Single strand splint leash and method of use

Abstract

The disclosure provides compositions, including kits, that include nucleic acid single-stranded splint strands, and methods of using the single-stranded splint strands, which can hybridize to portions of linear library molecules to form circularized library-splint complexes with nicks, which can be ligated to form covalently closed circular molecules, which can be subjected to downstream amplification and sequencing workflows.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 316,790, filed March 4, 2022, and U.S. Patent Application No. 17 / 725,042, filed April 20, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Incorporation by Reference of Sequence Listing 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 their entirety.

[0003] The present disclosure provides compositions comprising nucleic acid single-stranded splint strands and methods for using the single-stranded splint strands, which can hybridize to portions of library molecules to form library-splint complexes having nicks, which can be ligated to form covalently closed circular molecules that can be subjected to downstream amplification and sequencing workflows. [Background technology]

[0004] The present disclosure relates to the preparation and sequencing of polynucleotides from libraries of covalently closed circular molecules. Improvements in next-generation sequencing technology have greatly increased sequencing speed and data output, resulting in high sample throughput for current sequencing platforms. Efficient preparation of closed circular library molecules with target sequences is important for downstream amplification and sequencing workflows. Another way to increase sequencing throughput is to add unique index sequences to DNA fragments during library preparation, which allows multiple libraries to be pooled and sequenced simultaneously during each sequencing run. Therefore, there is a need for alternative methods for producing and sequencing circular library molecules containing target sequences and unique index sequences that are compatible with downstream next-generation sequencing technologies. Compositions, methods, and kits that address this need are provided herein. Summary of the Invention

[0005] The present disclosure provides a library-sprint 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 strand (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 circularizing the library molecule to generate a library-sprint complex (300) having a nick between the 5' end of the library molecule and the 3' end of the library molecule.

[0006] In some embodiments, the nucleic acid library molecule (100) further comprises a second left universal adaptor sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises a second right universal adaptor 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 identifier sequence (180). In some embodiments, the nucleic acid library molecule (100) further comprises a first right unique identifier sequence (190).

[0007] In some embodiments, the nucleic acid library molecule (100) further comprises any one or any combination of two or more of: (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 identifier sequence (180), and / or (vi) a first right unique identifier sequence (190).

[0008] In some embodiments, the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) comprise (i) a universal binding sequence for the forward sequencing primer, (ii) a universal binding sequence for the reverse sequencing primer, (iii) a universal binding sequence for the first surface primer, (iv) a universal binding sequence for the second surface primer, (v) a universal binding sequence for the forward amplification primer, (vi) a universal binding sequence for the reverse amplification primer, and / or (vii) a universal binding sequence for the compaction oligonucleotide.

[0009] In some embodiments, the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) comprise (i) a universal binding sequence for the forward sequencing primer, (ii) a universal binding sequence for the reverse sequencing primer, (iii) a universal binding sequence for the first surface primer, (iv) a universal binding sequence for the second surface primer, (v) a universal binding sequence for the forward amplification primer, (vi) a universal binding sequence for the reverse amplification primer, and / or (vii) a universal binding sequence for the compaction oligonucleotide.

[0010] 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), each of which comprises 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 b) providing a plurality of single-stranded splint strands (200), each of which comprises a first region (210) capable of hybridizing with at least the first left universal adaptor sequence (120) of an individual library molecule and a second region (220) capable of hybridizing with at least the first right universal adaptor sequence (130) of an individual library molecule. c) hybridizing a plurality of single-stranded splint strands (200) with a plurality of single-stranded nucleic acid library molecules (100) such that a first region of one of the single-stranded splint strands (210) anneals to at least a first left universal adaptor sequence (120) of the library molecule and a second region of the single-stranded splint strand (220) anneals to at least a first right universal sequence (130) of the library molecule, thus circularizing individual library molecules to form a plurality of library-splint complexes (300) having a nick between the 5' and 3' ends of the library molecule, wherein the nick is enzymatically ligatable; and d) ligating the nicks in the plurality of library-splint complexes (300), thereby generating a plurality of covalently closed circular library molecules (400).

[0011] 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 thereon under conditions suitable for hybridizing each of the covalently closed circular library molecules (400) to each of the immobilized surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (400) to the support.

[0012] In some embodiments, the method further comprises (f) contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand-displacing 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 the plurality of covalently closed circular library molecules (400) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules.

[0013] In some embodiments, the method further comprises sequencing the plurality of immobilized concatemeric nucleic acid molecules. In some embodiments, the sequencing comprises a) contacting the plurality of immobilized concatemeric 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 multiplexed polymerases, each of the multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, and the nucleic acid duplex comprising the concatemeric molecules hybridized to the soluble sequencing primer; and b) contacting the plurality of multiplexed sequencing polymerases with a plurality of nucleotides and at least one nucleotide. a) contacting a plurality of nucleotides comprising at least one nucleotide analog, at least one nucleotide analog labeled with a fluorophore and having a removable chain-terminating moiety at the sugar 3' position, with a multiplexed sequencing polymerase under conditions suitable for binding of the plurality of nucleotides to a multiplexed sequencing polymerase; b) incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; and c) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0014] In some embodiments, sequencing comprises a) contacting a plurality of immobilized concatemeric 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 multiplexed polymerases, each of the plurality of first multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, and the nucleic acid duplexes comprising concatemeric molecules hybridized to the soluble sequencing primers; and b) contacting the plurality of multiplexed sequencing polymerases with a plurality of detectably labeled multivalent molecules, wherein complementary nucleotide units of the multivalent molecules are hybridized to at least one of the plurality of first multiplexed polymerases. and contacting the first and second polymerases under conditions suitable for binding the first and second polymerases together to form a plurality of multivalent composite polymerases, where the conditions inhibit the incorporation of complementary nucleotide units into the sequencing primers of the plurality of multivalent composite polymerases, and where each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit; c) detecting the plurality of multivalent composite polymerases; and d) identifying the nucleobases of the complementary nucleotide units bound to a plurality of first composite polymerases in the plurality of multivalent composite polymerases, thereby determining the sequence of the nucleic acid template.

[0015] In some embodiments, the sequencing comprises: e) dissociating the plurality of multivalent composite polymerases to remove the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; and 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 of the plurality of second composite polymerases being a retained nucleic acid duplex. and g) contacting the plurality of second multiplexed polymerases with the plurality of nucleotides, wherein the contacting is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second multiplexed polymerases of step (f), thereby forming a plurality of nucleotide-complexed polymerases, and the conditions are suitable for promoting incorporation of the bound complementary nucleotides into the sequencing primer by the nucleotide-complexed polymerases.

[0016] In some embodiments, the sequencing further comprises h) detecting a complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase. In some embodiments, the sequencing further comprises h) detecting a complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase, and i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.

[0017] The present disclosure provides a method for forming at least one avidity 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 concatemeric molecule of claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first DNA polymerase; and b) binding a second universal nucleic acid primer, a second DNA polymerase, and the first multivalent molecule to a second portion of the same concatemeric template molecule, thereby forming a first binding complex. forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to a second DNA polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex, wherein the first multivalent molecule comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit, and the concatemeric molecule comprises a sequence of interest (110) and two or more tandem repeat sequences of universal primer binding sites that bind to the first and second universal nucleic acid primers.

[0018] The present disclosure provides a method for sequencing by forming at least one avidity complex, the method comprising: a) binding a first universal nucleic acid primer, a first DNA polymerase, and a first multivalent molecule to a first portion of a concatemeric molecule of claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first DNA polymerase; and b) binding a second universal nucleic acid primer, a second DNA polymerase, and the first multivalent molecule to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second DNA polymerase. The first and second binding complexes comprising the same multivalent molecule form an avidity complex, wherein the first multivalent molecule binds to a plurality of nucleotide arms. a) forming a concatemeric template molecule comprising a sequence of interest (110) and two or more tandem repeat sequences of universal primer binding sites that bind to first and second universal nucleic acid primers; b) detecting the first and second binding complexes on the same concatemeric template molecule; and c) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of a first portion of the concatemeric template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of a second portion of the concatemeric template molecule.

[0019] In some embodiments, the nucleic acid library molecule (100) further comprises a second left universal adaptor sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises a second right universal adaptor 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 identifier sequence (180). In some embodiments, the nucleic acid library molecule (100) further comprises a first right unique identifier sequence (190).

[0020] In some embodiments, the nucleic acid library molecule (100) further comprises any one or any combination of two or more of: (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 identifier sequence (180), and / or (vi) a first right unique identifier sequence (190).

[0021] In some embodiments, the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) comprise (i) a universal binding sequence for the forward sequencing primer, (ii) a universal binding sequence for the reverse sequencing primer, (iii) a universal binding sequence for the first surface primer, (iv) a universal binding sequence for the second surface primer, (v) a universal binding sequence for the forward amplification primer, (vi) a universal binding sequence for the reverse amplification primer, and / or (vii) a universal binding sequence for the compaction oligonucleotide.

[0022] In some embodiments, the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) comprise (i) a universal binding sequence for the forward sequencing primer, (ii) a universal binding sequence for the reverse sequencing primer, (iii) a universal binding sequence for the first surface primer, (iv) a universal binding sequence for the second surface primer, (v) a universal binding sequence for the forward amplification primer, (vi) a universal binding sequence for the reverse amplification primer, and / or (vii) a universal binding sequence for the compaction oligonucleotide.

[0023] The present disclosure provides a method for sequencing nucleic acid molecules, comprising: a) providing a plurality of nucleic acid template molecules immobilized at random locations on a support, each immobilized template molecule comprising an insert sequence region and one sample index, each sample index comprising a three-base random sequence linked to a universal sample index sequence that identifies the sample source of the insert sequence, different immobilized template molecules having different three-base random sequences and the same universal sample index sequence, and the immobilized template molecules having different insert sequences; and b) performing three cycles of polymerase-mediated sequencing reactions of the three-base random sequences of the plurality of immobilized template molecules using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleic acid bases A, G, C, and T / U. the nucleotide reagents comprise different detectable color labels corresponding to each different type of nucleobase, and three cycles of sequencing comprise detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to the immobilized template molecules, thereby determining the sequence of the three-base long random sequence in each of the plurality of immobilized template molecules, wherein a balanced diversity of A, G, C, and T / U nucleobases is detected and imaged among the plurality of immobilized template molecules in each of the first, second, and third sequencing cycles; and c) generating a map of the locations of the plurality of immobilized template molecules using the images obtained in step (b), wherein the sequence of the insertion region is not used to generate the map. In some embodiments, the balanced diversity in step (b) is 25-75% of each of the A, G, C, and T / U nucleobases detected and imaged in each of the first, second, and third sequencing cycles.

[0024] In some embodiments, the method further includes d) sequencing the universal sample index sequences of the plurality of immobilized template molecules; e) sequencing the insert sequence regions of the plurality of immobilized 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.

[0025] In some embodiments, the plurality of nucleic acid template molecules further comprises a second sample index, wherein the second sample index comprises a second universal sample index sequence that identifies the sample source of the insert sequence, and the second sample index lacks random sequence.

[0026] In some embodiments, the method further includes g) sequencing the 3-base long 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, second, and third sequencing cycles to generate a map of the locations of the plurality of immobilized template molecules; h) sequencing the first universal sample index sequence of the plurality of immobilized template molecules; i) sequencing the second universal sample index sequence of the plurality of immobilized template molecules; j) sequencing the insert sequence region of the plurality of immobilized template molecules; and k) assigning the insert sequence of a given template molecule obtained in step (c) to the first and second universal sample index sequences from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insert sequence.

[0027] The present disclosure provides a method for sequencing nucleic acid molecules, comprising: a) providing a plurality of nucleic acid template molecules immobilized at random locations on a support, each immobilized template molecule comprising an insert sequence region and one sample index, each sample index comprising a three-base random sequence linked to a universal sample index sequence that identifies the sample source of the insert sequence, the universal sample index sequence comprising 3 to 20 nucleotides, different immobilized template molecules having different three-base random sequences and the same universal sample index sequence, and immobilized template molecules having different insert sequences; and b) performing four cycles of polymerase-mediated sequencing reactions of the three-base random sequences of the plurality of immobilized template molecules and first base positions of the universal sample index sequence using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleic acid bases: A, G, C, and T / U. wherein the nucleotide reagents comprise different detectable color labels corresponding to each different type of nucleobase, and four cycles of sequencing comprise detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to the immobilized template molecules, thereby determining the sequence of a first base position of the 3-base long random sequence and the universal sample index sequence in each template molecule of the plurality of immobilized template molecules, wherein a balanced diversity of A, G, C, and T / U nucleobases is detected and imaged among the plurality of immobilized template molecules in each of the first, second, third, and fourth sequencing cycles; and c) generating a map of the positions of the plurality of immobilized template molecules using the images of the four cycles of the polymerase-mediated sequencing reaction obtained in step (b), wherein the sequence of the insertion 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, third, and fourth sequencing cycles.

[0028] In some embodiments, the method further comprises: a) sequencing the remaining base positions of the universal sample index sequence of the plurality of immobilized template molecules; b) sequencing the insert sequence region of the plurality of immobilized template molecules; and c) 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 a sample source of the given insert sequence. In some embodiments, the plurality of nucleic acid template molecules further comprise a second sample index, wherein the second sample index comprises a second universal sample index sequence that identifies the sample source of the insert sequence, and the second sample index lacks random sequence.

[0029] In some embodiments, the method further includes: a) sequencing the first base position of the 3-base long random sequence and the universal sample index sequence 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, second, third, and fourth sequencing cycles to generate a map of the positions of the plurality of immobilized template molecules; b) sequencing the remaining base positions of the first universal sample index sequence of the plurality of immobilized template molecules; c) sequencing the second universal sample index sequence of the plurality of immobilized template molecules; d) sequencing the insert sequence region of the plurality of immobilized template molecules; and e) assigning the insert sequence of a given template molecule obtained in step (c) to the first and second universal sample index sequences from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insert sequence.

[0030] In some embodiments, the support comprises a glass or plastic substrate. In some embodiments, the support is configured on a flow cell channel, a flow cell, or a capillary lumen. In some embodiments, the support is passivated with at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less.

[0031] 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(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the at least one hydrophilic polymer coating comprises a branched hydrophilic polymer molecule having at least four branches. In some embodiments, the at least one hydrophilic polymer coating comprises a polymer molecule having a molecular weight of at least 1000 Daltons.

[0032] In some embodiments, the immobilized template molecules comprise a plurality of immobilized concatemeric molecules having tandem repeat sequences of the insert sequence and one sample index. In some embodiments, the immobilized template molecules comprise a plurality of different clustered template molecules having one copy of the insert sequence and one copy of one sample index, wherein the clustered template molecules are generated via bridge amplification.

[0033] The density of immobilized nucleic acid template molecules located at random positions on the support is 1 mm 2 10 per 4 ~10 8 In some embodiments, the sample source of the insert sequence is genomic DNA, double-stranded cDNA, or cell-free circulating DNA.

[0034] In some embodiments, the detectably labeled nucleotide reagent comprises nucleotides, each comprising an aromatic nucleobase, a five-carbon sugar moiety, one to ten phosphate groups, and a fluorophore. In some embodiments, the detectably labeled nucleotide reagent comprises nucleotides, each comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain-terminating group at the 3'-carbon sugar position, one to ten phosphate groups, and a fluorophore.

[0035] In some embodiments, the detectably labeled nucleotide reagent comprises a multivalent molecule, each of which 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 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, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0036] In some embodiments, the detectably labeled nucleotide reagents bound to immobilized template molecules include individual immobilized template molecules, each of which is hybridized to a sequencing primer to form a duplex, the duplex being bound to a polymerase to form a hybridized polymerase, and the hybridized polymerase being bound to the detectably labeled nucleotide reagent.

[0037] In some embodiments, the hybridized polymerase is coupled to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the hybridized polymerase and for incorporation of the detectably labeled nucleotide into a hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety, one to ten phosphate groups, and a fluorophore. In some embodiments, the hybridized polymerase is coupled to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the hybridized polymerase and for incorporation of the detectably labeled nucleotide into a hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain-terminating group at the 3'-carbon sugar position, one to ten phosphate groups, and a fluorophore.

[0038] In some embodiments, the hybridized polymerase is coupled to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the hybridized polymerase, the conditions being suitable for inhibiting nucleotide incorporation, and the detectably labeled nucleotide reagent comprises a multivalent molecule, the multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, each nucleotide arm comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0039] In some embodiments, the immobilized template molecule comprises immobilized concatemeric molecules, the immobilized concatemeric molecules hybridized to a plurality of sequencing primers, forming at least first and second duplexes on the same concatemeric molecule, the first and second duplexes bound to a first polymerase and the second duplex bound to a second polymerase, forming first and second hybridized polymerases, and the method comprises: a) hybridizing the plurality of multivalent molecules to the first and second hybridized polymerases on the same concatemeric template molecule; and contacting a single multivalent molecule from the plurality of multivalent molecules with a first and second conjugated polymerase, 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 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, the spacer is attached to the linker, and the linker is attached to the nucleotide unit; and wherein the contacting separates a single multivalent molecule from the plurality of multivalent molecules into a first and second conjugated polymerase. a) contacting a first nucleotide unit of the single multivalent molecule to a first multiplexed polymerase comprising a first sequencing primer hybridized to a first portion of the concatemeric template molecule, thereby forming a first binding complex, a second nucleotide unit of the single multivalent molecule to a second multiplexed polymerase comprising a second sequencing primer hybridized to a second portion of the concatemeric template molecule, thereby forming a second binding complex, the first and second binding complexes bound to the same multivalent molecule forming an avidity complex, the contacting being carried out under conditions suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes; b) detecting the first and second binding complexes on the same concatemeric template molecule; and c) imaging the optical color signals emitted from the detectably labeled multivalent molecules that form the first and second binding complexes on the same concatemeric template molecule.d) identifying a first nucleotide unit in the first binding complex, thereby determining the sequence of a first portion of the concatemeric template molecule, and identifying a second nucleotide unit in the second binding complex, thereby determining the sequence of a second portion of the concatemeric template molecule.

[0040] The present disclosure provides a method for sequencing nucleic acid molecules, comprising: a) providing a first plurality of library molecules, each molecule in the first plurality of library molecules comprising: (i) an insert sequence region derived from a first sample source; (ii) a first sample index having a three-base long random sequence linked to a first universal sample index sequence; and (iii) a second sample index having a second universal sample index sequence lacking the random sequence, wherein a combination of the first and second universal sample index sequences uniquely identifies the first sample source of the insert sequence, and different first library molecules have different three-base long random sequences and different insert sequences; and b) providing a second plurality of library molecules, each molecule in the second plurality of library molecules comprising: (i) an insert sequence region derived from a second sample source; (ii) a third sample index having a three-base long random sequence linked to a third universal sample index sequence; and (iii) a fourth sample index having a fourth universal sample index sequence lacking the random sequence. a) providing a library molecule having a first and a second plurality of library molecules, the first and second plurality of library molecules having a third and a fourth universal sample index sequence, the combination of the third and fourth universal sample index sequences uniquely identifying a second sample source of insert sequences, the different second library molecules having different three-base long random sequences and having different insert sequences; b) pooling the first and second plurality of library molecules; c) 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; and e) performing three cycles of polymerase-mediated sequencing reactions of the three-base long random sequences of the first and third sample indexes using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C, and T / U, the nucleotide reagents comprising a different detectable color label corresponding to each different type of nucleobase, the three cycles of sequencing including detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to the immobilized amplified template molecules, thereby(f) determining the sequence of a 3-base random sequence in each of the plurality of immobilized template molecules, wherein a balanced diversity of A, G, C, and T / U nucleobases is detected and imaged in each of the first, second, and third sequencing cycles of the plurality of immobilized amplified template molecules; and (f) generating a map of the locations of the plurality of immobilized template molecules using the image obtained in step (e), wherein the sequence of the insertion region is not used to generate the map. In some embodiments, the balanced diversity in step (e) is 25-75% of each of the A, G, C, and T / U nucleobases detected and imaged in each of the first, second, and third sequencing cycles.

[0041] In some embodiments, the method further includes g) sequencing the first universal sample index sequence of the plurality of immobilized template molecules; h) sequencing the second universal sample index sequence of the plurality of immobilized template molecules; i) sequencing insert sequence regions of the plurality of immobilized template molecules derived from the first library molecule; and j) assigning the insert sequence of a given template molecule obtained in step (c) to the first and second universal sample index sequences from the same given template molecule, thereby identifying a first sample source of the given insert sequence.

[0042] In some embodiments, the method further includes g) sequencing a third universal sample index sequence of the plurality of immobilized template molecules; h) sequencing a fourth universal sample index sequence of the plurality of immobilized template molecules; i) sequencing an insert sequence region of the plurality of immobilized 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 and fourth universal sample index sequences from the same given template molecule, thereby identifying a second sample source of the given insert sequence.

[0043] The present disclosure provides a method for sequencing nucleic acid molecules, comprising: a) providing a first plurality of library molecules, each molecule in the first plurality of library molecules comprising: (i) an insert sequence region derived from a first sample source; (ii) a first sample index having a three-base long random sequence linked to a first universal sample index sequence; and (iii) a second sample index having a second universal sample index sequence lacking the random sequence, wherein a combination of the first and second universal sample index sequences uniquely identifies the first sample source of the insert sequence, the first universal sample index sequence comprising between 3 and 20 nucleotides, and different first library molecules having different three-base long random sequences and different insert sequences; and b) providing a second plurality of library molecules, each molecule in the second plurality of library molecules comprising: (i) an insert sequence region derived from a second sample source; (ii) a third sample index having a three-base long random sequence linked to a third universal sample index sequence; and (iii) a fourth sample index having a three-base long random sequence lacking the random sequence. a fourth sample index having a universal sample index sequence, wherein the combination of the third and fourth universal sample index sequences uniquely identifies a second sample source of the insert sequence, the third universal sample index sequence comprising 3 to 20 nucleotides, and different second library molecules having different three-base long random sequences and different insert sequences; c) pooling the first and second plurality of library molecules; d) distributing the pooled library molecules onto a support and conducting an amplification reaction to generate a plurality of clonally amplified template molecules immobilized at random positions on the support; e) conducting four cycles of polymerase-mediated sequencing reactions of the three-base long random sequences of the first and third sample indexes and sequencing of the first base positions of the first and third universal sample index sequences 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;The method includes: performing three cycles of sequencing, detecting and imaging optical color signals emitted from detectably labeled nucleotide reagents bound to immobilized amplified template molecules, thereby determining the sequence of a three-base-long random sequence in each of the plurality of immobilized template molecules, wherein a balanced diversity of A, G, C, and T / U nucleobases is detected and imaged in each of the first, second, third, and fourth sequencing cycles among the plurality of immobilized amplified template molecules; and f) generating a map of the locations of the plurality of immobilized template molecules using the images obtained in step (e), wherein the sequence of the insertion region is not used to generate the map. In some embodiments, the balanced diversity in step (e) is 25-75% of each of the A, G, C, and T / U nucleobases detected and imaged in each of the first, second, third, and fourth sequencing cycles.

