RU2788402C2 - Methods and compositions for obtainment of nucleic acid libraries - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to obtainment of nucleic acid libraries. A method for obtainment of a nucleic acid library includes obtainment of a set of single-stranded nucleic acids or a set of double-stranded nucleic acids with subsequent denaturation of the latter to obtain the set of single-stranded nucleic acids, and ligation of the first and the second adapters.

EFFECT: implementation of the group of inventions allows for simultaneous fragmentation of a genome and ligation of adapters to fragments, thereby simplifying obtainment of a nucleic acid library.

50 cl, 27 dwg, 4 ex

Description

RELATED APPLICATIONS

[0001] This application claims priority based on U.S. Provisional Application No. 62/473595 filed March 20, 2017 titled "METHODS AND COMPOISTIONS FOR PREPARING NUCLEIC ACID LIBRARIES"), description which is incorporated herein by reference in its entirety.

SEQUENCE LIST

[0002] This application includes a sequence listing in electronic format. This sequence listing is provided as a file named ILLINC362WOSEQLIST created on March 15, 2018, approximately 4 kB in size. The information contained in the electronic sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The systems, methods, and compositions provided herein relate to the production of nucleic acid libraries. Some implementation options include obtaining libraries of nucleic acids through ligation of single-stranded nucleic acids.

BACKGROUND OF THE INVENTION

[0004] Several next-generation sequencing technologies exist for the rapid and cost-effective determination of a complete genome sequencing. Typically, prior to sequencing, a library of template nucleic acids is obtained from a double-stranded target genomic DNA sample. Sample collection typically includes a DNA fragmentation step that breaks large DNA strands into smaller fragments more suitable for next-generation sequencing technologies. Often, adapters are attached to the ends of DNA fragments, which can be done by DNA end repair followed by adapter ligation, or, more recently, using the transposome system. The use of transposomes, which are a complex of transposase and transposon sequences, makes it possible to simultaneously fragment the genome and ligate adapters to the fragments, thereby simplifying the preparation of the library. Methods for obtaining libraries are usually laborious and require several steps performed by the operator at various stages.

BRIEF DESCRIPTION OF THE INVENTION

[0005] Some embodiments include a method for obtaining a nucleic acid library, comprising: (a) obtaining a plurality of nucleic acids, said plurality of nucleic acids being single-stranded nucleic acids; (b) dephosphorylation of the 5' ends of single-stranded nucleic acids; (c) ligating the first adapter to the 3' ends of the single stranded nucleic acids in the presence of a ligase, wherein the 3' end of the first adapter contains a blocking group; (d) phosphorylation of the 5' ends of the ligated single-stranded nucleic acids; and (e) ligating the second adapter to the 5' ends of the phosphorylated ligated single stranded nucleic acids in the presence of a ligase, whereby a nucleic acid library is obtained.

[0006] In some embodiments, the 5' end of the second adapter is non-phosphorylated.

[0007] In some embodiments, the second adapter is attached to the substrate. In some embodiments, the support includes a bead or flow cell.

[0008] Some implementation options also include the removal of unligated single-stranded first adapters prior to step (e).

[0009] Some implementations also include the hybridization of unligated single-stranded first adapters with a capture probe. In some embodiments, the capture probe contains a sequence that is complementary to at least a portion of the first adapter. In some embodiments, the 3' end of the capture probe contains a blocking group. In some embodiments, the 5' end of the capture probe contains a blocking group.

[0010] Some embodiments also include hydrolysis of non-ligated single stranded first adapters. In some embodiments, hydrolysis of the unligated single stranded first adapters comprises contacting the unligated single stranded first adapters with a 5'-phosphate dependent exonuclease. In some embodiments, hydrolysis of the unligated single strand first adapters comprises contacting the unligated single strand first adapters with a 5' phosphate dependent exonuclease and a 5' deadenylase.

[0011] In some embodiments, phosphorylation of the 5' end of the ligated single-stranded nucleic acids comprises bringing the ligated single-stranded nucleic acids into contact with a kinase.

[0012] Some embodiments include a method for obtaining a nucleic acid library, comprising: (a) obtaining a plurality of nucleic acids, said plurality of nucleic acids being double-stranded nucleic acids; (b) contacting the double-stranded nucleic acids with a 5'-exonuclease to produce a plurality of modified double-stranded nucleic acids with single-stranded 3' overhangs; (c) ligating the first adapter to the 3' ends of the modified double-stranded nucleic acids in the presence of a ligase, wherein the 3' end of the first adapter contains a blocking group; (d) denaturing the modified double-stranded nucleic acids ligated to the first adapters to produce a plurality of single-stranded nucleic acids; and (e) ligating the second adapter to the 5' ends of the single stranded nucleic acids in the presence of a ligase, whereby a nucleic acid library is obtained.

[0013] In some embodiments, the 5' end of the second adapter is non-phosphorylated.

[0014] Some embodiments include a method for obtaining a nucleic acid library, comprising: (a) obtaining a plurality of nucleic acids, said plurality of nucleic acids being single-stranded nucleic acids; (b) dephosphorylation of the 5' ends of single-stranded nucleic acids; (c) ligating the first adapter to the 3' ends of the single stranded nucleic acids in the presence of a ligase, wherein the 3' end of the first adapter contains a blocking group; (d) hybridizing the ligated first adapter to the capture probe; and (e) extending the capture probe and ligating the second adapter to the 3' end of the extended capture probe in the presence of a ligase, wherein the 3' end of the second adapter contains a blocking group, thereby obtaining a nucleic acid library.

[0015] Some embodiments also include removing the hybridized ligated first adapter from the extended capture probe.

[0016] In some embodiments, the capture probe is attached to the substrate. In some embodiments, the support includes a bead or flow cell. In some embodiments, the capture probe contains an index of the capture probe. In some embodiments, the capture probe contains a cleavable linker. Some embodiments also include cleavage of the cleavable linker.

[0017] Some embodiments include a method for obtaining a nucleic acid library, including: (a) obtaining a plurality of nucleic acids, said plurality of nucleic acids being single-stranded nucleic acids; (b) dephosphorylation of the 5' ends of single-stranded nucleic acids; (c) ligating the linker to the 3' ends of the single stranded nucleic acids in the presence of a ligase, said linker comprising a first adapter and a second adapter, and the 3' end of the linker containing a blocking group; (d) phosphorylation of the 5' ends of the ligated single-stranded nucleic acids; and (e) deprotection of the 3' ends of the phosphorylated nucleic acids and ring closure of the deprotected nucleic acids, whereby a nucleic acid library is obtained.

[0018] In some implementations, the linker contains a cleavable site between the first adapter and the second adapter.

[0019] Some embodiments also include linearization of circular nucleic acids by cleavage at a cleavable linker. In some embodiments, the cleavage site contains a uracil residue. Some embodiments also include bringing the circular nucleic acids into contact with a uracil-specific excision reagent. In some embodiments, the uracil-specific excision reagent contains an enzyme selected from uracil DNA glycosylase (UDG) and VTII endonuclease DNA glycosylase lyase.

[0020] Some embodiments also include amplifying circular nucleic acids by a method comprising hybridizing at least a primer to a first adapter or a second adapter. In some embodiments, the amplification is selected from NRP and rolling ring amplification (RCA). In some embodiments, amplification comprises contacting the circular nucleic acids with a polymerase that forms a linear product upon contact with a uracil residue in the template.

[0021] In some embodiments, dephosphorylation of the 5' end of single-stranded nucleic acids comprises contacting the single-stranded nucleic acids with a phosphatase.

[0022] In some embodiments, steps (b)-(e) are performed in a single reaction volume.

[0023] In some embodiments, steps (b)-(e) are performed in a single reaction vessel.

[0024] In some embodiments, the first adapter and/or the second adapter comprises a sequencing primer binding site.

[0025] In some embodiments, the first adapter comprises a P7 sequence, a P5 sequence, or their complement or reverse complement. In some embodiments, the second adapter contains a P7 sequence, a P5 sequence, or their complement or reverse complement.

[0026] In some embodiments, the first adapter and/or the second adapter contains an adapter index. In some implementations, the index of the first adapter is different from the index of the second adapter. In some embodiments, the adapter index indicates the source of the plurality of nucleic acids.

[0027] In some embodiments, the blocking group contains a C3 3' spacer or dideoxynucleotide.

[0028] In some embodiments, the ligase comprises a single-stranded nucleic acid ligase.

In some embodiments, ligation of the first adapter and/or ligation of the second adapter is performed in the presence of a volume excluding agent. In some embodiments, the exclusion agent is selected from the group consisting of polyethylene glycol (PEG), dextran, hydroxyethyl starch, ficoll, and polyvinylpyrrolidone. In some embodiments, the first ligation step and/or the second ligation step is performed in a reaction volume containing at least about 37% (w/v) PEG. In some embodiments, the first ligation step and/or the second ligation step is performed in a reaction volume containing at least about 60% (w/v) PEG.

[0030] In some embodiments, a plurality of nucleic acids comprise RNA. In some embodiments, the plurality of nucleic acids comprise cDNA or genomic DNA.

[0031] In some embodiments, the average nucleic acid size of a plurality of nucleic acids is less than about 200 nucleotides.

[0032] In some embodiments, a plurality of nucleic acids are obtained from a low quality nucleic acid source. In some embodiments, a plurality of nucleic acids are obtained from a fixed sample.

[0033] Some embodiments also include amplification of a nucleic acid library.

[0034] Some embodiments also include obtaining sequence data from a nucleic acid library.

[0035] Some embodiments include a nucleic acid library obtained by a method according to any of the above embodiments.

[0036] Some embodiments include a set comprising: a first adapter, the 3' end of said first adapter having a blocking group, the first adapter having a P7 sequence, a P5 sequence, or their complement or reverse complement; ligase; and a component selected from the group consisting of a volume exclusion agent, a kinase, a phosphatase, a 5'-phosphate dependent exonuclease, a 5'-deadenylase, a polymerase forming a linear product upon contact with a uracil residue in a matrix, a uracil-specific excision a reagent, and a second adapter, said second adapter comprising a P7 sequence, a P5 sequence, or their complement or reverse complement. In some embodiments, the linker includes first and second adapters.

