Methods and compositions for analyzing nucleic acids

Abstract

The present technology relates in part to methods and compositions for analyzing nucleic acids. In some aspects, the present technology relates to methods and compositions for preparing a nucleic acid library from single-stranded nucleic acid fragments. The method includes combining (i) a nucleic acid composition comprising single-stranded nucleic acids (ssNA), (ii) a first oligonucleotide, and (iii) a plurality of first scaffold polynucleotide species, wherein (a) each polynucleotide in the plurality of first scaffold polynucleotide species may comprise an ssNA hybridization region and a first oligonucleotide hybridization region.

Description

[Technical Field]

[0001] Related patent applications This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 830,211, filed April 5, 2019, entitled "METHODS AND COMPOSITIONS FOR ANALYZING NUCLEIC ACID," naming Kelly M. Harkins Kincaid et al. as inventors, and designated by attorney docket number CBS-2002-PV. This patent application also claims the benefit of U.S. Provisional Patent Application No. 62 / 861,594, filed June 14, 2019, entitled "METHODS AND COMPOSITIONS FOR ANALYZING NUCLEIC ACID," naming Kelly M. Harkins Kincaid et al. as inventors, and designated by attorney docket number CBS-2002-PV2. This patent application also claims the benefit of U.S. Provisional Patent Application No. 62 / 925,132, filed October 23, 2019, entitled "METHODS AND COMPOSITIONS FOR ANALYZING NUCLEIC ACID," naming Kelly M. HARKINS KINCAID et al. as inventors, and designated by attorney docket number CBS-2002-PV3. The entire contents of the above-referenced application, including all text, tables, and drawings, are incorporated herein by reference.

[0002] Field The present technology relates, in part, to methods and compositions for analyzing nucleic acids. In some aspects, the present technology relates to methods and compositions for preparing nucleic acid libraries from single-stranded nucleic acid fragments. [Background technology]

[0003] background The genetic information of living organisms (e.g., animals, plants, and microorganisms) and other forms of replicating genetic information (e.g., viruses) is encoded in nucleic acids (i.e., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). Genetic information is a sequence of nucleotides or modified nucleotides that corresponds to the primary structure of a chemical or hypothetical nucleic acid.

[0004] Various high-throughput sequencing platforms are used to analyze nucleic acids. For example, the ILLUMINA platform involves clonal amplification of adaptor-ligated DNA fragments. Another platform is nanopore-based sequencing, which relies on the translocation of nucleic acid molecules or individual nucleotides through small channels. Library preparation for a particular sequencing platform often involves DNA fragmentation, fragment end modification, and adaptor ligation, and may involve amplification of nucleic acid fragments (e.g., PCR amplification).

[0005] The selection of an appropriate sequencing platform for a specific type of nucleic acid analysis requires a detailed understanding of available technologies, including error sources, error rates, and sequencing speed and cost. Although sequencing costs have decreased, the throughput and cost of library preparation can be a limiting factor. One aspect of library preparation involves modifying the ends of nucleic acid fragments so that they are suitable for a specific sequencing platform. Nucleic acid ends can contain useful information. Therefore, a method for modifying nucleic acid ends (for example, for library preparation) while preserving the information contained in nucleic acid ends is expected to be useful in nucleic acid processing and analysis.

[0006] Another aspect of library preparation involves capturing single-stranded nucleic acid fragments. In certain cases, single-stranded library preparation methods can produce better and more complex libraries compared to traditional double-stranded DNA (dsDNA) preparation methods.

[0007] The drawbacks of generating single-stranded DNA (ssDNA) libraries include labor-intensive, costly, and time-consuming protocols and the requirement for novel or custom reagents. Therefore, a method for capturing single-stranded nucleic acids (e.g., for library preparation) without the need for labor-intensive, costly, and time-consuming protocols and / or novel or custom reagents is expected to be useful for processing and analyzing nucleic acids (e.g., single-stranded nucleic acids, denatured double-stranded nucleic acids, or mixtures containing single-stranded nucleic acids).

[0008] Another aspect of library preparation involves capturing single-stranded RNA fragments. Generally, existing RNA library preparation methods require not only first-strand DNA synthesis from RNA using reverse transcriptase, but also second-strand synthesis to generate cDNA molecules compatible with downstream double-stranded sequencing adapter ligation. In many cases, it is desirable to generate a stranded RNA sequencing library so that accurate assessment can be made of the genomic DNA strand from which the transcript will be transcribed. To create a stranded RNA sequencing library, the method may include a step of degrading the second DNA strand after sequencing adapter ligation. However, performing second-strand DNA synthesis before pre-degrading the RNA is problematic because it can create third and sometimes fourth-strand synthesis by-products that convolute the resulting RNA sequencing data, partially obscuring the number of strands in the library. Therefore, a method for capturing single-stranded RNA (e.g., for library preparation) without the need for second-strand synthesis is expected to be useful in processing and analyzing nucleic acids containing RNA. Summary of the Invention [Means for solving the problem]

[0009] Abstract In some aspects, a method for generating a nucleic acid library is provided, comprising combining (i) a nucleic acid composition comprising a single-stranded nucleic acid (ssNA), (ii) a first oligonucleotide, and (iii) a plurality of first scaffold polynucleotide species, wherein (a) each polynucleotide in the plurality of first scaffold polynucleotide species comprises an ssNA hybridization region and a first oligonucleotide hybridization region; and (b) the nucleic acid composition, the first oligonucleotide, and the plurality of first scaffold polynucleotide species are combined under conditions such that molecules of the first scaffold polynucleotide species hybridize with (i) the first ssNA terminal region and (ii) molecules of the first oligonucleotide, thereby forming hybridization products in which the ends of the molecules of the first oligonucleotide are adjacent to the ends of the first ssNA terminal region. In some embodiments, the method includes, prior to the binding step, contacting the first oligonucleotide and / or the plurality of first scaffold polynucleotide species with an agent having phosphatase activity under conditions in which the first oligonucleotide and / or the plurality of first scaffold polynucleotide species are dephosphorylated, thereby generating dephosphorylated first oligonucleotides and / or dephosphorylated first scaffold polynucleotide species. In some embodiments, the method is an SSB-free method for generating a library from ssNAs.

[0010] In some aspects, compositions are also provided that include a nucleic acid composition comprising a single-stranded nucleic acid (ssNA), a first oligonucleotide, and a plurality of first scaffold polynucleotide species, each of which comprises an ssNA hybridization region and a first oligonucleotide hybridization region.

[0011] In some embodiments, a kit is also provided that includes a first oligonucleotide, a plurality of first scaffold polynucleotide species, each of which comprises an ssNA hybridization region and a first oligonucleotide hybridization region, and instructions for using the first oligonucleotide and the plurality of first scaffold polynucleotide species to generate a nucleic acid library.

[0012] In some aspects, methods for generating a nucleic acid library are provided, comprising combining (i) a nucleic acid composition comprising single-stranded ribonucleic acid (ssRNA) or single-stranded complementary deoxyribonucleic acid (sscDNA), (ii) a first oligonucleotide, and (iii) a plurality of first scaffold polynucleotide species, wherein (a) each polynucleotide in the plurality of first scaffold polynucleotide species comprises an ssRNA or sscDNA hybridization region and a first oligonucleotide hybridization region; and (b) the nucleic acid composition, the first oligonucleotide, and the plurality of first scaffold polynucleotide species are combined under conditions such that molecules of the first scaffold polynucleotide species hybridize with (i) the first ssRNA or sscDNA terminal region and (ii) molecules of the first oligonucleotide, thereby forming hybridization products in which the ends of the molecules of the first oligonucleotide are adjacent to the ends of the first ssRNA or sscDNA terminal region. In some embodiments, the method includes, prior to the binding step, contacting the first oligonucleotide and / or the plurality of first scaffold polynucleotide species with an agent having phosphatase activity under conditions in which the first oligonucleotide and / or the plurality of first scaffold polynucleotide species are dephosphorylated, thereby generating dephosphorylated first oligonucleotides and / or dephosphorylated first scaffold polynucleotide species. In some embodiments, the method is an SSB-free method for generating a library from ssRNA or sscDNA.

[0013] In some embodiments, compositions are also provided that include a nucleic acid composition comprising a single-stranded ribonucleic acid (ssRNA) or a single-stranded complementary deoxyribonucleic acid (sscDNA), a first oligonucleotide, and a plurality of first scaffold polynucleotide species, each of which comprises an ssRNA or sscDNA hybridization region and a first oligonucleotide hybridization region.

[0014] In some embodiments, kits are also provided that include a first oligonucleotide, a plurality of first scaffold polynucleotide species, each of which comprises an ssRNA or sscDNA hybridization region and a first oligonucleotide hybridization region, and instructions for generating a nucleic acid library from ssRNA or sscDNA using the first oligonucleotide and the plurality of first scaffold polynucleotide species.

[0015] Certain embodiments are further described in the following description, examples, claims and drawings.

[0016] The drawings are illustrative of certain embodiments of the present technology, but are not limiting. For clarity and ease of illustration, the drawings have not been made to scale, and in some instances, various aspects may be shown exaggerated or enlarged to facilitate understanding of particular embodiments. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 shows the general workflow for one particular library preparation method described herein. The solid chevrons at the ends of the P5 adaptors, the P7 adaptors, and the P5 / P7 scaffold polynucleotides represent blocking modifications.

[0018] [Figure 2A] FIG. 2A shows molecular QC metrics for ssDNA libraries generated with and without the hyperthermostable single-stranded DNA binding protein (ET SSB). [Figure 2B] Figure 2B shows ILLUMINA HISEQ sequencing metrics for ssDNA libraries generated with and without ET SSB. A two-sample equal variance t-test showed no significant differences in sequencing metrics.

[0019] [Figure 3A] Figure 3A shows the ssDNA final library product size distribution from 1 ng of cell-free DNA generated with (top) and without (bottom) the presence of SSB. [Figure 3B] Figure 3B shows the overlap of insert length distributions inferred from sequencing data of two cfDNA libraries (generated with and without SSB).

[0020] [Figure 4] Figure 4 shows the library yield (top) and % adapter dimers (bottom) measured by Qubit fluorometer after pretreatment with rapid shrimp alkaline phosphatase (rSAP) to dephosphorylate the P5 / P7 adapters. After rSAP treatment, the library yield increases and the amount of artifacts due to adapter dimers decreases.

[0021] [Figure 5A] Figures 5A and 5B show a PCR-free approach involving ligation of fully indexed sequencing adapters and scaffolds during the ssDNA library ligation step. Such adapters may or may not contain unique molecular identifiers (UMIs). [Figure 5B] Figures 5A and 5B show a PCR-free approach involving ligation of fully indexed sequencing adapters and scaffolds during the ssDNA library ligation step. Such adapters may or may not contain unique molecular identifiers (UMIs).

[0022] [Figure 6A] Figures 6A and 6B show scaffold adapter configurations with unique molecular identifiers (UMIs). Figure 6A shows a configuration in which the UMI is adjacent to the template. Figure 6B shows a configuration in which the UMI is adjacent to the index polynucleotide. [Figure 6B] Figures 6A and 6B show scaffold adapter configurations with unique molecular identifiers (UMIs). Figure 6A shows a configuration in which the UMI is adjacent to the template. Figure 6B shows a configuration in which the UMI is adjacent to the index polynucleotide.

[0023] [Figure 7] Figure 7 shows an example of incorporation of a unique molecular identifier (UMI) and P7 indexing adapter after scaffold adapter ligation prior to index PCR using single primer extension.

[0024] [Figure 8] FIG. 8 shows an example of a hairpin scaffold adaptor design.

[0025] [Figure 9] Figure 9 shows an example workflow with stepwise ligation and enzymatic delay. X, blocking modification; 5'P, 5' phosphate; 5'OH, 5' hydroxyl; 3'OH, 3' hydroxyl; N, any nucleotide.

[0026] [Figure 10] Figure 10 shows an example of a workflow using stepwise ligation using a P7 scaffold adapter with a 5'App modification and an ATP delay. X, blocking modification. 5'App, 5'-adenylated DNA. 5'P, 5' phosphate. 3'OH, 3' hydroxyl. N, any nucleotide.