[0044] In some embodiments, the method further includes g) sequencing the remaining base positions of the first universal sample index sequence of the plurality of immobilized template molecules; h) sequencing the second universal sample index sequence of the plurality of immobilized template molecules; i) sequencing the insert sequence region of the plurality of immobilized template molecules derived from the first library molecule; and j) assigning the insert sequence of a given template molecule obtained in step (c) to the first and second universal sample index sequences from the same given template molecule, thereby identifying a first sample source of the given insert sequence.

[0045] In some embodiments, the method further includes g) sequencing the remaining base positions of the third universal sample index sequence of the plurality of immobilized template molecules; h) sequencing the fourth universal sample index sequence of the plurality of immobilized template molecules; i) sequencing the insert sequence region of the plurality of immobilized template molecules from the second library molecule; and j) assigning the insert sequence of a given template molecule obtained in step (c) to the third and fourth universal sample index sequences from the same given template molecule, thereby identifying a second sample source of the given insert sequence.

[0046] In some embodiments, the method further comprises: a) sequencing the remaining base positions of the universal sample index sequence of the plurality of immobilized template molecules; b) sequencing the insert sequence region of the plurality of immobilized template molecules; and c) 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 a sample source of the given insert sequence. In some embodiments, the plurality of nucleic acid template molecules further comprise a second sample index, wherein the second sample index comprises a second universal sample index sequence that identifies the sample source of the insert sequence, and the second sample index lacks random sequence.

[0047] In some embodiments, the method further includes: a) sequencing the first base position of the 3-base long random sequence and the universal sample index sequence 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, second, third, and fourth sequencing cycles to generate a map of the positions of the plurality of immobilized template molecules; b) sequencing the remaining base positions of the first universal sample index sequence of the plurality of immobilized template molecules; c) sequencing the second universal sample index sequence of the plurality of immobilized template molecules; d) sequencing the insert sequence region of the plurality of immobilized template molecules; and e) assigning the insert sequence of a given template molecule obtained in step (c) to the first and second universal sample index sequences from the same given template molecule obtained in steps (a) and (b), thereby identifying the sample source of the given insert sequence.

[0048] In some embodiments, the support comprises a glass or plastic substrate. In some embodiments, the support is configured on a flow cell channel, a flow cell, or a capillary lumen. In some embodiments, the support is passivated with at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less.

[0049] 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(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the at least one hydrophilic polymer coating comprises a branched hydrophilic polymer molecule having at least four branches. In some embodiments, the at least one hydrophilic polymer coating comprises a polymer molecule having a molecular weight of at least 1000 Daltons.

[0050] In some embodiments, the immobilized template molecules comprise a plurality of immobilized concatemeric molecules having tandem repeat sequences of the insert sequence and one sample index. In some embodiments, the immobilized template molecules comprise a plurality of different clustered template molecules having one copy of the insert sequence and one copy of one sample index, wherein the clustered template molecules are generated via bridge amplification.

[0051] The density of immobilized nucleic acid template molecules located at random positions on the support is 1 mm 2 10 per 4 ~10 8 In some embodiments, the sample source of the insert sequence is genomic DNA, double-stranded cDNA, or cell-free circulating DNA.

[0052] In some embodiments, the detectably labeled nucleotide reagent comprises nucleotides, each comprising an aromatic nucleobase, a five-carbon sugar moiety, one to ten phosphate groups, and a fluorophore. In some embodiments, the detectably labeled nucleotide reagent comprises nucleotides, each comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain-terminating group at the 3'-carbon sugar position, one to ten phosphate groups, and a fluorophore.

[0053] In some embodiments, the detectably labeled nucleotide reagent comprises a multivalent molecule, each of which 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 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, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0054] In some embodiments, the detectably labeled nucleotide reagents bound to immobilized template molecules include individual immobilized template molecules, each of which is hybridized to a sequencing primer to form a duplex, the duplex being bound to a polymerase to form a hybridized polymerase, and the hybridized polymerase being bound to the detectably labeled nucleotide reagent.

[0055] In some embodiments, the hybridized polymerase is coupled to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the hybridized polymerase and for incorporation of the detectably labeled nucleotide into a hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety, one to ten phosphate groups, and a fluorophore. In some embodiments, the hybridized polymerase is coupled to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the hybridized polymerase and for incorporation of the detectably labeled nucleotide into a hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain-terminating group at the 3'-carbon sugar position, one to ten phosphate groups, and a fluorophore.

[0056] In some embodiments, the hybridized polymerase is coupled to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the hybridized polymerase, the conditions being suitable for inhibiting nucleotide incorporation, and the detectably labeled nucleotide reagent comprises a multivalent molecule, the multivalent molecule comprising (1) a core, (2) a plurality of nucleotide arms, and (3) at least one fluorophore, each nucleotide arm comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, the spacer is attached to the linker, and the linker is attached to the nucleotide unit.

[0057] In some embodiments, the immobilized template molecule comprises immobilized concatemeric molecules, the immobilized concatemeric molecules hybridized to a plurality of sequencing primers, forming at least first and second duplexes on the same concatemeric molecule, the first and second duplexes bound to a first polymerase and the second duplex bound to a second polymerase, forming first and second hybridized polymerases, and the method comprises: a) hybridizing the plurality of multivalent molecules to the first and second hybridized polymerases on the same concatemeric template molecule; and contacting a single multivalent molecule from the plurality of multivalent molecules with a first and second conjugated polymerase, 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 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, the spacer is attached to the linker, and the linker is attached to the nucleotide unit; and wherein the contacting separates a single multivalent molecule from the plurality of multivalent molecules into a first and second conjugated polymerase. a) contacting a first nucleotide unit of the single multivalent molecule to a first multiplexed polymerase comprising a first sequencing primer hybridized to a first portion of the concatemeric template molecule, thereby forming a first binding complex, a second nucleotide unit of the single multivalent molecule to a second multiplexed polymerase comprising a second sequencing primer hybridized to a second portion of the concatemeric template molecule, thereby forming a second binding complex, the first and second binding complexes bound to the same multivalent molecule forming an avidity complex, the contacting being carried out under conditions suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes; b) detecting the first and second binding complexes on the same concatemeric template molecule; and c) imaging the optical color signals emitted from the detectably labeled multivalent molecules that form the first and second binding complexes on the same concatemeric template molecule.d) identifying a first nucleotide unit in the first binding complex, thereby determining the sequence of a first portion of the concatemeric template molecule, and identifying a second nucleotide unit in the second binding complex, thereby determining the sequence of a second portion of the concatemeric template molecule.

[0058] 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 library molecule in the plurality of library molecules comprises, in 5' to 3' order, regions of: (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); (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; b) providing a plurality of single-stranded splint strands (200), wherein each single-stranded splint strand (200) in the plurality of single-stranded splint strands (200) is a region arranged in 5' to 3' order; (i) a first region (210) having a universal binding sequence that hybridizes to the first right universal adaptor sequence (120) of the linear single-stranded library molecule (100), and (ii) a second region (220) having a universal binding sequence that hybridizes to the first right universal adaptor sequence (130) of the linear single-stranded library molecule (100); c) providing a plurality of single-stranded splint strands (200) comprising a plurality of single-stranded nucleic acid library molecules (100) and a second region (220) having a universal binding sequence that hybridizes to the first left universal adaptor sequence (120) of the linear single-stranded library molecule (100); and hybridizing a second region (220) of the single-stranded splint strand (200) to a first right universal adaptor sequence (130) of the linear single-stranded library molecule (100) under conditions suitable for hybridizing the first region (220) of the single-stranded splint strand (200) to a first right universal adaptor sequence (130) of the linear single-stranded library molecule (100), thereby circularizing the individual library molecules to form a plurality of library-splint complexes (300) having nicks between the 5' and 3' ends of the library molecules, wherein the nicks are enzymatically ligatable.

[0059] 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 identifier sequence (180) and / or a first right unique identifier sequence (190).

[0060] In some embodiments, the method further comprises d) ligating nicks 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 enzyme to remove a plurality of single-stranded splint strands (200) and retain a plurality of covalently closed circular library molecules (400).

[0061] In some embodiments, each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules (400) comprises a first right universal adaptor 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 on which the plurality of first surface primers are immobilized 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.

[0062] In some embodiments, the method further comprises: g) contacting the plurality of immobilized covalently closed circular library molecules (400) with a plurality of strand-displacing 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 generating a plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0063] In some embodiments, the plurality of immobilized first surface capture primers on the support are located at predetermined or random positions on the support. In some embodiments, the plurality of immobilized second surface capture primers on the support are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes, nucleotides, divalent cations, etc.) to flow over the support, thereby allowing the plurality of immobilized first surface primers to react with the reagents essentially simultaneously in a massively parallel manner. In some embodiments, the density of the plurality of immobilized nucleic acid concatemer molecules on the support is greater than 1 mm 2 10 per 4 ~10 8 is.

[0064] In some embodiments, the method further comprises a) sequencing the plurality of immobilized concatemer molecules, 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 multiplexed polymerases, each of the multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, and the nucleic acid duplex comprising the concatemer molecules hybridized to the soluble sequencing primers; and c) contacting the plurality of multiplexed sequencing polymerases with a plurality of soluble sequencing primers. with at least one nucleotide under conditions suitable for binding to a multiplexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog, at least one nucleotide analog being labeled with a fluorophore and having a removable chain-terminating moiety at the sugar 3' position; d) incorporating the 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.

[0065] In some embodiments, the plurality of nucleotides comprises a removable chain-terminating moiety at the 3' sugar group, the removable chain-terminating moiety comprising an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, the removable chain-terminating moiety being cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group. In some embodiments, the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of 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.

[0066] 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 multiplexed polymerases, each of the plurality of first multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, and the nucleic acid duplexes comprising concatemer molecules hybridized to the soluble sequencing primers; and b) contacting the plurality of multiplexed sequencing polymerases with a plurality of detectably labeled multivalent molecules to synthesize complementary nucleotide units of the multivalent molecules. a) contacting the plurality of first multiplex polymerases with at least two of the plurality of first multiplex polymerases under conditions suitable for binding to the plurality of first multiplex polymerases, thereby forming a plurality of multivalent multiplex polymerases, wherein the conditions inhibit incorporation of complementary nucleotide units into the sequencing primers of the plurality of multivalent multiplex polymerases, and wherein each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit; b) detecting the plurality of multivalent multiplex polymerases; and d) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first multiplex polymerases in the plurality of multivalent multiplex polymerases, thereby determining the sequence of the nucleic acid template.

[0067] In some embodiments, the method includes: e) dissociating the plurality of multivalent multiplexed polymerases to remove the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; and 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 multiplexed polymerases, each of which binds the second sequencing polymerases to the retained nucleic acid duplexes. and g) contacting a plurality of second multiplexed polymerases with a plurality of nucleotides comprising at least one nucleotide analog having a removable chain-terminating moiety at the 3' sugar position, wherein the contacting is performed under conditions suitable for binding a complementary nucleotide from the plurality of nucleotides to at least two of the second multiplexed polymerases of step (f), thereby forming a plurality of nucleotide-complexed polymerases, and wherein the conditions are suitable for promoting the incorporation of the bound complementary nucleotide into the sequencing primer by the nucleotide-complexed polymerase.

[0068] In some embodiments, the method further comprises h) detecting a complementary nucleotide incorporated into the sequencing primer for the nucleotide-complexed polymerase. In some embodiments, the method further comprises h) detecting a complementary nucleotide incorporated into the sequencing primer for the nucleotide-complexed polymerase, and i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer for the nucleotide-complexed polymerase.

[0069] The present disclosure provides a method for sequencing by forming at least one avidity 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 concatemeric molecule of claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and b) binding a second universal sequencing primer, a second sequencing polymerase, and the first detectably labeled multivalent molecule to a second portion of the same concatemeric molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase. wherein the first detectably labeled multivalent molecule binds to a second sequencing polymerase, and first and second binding complexes comprising the same multivalent molecule form an avidity complex, wherein the first detectably labeled multivalent molecule comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit, and the concatemeric molecule comprises a sequence of interest (110) and two or more tandem repeat sequences of universal primer binding sites that bind to first and second universal sequencing primers, and the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes; and c) detecting the first and second binding complexes on the same concatemeric molecule.

[0070] In some embodiments, the nucleotide arms attached to the core of each multivalent molecule have the same type of nucleotide unit, where 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, where each of the two or more types of multivalent molecules has a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0071] In some embodiments, the plurality of nucleotides comprises a removable chain-terminating moiety at the 3' sugar group, the removable chain-terminating moiety comprising an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, the removable chain-terminating moiety being cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group. In some embodiments, the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of 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. 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 45 degrees or less. 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(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.

[0072] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0073] [Figure 1] 1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridizing to a single-stranded splint molecule / strand (200), thereby circularizing the library molecule and forming a nicked library-splint complex (300). The library molecule (100) comprises a sequence of interest (110) flanked on one side by a first left universal adaptor sequence (120) and on the other side by a first right universal adaptor sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence at one end of the linear, single-stranded library molecule and a second region (220) that hybridizes to a sequence at the other end of the linear, single-stranded library molecule. [Figure 2] 1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridizing to a single-stranded splint molecule / strand (200), thereby circularizing the library molecule and forming a nicked library-splint complex (300). The library molecule (100) comprises a sequence of interest (110) flanked on one side by a first left universal adaptor sequence (120) and a second left universal adaptor sequence (140) and on the other side by a second right universal adaptor sequence (150) and a first right universal adaptor sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence at one end of the linear, single-stranded library molecule and a second region (220) that hybridizes to a sequence at the other end of the linear, single-stranded library molecule. [Figure 3]1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridizing to a single-stranded splint molecule / strand (200), thereby circularizing the library molecule and forming a nicked library-splint complex (300). The exemplary library molecule (100) includes a first left universal adaptor sequence (120), an optional first left unique identifier sequence (180), a first left index sequence (160), a second left universal adaptor sequence (140), a sequence of interest (110), a second right universal adaptor sequence (150), a first right index sequence (170), and a first right universal adaptor sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence at one end of a linear single-stranded library molecule, and a second region (220) that hybridizes to a sequence at the other end of the linear single-stranded library molecule. [Figure 4] 1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridizing to a single-stranded splint molecule / strand (200), thereby circularizing the library molecule and forming a nicked library-splint complex (300). The exemplary library molecule (100) includes a first left universal adaptor sequence (120), a first left index sequence (160), a second left universal adaptor sequence (140), a sequence of interest (110), a second right universal adaptor sequence (150), a first right index sequence (170), an optional first right unique identifier sequence (190), and a first right universal adaptor sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence at one end of a linear single-stranded library molecule, and a second region (220) that hybridizes to a sequence at the other end of the linear single-stranded library molecule. [Figure 5]Schematic diagram showing an exemplary linear single-stranded library molecule (100) hybridizing to a single-stranded splint molecule / strand (200), thereby circularizing the library molecule and forming a nicked library-splint complex (300). The library molecule (100) comprises a first left universal adaptor sequence (120), a first left junction adaptor sequence (125), a first left index sequence (160), a second left junction adaptor sequence (165), a second left universal adaptor sequence (140), a third left junction adaptor sequence (145), a sequence of interest (110), a third right junction adaptor sequence (155), a second right universal adaptor sequence (150), a second right junction adaptor sequence (175), a first right index sequence (170), a first right junction adaptor sequence (135), an optional first right unique identifier sequence (190), and a first right universal adaptor sequence (130). The single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence at one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence at the other end of the linear single-stranded library molecule. For simplicity, the library-sprint complex (300) does not show any of the junction adaptors. Those skilled in the art will recognize that the library-sprint complex (300) can include any one or any combination of two or more of the junction adaptors present in the library molecule (100). [Figure 6]Three schematic diagrams of exemplary covalently closed circular library molecules are shown, each hybridized to a single splint strand (200). The top schematic diagram (A) shows a covalently closed circular library molecule (400) having a sequence of interest (110), a first right universal adaptor sequence (130), and a first left universal adaptor sequence (120). The middle schematic diagram (B) shows a covalently closed circular library molecule (400) having a sequence of interest (110), a second right universal adaptor sequence (150), a first right universal adaptor sequence (130), a first left universal adaptor sequence (120), and a second left universal adaptor sequence (140). The bottom schematic (C) shows a covalently closed circular library molecule (400) having a sequence of interest (110), a second right universal adaptor sequence (150), a first right index sequence (170), a first right universal adaptor sequence (130), a first left universal adaptor sequence (120), an optional first left unique identifier sequence (180), a first left index sequence (160), and a second left universal adaptor sequence (140). [Figure 7] 1 is a schematic diagram showing an exemplary library-splint complex that is subjected to a ligation reaction to seal the nick and form a covalently closed circular library molecule (400) that is hybridized to a single-stranded splint strand (200), which is used as an amplification primer to perform a rolling circle amplification reaction. The dotted line represents the nascent extension product. [Figure 8] The nucleotide sequence of an exemplary single-stranded splint molecule / strand (200) is shown. The exemplary single-stranded splint strand includes a first region (210; SEQ ID NO: 193), a second region (220; SEQ ID NO: 194). [Figure 9A] Table 1 (six sheets) lists the arrangement of an exemplary first left index array (160) and an exemplary first right index array (170) having a short random array (e.g., NNN). [Figure 9B]Table 1 (six sheets) lists the arrangement of an exemplary first left index array (160) and an exemplary first right index array (170) having a short random array (e.g., NNN). [Figure 9C] Table 1 (six sheets) lists the arrangement of an exemplary first left index array (160) and an exemplary first right index array (170) having a short random array (e.g., NNN). [Figure 9D] Table 1 (six sheets) lists the arrangement of an exemplary first left index array (160) and an exemplary first right index array (170) having a short random array (e.g., NNN). [Figure 9E] Table 1 (six sheets) lists the arrangement of an exemplary first left index array (160) and an exemplary first right index array (170) having a short random array (e.g., NNN). [Figure 9F] Table 1 (six sheets) lists the arrangement of an exemplary first left index array (160) and an exemplary first right index array (170) having a short random array (e.g., NNN). [Figure 10] 10 is a schematic diagram showing exemplary Y-shaped adapters, each of which has a double-stranded annealing region with an end that is blunt, or the end can have a 5' overhang region or a 3' overhang region. Figure 10 also shows exemplary Y-shaped adapters with mismatched portions of different lengths. [Figure 11] 11 is a schematic diagram of an exemplary Y-shaped adapter having a first strand containing a binding sequence for a first sequencing primer and a binding sequence for a first surface primer. Figure 11 also shows an exemplary Y-shaped adapter having a second strand containing a binding sequence for a second sequencing primer and a binding sequence for a second surface primer. The first strand optionally contains a first sample index sequence. The second strand optionally contains a second sample index sequence. [Figure 12]12 is a schematic diagram of an exemplary Y-shaped adapter having a first strand containing a binding sequence for a first sequencing primer and a binding sequence for a first surface primer. FIG. 12 also shows an exemplary Y-shaped adapter having a second strand containing a binding sequence for a second sequencing primer and a binding sequence for a second surface primer. The first strand optionally contains a first sample index sequence having a short random sequence (e.g., NNN). The second strand optionally contains a second sample index sequence having a short random sequence (e.g., NNN). [Figure 13] 1 is a schematic diagram illustrating an exemplary library preparation workflow in which nucleic acid fragments are ligated at both ends to Y-shaped adaptors. The Y-shaped adaptors include a first strand that includes 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 adaptors include a second strand that includes a binding sequence for a second sequencing primer, a second sample index sequence, and a binding sequence for a second surface primer. [Figure 14] 1 is a schematic diagram illustrating an exemplary library preparation workflow in which nucleic acid fragments are ligated at both ends to Y-shaped adaptors. The Y-shaped adaptors include a first strand containing 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 adaptors include a second strand containing a binding sequence for a second sequencing primer, a second sample index sequence, and a binding sequence for a second surface primer. [Figure 15]1 is a schematic diagram illustrating an exemplary library preparation workflow in which a nucleic acid fragment is ligated at both ends to a Y-shaped adapter to generate an adapter-insert-adapter molecule. The Y-shaped adapter comprises a first strand that includes a binding sequence for a first sequencing primer. The Y-shaped adapter comprises a second strand that includes 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 that carries the binding sequence for the first surface primer to generate a first extension product. The first extension product is subjected to a second primer extension reaction using a second tailed PCR primer that carries the binding sequence for the second surface primer to generate a second extension product. [Figure 16] 1 is a schematic diagram illustrating an exemplary library preparation workflow in which a nucleic acid fragment is ligated at both ends to a Y-shaped adaptor to generate an adaptor-insert-adaptor molecule. The Y-shaped adaptor comprises a first strand comprising a binding sequence for a first sequencing primer. The Y-shaped adaptor comprises a second strand comprising a binding sequence for a second sequencing primer. The adaptor-insert-adaptor molecule is subjected to a first primer extension reaction using a first tailed PCR primer bearing the binding sequence for the first surface primer, a first sample index sequence, and the binding sequence for the first surface primer to generate a first extension product. The first extension product is subjected to a second primer extension reaction using a second tailed PCR primer bearing the binding sequence for the second surface primer, a second sample index sequence, and the binding sequence for the second surface primer to generate a second extension product. [Figure 17]1 is a schematic diagram illustrating an exemplary library preparation workflow in which a nucleic acid fragment is ligated at both ends to a Y-shaped adapter to generate an adapter-insert-adapter molecule. The Y-shaped adapter comprises a first strand comprising a binding sequence for a first sequencing primer. The Y-shaped adapter comprises 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 bearing the binding sequence for the first surface primer, a first sample index sequence having a short random sequence (e.g., NNN), and the binding sequence for the first surface primer to generate a first extension product. The first extension product is subjected to a second primer extension reaction using a second tailed PCR primer bearing the binding sequence for the second surface primer, a second sample index sequence, and the binding sequence for the second surface primer to generate a second extension product. [Figure 18] 1 is a schematic diagram of an exemplary low-binding support comprising a glass substrate and alternating layers of hydrophilic coating, the alternating layers of hydrophilic coating being covalently or non-covalently adhered to the glass and further comprising chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides). In alternative embodiments, the support can be made from any material, such as glass, plastic, or a polymeric material. [Figure 19] Schematic diagrams of various exemplary configurations of multivalent molecules. Left (Class I): Schematic diagram of a multivalent molecule with a "starburst" or "helter-skelter" configuration. Center (Class II): Schematic diagram of a multivalent molecule with a dendrimer configuration. Right (Class III): Schematic diagram of multiple multivalent molecules formed by reacting streptavidin with a 4-arm or 8-arm PEG-NHS bearing biotin and dNTPs. Nucleotide units are represented as "N," biotin is represented as "B," and streptavidin is represented as "SA." [Figure 20]FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a generic core attached to multiple nucleotide arms. [Figure 21] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms. [Figure 22] 1 shows a schematic diagram of an exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, the nucleotide arms comprising biotin, spacers, linkers, and nucleotide units. [Figure 23] FIG. 1 is a schematic diagram of an exemplary nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit. [Figure 24] The chemical structures of an exemplary spacer (top) and various exemplary linkers (bottom) are shown, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker. [Figure 25] 1 shows the chemical structures of various exemplary linkers, including linkers 1-9. [Figure 26] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 27] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 28] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 29] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 30] 1 shows the chemical structure of an exemplary biotinylated nucleotide arm. In this example, the nucleotide unit is connected to the linker via a propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base. [Figure 31]1 is a graph showing the nucleotide base diversity of a right sample index sequence (170) including a universal right sample index and a three-base random sequence (NNN). The graph shows the nucleotide diversity of the three-base random sequence (NNN) of approximately 30% for A and T base calls and approximately 20% for C and G base calls. [Figure 32] 1 is a graph showing the nucleotide base diversity of the left sample index sequence (160) lacking the 3-base long random sequence (NNN). The graph shows approximately 40% nucleotide diversity for A and T base calls, approximately 15% for C base calls, and approximately 5% for G base calls. DETAILED DESCRIPTION OF THE INVENTION

[0074] Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of these publications, patents, and / or patent applications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0075] Definition: The headings provided herein are not limitations of various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Generally, terms relating to molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization techniques described herein are well known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with, and the experimental procedures and techniques described herein are well known and commonly used in the art.

