Double stranded splint adapter and method of use

Abstract

The present disclosure provides compositions, including kits, that include nucleic acid double-stranded splint adapters, and methods of using double-stranded splint adapters. The double-stranded splint adapters (200) can be used in a one-pot multi-enzyme reaction to introduce one or more new adapter sequences into library molecules. The double-stranded splint adapters (200) include a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)), where the first and second splint strands hybridize together to form a double-stranded splint adapter (200) having a double-stranded region and two adjacent single-stranded regions. The second splint strand (400) carries the new adapter sequence(s) to be introduced, e.g., a universal binding sequence and / or an index sequence.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 316,784, filed March 4, 2022, and U.S. Patent Application No. 17 / 725,065, 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-003_001WO_SequenceListing_ST26.xml, size: 65,027 bytes, and creation date: March 3, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors and methods for using the double-stranded splint adaptors, which can hybridize to a portion of a library molecule to form a library-splint complex having a nick, which can be ligated to form a covalently closed circular molecule 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 nucleic acid library molecule (100) 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 double-stranded splint adapter (200) comprising a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)), wherein the double-stranded splint adapter (200) comprises a double-stranded region and two adjacent single-stranded regions, and the first splint strand comprises a first region (320), an internal region (310), and a second region (330). and a left adaptor (200), wherein an internal region of the first splint strand (310) hybridizes to the second splint strand (400), a first region of the first splint strand (320) hybridizes to at least a first left universal adaptor sequence (120) of the library molecule, and a second region of the first splint strand (330) 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 (500).

[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 index sequence (160). In some embodiments, the nucleic acid library molecule (100) further comprises a first right 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] In some embodiments, the second splint strand (400) comprises at least two subregions, the first subregion comprising a universal binding sequence for the third surface primer, and the second subregion comprising a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to (or at least exhibit very little hybridization to) the first and second surface primers.

[0011] In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identifier sequence (e.g., NN) having 2-10 or more bases.

[0012] In some embodiments, the first splint strand (300) comprises an internal region (310) comprising at least two subregions, the fourth subregion comprising a universal binding sequence for a third surface primer, the fourth subregion hybridizing to a first subregion of the second splint strand (400), the fifth subregion comprising a universal binding sequence for a fourth surface primer, the fifth subregion hybridizing to a second subregion of the second splint strand (400), and the fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the first splint strand (300) comprises an internal region (310) further comprising a sixth subregion, the sixth subregion comprising a sample index sequence having 5 to 20 bases and / or a unique identification sequence (e.g., NN) having 2 to 10 or more bases, and the sixth subregion hybridizes to a third subregion of the second splint strand (400).

[0013] The present disclosure provides a method for chromatin ligation comprising: a) a sequence of interest (110), the sequence of interest (110) being a first left universal adapter sequence (120) having a binding sequence for a first surface primer (120); (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 binding sequence for a second surface primer (130). a) a single-stranded nucleic acid library molecule (100) comprising a first right universal adaptor sequence (130) having a first region (320), an internal region (310), and a second region (330) arranged in 5' to 3' order; and c) a second splint strand (400) comprising subregions arranged in 3' to 5' order, a first subregion having a universal binding sequence for a third surface primer, and a second subregion having a universal binding sequence for a fourth surface primer. and a library-sprint complex (500) comprising a first splint strand (300) hybridized to a portion of the library molecule (100), thereby circularizing the library molecule to produce a library-sprint complex (500), whereby a first region (320) of the first splint strand hybridizes to a binding sequence for the first surface primer (120) and a third region (330) of the first splint strand hybridizes to a binding sequence for the second surface primer (130). The method provides a library-sprint complex (500), wherein the first splint strand (300) is hybridized to an internal region (310) of the second splint strand (400), the second splint strand (400) hybridizes to an internal region (310) of the first splint strand (300), the library-sprint complex (500) comprises a first nick between the 5' end of the library molecule and the 3' end of the second splint strand, and the library-sprint complex (500) comprises a second nick between the 5' end of the second splint strand and the 3' end of the library molecule, and the first and second nicks are enzymatically ligatable.

[0014] In some embodiments, in a library-sprint complex (500) further comprising a plurality of library-sprint complexes (500), 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.

[0015] The present disclosure provides a reaction mixture comprising a plurality of library-splint complexes (500) of the present disclosure and T4 polynucleotide kinase. The present disclosure provides a reaction mixture comprising a plurality of library-splint complexes (500) of the present disclosure and a ligase enzyme. The present disclosure provides a reaction mixture comprising a plurality of library-splint complexes (500) of the present disclosure, T4 polynucleotide kinase, and a ligase enzyme. In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0016] The present disclosure provides a covalently closed circular library molecule (600) comprising a sequence of interest (110), at least a first left universal adaptor sequence (120), at least a first right universal adaptor sequence (130), and a second splint strand sequence (400).

[0017] In some embodiments, the covalently closed circular library molecule (600) of the present disclosure further comprises a second left universal adaptor sequence (140). In some embodiments, the covalently closed circular library molecule (600) of the present disclosure further comprises a second right universal adaptor sequence (150). In some embodiments, the covalently closed circular library molecule (600) of the present disclosure further comprises a first left indexing sequence (160). In some embodiments, the covalently closed circular library molecule (600) of the present disclosure further comprises a first right indexing sequence (170). In some embodiments, the covalently closed circular library molecule (600) of the present disclosure further comprises a first left unique identifier sequence (180). In some embodiments, the covalently closed circular library molecule (600) of the present disclosure further comprises a first right unique identifier sequence (190).

[0018] In some embodiments, the covalently closed circular library molecule (600) of the present disclosure 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 indexing sequence (160), (iv) a first right indexing sequence (170), (v) a first left unique identifier sequence (180), and / or (vi) a first right unique identifier sequence (190).

[0019] 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.

[0020] 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.

[0021] In some embodiments, a covalently closed circular library molecule (600) of the present disclosure hybridizes to a first splint strand (long splint strand (300)) comprising a first region (320), an internal region (310), and a second region (330), wherein the second splint strand sequence (400) of the covalently closed circular library molecule hybridizes to the internal region (310) of the first splint strand, the first region of the first splint strand (320) hybridizes to at least a first left universal adapter sequence (120) of the library molecule, and the second region of the first splint strand (330) hybridizes to at least a first right universal sequence (130) of the library molecule.

[0022] In some embodiments, the second splint strand (400) comprises at least two subregions, the first subregion comprising a universal binding sequence for the third surface primer, and the second subregion comprising a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to (or at least exhibit very little hybridization to) the first and second surface primers.

[0023] In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identifier sequence (e.g., NN) having 2-10 or more bases.

[0024] In some embodiments, the first splint strand (300) comprises an internal region (310) and comprises at least two subregions, the fourth subregion comprising a universal binding sequence for the third surface primer, the fourth subregion hybridizing to the first subregion of the second splint strand (400), the fifth subregion comprising a universal binding sequence for the fourth surface primer, the fifth subregion hybridizing to the second subregion of the second splint strand (400), and the fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the first splint strand (300) comprises an internal region (310) further comprising a sixth subregion, the sixth subregion comprising a sample index sequence having 5 to 20 bases and / or a unique identification sequence (e.g., NN) having 2 to 10 or more bases, and the sixth subregion hybridizes to a third subregion of the second splint strand (400).

[0025] The present disclosure provides a single-stranded covalently closed circular molecule (600) comprising: (i) a first left universal adapter sequence (120) having a binding sequence for a first surface primer (120); (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 for a second surface primer (130); and (vi) a second splint strand (400), wherein the covalently closed circular molecule (600) is optionally hybridized to the first splint strand (300).

[0026] In some embodiments, the covalently closed circular library molecule (600) further comprises a plurality of covalently closed circular library molecules (600), wherein the target sequences (110) of the individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) comprise the same target sequence or different target sequences.

[0027] The present disclosure provides a reaction mixture comprising a plurality of covalently closed circular library molecules (600) of the present disclosure, wherein each covalently closed circular library molecule (600) is hybridized to a first splint strand (300). In some embodiments, the reaction mixture comprises the plurality of covalently closed circular library molecules (600) and at least one exonuclease enzyme. In some embodiments, the exonuclease enzyme comprises exonuclease I, thermolabile exonuclease I, or T7 exonuclease.

[0028] The present disclosure provides a kit comprising a double-stranded splint adapter (200), wherein the double-stranded splint adapter (200) comprises (i) a first splint strand (long splint strand (300)), and (ii) a second splint strand (short splint strand (400)), wherein the first splint strand (300) can hybridize to the second splint strand (400) to form a double-stranded region and two adjacent single-stranded regions, and wherein the first splint strand (300) comprises a first region (320), an internal region (310), and a second region (330), wherein the internal region of the first splint strand (310) can hybridize to the second splint strand (400). In some embodiments, the first splint strand (300) and the second splint strand (400) are hybridized to each other to form a double-stranded splint adapter (200) having a double-stranded region and two adjacent single-stranded regions, and the internal region of the first splint strand (310) can hybridize to the second splint strand (400).

[0029] In some embodiments, the kits of the present disclosure are for use in circularizing single-stranded nucleic acid library molecules (100) 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, a first region of the first splint strand (320) is capable of hybridizing to at least the first left universal adaptor sequence (120) of the library molecule (100), and a second region of the first splint strand (330) is capable of hybridizing to at least the first right universal sequence (130) of the library molecule. In some embodiments, at least the first left universal adapter sequence (120) of the library molecule (100) comprises (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.

[0030] In some embodiments, at least the first right universal adapter sequence (130) of the library molecule (100) comprises (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.

[0031] In some embodiments, the second splint strand (400) comprises at least two subregions, the first subregion comprising a universal binding sequence for the third surface primer and the second subregion comprising a universal binding sequence for the fourth surface primer, the first and second subregions not hybridizing to (or at least exhibiting very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, the optional third subregion comprising a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases.

[0032] In some embodiments, the first splint strand (300) comprises an internal region (310) comprising at least two subregions, the fourth subregion comprising a universal binding sequence for a third surface primer, the fourth subregion hybridizing to a first subregion of the second splint strand (400), the fifth subregion comprising a universal binding sequence for a fourth surface primer, the fifth subregion hybridizing to a second subregion of the second splint strand (400), and the fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the first splint strand (300) comprises an internal region (310) further comprising a sixth subregion, the sixth subregion comprising a sample index sequence having 5 to 20 bases and / or a unique identification sequence (e.g., NN) having 2 to 10 or more bases, and the sixth subregion hybridizes to a third subregion of the second splint strand (400).

[0033] In some embodiments, the kits of the present disclosure further comprise T4 polynucleotide kinase. In some embodiments, the kits of the present disclosure further comprise a ligase enzyme. In some embodiments, the kits of the present disclosure further comprise T4 polynucleotide kinase and a ligase enzyme. In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0034] The present disclosure provides a method for forming a plurality of library-sprint complexes (500), comprising: a) providing a plurality of double-stranded splint adapters, each double-stranded splint adapter (200) comprising a first splint strand (300) hybridized to a second splint strand (400), the double-stranded splint adapter comprising a double-stranded region and two adjacent single-stranded regions, the first splint strand comprising a first region (320), an internal region (310), and a second region (330), the internal region of the first splint strand (310) hybridizing to the second splint strand (400); and b) hybridizing the plurality of double-stranded splint adapters to a plurality of single-stranded nucleic acid library molecules (100), each library molecule comprising: wherein the hybridization is carried out under conditions suitable for hybridizing a first region of the first splint strand (320) to at least the first left universal adapter sequence (120) of the library molecule, and conditions suitable for hybridizing a second region of the first splint strand (330) to at least the first right universal sequence (130) of the library molecule, thereby circularizing the plurality of library molecules to form a plurality of library-sprint complexes (500).

[0035] In some embodiments, each individual library-sprint complex (500) in the plurality of library-sprint complexes (500) comprises a first nick between the 5' end of the library molecule and the 3' end of the second splint strand, and each individual library-sprint complex (500) in the plurality of library-sprint complexes (500) comprises a second nick between the 5' end of the second splint strand and the 3' end of the library molecule. In some embodiments, each individual nucleic acid library molecule (100) in the plurality of nucleic acid library molecules (100) further comprises a second left universal adaptor sequence (140). In some embodiments, each individual nucleic acid library molecule (100) in the plurality of nucleic acid library molecules (100) further comprises a second right universal adaptor sequence (150). In some embodiments, each individual nucleic acid library molecule (100) in the plurality of nucleic acid library molecules (100) further comprises a first left index sequence (160). In some embodiments, each individual nucleic acid library molecule (100) in the plurality of nucleic acid library molecules (100) further comprises a first right index sequence (170). In some embodiments, each individual nucleic acid library molecule (100) in the plurality of nucleic acid library molecules (100) further comprises a first left unique identifier sequence (180). In some embodiments, each individual nucleic acid library molecule (100) in the plurality of nucleic acid library molecules (100) further comprises a first right unique identifier sequence (190).

[0036] In some embodiments, each individual nucleic acid library molecule (100) in the plurality of nucleic acid library molecules (100) further comprises any combination of one or more of: (i) a second left universal adapter sequence (140), (ii) a second right universal adapter sequence (150), (iii) a first left index sequence (160), (iv) a first right index sequence (170), (v) a first left unique identifier sequence (180), and / or (vi) a first right unique identifier sequence (190). In some embodiments, the first and / or second left universal adapter sequence comprises: (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.

[0037] In some embodiments, the first and / or second right universal adapter sequence comprises (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.

[0038] In some embodiments, the second splint strand (400) comprises at least two subregions, the first subregion comprising a universal binding sequence for the third surface primer and the second subregion comprising a universal binding sequence for the fourth surface primer, the first and second subregions not hybridizing to (or at least exhibiting very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, the optional third subregion comprising a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases.

[0039] In some embodiments, the first splint strand (300) comprises an internal region (310) comprising at least two subregions, the fourth subregion comprising a universal binding sequence for a third surface primer, the fourth subregion hybridizing to a first subregion of the second splint strand (400), the fifth subregion comprising a universal binding sequence for a fourth surface primer, the fifth subregion hybridizing to a second subregion of the second splint strand (400), and the fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the first splint strand (300) comprises an internal region (310) further comprising a sixth subregion, the sixth subregion comprising a sample index sequence having 5 to 20 bases and / or a unique identification sequence (e.g., NN) having 2 to 10 or more bases, and the sixth subregion hybridizes to a third subregion of the second splint strand (400).

[0040] The present disclosure provides a method for forming a plurality of library-sprint complexes (500), comprising: a) providing a plurality of double-stranded splint adaptors (200), each of which comprises a first splint strand (300) hybridized to a second splint strand (400), the first splint strand (300) comprising a first region (320), an internal region (310), and a second region (330), arranged in 5' to 3' order, the internal region of the first splint strand (310) being hybridized to a second splint strand (400). (400), wherein the second splint strand comprises (i) a second subregion having a universal binding sequence for a fourth surface primer, and (ii) a first subregion having a universal binding sequence for a third surface primer, which are regions arranged in 5' to 3' order; and b) hybridizing a plurality of double-stranded splint adapters to a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule comprises a region arranged in 5' to 3' order, wherein (i) the first surface primer comprises a second subregion having a universal binding sequence for a fourth surface primer, and (ii) a first subregion having a universal binding sequence for a third surface primer. (ii) a first left universal adapter sequence (120) having a binding sequence for a first sequencing primer (120), (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 (130), wherein hybridizing the first sequence to the second universal adapter sequence (120) results in a first sequence to the second universal adapter sequence (140). This is done under conditions suitable for hybridizing a portion of the splint strand (300) to a portion of the library molecule (100), thereby circularizing the library molecule and producing a library-sprint complex (500), whereby a first region (320) of the first splint strand hybridizes to the binding sequence for the first surface primer (120), and a third region (330) of the first splint strand hybridizes to the binding sequence for the second surface primer (130), and the library-sprint complex (500) ishybridizing the library molecule (100) to a first nick between the 5' end of the library molecule and the 3' end of the second splint strand (300), and the library-sprint complex (500) comprises a second nick between the 5' end of the second splint strand (300) and the 3' end of the library molecule (100), the first and second nicks being enzymatically ligatable.