[0037] Some embodiments include a reaction vessel containing a reaction volume containing: a plurality of nucleic acids; the first adapter, and the 3'-end of the first adapter contains a blocking group; ligase; and an agent causing a volume exclusion effect.

[0038] In some embodiments, the plurality of nucleic acids are single-stranded nucleic acids.

[0039] In some embodiments, the first adapter is ligated to the 3' ends of a plurality of single-stranded nucleic acids, thereby forming a plurality of modified single-stranded nucleic acids.

[0040] In some embodiments, the unligated first adapter is hybridized to a capture probe. In some embodiments, the capture probe contains a sequence that is complementary to at least a portion of the first adapter.

[0041] In some embodiments, the plurality of nucleic acids are double-stranded nucleic acids with 3' overhangs. In some embodiments, the first adapter is ligated to the 3' ends of a plurality of double-stranded nucleic acids with 3' overhangs, thereby forming a plurality of modified double-stranded nucleic acids.

[0042] Some implementations also include a second adapter. In some embodiments, the 5' end of the second adapter is non-phosphorylated.

[0043] Some embodiments also include a dephosphorylating agent. In some embodiments, the dephosphorylating agent comprises a phosphatase. In some embodiments, said phosphatase is inactivated.

[0044] In some embodiments, the exclusion agent is selected from the group consisting of polyethylene glycol (PEG), dextran, hydroxyethyl starch, ficoll, and polyvinylpyrrolidone. In some embodiments, the reaction volume contains at least about 37% (w/v) PEG. In some embodiments, the reaction volume contains at least about 60% (w/v) PEG.

[0045] In some embodiments, the first adapter and/or the second adapter comprises a sequencing primer binding site.

[0046] In some embodiments, the first adapter and/or the second adapter contains an adapter index. In some implementations, the index of the first adapter is different from the index of the second adapter. In some embodiments, the adapter index indicates the source of the plurality of single-stranded nucleic acids.

[0047] In some embodiments, the blocking group contains a C3 3' spacer or a dideoxynucleotide. In some embodiments, the blocking group contains a C3 3' spacer.

[0048] In some embodiments, the ligase comprises a single-stranded nucleic acid ligase.

[0049] In some embodiments, a plurality of nucleic acids comprise RNA. In some embodiments, the plurality of nucleic acids comprise cDNA or genomic DNA.

[0050] In some embodiments, a plurality of single-stranded nucleic acids are obtained from a low quality source of nucleic acid. In some embodiments, a plurality of nucleic acids are obtained from a fixed sample.

[0051] Some embodiments include a flow cell containing a reaction vessel according to any of the above embodiments.

[0052] Some embodiments include a system comprising a reaction vessel according to any of the above embodiments and a detector for obtaining sequencing data.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 is a diagram of a method for obtaining a single-stranded nucleic acid library, in accordance with one embodiment. The double-stranded nucleic acid is denatured to a single-stranded nucleic acid, and a library can be obtained by dephosphorylation of the 5' end of the single-stranded nucleic acid, ligating a P7' adapter containing a 3' blocking group to the 3' end of the single-stranded nucleic acid, re-phosphorylating the 5' - end of the ligated single-stranded nucleic acid and ligating the P5 adapter containing the non-phosphorylated 5' end to the 5' end of the ligated single-stranded nucleic acid.

[0054] FIG. 2 is a diagram of a method for obtaining a single-stranded nucleic acid library, in accordance with one embodiment. The double-stranded nucleic acid is partially hydrolyzed with 5'-exonuclease to form a double-stranded nucleic acid with 3'-overhanging ends, denatured to form a single-stranded nucleic acid, the P7' adapter containing a 3'-blocking group can be ligated to the 3' ends of the double-stranded nucleic acid with 3' overhang, a ligated 3' overhang double stranded nucleic acid can be denatured to form a single stranded nucleic acid, and a P5 adapter containing a non-phosphorylated 5' end can be ligated to the 5' end of the ligated single stranded nucleic acid.

[0055] FIG. 3 is a diagram of a method for obtaining a single-stranded nucleic acid library using beads, in accordance with one embodiment. The library can be obtained by dephosphorylation of the 5' end of the single stranded nucleic acid, ligation of the P5' adapter containing the 3' blocking group to the 3' end of the single stranded nucleic acid, hybridization of the first ligation product with a P5 capture probe attached to the bead, extension of the probe capture, removing the hybridized first ligation from the extended capture probe, and ligating the P7' adapter containing the 3' blocking group to the 3' end of the extended capture probe.

[0056] FIG. 4A is a diagram of a method for preparing a library of circular single-stranded nucleic acids, in accordance with one embodiment. The double-stranded nucleic acid is denatured to a single-stranded nucleic acid, and a library can be prepared by dephosphorylating the 5' end of the single-stranded nucleic acid, ligating a linker containing a P7' adapter, a cleavage site ("U"), a P5 adapter containing a 3' blocking group ("R"), with the 3' end of a single-stranded nucleic acid; rephosphorylating the 5' end of the ligated single stranded nucleic acid; deprotecting the 3' end of the ligated single stranded nucleic acid; and circulating the deprotected nucleic acids to form a library member containing circular single stranded nucleic acids.

[0057] FIG. 4B is a diagram in which the circular nucleic acid of FIG. 4B can be linearized by cleavage site ("U") cleavage, or amplified by PCR or rolling ring amplification (RCA).

[0058] FIG. 5 is a diagram of a method for obtaining a library of single-stranded nucleic acids, including the embodiment shown in FIG. 1. In FIG. 5, a capture probe containing the P7 sequence is hybridized to the P7' adapter ligated to the target nucleic acid and to an excess of unligated P7' adapters. Hybridization of the capture probe with unligated P7' adapters inhibits ligation of unligated P7' adapters with other nucleic acids.

[0059] FIG. 6 is a diagram of a method for obtaining a single-stranded nucleic acid library, in accordance with one embodiment. The double-stranded nucleic acid is denatured to a single-stranded nucleic acid, and a library can be prepared by dephosphorylation of the 5' end of the single-stranded nucleic acid, ligating the P7' adapter containing the 3'-blocking group to the 3'-end of the single-stranded nucleic acid, removing excess unligated single-stranded nucleic acids. P7' adapters by hydrolyzing 5'-phosphate dependent exonuclease and 5'-deadenylase, rephosphorylating the 5' end of the ligated single stranded nucleic acid and ligating the P5 adapter containing the unphosphorylated 5' end to the 5' end of the ligated single stranded nucleic acid.

[0060] FIG. 7 shows the results of an experiment to dephosphorylate the 5' end of a 60-mer single stranded nucleic acid (3'OH-60-Phos 5') and confirm that the products (3'OH-60-OH 5') cannot form concatemers. The top panel is a diagram showing dephosphorylation. The table shows the conditions for carrying out reactions and analysis in the gel shown in the bottom panel.

[0061] FIG. 8 shows the results of an experiment on the ligation of a 64-mer adapter P7' (3'C3-P7-phos 5') containing a 3'-blocking group, with the 3'-end of a dephosphorylated 60-mer single-stranded nucleic acid (3'OH-60- OH 5'). The top panel is a diagram showing ligation. The table shows the conditions for carrying out reactions and analysis in the gel shown in the bottom panel.

[0062] In FIG. 9 shows the results of a 5' rephosphorylation experiment on a 124-mer dephosphorylated single-stranded nucleic acid containing a ligated 64-mer adapter with a 3' blocking group (3'C3-P7-60-OH). The top panel is a diagram showing the kinase reaction. The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0063] FIG. 10 shows the results of an experiment ligating a 60-mer P5 adapter containing a non-phosphorylated 5' end (5'P5-OH) to the 5' end of a 124-mer single-stranded nucleic acid (3'C3-60-B2-Phos). The 124-mer single-stranded nucleic acid contains a ligated 64-mer adapter with a 3' blocking group (3'C3-P7-phos 5'). The top panel is a diagram showing ligation. The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0064] FIG. 11 shows the results of an experiment for ligating a 64-mer P7' adapter containing a 3' blocking group (3'C3-P7-phos 5') to the 3' end of single-stranded dephosphorylated cell-free DNA (bcDNA). The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0065] FIG. 12 shows the results of an experiment in which dephosphorylated bcDNA (bcDNA [Apex]) was treated with kinase (PNK) and a 64-mer P7' adapter containing a 3' blocking group (3'C3-P7-phos 5') was ligated to 3' -end of the rephosphorylated bcDNA. The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0066] FIG. 13 shows the results of an experiment in which a 64-mer P7' adapter containing a 3' blocking group (3'C3-P7-phos 5') was ligated to the 3' end of the dephosphorylated bcDNA, resulting in the first ligated product (3'C3- P7-bcDNA-phos 5') was treated with kinase (PNK) and a 60-mer P5 adapter with a non-phosphorylated 5'-end (5P5'-OH) was ligated to the 3'-end of the first ligation product to form a second ligation product (P7'-bcDNA -P5). The table shows the conditions for carrying out reactions and analysis in the gel shown in the bottom panel.

[0067] FIG. 14 shows gels showing the ligations from Example 2 and amplification products of the ligations "Extract B1" and "Extract B2". In lane 'Ref, extract B1 is marked with two black boxes and extract B2 with one white box.

[0068] FIG. 15 shows the electropherogram of the amplified ligation products "Extract B1" and "Extract B2". The peaks are a 126 bp primer-dimer. (P7'-P5), varieties 292 bp long. (P7'-bcDNA-P5) and 480 bp. (P7'-bcDNA-P5).

[0069] FIG. 16 shows gels showing the results of using SPRI pellets at different ratios to extract amplification products.

[0070] FIG. 17 shows the results of an experiment for ligating a 63-mer P7' adapter containing a 3' blocking group (3'C3-P7-Phos 5') to the 3' end of a single-stranded dephosphorylated bcDNA. The table shows the conditions for carrying out reactions and analysis in the gel shown in the bottom panel.

[0071] In FIG. 18 shows the results of an experiment on the ligation of a 60-mer P5 adapter containing a non-phosphorylated 5' end (5'P5-OH) with the 3' end of the first ligation product (3'C3-P7'-bcDNA-phos5') in the presence of 63 α-membered capture probe P7 (3'C3-P7-C35') containing 5'- and 3'-blocking groups. The top panel is a diagram showing ligation. The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0072] FIG. 19 shows the analysis of the electrophoregram of the amplified ligation products "Extract B1" and "Extract B2". The peaks are a 126 bp primer-dimer. (P7'-P5), varieties 292 bp long. (P7'-bcDNA-P5) and 480 bp. (P7'-bcDNA-P5).