[0077] Unless otherwise required by context herein, singular terms include pluralities and plural terms include the singular. The singular forms "a," "an," and "the," as well as use of the singular form of any word, include plural references unless expressly and unambiguously limited to a reference to one.

[0078] The use of alternative terms (eg, "or") is understood to mean either one or both of the alternatives, or any combination thereof.

[0079] As used herein, the term "and / or" should be understood to mean a specific disclosure that each of the specified features or components may or may not have the other. For example, when used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (A alone), and "B" (B alone). In a similar manner, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following embodiments: "A, B, and C," "A, B, or C," "A or C," "A or B," "B or C," "A and B," "B and C," "A and C," "A" (alone), "B" (alone), and "C" (alone).

[0080] As used in this specification and the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be open-ended so that one or more items in a list do not exclude other items that may be substituted for or added to the listed items. Wherever an embodiment is described herein with the term "comprising," it is understood that alternative, similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0081] As used herein, the term "about" or "approximately" refers to a value or composition that is within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art. The acceptable error range depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) or more, depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean up to one order of magnitude or up to five times the value. When a particular value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be considered to be within an acceptable error range for that particular value or composition. Additionally, when ranges and / or subranges of values ​​are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0082] The terms "peptide," "polypeptide," and "protein," as well as other related terms used herein, are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides can contain natural and unnatural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not yet undergone post-translational modifications, such as proteolytic cleavage, ribosomal skipping cleavage, 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 of protein sequences (such as muteins, variants, chimeric proteins, and fusion proteins), as well as proteins that are post-translationally or otherwise covalently or non-covalently modified.

[0083] The term "cellular biological sample" refers to a single cell, multiple cells, tissue, organ, organism, or section of any of these cellular biological samples. A cellular biological sample may be extracted from an organism (e.g., biopsy) or obtained from a cell culture growing in liquid or on a culture dish. A cellular biological sample includes a fresh sample, a frozen sample, a fresh frozen sample, or an archived sample (e.g., formalin-fixed paraffin-embedded; FFPE) sample. A cellular biological sample may be embedded in wax, resin, epoxy, or agar. A cellular biological sample may be fixed, for example, in any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton, or glutaraldehyde. A cellular biological sample may or may not be sectioned. A cellular biological sample may be stained, destained, or unstained.

[0084] Nucleic acids of interest can be extracted from cells or cellular biological samples using any of several techniques known to those skilled in the art. For example, a typical DNA extraction procedure includes: (i) collecting a cell or tissue sample from which DNA is to be extracted; (ii) disrupting cell membranes (i.e., lysing cells) to release DNA and other cytoplasmic components; (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA; and (iv) purifying DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during cell membrane lysis. A variety of suitable commercially available nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolation of genomic DNA from human samples) and the DNAeasy kit (for isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI).

[0085] As used herein, the term "polymerase" and variations thereof include enzymes that contain a nucleotide (or nucleoside)-binding domain, and the polymerase can form a complex with a template nucleic acid and a complementary nucleotide. A 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. A polymerase can be any enzyme that can catalyze the polymerization of nucleotides (including their analogs) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase contains one or more active sites, and nucleotide binding and / or catalysis of nucleotide polymerization can occur in one or more active sites. In some embodiments, a polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacement activity. Polymerases can include, but are not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes that contain a nucleotide-binding domain. In some embodiments, polymerases may be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases may be expressed in prokaryotic, eukaryotic, viral, or phage organisms. In some embodiments, polymerases may be post-translationally modified proteins or fragments thereof. Polymerases may be derived from prokaryotic, eukaryotic, viral, or phage organisms.Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0086] The term "strand displacement" refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acid and synthesize a new strand in a template-based manner. Strand-displacing polymerases displace a complementary strand from the template strand and catalyze new strand synthesis. Strand-displacing polymerases include mesophilic and thermophilic polymerases. Strand-displacing polymerases include wild-type enzymes and variants, including exonuclease-minus mutants, mutated versions, chimeric enzymes, and truncated enzymes. Examples of strand-displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0087] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms, are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic 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, where the nucleotides comprise natural or non-natural bases and / or sugars. Nucleic acids comprise naturally occurring internucleoside linkages, such as phosphodiester linkages. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0088] As used herein, the terms "operably linked" and "operably linked," or related terms, refer to the juxtaposition of components. Juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically ligated together, where the bond linking the two components together comprises a phosphodiester bond. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion capable of binding to the primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) can be ligated into a vector, where the bond allows for expression or function of the transgene sequence contained within the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, etc. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of expression of the transgene.

[0089] The terms "bound," "linked," "attached," and "appended," and variations thereof, include any type of fusion, bond, adhesion, or association between any combination of compounds or molecules that is stable enough to withstand use in a particular procedure. Procedures can include, but are not limited to, nucleotide binding, nucleotide incorporation, deblocking (e.g., removal of chain-terminating moieties), washing, removal, flow, detection, imaging, and / or identification. Such bonds can include, for example, covalent bonds, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonds, bonds or associations involving van der Waals forces, and mechanical bonds. In some embodiments, such bonds occur intramolecularly, such as by joining the ends of a single- or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonds can occur between different molecular combinations or between molecules and non-molecules, including, but not limited to, bonds between nucleic acid molecules and solid surfaces, bonds between proteins and detectable reporter moieties, and bonds between nucleotides and detectable reporter moieties. Some examples of conjugation can be found, for example, in Hermanson, G., "Bioconjugate Techniques", Second Edition (2008), Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998), Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998).

[0090] As used herein, the term "primer" and related terms refer to an oligonucleotide capable of hybridizing to a DNA and / or RNA polynucleotide template to form a duplex molecule. Primers may be single-stranded along their entire length or may have single-stranded and double-stranded portions. Primers contain natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers can be of any length but typically range from 4 to 50 nucleotides. Typical primers contain a 5' end and a 3' end. The 3' end of a primer can contain a 3' OH moiety that serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of a primer can lack a 3' OH moiety or contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one or more nucleotides along the length of a primer can be labeled with a detectable reporter moiety. The primers may be in solution (eg, soluble primers) or immobilized on a support (eg, capture primers).

[0091] The terms "template nucleic acid," "template polynucleotide," "target nucleic acid," "target polynucleotide," "template strand," and other variations thereof, refer to a nucleic acid strand that serves as the base nucleic acid molecule for any of the amplification and / or sequencing methods described herein. A template nucleic acid may be single-stranded or double-stranded, or may have single-stranded or double-stranded portions. A template nucleic acid may be obtained from a naturally occurring source or recombinant form, or may be chemically synthesized to include any type of nucleic acid analog. A template nucleic acid may be linear, concatemeric, circular, or in other forms.

[0092] The term "adapter" and related terms refer to an oligonucleotide that can be operably attached to (applied to) a target polynucleotide, and the adapter confers a function to the co-ligated adapter-target molecule. Adapters include DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adapters can contain at least one ribonucleoside residue. Adapters can be single-stranded or double-stranded, or can have single-stranded and / or double-stranded portions. Adapters can be configured to be linear, stem-loop, hairpin, or Y-shaped. Adapters can be any length, from 4 to 100 nucleotides or more. Adapters can have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' overhanging ends and 3' overhanging ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, can have a 5' phosphate group or lack a 5' phosphate group. The adapter may include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tailed adapter), or the adapter may be tailless. At least a portion of the adapter includes a known and predetermined sequence. The adapter may include a sequence complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter may include a random or degenerate sequence. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, phosphorothiolate, and / or phosphoramidate linkage. The adapter may include at least one barcode sequence, which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex assays. The adapter may include at least one unique identification sequence (e.g., a molecular tag), which 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 to 12 or more nucleotides having a known sequence. For example, the unique identification sequence comprises a known random sequence, where the nucleotides at each position in the known random sequence are randomly selected from nucleotides having the bases A, G, C, T, or U. The adaptor can comprise at least one restriction enzyme recognition sequence, where the at least one restriction enzyme recognition sequence comprises 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, and type IIB.

[0093] The term "universal sequence" and related terms refer to a sequence in a nucleic acid molecule that is common between two or more polynucleotide molecules. For example, an adapter having a universal sequence can be operably linked to multiple polynucleotides, such that a population of co-linked molecules possesses the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0094] When used in reference to nucleic acid molecules, the terms "hybridize" or "hybridizing" or "hybridization," or other related terms, refer to hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a double-stranded region. Hybridization can involve Watson-Crick or Hoogstein binding to form a double-stranded double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. A double-stranded nucleic acid, or two different regions of a single nucleic acid, can be fully complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other throughout their entire length. Complementary base pairing can be standard AT or CG base pairing, or other forms of base-pairing interactions. A double-stranded nucleic acid can contain mismatched base-pairing nucleotides.

[0095] When used with respect to nucleic acids, the terms "extend," "extending," "extension," and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides to the terminal 3'OH terminus of a nucleic acid chain, resulting in the elongation of the nucleic acid chain. Nucleotide incorporation can be performed with natural nucleotides and / or nucleotide analogs. Typically, although not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending a nucleic acid molecule can be used, and suitable methods include primer extension catalyzed by DNA polymerase or RNA polymerase.

[0096] The term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or non-standard nucleotides are consistent with the use of this term. In some embodiments, a nucleotide comprises a monophosphate, diphosphate, or triphosphate, or the corresponding phosphate analog. 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.

[0097] Nucleotides (and nucleosides) typically contain a heterocyclic base containing a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, which are commonly found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants, or analogs thereof. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases are purines and pyrimidines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N, N-acetyl-p-tolyl-2-one ... 6 -Δ 2 -Isopentenyladenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -Methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine, and O 6 -methylguanine; 7-deaza-purines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4Examples of bases include, but are not limited to, 1-methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosine; hydroxymethylcytosine; 5-methycytosine; base (Y); and methylated, glycosylated, and acylated base moieties. Additional exemplary bases can be found in Fasman, 1989, "Practical Handbook of Biochemistry and Molecular Biology," pp. 385-394, CRC Press, Boca Raton, Fla.

[0098] Nucleotides (and nucleosides) typically include 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 another sugar moiety (Joeng, et al., 1993 J. Med. Chem. 36:2627-2638; Kim, et al., 1993 J. Med. Chem. 36:30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). Sugar moieties include ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic, or other modified sugars.

[0099] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramido linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise substituted side chain groups including O, S, or BH3. In some embodiments, the chain comprises phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoramidite groups.

[0100] The term "rolling circle amplification" generally refers to an amplification method using 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. A 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 repeat sequences of any adapter sequences present in the circularized template molecule and the original circularized nucleic acid template molecule. The concatemers can self-collapse into nucleic acid nanoballs. The shape and size of the nanoballs can be further compacted by including a pair of inverted repeat sequences within the circular template molecule or by performing the rolling circle amplification reaction with one or more compaction oligonucleotides. One advantage of using rolling circle amplification to generate clonal amplicons for sequencing workflows is that the repeated copies of the target sequence in the nanoballs can be simultaneously sequenced, increasing signal intensity. In some embodiments, a rolling circle amplification reaction can be performed in the presence of multiple compaction oligonucleotides having at least four consecutive guanines. The rolling circle amplification reaction generates concatemers containing repeated copies of the universal binding sequence for the compaction oligonucleotides. At least one compaction oligonucleotide can form a G-quadruplex and hybridize to the universal binding sequence for the compaction oligonucleotide, and the resulting concatemers can fold to form an intramolecular G-quadruplex structure. The concatemers can self-collapse to form compact nanoballs.The formation of G-quadruplexes and G-quadruplexes in nanoballs can increase the stability of the nanoballs, allowing them to retain their compact size and shape, which can withstand repeated flows of reagents to perform any of the sequencing workflows described herein.

[0101] The terms "amplify," "amplifying," "amplification," and other related terms, when used with respect to nucleic acids, include producing multiple copies of an original polynucleotide template molecule, where the copies contain sequences that are complementary to the template sequence and / or where the copies contain sequences that are identical to the template sequence. In some embodiments, the copies contain sequences that are substantially identical to the template sequence and / or sequences that are substantially identical to the sequence that is complementary to the template sequence.

[0102] The terms "reporter moiety," "reporter moieties," or related terms refer to a compound that produces or can be caused to produce a detectable signal. Reporter moieties are often referred to as "labels." Any suitable reporter moiety can be used, and suitable reporter moieties include luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. A reporter moiety produces a detectable signal that results from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity, or a proximity event). A proximity event involves two reporter moieties coming into close proximity with, associating with, or binding to each other. It is well known to those skilled in the art to select reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties, allowing for the monitoring of the presence of different reporter moieties in the same or different reactions. Two or more different reporter moieties may be selected that have spectrally distinct emission profiles or that have minimal overlapping spectral emission profiles. The reporter moiety may be bound (e.g., operably bound) to a nucleotide, a nucleoside, a nucleic acid, an enzyme (e.g., a polymerase or reverse transcriptase), or a support (e.g., a surface).

[0103] The reporter moiety (or label) comprises a fluorescent label or fluorophore. Exemplary fluorescent moieties that can function as fluorescent labels or fluorophores include fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine and rhodamine derivatives, such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonylhydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives, such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanines and derivatives, such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5,Lanthanide chelates and derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbenes, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition, Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes can exist in either sulfonated or non-sulfonated form and consist of two indolenine, benzoindolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which is 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium, may include 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may include1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-yne dol-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-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl) Cy7 (which may include 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-5-sulfonate), and Cy8 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for "cyanine" and the first number identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0104] In some embodiments, the reporter moieties can be FRET pairs, allowing multiple classifications to be performed under a single excitation and imaging step. As used herein, FRET can include excitation exchange (Förster) transfer or electron exchange (Dexter) transfer.

[0105] As used herein, the term "support" refers to a substrate designed for the deposition of biomolecules or biological samples for assay and / or analysis. Examples of biomolecules deposited on a support include nucleic acids (e.g., DNA, RNA), polypeptides, sugars, lipids, single cells, or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

[0106] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, comprising a capillary or the inner surface of a capillary.

[0107] In some embodiments, the surface of the support can be substantially smooth, hi some embodiments, the support can be regularly or irregularly textured, including ridges, etchings, pores, three-dimensional scaffolds, or any combination thereof.

[0108] In some embodiments, the support comprises beads having any shape, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disk-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0109] The support can be made of any material, including, but not limited to, glass, fused silica, silicon, polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0110] The present disclosure provides a plurality (e.g., two or more) of nucleic acid template molecules immobilized on a support. In some embodiments, the immobilized plurality of nucleic acid template molecules have the same sequence or different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid template molecules are immobilized at different sites on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at sites on the support.

[0111] The term "array" refers to a support comprising a plurality of sites located at predetermined locations on the support, forming an array of sites. The sites may be dispersed and separated by interstitial regions. In some embodiments, the predetermined sites on the support may be arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, the plurality of predetermined sites are arranged in an organized manner on the support. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, including linear, hexagonal, lattice, patterns with reflection symmetry, or patterns with rotational symmetry. The pitch between different pairs of sites may be the same or may vary. In some embodiments, the support has at least 10 2 at least 10 sites 3 at least 10 sites 4at least 10 sites 5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 sites, or at least 10 15 In some embodiments, the substrate comprises a plurality of predetermined sites (e.g., 10 or more sites), the sites being located at predetermined locations on the substrate. 2 ~10 15 In some embodiments, the nucleic acid template molecules are immobilized at a plurality of predetermined sites by hybridization to 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, e.g., 10 2 ~10 15 In some embodiments, the immobilized nucleic acid template molecule is clonally amplified to generate immobilized nucleic acid clusters at a plurality of predetermined sites. In some embodiments, each immobilized nucleic acid cluster comprises a linear cluster or a single- or double-stranded concatemer.

[0112] 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 located sites thereon. The locations of the randomly located sites on the support are not predetermined locations. The plurality of randomly located sites are arranged on the support in a non-ordered and / or unpredictable manner. In some embodiments, the support comprises at least 10 2 at least 10 sites 3 at least 10 sites 4 at least 10 sites 5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 sites, or at least 10 15 In some embodiments, the substrate comprises a plurality of randomly positioned sites (e.g., 10 or more sites), where the sites are randomly located on the substrate. 2 ~10 15 In some embodiments, the nucleic acid template molecule is immobilized at a plurality of randomly located sites by hybridization to an immobilized surface capture primer, or the nucleic acid template molecule is covalently attached to a surface capture primer. In some embodiments, the nucleic acid template molecule is immobilized at a plurality of randomly located sites, e.g., 10 2 ~10 15In some embodiments, the immobilized nucleic acid template is clonally amplified to generate immobilized nucleic acid clusters at multiple randomly located sites. In some embodiments, each immobilized nucleic acid cluster comprises a linear cluster or a single- or double-stranded concatemer.

[0113] In some embodiments, multiple immobilized surface capture primers on a support (e.g., located at predetermined or random locations on the support) are in fluid communication with each other, allowing solutions of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, etc.) to flow over the support, thereby allowing the multiple immobilized surface capture primers on the support to react with the reagents essentially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of multiple immobilized surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) essentially simultaneously on the multiple immobilized surface capture primers.

[0114] In some embodiments, multiple immobilized nucleic acid clusters on a support are in fluid communication with each other, allowing solutions of reagents (e.g., enzymes, nucleotides, divalent cations, etc.) to flow over the support, thereby allowing multiple immobilized nucleic acid clusters on a support to react with reagents essentially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of multiple immobilized nucleic acid clusters can be used to perform nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) substantially simultaneously on multiple immobilized nucleic acid clusters, and optionally, to perform detection and imaging for massively parallel sequencing.

[0115] In some embodiments, the term "immobilized" and related terms refer to nucleic acid molecules that are attached to a support via covalent or non-covalent interactions, or that are attached to a coating on a support, or that are embedded within a matrix formed by a coating on a support, where the nucleic acid molecules include a surface capture primer, a nucleic acid template molecule, and an extension product of the capture primer. The extension product of the capture primer comprises a nucleic acid concatemer (e.g., a nucleic acid cluster). The nucleic acid molecules can be immobilized at predetermined or random locations on the support. The nucleic acid molecules can be immobilized at predetermined or random locations on or within a passivated coating on the support.

[0116] In some embodiments, the term "immobilized" and related terms refer to an enzyme (e.g., a polymerase) that is attached to a support via covalent or non-covalent interactions, or that is attached to a coating on a support, or that is embedded within a matrix formed by a coating on a support. The enzyme can be immobilized at predetermined or random locations on the support. The enzyme can be immobilized at predetermined or random locations on or within a passivated coating on the support.

[0117] In some embodiments, one or more nucleic acid template molecules are immobilized on a support, e.g., immobilized 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 off-support (e.g., in solution) and then deposited on a support and immobilized thereon. In some embodiments, a clonal amplification reaction of one or more nucleic acid template molecules is performed on the support, resulting in immobilization on the support. In some embodiments, one or more nucleic acid template molecules are clonally amplified using a nucleic acid amplification reaction (e.g., in solution or on a support), where the nucleic acid amplification reaction includes any one or any combination of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-strand binding (SSB) protein-dependent amplification.