[0041] 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).

[0042] In some embodiments, the method further comprises contacting the plurality of double-stranded splint adaptors (200) and the plurality of single-stranded nucleic acid library molecules (100) with a T4 polynucleotide kinase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of double-stranded splint adaptors (200) and / or the plurality of single-stranded nucleic acid library molecules (100).

[0043] In some embodiments, the method further includes contacting the plurality of double-stranded splint adaptors (200) and the plurality of single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatic ligation of the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600) each hybridized to the first splint strand (300).

[0044] In some embodiments, the method further comprises the sequential steps of: a) contacting a plurality of double-stranded splint adapters (200) and a plurality of single-stranded nucleic acid library molecules (100) with a T4 polynucleotide kinase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of double-stranded splint adapters (200) and the plurality of single-stranded nucleic acid library molecules (100); and b) contacting the phosphorylated double-stranded splint adapters (200) and the phosphorylated single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatic ligation of the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600), each hybridized to a first splint strand (300).

[0045] In some embodiments, the method further comprises performing the essentially simultaneous steps of contacting the plurality of double-stranded splint adapters (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 to phosphorylate the 5' ends of the plurality of double-stranded splint adapters (200) and the plurality of single-stranded nucleic acid library molecules (100), wherein the conditions are suitable to enzymatically ligate the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600), each hybridized to a first splint strand (300).

[0046] In some embodiments, the method further comprises contacting the plurality of covalently closed circular library molecules (600) with at least one exonuclease enzyme to remove the plurality of first splint strands (300) and retain the plurality of covalently closed circular library molecules (600). In some embodiments, the at least one exonuclease enzyme comprises Exonuclease I, Thermolabile Exonuclease I, and / or T7 Exonuclease.

[0047] In some embodiments, each covalently closed circular library molecule (600) in the plurality of covalently closed circular library molecules (600) comprises a second splint strand region (400) comprising a universal binding sequence for a third surface primer, and the method further comprises distributing the retained plurality of covalently closed circular library molecules (600) onto a support on which a plurality of third surface primers have been immobilized under conditions suitable for hybridizing each of the covalently closed circular library molecules (600) to each of the immobilized third surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (600).

[0048] In some embodiments, the method further includes contacting the plurality of immobilized covalently closed circular library molecules (600) 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 third surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the plurality of nucleotides includes dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0049] In some embodiments, the first left universal adapter sequence (120) or the second left universal adapter sequence (140) of each library molecule in the plurality of library molecules comprises a universal binding sequence for a forward amplification primer, and the method further comprises: a) hybridizing a plurality of soluble forward amplification primers to the first or second left universal adapter sequence comprising a universal binding sequence for the forward amplification primer; and b) performing a rolling circle amplification reaction, thereby generating a plurality of nucleic acid concatemer molecules.

[0050] In some embodiments, the first right universal adaptor sequence (130) or the second right universal adaptor sequence (150) of an individual library molecule in the plurality of library molecules comprises a universal binding sequence for a forward amplification primer, and the method further comprises: a) hybridizing a plurality of soluble forward amplification primers to the first or second right universal adaptor sequence comprising the universal binding sequence for the forward amplification primer; and b) performing a rolling circle amplification reaction, thereby generating a plurality of nucleic acid concatemer molecules, each hybridized to a covalently closed circular library molecule (600).

[0051] In some embodiments, each concatemer molecule in the plurality of concatemer molecules comprises a second splint strand region (400) having a universal binding sequence for a third surface primer, and each concatemer molecule is hybridized to a covalently closed circular library molecule (600), and the method further comprises distributing the plurality of concatemer molecules onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to at least a portion of the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules.

[0052] In some embodiments, the method further comprises contacting the immobilized plurality of concatemer molecules (hybridized to the covalently closed circular library molecules (600)) 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 covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0053] In some embodiments, each covalently closed circular library molecule (600) in the plurality of covalently closed circular library molecules (600) is hybridized to a first splint strand (300), and the method further includes contacting the plurality of covalently closed circular library molecules (600) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction using the first splint strand (300) as an amplification primer, thereby generating a plurality of concatemeric molecules.

[0054] In some embodiments, each concatemer molecule in the plurality of concatemer molecules comprises a second splint strand region (400) comprising a universal binding sequence for a third surface primer, and the method further comprises distributing the plurality of concatemer molecules hybridized to the covalently closed circular library molecule (600) onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to at least a portion of the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules hybridized to the covalently closed circular library molecule (600).

[0055] In some embodiments, the method further comprises 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 rolling circle amplification reaction on the support using the plurality of covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, the support further comprises a plurality of fourth surface primers immobilized on the support.

[0056] In some embodiments, the plurality of immobilized nucleic acid concatemer molecules comprises at least two second splint strand regions (400), each having a universal binding sequence for a third and fourth surface primer, and the method further comprises incubating the plurality of immobilized nucleic acid concatemer molecules under conditions suitable for hybridizing at least one second splint strand region (400) of at least one concatemer molecule to the immobilized fourth surface primer, thereby pressing at least one portion of the immobilized concatemer molecule against the support.

[0057] In some embodiments, the method further includes sequencing the plurality of immobilized concatemer molecules, the method comprising: a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of 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 b) contacting the plurality of multiplexed sequencing polymerases with the plurality of soluble sequencing primers. a) contacting a plurality of nucleotides 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; b) 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 c) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0058] In some embodiments, the method further comprises sequencing the plurality of immobilized concatemer molecules, the method comprising: a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases, and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of first 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 and complementary nucleotide units of the multivalent molecules to form a plurality of first multiplexed polymerases. a) contacting a plurality of multivalent composite polymerases with at least two of the first and second composite polymerases under conditions suitable for binding to the first and second composite polymerases, thereby forming a plurality of multivalent composite polymerases, wherein the conditions inhibit incorporation of complementary nucleotide units into the sequencing primers of the plurality of multivalent composite 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 composite polymerases; and c) identifying the nucleobase of the complementary nucleotide unit bound to a plurality of first composite polymerases in the plurality of multivalent composite polymerases, thereby determining the sequence of the nucleic acid template.

[0059] In some embodiments, the method comprises: e) dissociating the plurality of multivalent multiplexed polymerases, removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; f) contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contacting is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second multiplexed polymerases, each of which binds to a retained nucleic acid duplex. and (g) contacting the plurality of second multiplexed 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 multiplexed polymerases of step (f), thereby forming a plurality of nucleotide-multiplexed polymerases, and the conditions are suitable for promoting incorporation of the bound complementary nucleotides into the sequencing primer by the nucleotide-multiplexed polymerases. In some embodiments, 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.

[0060] 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.

[0061] 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 any of the concatemeric molecules of claims 80, 85, 89, or 92, 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. and combining the first and second multivalent molecules, thereby 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.

[0062] The present disclosure provides 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 any of the concatemeric molecules of the disclosure, 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; and the first and second binding complexes comprising the same multivalent molecules form an avidity complex, wherein the first multivalent molecule binds to a plurality of nucleotide arms. a) forming a concatemeric template molecule comprising a core attached to a nucleotide arm, each nucleotide arm being attached to a nucleotide unit, wherein 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 nucleic acid primers, wherein 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; 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 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.

[0063] The present disclosure provides a method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources, comprising: 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 first 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 second sequencing primer, (iv) a second left universal adapter sequence (160) having a binding sequence for a first surface primer, (v) a second left universal adapter sequence (170) having a binding sequence for a second sequencing primer, (vi) a first left universal adapter sequence (180) having a binding sequence for a second sequencing primer, (vii) a second left universal adapter sequence (190) having a binding sequence for a second sequencing primer, (viii) a second left universal adapter sequence (190) having a binding sequence for a second sequencing primer, (viii) a second left universal adapter sequence (190) having a binding sequence for a second sequencing primer, (viii) a first left universal adapter sequence (190) having a binding sequence for a second sequencing primer, (viii) a second ... (iii) a sequence of interest (110) isolated from a sample source; (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer; (v) a first right universal adapter sequence (130) having a binding sequence for a second 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, are associated with the sample source from which the sequence of interest (110) was isolated. and b) providing a plurality of double-stranded splint adapters (200), each double-stranded splint adapter (200) comprising a first splint strand (300) hybridized to a second splint strand (400), the first splint strand (300) comprising regions arranged in 5' to 3' order: a first region (320), an internal region (310), and a second region (330), the internal region of the first splint strand (310) hybridizing to the second splint strand (400), the second splint strand comprising: providing regions arranged in 5' to 3' order, the regions including (i) a second subregion having a universal binding sequence for a fourth surface primer, and (ii) a first subregion having a universal binding sequence for a third surface primer; and c) contacting, in separate compartments, the population of single-stranded nucleic acid library molecules (100) with a plurality of double-stranded splint adapters (200), wherein the contacting is performed under conditions suitable for hybridizing a portion of the first splint strand (300) to a portion of the library molecules (100);The library molecules are thereby circularized to produce a population of library-sprint complexes (500), whereby a first region (320) of each first splint strand hybridizes to the binding sequence for the first surface primer (120) of each library molecule (100), a third region (330) of each first splint strand hybridizes to the binding sequence for the second surface primer (130) of each library molecule (100), each library-sprint complex (500) comprising a first nick between the 5' end of the library molecule and the 3' end of the second splint strand (300), and each library-sprint complex (500) comprising a first nick between the 5' end of the second splint strand (300) and the 3' end of the library molecule (100). a second nick between the ends of the first splint strand (300), wherein the first and second nicks are enzymatically ligatable; d) in separate compartments, contacting the population of library-splint complexes (500) with a ligase under conditions suitable for enzymatic ligation of the first and second nicks, thereby producing two or more populations of covalently closed circular library molecules (600), each hybridized to the first splint strand (300); and e) pooling together the two or more populations of covalently closed circular library molecules (600) from the separate compartments to produce a multiplex mixture of covalently closed circular library molecules (600), comprising a multiplex mixture of sequences of interest isolated from multiple sample sources.

[0064] In some embodiments, each covalently closed circular library molecule (600) in the multiplex mixture of step (e) comprises a second splint strand region (400) comprising a universal binding sequence for a third surface primer, and the method further comprises: a) distributing the multiplex mixture of covalently closed circular library molecules (600) onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing each covalently closed circular library molecule (600) to each immobilized third surface primer, thereby immobilizing the plurality of covalently closed circular library molecules (600). and b) contacting the multiplex mixture of immobilized covalently closed circular library molecules (600) 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 third surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby producing a plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0065] In some embodiments, the method further includes performing a rolling circle amplification reaction by: a) contacting two or more populations of covalently closed circular library molecules (600) hybridized to the first splint strand (300) of step (d) or (e) with at least one exonuclease enzyme under conditions suitable for removing the plurality of first splint strands (300), thereby retaining the populations of covalently closed circular library molecules (600); and b) contacting the retained two or more populations of covalently closed circular library molecules (600) with a plurality of soluble amplification primers, a plurality of strand-displacing polymerases, and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction, thereby producing a plurality of nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0066] In some embodiments, the method further comprises: a) distributing a plurality of concatemer molecules onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to at least a portion of the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules; and b) contacting the immobilized plurality of concatemer molecules (hybridized to the covalently closed circular library molecules (600)) 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 covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0067] In some embodiments, the method further includes performing a rolling circle amplification reaction by contacting two or more populations of covalently closed circular library molecules (600) of step (d) or (e), each hybridized to a first splint strand (300), with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction, thereby generating a plurality of concatemeric molecules, wherein the plurality of nucleotides includes dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0068] In some embodiments, the method further comprises: a) distributing a plurality of concatemer molecules hybridized to the covalently closed circular library molecules (600) onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to at least a portion of the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules hybridized to the covalently closed circular library molecules (600); and b) contacting the plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on the support using the plurality of covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0069] In some embodiments, the method further comprises sequencing the insertion region (110) and the first left index sequence (160) and / or the first right index sequence (170) of the plurality of immobilized nucleic acid concatemer molecules, wherein the sequence of the insertion region (110), together with the first left index sequence (160) and / or the first right index sequence (170), can be used to identify the sample source of the sequence of interest.

[0070] In some embodiments, the sequencing reaction comprises: a) combining a plurality of immobilized nucleic acid concatemer molecules with a plurality of sequencing primers, a plurality of sequencing polymerases, and a plurality of detectably labeled nucleotide analogs, each comprising a 2' or 3' chain-terminating moiety, under conditions suitable for incorporation of the detectably labeled nucleotide analogs into the 3' ends of the sequencing primers; b) detecting the bound detectably labeled nucleotide analogs; and c) identifying the nucleobases of the detectably labeled nucleotide analogs. In some embodiments, the sequencing reaction uses a two-step sequencing reaction, the first step comprising: a) combining a plurality of immobilized nucleic acid concatemer molecules with a plurality of sequencing primers, a plurality of first sequencing polymerases, and a plurality of detectably labeled multivalent molecules under conditions suitable for binding of each concatemer molecule to the sequencing primers, the first sequencing polymerase, and the detectably labeled multivalent molecules (e.g., forming a plurality of multivalent hybrid polymerases); b) detecting the bound detectably labeled multivalent molecules; and c) identifying the nucleobases of the detectably labeled multivalent molecules. In some embodiments, the binding of the detectably labeled multivalent molecules to the plurality of immobilized nucleic acid concatemer molecules forms at least one avidity complex.

[0071] In some embodiments, the method further comprises performing a second step comprising: d) removing the plurality of first sequencing polymerases and the plurality of bound multivalent molecules from the plurality of immobilized nucleic acid concatemer molecules, retaining the immobilized concatemer molecules (nucleic acid duplexes), each hybridized to a sequencing primer; and e) contacting the retained immobilized concatemer molecules and the hybridized sequencing primers with a plurality of second polymerases and a plurality of nucleotides under conditions suitable for incorporating nucleotides into the 3' ends of the sequencing primers.

[0072] The present disclosure provides a method for preparing a multiplex mixture of sequences of interest isolated from multiple sample sources, comprising: 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 comprise: (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) isolated from the sample source; (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer; (v) a first right universal adapter sequence (130) having a binding sequence for a second surface primer; and (vi) 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) are contained within a separate compartment. b) pooling two or more populations of single-stranded nucleic acid library molecules (100) together in the same compartment; and c) adding a plurality of double-stranded splint adapters (200) to the compartment of step (b), each double-stranded splint adapter (200) comprising a first splint strand (300) hybridized to a second splint strand (400), and wherein the first splint strand (300) is hybridized to a second splint strand (400). adding a splint strand (300) comprising a first region (320), an internal region (310), and a second region (330) arranged in 5' to 3' order, the internal region of the first splint strand (310) hybridizing to a second splint strand (400), the second splint strand comprising (i) a second subregion having a universal binding sequence for a fourth surface primer, and (ii) a region arranged in 5' to 3' order of the first subregion having a universal binding sequence for a third surface primer;d) incubating the population of single-stranded nucleic acid library molecules (100) and a plurality of double-stranded splint adapters (200) in the same compartment of step (c), wherein the incubation is carried out under conditions suitable for hybridizing a portion of the first splint strand (300) to a portion of the library molecules (100), thereby circularizing the library molecules and generating a population of library-sprint complexes (500), whereby a first region (320) of each first splint strand hybridizes to a binding sequence for a first surface primer (120) of each library molecule (100), and a third region (330) of each first splint strand hybridizes to a binding sequence for a second surface primer (130) of each library molecule (100), and each library-sprint complex (500) is composed of five or more double-stranded splint adapters (200) of the library molecules. and (d) incubating the library-sprint complexes (500) in the compartments of step (d) with a ligase under conditions suitable for enzymatically ligating the first and second nicks, thereby producing a population of covalently closed circular library molecules (600) each hybridized to the first splint strand (300), thereby producing a multiplex mixture of covalently closed circular library molecules (600) comprising a multiplex mixture of sequences of interest isolated from a plurality of sample sources.

