm 2 .

76. The method of any one of claims 59 to 66, wherein the sample source of the insert sequence is genomic DNA, double-stranded cDNA, or cell-free circulating DNA.

77. The method of any one of claims 59 to 66, wherein the detectably labeled nucleotide reagents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups, and a fluorophore.

78. The method of any one of claims 59 to 66, wherein the detectably labeled nucleotide agents comprise nucleotides each comprising an aromatic nucleobase, a pentose moiety having a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups, and a fluorophore.

79. A method according to any one of claims 59 to 66, wherein the detectably labeled nucleotide agent comprises a multivalent molecule, each multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

80. A method according to any one of claims 59 to 66, wherein the detectably labeled nucleotide reagent bound to the fixed template molecule comprises individual fixed template molecules that hybridize with a sequencing primer to form a duplex, and the duplex is bound to a polymerase to form a composite polymerase, and the composite polymerase is bound to the detectably labeled nucleotide reagent.

81. A method according to claim 80, wherein the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into a hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety, 1-10 phosphate groups and a fluorophore.

82. The method of claim 80, wherein the composite polymerase binds to the detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the composite polymerase and incorporating the detectably labeled nucleotide into a hybridized sequencing primer, wherein the detectably labeled nucleotide reagent comprises an aromatic nucleobase, a pentose moiety having a chain terminating group at the 3' carbon sugar position, 1-10 phosphate groups, and a fluorophore.

83. The method of claim 80, wherein the composite polymerase binds to the detectably labeled nucleotide agent under conditions suitable for binding the detectably labeled nucleotide agent to the composite polymerase, and the conditions are suitable for inhibiting nucleotide incorporation, wherein the detectably labeled nucleotide agent comprises a multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

84. The method of claim 80, wherein the immobilized template molecules comprise immobilized concatemer molecules that hybridize to a plurality of sequencing primers to form at least a first duplex and a second duplex on the same concatemer molecule, wherein the first and second duplexes bind to a first polymerase and the second duplexes bind to a second polymerase to form a first composite polymerase and a second composite polymerase, and wherein the method comprises: a) contacting a plurality of multivalent molecules with the first composite polymerase and the second composite polymerase on the same concatameric template molecule, wherein each multivalent molecule comprises (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, wherein each nucleotide arm comprises (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, and the spacer is attached to the linker, and the linker is attached to the nucleotide unit, wherein the contacting is performed under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first composite polymerase and the second composite polymerase, wherein a first nucleotide unit of the single multivalent molecule binds to the first composite polymerase comprising the first sequencing primer hybridized to a first portion of the concatameric template molecule, thereby forming a first bound complex, and wherein a second nucleotide unit of the single multivalent molecule binds to the second composite polymerase comprising the second sequencing primer hybridized to a second portion of the concatameric template molecule, thereby forming a second bound complex, and wherein said first binding complex and said second binding complex bound to the same multivalent molecule form an affinity complex, wherein said contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first nucleotide unit and the second nucleotide unit in said first binding complex and said second binding complex; b) detecting the first binding complex and the second binding complex on the same concatemeric template molecule; c) imaging the optical color signal emitted from the detectably labeled multivalent molecule that forms the first binding complex and the second binding complex on the same concatemer template molecule; as well as d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule.

85. A method for generating a plurality of library-splint complexes (300), comprising: a) providing a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule in the plurality of single-stranded nucleic acid libraries comprises regions arranged in 5' to 3' order: (i) a first left universal adapter sequence (120) having a binding sequence for a first surface primer; (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer; (iii) a target sequence (110); (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer; and (v) a first right universal adapter sequence (130) having a binding sequence for a second surface primer; b) providing a plurality of single-stranded splint chains (200), wherein each single-stranded splint chain (200) in the plurality of single-stranded splint chains comprises regions arranged in 5′ to 3′ order: (i) a first region (210) having a universal binding sequence, which hybridizes with the first left universal adapter sequence (120) of the linear single-stranded library molecule (100), and (ii) a second region (220) having a universal binding sequence, which hybridizes with the first right universal adapter sequence (130) of the linear single-stranded library molecule (100); c) hybridizing the multiple single-stranded splint chains (200) with the multiple single-stranded nucleic acid library molecules (100) under conditions suitable for hybridizing the first region (210) of the single-stranded splint chain (200) with the first left universal adapter sequence (120) of the linear single-stranded library molecule (100) and suitable for hybridizing the second region (220) of the single-stranded splint chain (200) with the first right universal adapter sequence (130) of the linear single-stranded library molecule (100), thereby circularizing each library molecule to form a plurality of library-splint complexes (300), wherein the plurality of library-splint complexes have a gap between the 5' and 3' ends of the library molecules, wherein the gap is enzymatically ligatable.

86. The method of claim 85, wherein the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left index sequence (160) and / or a first right index sequence (170).

87. The method according to claim 85, wherein the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left unique identification sequence (180) and / or a first right unique identification sequence (190).

88. The method of claim 85, further comprising: d) ligating the gaps in each library-splint complex (300), thereby generating a plurality of covalently closed circular library molecules (400).

89. The method of claim 88, further comprising: e) contacting the plurality of covalently closed circular library molecules (400) with at least one exonuclease to remove the plurality of single-stranded splint chains (200) and retain the plurality of covalently closed circular library molecules (400).