[0118] The term "surface primer" and related terms refer to a single-stranded oligonucleotide that is immobilized to a support and includes a sequence that can hybridize to at least a portion of a nucleic acid template molecule. Surface capture primers can be used to immobilize template molecules to a support via hybridization. Surface capture primers can be immobilized to a support in a manner that resists primer removal during flow, washing, aspiration, and changes in temperature, pH, salt, chemical, and / or enzymatic conditions. Typically, although not necessarily, the 5' end of a surface capture primer can be immobilized to (or embedded within) a coating on) the support or a coating on the support. Alternatively, an internal portion or 3' end of a surface capture primer can be immobilized to the support.

[0119] The sequences of the surface capture primers can be wholly or partially complementary to at least a portion of the nucleic acid template molecule along their length. The support can contain multiple immobilized surface capture primers having the same sequence or two or more different sequences. The surface capture primers can be any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

[0120] A surface capture primer can have a terminal 3' nucleotide with a sugar 3' OH moiety that is extendible for nucleotide polymerization (e.g., polymerase-catalyzed polymerization). A surface capture primer can have a terminal 3' nucleotide with a 3' sugar moiety attached to a chain-terminating moiety that inhibits nucleotide polymerization. The 3' chain-terminating moiety can be removed (e.g., deblocked) using a deblocking agent to convert the 3' end to an extendible 3' OH end. Examples of chain-terminating moieties include alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl groups. Azido-type chain-terminating moieties include azide, azido, and azidomethyl groups. Examples of deblocking agents include phosphine compounds, such as tris(2-carboxyethyl)phosphine (TCEP) and bis-sulfotriphenylphosphine (BS-TPP), for chain-terminating azide, azido, and azidomethyl groups. Examples of deblocking agents include tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) for chain-terminating alkyl, alkenyl, alkynyl, and aryl groups. Examples of deblocking agents include Pd / C for chain-terminating aryl and benzyl groups. Examples of deblocking agents include phosphines, beta-mercaptoethanol, or dithiothreitol (DTT) for chain-terminating amine, amide, keto, isocyanate, phosphate, thio, and disulfide groups. Examples of deblocking agents include potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn(AcOH) in acetic acid for carbonate chain terminating groups. Examples of deblocking agents include tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, and triethylamine trihydrofluoride for urea and silyl chain terminating groups.

[0121] The term "sequencing" and related terms refer to methods for obtaining nucleotide sequence information from a nucleic acid molecule, typically by determining the identities of at least some nucleotides (including their nucleobase components) within the nucleic acid molecule. In some embodiments, sequence information for a given region of a nucleic acid molecule includes identifying every single nucleotide within the sequenced region. In some embodiments, the sequencing information determines only some of the region's nucleotides, with the identities of some nucleotides remaining undetermined or incorrectly determined. Any suitable sequencing method 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 nucleotide or fluorescent-based nucleotide sequencing methods. In some embodiments, sequencing can include polony-based sequencing or bridge sequencing methods. In some embodiments, sequencing uses a polymerase and a multivalent molecule to generate at least one avidity complex, each multivalent molecule comprising a plurality of nucleotide units tethered to a core. In some embodiments, sequencing uses a polymerase and free nucleotides to perform sequencing by synthesis, hi some embodiments, sequencing uses a ligase enzyme and multiple sequence-specific oligonucleotides to perform sequencing by ligation.

[0122] Detailed Description Introduction: Circularization using single-stranded splint strands The present disclosure provides compositions, including kits, that include nucleic acid single-stranded splint strands, and methods for using the single-stranded splint strands.

[0123] Single-stranded splint strands (200) can be used to circularize linear library molecules in a one-pot multi-enzyme reaction (see, e.g., Figures 1-5). The single-stranded splint strands (200) include a first region (210) that hybridizes to a sequence at one end of a linear single-stranded library molecule and a second region (220) that hybridizes to a sequence at the other end of the linear single-stranded library molecule. The two regions of the single-stranded splint strand (e.g., (210) and (220)) are designed to increase the efficiency of circularization of linear nucleic acid molecules. Single-stranded splint strands and methods using them offer the advantage of providing highly efficient circularization with as little as 0.25 pmol of library molecule.

[0124] The methods described herein may be performed manually or adapted for automation, as annealing and multi-enzyme reactions can be performed in a single reaction vessel (one-pot) by combining several enzymatic reactions (e.g., phosphorylation and ligation) and adding subsequent enzymes (e.g., exonucleases) without alcohol precipitation or organic extraction.

[0125] Single Strand Splint Chain The present disclosure provides a nucleic acid single-stranded splint strand (200) comprising a first region (210) that hybridizes to a sequence at one end of a linear single-stranded library molecule and a second region (220) that hybridizes to a sequence at the other end of the linear single-stranded library molecule. The two regions (e.g., (210) and (220)) of the single-stranded splint strand are designed to hybridize to universal adaptor sequences at the ends of a single-stranded linear library molecule (100) having a sequence of interest (110). For example, the first region of the single-stranded splint strand (210) hybridizes to one end of the library molecule, and the second region of the single-stranded splint strand (220) hybridizes to the other end of the library molecule, thereby circularizing the library molecule to generate a nick-containing library-splint complex (300) (see, e.g., Figures 1-5). The nicks can be enzymatically ligated to generate a covalently closed circular molecule (400) in which the ends of the library molecules are covalently linked.

[0126] The terminal sequences of the linear nucleic acid library molecule comprise at least a first and a second universal adaptor sequence, respectively. In some embodiments, the first and second universal adaptor sequences of the linear library molecule comprise binding sequences for first and second capture primers, respectively, immobilized on a support.

[0127] In some embodiments, the first region of the single-stranded splint strand (210) comprises a first universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0128] In some embodiments, the second region of the single-stranded splint strand (220) comprises a second universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0129] In some embodiments, the single-stranded splint strands (200) comprise one or more phosphorothioate linkages at their 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the single-stranded splint strands (200) comprise one or more phosphorothioate linkages at internal positions to confer endonuclease resistance. In some embodiments, the single-stranded splint strands (200) comprise one or more 2'-O-methylcytosine bases at their 5' and / or 3' ends or at internal positions. In some embodiments, the 5' end of the single-stranded splint strands (200) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the single-stranded splint strands (200) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0130] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, wherein the first region (210) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193) (e.g., Figures 7A-7C).

[0131] In some embodiments, the first left universal adaptor sequence (120) in a library molecule comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199).

[0132] 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, and the second region (220) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194) (e.g., Figures 7A-7C).

[0133] In some embodiments, the first right universal adaptor sequence (130) in a library molecule comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200).

[0134] In some embodiments, the single-stranded splint strand (200) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195) (e.g., Figures 7A-7C).

[0135] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and the first region (210) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0136] 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, and the second region (220) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0137] In some embodiments, the single-stranded splint strand (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0138] Library-Sprint Complex The present disclosure provides a library-sprint 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) the single-stranded splint strand (200) comprises a first region (210) that hybridizes to a sequence at one end of the linear single-stranded library molecule and a second region (220) that hybridizes to a sequence at the other end of the linear single-stranded library molecule. In the library-sprint complex (300), a first region of the single-stranded splint strand (210) hybridizes to at least a first left universal adaptor sequence (120) of the library molecule, and a second region of the single-stranded splint strand (220) hybridizes to at least a first right universal sequence (130) of the library molecule, thereby circularizing the library molecule to generate the library-sprint complex (300) (see, e.g., Figures 1-5).

[0139] In the library-splint complex (300), the first region of the single-stranded splint strand (210) comprises a first universal adaptor sequence capable of hybridizing to a first universal binding sequence at one end of a linear nucleic acid library molecule (see, e.g., Figures 1-5). In some embodiments, the first region of the single-stranded splint strand (210) comprises a first universal adaptor sequence comprising a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the single-stranded splint strand (200) can be 20-150 nucleotides in length, or 60-100 nucleotides in length, or 70-90 nucleotides in length, or 60-80 nucleotides in length. In some embodiments, the single-stranded splint strand (200) comprises one or more phosphorothioate linkages at the 5' and / or 3' end to confer exonuclease resistance. In some embodiments, the single-stranded splint strand (200) comprises one or more phosphorothioate linkages at an internal position to confer endonuclease resistance. In some embodiments, the single-stranded splint strand (200) comprises 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 strand (200) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the single-stranded splint strand (200) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0140] The second region of the single-stranded splint strand (220) 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 (see, e.g., Figures 1-5). In some embodiments, the second region of the single-stranded splint strand (220) comprises a second universal adapter sequence that comprises a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0141] In the library-splint complex (300), a first region of the single-stranded splint strand (210) hybridizes to at least the first left universal adaptor sequence (120) of the library molecule, and a second region of the single-stranded splint strand (220) hybridizes to at least the first right universal sequence (130) of the library molecule, thereby circularizing the library molecule to generate the library-splint complex (300). The library-splint complex (300) comprises a nick between the 5' end of the library molecule and the 3' end of the library molecule (see, e.g., Figures 1-5). In some embodiments, the nick is enzymatically ligatable.

[0142] In the library-sprint complex (300), a first region of the single-stranded splint strand (210) can hybridize to the sense or antisense strand of a double-stranded nucleic acid library molecule. In the library-sprint complex (300), a second region of the single-stranded splint strand (220) can hybridize to the sense or antisense strand of a double-stranded nucleic acid library molecule. The double-stranded nucleic acid library molecule can be denatured to generate single-stranded sense and antisense library strands. The single-stranded library molecule (100) can be the sense or antisense strand.

[0143] In the library-sprint complex (300), a first region of the single-stranded splint strand (210) does not hybridize to the sequence of interest (110), and a second region of the single-stranded splint strand (220) does not hybridize to the sequence of interest (110).

[0144] In some embodiments, the 5' ends of the single-stranded library molecules (100) in the library-sprint complex (300) are phosphorylated or lack a phosphate group. In some embodiments, the 3' ends of the single-stranded library molecules include a terminal 3' OH group or a terminal 3' blocking group.

[0145] In some embodiments, the nucleic acid library molecule (100) further comprises a second left universal adaptor sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises a second right universal adaptor sequence (150). An exemplary library-splint complex (300) is shown in Figures 1-5. In some embodiments, the nucleic acid library molecule (100) can further comprise additional left and / or right universal adaptor sequences.

[0146] 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 first right index sequence (170) can be 3 to 20 nucleotides in length. In some embodiments, the first left index sequence (160) comprises a sample index sequence. The first left index sequence (160) can be 3 to 20 nucleotides in length. In some embodiments, the first right index sequence (170) comprises 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 in a multiplex assay to distinguish sequences of interest obtained from different sample sources. An exemplary library-sprint complex (300) is shown in Figures 1-5.

[0147] A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Table 1 in Figures 9A-9F. The first right index sequence (170) may include a short random sequence (e.g., NNN) or may lack a short random sequence. The short random sequence can be 3 to 20 nucleotides in length.

[0148] In some embodiments, the nucleic acid library molecule (100) further comprises at least one junction adaptor sequence located between any of the universal adaptor sequences described herein (see, e.g., FIG. 5). For example, a first left junction adaptor sequence (125) can be located between the first left universal adaptor sequence (120) and the first left index sequence (160). A second left junction adaptor sequence (165) can be located between the first left index sequence (160) and the second left universal adaptor sequence (140). A third junction adaptor sequence (145) can be located between the second left universal adaptor sequence (140) and the sequence of interest (110). A first right junction adaptor sequence (135) can be located between the first right universal sequence (130) and the first right index sequence (170). A second right junction adapter sequence (175) can be located between the first right index sequence (170) and the second right universal adapter sequence (150). A third right junction adapter sequence (155) can be located between the second right universal adapter sequence (150) and the sequence of interest (110). Any of the junction adapter sequences can include any sequence and be 3 to 60 nucleotides in length. Any of the junction adapter sequences includes a universal sequence or a unique sequence. Any of the junction adapter sequences includes a random sequence (e.g., NNN) having 3 to 20 nucleotides. Any of the junction adapter sequences includes a binding sequence for an amplification primer, a sequencing primer, or a compaction oligonucleotide. Any of the junction adapter sequences includes a binding sequence for an immobilized surface primer (e.g., a capture primer). Any of the junction adapter sequences includes a sample index sequence. Any of the junction adapter sequences includes a unique identification sequence. One of the junction adapter sequences, in particular junction adapter sequence (145), contains the Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 209).Any of the junction adapter sequences, particularly junction adapter sequence (155), comprises the Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 210). The Tn5 transposon end sequence can be introduced into library molecule (100) via a transposase-mediated reaction, which comprises contacting double-stranded input DNA (e.g., genomic DNA) with a Tn5-type transposase enzyme and a double-stranded oligonucleotide comprising the Tn transposon end sequence (SEQ ID NO: 209) linked to a universal adapter sequence or a sample index sequence under conditions suitable for forming a transposon synaptic complex. 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 sample index sequence.

[0149] Multiplex workflows are enabled by preparing sample-indexed libraries using one or both index sequences (e.g., left index sequence and / or right index sequence). The first left index sequence (160) and / or the first right index sequence (170) can be used to prepare separate sample-indexed libraries using input nucleic acids isolated from different sources. The sample-indexed libraries can be pooled together to generate a multiplexed library mixture, and the pooled libraries can be circularized, amplified, and / or sequenced. The sequence of the insert region, along 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 sample-indexed libraries can be pooled together, for example, 2 to 10, or 10 to 50, or 50 to 100, or 100 to 200, or more than 200 sample-indexed libraries can be pooled. Exemplary nucleic acid sources include naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed, paraffin-embedded; FFPE) sources. Those skilled in the art 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.

[0150] 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 used to uniquely identify an individual sequence of interest (e.g., an insert sequence) to which a unique adapter has been added in a population of other sequences of molecules of interest. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular tagging. In some embodiments, the unique identification sequence (180) and / or (190) comprises two to twelve or more nucleotides having a known sequence. For example, the unique identification sequence comprises a known random sequence, in which the nucleotides at each position are 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 molecular tagging procedures. An exemplary library-sprint complex 300 is shown in Figures 1-5.

[0151] In some embodiments, the nucleic acid library molecule (100) comprises any one or any combination of two or more of a first left universal adaptor sequence (120), a second left universal adaptor sequence (140), a first left index sequence (160), a first left unique identifier sequence (180), a first right universal adaptor sequence (130), a second right universal adaptor sequence (150), a first right index sequence (170), and / or a first right unique identifier sequence (190). Figures 1-5 show some exemplary library-splint complexes (300).

[0152] In some embodiments, the first left universal adapter sequence (120) and / or the second left universal adapter sequence (140) comprise a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) can further comprise additional left universal adapter sequences.

[0153] In some embodiments, the first right universal adapter sequence (130) and / or the second right universal adapter sequence (150) comprise a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) can further comprise additional right universal adapter sequences.

[0154] In some embodiments, an exemplary library-splint complex (300) includes (a) a single-stranded nucleic acid library molecule (100) and (b) a single-stranded splint strand (200).

[0155] In the exemplary library-sprint complex (300), the single-stranded nucleic acid library molecule (100) includes the following 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 sequence of interest (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.

[0156] In the exemplary library-sprint complex (300), the single-stranded splint strand (200) comprises a first region (210) and a second region (220), components arranged in 5' to 3' order.

[0157] In an exemplary library-sprint complex (300), a portion of the single-stranded splint strand (200) hybridizes to a portion of the library molecule (100), thereby circularizing the library molecule to generate a library-sprint complex (300), whereby a first region (210) of the single-stranded splint strand hybridizes to the binding sequence (120) for the second surface primer, and a second region (220) of the single-stranded splint strand hybridizes to the binding sequence (130) for the first surface primer. The library-sprint complex (300) includes a nick between the 5' end of the library molecule and the 3' end of the library molecule, the nick being enzymatically ligatable. An exemplary library-sprint complex (300) is shown in Figures 1-5.

[0158] In the exemplary library-sprint complex (300), a first region of the single-stranded splint strand (210) does not hybridize to the sequence of interest (110), and a second region of the single-stranded splint strand (220) does not hybridize to the sequence of interest (110).

[0159] In some embodiments, any of the library-sprint complexes (300) described herein comprises a plurality of library-sprint complexes (300), wherein the target sequences (110) of the individual library-sprint complexes in the plurality of library-sprint complexes comprise the same target sequence or different target sequences.

[0160] In some embodiments, the first left universal adaptor sequence (120) in a library molecule comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199).

[0161] In some embodiments, the first left universal adaptor sequence (120) in a library molecule comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203).

[0162] In some embodiments, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 201).

[0163] In some embodiments, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 205).

[0164] In some embodiments, the second left universal adaptor sequence (140) in the library molecule comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 206).

[0165] In some embodiments, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 202).

[0166] In some embodiments, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 207).

[0167] In some embodiments, the second right universal adaptor sequence (150) in the library molecule comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 208).

[0168] In some embodiments, the first right universal adaptor sequence (130) in a library molecule comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200).

[0169] In some embodiments, the first right universal adaptor sequence (130) in a library molecule comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 204).

[0170] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and the first region (210) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0171] 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, and the second region (220) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0172] In some embodiments, the single-stranded splint strand (200) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195). See, e.g., Figures 7A-7C.

[0173] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and the first region (210) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0174] 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, and the second region (220) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0175] In some embodiments, in the method, the single-stranded splint strand (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0176] The present disclosure provides a reaction mixture comprising a plurality of any of the library-sprint complexes (300) described herein. In some embodiments, the reaction mixture comprises a plurality of any of the library-sprint complexes (300) described herein and T4 polynucleotide kinase. In some embodiments, the reaction mixture comprises a plurality of any of the library-sprint complexes (300) described herein and a ligase enzyme. In some embodiments, the reaction mixture comprises a plurality of any of the library-sprint complexes (300) described herein, T4 polynucleotide kinase, and a ligase enzyme. In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0177] Covalent closed ring molecule The present disclosure provides a covalently closed circular library molecule (400) comprising a sequence of interest (110), at least a first left universal adaptor sequence (120), and at least a first right universal adaptor sequence (130). Exemplary covalently closed circular library molecules are shown in Figures 6A-6C. In some embodiments, the covalently closed circular library molecule (400) further comprises a second left universal adaptor sequence (140). In some embodiments, the covalently closed circular molecule (400) further comprises a second right universal adaptor sequence (150). In some embodiments, the covalently closed circular molecule (400) further comprises additional left and / or right universal adaptor sequences.

[0178] 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 first left index sequence (160) can be 3 to 20 nucleotides in length. The first right index sequence (170) can be 3 to 20 nucleotides in length. The sample index sequence can be used in a multiplex assay to distinguish sequences of interest obtained from different sample sources. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Table 1 in Figures 9A-9F. The first right index sequence (170) and / or the first left index sequence (160) can include a short random sequence (e.g., NNN) or can lack a short random sequence. The short random sequence can be 3 to 20 nucleotides in length.

[0179] Multiplex workflows are enabled by preparing sample-indexed libraries using one or both index sequences (e.g., left index sequence and / or right index sequence). The first left index sequence (160) and / or the first right index sequence (170) can be used to prepare separate sample-indexed libraries using input nucleic acids isolated from different sources. The sample-indexed libraries can be pooled together to generate a multiplexed library mixture, and the pooled libraries can be circularized, amplified, and / or sequenced. The sequence of the insert region, along 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 sample-indexed libraries can be pooled together, for example, 2 to 10, or 10 to 50, or 50 to 100, or 100 to 200, or more than 200 sample-indexed libraries can be pooled. Exemplary nucleic acid sources include naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed, paraffin-embedded; FFPE) sources. Those skilled in the art 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.

[0180] 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 used to uniquely identify an individual sequence of interest (e.g., an insert sequence) to which a unique adaptor has been added in a population of other sequences of molecules of interest (e.g., molecular tagging).

[0181] In some embodiments, the covalently closed circular molecule (400) comprises any one or any combination of two or more of the following: a first left universal adaptor sequence (120), a second left universal adaptor sequence (140), a first left index sequence (160), a first left unique identifier sequence (180), a first right universal adaptor sequence (130), a second right universal adaptor sequence (150), a first right index sequence (170), and / or a first right unique identifier sequence (190). In some embodiments, the first left index sequence (160) comprises a sample index sequence. In some embodiments, the first right index sequence (170) comprises another sample index sequence. The sequences of the right sample index sequence and the left sample index sequence (e.g., (170) and (160)) may be the same as or different from each other. The sample index sequence may be used in a multiplex assay to distinguish sequences of interest obtained from different sample sources. In some embodiments, the first left unique identification sequence (180) and the first right unique identification sequence (190) each comprise a sequence used to uniquely identify an individual sequence of interest (e.g., an insert sequence) to which a unique adaptor has been added in a population of other sequences of molecules of interest. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular tagging. In some embodiments, the unique identification sequence comprises two to twelve or more nucleotides having a known sequence. For example, the unique identification sequence (180) and / or (190) comprises a known random sequence, in which the nucleotide at each position in the unique identification sequence is randomly selected from nucleotides having the nucleic acid bases A, G, C, T, or U. The unique identification sequence (180) and / or (190) can be used for molecular tagging procedures. Exemplary covalently closed circular library molecules are shown in Figures 6A-6C.

[0182] 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 or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the covalently closed circular molecule (400) can further comprise an additional left universal adapter sequence.

[0183] 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 comprise a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the covalently closed circular molecule 400 can further comprise an additional right universal adapter sequence.