[0073] In some embodiments, each covalently closed circular library molecule (600) in the multiplex mixture of step (e) comprises a second splint strand region (400) comprising a universal binding sequence for a third surface primer, and the method further comprises: a) distributing a plurality of covalently closed circular library molecules (600) onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing each of the covalently closed circular library molecules (600) to each of the immobilized third surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (600). and b) contacting the plurality of immobilized covalently closed circular library molecules (600) 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 third surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby producing a plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0074] In some embodiments, the method further includes performing a rolling circle amplification reaction by: a) contacting the population of covalently closed circular library molecules (600) hybridized to the first splint strand (300) of step (d) or (e) with at least one exonuclease enzyme under conditions suitable for removing a plurality of first splint strands (300), thereby retaining the population of covalently closed circular library molecules (600); and b) contacting the retained population of covalently closed circular library molecules (600) with a plurality of soluble amplification primers, a plurality of strand-displacing polymerases, and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction, thereby generating a plurality of nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0075] In some embodiments, the method further comprises: c) distributing the plurality of concatemer molecules onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to at least a portion of the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules; and d) contacting the immobilized plurality of concatemer molecules (hybridized to the covalently closed circular library molecules (600)) 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 covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0076] In some embodiments, the method further includes performing a rolling circle amplification reaction by contacting the population of covalently closed circular library molecules (600) of step (d) or (e), each hybridized to a first splint strand (300), with a plurality of strand-displacing polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction, thereby generating a plurality of concatemeric molecules, wherein the plurality of nucleotides includes dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0077] In some embodiments, the method further comprises: c) distributing the plurality of concatemer molecules hybridized to the covalently closed circular library molecules (600) onto a support having a plurality of third surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemers to at least a portion of the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules hybridized to the covalently closed circular library molecules (600); and d) contacting the plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on the support using the plurality of covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0078] In some embodiments, the method further comprises sequencing the insertion region (110) and the first left index sequence (160) and / or the first right index sequence (170) of the plurality of immobilized nucleic acid concatemer molecules, wherein the sequence of the insertion region (110), together with the first left index sequence (160) and / or the first right index sequence (170), can be used to identify the sample source of the sequence of interest.

[0079] In some embodiments, the sequencing reaction comprises: a) combining a plurality of immobilized nucleic acid concatemer molecules with a plurality of sequencing primers, a plurality of sequencing polymerases, and a plurality of detectably labeled nucleotide analogs, each comprising a 2' or 3' chain-terminating moiety, under conditions suitable for incorporation of the detectably labeled nucleotide analogs into the 3' ends of the sequencing primers; b) detecting the bound detectably labeled nucleotide analogs; and c) identifying the nucleobases of the detectably labeled nucleotide analogs.

[0080] In some embodiments, the sequencing reaction uses a two-step sequencing reaction, the first step comprising: a) combining a plurality of immobilized nucleic acid concatemer molecules with a plurality of sequencing primers, a plurality of first sequencing polymerases, and a plurality of detectably labeled multivalent molecules under conditions suitable for binding of each concatemer molecule to the sequencing primers, the first sequencing polymerase, and the detectably labeled multivalent molecules (e.g., forming a plurality of multivalent hybrid polymerases); b) detecting the bound detectably labeled multivalent molecules; and c) identifying the nucleic acid bases of the detectably labeled multivalent molecules. In some embodiments, the detectably labeled multivalent molecules to the plurality of immobilized nucleic acid concatemer molecules form at least one avidity complex.

[0081] In some embodiments, the method further comprises performing a second step comprising: d) removing the plurality of first sequencing polymerases and the plurality of bound multivalent molecules from the plurality of immobilized nucleic acid concatemer molecules, retaining the immobilized concatemer molecules (nucleic acid duplexes), each hybridized to a sequencing primer; and e) contacting the retained immobilized concatemer molecules and the hybridized sequencing primers with a plurality of second polymerases and a plurality of nucleotides under conditions suitable for incorporating nucleotides into the 3' ends of the sequencing primers.

[0082] The present disclosure provides a method for adding additional universal adapter sequences to nucleic acid library molecules, comprising: a) providing a plurality of nucleic acid library molecules (100), each of the plurality of nucleic acid library molecules (100) comprising, arranged in 5' to 3' order, the following sequences: (i) a first left universal adapter sequence (120), (ii) a second left universal adapter sequence (140), (iii) a sequence of interest (110), (iv) a second right universal adapter sequence (150), and (v) a first right universal adapter sequence ( b) hybridizing each individual library molecule of step (a) with a first tailed primer and performing a first primer extension reaction (or first PCR), wherein the first tailed primer comprises a 5' region bearing a third left universal adapter sequence and a 3' region that is complementary to at least a portion of the first left universal adapter sequence (120) of the library molecule (100), and the first primer extension reaction (or first PCR) produces a plurality of first modified library molecules. a third left universal adapter sequence attached at one end to each of the plurality of first modified library molecules, wherein the third left universal adapter sequence of the first tailed primer 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, and / or a universal binding sequence for a compaction oligonucleotide; and c) hybridizing each of the first modified library molecules of step (a) with a second tailed primer and performing a second primer extension reaction (or second PCR), wherein the second tailed primer comprises a 5' region bearing the third right universal adapter sequence and a 3' region that is complementary to at least a portion of the first right universal adapter sequence (130) of the first modified library molecule, wherein the second primer extension reaction (or second PCR) produces a plurality of second modified library molecules;and performing a method comprising: adding a third right universal adapter sequence to each of the plurality of second modified library molecules at one end, the third right universal adapter sequence of the second tailed primer 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, and / or a universal binding sequence for a compaction oligonucleotide.

[0083] In some embodiments, the nucleic acid library molecules each comprise, in 5' to 3' order, the following sequences: (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.

[0084] In some embodiments, the method further comprises: d) hybridizing each second modified library molecule of step (c) with a third tailed primer and performing a third primer extension reaction (or third PCR), wherein the third tailed primer comprises a 5' region bearing a fourth left universal adapter sequence and a 3' region that is complementary to at least a portion of the third left universal adapter sequence of the second modified library molecule; and wherein the third primer extension reaction (or third PCR) generates a plurality of third modified library molecules, each of the plurality of third modified library molecules having a fourth left universal adapter sequence appended at one end, wherein the fourth left universal adapter sequence of the third tailed primer 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, and / or a universal binding sequence for a compaction oligonucleotide. and e) hybridizing each third modified library molecule of step (d) with a fourth tailed primer and performing a fourth primer extension reaction (or fourth PCR), wherein the fourth tailed primer comprises a 5' region bearing a fourth right universal adapter sequence and a 3' region that is complementary to at least a portion of the third right universal adapter sequence of the third modified library molecule, wherein the fourth primer extension reaction (or fourth PCR) generates a plurality of fourth modified library molecules, each of the plurality of fourth modified library molecules having a fourth right universal adapter sequence appended at one end, wherein the fourth right universal adapter sequence of the fourth tailed primer 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, and / or a universal binding sequence for a compaction oligonucleotide.

[0085] In some embodiments, the method further comprises: f) hybridizing each fourth modified library molecule of step (e) with a fifth tailed primer and performing a fifth primer extension reaction (or fifth PCR), wherein the fifth tailed primer comprises a 5' region bearing a fifth left universal adapter sequence and a 3' region that is complementary to at least a portion of the fourth left universal adapter sequence of the fourth modified library molecule; wherein the fifth primer extension reaction (or fifth PCR) generates a plurality of fifth modified library molecules, each of the plurality of fifth modified library molecules having a fifth left universal adapter sequence appended at one end, wherein the fifth left universal adapter sequence of the fifth tailed primer 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, and / or a universal binding sequence for a compaction oligonucleotide; and g) hybridizing each fifth modified library molecule of step (f) with a sixth tailed primer and performing a sixth primer extension reaction (or sixth PCR), wherein the sixth tailed primer comprises a 5' region bearing a sixth right universal adapter sequence and a 3' region that is complementary to at least a portion of the fourth right universal adapter sequence of the fifth modified library molecule, wherein the sixth primer extension reaction (or sixth PCR) generates a plurality of sixth modified library molecules, each of the plurality of sixth modified library molecules having a sixth right universal adapter sequence appended at one end, wherein the sixth right universal adapter sequence of the sixth tailed primer comprises a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for the 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.

[0086] In some embodiments, the first tailed primer, the third tailed primer, and / or the fifth tailed primer further comprise a left index sequence. In some embodiments, the first tailed primer, the third tailed primer, and / or the fifth tailed primer further comprise a left unique identifier sequence. In some embodiments, the second tailed primer, the fourth tailed primer, and / or the sixth tailed primer further comprise a right index sequence. In some embodiments, the second tailed primer, the fourth tailed primer, and / or the sixth tailed primer further comprise a right unique identifier sequence. In some embodiments, the third left universal adapter sequence added in step (b) can bind to the first region (320) of the first splint strand. In some embodiments, the third right universal adapter sequence added in step (c) can bind to the second region (330) of the first splint strand. In some embodiments, the fourth left universal adaptor sequence added in step (d) can bind to the first region of the first splint strand (320). In some embodiments, the fourth right universal adaptor sequence added in step (e) can bind to the second region of the first splint strand (330).

[0087] In some embodiments, the fifth left universal adaptor sequence added in step (f) can bind to a first region (320) of the first splint strand, and in some embodiments, the fifth right universal adaptor sequence added in step (g) can bind to a second region (330) of the first splint strand.

[0088] The present disclosure provides a method for forming a plurality of library-sprint complexes (500), comprising: a) providing a plurality of double-stranded splint adaptors (200), each double-stranded splint adaptor (200) comprising a first splint strand (300) hybridized to a second splint strand (400), the first splint strand (300) comprising a first region (320), an internal region (310), and a second region (330) arranged in 5' to 3' order, the internal region of the first splint strand (310) being hybridized to a second splint strand (400). a) providing a splint adapter (400) hybridized to a second splint strand, the second splint strand comprising, arranged in 5' to 3' order, (i) a second sub-region having a universal binding sequence for a fourth surface primer, and (ii) a first sub-region having a universal binding sequence for a third surface primer; and b) hybridizing a plurality of double-stranded splint adapters to a plurality of single-stranded nucleic acid library molecules (100), the individual library molecules comprising, arranged in 5' to 3' order, (i) a second sub-region having a universal binding sequence for a fourth surface primer, and (ii) a first sub-region having a universal binding sequence for a third surface primer. (ii) a first left universal adapter sequence (120) having a binding sequence for a 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 fourth surface primer, wherein hybridizing to the first sequence of interest is The hybridization is carried out under conditions suitable for hybridizing a portion of the splint strand (300) to a portion of the library molecule (100), thereby circularizing the library molecule to produce a library-sprint complex (500), whereby a first region (320) of the first splint strand hybridizes to the binding sequence for the third surface primer (120), and a third region (330) of the first splint strand hybridizes to the binding sequence for the fourth surface primer (130), and the library-sprint complex (500) isand hybridizing the library molecule (100) to a first splint strand (400) comprising a first nick between the 5' end of the library molecule and the 3' end of the second splint strand (400), and the library-sprint complex (500) comprising a second nick between the 5' end of the second splint strand (400) and the 3' end of the library molecule (100), the first and second nicks being enzymatically ligatable.

[0089] 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).

[0090] In some embodiments, the method further comprises contacting the plurality of double-stranded splint adaptors (200) and the plurality of single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatic ligation of the first and second nicks, thereby producing a plurality of covalently closed circular library molecules (600), each hybridized to the first splint strand (300). In some embodiments, the method further comprises contacting the plurality of covalently closed circular library molecules (600) with at least one exonuclease enzyme to remove the plurality of first splint strands (300) and retain the plurality of covalently closed circular library molecules (600).

[0091] In some embodiments, each covalently closed circular library molecule (600) in the plurality of covalently closed circular library molecules (600) comprises a second splint strand region (400) comprising a universal binding sequence for a second surface primer, and the method further comprises distributing the retained plurality of covalently closed circular library molecules (600) onto a support on which a plurality of second surface primers have been immobilized under conditions suitable for hybridizing each covalently closed circular library molecule (600) to each immobilized second surface primer, thereby immobilizing the plurality of covalently closed circular library molecules (600).

[0092] In some embodiments, the method further includes contacting the plurality of immobilized covalently closed circular library molecules (600) 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 second surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the plurality of nucleotides includes dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0093] In some embodiments, the plurality of immobilized second 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 to flow over the support, thereby allowing the plurality of immobilized second surface primers to react essentially simultaneously in a massively parallel manner with the reagents, optionally the solution of reagents comprising enzymes, nucleotides, and divalent cations. 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.

[0094] In some embodiments, the method further includes sequencing the plurality of immobilized concatemer molecules, wherein sequencing comprises: a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases, and (ii) a plurality of soluble sequencing primers, wherein the contacting is performed under conditions suitable for forming a plurality of 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 b) contacting the plurality of multiplexed sequencing polymerases with a plurality of soluble sequencing primers. contacting at least one nucleotide with a 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; c) 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 d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0095] 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.

[0096] In some embodiments, the method further includes sequencing the plurality of immobilized concatemer molecules, wherein 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 soluble universal sequencing primers. contacting the first multiplexed polymerase with a plurality of detectably labeled multivalent molecules under conditions suitable for binding complementary nucleotide units of the multivalent molecules to at least two of the plurality of first multiplexed polymerases, thereby forming a plurality of multivalent multiplexed polymerases, wherein the conditions inhibit incorporation of the complementary nucleotide units into the sequencing primers of the plurality of multivalent multiplexed polymerases, and each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit; and c) detecting the plurality of multivalent multiplexed polymerases.

[0097] In some embodiments, the method comprises: d) dissociating the plurality of multivalent multiplexed polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, retaining the plurality of nucleic acid duplexes; e) contacting the plurality of retained nucleic acid duplexes of step d) 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 a second sequencing polymerase bound to the retained nucleic acid duplexes. f) 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 e), thereby forming a plurality of nucleotide multiplexed polymerases, and wherein the conditions are suitable for promoting the incorporation of the bound complementary nucleotide into the sequencing primer by the nucleotide multiplexed polymerase.

[0098] In some embodiments, the method further comprises g) detecting a complementary nucleotide incorporated into the sequencing primer for the nucleotide-complexed polymerase. In some embodiments, the method further comprises g) detecting a complementary nucleotide incorporated into the sequencing primer for the nucleotide-complexed polymerase, and h) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer for the nucleotide-complexed polymerase.

[0099] 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 second sequencing polymerase; and the first and second binding complexes comprising the same multivalent molecule form an avidity complex, wherein the first detectably labeled multivalent molecule binds to the first sequencing polymerase. a) forming a concatemeric molecule, the concatemeric 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 sequencing primers; b) detecting the first and second binding complexes on the same concatemeric 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.

[0100] 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.

[0101] In some embodiments, the plurality of nucleotides comprise 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, wherein the removable chain-terminating moiety is cleavable with a chemical compound to generate an extendable 3' OH moiety on the sugar group.

[0102] In some embodiments, the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, hi 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.

[0103] 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.