[0073] FIG. 20 shows gels of ligation products before amplification, after amplification, and after extraction with SPRI beads.

[0074] FIG. 21 shows the results of an experiment on the ligation of 39-mer oligonucleotide 1 with a dephosphorylated 5'-end (5'OH-oligo 1-OH3'), with a 37-mer oligonucleotide 2 with a phosphorylated 3'-blocking group (5'phos-Oligo 2- phos3') in the presence of various concentrations of polyethylene glycol (PEG). The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0075] FIG. 22 shows the results of the 39 bp oligonucleotide 1 ligation experiment. with dephosphorylated 5'-end (5'OH-oligo 1-OH3'), with 37 bp oligonucleotide 2 with a phosphorylated 3'-blocking group (5'phos-Oligo 2-phos3') at different ratios of "oligonucleotide 1 : oligonucleotide 2". The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0076] FIG. 23 shows the results of the 39 bp oligonucleotide 1 ligation experiment. with dephosphorylated 5'-end (5'OH-oligo 1-OH3'), with 37 bp oligonucleotide 2 with a phosphorylated 3'-blocking group (5'phos-Oligo 2-phos3') with different amounts of ligase (CIRCLIGASE). The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0077] FIG. 24 shows the results of an experiment on the ligation of 37-mer oligonucleotide 2 with a phosphorylated 3' blocking group (5'phos-Oligo 2-phos3') to a 39-mer oligonucleotide 1 with a dephosphorylated 5'-end (5'OH-oligo 1-OH3 ') conjugated to the beads. The ligation products were analyzed by cleavage from the beads using uracil-DNA glycosylase (UDG), which cleaved the uracil site in oligonucleotide 1. The table shows the reaction and analysis conditions in the gel shown in the bottom panel.

[0078] FIG. 25 shows the results of an experiment on the ligation of 37-mer oligonucleotide 2 with a phosphorylated 3' blocking group (5'phos-Oligo 2-phos3') to a 39-mer oligonucleotide 1 with a dephosphorylated 5'-end (5'OH-oligo 1-OH3 ') conjugated to the beads in a final reaction volume containing 45% PEG. The table shows the conditions for conducting reactions and analysis in the gel shown in the bottom panel.

[0079] FIG. 26 is a diagram of a method for preparing a single-stranded nucleic acid library using beads, in accordance with one embodiment. The library can be obtained by dephosphorylation of the 5' end of the single stranded nucleic acid, ligation of the P5' adapter containing the 3' blocking group to the 3' end of the single stranded nucleic acid, hybridization of the first ligation product with a P5 capture probe attached to the flow cell, extension capture probe, removing the hybridized first ligation from the extended capture probe, hybridizing the complementary primers, ligating the P7' adapter containing the 3' blocking group to the 3' end of the extended capture probe, and denaturing the complementary primers.

DETAILED DESCRIPTION

[0080] Embodiments of the systems, methods, and compositions provided herein relate to the production of nucleic acid libraries. Some implementations include the ligation of single-stranded nucleic acids with adapters to obtain libraries of nucleic acids from small amounts of original nucleic acids. Some implementations include the ligation of double-stranded nucleic acids with 3'-protruding ends with adapters to obtain libraries of nucleic acids from small amounts of original nucleic acids. Some implementations include the ligation of single-stranded nucleic acids with linkers containing adapters to obtain libraries of circular nucleic acids from small amounts of original nucleic acids.

[0081] In one embodiment, single-stranded nucleic acids are produced by reducing the likelihood of self-concatemerization of reacting compounds. For example, to ligate a 3' adapter to the 3' end of a single stranded nucleic acid, the 3' adapter may contain a blocking group that inhibits self-concatemerization. Similarly, the 5' end of a single stranded nucleic acid can be dephosphorylated to inhibit self-concatemerization. The products of this ligation reaction may include a ligated single stranded nucleic acid containing a 3' blocking group. In some embodiments, ligation of the 3' adapter to the 3' end of a single stranded nucleic acid is found to be highly efficient and is performed in the presence of a volume exclusion agent. The 5' end of a ligated single stranded nucleic acid containing a 3' blocking group can be dephosphorylated so that a 5' adapter can be ligated to its 5' end. For ligation of the 5' adapter to the 5' end of a rephosphorylated ligated single stranded nucleic acid containing a 3' blocking group, the 5' adapter may contain a dephosphorylated 5' end to inhibit self-concatemerization. The product of this ligation reaction may include a single stranded nucleic acid containing a 5' adapter and a 3' adapter. Adapters may contain sequencing primer sites, amplification primer sites, and indices. As used herein, the term "index" may include a nucleotide sequence that can be used as a molecular identifier and/or a barcode to label a nucleic acid and/or to identify a nucleic acid source. In some embodiments, the index can be used to identify a single nucleic acid or a subset of nucleic acids. In some embodiments, single-stranded nucleic acid libraries can be prepared in a single reaction vessel in a single volume. In addition, the embodiments provided herein may reduce the likelihood of primer-dimer products being generated in subsequent amplification reactions.

[0082] An example of a method according to one embodiment is shown in FIG. 1. As illustrated, the 5' ends of single-stranded target nucleic acids are dephosphorylated to avoid formation of concatemers in subsequent ligation steps. The first adapters are ligated to the 3' ends of the dephosphorylated targets using single strand ligase. To avoid the formation of concatemers of the first adapters, the 3' ends of the first adapters can be blocked. The 5' ends of the ligated targets are rephosphorylated with a kinase, and a second adapter is ligated to the 5' ends of the dephosphorylated targets using a single strand ligase, thereby forming a nucleic acid library. To avoid the formation of second adapter concatemers, the 5' ends of the second adapters may not be phosphorylated.

[0083] Another example of a method according to one embodiment is shown in FIG. 2 where the adapters are ligated to double-stranded nucleic acids with 3' overhangs. In some embodiments, the first adapter may be ligated to the 3' ends of double-stranded nucleic acids with 3' overhangs. To avoid the formation of concatemers of the first adapters, the 3' ends of the first adapters can be blocked. Double-stranded nucleic acids ligated to the first adapters can be denatured to produce a plurality of single-stranded nucleic acids. The second adapter can be ligated to the 5' ends of single stranded nucleic acids.

[0084] Another example of a method according to one implementation variant is shown in Fig.3, in which a library of nucleic acids can be obtained using adapters attached to the beads. In some such embodiments, the first adapter is ligated to the 3' ends of the dephosphorylated targets using a single strand ligase. The cited first adapters can be hybridized with capture probes attached to beads. Capture probes can be extended and a second adapter can be ligated with the extended capture probe. Unligated single strand adapters can be removed by washing off the beads. Some embodiments of obtaining a nucleic acid library, including the use of adapters attached to beads, can be used to add indexes to nucleic acid libraries. For example, a first adapter containing a first index may be attached to the bead, and a second adapter containing a second index and a capture probe may be ligated to the first adapter. A target nucleic acid hybridized to a capture probe can be extended by including a first and a second index.

[0085] Another example of a method according to one embodiment is shown in FIG. 4A, on which a library of single-stranded circular nucleic acids can be generated using a linker containing first and second adapters. In some such embodiments, the linker may include a cleavage site between the first adapter and the second adapter such that cleavage of the cleavage site of the circular nucleic acid may result in a linear nucleic acid having the first adapter at one end and the second adapter at the other end of the linear nucleic acid.

[0086] In some embodiments, a nucleic acid library can be amplified using primer sites in adapter sequences. In some embodiments, the efficiency of subsequent amplification steps may be reduced by the formation of primer-dimers. To increase the efficiency of subsequent amplification steps, unligated single strand adapters can be removed from the ligation products. The example shown in FIG. 5 and described hereinafter involves the removal of unligated single stranded first adapters by hybridization with capture probes. Another example shown in FIG. 6 and described hereinafter, involves the removal of unligated single stranded first adapters by hydrolysis using a 5'-phosphate dependent exonuclease and a 5'-deadenylase.

Obtaining single-stranded libraries

[0087] Some embodiments of the systems, methods, and compositions provided herein include methods in which adapters are ligated to target nucleic acids. Some of the embodiments provided herein for preparing the nucleic acid libraries provided herein can be successfully performed in a single reaction volume.

[0088] Adapters contain nucleic acids, for example, single-stranded nucleic acids. The adapters may contain short nucleic acids that are less than, greater than, or equal to about 5 nucleotides, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, or are in length. range between any two of the above sizes. Adapters may contain sequencing primer binding sites, amplification primer binding sites, and/or indices. For example, the adapter may contain a P5 sequence, a P7 sequence, or a complement thereof. Indexes can be used to identify the source of the nucleic acid molecule. Examples of the use and derivation of indices that can be used in the implementations proposed herein can be found in International Publications Nos. WO 2012/061832 and WO 2014/142850; and US Pat. Nos. 9,074,251, 8,829,171, each of which is incorporated herein by reference in its entirety. In some embodiments, the adapter may be modified to avoid the formation of concatemers, for example by adding blocking groups that prevent the adapter from extending at one or both ends. A blocking group is any chemical group that blocks the reaction of the compound to which it is attached in subsequent steps in the workflow, such as blocking adapter activation by ligase, reaction with phosphate, and reaction with itself. Examples of 3' blocking groups include the C3 3' spacer, dideoxynucleotide, and support attachment. Additional examples of 3' blocking groups include alkyl optionally substituted with one or more hydroxyl groups, thiol, azide, or alkyne. In some aspects, the blocking groups may have functionality that can be used in subsequent operations, such as enrichment, purification, further functionalization, and the like. Examples of 5' blocking groups include a dephosphorylated 5' nucleotide and attachment to a support (eg, where the blocking group is a support, optionally attached via a linker).