[0184] In some embodiments, the covalently closed circular molecule (400) further comprises at least one junction adaptor sequence located between any of the universal adaptor sequences described herein (see, e.g., FIG. 5). For example, the first left junction adaptor sequence (125) can be located between the first left universal adaptor sequence (120) and the first left index sequence (160). The second left junction adaptor sequence (165) can be located between the first left index sequence (160) and the second left universal adaptor sequence (140). The third junction adaptor sequence (145) can be located between the second left universal adaptor sequence (140) and the sequence of interest (110). The first right junction adaptor sequence (135) can be located between the first right universal sequence (130) and the first right index sequence (170). A second right junction adapter sequence (175) can be located between the first right index sequence (170) and the second right universal adapter sequence (150). A third right junction adapter sequence (155) can be located between the second right universal adapter sequence (150) and the sequence of interest (110). Any of the junction adapter sequences can include any sequence and be 3 to 60 nucleotides in length. Any of the junction adapter sequences includes a universal sequence or a unique sequence. Any of the junction adapter sequences includes a random sequence (e.g., NNN) having 3 to 20 nucleotides. Any of the junction adapter sequences includes a binding sequence for an amplification primer, a sequencing primer, or a compaction oligonucleotide. Any of the junction adapter sequences includes a binding sequence for an immobilized surface primer (e.g., a capture primer). Any of the junction adapter sequences includes a sample index sequence. Any of the junction adapter sequences includes a unique identification sequence. One of the junction adapter sequences, in particular junction adapter sequence (145), contains the Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 209).Any of the junction adapter sequences, particularly junction adapter sequence (155), comprises the Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 210). The Tn5 transposon end sequence can be introduced into library molecule (100) via a transposase-mediated reaction, which comprises contacting double-stranded input DNA (e.g., genomic DNA) with a Tn5-type transposase enzyme and a double-stranded oligonucleotide comprising the Tn transposon end sequence (SEQ ID NO: 209) linked to a universal adapter sequence or a sample index sequence under conditions suitable for forming a transposon synaptic complex. 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 sample index sequence.

[0185] In some embodiments, an exemplary covalently closed circular molecule (400) comprises: (i) a first left universal adapter sequence (120) having a binding sequence (120) 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); (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 (130) for a first surface primer; and (vi) optionally, the covalently closed circular molecule (400) is hybridized to a single-stranded splint strand (200) (see, e.g., Figures 6A-6C).

[0186] In the exemplary covalently closed ring molecule (400), the first and second regions of the single-stranded splint strand (210 and 220, respectively) do not hybridize to the sequence of interest (110).

[0187] In some embodiments, any of the covalently closed circular molecules (400) described herein further comprises a plurality of covalently closed circular molecules (400), wherein the target sequences (110) of the individual covalently closed circular molecules (400) in the plurality of covalently closed circular molecules (400) comprise the same target sequence or different target sequences.

[0188] 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).

[0189] 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).

[0190] 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).

[0191] 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).

[0192] 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).

[0193] 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).

[0194] 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).

[0195] 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).

[0196] 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).

[0197] 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).

[0198] In some embodiments, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and the first region (210) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0199] 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, and the second region (220) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0200] In some embodiments, the single-stranded splint strand (200) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195). See, e.g., Figures 7A-7C.

[0201] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and the first region (210) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0202] 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, and the second region (220) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0203] In some embodiments, in the method, the single-stranded splint strand (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0204] The present disclosure provides a reaction mixture comprising a plurality of any of the covalently closed circular molecules (400) described herein and at least one exonuclease enzyme, in some embodiments, the exonuclease enzyme comprises any one or any combination of two or more of Exonuclease I, Thermolabile Exonuclease I, and / or T7 Exonuclease.

[0205] Kit containing single-stranded splint strands The present disclosure provides at least one kit for circularizing linear nucleic acid library molecules to form a plurality of library-splint complexes (300), each having a nick. In some embodiments, the kit can be used to circularize single-stranded linear nucleic acid library molecules having 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). In some embodiments, the circularized library molecules can be converted into covalently closed circular molecules, which can be subjected to a rolling circle amplification (RCA) reaction to generate nucleic acid concatemers. The concatemers can be immobilized on a support. The immobilized concatemers can be subjected to a massively parallel sequencing reaction.

[0206] The present disclosure provides kits including nucleic acid single-stranded splint strands (200), each including a first region (210) and a second region (220). The first region (210) includes a first universal adaptor sequence capable of hybridizing to a first universal binding sequence at one end of a linear nucleic acid library molecule. The second region (220) includes a second universal adaptor sequence capable of hybridizing to a second universal binding sequence at the other end of the linear nucleic acid library molecule. In some embodiments, the first region of the single-stranded splint strand (210) includes a first universal adaptor sequence that includes a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the second region of the single-stranded splint strand (220) comprises a second universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the single-stranded splint strand (200) can be 20 to 150 nucleotides in length, or 60 to 100 nucleotides in length, or 70 to 90 nucleotides in length, or 60 to 80 nucleotides in length. In some embodiments, the single-stranded splint strand (200) comprises one or more phosphorothioate linkages at the 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the single-stranded splint strand (200) comprises one or more phosphorothioate linkages at internal positions to confer endonuclease resistance.In some embodiments, the single-stranded splint strand (200) comprises 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 strand (200) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the single-stranded splint strand (200) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0207] In some embodiments, the kit comprises a single-stranded splint strand (200) having a first region (210) comprising the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0208] In some embodiments, the kit comprises a single-stranded splint strand (200) having a second region (220) comprising the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0209] In some embodiments, the kit includes a single-stranded splint strand (200) comprising the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195).

[0210] In some embodiments, the kit further includes an adapter having a left universal adapter sequence (120) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199) capable of binding to a surface primer. In some embodiments, the adapter having the left universal adapter sequence (120) also 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. See, e.g., Figures 12A, B, and C.

[0211] In some embodiments, the kit further includes an adapter having a right universal adapter sequence (130) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 200) capable of binding to a surface primer. In some embodiments, the adapter having the right universal adapter sequence (130) also 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. See, e.g., Figures 12A, B, and C.

[0212] In some embodiments, the kit further includes an adapter having a left universal adapter sequence (140) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 201) capable of binding to a sequencing primer. In some embodiments, the adapter having the left universal adapter sequence (140) also 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. See, e.g., Figures 12A, B, and C.

[0213] In some embodiments, the kit further includes an adapter having a right universal adapter sequence (150) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 202) capable of binding to a sequencing primer. In some embodiments, the adapter having the right universal adapter sequence (150) also 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. See, e.g., Figures 12A, B, and C.

[0214] In some embodiments, the kit comprises a single-stranded splint strand (200) having a first region (210) comprising the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0215] In some embodiments, the kit comprises a single-stranded splint strand (200) having a second region (220) comprising the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0216] In some embodiments, the kit includes a single-stranded splint strand (200) comprising the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0217] In some embodiments, the kit further comprises an adapter having a left universal adapter sequence (120) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203) capable of binding to a surface primer. In some embodiments, the adapter having the left universal adapter sequence (120) also 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.

[0218] In some embodiments, the kit further comprises an adapter having a right universal adapter sequence (130) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 204) capable of binding to a surface primer. In some embodiments, the adapter having the right universal adapter sequence (130) also 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.

[0219] In some embodiments, the kit further includes an adapter having a left universal adapter sequence (140) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 205) capable of binding to a sequencing primer. In some embodiments, the adapter having the left universal adapter sequence (140) also 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.

[0220] In some embodiments, the kit further includes an adapter having a right universal adapter sequence (150) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 207) capable of binding to a sequencing primer. In some embodiments, the adapter having the right universal adapter sequence (150) also 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.

[0221] In some embodiments, the kit further includes an adapter having a left universal adapter sequence (140) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 206) capable of binding to a sequencing primer. In some embodiments, the adapter having the left universal adapter sequence (140) also 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.

[0222] In some embodiments, the kit further includes an adapter having a right universal adapter sequence (150) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 208) capable of binding to a sequencing primer. In some embodiments, the adapter having the right universal adapter sequence (150) also 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.

[0223] In some embodiments, the kit further includes a plurality of sample indexes including a plurality of first left indexes (160) and / or a plurality of first right index arrays (170). The first left index array (160) can be 3 to 20 nucleotides in length. The first right index array (170) can be 3 to 20 nucleotides in length. In some embodiments, the kit can include separate containers or wells of a multiwell plate (e.g., a 96-well plate) that hold individual first left indexes (160) or individual first right indexes (170). In some embodiments, the kit can include separate containers or wells of a multiwell plate that hold pairs of individual first left indexes (160) and individual first right indexes (170). In some embodiments, the kit contains the first left indexes (160) and / or the plurality of first right index arrays (170) within a multiwell 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 Table 1 in Figures 9A-9F. The first left index sequence (160) can include a short random sequence (e.g., NNN) or lack a short random sequence. The first right index sequence (170) can include a short random sequence (e.g., NNN) or lack a short random sequence. The short random sequence can be 3 to 20 nucleotides in length.

[0224] In some embodiments, the kit includes a plurality of tailed PCR primers each comprising a first left index sequence (160). The tailed PCR primers can 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), and (iii) a second left universal adapter sequence (140) having a binding sequence for a first or second sequencing primer. The first left index sequence (160) can also include a random sequence (e.g., NNN). See, for example, Figure 17.

[0225] In some embodiments, the kit includes a plurality of tailed PCR primers each comprising a first right index sequence (170). The tailed PCR primers 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 or second sequencing primer. The first right index sequence (170) can also include a random sequence (e.g., NNN). See, for example, Figure 17.

[0226] In some embodiments, the kit includes a nucleic acid single-stranded splint strand (200) and further includes T4 polynucleotide kinase. In some embodiments, the kit further includes a ligase enzyme, wherein the ligase enzyme includes T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. In some embodiments, the kit further includes at least one endonuclease, wherein the at least one endonuclease includes any one or any combination of two or more of exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease.

[0227] In some embodiments, the kit includes at least one buffer for hybridizing a plurality of single-stranded splint strands (200) with 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, the multiple enzymatic reactions including any combination of (i) phosphorylating the 5' ends of the single-stranded splint strands (200), (ii) ligating nicks in the library-splint complex (300), and / or (iii) exonuclease digestion of the single-stranded splint strands (200) from the covalently closed circular molecule (400). Alternatively, the kit includes two or more separate buffers, where 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.

[0228] In some embodiments, the kit includes one or more containers containing any of the single-stranded splint strands (200) described herein. 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 contained in a single container, or in separate containers, or any combination thereof.

[0229] The kit can include instructions for using the kit to perform a reaction to circularize a linear nucleic acid library molecule to form a plurality of library-sprint complexes (300), each having a nick. The kit can include instructions for using the kit to generate a covalently closed circular library molecule (400) by ligating the nicks in the library-sprint complexes (300).

[0230] Kit for library preparation, including adapter and tailed PCR primers 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., insert region (110)) to Y-shaped adapters to generate a plurality of adapter-insert-adapter constructs, and adding additional adapter sequences to the adapter-insert-adapter constructs, where the adding is performed by performing a primer extension or PCR reaction using first and second tailed PCR primers (e.g., Figure 17). The resulting linear library molecules can be circularized to form covalently closed circular molecules using any of the single-stranded splint molecules described herein (e.g., Figures 3 and 4).

[0231] In some embodiments, the library preparation kit includes a Y-shaped adaptor. The Y-shaped adaptor comprises a first and a second nucleic acid strand, where a portion of the two strands are perfectly complementary to each other and annealed together, and another portion of the two strands is not complementary to each other and is mismatched. In some embodiments, the ligation end of the Y-shaped adaptor comprises an annealing portion that forms a blunt end or an overhanging end (e.g., a 5' overhanging end or a 3' overhanging end). In some embodiments, in the library preparation kit, the first strand of the Y-shaped adaptor comprises an oligonucleotide having the sequence 5'-TGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 211). In some embodiments, in the library preparation kit, the second strand of the Y-shaped adaptor comprises an oligonucleotide having the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 212).

[0232] In some embodiments, the library preparation kit comprises a sequence [Sequence Table 1] JPEG2025509201000002.jpg10140, where 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 of the right sample index. In some embodiments, the last three "NNN"s (underlined) in the 12-base N sequence comprise a 3-base long random sequence designed to provide nucleotide diversity and color balance in each sequencing cycle.

[0233] In some embodiments, the library preparation kit includes a second tailed PCR primer comprising the sequence 5'-CATGTAATGCACGTACTTTCAGGGTNNNNNNNNNN CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 214), where 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.

[0234] Methods for forming multiple library-splint complexes 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), each of which comprises 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) (see, e.g., Figures 1-5).

[0235] The method for forming a plurality of library-splint complexes (300) further includes step (b): providing a plurality of single-stranded splint strands (200), wherein each single-stranded splint strand (200) in the plurality of single-stranded splint strands (200) comprises a first region (210) capable of hybridizing with at least a first left universal adaptor sequence (120) of an individual library molecule and a second region (220) capable of hybridizing with at least a first right universal adaptor sequence (130) of an individual library molecule. Exemplary single-stranded splint strands (200) are shown in Figures 1-5. In some embodiments, the single-stranded splint strands (200) can be 20-150 nucleotides in length, or 60-100 nucleotides in length, or 70-90 nucleotides in length, or 60-80 nucleotides in length.

[0236] The method for forming a plurality of library-splint complexes (300) further includes step (c): hybridizing the plurality of single-stranded splint strands (200) to a plurality of single-stranded nucleic acid library molecules (100). The hybridizing is performed under conditions suitable for hybridizing individual library molecules with individual single-stranded splint strands, such that a first region of one of the single-stranded splint strands (210) anneals to at least the first left universal adaptor sequence (120) of the library molecule and a second region of the single-stranded splint strand (220) anneals to at least the first right universal sequence (130) of the library molecule, thus circularizing the individual library molecules to form a plurality of library-splint complexes (300). In some embodiments, the library-splint complexes (300) include a nick between the 5' and 3' ends of the library molecule (e.g., Figures 1-5). In some embodiments, the nick is enzymatically ligatable.

[0237] In some embodiments, in a method for forming a plurality of library-splint complexes (300), a first region of the single-stranded splint strand (210) comprises a first universal adaptor sequence capable of hybridizing to a first universal binding sequence (120) at one end of a linear nucleic acid library molecule. In some embodiments, the first region of the single-stranded splint strand (210) comprises a first universal adaptor sequence comprising a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0238] In some embodiments, in a method for forming a plurality of library-splint complexes (300), the second region of the single-stranded splint strand (220) comprises a second universal adaptor sequence capable of hybridizing to a second universal binding sequence at the other end of the linear nucleic acid library molecule. In some embodiments, the second region of the single-stranded splint strand (220) comprises a second universal adaptor sequence comprising a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0239] In some embodiments, in a method for forming a plurality of library-splint complexes (300), the single-stranded splint strands (200) comprise one or more phosphorothioate linkages at their 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the single-stranded splint strands (200) comprise one or more phosphorothioate linkages at internal positions to confer endonuclease resistance. In some embodiments, the single-stranded splint strands (200) comprise one or more 2'-O-methylcytosine bases at their 5' and / or 3' ends or at internal positions. In some embodiments, the 5' end of the single-stranded splint strands (200) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the single-stranded splint strands (200) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0240] In some embodiments, in a method for forming a plurality of library-splint complexes (300), a first region of the single-stranded splint strand (210) can hybridize to the sense or antisense strand of a double-stranded nucleic acid library molecule. In the library-splint complexes (300), a second region of the single-stranded splint strand (220) can hybridize to the sense or antisense strand of a double-stranded nucleic acid library molecule. The double-stranded nucleic acid library molecules can be denatured to generate single-stranded sense and antisense library strands. The double-stranded nucleic acid library molecules can be denatured to generate the single-stranded nucleic acid library molecules (100) of step (a).

[0241] In some embodiments, in a method for forming a plurality of library-sprint 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).

[0242] In some embodiments, in the method for forming a plurality of library-splint complexes (300), the nucleic acid library molecule (100) further comprises a second left universal adaptor sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises a second right universal adaptor sequence (150). In some embodiments, the nucleic acid library molecule (100) can further comprise additional left and / or right universal adaptor sequences.

[0243] In some embodiments, in a method for forming a plurality of 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 first left index sequence (160) can be 3 to 20 nucleotides in length. In some embodiments, the first right index sequence (170) comprises another sample index sequence. The first right index sequence (170) can be 3 to 20 nucleotides in length. 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 sequences of interest obtained from different sample sources in a multiplex assay. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Table 1 in Figures 9A-9F. The first left index sequence (160) may include a short random sequence (e.g., NNN) or may lack a short random sequence. The first right index sequence (170) may include a short random sequence (e.g., NNN) or may lack a short random sequence. The short random sequence (e.g., NNN) can be 3 to 20 nucleotides in length.

[0244] Multiplex workflows are enabled by preparing sample-indexed libraries using one or both index sequences (e.g., left index sequence and / or right index sequence). The first left index sequence (160) and / or the first right index sequence (170) can be used to prepare separate sample-indexed libraries using input nucleic acids isolated from different sources. The sample-indexed libraries can be pooled together to generate a multiplexed library mixture, and the pooled libraries can be circularized, amplified, and / or sequenced. The sequence of the insert region, along 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 sample-indexed libraries can be pooled together, for example, 2 to 10, or 10 to 50, or 50 to 100, or 100 to 200, or more than 200 sample-indexed libraries can be pooled. Exemplary nucleic acid sources include naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed, paraffin-embedded; FFPE) sources. Those skilled in the art 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.

[0245] In some embodiments, in the 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 used to uniquely identify an individual sequence of interest (e.g., an insert sequence) to which a unique adapter has been added in a population of other sequences of molecules of interest. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular tagging. In some embodiments, the unique identification sequence comprises two to twelve or more nucleotides having a known sequence. For example, the unique identification sequence comprises a known random sequence, in which the nucleotides at each position are 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 molecular tagging procedures.

[0246] In some embodiments, in a method for forming a plurality of library-sprint complexes (300), the nucleic acid library molecule (100) comprises any combination of one or more of a first left universal adaptor sequence (120), a second left universal adaptor sequence (140), a first left index sequence (160), a first left unique identifier sequence (180), a first right universal adaptor sequence (130), a second right universal adaptor sequence (150), a first right index sequence (170), and / or a first right unique identifier sequence (190) in any order.

[0247] In some embodiments, in the 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) comprise a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) can further comprise additional left universal adapter sequences.

[0248] In some embodiments, in the 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) comprise a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward or reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) can further comprise additional right universal adapter sequences.

[0249] 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 adaptor sequence located between any of the universal adaptor sequences described herein (see, e.g., FIG. 5). For example, a first left junction adaptor sequence (125) can be located between the first left universal adaptor sequence (120) and the first left index sequence (160). A second left junction adaptor sequence (165) can be located between the first left index sequence (160) and the second left universal adaptor sequence (140). A third junction adaptor sequence (145) can be located between the second left universal adaptor sequence (140) and the sequence of interest (110). A first right junction adaptor sequence (135) can be located between the first right universal sequence (130) and the first right index sequence (170). A second right junction adapter sequence (175) can be located between the first right index sequence (170) and the second right universal adapter sequence (150). A third right junction adapter sequence (155) can be located between the second right universal adapter sequence (150) and the sequence of interest (110). Any of the junction adapter sequences can include any sequence and be 3 to 60 nucleotides in length. Any of the junction adapter sequences includes a universal sequence or a unique sequence. Any of the junction adapter sequences includes a random sequence (e.g., NNN) having 3 to 20 nucleotides. Any of the junction adapter sequences includes a binding sequence for an amplification primer, a sequencing primer, or a compaction oligonucleotide. Any of the junction adapter sequences includes a binding sequence for an immobilized surface primer (e.g., a capture primer). Any of the junction adapter sequences includes a sample index sequence. Any of the junction adapter sequences includes a unique identification sequence.Any of the junction adapter sequences, particularly junction adapter sequence (145), comprises the Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 209). Any of the junction adapter sequences, particularly junction adapter sequence (155), comprises the Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 210). The Tn5 transposon end sequence can be introduced into library molecule (100) via a transposase-mediated reaction, which comprises contacting double-stranded input DNA (e.g., genomic DNA) with a Tn5-type transposase enzyme and a double-stranded oligonucleotide comprising the Tn transposon end sequence (SEQ ID NO: 209) linked to a universal adapter sequence or sample index sequence, under conditions suitable for forming a transposon synaptic complex. In the double-stranded oligonucleotide, the Tn transposon end sequence (SEQ ID NO: 209) can be located 5' or 3' to the universal adapter sequence or the sample index sequence.

[0250] The present disclosure provides a method for forming a plurality of library-splint complexes (300), the method comprising: (a) providing a plurality of single-stranded splint strands (200), each of which comprises, arranged in 5' to 3' order, (i) a first region (210) having a universal binding sequence that hybridizes to a sequence (e.g., 120) at one end of a linear single-stranded library molecule, and (ii) a second region (220) having a universal binding sequence that hybridizes to a sequence (e.g., 130) at the other end of the linear single-stranded library molecule. The method for forming a plurality of library-splint complexes (300) includes step (b): hybridizing a plurality of single-stranded splint strands (200) with a plurality of single-stranded nucleic acid library molecules (100), each library molecule comprising a region 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 or second sequencing primer; (iii) a sequence of interest (110); (iv) a second right universal adapter sequence (150) having a binding sequence for a second or first sequencing primer; and (v) a first right universal adapter sequence (160) having a binding sequence for a first surface primer. The method further includes hybridizing the single-stranded splint strand (200) to the library molecule (100) under conditions suitable for hybridizing the single-stranded splint strand (200) to the library molecule (100), thereby circularizing the library molecule to produce a library-splint complex (300), whereby a first region (210) of the single-stranded splint strand hybridizes to the binding sequence (120) for the second surface primer and a second region (220) of the single-stranded splint strand hybridizes to the binding sequence (130) for the first surface primer, and the library-splint complex (300) comprises a nick between the 5' and 3' ends of the library molecule, the nick being enzymatically ligatable (see, e.g., Figure 2).In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprise a first left index sequence (160) and / or a first right index sequence (170) (see, e.g., Figures 3 and 4). In some embodiments, in a given library-sprint complex (300) of the plurality of library-sprint 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 first left index sequence (160) can be 3 to 20 nucleotides in length. The first right index sequence (170) can be 3 to 20 nucleotides in length. The first left index sequence (160) and / or the first right index sequence (170) can comprise a short random sequence (e.g., NNN). The short random sequence can be 3 to 20 nucleotides in length. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Table 1 in Figures 9A-9F. In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left unique identifier sequence (180) and / or a first right unique identifier sequence (190) that can be used for molecular tagging (see, e.g., Figures 3 and 4).

[0251] 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).

[0252] 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).

[0253] 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).

[0254] 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).

[0255] 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).

[0256] 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).

[0257] 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).

[0258] In some embodiments, the method comprises a second right universal adaptor sequence (150) in the library molecule comprising the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 208).

[0259] 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).

[0260] 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).

[0261] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and the first region (210) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 193).