[0104] 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]

[0105] [Figure 1]1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridized to a double-stranded splint molecule (200), thereby circularizing the library molecule and forming a library-splint complex (500) with two nicks. 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 double-stranded splint molecule comprises a first splint strand (long strand (300)) hybridized to a second splint strand (short strand (400)). The first splint strand comprises a first sequence (320) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second sequence (330) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. An internal region (310) of the first splint strand hybridizes to the second splint strand (400). [Figure 2] 1, with further details regarding the internal region (310) of the first splint strand (300) and the second splint strand (400) embodiments. The second splint strand (400) comprises two subregions, the first subregion comprising a universal binding sequence for a third surface primer and the second subregion comprising a universal binding sequence for a fourth surface primer. The internal region (310) of the first splint strand (300) comprises two subregions, the fourth subregion hybridizing to the first subregion of the second splint strand (400) and the fifth subregion hybridizing to the second subregion of the second splint strand (400). [Figure 3]1 with further details regarding an embodiment of the internal region (310) of the first splint strand (300) and the second splint strand (400). The second splint strand (400) comprises three subregions: the first subregion comprises a universal binding sequence for the third surface primer, the second subregion comprises a universal binding sequence for the fourth surface primer, and the third subregion comprises a sample index sequence having 5-20 bases and / or a unique identifier sequence (e.g., NN) having 2-10 or more bases. The internal region (310) of the first splint strand (300) includes three subregions, the fourth subregion hybridizes to the first subregion of the second splint strand (400), the fifth subregion hybridizes to the second subregion of the second splint strand (400), and the sixth subregion hybridizes to the third subregion of the second splint strand (400). [Figure 4] 1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridizing to a double-stranded splint molecule (200), thereby circularizing the library molecule and forming a library-splint complex (500) with two nicks. 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 double-stranded splint molecule comprises a first splint strand (long strand (300)) hybridized to a second splint strand (short strand (400)). The first splint strand comprises a first sequence (320) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second sequence (330) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. An internal region (310) of the first splint strand hybridizes to the second splint strand (400). [Figure 5]1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridizing to a double-stranded splint molecule (200), thereby circularizing the library molecule and forming a library-splint complex (500) with two nicks. The library molecule (100) comprises a first left universal adaptor sequence (120), a 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 double-stranded splint molecule comprises a first splint strand (long strand (300)) hybridized to a second splint strand (short strand (400)). The first splint strand comprises a first sequence (320) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second sequence (330) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. An internal region (310) of the first splint strand hybridizes to the second splint strand (400). [Figure 6]1 is a schematic diagram showing an exemplary linear, single-stranded library molecule (100) hybridizing to a double-stranded splint molecule (200), thereby circularizing the library molecule and forming a library-splint complex (500) with two nicks. The library molecule (100) comprises 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), a first right unique identifier sequence (190), and a first right universal adaptor sequence (130). The double-stranded splint molecule comprises a first splint strand (long strand (300)) hybridized to a second splint strand (short strand (400)). The first splint strand comprises a first sequence (320) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second sequence (330) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. An internal region (310) of the first splint strand hybridizes to the second splint strand (400). [Figure 7]Schematic diagram showing an exemplary linear single-stranded library molecule (100) hybridizing to a double-stranded splint molecule (200), thereby circularizing the library molecule to form a library-splint complex (500) with two nicks. The library molecule (100) comprises a first added left universal adaptor sequence (121), 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), a first right universal adaptor sequence (130), and a first added right universal adaptor sequence (131). The double-stranded splint molecule comprises a first splint strand (long strand (300)) hybridized to a second splint strand (short strand (400)). The first splint strand comprises a first sequence (320) that hybridizes to a sequence on one end of the linear single-stranded library molecule and a second sequence (330) that hybridizes to a sequence on the other end of the linear single-stranded library molecule. The internal region (310) of the first splint strand hybridizes to the second splint strand (400). For simplicity, the library-sprint complex (500) does not show either the junction adaptor sequences or the added universal adaptor sequences. Those skilled in the art will recognize that the linear library molecule (100) can comprise any one of the junction adaptors, or any combination of two or more of the junction adaptors, with or without one or both of the added universal adaptor sequences.Those skilled in the art will recognize that the library-sprint complex (500) can include any combination of one or more of the junction adapters, with or without one or both of the added universal adapter sequences present in the library molecule (100). [Figure 8] Three schematic diagrams of exemplary covalently closed circular library molecules are shown, each hybridized to a first splint strand (300). The top schematic diagram shows a covalently closed circular library molecule (600) having a sequence of interest (110), a first right universal adaptor sequence (130), a second splint strand sequence (400), and a first left universal adaptor sequence (120). The middle schematic diagram shows a covalently closed circular library molecule (600) having a sequence of interest (110), a second right universal adaptor sequence (150), a first right universal adaptor sequence (130), a second splint strand sequence (400), a first left universal adaptor sequence (120), and a second left universal adaptor sequence (140). The bottom schematic shows a covalently closed circular library molecule (600) 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 second splint strand sequence (400), a first left universal adaptor sequence (120), a first left unique identifier sequence (180), a first left index sequence (160), and a second left universal adaptor sequence (140). [Figure 9] 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 (600) that is hybridized to a first splint strand (300), which is used as an amplification primer to perform a rolling circle amplification reaction. The dotted line represents the nascent extension product. [Figure 10]1 shows the nucleotide sequence of an exemplary double-stranded splint molecule (200) having a first splint strand (300) and a second splint strand (400). The exemplary first splint strand includes a first region (320, SEQ ID NO:4), a second region (330, SEQ ID NO:5), and an internal region (310) having a fourth subregion (SEQ ID NO:6) and a fifth subregion (SEQ ID NO:7). The exemplary second splint strand (400) includes a first subregion (SEQ ID NO:1) and a second subregion (SEQ ID NO:2). [Figure 11] 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 attached to the glass and further comprising chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides and circularization oligonucleotides). In alternative embodiments, the support can be made from any material, such as glass, plastic, or a polymeric material. [Figure 12] Schematic diagrams of various exemplary configurations of multivalent molecules. Left: Schematic diagram of a multivalent molecule with a starburst or helter-skelter configuration. Center: Schematic diagram of a multivalent molecule with a dendrimer configuration. Right: Schematic diagram of multiple multivalent molecules formed by reacting streptavidin with 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 13] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a generic core attached to multiple nucleotide arms. [Figure 14] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms. [Figure 15] 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 16]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 17] 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 18] 1 shows the chemical structures of various exemplary linkers, including linkers 1-9. [Figure 19] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 20] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 21] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 22] 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 23A] 1 is Table 1 (six sheets) listing exemplary first left index array (160) and first right index array (170) arrangements. [Figure 23B] 1 is Table 1 (six sheets) listing exemplary first left index array (160) and first right index array (170) arrangements. [Figure 23C] 1 is Table 1 (six sheets) listing exemplary first left index array (160) and first right index array (170) arrangements. [Figure 23D] 1 is Table 1 (six sheets) listing exemplary first left index array (160) and first right index array (170) arrangements. [Figure 23E] 1 is Table 1 (six sheets) listing exemplary first left index array (160) and first right index array (170) arrangements. [Figure 23F]1 is Table 1 (six sheets) listing exemplary first left index array (160) and first right index array (170) arrangements. [Figure 24] 1 is a bar graph showing the average percent recovery of covalently closed circular library molecules using input DNA from various species as determined by qPCR. Lane 1: Haemophilus influenzae (38% GC), Lane 2: E. coli (51% GC), Lane 3: Rhodopseudomonas palustris (65% GC), Lane 4: PhiX, Lane 5: human, Lane 6: human exome, Lane 7: human mRNA. See Examples 1-3. [Figure 25] 1 is a bar graph showing the average polony density obtained by distributing covalently closed circular library molecules onto a support and performing on-support rolling circle amplification. The covalently closed circular library molecules were prepared from input DNA from various species. Lane 1: cell-free DNA, Lane 2: E. coli, Lane 3: human, Lane 4: metagenomic DNA, and Lane 5: PhiX. See Example 4. [Figure 26] 1 is a graph showing the nucleotide base diversity of the right sample index sequence (170) containing 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 27] 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

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] The use of alternative terms (eg, "or") is understood to mean either one or both of the alternatives, or any combination thereof.

[0111] 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).

[0112] 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.

[0113] 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.

[0114] 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.

[0115] The term "cellular biological sample" refers to a single cell, multiple cells, tissue, organ, organism, or section of any of these cellular biological samples. Cellular biological samples may be extracted from an organism (e.g., biopsy) or obtained from cell cultures growing in liquid or in culture dishes. Cellular biological samples include fresh samples, frozen samples, fresh frozen samples, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) samples. Cellular biological samples may be embedded in wax, resin, epoxy, or agar. Cellular biological samples may be fixed in, for example, any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton®, or glutaraldehyde. Cellular biological samples may or may not be sectioned. Cellular biological samples may be stained, destained, or unstained.

[0116] 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).

[0117] 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.

[0118] 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).

[0119] 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.

[0120] 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.

[0121] 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).

[0122] 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 functions 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. Primers can be in solution (e.g., soluble primers) or immobilized on a support (e.g., capture primers).

[0123] 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.

[0124] The term "adapter" and related terms refer to an oligonucleotide that can be operably attached to (appended 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 or more nucleotides. Adapters can have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' overhangs and 3' overhanging ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, can have a 5' phosphate group or 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 comprises 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 / index sequence, which may be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. The adapter may include at least one unique identification sequence (e.g., a molecular tag), which may be used to uniquely identify the nucleic acid molecule to which the adapter is attached.In some embodiments, the unique identification sequence 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.

[0125] 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).

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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).

[0135] Fluorescent reporter moieties (or labels) include fluorescent labels or fluorophores. 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.

[0136] In some embodiments, the reporter moieties can be fluorescence resonance energy transfer (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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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 At each of the plurality of predetermined sites (e.g., 10 or more sites), nucleic acid template molecules are immobilized to form a nucleic acid template array. In some embodiments, the nucleic acid template molecules are immobilized at the 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 the 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.

[0144] 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 At each of the plurality of randomly located sites (e.g., 10 or more sites), a nucleic acid template molecule is immobilized. 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] Introduction: Two-strand splint adapter The present disclosure provides compositions, including kits, that include nucleic acid double-stranded splint adaptors, and methods of using double-stranded splint adaptors.

[0155] The double-stranded splint adapter (200) can be used in a one-pot multi-enzyme reaction to introduce one or more new adapter sequences into library molecules. The double-stranded splint adapter (200) comprises a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)), which hybridize together to form the double-stranded splint adapter (200) having a double-stranded region and two adjacent single-stranded regions (see, e.g., Figures 1-7). The second splint strand (400) carries the new adapter sequence(s) to be introduced, e.g., a new universal binding sequence and / or a new index sequence. The first splint strand comprises a first region (320), an internal region (310), and a second region (330). The internal region of the first splint strand (310) hybridizes to the second splint strand (400). Two adjacent single-stranded regions (e.g., (320) and (330)) of the double-stranded splint adaptor 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, a first region of the first splint strand (320) hybridizes to one end of the library molecule, and a second region of the first splint strand (330) hybridizes to the other end of the library molecule, thereby circularizing the library molecule to generate a library-sprint complex (500) containing two nicks (see, e.g., Figures 1-7). The nicks can be enzymatically ligated to generate a covalently closed circular molecule (600) in which the second splint strand (400) is covalently linked to the library molecule at both ends, thereby introducing a new adapter sequence into the library molecule.

[0156] Thus, the double-stranded splint adapters and methods described herein can be used to convert any linear library molecule into a covalently closed circular molecule, which can be used for different workflows, e.g., different massively parallel sequencing platforms. The double-stranded splint adapter offers flexibility because the two adjacent single-stranded regions (e.g., (320) and (330)) and the second splint strand (400) can be designed to contain any combination of universal adapter sequences. For example, the two adjacent single-stranded regions (e.g., (320) and (330)) can contain universal binding sequences (or their complementary sequences) for P5 and P7 sequences that bind to surface primers immobilized on a support (e.g., a flow cell), which are typically used to construct library molecules for Illumina sequencing platforms. The second splint strand (400) can include at least one new universal adapter sequence (e.g., a new surface primer sequence) not found in Illumina sequencing platforms, thereby enabling the use of the covalently closed circular molecule (600) in non-Illumina sequencing platforms.

[0157] The methods described herein also offer the advantage of introducing new adapter sequences using a ligation reaction rather than a gap-filling reaction, which results in highly efficient circularization with as few as 0.25 pmol of library molecules.

[0158] 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.

[0159] Two-strand splint adapter The present disclosure provides a nucleic acid double-stranded splint adapter (200) comprising (i) a first splint strand (long splint strand (300)) hybridized to (ii) a second splint strand (short splint strand (400)) (see, e.g., Figures 1-7). The first splint strand comprises a first region (320), an internal region (310), and a second region (330). The internal region of the first splint strand (310) hybridizes to the second splint strand (400) to form a double-stranded splint adapter (200) having a double-stranded region and two adjacent single-stranded regions. The two adjacent single-stranded regions of the double-stranded splint adapter (200) are designed to hybridize to end sequences of a linear nucleic acid library molecule. The terminal sequences of the linear nucleic acid library molecule each comprise a first and a second universal adapter sequence, respectively. In some embodiments, the first and second universal adapter sequences of the linear library molecule each comprise a binding sequence for a first and a second capture primer immobilized on a support.

[0160] The first region of the first splint strand (320) comprises a first universal adapter sequence capable of hybridizing to a first universal binding sequence at one end of a linear nucleic acid library molecule (see, e.g., Figures 1-7). The second region of the first splint strand (330) comprises a second universal adapter sequence capable of hybridizing to a second universal binding sequence at the other end of the linear nucleic acid library molecule (see, e.g., Figures 1-7). In some embodiments, the first region of the first splint strand (320) 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. In some embodiments, the second region of the first splint strand (330) 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 5' end of the first splint strand (300) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the first splint strand (300) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0161] In some embodiments, the second splint strand (400) comprises at least two subregions, including a first and a second subregion (see, e.g., Figures 2 and 3). In some embodiments, the first subregion comprises a universal binding sequence for the third or fourth surface primer, and the second subregion comprises a universal binding sequence for the fourth or third surface primer, and the first and second subregions do not hybridize to (or at least show very little hybridization to)) the first and second surface primers. In some embodiments, the first subregion comprises a universal binding sequence for the third surface primer, and the second subregion comprises a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases (see, e.g., FIG. 3 ). In some embodiments, the second splint strand (400) comprises only one subregion, lacking the second and third subregions, and the first subregion comprises a sample index sequence having 5-20 bases. In some embodiments, the sample index sequence can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. In some embodiments, the unique identification sequence comprises a random sequence. The unique identification sequence can be designed to exhibit reduced hybridization to, or no hybridization to, the first, second, third, and fourth surface primers. An exemplary arrangement of the subregions in the second splint strand (400) in the 5' to 3' orientation includes: 5'-[second subregion]-[first subregion]-3'.Another exemplary arrangement of subregions in the second splint strand (400) in the 5' to 3' direction includes 5'-[third subregion]-[second subregion]-[first subregion]-3' (see, e.g., Figures 2 and 3). In some embodiments, the second splint strand (400) can be 20 to 100 nucleotides in length, or 30 to 80 nucleotides in length, or 40 to 60 nucleotides in length. In some embodiments, the 5' end of the second splint strand (400) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the second splint strand (400) includes a terminal 3' OH group or a terminal 3' blocking group. In some embodiments, the second splint strand (400) includes one or more phosphorothioate linkages at the 5' and / or 3' end to confer exonuclease resistance. In some embodiments, the second splint strand (400) contains one or more phosphorothioate linkages at internal positions to confer endonuclease resistance, hi some embodiments, the second splint strand (400) contains one or more 2'-O-methylcytosine bases at the 5' and / or 3' end or at internal positions.

[0162] In some embodiments, the first splint strand (300) comprises an internal region (310) comprising at least two subregions, including a fourth and fifth subregion (see, e.g., Figures 2 and 3). The fourth subregion hybridizes to the first subregion of the second splint strand (400). The fifth subregion hybridizes to the second subregion of the second splint strand (400). The fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the internal region (310) of the first splint strand further comprises an optional sixth subregion that hybridizes to the third subregion of the second splint strand (400) (see, e.g., Figure 3). An exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes 5'-[fourth subregion]-[fifth subregion]-3'. Another exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes 5'-[fourth subregion]-[fifth subregion]-[sixth subregion]-3' (see, e.g., Figures 2 and 3). In some embodiments, the first splint strand (300) can be 50 to 150 nucleotides in length, or 60 to 100 nucleotides in length, or 70 to 90 nucleotides in length. In some embodiments, the first splint strand (300) includes one or more phosphorothioate linkages at the 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the first splint strand (300) contains one or more phosphorothioate linkages at internal positions to confer endonuclease resistance, hi some embodiments, the first splint strand (300) contains one or more 2'-O-methylcytosine bases at the 5' and / or 3' end or at internal positions.