[0027] [Figure 11]Figure 11 shows an example of a workflow using stepwise ligation using a single-stranded P5 adapter with a 3' phosphorus strand. X, blocking modification. 3'P, 3' phosphate. 5'P, 5' phosphate. 3'OH, 3' hydroxyl. 5'OH, 5' hydroxyl. N, any nucleotide.

[0028] [Figure 12-1] Figure 12 shows tapestry traces of four ssDNA libraries generated from first-strand cDNA synthesized using random hexamer, octamer, and anchored poly-T primers and Mu-MLV reverse transcriptase (in-house protocol) or the New England Biolabs Ultra II RNA First Strand Synthesis Module (commercial protocol). i and ii are technical replicates of the in-house protocol, and iii and iv are technical replicates of the commercial protocol. [Figure 12-2] Figure 12 shows tapestry traces of four ssDNA libraries generated from first-strand cDNA synthesized using random hexamer, octamer, and anchored polyT primers and Mu-MLV reverse transcriptase (in-house protocol) or the New England Biolabs Ultra II RNA First Strand Synthesis Module (commercial protocol). i and ii are technical replicates of the in-house protocol, and iii and iv are technical replicates of the commercial protocol. [Figure 12-3] Figure 12 shows tapestry traces of four ssDNA libraries generated from first-strand cDNA synthesized using random hexamer, octamer, and anchored polyT primers and Mu-MLV reverse transcriptase (in-house protocol) or the New England Biolabs Ultra II RNA First Strand Synthesis Module (commercial protocol). i and ii are technical replicates of the in-house protocol, and iii and iv are technical replicates of the commercial protocol. [Figure 12-4]Figure 12 shows tapestry traces of four ssDNA libraries generated from first-strand cDNA synthesized using random hexamer, octamer, and anchored polyT primers and Mu-MLV reverse transcriptase (in-house protocol) or the New England Biolabs Ultra II RNA First Strand Synthesis Module (commercial protocol). i and ii are technical replicates of the in-house protocol, and iii and iv are technical replicates of the commercial protocol.

[0029] [Figure 13] Figure 13 shows molecular metrics of four ssDNA libraries generated from first-strand cDNA synthesized using random hexamer, octamer, and anchored poly-T primers and Mu-MLV reverse transcriptase (in-house protocol) or the New England Biolabs Ultra II RNA First Strand Synthesis Module (commercial protocol). Top panel: Gel image of the final RNA-Seq library. Bottom panel: Table showing library yield (ng / μl) and size (bp). i and ii are technical replicates of the in-house protocol, and iii and iv are technical replicates of the commercial protocol.

[0030] [Figure 14]Figure 14 provides a table showing sequencing metrics generated using STAR Aligner for ssDNA libraries generated from first-strand cDNA synthesized using either an in-house protocol (random hexamer, octamer, and anchored poly-T primers and Mu-MLV reverse transcriptase) or a commercial protocol (New England Biolabs Ultra II RNA First Strand Synthesis Module). Typical RNA-Seq metrics for high-quality libraries may include a) uniquely mapped reads for 70-90% of the library and b) approximately 5% of reads mapped to multiple loci (such metrics are shown in gray). The data provided in the table demonstrate successful RNA-seq library generation.

[0031] [Figure 15] Figure 15 shows a schematic overview of the method for first-strand preparation of RNA-Seq, as well as details of the single-strand scaffold adaptor ligation technique after first-strand cDNA synthesis.

[0032] [Figure 16] FIG. 16 shows a schematic overview of the method for single-strand preparation of RNA-Seq, as well as details of the single-stranded scaffold adapter ligation technique to create DNA / RNA hybrids, followed by cDNA first-strand synthesis.

[0033] [Figure 17] FIG. 17 illustrates various workflows that can be performed before or after the ssDNA / ssRNA ligation reactions described herein.

[0034] [Figure 18] FIG. 18 shows the results of a sequential 1.2× solid phase reversible immobilization (SPRI) clean for samples with a high percentage of adapter dimers.

[0035] [Figure 19]FIG. 19 shows the results of sequential 0.6×SPRI+0.6×SPRIclean.

[0036] [Figure 20] Figure 20 shows an example of putative adapter dimer formation, the single-stranded form of the adapter dimer, and the addition of an oligo that anneals only to such a single-stranded adapter dimer. In one example, an XbaI recognition site forms when the double-stranded hybridization product is formed.

[0037] [Figure 21] Figure 21 shows an example workflow for a directed RNA-Seq library preparation NGS assay.

[0038] [Figure 22] Figure 22 shows library yields from 1-20 ng mRNA input for the RNA single-stranded library preparation (ssPrep) described herein. A representative value from replicates is shown for each input concentration. All libraries were amplified for 9 cycles except for the 1 ng input, which was amplified for 11 PCR cycles. Libraries were quantified using the QUANT-IT dsDNA High Sensitivity Kit.

[0039] [Figure 23] Figure 23 provides a table comparing the RNA single-stranded library preparation (ssPrep) described herein with three commercially available double-stranded library preparation (dsPrep) kits (i.e., the NEBNEXT ULTRA II Directional RNA-Seq Kit; the NUGEN Universal mRNA Library Kit; and the TRUSEQ mRNA Stranded Library Kit). A comparison of input ranges, workflow times, and PCR cycles is shown.

[0040] [Figure 24]Figure 24 provides a table showing a comparison of yield and mapping metrics for human reads generated from the RNA single-stranded library preparation (ssPrep) described herein or a commercially available double-stranded library preparation (dsPrep) kit (i.e., NEBNEXT ULTRA II Directional RNA-Seq Kit). Sequencing data from ssPrep and dsPrep replicate libraries were mapped to the human reference genome (hg19). The ssPrep library had shorter mapped lengths, likely due to the lack of end-polishing.

[0041] [Figure 25] Figure 25 shows performance metrics for single-stranded library preparation (ssPrep) of RNA described herein versus a commercially available double-stranded library preparation (dsPrep) kit (i.e., the NEBNEXT ULTRA II Directional RNA-Seq Kit). Top panel: Representative mapping metrics for replicate libraries generated by both methods. Both libraries had approximately 90% uniquely mapped reads and approximately 5% ribosomal reads. Approximately 93% of reads mapped to the correct strand. Bottom panel: A Spearman correlation coefficient of ρ approximately 0.95 was observed between the normalized read counts (human) of replicates for both methods and with each other. Pairwise correlation coefficients are provided in the boxes.

[0042] [Figure 26-1]Figure 26 shows data comparing the single-stranded RNA library preparation (ssPrep) described herein to a commercially available double-stranded library preparation (dsPrep) kit (i.e., the NEBNEXT ULTRA II Directional RNA-Seq Kit). Top panel: Gene body coverage was calculated using Picard Tools CollectRNASeqMetrics; normalized coverage across the entire length of the transcript is shown for ssPrep and dsPrep replicate libraries. Middle panel: Genomic distribution of reads was calculated using Picard Tools CollectRNASeqMetrics, and the average composition of coding, untranslated (UTR), intronic, and intergenic regions was plotted. Both methods captured the least intergenic regions. ssPrep captured more untranslated regions due to more uniform gene body coverage. Bottom panel: Differential GC composition captured by the two methods. The GC distribution of the External RNA Standards Consortium (ERCC) spike-in control is shown in the inset. Percentages indicate the observed GC composition for each library. [Figure 26-2]Figure 26 shows data comparing the single-stranded RNA library preparation (ssPrep) described herein to a commercially available double-stranded library preparation (dsPrep) kit (i.e., the NEBNEXT ULTRA II Directional RNA-Seq Kit). Top panel: Gene body coverage was calculated using Picard Tools CollectRNASeqMetrics; normalized coverage across the entire length of the transcript is shown for ssPrep and dsPrep replicate libraries. Middle panel: Genomic distribution of reads was calculated using Picard Tools CollectRNASeqMetrics, and the average composition of coding, untranslated (UTR), intronic, and intergenic regions was plotted. Both methods captured the least intergenic regions. ssPrep captured more untranslated regions due to more uniform gene body coverage. Bottom panel: Differential GC composition captured by the two methods. The GC distribution of the External RNA Standards Consortium (ERCC) spike-in control is shown in the inset. Percentages indicate the observed GC composition for each library. [Figure 26-3]Figure 26 shows data comparing the single-stranded RNA library preparation (ssPrep) described herein to a commercially available double-stranded library preparation (dsPrep) kit (i.e., the NEBNEXT ULTRA II Directional RNA-Seq Kit). Top panel: Gene body coverage was calculated using Picard Tools CollectRNASeqMetrics; normalized coverage across the entire length of the transcript is shown for ssPrep and dsPrep replicate libraries. Middle panel: Genomic distribution of reads was calculated using Picard Tools CollectRNASeqMetrics, and the average composition of coding, untranslated (UTR), intronic, and intergenic regions was plotted. Both methods captured the least intergenic regions. ssPrep captured more untranslated regions due to more uniform gene body coverage. Bottom panel: Differential GC composition captured by the two methods. The GC distribution of the External RNA Standards Consortium (ERCC) spike-in control is shown in the inset. Percentages indicate the observed GC composition for each library.

[0043] [Figure 27] Figure 27 provides a table showing the number of reads mapping to the ERCC reference genome and the percent mapping to the correct strand for libraries generated using the RNA single-stranded library preparation (ssPrep) described herein or a commercially available double-stranded library preparation (dsPrep) kit (i.e., NEBNEXT ULTRA II Directional RNA-Seq Kit). Both types of libraries had >300,000 control reads, with >99% of reads mapping to the correct strand.

[0044] [Figure 28]Figure 28 shows a schematic overview of the single-stranded library preparation (ssPrep) method. A DNA input pool of diverse template molecules is heat-denatured, quenched, and maintained as single-stranded molecules by the use of a thermostable single-stranded DNA-binding protein (SSB). The template DNA is phosphorylated, and ssPrep scaffold adapters with randomized 7-bp single-stranded scaffold overhangs and ligation-blocking modifications at all ends, except those that facilitate correctly oriented library molecules, are ligated in a combined phosphorylation / ligation reaction. After cleanup, the molecules are ready for index PCR.