[0262] 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, and the second region (220) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194).

[0263] In some embodiments, in the method, the single-stranded splint strand (200) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 195). See, e.g., Figures 7A-7C.

[0264] In some embodiments, in the method, the first region of the single-stranded splint strand (210) comprises a universal binding sequence for the first left universal adaptor sequence (120) of the library molecule, and the first region (210) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 196).

[0265] 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, and the second region (220) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197).

[0266] In some embodiments, in the method, the single-stranded splint strand (200) comprises the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 198).

[0267] In some embodiments, any of the methods for forming a plurality of library-splint complexes (300) described herein can further include at least one enzymatic reaction, including a phosphorylation reaction, a ligation reaction, and / or an exonuclease reaction. The enzymatic reactions can be performed sequentially or essentially 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, in which a second enzymatic reaction is performed, then transferred to a third reaction vessel, in which a third enzymatic reaction is performed.

[0268] 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, wherein the separate and sequential phosphorylation and ligation reactions are 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 strands (200) and the plurality of single-stranded nucleic acid library molecules (100) in a first reaction vessel with T4 polynucleotide kinase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of single-stranded splint strands (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, in a second reaction vessel, the plurality of phosphorylated single-stranded splint strands (200) and the plurality of phosphorylated single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatic ligation of nicks, thereby producing a plurality of covalently closed circular library molecules (400), each hybridized to a single-stranded splint strand (200). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0269] 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, wherein the sequential phosphorylation and ligation reactions are 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, in a first reaction vessel, the plurality of single-stranded splint strands (200) and the plurality of single-stranded nucleic acid library molecules (100) with T4 polynucleotide kinase enzyme 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, in the same first reaction vessel, the phosphorylated single-stranded splint strand (200) and the phosphorylated single-stranded nucleic acid library molecule (100) with a ligase under conditions suitable for enzymatic ligation of nicks, thereby producing a plurality of covalently closed circular library molecules (400), each hybridized to the single-stranded splint strand (200). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0270] In some embodiments, any of the methods for forming a plurality of library-splint complexes (300) described herein further includes performing essentially simultaneous phosphorylation and ligation reactions, wherein the essentially simultaneous phosphorylation and ligation reactions are performed together in the same reaction vessel. In some embodiments, the method for forming a plurality of library-splint complexes (300) further includes step (c3): contacting, in a first reaction vessel, the plurality of single-stranded splint strands (200) and the plurality of single-stranded nucleic acid library molecules (100) with (i) a T4 polynucleotide kinase enzyme and (ii) a ligase enzyme 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), wherein the conditions are suitable for enzymatically ligating nicks, thereby generating a plurality of covalently closed circular library molecules (400), each hybridized to a single-stranded splint strand (200). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0271] In some embodiments, any of the methods for forming a plurality of library-splint complexes (300) described herein further includes an optional step of enzymatically removing a plurality of single-stranded splint strands (200) from a plurality of covalently closed circular library molecules (400), comprising contacting the plurality of covalently closed circular library molecules (400) with at least one exonuclease enzyme 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 exonuclease reaction can be performed in the same reaction buffer used to perform the phosphorylation and / or ligation reactions, or in a different reaction buffer. In some embodiments, the phosphorylation reaction can be performed in a first reaction vessel (see step c1 above) and the ligation reaction can be performed in a second reaction vessel (see step d1 above), after which the exonuclease reaction can be performed in a third reaction vessel. In some embodiments, a phosphorylation reaction may be performed in a first reaction vessel (see step c2 above), a sequential ligation reaction may be performed in a first reaction vessel (see step d2 above), and then an exonuclease reaction may be performed in the first reaction vessel. In some embodiments, an essentially simultaneous phosphorylation and ligation reaction may be performed in a first reaction vessel (see step c3 above), and then an exonuclease reaction may be performed in the first reaction vessel. In some embodiments, the at least one exonuclease enzyme comprises any combination of two or more of Exonuclease I, Thermolabile Exonuclease I, and / or T7 Exonuclease.

[0272] Methods for Rolling Circle Amplification The present disclosure provides methods for performing a rolling circle amplification reaction on a covalently closed circular library molecule (400). The rolling circle amplification reaction can be performed after a phosphorylation and ligation reaction or after a ligation reaction. In some embodiments, the rolling circle amplification reaction can be performed on a covalently closed circular library molecule (400) hybridized to a single-stranded splint strand (200). In some embodiments, the rolling circle amplification reaction can be performed on a covalently closed circular library molecule (400) that is no longer hybridized to a single-stranded splint strand (200), for example, after an exonuclease reaction. In some embodiments, the covalently closed circular library molecule (400) can be dispensed onto a support and then subjected to a rolling circle amplification reaction. In some embodiments, the covalently closed circular library molecule (400) can be subjected to a rolling circle amplification reaction in solution and then dispensed onto a support. In some embodiments, the rolling circle amplification reaction may use the retained single-stranded splint strand (200) as an amplification primer, or the single-stranded splint strand (200) may be removed (e.g., via exonuclease digestion) and replaced with a soluble amplification primer.

[0273] On-support rolling circle amplification In some embodiments, in a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules lacking a hybridized single-stranded splint strand (200), each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules (400) comprises a universal binding sequence for a first surface primer, the method comprising the step (a): distributing the 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 of the covalently closed circular library molecules (400) to each of the immobilized first surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (400) to the support.

[0274] In some embodiments, in the method for 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).

[0275] In some embodiments, in the method for 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).

[0276] Each first surface primer can hybridize to a covalently closed circular library molecule (400) that has a universal binding sequence for the first surface primer.

[0277] 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-displacing 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 the plurality of covalently closed circular library molecules (400) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules immobilized to 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 linked to each first surface primer. In some embodiments, each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules (400) comprises a universal binding sequence for the first and second surface primers (e.g., (130) and (120), respectively), such that the rolling circle amplification reaction generates concatemer molecules having multiple tandem copies of the universal binding sequences for the first and second surface primers. In some embodiments, the support further comprises multiple second surface primers. In some embodiments, the immobilized second surface primers function to hold at least one portion of the concatemer molecules down to the support. In some embodiments, the immobilized second surface primers have a non-extendable 3' end and are not used for amplification. In some embodiments, the immobilized concatemers can be subjected to a sequencing reaction.

[0278] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199) or a complementary sequence thereof.

[0279] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203 or a complementary sequence thereof).

[0280] Each second surface primer can hybridize to a portion of the concatemer molecule that has a universal binding sequence for the second surface primer. In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule to the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0281] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of covalently closed circular library molecules (400) can be distributed on a support coated with one or more compounds to produce a passivated layer on the support (e.g., FIG. 18). In some embodiments, the passivated 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 passivated layer for immobilization to the support. In some embodiments, the support comprises a low non-specific binding surface, which allows for improved nucleic acid hybridization and amplification performance on the support. Generally, the support can comprise one or more layers of covalently or non-covalently attached low-binding chemically modified layers, e.g., silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize a plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support comprises a functionalized polymer coating layer covalently bonded to at least a portion of the support via chemical groups on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating layer can comprise polyethylene glycol (PEG). The hydrophilic polymer coating layer can comprise a branched PEG having at least four branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, where the water contact angle is 45 degrees or less.In some embodiments, the density of the covalently closed circular library molecules (400) immobilized to the support or to a coating on the support is 1 mm. 2 Approximately 10 per 2 ~10 6 , or 1 mm 2 Approximately 10 per 6 ~10 9 , or 1 mm 2 Approximately 10 per 9 ~10 12 In some embodiments, the plurality of covalently closed circular library molecules (400) are immobilized to a support, to a coating on a support, at predetermined sites on the support (or coating on the support), or to a coating on a support, at random sites on the support (or coating on the support).

[0282] In some embodiments, in the method for 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, the high-efficiency hybridization buffer comprising: (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less, the second polar aprotic solvent being present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0283] In-solution rolling circle amplification using soluble amplification primers In some embodiments, a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules (400) lacking a hybridized single-stranded splint strand (200), wherein each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules (400) 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 a plurality of soluble forward amplification primers in 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-displacing polymerases and a plurality of nucleotides using the plurality of forward amplification primers and the plurality of covalently closed circular library molecules (400) as template molecules under conditions suitable for performing a rolling circle amplification reaction in solution, thereby generating a plurality of nucleic acid concatemer molecules having a portion still hybridized to the covalently closed circular library molecule (400). In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (c): distributing the plurality of concatemer molecules onto a support having a plurality of first surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to the plurality of immobilized first surface primers, thereby immobilizing the plurality of concatemer molecules. The plurality of immobilized 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 step (d): contacting the immobilized plurality of concatemer molecules with a plurality of strand-displacing polymerases and a plurality of nucleotides under conditions suitable for performing a second rolling circle amplification reaction on the support using the plurality of covalently closed circular library molecules (400) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules.In some embodiments, the first and / or second rolling circle amplification reaction can be performed with a plurality of nucleotides containing any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, each immobilized concatemer is hybridized to a respective first surface primer. In some embodiments, each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules (400) contains universal binding sequences for the first and second surface primers (e.g., (130) and (120), respectively), such that the in-solution rolling circle amplification reaction generates concatemer molecules having multiple tandem copies of the universal binding sequences for the first and second surface primers. In some embodiments, the support further comprises a plurality of second surface primers. In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule down to the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemers can be subjected to a sequencing reaction.

[0284] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support comprise the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194). Each first surface primer is capable of hybridizing to a covalently closed circular library molecule (400) having a universal binding sequence for the first surface primer.

[0285] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support comprise the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197). Each first surface primer is capable of hybridizing to a covalently closed circular library molecule (400) having a universal binding sequence for the first surface primer.

[0286] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199) or a complementary sequence thereof. Each second surface primer can hybridize to a portion of a concatemer molecule that has a universal binding sequence for the second surface primer.

[0287] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203 or a complementary sequence thereof). Each second surface primer can hybridize to a portion of a concatemer molecule that has a universal binding sequence for the second surface primer.

[0288] In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule to the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0289] In some embodiments, the plurality of concatemer molecules of step (c) can be distributed on a support coated with one or more compounds to produce a passivated layer on the support (e.g., Figure 18). In some embodiments, the passivated 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 passivated layer for immobilization to the support. In some embodiments, the support comprises a low-nonspecific binding surface, which allows for improved nucleic acid hybridization and amplification performance on the support. Generally, the support can comprise one or more layers of covalently or non-covalently attached low-binding chemically modified layers, e.g., silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize the plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can comprise, at least in part, a functionalized polymer coating layer covalently bonded via chemical groups on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating, the surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating layer can comprise polyethylene glycol (PEG). The hydrophilic polymer coating layer can comprise a branched PEG having at least four branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, and the water contact angle is 45 degrees or less. In some embodiments, the density of concatemeric molecules immobilized to the support or to the coating on the support is less than 1 mm 2 Approximately 10 per 2 ~106 , or 1 mm 2 Approximately 10 per 6 ~10 9 , or 1 mm 2 Approximately 10 per 9 ~10 12 In some embodiments, the plurality of concatemeric molecules are immobilized to a support, to a coating on a support, at predetermined sites on the support (or coating on the support), or to a coating on a support, at random sites on the support (or coating on the support).

[0290] 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 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0291] In-solution rolling circle amplification using single-stranded splint strands In some embodiments, in a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules hybridized to a single-stranded splint strand (200), each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules (400) 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 strand (200) with a plurality of strand-displacing polymerases and a plurality of nucleotides in solution under conditions suitable for performing a first rolling circle amplification reaction using the single-stranded splint strand (200) as an amplification primer, thereby generating a plurality of concatemeric molecules still hybridized to the covalently closed circular library molecule (400) (see, e.g., Figures 6A-6C).

[0292] In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (b): distributing the plurality of concatemer molecules hybridized to the covalently closed circular library molecules (400) onto a support having a plurality of first surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to the plurality of immobilized first surface primers, thereby immobilizing the plurality of concatemer molecules, the plurality of immobilized concatemer molecules still hybridized to their covalently closed circular library molecules (400).

[0293] 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-displacing polymerases and a plurality of nucleotides under conditions suitable for performing a second rolling circle amplification reaction on the support using the plurality of covalently closed circular library molecules (400) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules.

[0294] In some embodiments, the first and / or second rolling circle amplification reaction can be performed with a plurality of nucleotides containing any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, each immobilized concatemer is hybridized to a respective first surface primer. In some embodiments, each covalently closed circular library molecule (400) in the plurality of covalently closed circular library molecules (400) contains universal binding sequences for the first and second surface primers (e.g., (130) and (120), respectively), such that the in-solution rolling circle amplification reaction generates concatemer molecules having multiple tandem copies of the universal binding sequences for the first and second surface primers. In some embodiments, the support further comprises a plurality of second surface primers. In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule down to the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemers can be subjected to a sequencing reaction.

[0295] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support comprise the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 194). Each first surface primer is capable of hybridizing to a covalently closed circular library molecule (400) having a universal binding sequence for the first surface primer.

[0296] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support comprise the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 197). Each first surface primer is capable of hybridizing to a covalently closed circular library molecule (400) having a universal binding sequence for the first surface primer.

[0297] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 199) or a complementary sequence thereof. Each second surface primer can hybridize to a portion of a concatemer molecule that has a universal binding sequence for the second surface primer.

[0298] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203 or a complementary sequence thereof). Each second surface primer can hybridize to a portion of a concatemer molecule that has a universal binding sequence for the second surface primer.

[0299] In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule to the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0300] In some embodiments, the plurality of concatemer molecules of step (b) can be distributed on a support coated with one or more compounds to produce a passivated layer on the support (e.g., Figure 18). In some embodiments, the passivated layer forms a porous or semi-porous layer. In some embodiments, surface primers, concatemer template molecules, and / or polymerase can be attached to the passivated layer for immobilization to the support. In some embodiments, the support comprises a low-nonspecific binding surface, which allows for improved nucleic acid hybridization and amplification performance on the support. Generally, the support can comprise one or more covalently or non-covalently attached low-binding chemically modified layers, e.g., silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize the plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can comprise, at least in part, a functionalized polymer coating layer covalently bonded via chemical groups on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating, the surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating layer can comprise polyethylene glycol (PEG). The hydrophilic polymer coating layer can comprise a branched PEG having at least four branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, and the water contact angle is 45 degrees or less. In some embodiments, the density of concatemeric molecules immobilized to the support or to the coating on the support is less than 1 mm 2 Approximately 10 per 2 ~10 6, or 1 mm 2 Approximately 10 per 6 ~10 9 , or 1 mm 2 Approximately 10 per 9 ~10 12 In some embodiments, the plurality of concatemeric molecules are immobilized to a support, to a coating on a support, at predetermined sites on the support (or coating on the support), or to a coating on a support, at random sites on the support (or coating on the support).

[0301] 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 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0302] Methods for sequencing The present disclosure provides a method for sequencing any of the immobilized concatemer molecules described herein. Any of the methods for performing a rolling circle amplification reaction described herein can be used to generate a plurality of concatemer molecules immobilized on a support, and the immobilized concatemers can be subjected to a sequencing reaction. In some embodiments, the sequencing reaction uses detectably labeled nucleotide analogs. In some embodiments, the sequencing reaction uses a two-step sequencing reaction, which includes binding to 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.

[0303] In some embodiments, any of the rolling circle amplification reactions described herein (e.g., RCA performed on a support or in solution) can be used to generate immobilized concatemers, each containing tandem repeat units of a sequence of interest and any adapter sequences present in the covalently closed circular library molecule (400). For example, the tandem repeat units include (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) the sequence of interest (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., Figures 3 and 4). In some embodiments, the tandem repeat unit further comprises a first left unique identifier sequence (180) and / or a first right unique identifier sequence (190).

[0304] Immobilized concatemers can self-collapse into compact nucleic acid nanoballs. Including one or more compaction oligonucleotides in an RCA reaction can further compact the size and / or shape of the nanoballs. Increasing the number of tandem repeat units in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers (e.g., sequencing primers with universal sequences) that serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction uses detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., with nucleotide units), signals emitted by nucleotides or nucleotide units participating in parallel sequencing reactions along the concatemer result in increased signal intensity for each concatemer. Multiple portions of a given concatemer can be sequenced simultaneously. Furthermore, multiple binding complexes can be formed along a particular concatemeric molecule, each containing a sequencing polymerase bound to a multivalent molecule, and the multiple binding complexes remain stable without dissociation, resulting in increased duration, which increases signal intensity and reduces imaging time.

[0305] Methods for sequencing using nucleotide analogs The present disclosure provides a method for sequencing any of the immobilized concatemer molecules described herein, comprising: step (a): contacting a sequencing polymerase with (i) the nucleic acid concatemer molecules and (ii) a nucleic acid sequencing primer, wherein the contacting is performed under conditions suitable for the sequencing polymerase to bind to the nucleic acid concatemer molecules hybridized to the nucleic acid primer, such that the nucleic acid concatemer molecules hybridized to the nucleic acid primer form a nucleic acid duplex. In some embodiments, the sequencing polymerase comprises a recombinant mutant sequencing polymerase capable of binding to and incorporating nucleotide analogs. In some embodiments, the sequencing primer comprises a 3' extendable end.

[0306] In some embodiments, in the method for sequencing concatemer molecules, the sequencing primer comprises a 3' extendable end or a 3' non-extendable end. In some embodiments, the plurality of nucleic acid concatemer molecules comprises amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, the plurality of nucleic acid concatemer molecules comprises one copy of a target sequence of interest. In some embodiments, the plurality of nucleic acid molecules comprises two or more tandem copies (e.g., concatemers) of a target sequence of interest. In some embodiments, the nucleic acid concatemer molecules in the plurality of nucleic acid concatemer molecules comprise the same target sequence of interest or different target sequences of interest. In some embodiments, the plurality of nucleic acid concatemer molecules and / or the plurality of nucleic acid primers are in solution or immobilized on a support. In some embodiments, when the plurality of nucleic acid concatemer molecules and / or the plurality of nucleic acid primers are immobilized on a support, binding with a first sequencing polymerase generates a plurality of immobilized first hybrid polymerases. In some embodiments, the plurality of nucleic acid concatemer molecules and / or nucleic acid primers are 10 2 ~10 15In some embodiments, the binding of the plurality of concatemer molecules and nucleic acid primers to the plurality of first sequencing polymerases is performed at 10 different sites on the support. 2 ~10 15 In some embodiments, the plurality of immobilized first multiplexed polymerases on the support are immobilized at predetermined or random sites on the support. In some embodiments, the plurality of immobilized first multiplexed polymerases are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes, including sequencing polymerases, multivalent molecules, nucleotides, and / or divalent cations) to flow over the support, thereby allowing the plurality of immobilized multiplexed polymerases on the support to react with the solution of reagents in a massively parallel manner.

[0307] In some embodiments, the method for sequencing further comprises step (b): contacting a sequencing polymerase with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the sequencing polymerase bound to the nucleic acid duplex and suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase contacts the 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-terminating moiety at the 2'- or 3'-sugar position. In some embodiments, the chain-terminating moiety is removable from the 2'- or 3'-sugar position to convert the chain-terminating moiety to an OH or H group. In some embodiments, the plurality of nucleotides comprises at least one nucleotide lacking a chain-terminating moiety. In some embodiments, at least one nucleotide is labeled with a detectable reporter moiety (e.g., a fluorophore).

[0308] 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 the incorporation of at least one nucleotide. In some embodiments, the conditions suitable for nucleotide binding to the polymerase and nucleotide incorporation may be the same or different. In some embodiments, the conditions suitable for nucleotide incorporation comprise the inclusion of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, at least one nucleotide binds to the sequencing polymerase and is incorporated into the 3' end of the extendable primer. In some embodiments, incorporating a nucleotide into the 3' end of the primer in step (c) comprises a primer extension reaction.

[0309] In some embodiments, the method for sequencing further comprises step (d): repeating steps (b) and (c) of incorporating at least one nucleotide into the 3' end of the extendable primer at least once. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. 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, the linker being cleavable / removable from the base. In some embodiments, at least one of the nucleotides in the plurality of nucleotides is not labeled with a detectable reporter moiety. In some embodiments, the particular detectable reporter moiety (e.g., fluorophore) attached to the nucleotide can correspond to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to enable detection and identification of the nucleotide base. In some embodiments, the method further comprises detecting at least one incorporated nucleotide in steps (c) and / or (d). In some embodiments, the method further comprises identifying at least one incorporated nucleotide in steps (c) and / or (d). In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying the nucleotide bound to the sequencing polymerase, thereby determining the sequence of the concatemer molecule. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying the nucleotide incorporated within the 3' end of the primer, thereby determining the sequence of the concatemer molecule.

[0310] In some embodiments, the method for sequencing includes a method of sequencing a plurality of sequencing polymerases bound to a nucleic acid duplex, the plurality of sequencing polymerases comprising at least a first and a second multiplexed polymerase, wherein (a) the first multiplexed polymerase comprises a first sequencing polymerase bound to a first nucleic acid duplex comprising a first nucleic acid template sequence hybridized to a first nucleic acid primer, (b) the second multiplexed polymerase comprises a second sequencing polymerase bound to a second nucleic acid duplex comprising a second nucleic acid template sequence hybridized to a second nucleic acid primer, (c) the first and second nucleic acid template sequences comprise the same or different sequences, (d) the first and second nucleic acid concatemers are clonally amplified, (e) the first and second primers comprise an extendable 3' end or a non-extendable 3' end, and (f) the plurality of multiplexed polymerases are immobilized on a support. In some embodiments, the density of the plurality of multiplexed polymerases is greater than 1 mm 2 Approximately 10 per 2 ~10 15 The polymerase is a composite polymerase immobilized on a support.

[0311] A two-step method for nucleic acid sequencing The present disclosure provides a two-step method for sequencing any of the immobilized concatemeric molecules described herein. In some embodiments, the first step generally comprises binding a multivalent molecule to a composite polymerase to form a multivalent composite polymerase and detecting the multivalent composite polymerase.