[0163] In some embodiments, the first subregion of the second splint strand (400) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200). In some embodiments, the second subregion of the second splint strand (400) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201). In some embodiments, the second splint strand (400) comprises first and second subregions comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATCCATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 202). See Figure 10. In some embodiments, the 5' end of the second splint strand (400) can be phosphorylated or non-phosphorylated.

[0164] In some embodiments, the first region of the first splint strand (320) comprises a first universal adapter sequence that includes a universal binding sequence for a first surface primer (or its complementary sequence), and the first region (320) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 193). For example, the first region of the first splint strand (320) can hybridize to a P5 surface primer or the complementary sequence of a P5 surface primer. For example, a P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203; short P5), or a P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGAGATC-3' (SEQ ID NO: 194; long P5). In some embodiments, the second region of the first splint strand (330) comprises a second universal adapter sequence that includes a universal binding sequence for a second surface primer (or its complementary sequence), and the second region (330) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195). For example, the second region of the first splint strand (330) can hybridize to a P7 surface primer or the complementary sequence of a P7 surface primer. For example, the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195; short P7), or the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 196; long P7). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fourth subregion having the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fifth subregion having the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 198).In some embodiments, the first splint strand (300) comprises a first region (320), an internal region (310) having fourth and fifth subregions, and a second region (330) having the sequence 5'-TCGGTGGTCGCCGTATCATTACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 199). See Figure 10. In some embodiments, the 5' end of the first splint strand (300) can be phosphorylated or unphosphorylated. In some embodiments, the first subregion of the second splint strand (400) can hybridize to the fourth subregion of the first splint strand (300). In some embodiments, the second subregion of the second splint strand (400) can hybridize to the fifth subregion of the first splint strand (300).

[0165] Library-Sprint Complex The present disclosure provides a nucleic acid library molecule (100) 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 double-stranded splint adaptor (200) comprising a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)), wherein the first splint strand and a double-stranded splint adapter (200) wherein the splint strand comprises a first region (320), an internal region (310), and a second region (330), wherein the internal region of the first splint strand (310) hybridizes to the second splint strand (400) to form a double-stranded splint adapter (200) having a double-stranded region and two adjacent single-stranded regions. In the library-sprint complex (500), a first region of the first splint strand (320) hybridizes to at least the first left universal adaptor sequence (120) of the library molecule, and a second region of the first splint strand (330) hybridizes to at least the first right universal sequence (130) of the library molecule, thereby circularizing the library molecule to generate the library-sprint complex (500) (see, for example, Figures 1-7).

[0166] In the library-splint complex (500), the first region of the first splint strand (320) 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 1-7). In some embodiments, the first region of the first splint strand (320) 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 first splint strand (300) can be 50-150 nucleotides in length, or 60-100 nucleotides in length, or 70-90 nucleotides in length. In some embodiments, the first splint strand (300) comprises one or more phosphorothioate linkages at the 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the first splint strand (300) comprises one or more phosphorothioate linkages at an internal position to confer endonuclease resistance. In some embodiments, the first splint strand (300) 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 first splint strand (300) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the first splint strand (300) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0167] The second region of the first splint strand (330) comprises a second universal adapter sequence capable of hybridizing to a second universal binding sequence at the other end of the linear nucleic acid library molecule (see, e.g., Figures 1-7). In some embodiments, the second region of the first splint strand (330) 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 5' end of the second splint strand (400) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the second splint strand (400) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0168] In the library-sprint complex (500), a first region of the first splint strand (320) hybridizes to at least the first left universal adaptor sequence (120) of the library molecule, and a second region of the first splint strand (330) hybridizes to at least the first right universal sequence (130) of the library molecule, thereby circularizing the library molecule to generate the library-sprint complex (500). The library-sprint complex (500) includes a first nick between the 5' end of the library molecule and the 3' end of the second splint strand. The library-sprint complex (500) also includes a second nick between the 5' end of the second splint strand and the 3' end of the library molecule (see, e.g., Figures 1-7). In some embodiments, the first and second nicks are enzymatically ligatable.

[0169] In the library-sprint complex (500), a first region of the first splint strand (320) can hybridize to either the sense or antisense strand of a double-stranded nucleic acid library molecule. In the library-sprint complex (500), a second region of the first splint strand (330) can hybridize to either 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.

[0170] In the library-sprint complex (500), the second splint strand (400) does not hybridize to the sequence of interest (110), and the internal region of the first splint strand (310) does not hybridize to the sequence of interest (110).

[0171] In the library-sprint complex (500), a first region of the first splint strand (320) does not hybridize to the target sequence (110), and a second region of the first splint strand (330) does not hybridize to the target sequence (110).

[0172] In some embodiments, the 5' ends of the single-stranded library molecules (100) in the library-sprint complex (500) 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.

[0173] 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). Exemplary library molecules (100) are shown in Figures 4-7. In some embodiments, the nucleic acid library molecule (100) can further comprise additional left and / or right universal adaptor sequences.

[0174] 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). 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. In some embodiments, the nucleic acid library molecule (100) comprises a first left index sequence (160) and / or a first right index sequence (170). Sample index sequences can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. Exemplary library molecules (100) are shown in Figures 4-7. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Table 1 in Figures 23A-23F. The first left index array (160) may include a random sequence (e.g., NNN) or may lack a random sequence. The first right index array (170) may include a random sequence (e.g., NNN) or may lack a random sequence.

[0175] 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. 7). 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). The second right junction adaptor sequence (175) can be located between the first right index sequence (170) and the second right universal adaptor sequence (150). The third right junction adaptor sequence (155) can be located between the second right universal adaptor sequence (150) and the sequence of interest (110). In some embodiments, the nucleic acid library molecule (100) further comprises at least one to up to ten added universal adaptor sequences located 5' (upstream) relative to the first left universal adaptor sequence (120) (see, e.g., FIG. 7). In some embodiments, the nucleic acid library molecule (100) further comprises at least one to up to ten added universal adaptor sequences located 3' (downstream) relative to the first right universal sequence (130) (see, e.g., FIG. 7). Any of the junction adaptor sequences can be any sequence and can be 3 to 60 nucleotides in length and / or added universal adaptor sequences. Either the junction adapter sequence and / or the added universal adapter sequence comprises a universal sequence or a unique sequence.Any of the junction adapter sequences and / or the added universal adapter sequences include a binding sequence for an amplification primer, a sequencing primer, or a compaction oligonucleotide. Any of the junction adapter sequences and / or the added universal adapter sequences include a binding sequence for an immobilized surface primer (e.g., a capture primer). Any of the junction adapter sequences and / or the added universal adapter sequences include a sample index sequence. Any of the junction adapter sequences and / or the added universal adapter sequences include a unique identification sequence. Any of the junction adapter sequences and / or the added universal adapter sequences, particularly junction adapter sequence (145), include the Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 211). Any of the junction adapter sequences and / or the added universal adapter sequences, particularly junction adapter sequence (155), include the Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 212). The Tn5 transposon end sequence can be introduced into the library molecule (100) via a transposase-mediated reaction, which involves contacting double-stranded input DNA (e.g., genomic DNA) with a Tn5-type transposase enzyme and a double-stranded oligonucleotide comprising a Tn transposon end sequence (SEQ ID NO: 211) 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: 211) can be located 5' or 3' to the universal adapter sequence or sample index sequence.

[0176] Multiplex workflows are enabled by preparing sample-indexed libraries using one or both index sequences (e.g., left and / or right index sequences). 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 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.

[0177] 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 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. Exemplary library molecules (100) are shown in Figures 4-7.

[0178] 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). Exemplary library molecules (100) are shown in Figures 4-7.

[0179] 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.

[0180] 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.

[0181] In some embodiments, the second splint strand (400) comprises at least two subregions, including a first and a second subregion (see, e.g., Figures 2 and 3). The first subregion comprises a universal binding sequence for the third surface primer, and the second subregion comprises a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to (or at least exhibit very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases (see, e.g., Figures 2 and 3). In some embodiments, the second splint strand (400) comprises only one subregion, lacking the second and third subregions, and the first subregion comprises a sample index sequence having 5-20 bases. In some embodiments, the sample index sequence can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. In some embodiments, the unique identification sequence comprises a random sequence. The unique identification sequence can be designed to exhibit reduced or no hybridization to the first, second, third, and fourth surface primers. An exemplary arrangement of subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[second subregion]-[first subregion]-3'. Another exemplary arrangement of subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[third subregion]-[second subregion]-[first subregion]-3'. In some embodiments, the second splint strand (400) can be 20 to 100 nucleotides in length, or 30 to 80 nucleotides in length, or 40 to 60 nucleotides in length. In some embodiments, the second splint strand (400) contains one or more phosphorothioate linkages at the 5' and / or 3' ends to confer exonuclease resistance.In some embodiments, the second splint strand (400) comprises one or more phosphorothioate linkages at an internal position to confer endonuclease resistance. In some embodiments, the second splint strand (400) 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 second splint strand (400) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the second splint strand (400) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0182] In some embodiments, the first splint strand (300) comprises an internal region (310) comprising at least two subregions, including a fourth and fifth subregion (see, e.g., Figures 2 and 3). The fourth subregion hybridizes to the first subregion of the second splint strand (400). The fifth subregion hybridizes to the second subregion of the second splint strand (400). The fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the internal region (310) of the first splint strand further comprises an optional sixth subregion that hybridizes to the third subregion of the second splint strand (400) (see, e.g., Figures 2 and 3). An exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes: 5'-[fourth subregion]-[fifth subregion]-3'. Another exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes: 5'-[fourth subregion]-[fifth subregion]-[sixth subregion]-3'.

[0183] In some embodiments, an exemplary library-sprint complex (500) includes (a) a single-stranded nucleic acid library molecule (100), (b) a first splint strand (300), and (c) a second splint strand (400).

[0184] In the exemplary library-sprint complex (500), 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 first surface primer (120), (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 (130).

[0185] In the exemplary library-sprint complex (500), the first splint strand (300) comprises components arranged in 5' to 3' order: a first region (320), an internal region (310), and a second region (330).

[0186] In the exemplary library-splint complex (500), the second splint strand (400) includes subregions arranged in 3' to 5' order: a first subregion having a universal binding sequence for a third surface primer, and a second subregion having a universal binding sequence for a fourth surface primer.

[0187] In the exemplary library-sprint complex (500), a portion of the first splint strand (300) hybridizes to a portion of the library molecule (100), thereby circularizing the library molecule to generate the library-sprint complex (500), whereby a first region (320) of the first splint strand hybridizes to the binding sequence for the first surface primer (120) and a third region (330) of the first splint strand hybridizes to the binding sequence for the second surface primer (130). Additionally, a second splint strand (400) hybridizes to an internal region (310) of the first splint strand (300). The library-sprint complex (500) comprises a first nick between the 5' end of the library molecule and the 3' end of the second splint strand, and a second nick between the 5' end of the second splint strand and the 3' end of the library molecule, wherein the first and second nicks are enzymatically ligatable.

[0188] In the exemplary library-sprint complex (500), the second splint strand (400) does not hybridize to the sequence of interest (110), and the internal region of the first splint strand (310) does not hybridize to the sequence of interest (110).

[0189] In the exemplary library-sprint complex (500), a first region of the first splint strand (320) does not hybridize to the sequence of interest (110), and a second region of the first splint strand (330) does not hybridize to the sequence of interest (110).

[0190] In some embodiments, any of the library-sprint complexes (500) described herein comprises a plurality of library-sprint complexes (500), 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.

[0191] In some embodiments, in any of the library-splint complexes (500) described herein, the first subregion of the second splint strand (400) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200). In some embodiments, the second subregion of the second splint strand (400) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201). In some embodiments, the second splint strand (400) comprises first and second subregions comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATCCATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 202). See Figure 10. In some embodiments, the 5' end of the second splint strand (400) can be phosphorylated or unphosphorylated. In some embodiments, the second splint strand (400) comprises only one subregion, lacking the second and third subregions, and the first subregion comprises a sample index sequence having between 5 and 20 bases.

[0192] In some embodiments, in any of the library-splint complexes (500) described herein, the first region of the first splint strand (320) comprises a first universal adapter sequence that includes a universal binding sequence for a first surface primer (or its complementary sequence), and the first region (320) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 193). For example, the first region of the first splint strand (320) can hybridize to a P5 surface primer or the complementary sequence of a P5 surface primer. For example, the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203; short P5), or the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGAGATC-3' (SEQ ID NO: 194; long P5). In some embodiments, the second region of the first splint strand (330) comprises a second universal adapter sequence that includes a universal binding sequence for a second surface primer (or its complementary sequence), and the second region (330) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195). For example, the second region of the first splint strand (330) can hybridize to a P7 surface primer or the complementary sequence of a P7 surface primer. For example, the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195; short P7), or the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 196; long P7). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fourth subregion having the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fifth subregion having the sequence 5'-GATCAGGTGAGGCTGCGACGACT'3' (SEQ ID NO: 198).In some embodiments, the first splint strand (300) comprises a first region (320), an internal region (310) having fourth and fifth subregions, and a second region (330) having the sequence 5'-TCGGTGGTCGCCGTATCATTACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 199). See Figure 10. In some embodiments, the 5' end of the first splint strand (300) can be phosphorylated or unphosphorylated. In some embodiments, the first subregion of the second splint strand (400) can hybridize to the fourth subregion of the first splint strand (300). In some embodiments, the second subregion of the second splint strand (400) can hybridize to the fifth subregion of the first splint strand (300).

[0193] In some embodiments, in any of the library-sprint complexes (500) described herein, the first region of the first splint strand (320) comprises a sequence capable of binding to the first left universal adapter sequence (120) of the library molecule, and the first region of the first splint strand (320) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197) or a complementary sequence thereof.

[0194] In some embodiments, in any of the library-sprint complexes (500) described herein, the second region of the first splint strand (330) comprises a sequence capable of binding to the first right universal adapter sequence (130) of the library molecule, and the second region of the first splint strand (330) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (sequence number 198) or a complementary sequence thereof.

[0195] In some embodiments, in any of the library-splint complexes (500) described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first splint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (sequence number 203).

[0196] In some embodiments, in any of the library-splint complexes (500) described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first splint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200) or a complementary sequence thereof.

[0197] In some embodiments, in any of the library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 204).

[0198] In some embodiments, in any of the library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 207).

[0199] In some embodiments, in any of the library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 208).

[0200] In some embodiments, in any of the library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 205).

[0201] In some embodiments, in any of the library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 209).

[0202] In some embodiments, in any of the library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 210).

[0203] In some embodiments, in any of the library-sprint complexes (500) described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first splint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (sequence number 206).

[0204] In some embodiments, in any of the library-splint complexes (500) described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first splint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (sequence number 201) or a complementary sequence thereof.

[0205] The present disclosure provides a reaction mixture comprising a plurality of any of the library-sprint complexes (500) described herein. In some embodiments, the reaction mixture comprises a plurality of any of the library-sprint complexes (500) described herein and T4 polynucleotide kinase. In some embodiments, the reaction mixture comprises a plurality of any of the library-sprint complexes (500) described herein and a ligase enzyme. In some embodiments, the reaction mixture comprises a plurality of any of the library-sprint complexes (500) 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.

[0206] Covalent closed ring molecule The present disclosure provides a covalently closed circular library molecule (600) comprising a sequence of interest (110), at least a first left universal adaptor sequence (120), at least a first right universal adaptor sequence (130), and a second splint strand sequence (400). An exemplary covalently closed circular library molecule is shown in Figure 8. In some embodiments, the covalently closed circular library molecule (600) further comprises a second left universal adaptor sequence (140). In some embodiments, the covalently closed circular molecule (600) further comprises a second right universal adaptor sequence (150). In some embodiments, the covalently closed circular molecule (600) further comprises additional left and / or right universal adaptor sequences.

[0207] In some embodiments, the covalently closed circular molecule (600) further comprises a first left-indexing sequence (160) and / or a first right-indexing sequence (170). Sample indexing sequences can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. A list of exemplary first left-indexing sequences (160) and first right-indexing sequences (170) is provided in Table 1 in Figures 23A-23F. The first left-indexing sequence (160) may include a random sequence (e.g., NNN) or may lack a random sequence. The first right-indexing sequence (170) may include a random sequence (e.g., NNN) or may lack a random sequence.