[0089] Target nucleic acids include single-stranded nucleic acids and double-stranded nucleic acids. Methods for denaturing double-stranded nucleic acids to form single-stranded nucleic acids are well known in the art and include thermal or chemical methods. Examples of target nucleic acids include DNA, eg genomic DNA or cDNA; RNA, such as mRNA, short RNA or rRNA, or hybrids of DNA and RNA. The nucleic acid may contain phosphodiester linkages and may include other types of backbone including, for example, phosphoramide, thiophosphate, thiodiphosphate, O-methylphosphoroamidite, and peptide backbones and nucleic acid linkages. The nucleic acid may contain any combination of deoxyribo- and ribonucleotides and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxantanine, isocytosine, isoguanine, and base analogs such as nitropyrrole (including 3-nitropyrrole) and nitroindole (including 5-nitroindole), etc. In some embodiments, the nucleic acid may include at least one random base. A random base may be a base pair containing more than one of different types of bases and may be useful, for example, when included in oligonucleotide primers or inserts used for random hybridization in complex nucleic acid samples, for example, genomic DNA samples. An example of a random base includes inosine, which can pair with adenine, thymine, or cytosine. Other examples include hypoxanthine, 5-nitroindole, acyl 5-nitroindole, 4-nitropyrazole, 4-nitroimidazole, and 3-nitropyrrole. Random bases can be used that can pair with at least two, three, four or more types of bases.

[0090] Target nucleic acids may include a sample in which the average double-stranded nucleic acid size is less than, greater than, or equal to about 2 kb, 1 kb, 500 bp, 400 bp ., 200 bp, 100 bp, 50 bp or lies between any two of the above dimensions. In some embodiments, the average size of a single-stranded nucleic acid in a sample is less than, greater than, or equal to about 2000 nucleotides, 1000 nucleotides, 500 nucleotides, 400 nucleotides, 200 nucleotides, 100 nucleotides, 50 nucleotides, or between any two of the above size values. The sample may contain target nucleic acids in an amount less than, greater than or equal to approximately 50 μg, 10 μg, 5 μg, 1 μg, 500 ng, 400 ng, 200 ng, 100 ng, 50 ng, 10 ng, or in the range between any two of the above quantities. Target nucleic acids can be obtained from low quality sources of nucleic acids, such as a sample that has been degraded, such as a fixed sample and an ancient sample. Examples of fixed samples include samples fixed with compounds such as formalin, glutaraldehyde, alcohol, osmic acid, and paraformaldehyde. The fixed sample may include a formalin-fixed, paraffin-embedded (FFPE) sample. Ancient specimens may include specimens that are over 5 years old, 10 years old, 20 years old, 50 years old, 100 years old, 500 years old, 1000 years old, or between any two of the above ages.

[0091] Methods for dephosphorylation of nucleic acids, for example, the 5'-nucleotide of the nucleic acid, include bringing the nucleic acid into contact with a phosphatase. Examples of phosphatases include calf intestinal phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase, and APEX alkaline phosphatase (Epicenter, Madison, Wisconsin, USA).

[0092] Methods for ligating single-stranded nucleic acids include bringing the single-stranded nucleic acids into contact with a ligase. Examples of single strand ligases include T4 RNA ligase 1, T4 RNA ligase 2, RtcB ligase, Methanobacterium RNA ligase, and TS2126 RNA ligase (CIRCLIGASE; Epicenter, Madison, WI, USA). Methods for ligating two nucleic acids to each other can be carried out in a reaction volume in the presence of a volume exclusion agent. As used herein, the term "volume exclusion agent" may include agents that reduce the effective volume in which a reaction, such as a ligation reaction, can occur. Examples of volume exclusion agents include polymers, such as inert polymers such as polyethylene glycol (PEG), ficoll, dextran, hydroxyethyl starch, and polyvinylpyrrolidone. Examples of PEGs that can be used in the embodiments provided herein include PEG 600, PEG-800, PEG-1000, PEG-6000, and PEG-8000. The reaction volume may contain a concentration of the agent causing the volume exclusion effect, for example, in weight-volume percent, which is less than, greater than or equal to about 5%, 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 95%, or between any two of the above percentages.

[0093] Methods for phosphorylation of nucleic acids, for example, the 5'-nucleotide of the nucleic acid, include bringing the nucleic acid into contact with a kinase. Examples of kinases include T4 polynucleotide kinase.

[0094] Some embodiments provided herein can be performed in a single reaction volume. In some embodiments, the nucleic acid production method can be performed over a reduced period of time, such as less than about 12 hours, less than about 6 hours, or less than about 3 hours.

(SEQ ID NO: 02: tcggtggtcgccgtatcatt); последовательность праймера Р7 (SEQ ID NO: 03: caagcagaagacggcatacga); и последовательность праймера

(SEQ ID NO: 04: tcgtatgccgtcttctgcttg).[0095] FIG. 1 depicts an embodiment in which a double-stranded target DNA fragment is dephosphorylated and denatured to form a single-stranded target DNA. The first adapter, 64 bp, containing the phosphorylated 5' end, the P7' sequence, and the blocked 3' end, is ligated to the 3' end of the single-stranded target DNA to form ligated DNA using a ligase such as CIRCLIGASE™ (Lucigen Corporation, Middleton, Wisconsin, USA). The 5' end of the ligated DNA is phosphorylated using a kinase such as T4 polynucleotide kinase. A second 60 bp adapter containing the P5 sequence is ligated to the 5'-phosphorylated ligated DNA to form a member (5'-P5-ssDNA-insert-P7-3') of the DNA library. Examples of primer sequences may include: P5 primer sequence (SEQ ID NO: 01: aatgatacggcgaccaccga); primer sequence

(SEQ ID NO: 02: tcggtggtcgccgtatcatt); primer sequence P7 (SEQ ID NO: 03: caagcagaagacggcatacga); and primer sequence

(SEQ ID NO: 04: tcgtatgccgtcttctgcttg).

[0096] In some embodiments, the method for obtaining a nucleic acid library may include dephosphorylation of the 5' ends of single-stranded target nucleic acids to avoid the formation of concatemers in subsequent ligation steps; ligating the first adapters to the 3' ends of the dephosphorylated targets using single strand ligase, the 3' ends of the first adapters being blocked; rephosphorylation of the 5' ends of ligated targets; ligating the second adapter to the 5' ends of the dephosphorylated targets using a single strand ligase, the 5' ends of the second adapters being unphosphorylated, resulting in a nucleic acid library. In some embodiments, the second adapter is attached to a support. In some embodiments, the support includes a bead.

[0097] Some implementations involve removing the unligated single stranded first adapters before ligating the second adapter to the 5' ends of the dephosphorylated targets. In some embodiments, non-ligated single stranded first adapters can be hybridized to a capture probe. In some embodiments, the capture probe contains a sequence that is complementary to at least a portion of the first adapter. In some such embodiments, hybridization to the capture probe inhibits ligation of unligated first adapters to single stranded second adapters. In some embodiments, the 3' end of the capture probe contains a blocking group. In some embodiments, the 5' end of the capture probe contains a blocking group. In some embodiments, unligated single stranded first adapters can be removed by hydrolysis. In some embodiments, hydrolysis of the unligated single strand first adapters comprises contacting the unligated single strand first adapters with a 5'-phosphate dependent exonuclease, and optionally a 5'-deadenylase.

[0098] FIG. 2 depicts another embodiment in which a double-stranded target nucleic acid is partially hydrolyzed with a 5' exonuclease to form a double-stranded nucleic acid with single-stranded 3' overhangs. The first adapter, 64 bp, containing the phosphorylated 5' end, the P7' sequence and the blocked 3' end, is ligated to the 3' ends of the double-stranded DNA target to form ligated DNA using a ligase such as CIRCLIGASE™. The ligated DNA is denatured to form single-stranded nucleic acids. A second 60 bp adapter containing the P5 sequence is ligated to the 5'-phosphorylated ligated DNA to form a member (5'-P5-ssDNA-insert-P7-3') of the DNA library.

[0099] In some embodiments, a method for obtaining a nucleic acid library may include contacting double-stranded target nucleic acids with a 5' exonuclease to produce a plurality of modified double-stranded nucleic acids with single-stranded 3' overhangs; ligating the first adapter to the 3' ends of the modified double-stranded nucleic acids in the presence of a ligase and a volume exclusion agent, wherein the 3' end of the first adapter contains a blocking group; denaturing the modified double-stranded nucleic acids ligated to the first adapters to produce a plurality of single-stranded nucleic acids; and ligating the second adapter to the 5' ends of the single stranded nucleic acids in the presence of a ligase, whereby a nucleic acid library is obtained.

[0100] In FIG. 3 depicts an embodiment in which the single stranded DNA target is dephosphorylated using alkaline phosphatase. The first adapter, containing the phosphorylated 5' end, the P5' sequence and the 3' blocked end, is ligated to the 3' end of the single stranded target DNA to form ligated DNA using a ligase such as CIRCLIGASE™. The ligated DNA is hybridized through the first adapter to a capture probe containing the P5 sequence attached to the bead. The capture probe is extended to form the extended DNA, and the yogized DNA is removed from the extended DNA. The second adapter, containing the phosphorylated 5' end, the P7' sequence and the blocked 3' end, is ligated to the 3' end of the extended DNA using a ligase such as CIRCLIGASE™ to form a member (P5-insert'-P7') a DNA library attached to the bead.

[0101] In some embodiments, a method for obtaining a nucleic acid library may include dephosphorylation of the 5' ends of single-stranded target nucleic acids; ligating the first adapter to the 3' ends of the single stranded nucleic acids in the presence of a ligase and a volume exclusion agent, wherein the 3' end of the first adapter contains a blocking group; hybridizing the ligated first adapter to the capture probe; extending the capture probe; and ligating the second adapter to the 3' end of the extended capture probe in the presence of a ligase, wherein the 3' end of the second adapter contains a blocking group, whereby a nucleic acid library is obtained. Some embodiments also include removing the hybridized ligated first adapter from the extended capture probe. In some embodiments, the capture probe is attached to the substrate. In some embodiments, the capture probe substrate is a flow cell, bead, glass, controlled pore glass, plastic, silicon, quartz glass, silicon dioxide, silicon nitride, silicon derivatives, gallium arsenide, indium phosphide, aluminum, ceramic, polyimide , quartz, resins, or a polymer or copolymer (eg, polystyrene). The substrates may consist of layers of several materials or a mixture of materials. The substrates may be rigid or semi-rigid. The substrates may be flat, round or textured. In some embodiments, the substrates may be a rigid material, such as glass and/or silica derivatives, supported by one or more polymeric materials. In some embodiments, the support includes a bead. In some embodiments, the capture probe contains an index of the capture probe. In some embodiments, the capture probe index indicates the source of the capture probe. In some embodiments, the capture probe contains a cleavable linker. Some embodiments also include cleavage of the cleavable linker. In some embodiments, the capture probe is attached to a support, such as a flow cell (see FIG. 26). and its subsequent elongation occurs on the substrate. If the support is a flow cell, the resulting library can be directly sequenced. In some embodiments, the method further comprises hybridizing the complementary primers to the extended capture probe, ligating the P7' adapter containing the 3' blocking group to the 3' end of the extended capture probe, and denaturing the complementary primers as shown, for example, in FIG. 26.