[0045] [Figure 29-1]Figure 29 shows standard NGS metrics for merged reads from single-stranded library preparation (ssPrep) and commercial kit (dsPrep; i.e., NEBNEXT ULTRA II) libraries from healthy human cfDNA extracts (A, sample A; B, sample B). Unless otherwise noted, all libraries for each method were combined per cfDNA extract and filtered for PCR duplicates and quality scores equal to or greater than q20 before analysis. Panel A: Insert distribution plots for cfDNA extracts A and B, respectively. Panel B: Base percentage coverage across the human genome (hg19) for ssPrep and commercial kit per cfDNA extract. Combined libraries were subsampled to a similar read depth before coverage calculation. The subsampling depth was set to 295M reads, the limit of sequenced reads for ssPrep-B. Panel C: Normalized coverage as a function of GC content across the human genome on a 100-bp sliding scale for ssPrep and commercial kits per cfDNA extract. The shaded histogram represents human genome GC across a 100-bp sliding window. Panel D: Preseq complexity estimates for ssPrep and commercial kits per cfDNA extract. To obtain the most accurate preseq estimate possible, three libraries of equivalent sequencing depth were combined for each method, as more libraries were generated by ssPrep than by commercial kits. Files containing PCR duplicate reads were used to facilitate complexity estimation. Panel E: Normalized, log-transformed base composition at each position of the read end, starting 2 bp upstream and extending 34 bp downstream from the read start site. All reads were considered, regardless of insert length. [Figure 29-2]Figure 29 shows standard NGS metrics for merged reads from single-stranded library preparation (ssPrep) and commercial kit (dsPrep; i.e., NEBNEXT ULTRA II) libraries from healthy human cfDNA extracts (A, sample A; B, sample B). Unless otherwise noted, all libraries for each method were combined per cfDNA extract and filtered for PCR duplicates and quality scores equal to or greater than q20 before analysis. Panel A: Insert distribution plots for cfDNA extracts A and B, respectively. Panel B: Base percentage coverage across the human genome (hg19) for ssPrep and commercial kit per cfDNA extract. Combined libraries were subsampled to a similar read depth before coverage calculation. The subsampling depth was set to 295M reads, the limit of sequenced reads for ssPrep-B. Panel C: Normalized coverage as a function of GC content across the human genome on a 100-bp sliding scale for ssPrep and commercial kits per cfDNA extract. The shaded histogram represents human genome GC across a 100-bp sliding window. Panel D: Preseq complexity estimates for ssPrep and commercial kits per cfDNA extract. To obtain the most accurate preseq estimate possible, three libraries of equivalent sequencing depth were combined for each method, as more libraries were generated by ssPrep than by commercial kits. Files containing PCR duplicate reads were used to facilitate complexity estimation. Panel E: Normalized, log-transformed base composition at each position of the read end, starting 2 bp upstream and extending 34 bp downstream from the read start site. All reads were considered, regardless of insert length. [Figure 29-3]Figure 29 shows standard NGS metrics for merged reads from single-stranded library preparation (ssPrep) and commercial kit (dsPrep; i.e., NEBNEXT ULTRA II) libraries from healthy human cfDNA extracts (A, sample A; B, sample B). Unless otherwise noted, all libraries for each method were combined per cfDNA extract and filtered for PCR duplicates and quality scores equal to or greater than q20 before analysis. Panel A: Insert distribution plots for cfDNA extracts A and B, respectively. Panel B: Base percentage coverage across the human genome (hg19) for ssPrep and commercial kit per cfDNA extract. Combined libraries were subsampled to a similar read depth before coverage calculation. The subsampling depth was set to 295M reads, the limit of sequenced reads for ssPrep-B. Panel C: Normalized coverage as a function of GC content across the human genome on a 100-bp sliding scale for ssPrep and commercial kits per cfDNA extract. The shaded histogram represents human genome GC across a 100-bp sliding window. Panel D: Preseq complexity estimates for ssPrep and commercial kits per cfDNA extract. To obtain the most accurate preseq estimate possible, three libraries of equivalent sequencing depth were combined for each method, as more libraries were generated by ssPrep than by commercial kits. Files containing PCR duplicate reads were used to facilitate complexity estimation. Panel E: Normalized, log-transformed base composition at each position of the read end, starting 2 bp upstream and extending 34 bp downstream from the read start site. All reads were considered, regardless of insert length. [Figure 29-4]Figure 29 shows standard NGS metrics for merged reads from single-stranded library preparation (ssPrep) and commercial kit (dsPrep; i.e., NEBNEXT ULTRA II) libraries from healthy human cfDNA extracts (A, sample A; B, sample B). Unless otherwise noted, all libraries for each method were combined per cfDNA extract and filtered for PCR duplicates and quality scores equal to or greater than q20 before analysis. Panel A: Insert distribution plots for cfDNA extracts A and B, respectively. Panel B: Base percentage coverage across the human genome (hg19) for ssPrep and commercial kit per cfDNA extract. Combined libraries were subsampled to a similar read depth before coverage calculation. The subsampling depth was set to 295M reads, the limit of sequenced reads for ssPrep-B. Panel C: Normalized coverage as a function of GC content across the human genome on a 100-bp sliding scale for ssPrep and commercial kits per cfDNA extract. The shaded histogram represents human genome GC across a 100-bp sliding window. Panel D: Preseq complexity estimates for ssPrep and commercial kits per cfDNA extract. To obtain the most accurate preseq estimate possible, three libraries of equivalent sequencing depth were combined for each method, as more libraries were generated by ssPrep than by commercial kits. Files containing PCR duplicate reads were used to facilitate complexity estimation. Panel E: Normalized, log-transformed base composition at each position of the read end, starting 2 bp upstream and extending 34 bp downstream from the read start site. All reads were considered, regardless of insert length. [Figure 29-5]Figure 29 shows standard NGS metrics for merged reads from single-stranded library preparation (ssPrep) and commercial kit (dsPrep; i.e., NEBNEXT ULTRA II) libraries from healthy human cfDNA extracts (A, sample A; B, sample B). Unless otherwise noted, all libraries for each method were combined per cfDNA extract and filtered for PCR duplicates and quality scores equal to or greater than q20 before analysis. Panel A: Insert distribution plots for cfDNA extracts A and B, respectively. Panel B: Base percentage coverage across the human genome (hg19) for ssPrep and commercial kit per cfDNA extract. Combined libraries were subsampled to a similar read depth before coverage calculation. The subsampling depth was set to 295M reads, the limit of sequenced reads for ssPrep-B. Panel C: Normalized coverage as a function of GC content across the human genome on a 100-bp sliding scale for ssPrep and commercial kits per cfDNA extract. The shaded histogram represents human genome GC across a 100-bp sliding window. Panel D: Preseq complexity estimates for ssPrep and commercial kits per cfDNA extract. To obtain the most accurate preseq estimate possible, three libraries of equivalent sequencing depth were combined for each method, as more libraries were generated by ssPrep than by commercial kits. Files containing PCR duplicate reads were used to facilitate complexity estimation. Panel E: Normalized, log-transformed base composition at each position of the read end, starting 2 bp upstream and extending 34 bp downstream from the read start site. All reads were considered, regardless of insert length.

[0046] [Figure 30-1]Figure 30 shows coverage of double-stranded oligos containing single-stranded overhangs for single-stranded library preparation (ssPrep) and a commercial kit (dsPrep; i.e., NEBNEXT ULTRA II). Panel A: Schematic representation of a double-stranded synthetic oligo—one blunt end, a distinct 50-nt complementary region, and an overhang of a specific length and type. Panel B: Representative values ​​of base-by-base coverage across the length of all double-stranded oligos for three technical replicates at the 0 base coordinate for both the ssPrep and commercial kit methods. Technical replicates were not statistically different from each other (Student's t-test: ssPrep p=0.714, commercial kit p=0.985). More than 5,000 reads of each oligo were sequenced. [Figure 30-2] Figure 30 shows coverage of double-stranded oligos containing single-stranded overhangs for single-stranded library preparation (ssPrep) and a commercial kit (dsPrep; i.e., NEBNEXT ULTRA II). Panel A: Schematic representation of a double-stranded synthetic oligo—one blunt end, a distinct 50-nt complementary region, and an overhang of a specific length and type. Panel B: Representative values ​​of base-by-base coverage across the length of all double-stranded oligos for three technical replicates at the 0 base coordinate for both the ssPrep and commercial kit methods. Technical replicates were not statistically different from each other (Student's t-test: ssPrep p=0.714, commercial kit p=0.985). >5,000 reads of each oligo sequenced.

[0047] [Figure 31-1]Figure 31 shows single-stranded oligo analysis per ssPrep. Gray and black lines and dots represent technical replicates. Panel A: Insert distribution of equimolar pooled single-stranded oligo libraries. 20-120 nt oligos synthesized at 10 nt intervals were purified by standard desalting. Raw, unfiltered sequencing data. Panel B: Mapped sequencing data for technical replicates separated by oligo, presented as a function of oligo length. The black vertical bars and accompanying black and gray numbers indicate the percentage of full-length product for each oligo length present in the library pool. Each library was sequenced to a depth of approximately 100,000 read pairs (10,000 read pairs per oligo, excluding 20- and 30-nt lengths). Panel C: Effect of various purification methods on oligo purity as a function of oligo length for 60-nt synthesized oligos. The accompanying black and gray numbers indicate the percentage of full-length product per oligo. Data taken from panel B for a standard desalted 60 nt synthetic oligo. [Figure 31-2] Figure 31 shows single-stranded oligo analysis per ssPrep. Gray and black lines and dots represent technical replicates. Panel A: Insert distribution of equimolar pooled single-stranded oligo libraries. Oligos ranging from 20 to 120 nt, synthesized at 10 nt intervals, were purified by standard desalting. Raw, unfiltered sequencing data. Panel B: Mapped sequencing data for technical replicates separated by oligo, presented as a function of oligo length. The black vertical bars and accompanying black and gray numbers indicate the percentage of full-length product for each oligo length present in the library pool. Each library was sequenced to a depth of approximately 100,000 read pairs (10,000 read pairs per oligo, excluding 20- and 30-nt lengths). Panel C: Effect of various purification methods on oligo purity as a function of oligo length for 60-nt synthesized oligos. The accompanying black and gray numbers indicate the percentage of full-length product per oligo. Data taken from panel B for a standard desalted 60 nt synthetic oligo. [Figure 31-3]Figure 31 shows single-stranded oligo analysis per ssPrep. Gray and black lines and dots represent technical replicates. Panel A: Insert distribution of equimolar pooled single-stranded oligo libraries. Oligos ranging from 20 to 120 nt, synthesized at 10 nt intervals, were purified by standard desalting. Raw, unfiltered sequencing data. Panel B: Mapped sequencing data for technical replicates separated by oligo, presented as a function of oligo length. The black vertical bars and accompanying black and gray numbers indicate the percentage of full-length product for each oligo length present in the library pool. Each library was sequenced to a depth of approximately 100,000 read pairs (10,000 read pairs per oligo, excluding 20- and 30-nt lengths). Panel C: Effect of various purification methods on oligo purity as a function of oligo length for 60-nt synthesized oligos. The accompanying black and gray numbers indicate the percentage of full-length product per oligo. Data taken from panel B for a standard desalted 60 nt synthetic oligo.