90. The method of claim 89, wherein each covalently closed circular library molecule (400) of the plurality of covalently closed circular library molecules comprises a first right universal adaptor sequence (130) having a binding sequence for a second surface primer, and wherein the method further comprises: f) distributing the plurality of covalently closed circular library molecules (400) onto the support having the plurality of second surface primers immobilized on the support under conditions suitable for hybridizing each covalently closed circular library molecule (400) to each immobilized second surface primer, thereby immobilizing the plurality of covalently closed circular library molecules (400) to the support.

91. The method of claim 90, further comprising: g) contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on the support using the plurality of second surface primers as immobilized amplification primers and using the plurality of covalently closed circular library molecules (400) as template molecules, thereby producing a plurality of immobilized nucleic acid concatemer molecules.

92. The method of claim 91, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP and / or dUTP.

93. The method of claim 90, wherein the plurality of immobilized second surface capture primers on the support are located at predetermined or random positions on the support.

94. A method according to claim 90, wherein the multiple fixed second surface capture primers on the support are fluidically connected to each other to allow a solution of a reagent (e.g., an enzyme, a nucleotide, a divalent cation, etc.) to flow onto the support so that the multiple fixed second surface primers can react with the reagent in a massively parallel manner essentially simultaneously.

95. The method of claim 91, wherein the density of the plurality of immobilized nucleic acid concatemer molecules on the support is 10 4 -10 8 Pieces / mm 2 .

96. The method of claim 91, further comprising: The plurality of fixed concatemers are sequenced, wherein the sequencing comprises: a) contacting a plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of composite polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises concatemer molecules hybridized to the soluble sequencing primers; b) contacting the plurality of composite sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the composite sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the 3' position of the sugar; c) incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

97. A method according to claim 96, wherein the plurality of nucleotides comprises a removable chain-terminating portion at the 3' sugar group, wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azido 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 wherein the removable chain-terminating portion can be cleaved with a chemical compound to produce an extendable 3' OH portion on the sugar group.

98. The method of claim 96, wherein the plurality of nucleotides comprises a type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

99. The method of claim 96, wherein 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, dTTP and / or dUTP.

100. The method of claim 91, further comprising: The plurality of fixed concatemers are sequenced, wherein the sequencing comprises: a) contacting a plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble universal sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first composite polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises concatemer molecules hybridized to the soluble sequencing primers; b) contacting the plurality of composite sequencing polymerases with a plurality of detectably labeled multivalent molecules under conditions suitable for combining the complementary nucleotide units of the multivalent molecules with at least two of the plurality of first composite polymerases to form a plurality of multivalent-complex polymerases, thereby forming a plurality of multivalent-complex polymerases, and wherein the conditions inhibit incorporation of the complementary nucleotide units into sequencing primers of the plurality of multivalent-complex polymerases, wherein each multivalent molecule of the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; c) detecting the plurality of multivalent-complex polymerases; and d) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first complex polymerases among the plurality of multivalent-complex polymerases, thereby determining the sequence of the nucleic acid template.

101. The method of claim 100, further comprising: e) dissociating the plurality of multivalent-complex polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining a 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 composite polymerases each comprising a second sequencing polymerase bound to the retained nucleic acid duplexes; g) contacting a plurality of second composite polymerases with a plurality of nucleotides, said plurality of nucleotides comprising at least one nucleotide analog having a removable chain terminating portion at the 3' position of the sugar, wherein said contacting is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of said second composite polymerases of step (f), thereby forming a plurality of nucleotide-composite polymerases, and said conditions are suitable for promoting incorporation of the bound complementary nucleotides into said sequencing primers of said nucleotide-composite polymerases.

102. The method of claim 101, further comprising: h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase.

103. The method of claim 101, further comprising: h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase; as well as i) recognizing the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complex polymerase.

104. A method for sequencing by forming at least one affinity complex, comprising: a) combining a first universal sequencing primer, a first sequencing polymerase, and a first detectably labeled multivalent molecule with a first portion of the concatemer molecule of claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first sequencing polymerase; b) combining a second universal sequencing primer, a second sequencing polymerase, and the first detectably labeled multivalent molecule with a second portion of the same concatemer molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex, wherein the first detectably labeled multivalent molecule comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit, wherein the concatemer molecule comprises two or more tandem repeats of a sequence of interest (110) and a universal primer binding site that binds the first universal sequencing primer and the second universal sequencing primer, and wherein the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first nucleotide unit and the second nucleotide unit in the first binding complex and the second binding complex; and c) detecting the first binding complex and the second binding complex on the same concatemer molecule.

105. The method of claim 100, wherein the plurality of nucleotide arms attached to the core of the respective multivalent molecules have the same type of nucleotide units, and wherein the type of nucleotide units is selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

106. The method of claim 100, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

107. The method of claim 101, wherein the plurality of nucleotides comprises a removable chain-terminating portion at the 3' sugar group, wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azido 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 wherein the removable chain-terminating portion is cleavable with a chemical compound to produce an extendable 3' OH portion on the sugar group.

108. The method of claim 101, wherein the plurality of nucleotides comprises a type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

109. The method of claim 101, wherein 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, dTTP and / or dUTP.

110. The method of claim 90, wherein the support comprises a glass or plastic substrate.

111. The method of claim 90, wherein the support is passivated with at least one hydrophilic polymer coating having a water contact angle of no greater than 45 degrees.

112. The method of claim 111, wherein the at least one hydrophilic polymer coating comprises 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.

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