[0102] In FIG. 4A depicts an embodiment in which a double-stranded target DNA fragment is dephosphorylated and denatured to form a single-stranded target DNA. An adapter is provided, containing from the 5' end to the 3' end: a phosphorylated 5' end, a P7' sequence, a cleavable site ("U"), a P5 sequence, and a blocked 3' end ("R"). The adapter is ligated with a ligase to the 3' end of the single-stranded target DNA to form ligated DNA. The 5' end of the ligated DNA is phosphorylated by a kinase. The blocked 3' end of the ligated DNA is deprotected and the ligated DNA is closed in a ring by ligation with a ligase to form a library member containing circular single stranded DNA. In some embodiments, the implementation of the circular nucleic acid can be used to obtain single-stranded nucleic acids in any of a variety of ways. Some exemplary implementations are shown in FIG. 4B, in which a circular nucleic acid can be linearized by cleaving a cleavage site, which may contain a uracil residue. The circular nucleic acid can also be used to generate single stranded nucleic acids by amplification, such as PCR, using primer sites within an adapter. Circular nucleic acid can also be amplified by rolling ring amplification (RCA).

[0103] In some embodiments, a method for obtaining a nucleic acid library may include: dephosphorylation of the 5' ends of single-stranded target nucleic acids; ligation of the linker with the 3' ends of single-stranded nucleic acids, and the linker contains a first adapter and a second adapter, and the 3' end of the linker contains a blocking group; phosphorylation of the 5' ends of ligated single-stranded nucleic acids; deprotection of the 3' ends of the phosphorylated nucleic acids; and closure of the deprotected nucleic acids into a ring by ligation, whereby a library of circular nucleic acids is obtained.

[0104] In some implementations, the linker contains a cleavable site between the first adapter and the second adapter. In some such embodiments, cleavage of the cleavable site of a circular nucleic acid may result in a linear nucleic acid having a first adapter at one end and a second adapter at the other end of the linear nucleic acid. In some embodiments, cleavable sites include restriction sites, sites containing at least one uracil residue, and sites containing atypical bases such as 8-oxoguanine and dithiol groups. Some embodiments include linearizing circular nucleic acids by cleavage at a cleavable site. Examples of cleavage of a cleavable site include bringing said site into contact with a uracil-specific excision reagent, for example an enzyme selected from uracil DNA glycosylase (UDG) and DNA glycosylase lyase VTII endonuclease, or a mixture thereof, or bringing said site into contact with restriction endonuclease.

[0105] Some embodiments also include the amplification of circular nucleic acids by including hybridizable primers in the first and second adapters. Examples of amplification methods include PCR, rolling ring amplification (RCA), and cluster amplification. In some such embodiments, RCA-like amplification may be performed in solution, or circular single-stranded nucleic acids may be captured on a surface, such as the surface of a flow cell, and amplified. In some embodiments, amplification comprises contacting the circular nucleic acids with a polymerase that forms a linear product upon contact with a uracil residue in the template. Examples of polymerases include a proofreading polymerase like Pyrococcus polymerase, such as PHUSION DNA polymerase.

[0106] Some implementation options include a method for obtaining a library of single-stranded nucleic acids, including: (a) obtaining a plurality of single-stranded nucleic acids; (b) contacting the single-stranded nucleic acids with APEX alkaline phosphatase, thereby dephosphorylation of the 5' ends of the single-stranded nucleic acids; (c) ligation of the first adapter to the 3' ends of single-stranded nucleic acids in a reaction volume containing TS2126 RNA ligase (Lucigen Corporation, Middleton, WI, USA) and at least 45% (w/v) polyethylene glycol (PEG) , and the 3'-end of the first adapter contains a 3'-spacer blocking group C3; (d) contacting the ligated single-stranded nucleic acids with T4 polynucleotide kinase, thereby phosphorylation of the 5' ends of the ligated single-stranded nucleic acids; and (e) ligating the second adapter to the 5' ends of the phosphorylated ligated single stranded nucleic acids in the presence of ligase and PEG, with the 5' end of the second adapter not being phosphorylated, thereby obtaining a nucleic acid library.

[0107] Some implementation options include a method for obtaining a library of single-stranded nucleic acids, including: (a) obtaining a plurality of double-stranded nucleic acids; (b) contacting the double-stranded nucleic acids with a 5' exonuclease to produce a plurality of modified double-stranded nucleic acids with single-stranded 3' overhangs; (c) ligating the first adapter to the 3' ends of the modified double-stranded nucleic acids in a reaction volume containing TS2126 RNA ligase and at least 45% (w/v) polyethylene glycol (PEG), wherein the 3' end of the first adapter contains 3 '-spacer blocking group C3; (d) denaturing the modified double-stranded nucleic acids ligated to the first adapters to produce a plurality of single-stranded nucleic acids; and (e) ligating the second adapter to the 5' ends of the single stranded nucleic acids in the presence of a ligase and PEG, wherein the 5' end of the second adapter is not phosphorylated, thereby obtaining a nucleic acid library.

[0108] Some implementation options include a method for obtaining a library of single-stranded nucleic acids, including: (a) obtaining a plurality of single-stranded nucleic acids; (b) contacting the single-stranded nucleic acids with APEX alkaline phosphatase, thereby dephosphorylation of the 5' ends of the single-stranded nucleic acids; (c) ligating the first adapter to the 3' ends of single-stranded nucleic acids in a reaction volume containing TS2126 RNA ligase and at least 45% (w/v) polyethylene glycol (PEG), wherein the 3' end of the first adapter contains the 3' - spacer blocking group C3; (d) contacting the ligated single-stranded nucleic acids with T4 polynucleotide kinase, thereby phosphorylation of the 5' ends of the ligated single-stranded nucleic acids; (e) removing the unligated single-stranded first adapters from the ligated single-stranded nucleic acids by hybridizing the single-stranded first adapters to a capture probe, wherein the capture probe contains: at least a portion of the first adapter; and (f) ligating the second adapter to the 5' ends of the phosphorylated ligated single stranded nucleic acids in the presence of ligase and PEG, with the 5' end of the second adapter not being phosphorylated, thereby obtaining a nucleic acid library.

[0109] Some embodiments include a method for obtaining a library of nucleic acids attached to beads, including: (a) obtaining a plurality of single-stranded nucleic acids; (b) bringing the single-stranded nucleic acids into contact with APEX alkaline phosphatase, thereby dephosphorylation of the 5' ends of the single-stranded nucleic acids; (c) ligating the first adapter to the 3' ends of the single stranded nucleic acids in a reaction volume containing TS2126 RNA ligase and at least 45% (w/v) polyethylene glycol (PEG), wherein the 3' end of the first adapter contains the 3' - spacer blocking group C3; (d) hybridizing the ligated first adapter to a capture probe, wherein the capture probe is attached to the bead; (e) extending the capture probe; and (f) ligating the second adapter to the 3' end of the extended capture probe in the presence of ligase and PEG, the 5' end of the second adapter not being phosphorylated, thereby obtaining a nucleic acid library.

[0110] Some embodiments include a method for obtaining a library of indexed nucleic acids, comprising: (a) obtaining a plurality of first single-stranded nucleic acids containing a first index, wherein the 5' ends of the first single-stranded nucleic acids are attached to a plurality of beads, and a plurality of second single-stranded nucleic acids containing a second index and a capture probe, with the 3'-ends of the second single-stranded nucleic acids containing a 3'-spacer blocking group C3; (b) ligating the 3' ends of the first single stranded nucleic acids to the 5' ends of the second single stranded nucleic acids in a reaction volume containing TS2126 RNA ligase and at least 45% (w/v) polyethylene glycol (PEG); (c) hybridizing the target nucleic acid with the capture probe; and (d) extending the target nucleic acid to obtain a library of indexed nucleic acids.

Removal of unligated single strand adapters

[0111] Some embodiments of the systems, methods, and compositions provided herein involve the removal of single-stranded unligated adapters from ligated adapters, eg, adapters ligated to single-stranded nucleic acids. In some embodiments, the removal of single-stranded non-ligated adapters from ligated adapters may increase the efficiency of amplification of the resulting nucleic acid library, for example, reduce the likelihood of generation or the number of primer-dimers in subsequent amplification steps.

[0112] Some embodiments include systems or methods for removing single strand unligated adapters and may include hybridizing the single strand unligated adapters to a capture probe. In FIG. 5 depicts an embodiment in which a double-stranded target DNA fragment is dephosphorylated and denatured to form a single-stranded target DNA. The first adapter, 64 bp, containing the phosphorylated 5' end, the P7' sequence, and the blocked 3' end, is ligated to the 3' end of the single-stranded target DNA to form ligated DNA. The 5' end of the ligated DNA is phosphorylated using a kinase such as T4 polynucleotide kinase. The unligated first adapters are removed from the ligated DNA by hybridization with a capture probe containing the P7 sequence. A second 60 bp adapter containing the P5 sequence is ligated to the 5'-phosphorylated ligated DNA to form a member (5'-P5-ssDNA-insert-P7-3') of the DNA library. In some such embodiments, hybridization with a bank capture probe may inhibit ligation of unligated first adapters to single-stranded second adapters. In some embodiments, the capture probe may contain a complementary sequence of at least a fragment of a single-stranded non-ligated adapter. In some embodiments, the capture probe may include a 3' and/or 5' blocking group to avoid the formation of concatemers. In some embodiments, the capture probe may be attached to a support, such as a bead.