[0048] [Figure 32-1]Figure 32 shows cfDNA analysis. Panel A: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 100 bp ± (coordinates to which the reads were mapped). The read midpoint is centered at zero. Negative numbers indicate genomic regions upstream (5 prime) of the midpoint, and positive numbers indicate genomic regions downstream (3 prime) of the midpoint. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel B: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 9 bp regions into the read (positive numbers) and 10 bp out of the read (negative numbers). Read start and end coordinates are centered at zero. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel C: Same as Panel A, except for commercial kit data. Panel D: Same as Panel B, except for commercial kit data. Panel E: Normalized WPS values ​​for ssPrep data (120 bp window; 120-180 bp fragments) compared to sample CH01 at the same pericentromeric locus on chromosome 12 used to initially demonstrate WPS. Panel F: Representative normalized WPS scores within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window; 120-180 bp fragments) and short fragment length binned data (16 bp window; 35-80 bp fragments) of ssPrep data compared to sample CH01. [Figure 32-2]Figure 32 shows cfDNA analysis. Panel A: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 100 bp ± (coordinates to which the reads were mapped). The read midpoint is centered at zero. Negative numbers indicate genomic regions upstream (5 prime) of the midpoint, and positive numbers indicate genomic regions downstream (3 prime) of the midpoint. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel B: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 9 bp regions into the read (positive numbers) and 10 bp out of the read (negative numbers). Read start and end coordinates are centered at zero. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel C: Same as Panel A, except for commercial kit data. Panel D: Same as Panel B, except for commercial kit data. Panel E: Normalized WPS values ​​for ssPrep data (120 bp window; 120-180 bp fragments) compared to sample CH01 at the same pericentromeric locus on chromosome 12 used to initially demonstrate WPS. Panel F: Representative normalized WPS scores within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window; 120-180 bp fragments) and short fragment length binned data (16 bp window; 35-80 bp fragments) of ssPrep data compared to sample CH01. [Figure 32-3]Figure 32 shows cfDNA analysis. Panel A: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 100 bp ± (coordinates to which the reads were mapped). The read midpoint is centered at zero. Negative numbers indicate genomic regions upstream (5 prime) of the midpoint, and positive numbers indicate genomic regions downstream (3 prime) of the midpoint. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel B: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 9 bp regions into the read (positive numbers) and 10 bp out of the read (negative numbers). Read start and end coordinates are centered at zero. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel C: Same as Panel A, except for commercial kit data. Panel D: Same as Panel B, except for commercial kit data. Panel E: Normalized WPS values ​​for ssPrep data (120 bp window; 120-180 bp fragments) compared to sample CH01 at the same pericentromeric locus on chromosome 12 used to initially demonstrate WPS. Panel F: Representative normalized WPS scores within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window; 120-180 bp fragments) and short fragment length binned data (16 bp window; 35-80 bp fragments) of ssPrep data compared to sample CH01. [Figure 32-4]Figure 32 shows cfDNA analysis. Panel A: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 100 bp ± (coordinates to which the reads were mapped). The read midpoint is centered at zero. Negative numbers indicate genomic regions upstream (5 prime) of the midpoint, and positive numbers indicate genomic regions downstream (3 prime) of the midpoint. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel B: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 9 bp regions into the read (positive numbers) and 10 bp out of the read (negative numbers). Read start and end coordinates are centered at zero. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel C: Same as Panel A, except for commercial kit data. Panel D: Same as Panel B, except for commercial kit data. Panel E: Normalized WPS values ​​for ssPrep data (120 bp window; 120-180 bp fragments) compared to sample CH01 at the same pericentromeric locus on chromosome 12 used to initially demonstrate WPS. Panel F: Representative normalized WPS scores within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window; 120-180 bp fragments) and short fragment length binned data (16 bp window; 35-80 bp fragments) of ssPrep data compared to sample CH01. [Figure 32-5]Figure 32 shows cfDNA analysis. Panel A: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 100 bp ± (coordinates to which the reads were mapped). The read midpoint is centered at zero. Negative numbers indicate genomic regions upstream (5 prime) of the midpoint, and positive numbers indicate genomic regions downstream (3 prime) of the midpoint. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel B: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 9 bp regions into the read (positive numbers) and 10 bp out of the read (negative numbers). Read start and end coordinates are centered at zero. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel C: Same as Panel A, except for commercial kit data. Panel D: Same as Panel B, except for commercial kit data. Panel E: Normalized WPS values ​​for ssPrep data (120 bp window; 120-180 bp fragments) compared to sample CH01 at the same pericentromeric locus on chromosome 12 used to initially demonstrate WPS. Panel F: Representative normalized WPS scores within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window; 120-180 bp fragments) and short fragment length binned data (16 bp window; 35-80 bp fragments) of ssPrep data compared to sample CH01. [Figure 32-6]Figure 32 shows cfDNA analysis. Panel A: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 100 bp ± (coordinates to which the reads were mapped). The read midpoint is centered at zero. Negative numbers indicate genomic regions upstream (5 prime) of the midpoint, and positive numbers indicate genomic regions downstream (3 prime) of the midpoint. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel B: Normalized genomic dinucleotide occurrence frequency as a function of read length for ssPrep data of three distinct fragment lengths, including 9 bp regions into the read (positive numbers) and 10 bp out of the read (negative numbers). Read start and end coordinates are centered at zero. Input data is from the combined Sample A and Sample B ssPrep datasets. Panel C: Same as Panel A, except for commercial kit data. Panel D: Same as Panel B, except for commercial kit data. Panel E: Normalized WPS values ​​for ssPrep data (120 bp window; 120-180 bp fragments) compared to sample CH01 at the same pericentromeric locus on chromosome 12 used to initially demonstrate WPS. Panel F: Representative normalized WPS scores within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window; 120-180 bp fragments) and short fragment length binned data (16 bp window; 35-80 bp fragments) of ssPrep data compared to sample CH01.

[0049] [Figure 33A-1]Figures 33A and 33B show input cfDNA and representative ssPrep and dsPrep libraries. Figure 33A, top panel: Cell-free DNA extract (Sample A) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D5000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33A, bottom panel: Cell-free DNA extract (Sample B) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33B, top panel: One representative ssPrep library from a cfDNA extract (Sample A, Library ID: A') and one representative ssPrep library from a cfDNA extract (Sample B, Library ID: B') analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown. Figure 33B, bottom panel: One representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample A, Library ID: A") and one representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample B, Library ID: B") analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown. [Figure 33A-2]Figures 33A and 33B show input cfDNA and representative ssPrep and dsPrep libraries. Figure 33A, top panel: Cell-free DNA extract (Sample A) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D5000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33A, bottom panel: Cell-free DNA extract (Sample B) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33B, top panel: One representative ssPrep library from a cfDNA extract (Sample A, Library ID: A') and one representative ssPrep library from a cfDNA extract (Sample B, Library ID: B') analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown. Figure 33B, bottom panel: One representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample A, Library ID: A") and one representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample B, Library ID: B") analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown. [Figure 33B-1]Figures 33A and 33B show input cfDNA and representative ssPrep and dsPrep libraries. Figure 33A, top panel: Cell-free DNA extract (Sample A) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D5000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33A, bottom panel: Cell-free DNA extract (Sample B) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33B, top panel: One representative ssPrep library from a cfDNA extract (Sample A, Library ID: A') and one representative ssPrep library from a cfDNA extract (Sample B, Library ID: B') analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown. Figure 33B, bottom panel: One representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample A, Library ID: A") and one representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample B, Library ID: B") analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown. [Figure 33B-2]Figures 33A and 33B show input cfDNA and representative ssPrep and dsPrep libraries. Figure 33A, top panel: Cell-free DNA extract (Sample A) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D5000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33A, bottom panel: Cell-free DNA extract (Sample B) after plasma extraction / purification and before NGS library preparation. Analysis was performed using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. A gel image and electropherogram are shown. Figure 33B, top panel: One representative ssPrep library from a cfDNA extract (Sample A, Library ID: A') and one representative ssPrep library from a cfDNA extract (Sample B, Library ID: B') analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown. Figure 33B, bottom panel: One representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample A, Library ID: A") and one representative dsPrep (i.e., NEBNEXT ULTRA II) library from a cfDNA extract (Sample B, Library ID: B") analyzed after index PCR using a D1000 HS tape and accompanying Tapestation 4200 (Agilent) according to the manufacturer's instructions. Gel images and electropherograms are shown.

[0050] [Figure 34-1]Figure 34 shows the insert distribution for replicate libraries of Sample A and Sample B. Panel A: Insert distribution for all libraries made for Sample A cfDNA extract. Panel B: Insert distribution for all libraries made for Sample B cfDNA extract. The order of library IDs listed in each legend corresponds to the order of the traces shown in the legend. [Figure 34-2] Figure 34 shows insert distribution for replicate libraries of Sample A and Sample B. Panel A: Insert distribution for all libraries made for Sample A cfDNA extract. Panel B: Insert distribution for all libraries made for Sample B cfDNA extract. The order of library IDs listed in each legend corresponds to the order of the traces shown in the legend.

[0051] [Figure 35] Figure 35 shows the effect of post-index PCR DNA purification on fragment length retention. An ssPrep library of cfDNA (Sample A) was purified using either a 1.2x or 1.5x DNA purification bead volume:index PCR reaction volume ratio. Recovery of <100 bp fragments varied from 9.3% to 14.7% from the lower to the higher ratio.

[0052] [Figure 36] Figure 36 shows an example workflow for single-stranded DNA library preparation (ssPrep). ssPrep operates in a one-step combined phosphorylation / ligation step that simultaneously prepares template DNA molecules for ligation without end-polishing (i.e., end-repair) and ligates ILLUMINA adapters by utilizing the scaffold adapters described herein.

[0053] [Figure 37] Figure 37 shows an illustration of the cfDNA protection model.

[0054] [Figure 38] Figure 38 provides a table outlining the library preparation kits and input cfDNA. Sequencing libraries were generated from cfDNA extracts of two healthy individuals (Sample 1 and Sample 2) using ssPrep, two commercially available end-polished dsDNA library preparation kits, and a commercially available ssDNA kit. Libraries were sequenced 2x151bp on an ILLUMINA HISEQ X. Commercial kit 1, NEBNext® Ultra II™; commercial kit 2, Takara Bio Inc. ThruPLEX® Plasma-Seq; commercial kit 3, Swift Accel-NGS® 1S Plus.

[0055] [Figure 39] Figure 39 shows yields after index PCR. Libraries were quantified using Qubit 3.0 and 2 μl of the final purified library after index PCR. All libraries were indexed with 10 cycles of PCR using the polymerase master mix and primers provided with the kit. Prep kits are indicated in each histogram from left to right: ssPrep, Swift, Takara Bio Inc., NEB.

[0056] [Figure 40] Figure 40 shows mapping and short insert binning data. Libraries were trimmed / merged using SeqPrep 2.0, and merged reads <30 bp were discarded. Remaining reads were mapped to the human reference genome hg19 with BWA aln. Size binning was performed using a custom script. Prep kits are indicated in each histogram from left to right: ssPrep, Swift (Swift Accel-NGS® 1S Plus), Takara Bio Inc. (Swift Accel-NGS® 1S Plus), and NEB (NEBNext® Ultra II™).

[0057] [Figure 41]Figure 41 provides a table showing the yield and mapping status after index PCR. *Due to tailing artifacts generated during library preparation, commercial kit 3 recommends trimming 10 bp from forward and reverse reads before mapping. Based on this, the upper limit of the bin size from 30 to 100 bp was lowered to 90 bp instead of 100 bp. Commercial kit 1, NEBNext® Ultra II™; commercial kit 2, Takara Bio Inc. ThruPLEX® Plasma-Seq; commercial kit 3, Swift Accel-NGS® 1S Plus.

[0058] [Figure 42A] Figures 42A and 42B show the mapped insert length distributions. Figure 42A shows ssPrep versus dsDNA preparation: Kit 2, Takara Bio Inc. ThruPLEX® Plasma-Seq; Kit 1, NEBNext® Ultra II™. Figure 42B shows ssPrep versus a commercial ssDNA preparation kit (Swift Accel-NGS® 1S Plus). Read lengths for all molecules contained in each library were extracted from the correctly mapped and sorted bam files and then plotted. [Figure 42B] Figures 42A and 42B show the mapped insert length distributions. Figure 42A shows ssPrep versus dsDNA preparation: Kit 2, Takara Bio Inc. ThruPLEX® Plasma-Seq; Kit 1, NEBNext® Ultra II™. Figure 42B shows ssPrep versus a commercial ssDNA preparation kit (Swift Accel-NGS® 1S Plus). Read lengths for all molecules contained in each library were extracted from the correctly mapped and sorted bam files and then plotted.

[0059] [Figure 43]Figure 43 shows the complexity estimates. The complexity estimates output of the library prep kit were from the Preseq algorithm. The reads from the table in Figure 41 were used as input data, and the number of unique molecules was extrapolated to 300M read pairs.

[0060] [Figure 44] Figure 44 shows the GC coverage as normalized coverage of the library for each cfDNA extract as a function of the GC content of the human reference genome (hg19) for a 100 bp sliding window. The left Y-axis refers to cfDNA (line). The right axis refers to the reference genome (gray histogram). Data retrieved from Picard Tools CollectGcBiasMetrics.

[0061] [Figure 45] Figure 45 shows nucleosome occupancy. WPS calculations were generated and normalized to the highest absolute WPS value for the alpha satellite array subregion on chr12. Prior to WPS calculation, cfDNA data was obtained by combining ssPrep bam files for healthy cfDNA samples 1 and 2 and filtering reads for nucleosome-associated inserts.

[0062] [Figure 46] Figure 46 shows dinucleotide frequencies. Dinucleotide frequencies for AT and CG containing dinucleotides were plotted for the 5' and 3' ends of all reads corresponding to the major histone monomer peak of sample 1 at 1657 bp.

[0063] [Figure 47] Figure 47 shows a comparison of library preparation methods for damaged DNA.