[0208] Multiplex workflows are enabled by preparing sample-indexed libraries using one or both index sequences (e.g., left and / or right index sequences). 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 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.

[0209] In some embodiments, the covalently closed circular molecule (600) further comprises an optional first left unique identification sequence (180) and / or an optional first right unique identification sequence (190). While Figure 8 shows an exemplary covalently closed circular molecule (600) having a first left unique identification sequence (180), one skilled in the art will recognize that a linear library molecule (100), such as that shown in Figure 6, can be circularized using a double-stranded splint adaptor (200) to generate a covalently closed circular molecule (600) having a 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. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) may be used for molecular tagging.

[0210] In some embodiments, the covalently closed circular molecule (600) 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). 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 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) may be used for molecular tagging.

[0211] In some embodiments, in the covalently closed circular molecule (600), 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 (600) can further comprise an additional left universal adapter sequence.

[0212] In some embodiments, in the covalently closed circular molecule (600), 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 (600) can further comprise an additional right universal adapter sequence.

[0213] In some embodiments, the covalently closed circular molecule (600) further comprises at least one junction adaptor sequence located between any of the universal adaptor sequences described herein (see, e.g., FIG. 7). 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). The second right junction adaptor sequence (175) can be located between the first right index sequence (170) and the second right universal adaptor sequence (150). The third right junction adaptor sequence (155) can be located between the second right universal adaptor sequence (150) and the sequence of interest (110). In some embodiments, the covalently closed circular molecule (600) further comprises at least one to up to ten additional universal adaptor sequences located 5' (upstream) relative to the first left universal adaptor sequence (120) (see, e.g., Figure 7). In some embodiments, the covalently closed circular molecule (600) further comprises at least one to up to ten additional universal adaptor sequences located 3' (downstream) relative to the first right universal sequence (130) (see, e.g., Figure 7). Any of the junction adaptor sequences and / or the additional universal adaptor sequences can comprise any sequence and be 3 to 60 nucleotides in length. Either the junction adapter sequence and / or the added universal adapter sequence comprises a universal sequence or a unique sequence.Any of the junction adapter sequences and / or the added universal adapter sequences include a binding sequence for an amplification primer, a sequencing primer, or a compaction oligonucleotide. Any of the junction adapter sequences and / or the added universal adapter sequences include a binding sequence for an immobilized surface primer (e.g., a capture primer). Any of the junction adapter sequences and / or the added universal adapter sequences include a sample index sequence. Any of the junction adapter sequences and / or the added universal adapter sequences include a unique identification sequence. Any of the junction adapter sequences and / or the added universal adapter sequences, particularly junction adapter sequence (145), include the Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 211). Any of the junction adapter sequences and / or the added universal adapter sequences, particularly junction adapter sequence (155), include the Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 212). The Tn5 transposon end sequence can be introduced into the library molecule (100) via a transposase-mediated reaction, which involves contacting double-stranded input DNA (e.g., genomic DNA) with a Tn5-type transposase enzyme and a double-stranded oligonucleotide comprising a Tn transposon end sequence (SEQ ID NO: 211) 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: 211) can be located 5' or 3' to the universal adapter sequence or sample index sequence.

[0214] In some embodiments, the second splint strand sequence (400) of the covalently closed circular molecule comprises at least two subregions, including a first and a second subregion. The first subregion comprises a universal binding sequence for the third surface primer, and the second subregion comprises a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to (or at least exhibit very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases. In some embodiments, the second splint strand (400) comprises only one subregion, lacking the second and third subregions, and the first subregion comprises a sample index sequence having 5-20 bases. In some embodiments, the sample index sequence can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. In some embodiments, the unique identification sequence comprises a random sequence. The unique identification sequence can be designed to exhibit reduced or no hybridization to the first, second, third, and fourth surface primers. An exemplary arrangement of subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[second subregion]-[first subregion]-3'. Another exemplary arrangement of subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[third subregion]-[second subregion]-[first subregion]-3'.

[0215] In some embodiments, the second splint strand sequence (400) of the covalently closed circular library molecule (600) can hybridize to the first splint strand (300). In some embodiments, the first splint strand (300) comprises an internal region (310) comprising at least two subregions, including a fourth and fifth subregion. The fourth subregion hybridizes to the first subregion of the second splint strand (400). The fifth subregion hybridizes to the second subregion of the second splint strand (400). The fourth and fifth subregions do not hybridize to (or at least exhibit very little hybridization to) the first and second surface primers. In some embodiments, the internal region (310) of the first splint strand further comprises an optional sixth subregion that hybridizes to the third subregion of the second splint strand (400). An exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes: 5'-[fourth subregion]-[fifth subregion]-3'. Another exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes: 5'-[fourth subregion]-[fifth subregion]-[sixth subregion]-3'.

[0216] In some embodiments, an exemplary covalently closed circular molecule (600) comprises: (i) a first left universal adapter sequence (120) having a binding sequence for a first surface primer (120); (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 for a second surface primer (130); and (vi) a second splint strand sequence (400), wherein the covalently closed circular molecule (600) is optionally hybridized to the first splint strand (300).

[0217] In the exemplary covalently closed circular molecule (600), the second splint strand region (400) comprises at least two subregions, including a first and a second subregion. The first subregion comprises a universal binding sequence for the third surface primer, and the second subregion comprises a universal binding sequence for the fourth surface primer, with the first and second subregions not hybridizing to (or at least exhibiting very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases. In some embodiments, the second splint strand (400) comprises only one subregion, lacking the second and third subregions, and the first subregion comprises a sample index sequence having 5-20 bases. In some embodiments, the sample index sequence can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. In some embodiments, the unique identification sequence comprises a random sequence. The unique identification sequence can be designed to exhibit reduced or no hybridization to the first, second, third, and fourth surface primers. An exemplary arrangement of subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[second subregion]-[first subregion]-3'. Another exemplary arrangement of subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[third subregion]-[second subregion]-[first subregion]-3'.

[0218] In some embodiments, any of the covalently closed circular molecules (600) described herein further comprises a plurality of covalently closed circular molecules (600), wherein the target sequences (110) of the individual covalently closed circular molecules (600) in the plurality of covalently closed circular molecules (600) comprise the same target sequence or different target sequences.

[0219] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the first subregion of the second splint strand (400) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200). In some embodiments, the second subregion of the second splint strand (400) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201). In some embodiments, the second splint strand (400) comprises first and second subregions comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATCCATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 202). See Figure 10. In some embodiments, the 5' end of the second splint strand (400) can be phosphorylated or non-phosphorylated.

[0220] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the first region of the first splint strand (320) comprises a first universal adapter sequence comprising a universal binding sequence for a first surface primer (or its complementary sequence), wherein the first region (320) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 193). For example, the first region of the first splint strand (320) can hybridize to a P5 surface primer or the complementary sequence of a P5 surface primer. For example, the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203; short P5), or the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGAGATC-3' (SEQ ID NO: 194; long P5). In some embodiments, the second region of the first splint strand (330) comprises a second universal adapter sequence that includes a universal binding sequence for a second surface primer (or its complementary sequence), and the second region (330) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195). For example, the second region of the first splint strand (330) can hybridize to a P7 surface primer or the complementary sequence of a P7 surface primer. For example, the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195; short P7), or the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 196; long P7). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fourth subregion having the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fifth subregion having the sequence 5'-GATCAGGTGAGGCTGCGACGACT'3' (SEQ ID NO: 198).In some embodiments, the first splint strand (300) comprises a first region (320), an internal region (310) having fourth and fifth subregions, and a second region (330) having the sequence 5'-TCGGTGGTCGCCGTATCATTACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 199). See Figure 10. In some embodiments, the 5' end of the first splint strand (300) can be phosphorylated or unphosphorylated. In some embodiments, the first subregion of the second splint strand (400) can hybridize to the fourth subregion of the first splint strand (300). In some embodiments, the second subregion of the second splint strand (400) can hybridize to the fifth subregion of the first splint strand (300).

[0221] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the first region of the first splint strand (320) comprises a sequence capable of binding to the first left universal adapter sequence (120) of the library molecule, and the first region of the first splint strand (320) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197) or a complementary sequence thereof.

[0222] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the second region of the first splint strand (330) comprises a sequence capable of binding to the first right universal adapter sequence (130) of the library molecule, and the second region of the first splint strand (330) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (sequence number 198) or a complementary sequence thereof.

[0223] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (120) comprising the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203).

[0224] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first splint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200) or a complementary sequence thereof.

[0225] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 204).

[0226] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 207).

[0227] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 208).

[0228] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 205).

[0229] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 209).

[0230] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 210).

[0231] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a right universal binding sequence (130) comprising the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 206).

[0232] In some embodiments, in any of the covalently closed circular molecules (600) described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first splint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201) or a complementary sequence thereof.

[0233] The present disclosure provides a reaction mixture comprising a plurality of any of the covalently closed circular molecules (600) 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.

[0234] Kit containing a double-stranded splint adapter The present disclosure provides a kit for use in introducing one or more new adapter sequences into linear nucleic acid library molecules. In some embodiments, the kit can be used to circularize single-stranded nucleic acid library molecules having a sequence of interest (110) flanked on one side by at least a first left universal adapter sequence (120) and on the other side by at least a first right universal adapter sequence (130). 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 for massively parallel sequencing.

[0235] The present disclosure provides a kit including a nucleic acid double-stranded splint adapter (200), wherein the nucleic acid double-stranded splint adapter (200) includes (i) a first splint strand (long splint strand (300)), which is hybridized to (ii) a second splint strand (short splint strand (400)). The first splint strand includes a first region (320), an internal region (310), and a second region (330). The internal region of the first splint strand (310) hybridizes to the second splint strand (400) to form a double-stranded splint adapter (200) having a double-stranded region and two adjacent single-stranded regions. The second splint strand (400) includes a new adapter sequence that can be introduced into a linear nucleic acid library molecule. Exemplary double-stranded splint adapters are shown in Figures 1-7. The kit can include a container containing a first splint strand (300) hybridized to a second splint strand (400). The kit can include a first container containing the first splint strand (300) and a second container containing the second splint strand (400).

[0236] In some embodiments, in the kit, the second splint strand (400) comprises at least two subregions, including a first and a second subregion (see, e.g., Figures 2 and 3). The first subregion comprises a universal binding sequence for the third surface primer, and the second subregion comprises a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to (or at least exhibit very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases (see, e.g., Figure 3). In some embodiments, the second splint strand (400) includes only one subregion and lacks the second and third subregions, and the first subregion includes a sample index sequence having 5 to 20 bases. In some embodiments, the sample index sequence can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. In some embodiments, the unique identification sequence includes a random sequence. The unique identification sequence can be designed to exhibit reduced hybridization to, or no hybridization to, the first, second, third, and fourth surface primers. An exemplary arrangement of the subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[second subregion]-[first subregion]-3'. Another exemplary arrangement of the subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[third subregion]-[second subregion]-[first subregion]-3'. Exemplary first splint strand (300) and second splint strand (400) are shown in Figures 2 and 3. In some embodiments, the second splint strand (400) can be 20-100 nucleotides in length, or 30-80 nucleotides in length, or 40-60 nucleotides in length.In some embodiments, the second splint strand (400) comprises one or more phosphorothioate linkages at the 5' and / or 3' end to confer exonuclease resistance. In some embodiments, the second splint strand (400) comprises one or more phosphorothioate linkages at an internal position to confer endonuclease resistance. In some embodiments, the second splint strand (400) 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 second splint strand (400) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the second splint strand (400) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0237] In some embodiments, in the kit, the first splint strand (300) comprises a first region (320), a second region (330), and an internal region (310). The first region (320) comprises a first universal adapter sequence capable of hybridizing to a first universal binding sequence at one end of a linear nucleic acid library molecule. The second region (330) comprises a second universal adapter 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 first splint strand (320) 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. In some embodiments, the second region of the first splint strand (330) 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 first splint strand (300) can be 50 to 150 nucleotides in length, or 60 to 100 nucleotides in length, or 70 to 90 nucleotides in length. In some embodiments, the first splint strand (300) comprises one or more phosphorothioate linkages at the 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the first splint strand (300) comprises one or more phosphorothioate linkages at internal positions to confer endonuclease resistance. In some embodiments, the first splint strand (300) comprises one or more 2'-O-methylcytosine bases at the 5' and / or 3' ends or at an internal position.In some embodiments, the 5' end of the first splint strand (300) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the first splint strand (300) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0238] In some embodiments, in the kit, the first splint strand (300) comprises an internal region (310) comprising at least two subregions, including a fourth and fifth subregion (see, e.g., Figures 2 and 3). The fourth subregion hybridizes to the first subregion of the second splint strand (400). The fifth subregion hybridizes to the second subregion of the second splint strand (400). The fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the internal region (310) of the first splint strand further comprises an optional sixth subregion that hybridizes to the third subregion of the second splint strand (400). An exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes: 5'-[fourth subregion]-[fifth subregion]-3'. Another exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes: 5'-[fourth subregion]-[fifth subregion]-[sixth subregion]-3'. An exemplary first splint strand (300) is shown in Figures 2 and 3.

[0239] In some embodiments, in any of the kits described herein, the first subregion of the second splint strand (400) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200). In some embodiments, the second subregion of the second splint strand (400) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201). In some embodiments, the second splint strand (400) comprises first and second subregions comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATCCATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 202). See Figure 10. In some embodiments, the 5' end of the second splint strand (400) can be phosphorylated or non-phosphorylated.

[0240] In some embodiments, in any of the kits described herein, the first region of the first splint strand (320) comprises a first universal adapter sequence comprising a universal binding sequence for a first surface primer (or its complementary sequence), wherein the first region (320) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 193). For example, the first region of the first splint strand (320) can hybridize to a P5 surface primer or the complementary sequence of a P5 surface primer. For example, the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203; short P5), or the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGAGATC-3' (SEQ ID NO: 194; long P5). In some embodiments, the second region of the first splint strand (330) comprises a second universal adapter sequence that includes a universal binding sequence for a second surface primer (or its complementary sequence), and the second region (330) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195). For example, the second region of the first splint strand (330) can hybridize to a P7 surface primer or the complementary sequence of a P7 surface primer. For example, the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195; short P7), or the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 196; long P7). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fourth subregion having the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fifth subregion having the sequence 5'-GATCAGGTGAGGCTGCGACGACT'3' (SEQ ID NO: 198).In some embodiments, the first splint strand (300) comprises a first region (320), an internal region (310) having fourth and fifth subregions, and a second region (330) having the sequence 5'-TCGGTGGTCGCCGTATCATTACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 199). See Figure 10. In some embodiments, the 5' end of the first splint strand (300) can be phosphorylated or unphosphorylated. In some embodiments, the first subregion of the second splint strand (400) can hybridize to the fourth subregion of the first splint strand (300). In some embodiments, the second subregion of the second splint strand (400) can hybridize to the fifth subregion of the first splint strand (300).

[0241] In some embodiments, in any of the kits described herein, the first region of the first splint strand (320) comprises a sequence capable of binding to the first left universal adapter sequence (120) of the library molecule, and the first region of the first splint strand (320) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (sequence number 197) or a complementary sequence thereof.

[0242] In some embodiments, in any of the kits described herein, the second region of the first splint strand (330) comprises a sequence capable of binding to the first right universal adapter sequence (130) of the library molecule, and the second region of the first splint strand (330) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (sequence number 198) or a complementary sequence thereof.

[0243] In some embodiments, the kit further comprises an adaptor having a left universal binding sequence (120) that binds to a first region of a first splint strand (320) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0244] In some embodiments, in any of the kits described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first splint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200) or a complementary sequence thereof.