[0113] Some implementations with the removal of single-stranded non-ligated adapters may include hydrolysis of single-stranded non-ligated adapters. Some such implementation options may include contacting single-stranded unligated adapters with a nuclease, eg, a 5'-phosphate dependent exonuclease, eg, TERMINATOR (Epicentre, Madison, Wisconsin, USA). In FIG. 6 depicts an embodiment in which a double-stranded target DNA fragment is dephosphorylated and denatured to form a single-stranded target DNA. The first adapter, 64 bp, containing the phosphorylated 5' end, the P7' sequence, and the blocked 3' end, is ligated to the 3' end of the single-stranded target DNA to form ligated DNA. Unligated first adapters are removed by hydrolysis with 5'-phosphate dependent exonuclease and 5'-deadenylase. The 5' end of the ligated DNA is phosphorylated using a kinase such as T4 polynucleotide kinase. A second 60 bp adapter containing the P5 sequence is ligated to the 5'-phosphorylated ligated DNA to form a member (5'-P5-ssDNA-insert-P7-3') of the DNA library.

[0114] Some implementations with the removal of single-stranded non-ligated adapters may include the use of beads. The preferred use of the granules may include washing the reaction components from the granules and the components attached to the granules. In some embodiments, the first adapter may be ligated to the 3' ends of the dephosphorylated single stranded nucleic acids. The first adapter may contain a 3' blocking group and may contain target sequences. Target sequences can be hybridized to capture probes attached to a support. In some embodiments, the support may include a bead. The capture probe may contain sequencing primer sites, indexes, and amplification primer sites. Single stranded unligated adapters can be laundered from support and hybridized ligated single stranded nucleic acids. The capture probe can be extended. In some embodiments, hybridized non-ligated single-stranded nucleic acids can be removed from the extended capture probe. The second adapter can be ligated to the 5' ends of the extended capture probes. In some embodiments, the second adapters contain a blocking group at the 3' ends. The second adapter may contain sequencing primer sites, indexes, and amplification primer sites.

Getting Indexed Libraries

[0115] Some embodiments of the systems, methods, and compositions provided herein include methods for obtaining an indexed nucleic acid library. Capture adapters and/or probes may contain indexes. Examples of the use and derivation of indices that can be used in the implementations proposed herein can be found in International Publications Nos. WO 2012/061832 and WO 2014/142850; and US Pat. Nos. 9,074,251, 8,829,171, each of which is incorporated herein by reference in its entirety. In some embodiments, the index may be incorporated into the target nucleic acid by ligation. In some embodiments, the index can be incorporated into the nucleic acid by extending the capture probe, adapter, or target nucleic acid. Thus, indexed libraries are easy to obtain using the methods proposed in this document.

[0116] In some embodiments, a population of capture probes can be obtained. In some embodiments, the capture probe can be obtained by ligating the first adapter to the second adapter. Each adapter may contain an index. The first adapter can be attached to a support, such as a bead. The second adapter may contain a hybridization probe. The hybridization probe can be hybridized to single stranded target nucleic acids. Single-stranded target nucleic acids can be hybridized to a hybridization probe and extended, thereby including sequences that are complementary to an index of one or more adapters.

[0117] Some implementations may include obtaining a plurality of first adapters containing a first index, with the 5' ends of the first adapters containing a 5' blocking group, and a plurality of second adapters containing a second index and a capture probe, with the 3' ends of the second adapters contain a 3' blocking group. Some embodiments also include ligating the 3' ends of the first single stranded nucleic acids to the 5' ends of the second single stranded nucleic acids in the presence of a ligase. Some embodiments also include hybridization of the target nucleic acid with the capture probe. Some embodiments also include extension of target nucleic acids, thereby obtaining a library of indexed nucleic acids. Some embodiments also include amplification of extended target nucleic acids. In some embodiments, the 5' blocking group contains a dephosphorylated nucleotide or is attached to a support. In some embodiments, the support includes a plurality of beads.

Kits, Reaction Vessels and Flow Cells

[0118] Embodiments of the systems and methods provided herein include kits, reaction vessels, and flow cells containing any one or more of the components useful for generating nucleic acid libraries by ligating adapters to nucleic acids, e.g., single-stranded nucleic acids and /or double-stranded nucleic acids. Typical components include a first adapter, wherein the 3' end of the first adapter contains a blocking group, a ligase, a volume exclusion agent, a dephosphorylating agent, a second adapter, the 3' end of the second adapter contains a blocking group, sequencing primers, amplification primers , nucleic acids containing indexes, and 5'-exonucleases. In some embodiments, the kit may include a reagent for any of the methods provided herein. In some embodiments, the kit may include a reaction vessel. In some embodiments, the kit may include a flow cell containing a reaction vessel.

[0119] Some embodiments include a reaction vessel containing a reaction volume in which the method provided herein can be carried out. In some such embodiments, the reaction vessel may include components of any step or step of the method provided herein. In some embodiments, the reaction vessel may contain a plurality of nucleic acids, for example, single-stranded nucleic acids and/or double-stranded nucleic acids; the first adapter, and the 3'-end of the first adapter contains a blocking group; ligase; and an agent causing a volume exclusion effect. In some embodiments, the reaction vessel may also contain a second adapter. In some embodiments, the reaction vessel may also contain a dephosphorylating agent.

[0120] In some embodiments, the reaction vessel may contain a reaction volume. In some embodiments, the reaction volume may contain a plurality of nucleic acids, for example, single-stranded nucleic acids and/or double-stranded nucleic acids; the first adapter, and the 3'-end of the first adapter contains a blocking group; second adapter; dephosphorylating agent; ligase; and an agent causing a volume exclusion effect. In some embodiments, the reaction volume may contain a plurality of first single-stranded nucleic acids containing a first index, wherein the 5' ends of the first single-stranded nucleic acids contain a 5'-blocking group, a plurality of second single-stranded nucleic acids containing a second index and a capture probe, with 3' - the ends of the second single-stranded nucleic acids contain a 3'-blocking group; and ligase.

[0121] In some embodiments, the first adapter is ligated to the 3' ends of a plurality of single-stranded nucleic acids, thereby forming a plurality of modified single-stranded nucleic acids. In some embodiments, the unligated first adapter is hybridized to a capture probe. In some embodiments, the capture probe contains a sequence that is complementary to at least a portion of the first adapter.

[0122] In some embodiments, the 5' end of the second adapter is non-phosphorylated. In some embodiments, the second adapter is ligated to the 3' ends of the plurality of modified single stranded nucleic acids. In some embodiments, the dephosphorylating agent comprises a phosphatase. In some embodiments, said phosphatase is inactivated. Examples of phosphatases include calf intestinal phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase, and APEX alkaline phosphatase.

[0123] In some embodiments, the first adapter and/or the second adapter may comprise a primer binding site for sequencing. Examples of sequencing primer binding sites include the P7 sequence, its complement or reverse complement; and the P5 sequence, its complement or reverse complement.

[0124] In some embodiments, the first adapter and/or the second adapter may contain an adapter index. In some implementations, the index of the first adapter is different from the index of the second adapter. In some embodiments, the adapter index indicates the source of the plurality of single-stranded nucleic acids.

[0125] Some embodiments provided herein include a reaction vessel containing a reaction volume comprising: a plurality of first single-stranded nucleic acids containing a first index, the 5' ends of the first single-stranded nucleic acids containing single-stranded nucleic acids containing a second index and a capture probe, wherein the 3' ends of the second single-stranded nucleic acids contain a 3' blocking group; and ligase.

[0126] In some embodiments, the blocking group contains a C3 3' spacer, a dideoxynucleotide, or a phosphate group. In some embodiments, the second adapter may include attachment to the substrate. Examples of the support include beads.

[0127] In some embodiments, the exclusion agent is selected from the group consisting of polyethylene glycol (PEG), dextran, hydroxyethyl starch, ficoll, and polyvinylpyrrolidone. Examples of PEGs that can be used in the embodiments provided herein include PEG 600, PEG-800, PEG-1000, PEG-6000, and PEG-8000. The reaction volume may contain a concentration of the agent causing the volume exclusion effect, for example, in weight-volume percent, which is less than, greater than or equal to about 5%, 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 95%, or between any two of the above percentages.

[0128] In some embodiments, the ligase comprises a single-stranded nucleic acid ligase. Examples of single-stranded nucleic acid ligases include T4 RNA ligase 1, T4 RNA ligase 2, RtcB ligase, Methanobacterium RNA ligase, and TS2126 RNA ligase (CIRCLIGASE).

[0129] Some embodiments provided herein include a flow cell comprising a reaction vessel as provided herein. Some embodiments include a system containing a reaction vessel as provided herein and a detector for obtaining sequencing data.

[0130] The flow cell may include a chamber with a surface through which the flow of one or more of the liquid reactants can be directed. In general, a flow cell has an inlet and an outlet to facilitate fluid flow. Examples of flow cells and associated fluid systems and detection platforms that are readily applicable to the methods of the present invention are described, for example, in Bentley et al, Nature 456:53-59 (2008), WO 04/018497; US 7057026; W091/06678; WO 071123744; US 7329492; US 7211414; US 7315019; US 7405281 and US 2008/0108082, each of which is incorporated herein by reference in its entirety.

EXAMPLES

Example 1 - Ligation of Adapters to Single Stranded Target Nucleic Acids

[0131] A four-step method for obtaining a single-stranded DNA (ssDNA) library was performed using single-stranded DNA as the target nucleic acid. In FIG. 1 provides an overview of four steps that included 5'-dephosphorylation of the target nucleic acid; ligating the 3' adapter to the 3' end of the target nucleic acid, wherein the 3' end of the 3' adapter is blocked to inhibit self-concatemerization; rephosphorylation of the 5'-dephosphorylated ligated target nucleic acid; and ligating the 5' adapter to the 5' end of the ligated target nucleic acid, the 5' end of the 5' adapter being blocked to inhibit self-concatemerization.