[0064] [Figure 48A]Figures 48A-48D show a comparison of fragment length and adapter dimer percentage recovered after SPRI purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 48A shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer (i.e., 25 μl of ligation product added). Figure 48B shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 25 μl of isopropanol and 25 μl of Tris buffer. Figure 48C shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of isopropanol. Figure 48D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using SPRI bead lysis buffer containing 38% PEG. [Figure 48B]Figures 48A-48D show a comparison of fragment length and adapter dimer percentage recovered after SPRI purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 48A shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer (i.e., 25 μl of ligation product added). Figure 48B shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 25 μl of isopropanol and 25 μl of Tris buffer. Figure 48C shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of isopropanol. Figure 48D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using SPRI bead lysis buffer containing 38% PEG. [Figure 48C]Figures 48A-48D show a comparison of fragment length and adapter dimer percentage recovered after SPRI purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 48A shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer (i.e., 25 μl of ligation product added). Figure 48B shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 25 μl of isopropanol and 25 μl of Tris buffer. Figure 48C shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of isopropanol. Figure 48D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using SPRI bead lysis buffer containing 38% PEG. [Figure 48D]Figures 48A-48D show a comparison of fragment length and adapter dimer percentage recovered after SPRI purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 48A shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer (i.e., 25 μl of ligation product added). Figure 48B shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 25 μl of isopropanol and 25 μl of Tris buffer. Figure 48C shows adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of isopropanol. Figure 48D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using SPRI bead lysis buffer containing 38% PEG.

[0065] [Figure 49A]Figures 49A-49E show a comparison of the fragment length and adapter dimer percentage recovered after purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 49A shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer. Figure 49B shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using column purification. Figure 49C shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 5 μl of isopropanol and 45 μl of Tris buffer. Figure 49D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 10 μl of isopropanol and 40 μl of Tris buffer. Figure 49E shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 20 μl of isopropanol and 30 μl of Tris buffer. [Figure 49B]Figures 49A-49E show a comparison of the fragment length and adapter dimer percentage recovered after purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 49A shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer. Figure 49B shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using column purification. Figure 49C shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 5 μl of isopropanol and 45 μl of Tris buffer. Figure 49D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 10 μl of isopropanol and 40 μl of Tris buffer. Figure 49E shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 20 μl of isopropanol and 30 μl of Tris buffer. [Figure 49C]Figures 49A-49E show a comparison of the fragment length and adapter dimer percentage recovered after purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 49A shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer. Figure 49B shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using column purification. Figure 49C shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 5 μl of isopropanol and 45 μl of Tris buffer. Figure 49D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 10 μl of isopropanol and 40 μl of Tris buffer. Figure 49E shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 20 μl of isopropanol and 30 μl of Tris buffer. [Figure 49D]Figures 49A-49E show a comparison of the fragment length and adapter dimer percentage recovered after purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 49A shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer. Figure 49B shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using column purification. Figure 49C shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 5 μl of isopropanol and 45 μl of Tris buffer. Figure 49D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 10 μl of isopropanol and 40 μl of Tris buffer. Figure 49E shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 20 μl of isopropanol and 30 μl of Tris buffer. [Figure 49E]Figures 49A-49E show a comparison of the fragment length and adapter dimer percentage recovered after purification of fragment-scaffold adapter ligation products under various purification conditions. Figure 49A shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 50 μl of Tris buffer. Figure 49B shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using column purification. Figure 49C shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 5 μl of isopropanol and 45 μl of Tris buffer. Figure 49D shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 10 μl of isopropanol and 40 μl of Tris buffer. Figure 49E shows the adapter dimer and fragment length peaks for fragment-scaffold adapter ligation products purified using 18% PEG SPRI purification with the addition of 20 μl of isopropanol and 30 μl of Tris buffer.

[0066] [Figure 50] Figure 50 shows an example workflow for generating a sequencing library from single-stranded RNA.

[0067] [Figure 51] Figure 51 shows examples of scaffold adapter designs where some or all of the bases in the ssNA hybridization region are defined or known bases.

[0068] [Figure 52A] Figures 52A-52D show an example workflow for enrichment of modified nucleic acids. Enrichment of modified nucleic acids can be performed before (Figures 52A, 52B) or after (Figures 52C, 52D) denaturation of dsDNA. [Figure 52B] Figures 52A-52D show an example workflow for enrichment of modified nucleic acids. Enrichment of modified nucleic acids can be performed before (Figures 52A, 52B) or after (Figures 52C, 52D) denaturation of dsDNA. [Figure 52C] Figures 52A-52D show an example workflow for enrichment of modified nucleic acids. Enrichment of modified nucleic acids can be performed before (Figures 52A, 52B) or after (Figures 52C, 52D) denaturation of dsDNA. [Figure 52D] Figures 52A-52D show an example workflow for enrichment of modified nucleic acids. Enrichment of modified nucleic acids can be performed before (Figures 52A, 52B) or after (Figures 52C, 52D) denaturation of dsDNA.

[0069] [Figure 53A] Figures 53A and 53B show an example workflow for generating a library from nicked DNA. [Figure 53B] Figures 53A and 53B show an example workflow for generating a library from nicked DNA.

[0070] [Figure 54] Figure 54 shows examples of scaffold adaptor configurations that include DNA, RNA, or a combination thereof.

[0071] [Figure 55] Figure 55 shows an example workflow for generating a sequencing library from a sample containing pathogen RNA. DETAILED DESCRIPTION OF THE INVENTION

[0072] Detailed Description Methods and compositions useful for analyzing nucleic acids are provided herein. Methods and compositions useful for generating nucleic acid libraries are also provided herein. Methods and compositions useful for analyzing the ends of single-stranded nucleic acid fragments are also provided herein. In certain aspects, the method includes binding a sample nucleic acid containing single-stranded nucleic acid fragments to a designated adaptor. In some embodiments, the designated adaptor comprises a scaffold polynucleotide that can hybridize with the end of the single-stranded nucleic acid. The product of such hybridization can be useful, for example, for generating a nucleic acid library and / or for further analysis or processing.

[0073] Scaffolding Adapter Certain methods herein include a step of binding ssNAs to a scaffold adapter or a component thereof. A scaffold adapter generally includes a scaffold polynucleotide and an oligonucleotide. Accordingly, a "component" of a scaffold adapter may refer to a scaffold polynucleotide and / or an oligonucleotide, or a subcomponent or region thereof. The oligonucleotide and / or scaffold polynucleotide may be composed of pyrimidine (C, T, U) and / or purine (A, G) nucleotides. Additional components or subcomponents may include one or more of an index polynucleotide, a unique molecular identifier (UMI), a primer binding site (e.g., a sequencing primer binding site, a P5 primer binding site, a P7 primer binding site), a flow cell binding region, and the like, and components thereof. A scaffold adapter containing a P5 primer binding site may be referred to as a P5 adapter or a P5 scaffold adapter. A scaffold adapter containing a P7 primer binding site may be referred to as a P7 adapter or a P7 scaffold adapter.

[0074] A scaffold polynucleotide is a single-stranded component of a scaffold adaptor. As used herein, a polynucleotide generally refers to a single-stranded multimer of nucleotides, typically between 5 and 500 nucleotides, e.g., between 5 and 100 nucleotides. Polynucleotides can be synthetic or enzymatically generated and, in some embodiments, are approximately 5 to 50 nucleotides in length. Polynucleotides can contain ribonucleotide monomers (i.e., polyribonucleotides or "RNA polynucleotides"), deoxyribonucleotide monomers (i.e., polydeoxyribonucleotides or "DNA polynucleotides"), or a combination thereof. Polynucleotides can be, for example, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 100, 100 to 150, 150 to 200 nucleotides in length, or up to 500 nucleotides in length. The terms polynucleotide and oligonucleotide may be used interchangeably herein.

[0075] The scaffold polynucleotide may comprise an ssNA hybridization region (also referred to as a scaffold, a scaffold region, a single-stranded scaffold, or a single-stranded scaffold region) and an oligonucleotide hybridization region. The ssNA hybridization region and the oligonucleotide hybridization region may be referred to as subcomponents of the scaffold polynucleotide. The ssNA hybridization region typically comprises a polynucleotide that hybridizes or can hybridize to the ssNA terminal region. The oligonucleotide hybridization region typically comprises a polynucleotide that hybridizes or can hybridize to all or a portion of the oligonucleotide components of the scaffold adaptor.

[0076] The ssNA hybridization region of the scaffold polynucleotide may comprise a polynucleotide that is complementary or substantially complementary to the ssNA terminal region. In some embodiments, the ssNA hybridization region comprises a random sequence. In some embodiments, the ssNA hybridization region comprises a sequence complementary to the desired ssNA terminal region sequence (e.g., target sequence). In certain embodiments, the ssNA hybridization region comprises one or more nucleotides that are capable of non-specific base pairing with the bases in the ssNA. Nucleotides capable of non-specific base pairing are sometimes referred to as universal bases. A universal base is a base that can indiscriminately base pair with each of the four standard nucleotide bases: A, C, G, and T. Universal bases that can be incorporated into the ssNA hybridization region include, but are not limited to, inosine, deoxyinosine, 2'-deoxyinosine (dI, dInosine), nitroindole, 5-nitroindole, and 3-nitropyrrole. In certain embodiments, the ssNA hybridization region contains one or more degenerate / wobble bases that can replace two or three (but not all) of the four typical bases (e.g., the unnatural bases P and K).

[0077] The ssNA hybridization region of the scaffold polynucleotide can have any suitable length and sequence. In some embodiments, the length of the ssNA hybridization region is 10 nucleotides or less. In certain aspects, the ssNA hybridization region is 4 to 100 nucleotides in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In certain aspects, the ssNA hybridization region is 4 to 20 nucleotides in length, for example, 5 to 15, 5 to 10, 5 to 9, 5 to 8, or 5 to 7 (e.g., 6 or 7) nucleotides in length. In some embodiments, the ssNA hybridization region is 7 nucleotides in length. In some embodiments, the ssNA hybridization region comprises or consists of a random nucleotide sequence; thus, when a plurality of heterogeneous scaffold polynucleotides having different random ssNA hybridization regions are used, the group can act as a scaffold polynucleotide for a heterogeneous population of ssNAs, regardless of the sequences of the terminal regions of the ssNAs. Each scaffold polynucleotide having a unique ssNA hybridization region sequence may be referred to as a scaffold polynucleotide species, and a group of multiple scaffold polynucleotide species may be referred to as multiple scaffold polynucleotide species (e.g., for a scaffold polynucleotide designed to have seven random bases in the ssNA hybridization region, the multiple scaffold polynucleotide species may be a group of 4 7(The scaffold adaptor species will contain a unique ssNA hybridization region sequence.) Thus, each scaffold adaptor having a unique scaffold polynucleotide (i.e., containing a unique ssNA hybridization region sequence) may be referred to as a scaffold adaptor species, and a collection of multiple scaffold adaptor species may be referred to as multiple scaffold adaptor species. A scaffold polynucleotide species generally has characteristics that are unique relative to other scaffold polynucleotide species. For example, a scaffold polynucleotide species can have unique sequence characteristics. Unique sequence characteristics can include a unique sequence length, a unique nucleotide sequence (e.g., a unique random sequence, a unique target sequence), or a combination of a unique sequence length and nucleotide sequence.

[0078] A scaffold polynucleotide may include one or more additional subcomponents, including an index polynucleotide, a unique molecular identifier (UMI), a primer binding site (e.g., a P5 primer binding site, a P7 primer binding site), a flow cell binding region, etc., or complementary polynucleotides thereof. A scaffold polynucleotide may also include a primer binding site (or a polynucleotide complementary to a primer binding site). A scaffold polynucleotide (or its complement) comprising a P5 primer binding site may be referred to as a P5 scaffold or a P5 scaffold polynucleotide. A scaffold polynucleotide (or its complement) comprising a P7 primer binding site may be referred to as a P7 scaffold or a P7 scaffold polynucleotide.

[0079] An oligonucleotide can be an additional single-stranded component of a scaffold adaptor. An oligonucleotide, as used herein, generally refers to a single-stranded multimer of nucleotides, ranging from 5 to 500 nucleotides, e.g., from 5 to 100 nucleotides. Oligonucleotides can be synthetic or enzymatically generated and, in some embodiments, are 5 to 50 nucleotides in length. Oligonucleotides can contain ribonucleotide monomers (i.e., oligoribonucleotides or "RNA oligonucleotides"), deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides or "DNA oligonucleotides"), or a combination thereof. Oligonucleotides can be, for example, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 100, 100 to 150, 150 to 200 nucleotides in length, or up to 500 nucleotides in length. The terms oligonucleotide and polynucleotide can be used interchangeably herein.