[0312] 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 sequencing primers, wherein the contacting is carried out 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 multiplexed polymerases, each of the plurality of first multiplexed polymerases comprising a first sequencing polymerase bound to a nucleic acid duplex, and the nucleic acid duplex comprising the nucleic acid concatemer molecules hybridized to the nucleic acid primers. In some embodiments, the first polymerase comprises a recombinant mutant sequencing polymerase. In some embodiments, the sequencing primer comprises a 3' extendable end.

[0313] In some embodiments, in the method for sequencing concatemer molecules, the sequencing primer comprises a 3' extendable end or a 3' non-extendable end. In some embodiments, the plurality of nucleic acid concatemer molecules comprises amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, the plurality of nucleic acid concatemer molecules comprises one copy of a target sequence of interest. In some embodiments, the plurality of nucleic acid molecules comprises two or more tandem copies (e.g., concatemers) of a target sequence of interest. In some embodiments, the nucleic acid concatemer molecules in the plurality of nucleic acid concatemer molecules comprise the same target sequence of interest or different target sequences of interest. In some embodiments, the plurality of nucleic acid concatemer molecules and / or the plurality of nucleic acid primers are in solution or immobilized on a support. In some embodiments, when the plurality of nucleic acid concatemer molecules and / or the plurality of nucleic acid primers are immobilized on a support, binding with a first sequencing polymerase generates a plurality of immobilized first hybrid polymerases. In some embodiments, the plurality of nucleic acid concatemer molecules and / or nucleic acid primers are 10 2 ~1015 In some embodiments, the binding of the plurality of concatemer molecules and nucleic acid primers to the plurality of first sequencing polymerases is performed at 10 different sites on the support. 2 ~10 15 In some embodiments, the plurality of immobilized first multiplexed polymerases on the support are immobilized at predetermined or random sites on the support. In some embodiments, the plurality of immobilized first multiplexed polymerases are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes, including sequencing polymerases, multivalent molecules, nucleotides, and / or divalent cations) to flow over the support, thereby allowing the plurality of immobilized multiplexed polymerases on the support to react with the solution of reagents in a massively parallel manner.

[0314] In some embodiments, the method for sequencing further comprises step (b): contacting a plurality of first multiplexed polymerases with a plurality of multivalent molecules to form a plurality of multivalent multiplexed polymerases (e.g., bound complexes). In some embodiments, each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide (e.g., a nucleotide unit) (e.g., Figures 19-23). ​​In some embodiments, the contacting in step (b) is performed under conditions suitable for binding complementary nucleotide units of the multivalent molecule to at least two of the plurality of first multiplexed polymerases, thereby forming a plurality of multivalent multiplexed polymerases. In some embodiments, the conditions are suitable for inhibiting polymerase-catalyzed incorporation of complementary nucleotide units into primers of the plurality of multivalent multiplexed polymerases. In some embodiments, the plurality of multivalent molecules comprises at least one multivalent molecule having multiple nucleotide arms (e.g., Figures 19-23), each of the multiple nucleotide arms being attached to a nucleotide analog (e.g., a nucleotide analog unit) that comprises a chain-terminating moiety at the sugar 2' and / or 3' position. In some embodiments, the plurality of multivalent molecules comprises at least one multivalent molecule comprising multiple nucleotide arms, each of the multiple nucleotide arms being attached to a nucleotide unit lacking a chain-terminating moiety. In some embodiments, at least one of the multivalent molecules in the plurality of multivalent molecules is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the contacting in step (b) is performed in the presence of at least one non-catalytic cation comprising strontium, barium, and / or calcium.

[0315] In some embodiments, the method for sequencing further comprises step (c): detecting the plurality of multivalent hybrid polymerases. In some embodiments, detecting comprises detecting multivalent molecules bound to the hybrid polymerases, wherein complementary nucleotide units of the multivalent molecules are bound to the primers but incorporation of the complementary nucleotide units is inhibited. In some embodiments, the multivalent molecules are labeled with a detectable reporter moiety to enable detection. In some embodiments, the labeled multivalent molecule comprises a fluorophore attached to the core, linker, and / or nucleotide units of the multivalent molecule.

[0316] In some embodiments, the method for sequencing further comprises step (d): identifying the nucleobases of the complementary nucleotide units bound to the plurality of first multiplexed polymerases, thereby determining the sequence of the concatemeric molecule. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety corresponding to the particular nucleotide unit attached to the nucleotide arm, so as to enable identification of the complementary nucleotide units (e.g., the nucleotide bases adenine, guanine, cytosine, thymine, or uracil) bound to the plurality of first multiplexed polymerases.

[0317] In some embodiments, the second step of the two-step sequencing method generally comprises nucleotide incorporation. In some embodiments, the method for sequencing further comprises step (e): dissociating the plurality of multivalent composite polymerases, removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes.

[0318] In some embodiments, the method for sequencing further comprises step (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 multiplexed polymerases, each comprising a second sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the second sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[0319] In some embodiments, the plurality of first sequencing polymerases in step (a) have amino acid sequences that are 100% identical to the amino acid sequence of the plurality of second sequencing polymerases in step (f). In some embodiments, the plurality of first sequencing polymerases in step (a) have amino acid sequences that differ from the amino acid sequence of the plurality of second sequencing polymerases in step (f).

[0320] In some embodiments, the method for sequencing further includes step (g): contacting a plurality of second hybrid polymerases with a plurality of nucleotides, wherein the contacting is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second hybrid polymerases, thereby forming a plurality of nucleotide-hybridized polymerases. In some embodiments, the contacting in step (g) is performed under conditions suitable for promoting polymerase-catalyzed incorporation of the bound complementary nucleotides into the primer by the nucleotide-hybridized polymerase, thereby forming a plurality of nucleotide-hybridized polymerases. In some embodiments, the incorporation of the nucleotide into the 3' end of the primer in step (g) comprises a primer extension reaction. In some embodiments, the contacting in step (g) is performed in the presence of at least one catalytic cation, including 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 terminating moieties. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides that are unlabeled or labeled with a detectable reporter moiety. 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, which may be cleavable / removable from the base or may not be removable from the base. In some embodiments, at least one of the nucleotides in 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 the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for detection and identification of the nucleotide base.

[0321] In some embodiments, the method for sequencing further comprises step (h): detecting complementary nucleotides incorporated into the primer of the nucleotide-complexed polymerase. In some embodiments, the plurality of nucleotides are labeled with a detectable reporter moiety to enable detection. In some embodiments, in the method for sequencing concatemeric molecules, the detecting step (h) is omitted.

[0322] In some embodiments, the method for sequencing further comprises step (i): identifying the base of a complementary nucleotide incorporated into the primer of the nucleotide-complexed polymerase. In some embodiments, the identification of the incorporated complementary nucleotide in step (i) can be used to confirm the identity of the complementary nucleotide of the multivalent molecule bound to the plurality of first composite polymerases in step (d). In some embodiments, the identifying in step (i) can be used to determine the sequence of the nucleic acid concatemer molecule. In some embodiments, the identifying in step (i) is omitted in the method for sequencing the concatemer molecule.

[0323] In some embodiments, the method for sequencing further comprises step (j): removing a chain-terminating moiety from the incorporated nucleotide when step (g) is carried out by contacting a plurality of second hybrid polymerases with a plurality of nucleotides comprising at least one nucleotide having a 2' chain-terminating moiety and / or a 3' chain-terminating moiety.

[0324] In some embodiments, the method for sequencing further comprises step (k): repeating steps (a) through (g) and (j) at least once. In some embodiments, the method for sequencing further comprises repeating step (k): steps (a) through (j) at least once. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined in steps (c) and (d) by detecting and identifying multivalent molecules that bind to the sequencing polymerase but are not incorporated into the 3' end of the primer. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined (or confirmed) in steps (h) and (i) by detecting and identifying nucleotides incorporated into the 3' end of the primer.

[0325] In some embodiments, in any of the methods for sequencing nucleic acid molecules, binding of a plurality of first multiplexed polymerases to a plurality of multivalent molecules forms at least one avidity 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 concatemeric template molecule, thereby forming a first binding complex, wherein a 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 a first multivalent molecule to a second portion of the same concatemeric template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase; and the first and second binding complexes comprising the same multivalent molecule form an avidity 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 concatemeric template molecule comprises a tandem repeat sequence of a sequence of interest and at least one universal sequencing primer binding site. First and second nucleic acid primers can bind to the sequencing primer binding sites along the concatemeric template molecule. Exemplary multivalent molecules are shown in Figures 19-23.

[0326] In some embodiments, in any of the methods for sequencing nucleic acid molecules, the method comprises combining a plurality of first multiplexed polymerases with a plurality of multivalent molecules to form at least one avidity complex, the method comprising the steps of: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers with different portions of concatemeric nucleic acid concatemeric molecules to form at least first and second multiplexed polymerases on the same concatemeric template molecule; and (b) contacting a plurality of detectably labeled multivalent molecules with at least the first and second multiplexed polymerases on the same concatemeric template molecule under conditions suitable for binding of a single multivalent molecule from the plurality of multivalent molecules to the first and second multiplexed polymerases, wherein at least a first nucleotide unit of the single multivalent molecule is bound to the first multiplexed polymerase comprising a first primer hybridized to a first portion of the concatemeric template molecule, thereby forming a first binding complex (e.g., a first ternary complex) in which at least a second nucleotide unit of the single multivalent molecule is bound to a second multiplexed polymerase comprising a second primer hybridized to a second portion of the concatemeric template molecule, thereby forming a second binding complex (e.g., a second ternary complex); contacting under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes, such that the first and second binding complexes bound to the same multivalent molecule form an avidity complex; (c) detecting the first and second binding complexes 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 concatemeric template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemeric 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. Multiple 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.

[0327] Sequencing by ligation The present disclosure provides a method for sequencing any of the immobilized concatemeric molecules described herein, wherein the sequencing method comprises a sequencing-by-binding (SBB) procedure using unlabeled chain-terminating nucleotides. In some embodiments, the SBB method comprises the following steps: (a) sequentially contacting a primed template nucleic acid with at least two separate mixtures under ternary complex-stabilizing conditions, each of the at least two separate mixtures comprising a polymerase and a nucleotide, whereby the sequential contacting results in the primed template nucleic acid contacting nucleotide cognate for a first, second, and third base type in the template under ternary complex-stabilizing conditions; (b) examining the at least two separate mixtures to determine whether a ternary complex has formed; and (c) examining the primed template nucleic acid with at least two separate mixtures to determine whether a ternary complex has formed. (d) adding the next correct nucleotide to the primer of the primed template nucleic acid after step (b), thereby producing an extended primer; and (e) repeating steps (a)-(d) at least once on the primed template nucleic acid containing the extended primer. Exemplary sequencing-by-binding methods are described in U.S. Patent Nos. 10,246,744 and 10,731,141 (the contents of both patents are incorporated herein by reference in their entirety).

[0328] Multiplexed Workflow The present disclosure provides a multiplex workflow, which generally includes preparing separate populations of sample-indexed, covalently closed circular library molecules using a single-stranded splint strand (200) and a sample-indexed nucleic acid library bearing one or both index sequences (e.g., a left sample index sequence (160) and / or a right sample index sequence (170)). The first left index sequence (160) and the first right index sequence (170) are known sequences. The separate sample-indexed libraries can be prepared from input nucleic acids isolated from different sources, and the sample index sequences are used to distinguish between the different sources. A pooling step can be performed either after generating the sample-indexed, covalently closed circular library molecules or before generating the sample-indexed library-splint complexes. The pooled molecules can be subjected to downstream multiplex amplification and / or multiplex sequencing reactions.

[0329] Traditional pooling workflow for multiplexing In some embodiments, a method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources includes (a) providing two or more populations of single-stranded nucleic acid library molecules (100), each population of library molecules (100) contained within a separate compartment, wherein the nucleic acid library molecules in a given population are bound to (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 first left universal adapter sequence (150) having a binding sequence for a first sequencing primer, and (iv) a second left universal adapter sequence (160) having a binding sequence for a second surface primer, each of which is bound to ... (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 sequence (160) and / or the right index sequence (170), separately or in combination, identify the sample source from which the sequence of interest (110) was isolated (see, e.g., Figures 3 and 4). In some embodiments, the single-stranded nucleic acid library molecule (100) further comprises a first left unique identifier sequence (180) and / or a first right unique identifier sequence (190).

[0330] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further includes step (b): providing a plurality of single-stranded splint strands (200), each single-stranded splint strand (200) comprising a first region (210) and a second region (220), wherein the first region (210) is capable of hybridizing to a first left universal adapter sequence (120) of a single-stranded library molecule, and the second region (220) is capable of hybridizing to a first right universal adapter sequence (130) of a single-stranded library molecule.

[0331] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further comprises step (c): contacting, in separate compartments, the population of single-stranded nucleic acid library molecules (100) with a collection of multiple single-stranded splint strands (200), wherein the contacting is performed under conditions suitable for hybridizing a first region (210) of the single-stranded splint strand to a first left universal adaptor sequence (120) of the single-stranded library molecules and conditions suitable for hybridizing a second region (220) of the single-stranded splint strand to a first right universal adaptor sequence (130) of the single-stranded library molecules, thereby circularizing the library molecules and generating a population of library-splint complexes (300) having a nick between the 5' end of the library molecule and the 3' end of the library molecule (see, e.g., Figures 1-5). In some embodiments, the nick is enzymatically ligatable.

[0332] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further comprises step (d): contacting, in separate compartments, the population of library-splint complexes (300) with a ligase under conditions suitable for enzymatic ligation of nicks, thereby generating a population of covalently closed circular library molecules (400), each hybridized to a single-stranded splint strand (200) (see, e.g., Figures 6A-6C).

[0333] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further comprises step (e): pooling together the populations of covalently closed circular library molecules (400) from the separate compartments to produce a multiplex mixture of covalently closed circular library molecules (400), comprising a multiplex mixture of sequences of interest isolated from multiple sample sources.

[0334] In some embodiments, sequences of interest can be isolated from two or more different sample sources (e.g., 2 to 10, or 10 to 50, or 50 to 100, or 100 to 250, or more than 250 different sample sources). Exemplary nucleic acid sources include naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed, paraffin-embedded; FFPE) sources. Those of skill in the art will recognize that nucleic acids can be isolated from many other sources. The sequences of interest in a given population may have the same or different sequences.

[0335] In some embodiments, the number of populations of single-stranded nucleic acid library molecules (100) in step (a) can be 2 to 10, or 10 to 50, or 50 to 100, or 100 to 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 to 10, or 10 to 50, or 50 to 100, or 100 to 200, or more than 200 different populations of covalently closed circular library molecules (400) can be pooled together.

[0336] Those skilled in the art will recognize that any number of distinct compartments (e.g., 2-10, or 10-50, or 50-100, or 100-250, or more distinct compartments) (e.g., a multi-well plate, e.g., a 96-well plate) can be used in step (a).

[0337] In some embodiments, the 3' end of the single-stranded splint strand (200) hybridized to the covalently closed circular library molecule (400) of step (d) or (e) comprises an extendable 3' OH end that can serve as an initiation point for a primer extension reaction (e.g., a rolling circle amplification reaction).

[0338] 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 optionally be reacted with at least one exonuclease enzyme to remove a plurality of the single-stranded splint strands (200) and retain a plurality of covalently closed circular library molecules (400). In some embodiments, the at least one exonuclease enzyme comprises exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease.

[0339] In some embodiments, the single-stranded nucleic acid library molecules (100) of step (a) further comprise any one or any combination of two or more of a universal binding sequence for the forward amplification primer, a universal binding sequence for the reverse amplification primer, and / or a universal binding sequence for the compaction oligonucleotide.

[0340] Non-traditional pooling workflow for multiplexing In some embodiments, a method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources includes: 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 within a separate compartment, wherein the nucleic acid library molecules in a given population are bound to (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 second left universal adapter sequence (150) having a binding sequence for a second surface primer, (iv) a first left universal adapter sequence (160) having a binding sequence for a second sequencing primer, (v) a second left universal adapter sequence (170) having a binding sequence for a second surface primer, (vi) a second left universal adapter sequence (180) having a binding sequence for a second surface primer, (vii) a second left universal adapter sequence (180) having a binding sequence for a second surface primer, (viii) a second left universal adapter sequence (19 ... (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 sequence (160) and / or the right index sequence (170), separately or in combination, identify the sample source from which the sequence of interest (110) was isolated (see, e.g., Figures 3 and 4). In some embodiments, the single-stranded nucleic acid library molecule (100) further comprises a first left unique identifier sequence (180) and / or a first right unique identifier sequence (190).

[0341] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further comprises step (b): pooling two or more populations of single-stranded nucleic acid library molecules (100) together in the same compartment.

[0342] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further comprises step (c): adding to the compartment of step (b) a plurality of single-stranded splint strands (200), each single-stranded splint strand (200) comprising a first region (210) and a second region (220), wherein the first region (210) is capable of hybridizing to a first left universal adapter sequence (120) of a single-stranded library molecule and the second region (220) is capable of hybridizing to a first right universal adapter sequence (130) of a single-stranded library molecule.

[0343] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further comprises step (d): incubating the population of single-stranded nucleic acid library molecules (100) and the plurality of single-stranded splint strands (200) under conditions suitable for hybridizing a first region (210) of the single-stranded splint strand with a first left universal adaptor sequence (120) of the single-stranded library molecules, and conditions suitable for hybridizing a second region (220) of the single-stranded splint strand with a first right universal adaptor sequence (130) of the single-stranded library molecules, thereby circularizing the library molecules and generating a population of library-splint complexes (300) having a nick between the 5' end of the library molecule and the 3' end of the library molecule (see, e.g., Figures 1-5). In some embodiments, the nick is enzymatically ligatable.

[0344] In some embodiments, the method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources further comprises step (e): contacting the population of library-splint complexes (300) in the compartment of step (d) with a ligase under conditions suitable for enzymatic ligation of nicks, thereby producing a population of covalently closed circular library molecules (400) each hybridized to a single-stranded splint strand (200), thereby producing a multiplex mixture of covalently closed circular library molecules (400) comprising a multiplex mixture of sequences of interest isolated from multiple sample sources (see, e.g., Figures 6A-6C).

[0345] In some embodiments, sequences of interest can be isolated from two or more different sample sources (e.g., 2 to 10, or 10 to 50, or 50 to 100, or 100 to 250, or more than 250 different sample sources). Exemplary nucleic acid sources include naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed, paraffin-embedded; FFPE) sources. Those of skill in the art will recognize that nucleic acids can be isolated from many other sources. The sequences of interest in a given population may have the same or different sequences.

[0346] In some embodiments, the number of populations of single-stranded nucleic acid library molecules (100) in step (a) can be 2 to 10, or 10 to 50, or 50 to 100, or 100 to 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 to 10, or 10 to 50, or 50 to 100, or 100 to 200, or more than 200 different populations of covalently closed circular library molecules (400) can be pooled together.

[0347] Those skilled in the art will recognize that any number of distinct compartments (e.g., 2-10, or 10-50, or 50-100, or 100-250, or more distinct compartments) (e.g., a multi-well plate, e.g., a 96-well plate) can be used in step (a).

[0348] In some embodiments, the 3' end of the single-stranded splint strand (200) hybridized to the covalently closed circular library molecule (400) in step (e) comprises an extendable 3' OH end that can serve as an initiation point for a primer extension reaction (e.g., a rolling circle amplification reaction).

[0349] 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 enzyme to remove a plurality of the single-stranded splint strands (200) and retain a plurality of covalently closed circular library molecules (400). In some embodiments, the at least one exonuclease enzyme comprises exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease.

[0350] In some embodiments, the single-stranded nucleic acid library molecules (100) of step (a) further comprise any one or any combination of two or more of a universal binding sequence for the forward amplification primer, a universal binding sequence for the reverse amplification primer, and / or a universal binding sequence for the compaction oligonucleotide.

[0351] On-support multiplex rolling circle amplification reaction In some embodiments, any of the polyvalent mixtures of covalently closed circular library molecules (400) described herein can be distributed on a support, and the method includes distributing 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 of the covalently closed circular library molecules (400) to each of the immobilized first surface primers, 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-displacing 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 generating a plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides includes dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, the population of covalently closed circular library molecules is still hybridized to the single-stranded splint strand (200) and distributed on the support. In some embodiments, the single-stranded splint strand (200) is removed from the population of covalently closed circular library molecules (400) using at least one exonuclease enzyme under conditions suitable for removing a plurality of single-stranded splint strands (200...

Claims

1. (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); (ii) a single-stranded splint strand (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 circularizing the library molecule to generate a library-sprint complex (300) having a nick between the 5' end of the library molecule and the 3' end of the library molecule.

2. The library-sprint complex (300) of claim 1, wherein the nucleic acid library molecule (100) further comprises a second left universal adaptor sequence (140).

3. The library-sprint complex (300) of claim 1, wherein the nucleic acid library molecule (100) further comprises a second right universal adaptor sequence (150).

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

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

6. The library-sprint complex (300) of claim 1, wherein the nucleic acid library molecule (100) further comprises a first left unique identifier sequence (180).

7. The library-sprint complex (300) of claim 1, wherein the nucleic acid library molecule (100) further comprises a first right unique identifier sequence (190).

8. The nucleic acid library molecule (100) (i) a second left universal adaptor sequence (140); (ii) a second right universal adaptor sequence (150); (iii) a first left index array (160); (iv) a first right index array (170); (v) a first left unique identification sequence (180); and / or (vi) the library-sprint complex (300) of claim 1 further comprising any one or any combination of two or more of the first right unique identifier sequences (190).

9. The first left universal adaptor array (120) and / or the second left universal adaptor array (140) (i) a universal binding sequence for the forward sequencing primer; (ii) a universal binding sequence for the reverse sequencing primer; (iii) a universal binding sequence for the first surface primer; (iv) a universal binding sequence for the second surface primer; (v) a universal binding sequence for the forward amplification primer; (vi) a universal binding sequence for the reverse amplification primer, and / or (vii) The library-sprint complex (300) of claim 8, comprising a universal binding sequence for the compaction oligonucleotide.