[0132] To verify the dephosphorylation step, the 5' end of the 60-mer single-stranded nucleic acid (3'OH-60-Phos 5') was dephosphorylated using alkaline phosphatase, APEX phosphatase (Epicenter, Madison, Wisconsin, USA) in a reaction volume with 25% polyethylene glycol (PEG). The degree of 5'-dephosphorylation was analyzed using a single-stranded ligase - CIRCLIGASE™ ligase (Lucigen Corporation, Middleton, Wisconsin, USA) to confirm that the dephosphorylated products did not form concatemers. As shown in FIG. 7, the alkaline phosphatase dephosphorylation efficiency was approximately 100%.

[0133] To test the initial ligation step, a 64-mer adapter P7' (3'C3-P7-phos 5') containing a 3' blocking group was ligated to the 3' end of a dephosphorylated 60-mer single-stranded nucleic acid (3'OH -60-OH 5') with CIRCLIGASE in a reaction volume containing 25% PEG. The 3' end of the P7' adapter was blocked to inhibit self-concatemerization. As shown in FIG. 8, the yield of this ligation step (lane 2) was 97.5%.

[0134] To test the kinase step, the 5' end of the 124-mer dephosphorylated single-stranded nucleic acid containing a ligated 64-mer adapter with a 3' blocking group (3'C3-P7-60-OH) was rephosphorylated with T4 polynucleotide kinase (PNK ). As shown in FIG. 9, the rephosphorylation yield was approximately 99%.

[0135] To test the second ligation step, a 60-mer P5 adapter with a non-phosphorylated 5' end (5'P5-OH) was ligated to the 5' end of a 124-mer single-stranded nucleic acid (3'C3-60-B2-Phos) using CIRCLIGASE in a reaction volume containing 1.6% PEG. The 124-mer single-stranded nucleic acid contained a ligated 64-mer adapter with a 3' blocking group (3'C3-P7-phos 5'). As shown in Figure 10, the yield of this ligation step (lane 2) was >85%. Thus, the overall yield of the 4-step approach to obtaining the ssDNA library was >80%.

Example 2 - Ligation of Adapters to Cell-Free Target Nucleic Acids

[0136] The circuit shown in FIG. 1 was performed using cell-free DNA (bcDNA). Double-stranded target nucleic acids were dephosphorylated using APEX alkaline phosphatase and denatured to form single-stranded target nucleic acids. To test the initial ligation step, a 64-mer P7' adapter containing a 3'-blocking group (3'C3-P7-phos 5') was ligated to the 3'-end of single-stranded dephosphorylated cell-free DNA (cDNA) using CIRCLIGASE in the reaction volume, containing 22.5% PEG for 3 hours. As shown in FIG. 11, the yield of this ligation step (lane 2) was over 95%.

[0137] To test the kinase step, dephosphorylated bcDNA (bcDNA [Apex]) was treated with T4 polynucleotide kinase (PNK) and a 64-mer P7' adapter containing a 3' blocking group (3'C3-P7-phos 5') was ligated with 3 the '-end of the rephosphorylated bcDNA. The reaction was carried out in a volume containing 11.25% PEG for 1 hour (FIG. 12).

[0138] To test the second ligation step, a 64-mer P7' adapter containing a 3' blocking group (3'C3-P7-phos 5') was ligated to the 3' end of the dephosphorylated bcDNA, resulting in the first ligated product (3'C3 -P7-bcDNA-phos 5') was treated with kinase (PNK) and a 60-mer P5 adapter with a non-phosphorylated 5' end (5'P5-OH) was ligated to the 5' end of the first ligation product to form a second ligation product (P7' -bcDNA-P5). The reaction was performed using CIRCLIGASE in a reaction volume containing 3.8% PEG. In FIG. 13 shows the ligation products of (P7'-bcDNA-P5) and (P7'-P5).

Example 3 - analysis of ligation products

[0139] The ligations from Example 2 were gel separated, the ligation bands were extracted from the gel, the extracted ligations were amplified, and the amplified products were separated by capillary electrophoresis (BioAnalyzer, Agilent).

[0140] The ligation products were separated on the gel, the "Extract B1" and "Extract B2" bands were excised from the gel. As shown in FIG. 14, in lane 'REF' extract B1 is indicated by two black boxes and extract B2 by one white box. The extracts were amplified by PCR using P7 primers not intended for PCR (non-amplifiable) in a ratio to P7 PCR primers from 1:40 to 1:20 for 24 cycles. PCR products were extracted using solid phase reversible immobilization (SPRI) on beads in a ratio of 2:1. The results are shown in FIG. fourteen.

[0141] The amplified products of extract B1 and B2 were then analyzed by capillary electrophoresis (BioAnalyser, Agilent). As shown in FIG. 15, extract B2 predominantly contained 127 bp products. (P7'-P5) and extract B1 contained 292 bp product varieties. (P7'-bcDNA-P5) and 480 bp. (P7'-bcDNA-P5).

[0142] The number of SPRI beads used to purify the extracts from the gel was changed to determine the ratio that allowed effective removal of primer-dimers (Fig. 16). The ratio of SPRI granule : sample (v : v), equal to 1.2:1, most effectively made it possible to remove primers-dimers without losing a significant part of the bcDNA library.

Example 4 Capture of unligated P7' primers

[0143] Excess unligated P7' primers were removed as shown in FIG. 5. Briefly, the P7 primer complementary to the P7' adapter primer was added to the reaction volume after the P7' adapter primer was ligated to the 3' end of the target nucleic acid. Primer P7 contained blocking groups at the 5' and 3' ends. After primer P7 was added, the P5 adapter was ligated to the 5' end of the rephosphorylated ligated target nucleic acid.

[0144] The 63-mer P7' primer (3'C3-P7'-phos 5') was ligated to the 3' ends of the bcDNA in a reaction volume containing 25% PEG. As shown in Fig. 17, the yield of the ligated product was >95%. To test ligation in the presence of a capture probe, the 60-mer P5 adapter containing the unphosphorylated 5' end (5'P5-OH) was ligated to the 5' end of the first ligation product (3'C3-P7'-bcDNA-phos5') in the presence of a 63-mer P7 capture probe (3'C3-P7-C35') containing 5'- and 3'-blocking groups. The reaction was carried out in a reaction volume containing 6.5% PEG. In FIG. 18 shows the ligation products of (P7'-bcDNA-P5) and (P7'-P5).

[0145] The ligation products were then analyzed. As shown in FIG. 19, the ligation products were separated by size in the gel, some bands were extracted from the gel, extract B1 and extract B2 were amplified. The amplified products were analyzed by capillary electrophoresis; they contained 127 bp product varieties. (P7'-P5), 292 bp (P7'-bcDNA-P5) and 480 bp. (P7'-bcDNA-P5). In FIG. 20 shows gels of ligation products before amplification, after amplification, and after extraction with SPRI beads.

[0146] Inclusion of P7, not intended for PCR, to capture excess P7', significantly reduced the formation of primer-dimers. In addition, the formation of primer-dimers was significantly reduced when removed using 1.2X SPRI without significant loss of the bcDNA library.

Example 5 - Ligation of Single Stranded Bead-Conjugated Nucleic Acids

[0147] The efficiency of ligation of two oligomers (oligonucleotide 1 and oligonucleotide 2) was tested at different concentrations of PEG. Oligonucleotide 1 contained a blocked 3' end (OH) and oligonucleotide 2 contained a blocked 5' end (phos), thus ligation resulted in a single ligated product oligonucleotide 1-oligonucleotide 2. Reaction conditions included: 10 pmol of each oligonucleotide + 2 µl 10 × buffer + 1 µl 1 mM ATP, 1 µl 50 mM MnCl ₂ , 1 µl CIRCLIGASE (100 U/µl) + 15 µl 0, 25, 50 or 60% PEG in a final reaction volume of 20 µl at PEG concentration 0 , 19, 38 and 45%, respectively. The results are shown in FIG. 21. The reaction volume containing 45% PEG provided a 55.9% yield of ligated oligonucleotide 1 and oligonucleotide 2.

[0148] Performed a check on the efficiency of ligation of oligonucleotide 1 and oligonucleotide 2 at different concentrations of oligonucleotide 2. Reaction conditions included: 10 pmol of oligonucleotide 1 + 10 × pmol of oligonucleotide 2 + 2 μl of 10 × buffer + 1 μl of 1 mM ATP, 1 μl of 50 mM MnCl ₂ , 2 µl CIRCLIGASE (100 U/µl) + 15 µl 60% PEG, resulting in a final reaction volume of 20 µl at a PEG concentration of 45%. The results are shown in FIG. 22. Reaction volume containing 4X excess of oligonucleotide 2 and 2X CIRCLIGASE provided 79.6% yield of ligated oligonucleotide 1 and oligonucleotide 2.

[0149] The efficiency of ligation of oligonucleotide 1 and oligonucleotide 2 was tested at various concentrations of CIRCLIGASE. Reaction conditions included: 10 pmol of each oligonucleotide + 2 µl 10×buffer + 1 µl 1 mM ATP, 1 µl 50 mM MnCl ₂ , X µl CIRCLIGASE (100 U/µl) + 15 µl 60% PEG, resulting in a final reaction volume of 20 μl at a PEG concentration of 45%. The results are shown in FIG. 23. A reaction volume containing 8X the concentration of CIRCLIGASE provided a 75.3% yield of ligated oligonucleotide 1 and oligonucleotide 2.

[0150] Oligonucleotide 1 was conjugated to the beads via a hydrazine-aldehyde coupling reaction. The 39-mer oligonucleotide 1 contained two uracil bases that could be cleaved using uracil DNA glycosylase (UDG) to produce a shorter 27-mer oligonucleotide (oligonucleotide 1*).