[0080] The oligonucleotide component of the scaffold adaptor generally comprises a nucleic acid sequence that is complementary or substantially complementary to the oligonucleotide hybridization region of the scaffold polynucleotide. The oligonucleotide component of the scaffold adaptor may comprise one or more subcomponents that are useful for one or more downstream applications, such as, for example, PCR amplification of ssNA fragments or their derivatives, sequencing of ssNA fragments or their derivatives, etc. In some embodiments, the oligonucleotide subcomponent is a sequencing adaptor. The sequencing adapters may be compatible with a sequencing platform of interest, such as a sequencing platform offered by Illumina® (e.g., HiSeq™, MiSeq™, and / or Genome Analyzer™ sequencing systems); a sequencing platform offered by Oxford Nanopore™ Technologies (e.g., MinION™ sequencing system); a sequencing platform offered by Ion Torrent™ (e.g., Ion PGM™ and / or Ion Proton™ sequencing systems); a sequencing platform offered by Pacific Biosciences (e.g., Sequel or PACBIO RS II sequencing systems); a sequencing platform offered by Life Technologies™ (e.g., SOLiD™ sequencing system); a sequencing platform offered by Roche (e.g., 454 GS FLX+ and / or GS "Genapsys Junior sequencing system"); a sequencing platform offered by Genapsys; a sequencing platform offered by BGI; or any sequencing platform of interest. "Genapsys" generally refers to one or more nucleic acid domains comprising at least a portion of a nucleotide sequence (or its complement) used by Genapsys Junior sequencing system; a sequencing platform offered by Genapsys; a sequencing platform offered by BGI; or any sequencing platform of interest.

[0081] In some embodiments, the oligonucleotide component of the scaffold adapter comprises a domain (e.g., a "capture site" or "capture sequence") that specifically binds to a surface-bound sequencing platform oligonucleotide (e.g., a P5 or P7 oligonucleotide bound to the surface of a flow cell in an Illumina® sequencing system); a sequencing primer binding domain (e.g., a Read 1 or Read 2 oligonucleotide of an Illumina® platform); a domain to which two primers can bind; a unique identifier or index (e.g., a barcode or other domain that uniquely identifies the sample source of the ssNAs to be sequenced to enable sample multiplexing by marking every molecule from a given sample with a specific barcode or "tag"); a barcode sequencing primer binding domain (a domain to which a primer used to sequence the barcode binds); a molecular identification domain or unique molecular identifier (UMI) (e.g., a molecular index tag, such as a randomized tag of 4, 6, or other number of nucleotides) for uniquely marking a molecule of interest, e.g., to determine expression levels based on the number of instances of the unique tag that are sequenced); a component of any such domain; or any combination thereof. In some embodiments, the barcode domain (e.g., sample index tag) and the molecular identification domain (e.g., molecular index tag; UMI) can be included in the same nucleic acid. Sequencing platform oligonucleotides, sequencing primers, and their corresponding binding domains can be designed to be compatible with a variety of available sequencing platforms and technologies, including but not limited to those discussed herein.

[0082] When the oligonucleotide component of the scaffold adaptor comprises one or a portion of a sequencing adaptor, one or more additional sequencing adaptors and / or the remaining portion of the sequencing adaptor can be added using various approaches.For example, the additional sequencing adaptor and / or the remaining portion of the sequencing adaptor can be added by any one of ligation, reverse transcription, PCR amplification, etc.For PCR, an amplification primer pair can be used, including a first amplification primer comprising a 3' hybridization region (for example, for hybridizing with the adaptor region of the oligonucleotide) and a 5' region comprising the additional sequencing adaptor and / or the remaining portion of the sequencing adaptor, and a second amplification primer comprising a 3' hybridization region (for example, for hybridizing with the adaptor region of the second oligonucleotide added to the opposite end of the ssNA molecule) and, if necessary, a 5' region comprising the additional sequencing adaptor and / or the remaining portion of the sequencing adaptor.

[0083] The oligonucleotide component of the scaffold adapter may include one or more additional subcomponents, including an index polynucleotide, a unique molecular identifier (UMI), a primer binding site (e.g., a P5 primer binding site, a P7 primer binding site), a flow cell binding region, or a sequencing adapter, or their complementary polynucleotides. The oligonucleotide may also include a primer binding site (or a polynucleotide complementary to a primer binding site). An oligonucleotide (or its complement) containing a P5 primer binding site may be referred to as a P5 oligo or P5 oligonucleotide. An oligonucleotide (or its complement) containing a P7 primer binding site may be referred to as a P7 oligo or P7 oligonucleotide.

[0084] The scaffold polynucleotide can be hybridized with an oligonucleotide to form a duplex within the scaffold adapter. Thus, the scaffold adapter may be referred to as a scaffold duplex, a duplex adapter, a duplex oligonucleotide, or a duplex polynucleotide. Each scaffold duplex having a unique scaffold polynucleotide (i.e., containing a unique ssNA hybridization region sequence) may be referred to as a scaffold duplex species, and a group of multiple scaffold duplex species may be referred to as multiple scaffold duplex species. In some embodiments, the scaffold polynucleotide and the oligonucleotide are on separate DNA strands. In some embodiments, the scaffold polynucleotide and the oligonucleotide are on a single DNA strand (e.g., a single DNA strand that can form a hairpin structure).

[0085] Scaffold adapters can comprise DNA, RNA, or a combination thereof. Scaffold adapters can comprise DNA scaffold polynucleotides and DNA oligonucleotides, DNA scaffold polynucleotides and RNA oligonucleotides, RNA scaffold polynucleotides and DNA oligonucleotides, or RNA scaffold polynucleotides and RNA oligonucleotides. Examples of scaffold adapter compositions and designs are shown in Figure 54 (vertical shading indicates RNA, diagonal shading indicates DNA). Figure 54, top, shows a scaffold adapter comprising a DNA scaffold polynucleotide and a DNA oligonucleotide in association with an RNA sample nucleic acid; examples of ligases for use with such adapters include T4 RNA ligase 2 and T4 DNA ligase. Figure 54, middle, shows a scaffold adapter comprising a DNA scaffold polynucleotide and an RNA oligonucleotide in association with an RNA sample nucleic acid; examples of ligases for use with such adapters include T4 RNA ligase 1. Figure 54, bottom, shows a scaffold adapter comprising an RNA scaffold polynucleotide and an RNA oligonucleotide in association with an RNA sample nucleic acid; examples of ligases for use with such adapters include T4 RNA ligase 1. In some cases, the adaptor nucleotide composition is selected to provide uniformity between the sample nucleic acid and the scaffold adaptor nucleic acid (e.g., at least the oligonucleotide is uniform with the sample nucleic acid). In some cases, the adaptor nucleotide composition is selected to provide uniformity between the oligonucleotide and the sample nucleic acid, and also heterogeneity between the scaffold polynucleotide and the sample nucleic acid.

[0086] Conjugation of the scaffold adaptor or its components with ssNA The methods herein may include binding one or more scaffold adaptors or components thereof to a composition comprising a single-stranded nucleic acid (ssNA) to form one or more complexes. The scaffold polynucleotide is designed for simultaneous hybridization with the ssNA fragment and the oligonucleotide component, such that the end of the oligonucleotide component is adjacent to the end of the terminal region of the ssNA fragment upon complex formation. Typically, upon complex formation, the 5' end of the oligonucleotide component is adjacent to the 3' end of the terminal region of the ssNA, or the 5' end of the oligonucleotide component is adjacent to the 3' end of the terminal region of the ssNA. In cases where the scaffold adaptors are bound to both ends of the ssNA fragment upon complex formation, the 5' end of one oligonucleotide component is adjacent to the 3' end of one terminal region of the ssNA, and the 5' end of the second oligonucleotide component is adjacent to the 3' end of the second terminal region of the ssNA.

[0087] In some embodiments, the method includes forming a complex by combining an ssNA composition with an oligonucleotide and a plurality of heterogeneous scaffold polynucleotides having various random ssNA hybridization regions that can act as scaffolds for a heterogeneous population of ssNAs with terminal regions of uncertain sequence.

[0088] In some embodiments, the ssNA hybridization region comprises a known sequence designed to hybridize with the ssNA terminal region of a known sequence. In some embodiments, two or more heterogeneous scaffold polynucleotides with different ssNA hybridization regions of known sequences are designed to hybridize with each ssNA terminal region of a known sequence. The embodiment in which the ssNA hybridization region has a known sequence can be useful, for example, for generating a nucleic acid library from a subset of ssNAs with terminal regions of known sequences. Thus, in certain embodiments, the method herein comprises forming a complex by combining an ssNA composition with an oligonucleotide and one or more heterogeneous scaffold polynucleotides with one or more different ssNA hybridization regions of known sequences that can act as a scaffold for one or more ssNAs with one or more terminal regions of known sequences.

[0089] The ssNA fragment, oligonucleotide, and scaffold polynucleotide can be linked in various ways. In some configurations, linking comprises linking 1) a complex comprising a scaffold polynucleotide hybridized to an oligonucleotide component via an oligonucleotide hybridization region with 2) the ssNA fragment. In other configurations, linking comprises linking 1) a complex comprising a scaffold polynucleotide hybridized to an ssNA fragment via an ssNA hybridization region with 2) the oligonucleotide component. In other configurations, linking comprises linking 1) the ssNA fragment with 2) the oligonucleotide and 3) the scaffold polynucleotide, where none of the three components are pre-complexed or hybridized with another component prior to linking.

[0090] The binding can be carried out under hybridization conditions that form a complex comprising the scaffold polynucleotide hybridized with the terminal region of the ssNA fragment through the ssNA hybridization region and the scaffold polynucleotide hybridized with the oligonucleotide component through the oligonucleotide hybridization region.Whether specific hybridization occurs can be determined by factors such as the degree of complementarity between the hybridization regions of the scaffold polynucleotide, the terminal region of the ssNA fragment and the oligonucleotide component, as well as their lengths, salt concentration, and the temperature at which hybridization occurs, and the melting temperature (Tm) of the relevant region can provide information about the temperature at which hybridization occurs.

[0091] The complex can be formed such that the terminus of the oligonucleotide component is adjacent to the terminus of the terminal region of the ssNA fragment. "Adjacent to" refers to the terminal nucleotide of the oligonucleotide and the terminal nucleotide end of the terminal region of the ssNA fragment being close enough to each other that they can be covalently linked, for example, by chemical ligation, enzymatic ligation, etc. In some embodiments, the terminus is adjacent to each other because the terminal nucleotide of the oligonucleotide and the terminal nucleotide end of the terminal region of the ssNA are hybridized to adjacent nucleotides of the scaffold polynucleotide. The scaffold polynucleotide can be designed to ensure that the terminus of the oligonucleotide is adjacent to the terminus of the terminal region of the ssNA fragment.

[0092] The scaffold polynucleotide can be designed with one or more uracil bases instead of thymine. In some embodiments, one of the strands in the scaffold adaptor duplex can be degraded by generating multiple cleavage sites at the uracil bases, for example, by using uracil-DNA glycosylase and an endonuclease.

[0093] The scaffold adaptor, oligonucleotide component, and scaffold polynucleotide may be referred to herein as a first scaffold adaptor (or first scaffold duplex), a first oligonucleotide component (or first oligonucleotide), and a first scaffold polynucleotide; or a second scaffold adaptor (or second scaffold duplex), a second oligonucleotide component (or second oligonucleotide), and a second scaffold polynucleotide. The terms first and second generally refer to the scaffold adaptor, or a component thereof, hybridized to and / or covalently linked to the first and second ends (i.e., the 5' and 3' ends) of the ssNA fragment ends. The terms first and second ends do not necessarily refer to a particular orientation of the ssNA fragment. Thus, the first end of the ssNA end may be the 5' end or the 3' end, and the second end of the ssNA end may be the 5' end or the 3' end. The first scaffold adaptor, or component thereof, may refer to a P5 adaptor, or component thereof, or a P7 adaptor, or component thereof. The second scaffold adaptor, or component thereof, may refer to a P5 adaptor, or component thereof, or a P7 adaptor, or component thereof.