10. The first right universal adaptor array (130) and / or the second right universal adaptor array (150) (i) a universal binding sequence for the forward sequencing primer; (ii) a universal binding sequence for the reverse sequencing primer; (iii) a universal binding sequence for the first surface primer; (iv) a universal binding sequence for the second surface primer; (v) a universal binding sequence for the forward amplification primer; (vi) a universal binding sequence for the reverse amplification primer, and / or (vii) The library-sprint complex (300) of claim 8, comprising a universal binding sequence for the compaction oligonucleotide.

11. 1. 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), each library molecule in the plurality of library molecules 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); b) providing a plurality of single-stranded splint strands (200), each of the single-stranded splint strands (200) comprising a first region (210) capable of hybridizing with the at least a first left universal adaptor sequence (120) of an individual library molecule, and a second region (220) capable of hybridizing with the at least a first right universal adaptor sequence (130) of an individual library molecule; c) hybridizing the plurality of single-stranded splint strands (200) with a plurality of single-stranded nucleic acid library molecules (100) such that the first region of one of the single-stranded splint strands (210) anneals to the at least a first left universal adaptor sequence (120) of the library molecule and the second region of the single-stranded splint strand (220) anneals to the at least a first right universal sequence (130) of the library molecule, thus circularizing individual library molecules to form a plurality of library-sprint complexes (300) having a nick between the 5' and 3' ends of the library molecule, wherein the nick is enzymatically ligatable; d) ligating said nicks in said plurality of library-sprint complexes (300), thereby generating a plurality of covalently closed circular library molecules (400).

12. 12. The method of 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 thereon under conditions suitable for hybridizing each of the covalently closed circular library molecules (400) to each of the immobilized surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (400) to the support.

13. 13. The method of claim 12, further comprising: (f) contacting a plurality of the 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 the plurality of covalently closed circular library molecules (400) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules.

14. 14. The method of claim 13, further comprising sequencing the plurality of immobilized nucleic acid concatemer molecules.

15. 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, said contacting being performed under conditions suitable for forming a plurality of multiplexed polymerases, each of said multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, said nucleic acid duplexes comprising concatemer molecules hybridized to soluble sequencing primers; b) contacting the plurality of multiplexed sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the multiplexed sequencing polymerase, the plurality of nucleotides comprising at least one nucleotide analogue, the at least one nucleotide analogue being labeled with a fluorophore and having a removable chain-terminating moiety at the sugar 3′ position; c) incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

16. 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, said contacting being performed under conditions suitable for forming a plurality of first multiplexed polymerases, each of said plurality of first multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, said nucleic acid duplexes comprising concatemer molecules hybridized to a soluble sequencing primer; b) contacting the plurality of multiplexed 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 multiplexed polymerases, thereby forming a plurality of multivalent multiplexed polymerases, wherein the conditions inhibit incorporation of the complementary nucleotide units into sequencing primers of the plurality of multivalent multiplexed polymerases, and each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being 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 first multiplexed polymerases in the plurality of multivalent multiplexed polymerases, thereby determining the sequence of the nucleic acid template.

17. e) dissociating the plurality of multivalent composite polymerases and removing the plurality of first sequencing polymerases and their associated multivalent molecules, retaining a plurality of the nucleic acid duplexes; f) contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, said contacting being performed under conditions suitable for binding of said plurality of second sequencing polymerases to said plurality of retained nucleic acid duplexes, thereby forming a plurality of second multiplexed polymerases, each of said plurality of second multiplexed polymerases comprising a second sequencing polymerase bound to a retained nucleic acid duplex; 17. The method of claim 16, further comprising: g) contacting the plurality of second multiplexed polymerases with a plurality of nucleotides, said contacting being performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second multiplexed polymerases of step (f), thereby forming a plurality of nucleotide multiplexed polymerases, said conditions suitable for promoting incorporation of the bound complementary nucleotides into a sequencing primer by the nucleotide multiplexed polymerases.

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

19. h) detecting the complementary nucleotide incorporated into the sequencing primer by the nucleotide-complexed polymerase; 20. The method of claim 17, further comprising: i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-conjugated polymerase.

20. 1. A method for forming at least one avidity complex, comprising: a) binding a first universal nucleic acid primer, a first DNA polymerase, and a first multivalent molecule to a first portion of the concatemeric molecule of claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds 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 concatemeric template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second DNA polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex, wherein the first multivalent molecule comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit, and the concatemeric molecule comprises a sequence of interest (110), and two or more tandem repeat sequences of universal primer binding sites that bind to the first and second universal nucleic acid primers.

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

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

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

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

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

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

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

28. The nucleic acid library molecule (100) (vii) a second left universal adaptor sequence (140); (viii) a second right universal adaptor sequence (150); (ix) a first left index array (160); (x) a first right index array (170); (xi) a first left unique identifier sequence (180); and / or 12. The method of claim 11, further comprising: (xii) any one or any combination of two or more of the first right unique identification sequence (190).

29. The first left universal adaptor array (120) and / or the second left universal adaptor array (140) (viii) a universal binding sequence for the forward sequencing primer; (ix) a universal binding sequence for the reverse sequencing primer; (x) a universal binding sequence for the first surface primer; (xi) a universal binding sequence for the second surface primer; (xii) a universal binding sequence for the forward amplification primer; (xiii) a universal binding sequence for the reverse amplification primer, and / or (xiv) a universal binding sequence for the compaction oligonucleotide.

30. The first right universal adaptor array (130) and / or the second right universal adaptor array (150) (viii) a universal binding sequence for the forward sequencing primer; (ix) a universal binding sequence for the reverse sequencing primer; (x) a universal binding sequence for the first surface primer; (xi) a universal binding sequence for the second surface primer; (xii) a universal binding sequence for the forward amplification primer; (xiii) a universal binding sequence for the reverse amplification primer, and / or (xiv) a universal binding sequence for the compaction oligonucleotide.

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

32. 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, second, and third sequencing cycles.

33. d) sequencing the universal sample index sequences of the plurality of immobilized template molecules; and e) sequencing the insert sequence region of the plurality of immobilized template molecules; and 32. The method of claim 31, further comprising: f) assigning an insert sequence of a given template molecule obtained in step (b) to a universal sample index sequence from the same given template molecule obtained in step (a), thereby identifying a sample source of the given insert sequence.

34. 32. 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 that identifies the sample source of the insert sequence, and the second sample index lacks random sequence.

35. 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, second, and third sequencing cycles, and 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 immobilized template molecules; and i) sequencing the second universal sample index sequences of the plurality of immobilized template molecules; j) sequencing the insert sequence regions of the plurality of immobilized template molecules; and 35. The method of claim 34, further comprising: k) assigning an insert sequence of a given template molecule obtained in step (c) to the first and second universal sample index sequences from the same given template molecule obtained in steps (a) and (b), thereby identifying a sample source of the given insert sequence.

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

37. 37. The method of 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, second, third, and fourth sequencing cycles.

38. a) sequencing the remaining base positions of the universal sample index sequence of the plurality of immobilized template molecules; b) sequencing the insert sequence region of the plurality of immobilized template molecules; and 37. The method of claim 36, further comprising: c) assigning an insert sequence of a given template molecule obtained in step (b) to a universal sample index sequence from the same given template molecule obtained in step (a), thereby identifying a sample source of the given insert sequence.

39. 37. The method of claim 36, 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 that identifies the sample source of the insert sequence, and the second sample index lacks random sequence.

40. a) sequencing the 3-mer random sequence of the plurality of immobilized template molecules and the first base position 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, second, third, and fourth sequencing cycles to generate a map of the positions of the plurality of immobilized template molecules; b) sequencing the remaining base positions of the first universal sample index sequence of the plurality of immobilized template molecules; and c) sequencing the second universal sample index sequences of the plurality of immobilized template molecules; and d) sequencing the insert sequence region of the plurality of immobilized template molecules; and 40. The method of claim 39, further comprising: e) assigning an insert sequence of a given template molecule obtained in step (c) to first and second universal sample index sequences from the same given template molecule obtained in steps (a) and (b), thereby identifying a sample source of the given insert 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 configured on a flow cell channel, a flow cell, or a capillary lumen.

43. The method according to 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 45 degrees or less.

44. 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(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucosides, streptavidin, and dextran.

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

46. 44. 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. 41. The method of any one of claims 30 to 40, wherein the immobilized template molecule comprises a plurality of immobilized concatemeric molecules having the insert sequence and a tandem repeat sequence of one of the sample indexes.

48. 41. The method of any one of claims 30 to 40, wherein the immobilized 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, and the clustered template molecules are generated via bridge amplification.

49. The density of the immobilized nucleic acid template molecules located at random positions on the support is less than 1 mm ２ 10 per ４ ~10 ８ The method according to any one of claims 30 to 40, wherein

50. 41. 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. 41. The method of any one of claims 30-40, wherein the detectably labeled nucleotide reagent comprises nucleotides, each of the nucleotides comprising an aromatic nucleobase, a five-carbon sugar moiety, from one to ten phosphate groups, and a fluorophore.

52. 41. The method of any one of claims 30-40, wherein the detectably labeled nucleotide reagent comprises nucleotides, each of the nucleotides comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain terminating group at the 3' carbon sugar position, from one to ten phosphate groups, and a fluorophore.

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

54. 41. The method of any one of claims 30-40, wherein the detectably labeled nucleotide reagent bound to the immobilized template molecule comprises individual immobilized template molecules, the individual immobilized template molecules hybridized to a sequencing primer forming a duplex, the duplex bound to a polymerase forming a multiplexed polymerase, and the multiplexed polymerase bound to a detectably labeled nucleotide reagent.

55. 55. The method of claim 54, wherein the multiplexed polymerase binds a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the multiplexed polymerase and for incorporation of the detectably labeled nucleotide into the hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety, one to ten phosphate groups, and a fluorophore.

56. 55. The method of claim 54, wherein the multiplexed polymerase binds to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the multiplexed polymerase and for incorporation of the detectably labeled nucleotide into the hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain terminating group at the 3' carbon sugar position, from one to ten phosphate groups, and a fluorophore.

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

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

59. 1. A method for sequencing a nucleic acid molecule, comprising: a) providing a first plurality of library molecules, each molecule in the first plurality of library molecules comprising (i) an insert sequence region derived from a first sample source, (ii) a first sample index having a three base long random sequence linked to a first universal sample index sequence, and (iii) a second sample index having a second universal sample index sequence lacking the random sequence, wherein a combination of the first and second universal sample index sequences uniquely identifies the first sample source of the insert sequence, and different first library molecules have different three base long random sequences and have different insert sequences; b) providing a second plurality of library molecules, each molecule in the second plurality of library molecules comprising (i) an insert sequence region derived from a second sample source, (ii) a third sample index having a three base long random sequence linked to a third universal sample index sequence, and (iii) a fourth sample index having a fourth universal sample index sequence lacking random sequence, wherein the combination of the third and fourth universal sample index sequences uniquely identifies the second sample source of the insert sequence, and different second library molecules have different three base long random sequences and have different insert sequences; c) pooling the first and 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 reactions of the 3-base long random sequences of the first and third sample indexes using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C, and T / U, the nucleotide reagents comprising a different detectable color label corresponding to each different type of nucleobase, the three cycles of sequencing comprising detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to the immobilized amplified template molecules, thereby determining the sequence of the 3-base long random sequence in each template molecule of the plurality of immobilized template molecules, a balanced diversity of A, G, C, and T / U nucleobases being detected and imaged in each of the first, second, and third sequencing cycles among the plurality of immobilized amplified template molecules; f) generating a map of the locations of the plurality of immobilized template molecules using the image obtained in step (e), wherein the sequence of the insert region is not used to generate the map.

60. 2. 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, second, and third sequencing cycles.

61. g) sequencing a first universal sample index sequence of the plurality of immobilized template molecules; h) sequencing a second universal sample index sequence of the plurality of immobilized template molecules; and i) sequencing insert sequence regions of the plurality of immobilized template molecules derived from the first library molecule; 60. The method of claim 59, further comprising: j) assigning the insert sequence of the given template molecule obtained in step (c) to first and second universal sample index sequences from the same given template molecule, thereby identifying a first sample source of the given insert sequence.

62. g) sequencing a third universal sample index sequence of the plurality of immobilized template molecules; and h) sequencing a fourth universal sample index sequence of the plurality of immobilized template molecules; and i) sequencing insert sequence regions of the plurality of immobilized template molecules derived from the second library molecules; 60. The method of claim 59, further comprising: j) assigning the insert sequence of the given template molecule obtained in step (c) to a third and a fourth universal sample index sequence from the same given template molecule, thereby identifying a second sample source of the given insert sequence.

63. 1. A method for sequencing a nucleic acid molecule, comprising: a) providing a first plurality of library molecules, each molecule in the first plurality of library molecules comprising (i) an insert sequence region derived from a first sample source, (ii) a first sample index having a three base long random sequence linked to a first universal sample index sequence, and (iii) a second sample index having a second universal sample index sequence lacking the random sequence, wherein a combination of the first and second universal sample index sequences uniquely identifies the first sample source of the insert sequence, the first universal sample index sequence comprising between 3 and 20 nucleotides, and different first library molecules having different three base long random sequences and having different insert sequences; b) providing a second plurality of library molecules, each molecule in the second plurality of library molecules comprising (i) an insert sequence region derived from a second sample source, (ii) a third sample index having a three base long random sequence linked to a third universal sample index sequence, and (iii) a fourth sample index having a fourth universal sample index sequence lacking random sequence, wherein a combination of the third and fourth universal sample index sequences uniquely identifies the second sample source of insert sequence, the third universal sample index sequence comprising between 3 and 20 nucleotides, and wherein different second library molecules have different three base long random sequences and have different insert sequences; c) pooling the first and 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 of the 3-base long random sequences of the first and third sample indexes and sequencing of the first base positions of the first and third universal sample index sequences using a plurality of detectably labeled nucleotide reagents comprising a mixture of different types of nucleobases A, G, C, and T / U, the nucleotide reagents comprising a different detectable color label corresponding to each different type of nucleobase, the three cycles of sequencing comprising detecting and imaging optical color signals emitted from the detectably labeled nucleotide reagents bound to the immobilized amplified template molecules, thereby determining the sequence of the 3-base long random sequence in each template molecule of the plurality of immobilized template molecules, a balanced diversity of A, G, C, and T / U nucleobases being detected and imaged in each of the first, second, third, and fourth sequencing cycles among the plurality of immobilized amplified template molecules; f) generating a map of the locations of the plurality of immobilized template molecules using the image obtained in step (e), wherein the sequence of the insert region is not used to generate the map.

64. 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, second, third, and fourth sequencing cycles.

65. g) sequencing the remaining base positions of the first universal sample index sequence of the plurality of immobilized template molecules; and h) sequencing a second universal sample index sequence of the plurality of immobilized template molecules; and i) sequencing insert sequence regions of the plurality of immobilized template molecules derived from the first library molecule; 64. The method of claim 63, further comprising: j) assigning the insert sequence of the given template molecule obtained in step (c) to first and second universal sample index sequences from the same given template molecule, thereby identifying a first sample source of the given insert sequence.

66. g) sequencing the remaining base positions of a third universal sample index sequence of the plurality of immobilized template molecules; and h) sequencing a fourth universal sample index sequence of the plurality of immobilized template molecules; and i) sequencing insert sequence regions of the plurality of immobilized template molecules derived from the second library molecules; 60. The method of claim 59, further comprising: j) assigning the insert sequence of the given template molecule obtained in step (c) to a third and a fourth universal sample index sequence from the same given template molecule, thereby identifying a 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. 67. The method of any one of claims 59 to 66, wherein the support is configured on a flow cell channel, a flow cell, or a capillary lumen.

69. A method according to 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 45 degrees or less.

70. 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(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucosides, streptavidin, and dextran.

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

72. 70. 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. 67. The method of any one of claims 59 to 66, wherein the immobilized template molecule comprises a plurality of immobilized concatemeric molecules having tandem repeat sequences of the insert sequence and one of the sample indexes.

74. 67. The method of any one of claims 59 to 66, wherein the immobilized 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, and the clustered template molecules are generated via bridge amplification.

75. The density of the immobilized nucleic acid template molecules located at random positions on the support is less than 1 mm ２ 10 per ４ ~10 ８ The method according to any one of claims 59 to 66, wherein

76. 67. 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. 67. The method of any one of claims 59-66, wherein the detectably labeled nucleotide reagent comprises nucleotides, each of the nucleotides comprising an aromatic nucleobase, a five-carbon sugar moiety, from one to ten phosphate groups, and a fluorophore.

78. 67. The method of any one of claims 59-66, wherein the detectably labeled nucleotide reagent comprises nucleotides, each of said nucleotides comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain terminating group at the 3' carbon sugar position, from one to ten phosphate groups, and a fluorophore.

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

80. 67. The method of any one of claims 59-66, wherein the detectably labeled nucleotide reagent bound to the immobilized template molecule comprises individual immobilized template molecules, the individual immobilized template molecules hybridized to a sequencing primer forming a duplex, the duplex bound to a polymerase forming a multiplexed polymerase, and the multiplexed polymerase bound to a detectably labeled nucleotide reagent.

81. 81. The method of claim 80, wherein the multiplexed polymerase binds a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the multiplexed polymerase and for incorporation of the detectably labeled nucleotide into the hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety, one to ten phosphate groups, and a fluorophore.

82. 81. The method of claim 80, wherein the multiplexed polymerase binds to a detectably labeled nucleotide reagent under conditions suitable for binding the detectably labeled nucleotide reagent to the multiplexed polymerase and for incorporation of the detectably labeled nucleotide into the hybridized sequencing primer, the detectably labeled nucleotide reagent comprising an aromatic nucleobase, a five-carbon sugar moiety having a chain terminating group at the 3' carbon sugar position, from one to ten phosphate groups, and a fluorophore.

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

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

85. A method for generating a plurality of library-sprint complexes (300), comprising: a) providing a plurality of single-stranded nucleic acid library molecules (100), each library molecule in the plurality of library molecules comprising the following 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 sequence of interest (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 strands (200), each single-stranded splint strand (200) in the plurality of single-stranded splint strands (200) comprising regions arranged in 5' to 3' order: (i) a first region (210) having a universal binding sequence that hybridizes to the first left universal adaptor sequence (120) of the linear single-stranded library molecule (100); and (ii) a second region (220) having a universal binding sequence that hybridizes to the first right universal adaptor sequence (130) of the linear single-stranded library molecule (100); c) hybridizing the plurality of single-stranded splint strands (200) to the plurality of single-stranded nucleic acid library molecules (100) under conditions suitable for hybridizing the first region (210) of the single-stranded splint strands (200) to the first left universal adaptor sequence (120) of the linear single-stranded library molecule (100) and suitable for hybridizing the second region (220) of the single-stranded splint strands (200) to the first right universal adaptor sequence (130) of the linear single-stranded library molecule (100), thereby circularizing individual library molecules to form a plurality of library-sprint complexes (300) having a nick between the 5' and 3' ends of the library molecule, wherein the nick is enzymatically ligatable.

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

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

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

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

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

91. 91. The method of claim 90, further comprising: g) contacting a plurality of the 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 the plurality of covalently closed circular library molecules (400) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules.

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

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

94. 91. The method of claim 90, wherein the plurality of immobilized second surface capture primers on the support are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes, nucleotides, divalent cations, etc.) to flow over the support, thereby allowing the plurality of immobilized second surface primers to react with the reagents essentially simultaneously in a massively parallel manner.

95. The density of the plurality of immobilized nucleic acid concatemer molecules on the support is 1 mm ２ 10 per ４ ~10 ８ 92. The method of claim 91 , wherein:

96. The method further comprises sequencing the plurality of immobilized concatemers, wherein said 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, said contacting being performed under conditions suitable for forming a plurality of multiplexed polymerases, each of said multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, said nucleic acid duplexes comprising concatemer molecules hybridized to soluble sequencing primers; b) contacting the plurality of multiplexed sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the multiplexed sequencing polymerase, the plurality of nucleotides comprising at least one nucleotide analogue, the at least one nucleotide analogue being labeled with a fluorophore and having a removable chain-terminating moiety at the sugar 3′ position; c) incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

97. 97. The method of claim 96, wherein the plurality of nucleotides comprises a removable chain terminating moiety at a 3' sugar group, the removable chain terminating moiety comprising an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, the removable chain terminating moiety being cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group.

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

99. 97. 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 further comprises sequencing the plurality of immobilized concatemers, wherein said 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, said contacting being performed under conditions suitable for forming a plurality of first multiplexed polymerases, each of said plurality of first multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, said nucleic acid duplexes comprising concatemer molecules hybridized to a soluble sequencing primer; b) contacting the plurality of multiplexed 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 multiplexed polymerases, thereby forming a plurality of multivalent multiplexed polymerases, wherein the conditions inhibit incorporation of the complementary nucleotide units into sequencing primers of the plurality of multivalent multiplexed polymerases, and each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being 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 first multiplexed polymerases in the plurality of multivalent multiplexed polymerases, thereby determining the sequence of the nucleic acid template.

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

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

103. h) detecting the complementary nucleotide incorporated into the sequencing primer by the nucleotide-complexed polymerase; 102. The method of claim 101, further comprising: i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.

104. 1. A method for sequencing by forming at least one avidity 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 the concatemer molecule of claim 16, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; b) binding a second universal sequencing primer, a second sequencing polymerase, and the first detectably labeled multivalent molecule to a second portion of the same concatemeric molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex, the first detectably labeled multivalent molecule comprising a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit, the concatemeric molecule comprising a sequence of interest (110), and two or more tandem repeat sequences of universal primer binding sites that bind to the first and second universal sequencing primers, and the contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes; c) detecting said first and second binding complexes on the same concatemeric molecule.

105. 101. The method of claim 100, wherein the nucleotide arms attached to the core of each multivalent molecule have the same type of nucleotide unit, the type of nucleotide unit being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

106. 101. The method of claim 100, wherein said 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. 102. The method of claim 101, wherein the plurality of nucleotides comprises a removable chain terminating moiety at a 3' sugar group, the removable chain terminating moiety comprising an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, the removable chain terminating moiety being cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group.

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

109. 102. 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. 91. The method of claim 90, wherein the support comprises a glass or plastic substrate.

111. 91. The method of claim 90, wherein the substrate is passivated with at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less.

112. 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(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucosides, streptavidin, and dextran.

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