[0151] Various concentrations of oligonucleotide 1 conjugated to the beads were tested during the ligation reaction. Ligation of oligonucleotide 2 with conjugated oligonucleotide 1 was analyzed by post-treatment with UDG. The ligation reaction conditions assumed that the amount of oligonucleotide 1 used to attach to the beads was equal to the amount of oligonucleotide 1 on the beads. Four initial concentrations of total oligonucleotide 1 were tested: 0.5 nmol, 1 nmol, 2 nmol and 4 nmol. A 10% total bead solution was used, so the estimated amount of oligonucleotide 1 in each reaction was 0.1 nmol, 0.2 nmol, 0.3 nmol, and 0.4 nmol. Reaction mixture: 10 µmol oligonucleotide bead solution (2 x concentration), oligonucleotide 2 (0.4 nmol), 2 µl 10 x buffer, 1 µl 1 mM ATP, 1 µl 50 mM MnCl ₂ , 1 µl CIRCLIGASE (100 U/µl ) + 5 μl of 90% PEG, which provided a final reaction volume of 20 μl at a PEG concentration of 22.5%. The reaction mixture was incubated at 60°C for 3 h and inactivated at 80°C for 10 min. The ratio of oligonucleotide 1: oligonucleotide 2 in the reactions: 0.05 nmol (1:8); 0.1 nmol (1:4), 0.2 nmol (1:2), 0.4 nmol (1:1). Uracil digestion was performed using 10 μl LMX1 in a final reaction volume of 10 μl and incubated at 37° C. for 1 hour. The results are shown in FIG. 24. The reaction volume containing a 1:1 ratio of oligonucleotide 1:oligonucleotide 2 provided 85.2% yield of ligated oligonucleotide 1 and oligonucleotide 2. The analysis was repeated for final reaction volumes containing 45% PEG. The results are shown in FIG. 25. The reaction volume containing the ratio of oligonucleotide 1 : oligonucleotide 2, equal to 1:1, provided 84.5% yield of ligated oligonucleotide 1 and oligonucleotide 2.

[0152] The term "comprising", used herein as a synonym for the terms "comprising", "consisting" or "characterized", is open and does not exclude the presence of additional, not specified elements or steps of the method.

[0153] In the above description, several methods and materials according to the present invention are set forth. The present invention is subject to modifications in terms of methods and materials, as well as changes in terms of manufacturing methods and equipment. Such modifications are obvious to those skilled in the art upon consideration of the description or implementation of the present invention as described herein. Accordingly, the present invention is not to be limited to the specific embodiments described herein, but embraces all modifications and alternatives within the true spirit of the present invention.

[0154] All sources cited herein, including but not limited to published and unpublished applications, patents, and references, are incorporated herein by reference in their entirety, and form part of this document. To the extent that publications and patents or patent applications incorporated by reference conflict with the description contained herein, this document shall disregard and/or take precedence over any such conflicting material.

Claims (71)

1. A method for obtaining a library of nucleic acids, including: (a) obtaining a set of nucleic acids, and the specified set of nucleic acids is a single-stranded nucleic acids; (b) dephosphorylation of the 5' ends of single-stranded nucleic acids; (c) ligating the first adapter to the 3' ends of the single stranded nucleic acids in the presence of a ligase, wherein the 3' end of the first adapter contains a blocking group; (d) hybridization of unligated single stranded first adapters with a capture probe; (e) phosphorylation of the 5' ends of the ligated single-stranded nucleic acids; and (f) ligating the second adapter to the 5' ends of the phosphorylated ligated single stranded nucleic acids in the presence of a ligase, whereby a nucleic acid library is obtained. 2. The method according to claim 1, characterized in that the capture probe is in solution. 3. Process according to claim 1 or 2, characterized in that steps (b)-(f) are performed in a single reaction volume. 4. The method according to any one of paragraphs. 1-3, characterized in that the 5'-end of the second adapter is non-phosphorylated. 5. The method according to any one of paragraphs. 1-4, characterized in that the second adapter is attached to the substrate. 6. The method according to claim 5, characterized in that the substrate includes a bead or flow cell. 7. The method according to any one of paragraphs. 1-6, characterized in that the capture probe contains a sequence that is complementary to at least a portion of the first adapter. 8. The method according to any one of paragraphs. 1-7, characterized in that the 3' end of the capture probe contains a blocking group. 9. The method according to any one of paragraphs. 1-8, characterized in that the 5' end of the capture probe contains a blocking group. 10. The method according to any one of paragraphs. 1-9, characterized in that phosphorylation of the 5' end of the ligated single-stranded nucleic acids includes bringing the ligated single-stranded nucleic acids into contact with a kinase. 11. A method for obtaining a library of nucleic acids, including: (a) obtaining a plurality of nucleic acids, said plurality of nucleic acids being double-stranded nucleic acids; (b) contacting the double-stranded nucleic acids with a 5'-exonuclease to produce a plurality of modified double-stranded nucleic acids with single-stranded 3' overhangs; (c) ligating the first adapter to the 3' ends of the modified double-stranded nucleic acids in the presence of a ligase, wherein the 3' end of the first adapter contains a blocking group; (d) denaturing the modified double-stranded nucleic acids ligated to the first adapters to produce a plurality of single-stranded nucleic acids; and (e) ligating the second adapter to the 5' ends of the single stranded nucleic acids in the presence of a ligase, whereby a nucleic acid library is obtained. 12. The method of claim 11 wherein the 5' end of the second adapter is non-phosphorylated. 13. A method for obtaining a library of nucleic acids, including: (a) obtaining a set of nucleic acids, and the specified set of nucleic acids is a single-stranded nucleic acids; (b) dephosphorylation of the 5' ends of single-stranded nucleic acids; (c) ligating the first adapter to the 3' ends of the single stranded nucleic acids in the presence of a ligase, wherein the 3' end of the first adapter contains a blocking group; (d) hybridizing the ligated first adapter to the capture probe; (e) extending the capture probe and ligating the second adapter to the 3' end of the extended capture probe in the presence of a ligase, wherein the 3' end of the second adapter contains a blocking group, thereby obtaining a nucleic acid library. 14. The method of claim 13, comprising removing the hybridized ligated first adapter from the extended capture probe. 15. The method according to claim 13 or 14, characterized in that the capture probe is attached to the substrate. 16. The method of claim. 15, characterized in that the substrate includes a bead or flow cell. 17. The method according to any one of paragraphs. 13-16, characterized in that the capture probe contains an index of the capture probe. 18. The method according to any one of paragraphs. 13-17, characterized in that the capture probe contains a cleavable linker. 19. The method of claim 18, comprising cleavage of the cleavable linker. 20. A method for obtaining a library of nucleic acids, including: (a) obtaining a set of nucleic acids, and the specified set of nucleic acids is a single-stranded nucleic acids; (b) dephosphorylation of the 5' ends of single-stranded nucleic acids; (c) ligating the linker to the 3' ends of the single stranded nucleic acids, wherein the linker contains a first adapter and a second adapter, and the 3' end of the linker contains a blocking group; (d) phosphorylation of the 5' ends of the ligated single-stranded nucleic acids; (e) deprotection of the 3' ends of the phosphorylated nucleic acids and closure of the deprotected nucleic acids into a ring by ligation, whereby a library of circular nucleic acids is obtained. 21. The method of claim. 20, characterized in that the linker contains a cleavable site between the first adapter and the second adapter. 22. The method of claim 21, further comprising linearizing the circular nucleic acids by cleavage at a cleavable linker. 23. The method according to claim 21 or 22, characterized in that the cleavage site contains a uracil residue. 24. The method of claim 23, comprising contacting the circular nucleic acids with a uracil-specific excision reagent. 25. The method of claim 24, wherein the uracil-specific excision reagent contains an enzyme selected from uracil DNA glycosylase (UDG) and DNA glycosylase lyase endonuclease VIII. 26. The method according to any one of paragraphs. 20-25, further comprising amplifying the circular nucleic acids by a method comprising hybridizing at least a primer to a first adapter or a second adapter. 27. The method according to claim 26, characterized in that the amplification is selected from PCR and rolling ring amplification (RCA). 28. The method of claim 26, wherein the amplification comprises contacting the circular nucleic acids with a polymerase that forms a linear product upon contact with a uracil residue in the matrix. 29. The method according to any one of paragraphs. 20-28, characterized in that dephosphorylation of the 5' end of single-stranded nucleic acids involves bringing the single-stranded nucleic acids into contact with a phosphatase. 30. The method according to any one of paragraphs. 11-29, characterized in that steps (b)-(e) are performed in a single reaction volume. 31. The method according to any one of paragraphs. 11-30, characterized in that steps (b)-(e) are performed in a single reaction vessel. 32. The method according to any one of paragraphs. 1-31, characterized in that the first adapter and/or the second adapter contain a primer binding site for sequencing. 33. The method of claim. 32, characterized in that the first adapter contains a P7 sequence, a P5 sequence, or their complement or reverse complement. 34. The method of claim 32, wherein the second adapter contains a P7 sequence, a P5 sequence, or their complement or reverse complement. 35. The method according to any one of paragraphs. 1-34, characterized in that the first adapter and/or the second adapter contains an adapter index. 36. The method of claim. 35, characterized in that the index of the first adapter is different from the index of the second adapter. 37. The method according to claim 35 or 36, characterized in that the adapter index indicates the source of the plurality of nucleic acids. 38. The method according to any one of paragraphs. 1-37, characterized in that the blocking group contains a 3'-spacer C3 or dideoxynucleotide. 39. The method according to any one of paragraphs. 1-38, characterized in that the ligase contains a ligase of single-stranded nucleic acids. 40. The method according to any one of paragraphs. 1-39, characterized in that the ligation of the first adapter and/or the ligation of the second adapter is performed in the presence of an agent that causes a volume exclusion effect. 41. The method of claim 40, wherein the volume exclusion agent is selected from the group consisting of polyethylene glycol (PEG), dextran, hydroxyethyl starch, ficoll, and polyvinylpyrrolidone. 42. The method of claim 40, wherein the first ligation step and/or the second ligation step is performed in a reaction volume containing at least about 37% (w/v) PEG. 43. The method of claim 40, wherein the first ligation step and/or the second ligation step is performed in a reaction volume containing at least about 60% (w/v) PEG. 44. The method according to any one of paragraphs. 1-43, characterized in that many nucleic acids contain RNA. 45. The method according to any one of paragraphs. 1-43, characterized in that the set of nucleic acids contains cDNA or genomic DNA. 46. The method according to any one of paragraphs. 1-45, characterized in that the average nucleic acid size of the plurality of nucleic acids is less than about 200 nucleotides. 47. The method according to any one of paragraphs. 1-46, characterized in that a plurality of nucleic acids are obtained from a low quality source of nucleic acids. 48. The method according to claim 47, characterized in that a plurality of nucleic acids are obtained from a fixed sample. 49. The method according to any one of paragraphs. 1-48, further comprising amplifying the nucleic acid library. 50. The method according to any one of paragraphs. 1-49, further comprising obtaining sequence data from a nucleic acid library.

Images