[0094] In some cases, before binding the scaffold adapter or its components to the nucleic acid sample containing ssNA, the nucleic acid sample can be treated with a nuclease to remove undesired nucleic acids. For example, as disclosed herein, a double-strand-specific nuclease (e.g., T7 nuclease) can be used to digest some or all double-stranded DNA, and then the scaffold adapter can be used to prepare a sequencing library of the remaining nucleic acids. In some instances, the double-strand-specific nuclease is used to digest double-stranded nucleic acids in the sample, digesting double-stranded DNA from host organisms and / or bacteria while leaving intact single-stranded nucleic acids, such as those from single-stranded DNA viruses, single-stranded RNA viruses, and single-stranded DNA (e.g., damaged DNA).

[0095] Binding of the scaffold adaptor or its components to ssRNA or sscDNA The methods herein may include combining one or more scaffold adaptors, or components thereof, with a composition comprising a single-stranded ribonucleic acid (ssRNA) or a single-stranded complementary deoxyribonucleic acid (sscDNA) to form one or more complexes. The scaffold polynucleotide is designed for simultaneous hybridization with the ssRNA or sscDNA fragment and the oligonucleotide component, such that, upon complex formation, the termini of the oligonucleotide component are adjacent to the termini of the terminal regions of the ssRNA or sscDNA fragment, as described above for ssRNA.

[0096] In some embodiments, the nucleic acid composition comprises sscDNA. In some embodiments, the method includes generating sscDNA from single-stranded ribonucleic acid (ssRNA) prior to the binding step. Typically, when the nucleic acid composition comprises sscDNA, the methods herein use first-strand cDNA and do not require the generation of second-strand cDNA. Thus, in some embodiments, the nucleic acid composition comprises first-strand sscDNA. In some embodiments, the nucleic acid composition consists essentially of first-strand sscDNA. A nucleic acid composition "consisting essentially of" first-strand sscDNA generally comprises first-strand sscDNA and does not include additional protein or nucleic acid components. A nucleic acid composition consisting essentially of first-strand sscDNA generally does not include second-strand sscDNA. In addition, for example, a nucleic acid composition "consisting essentially of" first-strand sscDNA may not include double-stranded cDNA (dscDNA) or may include a low percentage of dscDNA (e.g., less than 10% dscDNA, less than 5% dscDNA, less than 1% dscDNA). A nucleic acid composition "consisting essentially of" first-strand sscDNA may be protein-free. For example, a nucleic acid composition "consisting essentially of" first-strand sscDNA may not contain single-strand binding protein (SSB) or other proteins useful for stabilizing first-strand sscDNA. A nucleic acid composition "consisting essentially of" first-strand sscDNA may contain chemical components typically present in nucleic acid compositions, such as buffers, salts, alcohols, crowding agents (e.g., PEG), and may also contain residual components (e.g., nucleic acids (e.g., residual RNA), proteins, cell membrane components) from the nucleic acid source (e.g., sample), nucleic acid extraction, or cDNA synthesis. A nucleic acid composition "consisting essentially of" first-strand sscDNA may contain first-strand sscDNA fragments having one or more phosphates (e.g., terminal phosphate, 5'-terminal phosphate). A nucleic acid composition "consisting essentially of" first-strand sscDNA may also contain first-strand sscDNA fragments containing one or more modified nucleotides.

[0097] In some embodiments, the step of generating sscDNA includes contacting the ssRNA with a primer and an agent having reverse transcriptase activity to generate a DNA-RNA duplex. In some embodiments, the step of generating sscDNA may further include contacting the DNA-RNA duplex with an agent having RNAse activity to digest the RNA and generate an sscDNA product. In some embodiments, the agent having reverse transcriptase activity also has RNAse activity. Thus, in some embodiments, reverse transcription and RNAse digestion are combined in one step. In some embodiments, the agent having reverse transcriptase activity and RNAse activity is M-MuLV reverse transcriptase (also referred to as M-MLV reverse transcriptase). The primer(s) may be any primer(s) suitable for use with a reverse transcriptase. The primer(s) may be selected from one or more of random primers (e.g., random hexamer primers, random octamer primers), and poly(T) primers. The sscDNA product can be purified by a suitable purification or washing method, for example, a purification or washing method described herein.

[0098] In some embodiments, the nucleic acid composition comprises ssRNA. In such embodiments, a scaffold adaptor is hybridized directly to the ssRNA fragment, and an oligonucleotide component is covalently linked to one or more of the ends of the ssRNA, thereby forming a hybridization product containing one or more scaffold adaptors and the ssRNA fragment. In some embodiments, the method further comprises generating a single-stranded ligation product from the hybridization product (e.g., by denaturing the hybridization product). In such embodiments, the single-stranded ligation product comprises an ssRNA fragment covalently linked to one or more oligonucleotide components. In some embodiments, the method further comprises contacting the single-stranded ligation product with a primer and an agent having reverse transcriptase activity to generate a DNA-RNA duplex. In some embodiments, the method further comprises contacting the DNA-RNA duplex with an agent having RNAse activity to digest the RNA and generate a single-stranded cDNA (sscDNA) product. In some embodiments, the agent having reverse transcriptase activity also has RNAse activity. Thus, in some embodiments, reverse transcription and RNAse digestion are combined into one step. In some embodiments, the agent having reverse transcriptase activity and RNAse activity is M-MuLV reverse transcriptase (also called M-MLV reverse transcriptase). The primer can be any primer suitable for use with reverse transcriptase. In some embodiments, the primer comprises a nucleotide sequence complementary to a sequence in the oligonucleotide component (i.e., the oligonucleotide component covalently linked to the ssRNA fragment). The sscDNA product can be purified by a suitable purification or washing method, for example, the purification or washing method described herein.

[0099] In some embodiments, the sscDNA product is amplified. The sscDNA product can be amplified by a suitable amplification method, such as those described herein. In some embodiments, the step of amplifying the sscDNA product can be performed simultaneously with the step of generating a DNA-RNA duplex and / or the step of generating the sscDNA product (e.g., performed simultaneously in a single step, reaction, vessel, and / or volume). Thus, reagents for generating the DNA-RNA duplex (e.g., one or more agents having reverse transcriptase activity), reagents for generating the sscDNA product (e.g., one or more agents having RNAse activity), and reagents for amplifying the sscDNA product (e.g., primers, agents having polymerase activity) can be combined for use in a single step, reaction, vessel, and / or volume. In some embodiments, the reagents for amplifying the sscDNA product include amplification primers that hybridize to components of the scaffold adapter described herein (e.g., first oligonucleotide). The amplification primers can be any primers suitable for use with a polymerase. In some embodiments, each primer comprises a nucleotide sequence complementary to a sequence in the sscDNA product corresponding to the oligonucleotide component (i.e., the oligonucleotide component covalently linked to the ssRNA fragment). The amplified sscDNA product can be purified by a suitable purification or wash method, such as those described herein.

[0100] In some embodiments, the methods herein include fragmenting the ssRNA to generate ssRNA fragments before binding the ssRNA to a scaffold adaptor or a component thereof, or before generating the sscDNA. Any suitable fragmentation method can be used, such as, for example, the fragmentation methods described herein. In some embodiments, the methods herein include depleting ribosomal RNA (rRNA) and / or enriching messenger RNA (mRNA) before binding the ssRNA to a scaffold adaptor or a component thereof, or before generating the sscDNA. Any suitable rRNA depletion and / or mRNA enrichment method can be used, such as, for example, the rRNA depletion and / or mRNA enrichment methods described herein.

[0101] Hybridization and ligation A ligation product is generated by binding a nucleic acid fragment (e.g., an ssNA fragment) to a scaffold adapter or a component thereof. Binding the ssNA fragment to the scaffold adapter or a component thereof can include hybridization and / or ligation (e.g., ligation of a hybridization product). The ligation product can include an ssNA fragment connected to (e.g., hybridized and / or ligated to) a scaffold adapter or a component thereof at one or both ends of the ssNA fragment. The ligation product can include an ssNA fragment hybridized to a scaffold adapter or a component thereof at one or both ends of the ssNA fragment, which fragment may be referred to as a hybridization product. The ligation product can include an ssNA fragment ligated to a scaffold adapter or a component thereof at one or both ends of the ssNA fragment, which fragment may be referred to as a ligation product. In some embodiments, the ligation product can be generated by binding the product from the cleavage step (i.e., the cleavage product) to a scaffold adapter or a component thereof. Certain methods herein include generating a set of ligation products (e.g., a first set of ligation products and a second set of ligation products). In some embodiments, the first set of ligation products comprises ssNAs connected to (e.g., hybridized and / or ligated with) scaffold adapters, or components thereof, from a first set of scaffold adapters, or components thereof. In some embodiments, the second set of ligation products comprises the first set of ligation products connected to (e.g., hybridized and / or ligated with) scaffold adapters, or components thereof, from a second set of scaffold adapters, or components thereof.

[0102] A hybridization product can be generated by binding the ssNA to the scaffold adapter or its components under hybridization conditions. In some embodiments, the scaffold adapter is provided as a pre-hybridized product, and the hybridization step comprises hybridizing the scaffold adapter to the ssNA. In some embodiments, the scaffold adapter components (i.e., the oligonucleotide and the scaffold polynucleotide) are provided as individual components, and the hybridization step comprises hybridizing the scaffold adapter components 1) to each other and 2) to the ssNA. In some embodiments, the scaffold adapter components (i.e., the oligonucleotide and the scaffold polynucleotide) are provided sequentially as individual components, and the hybridization step comprises 1) hybridizing the scaffold polynucleotide to the ssNA, and then 2) hybridizing the oligonucleotide to the oligonucleotide hybridization region of the scaffold polynucleotide. The conditions during the binding step are conditions under which the scaffold adapter, or a component thereof (e.g., a single-stranded scaffold region), specifically hybridizes with the ssNA having a terminal region or regions whose sequence is complementary to the single-stranded scaffold region. The conditions during the binding step can also include conditions under which the components of the scaffold adapter (e.g., an oligonucleotide and an oligonucleotide hybridization region in the scaffold polynucleotide) specifically hybridize or remain hybridized to each other.

[0103] Specific hybridization can be influenced or affected by factors such as the degree of complementarity between the single-stranded scaffold region and the ssNA terminal region or between the oligonucleotide and the oligonucleotide hybridization region, their lengths, and the temperature at which hybridization occurs, which can be determined from the melting temperature (Tm) of the single-stranded scaffold region. The melting temperature generally refers to the temperature at which half of the single-stranded scaffold region / ssNA terminal region remains hybridized and half of the single-stranded scaffold region / ssNA terminal region dissociates into single strands. The Tm of a duplex can be determined experimentally or can be calculated using the formula Tm = 81.5 + 16.6(log 10 [Na+]) + 0.41(fraction G+C) - (60 / N), where N is the chain length and [Na+] is less than 1 M. Additional models that depend on various parameters can be used to predict the Tm of related regions, which depend on various hybridization conditions. Approaches to achieving specific nucleic acid hybridization are described, for example, in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier (1993).

[0104] In some embodiments, the methods herein include exposing the hybridization product to conditions under which the end of the ssNA binds to the end of the scaffold adaptor to which it is hybridized. In particular, the methods herein may include exposing the hybridization product to conditions under which the end of the ssNA binds to the end of the oligonucleotide component of the scaffold adaptor to which it is hybridized. Binding can be achieved by any suitable approach that allows the ssNA to be covalently bound to the scaffold adaptor and / or the oligonucleotide component of the scaffold adaptor to which it is hybridized. When one end of the ssNA is bound to the end of the scaffold adaptor and / or the oligonucleotide component of the scaffold adaptor to which it is hybridized, one of the following two binding events usually occurs: 1) the 3' end of the ssNA binds to the 5' end of the oligonucleotide component of the scaffold adaptor, or 2) the 5' end of the ssNA binds to the 3' end of the oligonucleotide component of the scaffold adaptor. When both ends of the ssNA are each bound to the ends of the scaffold adapter and / or oligonucleotide component of the scaffold adapter to which it is hybridized, two binding events typically occur: 1) the binding event of the 3' end of the ssNA to the 5' end of the oligonucleotide component of the first scaffold adapter, and 2) the binding event of the 5' end of the ssNA to the 3' end of the oligonucleotide component of the second scaffold adapter.