[0245] In some embodiments, the kit further comprises an adaptor having a left universal binding sequence for a sequencing primer (140) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 204). In some embodiments, the adaptor having a left universal binding sequence for a sequencing primer (140) also comprises a sample index sequence (160). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0246] In some embodiments, the kit further comprises an adaptor having a left universal binding sequence for a sequencing primer (140) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 207). In some embodiments, the adaptor having a left universal binding sequence for a sequencing primer (140) also comprises a sample index sequence (160). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0247] In some embodiments, the kit further comprises an adaptor having a left universal binding sequence for a sequencing primer (140) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 208). In some embodiments, the adaptor having a left universal binding sequence for a sequencing primer (140) also comprises a sample index sequence (160). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0248] In some embodiments, the kit further comprises an adaptor having a right universal binding sequence (150) for a sequencing primer for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 205). In some embodiments, the adaptor having the right universal binding sequence (150) for a sequencing primer also comprises a sample index sequence (170). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0249] In some embodiments, the kit further comprises an adaptor having a right universal binding sequence (150) for a sequencing primer for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 209). In some embodiments, the adaptor having the right universal binding sequence (150) for the sequencing primer also comprises a sample index sequence (170). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0250] In some embodiments, the kit further comprises an adaptor having a right universal binding sequence (150) for a sequencing primer for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 210). In some embodiments, the adaptor having the right universal binding sequence (150) for a sequencing primer also comprises a sample index sequence (170). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0251] In some embodiments, the kit further comprises an adaptor having a right universal binding sequence (130) that binds to a first region of the first splint strand (330) for use in preparing a plurality of library molecules, the library molecules comprising the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 206). The adaptor can be a single-stranded adaptor (e.g., a PCR primer), a double-stranded adaptor, a bubble adaptor, or a Y-shaped adaptor.

[0252] In some embodiments, in any of the kits described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first splint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201) or a complementary sequence thereof.

[0253] In some embodiments, the kit further includes a plurality of first left indexes (160) and / or a plurality of first right index arrays (170). In some embodiments, the kit may include separate containers holding individual first left indexes (160) or individual first right indexes (170). In some embodiments, the kit may include separate containers holding 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) in a multiwell plate (e.g., a 96-well plate). A list of exemplary first left index arrays (160) and first right index arrays (170) is provided in Table 1 in Figures 23A-23F. The first left index array (160) may include a random sequence (e.g., NNN) or may lack a random sequence. The first right index sequence (170) may be random or may contain a sequence (eg, NNN), or may lack a random sequence.

[0254] In some embodiments, the kit includes a nucleic acid double-stranded splint adaptor (200) and further includes T4 polynucleotide kinase. In some embodiments, the kit further includes a ligase enzyme, wherein the ligase enzyme comprises 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 comprises any one or any combination of two or more of exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease.

[0255] In some embodiments, the kit includes at least one buffer for hybridizing a plurality of double-stranded splint adaptors (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 first and / or second splint strands (e.g., (300) and / or (400)), (ii) ligating nicks in the library-splint complex (500), and / or (iii) exonuclease digestion of the first splint strand (300) from the covalently closed circular molecule (600). 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.

[0256] In some embodiments, the kit includes one or more containers containing any of the double-stranded splint adapters (200) described herein, or any of the first and second splint strands (300) and (400) described herein. The kit can further include one or more containers containing T4 polynucleotide kinase, at least one ligase, and / or at least one exonuclease. The kit can include any of these components in any combination, and may be contained in a single container, or in separate containers, or any combination thereof.

[0257] The kit can include instructions for using the kit to perform the reaction to introduce one or more new adapter sequences into the linear nucleic acid library molecule.

[0258] Methods for forming multiple library-splint complexes The present disclosure provides a method for forming a plurality of library-sprint complexes (500), the method comprising: (a) providing a plurality of double-stranded splint adapters (200), each double-stranded splint adapter (200) in the plurality of double-stranded splint adapters (200) comprising a first splint strand (300) hybridized to a second splint strand (400), the double-stranded splint adapter comprising a double-stranded region and two adjacent single-stranded regions, the first splint strand comprising a first region (320), an internal region (310), and a second region (330), the internal region of the first splint strand (310) hybridizing to the second splint strand (400). Exemplary double-stranded splint adapters (200) are shown in Figures 1-7.

[0259] The method for forming a plurality of library-sprint complexes (500) further includes step (b): hybridizing a plurality of double-stranded splint adapters to a plurality of single-stranded nucleic acid library molecules (100), each library molecule comprising a sequence of interest (110) flanked on one side by at least a first left universal adapter sequence (120) and on the other side by at least a first right universal adapter sequence (130) (e.g., Figures 1-7). The hybridizing is performed under conditions suitable for hybridizing a first region of the first splint strand (320) to at least the first left universal adapter sequence (120) of the library molecule, and conditions suitable for hybridizing a second region of the first splint strand (330) to at least the first right universal sequence (130) of the library molecule, thereby circularizing the plurality of library molecules to form a plurality of library-sprint complexes (500).

[0260] In some embodiments, in a method for forming a plurality of library-splint complexes (500), a first region of the first splint strand (320) 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. In some embodiments, the first region of the first splint strand (320) 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 5' end of the first splint strand (300) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the first splint strand (300) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0261] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the second region of the first splint strand (330) comprises a second universal adaptor sequence capable of hybridizing to a second universal binding sequence at the other end of a linear nucleic acid library molecule. In some embodiments, the second region of the first splint strand (330) 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. In some embodiments, the 5' end of the second splint strand (400) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the second splint strand (400) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0262] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first region of the first splint strand (320) hybridizes to at least a first left universal adaptor sequence (120) of the library molecule, and a second region of the first splint strand (330) hybridizes to at least a first right universal sequence (130) of the library molecule, thereby circularizing the library molecule to generate a library-sprint complex (500). The library-sprint complex (500) comprises a first nick between the 5' end of the library molecule and the 3' end of the second splint strand (e.g., Figures 1-7). The library-sprint complex (500) also comprises a second nick between the 5' end of the second splint strand and the 3' end of the library molecule (e.g., Figures 1-7). In some embodiments, the first and second nicks are enzymatically ligatable.

[0263] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first region of the first splint strand (320) can hybridize to either the sense or antisense strand of a double-stranded nucleic acid library molecule. In the library-sprint complexes (500), a second region of the first splint strand (330) can hybridize to either 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.

[0264] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the second splint strand (400) does not hybridize to the sequence of interest (110), and the internal region of the first splint strand (310) does not hybridize to the sequence of interest (110).

[0265] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first region of the first splint strand (320) does not hybridize to the sequence of interest (110), and a second region of the first splint strand (330) does not hybridize to the sequence of interest (110).

[0266] In some embodiments, in the method for forming a plurality of library-splint complexes (500), the 5' ends of the single-stranded library molecules (100) 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.

[0267] In some embodiments, in the method for forming a plurality of library-splint complexes (500), 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.

[0268] In some embodiments, in a method for forming a plurality of library-splint complexes (500), 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). 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 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 23A-23F. The first left index sequence (160) may comprise a random sequence (e.g., NNN) or may lack a random sequence. The first right index sequence (170) may include a random sequence (eg, NNN) or may lack a random sequence.

[0269] Multiplex workflows are enabled by preparing sample-indexed libraries using one or both index sequences (e.g., left and / or right index sequences). 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 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.

[0270] In some embodiments, in the method for forming a plurality of library-splint complexes (500), the nucleic acid library molecule (100) further comprises a first left unique identification sequence (180). In some embodiments, the nucleic acid library molecule (100) further comprises a first right unique identification sequence (190). 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.

[0271] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the nucleic acid library molecules (100) comprise 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).

[0272] In some embodiments, in the method for forming a plurality of library-splint complexes (500), 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 an additional left universal adapter sequence.

[0273] In some embodiments, in the method for forming a plurality of library-splint complexes (500), 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.

[0274] In some embodiments, in a method for forming a plurality of library-splint complexes (500), 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. 7). 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). The second right junction adaptor sequence (175) can be located between the first right index sequence (170) and the second right universal adaptor sequence (150). The third right junction adaptor sequence (155) can be located between the second right universal adaptor sequence (150) and the sequence of interest (110). In some embodiments, the nucleic acid library molecule (100) further comprises at least one to up to ten added universal adaptor sequences located 5' (upstream) relative to the first left universal adaptor sequence (120) (see, e.g., Figure 7). In some embodiments, the nucleic acid library molecule (100) further comprises at least one to up to ten added universal adaptor sequences located 3' (downstream) relative to the first right universal sequence (130) (see, e.g., Figure 7). Any of the junction adaptor sequences and / or added universal adaptor sequences can comprise any sequence and can be 3 to 60 nucleotides in length. Either the junction adapter sequence and / or the added universal adapter sequence comprises a universal sequence or a unique sequence.Any of the junction adapter sequences and / or the added universal adapter sequences include a binding sequence for an amplification primer, a sequencing primer, or a compaction oligonucleotide. Any of the junction adapter sequences and / or the added universal adapter sequences include a binding sequence for an immobilized surface primer (e.g., a capture primer). Any of the junction adapter sequences and / or the added universal adapter sequences include a sample index sequence. Any of the junction adapter sequences and / or the added universal adapter sequences include a unique identification sequence. Any of the junction adapter sequences and / or the added universal adapter sequences, particularly junction adapter sequence (145), include the Tn5 transposon end sequence 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 211). Any of the junction adapter sequences and / or the added universal adapter sequences, particularly junction adapter sequence (155), include the Tn5 transposon end sequence 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 212). The Tn5 transposon end sequence can be introduced into the library molecule (100) via a transposase-mediated reaction, which involves contacting double-stranded input DNA (e.g., genomic DNA) with a Tn5-type transposase enzyme and a double-stranded oligonucleotide comprising a Tn transposon end sequence (SEQ ID NO: 211) 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: 211) can be located 5' or 3' to the universal adapter sequence or sample index sequence.

[0275] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the second splint strand (400) comprises at least two subregions, including a first and a second subregion (see, e.g., Figures 2 and 3). The first subregion comprises a universal binding sequence for a third surface primer, and the second subregion comprises a universal binding sequence for a fourth surface primer, and the first and second subregions do not hybridize to (or at least exhibit very little hybridization to) the first and second surface primers. In some embodiments, the second splint strand (400) further comprises an optional third subregion, which comprises a sample index sequence having 5-20 bases and / or a unique identification sequence (e.g., NN) having 2-10 or more bases (see, e.g., Figure 3). In some embodiments, the second splint strand (400) includes only one subregion and lacks the second and third subregions, and the first subregion includes a sample index sequence having 5 to 20 bases. In some embodiments, the sample index sequence can be used in multiplex assays to distinguish sequences of interest obtained from different sample sources. In some embodiments, the unique identification sequence includes a random sequence. The unique identification sequence can be designed to exhibit reduced hybridization to, or no hybridization to, the first, second, third, and fourth surface primers. An exemplary arrangement of the subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[second subregion]-[first subregion]-3'. Another exemplary arrangement of the subregions in the second splint strand (400) in the 5' to 3' direction includes: 5'-[third subregion]-[second subregion]-[first subregion]-3'. In some embodiments, the second splint strand (400) can be 20 to 100 nucleotides in length, or 30 to 80 nucleotides in length, or 40 to 60 nucleotides in length.In some embodiments, the second splint strand (400) comprises one or more phosphorothioate linkages at the 5' and / or 3' end to confer exonuclease resistance. In some embodiments, the second splint strand (400) comprises one or more phosphorothioate linkages at an internal position to confer endonuclease resistance. In some embodiments, the second splint strand (400) 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 second splint strand (400) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the second splint strand (400) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0276] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the first splint strand (300) comprises an internal region (310) comprising at least two subregions, including a fourth and fifth subregion (see, e.g., Figures 2 and 3). The fourth subregion hybridizes to the first subregion of the second splint strand (400). The fifth subregion hybridizes to the second subregion of the second splint strand (400). The fourth and fifth subregions do not hybridize to (or at least show very little hybridization to) the first and second surface primers. In some embodiments, the internal region (310) of the first splint strand further comprises an optional sixth subregion that hybridizes to the third subregion of the second splint strand (400) (see, e.g., Figure 3). An exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes 5'-[fourth subregion]-[fifth subregion]-3'. Another exemplary arrangement of the subregions of the first splint strand (300) in the 5' to 3' direction includes 5'-[fourth subregion]-[fifth subregion]-[sixth subregion]-3'. In some embodiments, the first splint strand (300) can be 50 to 150 nucleotides in length, or 60 to 100 nucleotides in length, or 70 to 90 nucleotides in length. In some embodiments, the first splint strand (300) includes one or more phosphorothioate linkages at the 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, the first splint strand (300) includes one or more phosphorothioate linkages at internal positions to confer endonuclease resistance. In some embodiments, the first splint strand (300) comprises one or more 2'-O-methylcytosine bases at the 5' and / or 3' ends or at an internal position.

[0277] The present disclosure provides a method for forming a plurality of library-sprint complexes (500), the method comprising: (a) providing a plurality of double-stranded splint adapters (200), each double-stranded splint adapter (200) comprising a first splint strand (300) hybridized to a second splint strand (400), the first splint strand (300) comprising a first region (320), an internal region (310), and a second region (330) arranged in 5' to 3' order, the internal region of the first splint strand (310) hybridizing to the second splint strand (400), the second splint strand comprising a region arranged in 5' to 3' order, the first region (320), an internal region (310), and a second region (330), the internal region of the first splint strand (310) hybridizing to the second splint strand (400), the second splint strand comprising (i) a second subregion having a universal binding sequence for a fourth surface primer, and (ii) a first subregion having a universal binding sequence for a third surface primer.The method for forming a plurality of library-splint complexes (500) includes step (b): hybridizing a plurality of double-stranded splint adapters to a plurality of single-stranded nucleic acid library molecules (100), each library molecule comprising 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 (130), wherein hybridizing converts the first splint strand (300) into a The method further includes hybridizing the first splint strand (300) under conditions suitable for hybridizing the first splint strand to the library molecule (100), thereby circularizing the library molecule to produce a library-sprint complex (500), whereby a first region (320) of the first splint strand hybridizes to the binding sequence for the first surface primer (120), a third region (330) of the first splint strand hybridizes to the binding sequence for the second surface primer (130), the library-sprint complex (500) comprising a first nick between the 5' end of the library molecule and the 3' end of the second splint strand (300), and the library-sprint complex (500) comprising a second nick between the 5' end of the second splint strand (300) and the 3' end of the library molecule (100), wherein the first and second nicks are enzymatically ligatable. In some embodiments, 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) (see, e.g., Figure 5). A list of exemplary first left indexing sequences (160) and first right indexing sequences (170) is provided in Table 1 in Figures 23A-23F.In some embodiments, the first left index sequence (160) comprises or lacks a short random sequence (e.g., NNN). In some embodiments, the first right index sequence (170) comprises or lacks a short random sequence (e.g., NNN). In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left unique identification sequence (180) and / or a first right unique identification sequence (190), each comprising 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. (See, e.g., Figure 6 ).

[0278] Multiplex workflows are enabled by preparing sample-indexed libraries using one or both index sequences (e.g., left and / or right index sequences). 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 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.

[0279] In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further comprises a first left unique identification sequence (180) and / or a first right unique identification sequence (190) (see, e.g., Figures 5 and 6). 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) may be used for molecular tagging.

[0280] In some embodiments, in any of the methods for forming a plurality of library-splint complexes (500) described herein, the first subregion of the second splint strand (400) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200). In some embodiments, the second subregion of the second splint strand (400) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201). In some embodiments, the second splint strand (400) comprises first and second subregions comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATCCATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 202). See Figure 10. In some embodiments, the 5' end of the second splint strand (400) can be phosphorylated or unphosphorylated.

[0281] In some embodiments, in any of the methods for forming a plurality of library-splint complexes (500) described herein, the first region of the first splint strand (320) comprises a first universal adapter sequence comprising a universal binding sequence for a first surface primer (or its complementary sequence), wherein the first region (320) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 193). For example, the first region of the first splint strand (320) can hybridize to a P5 surface primer or the complementary sequence of a P5 surface primer. For example, the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 203; short P5), or the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGAGATC-3' (SEQ ID NO: 194; long P5). In some embodiments, the second region of the first splint strand (330) comprises a second universal adapter sequence that includes a universal binding sequence for a second surface primer (or its complementary sequence), and the second region (330) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195). For example, the second region of the first splint strand (330) can hybridize to a P7 surface primer or the complementary sequence of a P7 surface primer. For example, the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 195; short P7), or the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 196; long P7). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fourth subregion having the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197). In some embodiments, the first splint strand (300) comprises an internal region (310) that includes a fifth subregion having the sequence 5'-GATCAGGTGAGGCTGCGACGACT'3' (SEQ ID NO: 198).In some embodiments, the first splint strand (300) comprises a first region (320), an internal region (310) having fourth and fifth subregions, and a second region (330) having the sequence 5'-TCGGTGGTCGCCGTATCATTACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 199). See Figure 10. In some embodiments, the 5' end of the first splint strand (300) can be phosphorylated or unphosphorylated. In some embodiments, the first subregion of the second splint strand (400) can hybridize to the fourth subregion of the first splint strand (300). In some embodiments, the second subregion of the second splint strand (400) can hybridize to the fifth subregion of the first splint strand (300).

[0282] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the first region of the first splint strand (320) comprises a sequence capable of binding to the first left universal adapter sequence (120) of the library molecule, and the first region of the first splint strand (320) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 197) or a complementary sequence thereof.

[0283] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the second region of the first splint strand (330) comprises a sequence capable of binding to the first right universal adapter sequence (130) of the library molecule, and the second region of the first splint strand (330) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (sequence number 198) or a complementary sequence thereof.

[0284] In some embodiments, in any of the methods for forming multiple library-splint complexes (500) described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first splint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (sequence number 203).

[0285] In some embodiments, in any of the methods for forming multiple library-splint complexes (500) described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first splint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 200) or a complementary sequence thereof.

[0286] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules comprise a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 204).

[0287] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules comprise a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 207).

[0288] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules comprise a left universal binding sequence (140) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 208).

[0289] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules comprise a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 205).

[0290] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules comprise a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 209).

[0291] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules comprise a left universal binding sequence (150) for the sequencing primer, wherein the left universal binding sequence comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 210).

[0292] In some embodiments, in any of the methods for forming multiple library-splint complexes (500) described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first splint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (sequence number 206).

[0293] In some embodiments, in any of the methods for forming multiple library-splint complexes (500) described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first splint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (sequence number 201) or a complementary sequence thereof.

[0294] In some embodiments, any of the methods for forming a plurality of library-sprint complexes (500) 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.

[0295] In some embodiments, any of the methods for forming a plurality of library-sprint complexes (500) 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-sprint complexes (500) further comprises step (c1): contacting, in a first reaction vessel, the plurality of double-stranded splint adaptors (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 double-stranded splint adaptors (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 (500) further comprises step (d1): contacting, in a second reaction vessel, a plurality of phosphorylated double-stranded splint adapters (200) and a plurality of phosphorylated single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatic ligation of the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600), each hybridized to the first splint strand (300). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0296] In some embodiments, any of the methods for forming a plurality of library-splint complexes (500) 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 (500) further comprises step (c2): contacting, in a first reaction vessel, the plurality of double-stranded splint adaptors (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 double-stranded splint adaptors (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 (500) further comprises step (d2): contacting, in the same first reaction vessel, the phosphorylated double-stranded splint adapters (200) and the phosphorylated single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatic ligation of the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600), each hybridized to the first splint strand (300). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0297] In some embodiments, any of the methods for forming a plurality of library-splint complexes (500) 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 (500) further includes step (c3): contacting, in a first reaction vessel, a plurality of double-stranded splint adapters (200) and a 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 double-stranded splint adapters (200) and the plurality of single-stranded nucleic acid library molecules (100), wherein the conditions are suitable for enzymatically ligating the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600), each hybridized to a first splint strand (300). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0298] In some embodiments, any of the methods for forming a plurality of library-splint complexes (500) described herein further includes an optional step of enzymatically removing a plurality of first splint strands (300) from a plurality of covalently closed circular library molecules (600), comprising contacting the plurality of covalently closed circular library molecules (600) with at least one exonuclease enzyme to remove the plurality of first splint strands (300) and retain the plurality of covalently closed circular library molecules (600). 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 (c1) and the ligation reaction can be performed in a second reaction vessel (d1), 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 (c2), a sequential ligation reaction may be performed in the first reaction vessel (d2), 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 the first reaction vessel (c3), 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.

[0299] Methods for Rolling Circle Amplification The present disclosure provides methods for performing a rolling circle amplification reaction on a covalently closed circular library molecule (600). 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 (600) that is no longer hybridized to the first splint strand (300) after an exonuclease reaction. In some embodiments, the rolling circle amplification reaction can be performed on a covalently closed circular library molecule (600) hybridized to the first splint strand (300). In some embodiments, the covalently closed circular library molecule (600) can be dispensed onto a support and then subjected to a rolling circle amplification reaction. In some embodiments, the covalently closed circular library molecule (600) 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 first splint strand (300) as an amplification primer, or the first splint strand (300) may be removed (e.g., via exonuclease digestion) and replaced with a soluble amplification primer.

[0300] 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 first splint strand (300), each covalently closed circular library molecule (600) in the plurality of covalently closed circular library molecules (600) comprises a second splint strand region (400) comprising a universal binding sequence for a fourth surface primer, the method comprising: (a) distributing the plurality of covalently closed circular library molecules (600) onto a support on which a plurality of fourth surface primers are immobilized under conditions suitable for hybridizing each of the covalently closed circular library molecules (600) to each of the immobilized fourth surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (600).

[0301] In some embodiments, a plurality of fourth surface primers immobilized on a support comprise the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 198). Each fourth surface primer can hybridize to a covalently closed circular library molecule (600) having a second splint strand region (400) comprising a universal binding sequence for the fourth surface primer, wherein the universal binding sequence for the fourth surface primer comprises a second subregion comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 201).

[0302] In some embodiments, a plurality of covalently closed circular library molecules (600) can be distributed on a support coated with one or more compounds to produce a passivated layer on the support (e.g., FIG. 11). 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 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 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 the covalently closed circular library molecules (600) 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 covalently closed circular library molecules (600) 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).

[0303] In some embodiments, the partitioning of step (a) 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.

[0304] 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 (600) 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 a plurality of fourth surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, t...

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 double-stranded splint adapter (200) comprising a first splint strand (300) and a second splint strand (400), wherein the double-stranded splint adapter (200) comprises a double-stranded region and two adjacent single-stranded regions, and the first splint strand comprises a region arranged in 5' to 3' order: (i) a first region (320), (ii) an internal region (310), and (iii) a second region (330); A library-sprint complex (500) comprising: The internal region of the first splint strand (310) hybridizes to the second splint strand (400), the first region of the first splint strand (320) hybridizes to the at least a first left universal adaptor sequence (120) of the single-stranded nucleic acid library molecule, and the second region of the first splint strand (330) hybridizes to the at least a first right universal sequence (130) of the single-stranded nucleic acid library molecule, thereby circularizing the single-stranded nucleic acid library molecule to generate a library-sprint complex (500). Library-Sprint Complex (500).

2. The single-stranded nucleic acid library molecule (100) (i) a second left universal adaptor sequence (140); (ii) a second right universal adapter 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) a first right unique identifier sequence (190), or any combination of two or more thereof.

3. The first left universal adapter array (120) and / or the second left universal adapter array (140) are (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 (500) of claim 2, comprising a universal binding sequence for the compaction oligonucleotide.

4. The first right universal adapter array (130) and / or the second right universal adapter array (150) are (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 (500) of claim 2, comprising a universal binding sequence for the compaction oligonucleotide.

5. A library-sprint complex (500) comprising: (i) said second splint strand (400) comprises at least two sub-regions; the first subregion comprises a universal binding sequence for the third surface primer; the second subregion comprises a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to, or show very little hybridization to, the first and second surface primers; or (ii) said first splint chain (300) comprises an interior region (310) comprising at least two sub-regions; a fourth subregion comprising a universal binding sequence for a third surface primer, said fourth subregion hybridizing to said first subregion of said second splint strand (400); a fifth subregion comprising a universal binding sequence for a fourth surface primer, said fifth subregion hybridizing to said second subregion of said second splint strand (400); and the fourth and fifth subregions do not hybridize to, or show very little hybridization to, the first and second surface primers; A library-sprint complex (500) according to claim 3 or 4.

6. The library-sprint complex described in claim 5, wherein the second splint strand (400) further includes a third subregion, and the third subregion includes a sample index sequence having 5 to 20 bases and / or a unique identification sequence having 2 to 10 or more bases.

7. The library-sprint complex (500) of claim 5, wherein the internal region (310) of the first splint strand includes a sixth subregion comprising a sample index sequence having 5 to 20 bases and / or a unique identification sequence having 2 to 10 or more bases, and the sixth subregion hybridizes to the third subregion of the second splint strand (400). a) a first nick between the 5' end of the single-stranded nucleic acid library molecule and the 3' end of the second splint strand; and / or b) a second nick between the 5' end of the second splint strand and the 3' end of the single-stranded nucleic acid library molecule; further comprising the first nick and / or the second nick are enzymatically ligatable; The library-sprint complex (500) of claim 1.

9. A method for forming a plurality of library-sprint composites (500), comprising: a) providing a plurality of double-stranded splint adapters, each double-stranded splint adapter (200) comprising a first splint strand (300) hybridized to a second splint strand (400), each double-stranded splint adapter comprising a double-stranded region and two adjacent single-stranded regions, the first splint strand comprising regions arranged in 5' to 3' order: (i) a first region (320), (ii) an internal region (310), and (iii) a second region (330), the internal region of the first splint strand (310) hybridizing to the second splint strand (400); b) hybridizing the plurality of double-stranded splint adapters to a plurality of single-stranded nucleic acid library molecules (100), each single-stranded nucleic acid library molecule comprising a sequence of interest (110) flanked on one side by at least a first left universal adapter sequence (120) and on the other side by at least a first right universal adapter sequence (130), wherein the hybridizing is performed under conditions suitable for hybridizing the first region of the first splint strand (320) to the at least a first left universal adapter sequence (120) of the single-stranded nucleic acid library molecule, and the conditions are suitable for hybridizing the second region of the first splint strand (330) to the at least a first right universal sequence (130) of the single-stranded nucleic acid library molecule, thereby circularizing the plurality of single-stranded nucleic acid library molecules to form a plurality of library-sprint complexes (500).

10. (a) each library-sprint complex (500) in the plurality of library-sprint complexes (500) comprises a first nick between the 5' end of the library molecule and the 3' end of the second splint strand, and a second nick between the 5' end of the second splint strand and the 3' end of the library molecule; or (b) each single-stranded nucleic acid library molecule (100) in the plurality of single-stranded nucleic acid library molecules (100) is (i) a second left universal adaptor sequence (140); (ii) a second right universal adapter 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) further comprising any one or any combination of two or more of the first right unique identification sequences (190); 10. The method of claim 9. (a) the first and / or second left universal adapter sequence comprises: (i) a universal binding sequence for a 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) comprises a universal binding sequence for compaction oligonucleotides; or (b) the first and / or second right universal adaptor sequences are: (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) comprising a universal binding sequence for compaction oligonucleotides; The method of claim 10.

12. the second splint chain (400) comprises at least two sub-regions; the first subregion comprises a universal binding sequence for the third surface primer; the second subregion comprises a universal binding sequence for the fourth surface primer, and the first and second subregions do not hybridize to, or show very little hybridization to, the first and second surface primers; The method of claim 11.

13. 13. The method of claim 12, wherein the second splint strand (400) further comprises a third subregion, the optional third subregion comprising a sample index sequence having 5 to 20 bases and / or a unique identification sequence having 2 to 10 or more bases.

14. the interior region (310) of the first splint chain comprises at least two sub-regions; a fourth subregion comprising a universal binding sequence for the third surface primer, the fourth subregion hybridizing to the first subregion of the second splint strand (400); a fifth subregion comprising a universal binding sequence for the fourth surface primer, the fifth subregion hybridizing to the second subregion of the second splint strand (400); and the fourth and fifth subregions do not hybridize to, or show very little hybridization to, the first and second surface primers; The method of claim 12.

15. 15. The method of claim 14, wherein the internal region (310) of the first splint strand further comprises a sixth subregion comprising a sample index sequence having 5 to 20 bases and / or a unique identification sequence having 2 to 10 or more bases, and the sixth subregion hybridizes to the third subregion of the second splint strand (400).

16. 10. The method of claim 9, further comprising contacting the plurality of library-splint complexes (500) with a ligase, thereby producing a plurality of covalently closed circular library molecules (600) hybridized to the first splint strand (300).

17. The method of claim 1, further comprising distributing the plurality of covalently closed circular library molecules (600) onto a support, the support comprising a plurality of surface capture primers immobilized thereon; and converting the plurality of covalently closed circular library molecules (600) into immobilized nucleic acid concatemer template molecules by performing a rolling circle amplification reaction on the support; 17. The method of claim 16, further comprising:

18. 17. The method of claim 16, further comprising contacting the plurality of covalently closed circular library molecules (600) with at least one exonuclease enzyme to remove the first splint strand (300) and retain the plurality of covalently closed circular library molecules (600).

19. The second splint strand (400) comprises a universal binding sequence for a third surface primer, and the method further comprises: c) distributing the plurality of covalently closed circular library molecules (600) onto the support on which the plurality of third surface primers are immobilized under conditions suitable for hybridizing each of the covalently closed circular library molecules (600) to each of the immobilized third surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (600).

20. The method of claim 18.

20. 20. The method of claim 19, further comprising contacting a plurality of the immobilized covalently closed circular library molecules (600) 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 third surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules.

21. 21. The method of claim 20, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP, and / or dUTP.

22. The method further comprises sequencing the plurality of immobilized nucleic acid concatemer molecules, the method comprising: a) contacting the plurality of immobilized nucleic acid concatemer molecules with (i) a plurality of sequencing polymerases, and (ii) a plurality of soluble sequencing primers, wherein said contacting is performed under conditions suitable for forming a plurality of multiplexed polymerases, wherein said plurality of multiplexed polymerases comprise sequencing polymerases bound to nucleic acid duplexes, and wherein said nucleic acid duplexes comprise nucleic acid 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 of at least one nucleotide to the multiplexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analogue, wherein the at least one nucleotide analogue is labeled with a fluorophore and has a removable chain-terminating moiety at the sugar 3' position; c) incorporating said at least one nucleotide into the 3' end of said soluble sequencing primer, thereby generating a plurality of nascent extended sequencing primers; d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

23. The method further comprises sequencing the plurality of immobilized nucleic acid concatemer molecules, the method comprising: a) contacting the plurality of immobilized nucleic acid concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein said contacting is performed under conditions suitable for forming a plurality of first multiplexed polymerases, wherein said plurality of first multiplexed polymerases comprise sequencing polymerases bound to nucleic acid duplexes, and wherein said nucleic acid duplexes comprise nucleic acid concatemer molecules hybridized to soluble sequencing primers; 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, and each nucleotide arm is attached to a nucleotide unit; c) detecting the plurality of multivalent complex polymerases; d) identifying the nucleobases of the complementary nucleotide units bound to the first of the plurality of multiplexed polymerases in the plurality of multivalent multiplexed polymerases, thereby determining the sequence of the nucleic acid template.

24. e) dissociating the plurality of multivalent composite polymerases, removing the plurality of first sequencing polymerases and bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; f) contacting the plurality of nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein said contacting is performed under conditions suitable for binding of said plurality of second sequencing polymerases to said plurality of nucleic acid duplexes, thereby forming a plurality of second multiplexed polymerases, wherein said plurality of second multiplexed polymerases comprises second sequencing polymerases bound to nucleic acid duplexes; and g) contacting the plurality of second multiplexed polymerases with a plurality of nucleotides, said contacting being carried out 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; 24. The method of claim 23, further comprising:

25. 25. The method of claim 24, wherein the plurality of nucleotides comprises at least one nucleotide analog, wherein the at least one nucleotide analog is labeled with a fluorophore and has a removable chain-terminating moiety at the sugar 3' position.

26. h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase; 25. The method of claim 24, further comprising: i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-conjugated polymerase.

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