[0105] In some embodiments, the methods herein include contacting the hybridization product with an agent having ligase activity under conditions in which the end of the ssNA is covalently ligated to the end of the scaffold adaptor and / or the oligonucleotide component of the scaffold adaptor to which the target nucleic acid (ssNA) is hybridized. Ligase activity may include, for example, blunt-end ligase activity, nick-sealing ligase activity, sticky-end ligase activity, circularization ligase activity, sticky-end ligase activity, DNA ligase activity, RNA ligase activity, single-stranded ligase activity, and double-stranded ligase activity. Ligase activity may include ligating the 5' phosphorylated end of one polynucleotide to the 3' OH end of another polynucleotide (5'P to 3'OH). Ligase activity may include ligating the 3' phosphorylated end of one polynucleotide to the 5' OH end of another polynucleotide (3'P to 5'OH). The ligase activity can include ligating the 5' end of the ssNA to the 3' end of the scaffold adapter hybridized therewith and / or the oligonucleotide component of the scaffold adapter hybridized therewith in a ligation reaction. The ligase activity can include ligating the 3' end of the ssNA to the 5' end of the scaffold adapter hybridized therewith and / or the oligonucleotide component of the scaffold adapter hybridized therewith in a ligation reaction. Suitable reagents (e.g., ligase) and kits for carrying out ligation reactions are known and available. For example, Instant Sticky-end Ligase Master Mix available from New England Biolabs (Ipswich, MA) can be used. Ligases that can be used include, but are not limited to, for example, T3 ligase, T4 DNA ligase (e.g., at low or high concentrations), T7 DNA ligase, E. coli DNA ligase, ElectroLigase®, RNA ligase, T4 RNA Ligase 2, Splint® ligase, RtcB ligase, Taq ligase, and the like, and combinations thereof.If required, a phosphate group can be added to the 5' end of an oligonucleotide component or ssNA fragment using a suitable kinase, such as, for example, T4 polynucleotide kinase (PNK). Such kinases and guidance for using them to phosphorylate 5' ends are available, for example, from New England BioLabs, Inc. (Ipswich, MA).

[0106] In some embodiments, the method comprises covalently linking the adjacent ends of the oligonucleotide component and the ssNA-terminal region, thereby generating a covalently linked hybridization product. In some embodiments, the covalently linking step comprises contacting the hybridization product (e.g., the ssNA fragment hybridized with at least one scaffold adaptor herein) with an agent having ligase activity under conditions in which the end of the ssNA-terminal region is covalently linked to the end of the oligonucleotide component. In some embodiments, the method comprises covalently linking the adjacent ends of the first oligonucleotide component and the first ssNA-terminal region, and covalently linking the adjacent ends of the second oligonucleotide component and the second ssNA-terminal region, thereby generating a covalently linked hybridization product. In some embodiments, the covalent ligation step comprises contacting the hybridization product (e.g., ssNA fragments each hybridizing to two scaffold adaptors herein) with an agent having ligase activity under conditions such that the end of the first ssNA terminal region is covalently ligated to the end of the first oligonucleotide component and the end of the second ssNA terminal region is covalently ligated to the end of the second oligonucleotide component. In some embodiments, the agent having ligase activity is T4 DNA ligase. In some embodiments, T4 DNA ligase is used in an amount of between about 1 unit / μl and about 50 units / μl. In some embodiments, T4 DNA ligase is used in an amount of between about 5 units / μl and about 30 units / μl. In some embodiments, T4 DNA ligase is used in an amount of between about 5 units / μl and about 15 units / μl. In some embodiments, T4 DNA ligase is used at about 10 units / μl. In some embodiments, T4 DNA ligase is used in an amount of less than 25 units / μl. In some embodiments, T4 DNA ligase is used in an amount of less than 20 units / μl. In some embodiments, T4 DNA ligase is used in an amount of less than 15 units / μl. In some embodiments, T4 DNA ligase is used in an amount of less than 10 units / μl.

[0107] In some embodiments, the hybridization product is contacted with a first agent having a first ligase activity and a second agent having a second ligase activity different from the first ligase activity. For example, the first ligase activity and the second ligase activity can be independently selected from blunt-end ligase activity, nick-sealing ligase activity, sticky-end ligase activity, circularization ligase activity, cohesive-end ligase activity, double-stranded ligase activity, single-stranded ligase activity, 5'P to 3'OH ligase activity, and 3'P to 5'OH ligase activity.

[0108] In some embodiments, the methods herein include attaching ssNAs to scaffold adaptors and / or oligonucleotide components of the scaffold adaptors via biocompatible bonds. The methods may include, for example, click chemistry or tagging, which include biocompatible reactions useful for attaching biomolecules. In some embodiments, the terminus of each of the oligonucleotide components includes a first chemically reactive moiety, and the terminus of each of the ssNAs includes a second chemically reactive moiety. In such embodiments, the first chemically reactive moiety is typically capable of reacting with the second chemically reactive moiety to form a covalent bond between the oligonucleotide component of the scaffold adaptor and the ssNA to which the scaffold adaptor is hybridized. In some embodiments, the methods herein include contacting the ssNA with one or more chemical agents under conditions such that a second chemically reactive moiety is incorporated into the terminus of each of the ssNA fragments. In some embodiments, the methods herein include exposing the hybridization product to conditions under which the first chemically reactive moiety reacts with the second chemically reactive moiety to form a covalent bond between the oligonucleotide component and the ssNA to which the scaffold adaptor is hybridized. In some embodiments, the first chemically reactive moiety can react with the second chemically reactive moiety to form a 1,2,3-triazole between the oligonucleotide component and the ssNA to which the scaffold adaptor is hybridized. In some embodiments, the first chemically reactive moiety can react with the second chemically reactive moiety under copper-containing conditions. The first and second chemically reactive moieties can comprise any suitable pairing. For example, the first chemically reactive moiety can be selected from an azide-containing moiety and 5-octadiynyldeoxyuracil, and the second chemically reactive moiety can independently be selected from an azide-containing moiety, hexynyl, and 5-octadiynyldeoxyuracil. In some embodiments, the azide-containing moiety is an N-hydroxysuccinimide (NHS) ester-azide.

[0109] Covalently linking the adjacent ends of the oligonucleotide and the ssNA fragment produces a covalently linked product, which may be referred to as a ligation product. A covalently linked product comprising an ssNA fragment covalently linked to an oligonucleotide component that remains hybridized to the scaffold polynucleotide may be referred to as a covalently linked hybridization product. The covalently linked hybridization product may be denatured (e.g., heat denatured) to separate the ssNA fragment covalently linked to the oligonucleotide component from the scaffold polynucleotide. A covalently linked product comprising an ssNA fragment covalently linked to an oligonucleotide component that is no longer hybridized to the scaffold polynucleotide (after denaturation) may be referred to as a single-stranded ligation product. In some cases, portions of the scaffold polynucleotide may be cleaved and / or modified, for example, by using uracil-DNA glycosylase or an endonuclease against one or more uracil bases in the scaffold polynucleotide.

[0110] Covalently linked hybridization products and / or single-stranded ligation products can be purified prior to use as input in a desired downstream application (e.g., amplification; sequencing). For example, covalently linked hybridization products and / or single-stranded ligation products can be purified from certain components present during the binding, hybridization, and / or covalent linking (ligation) steps (e.g., by solid-phase reversible immobilization (SPRI), column purification, and / or the like).

[0111] In some embodiments, when the method herein comprises combining an ssNA composition with a scaffold adaptor herein or a component thereof, and covalently linking adjacent ends of the oligonucleotide component and the ssNA fragment, the total duration of the combining and covalently linking steps can be 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. In some embodiments, the total duration of the combining and covalently linking steps is less than 1 hour.

[0112] In some embodiments, the methods herein are performed in a single container, a single chamber, and / or a single volume (i.e., a continuous volume), including but not limited to, a microfluidic device. In some embodiments, the steps of combining the ssNA composition with the scaffold adaptor herein or a component thereof, and covalently linking adjacent ends of the oligonucleotide component and the ssNA fragment, are performed in a single container, a single chamber, and / or a single volume (i.e., a continuous volume), including but not limited to, a microfluidic device. In some embodiments, the methods herein are performed in a series of wells, droplets, emulsions, compartments, or other reaction volumes, including but not limited to, a microfluidic device. In some embodiments, the steps of combining the ssNA composition with the scaffold adaptor herein or a component thereof, and covalently linking adjacent ends of the oligonucleotide component and the ssNA fragment, are performed in a series of wells, droplets, emulsions, compartments, or other reaction volumes, including but not limited to, a microfluidic device. In some cases, the series of reaction volumes are prepared such that most or all of the reaction volumes contain at most one ssNA. In some cases, a series of reaction volumes are prepared so that most or all of the reaction volumes contain at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 100000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 or more ssNAs. The distribution of one or a limited number of ssNAs to the reaction volumes can result in favorable reaction kinetics, such as increasing the library conversion of rare species of sample nucleic acids.

[0113] Adapter Dimer In some embodiments, the methods herein include one or more modifications and / or additional steps to prevent, reduce, or eliminate adapter dimers. Adapter dimers may be unintentionally formed during the methods described herein. An adapter dimer generally refers to two or more scaffold adapters, their components, or their parts that hybridize or hybridize and ligate with each other. An example of a specific adapter dimer configuration is provided in Figure 20.

[0114] In certain embodiments, the scaffold adapter, or a component thereof, is modified to prevent adapter dimer formation. Examples of modifications of the scaffold adapter include modified nucleotides that can block covalent linkage of the scaffold adapter, oligonucleotide component, or scaffold polynucleotide to another oligonucleotide, polynucleotide, or nucleic acid molecule (e.g., another scaffold adapter, oligonucleotide component, and / or scaffold polynucleotide). Examples of modified nucleotides are described below. Other / further modifications of the scaffold adapter include configurations such as a Y configuration or a hairpin configuration, which are described in more detail below. In some embodiments, the scaffold adapter, oligonucleotide component, and / or scaffold polynucleotide may include a phosphorothioate backbone modification (e.g., a phosphorothioate bond between the last two nucleotides on the strand).

[0115] In some embodiments, the method comprises a dephosphorylation step to prevent or reduce adapter dimer formation. In some embodiments, the method comprises, prior to the step of conjugating the scaffold adapter or a component thereof with the ssNA, contacting the scaffold adapter, oligonucleotide component, and / or scaffold polynucleotide with an agent having phosphatase activity under conditions in which the scaffold adapter, oligonucleotide component, and / or scaffold polynucleotide is dephosphorylated, thereby generating a dephosphorylated scaffold adapter, dephosphorylated oligonucleotide component, and / or dephosphorylated scaffold polynucleotide.

[0116] In some embodiments, the method includes one or more stepwise ligation approaches to prevent or reduce adapter dimer formation. In some embodiments, the method includes stepwise ligation, which includes delaying the addition of an agent having phosphoryl transfer activity (e.g., until after the hybridization product has formed) and / or delaying the addition of a second scaffold adapter or component thereof (see Figure 9). For example, the method may include contacting the oligonucleotide component with an agent having phosphoryl transfer activity under conditions in which a 5' phosphate is added to the 5' end of the oligonucleotide component after forming the hybridization product and before covalently linking the oligonucleotide component to the ssNA-terminal region. In another example, the method may include combining a first set of scaffold adapters with the ssNA. The first set of scaffold adapters may include oligonucleotide components having a 3' OH group. The first set of scaffold adapters is hybridized to the ssNA, and the 3' OH group of the oligonucleotide component is covalently linked to the 5' end (e.g., the 5' phosphorylated end) of the ssNA-terminal region. The product of this first round of hybridization and covalent linkage may be referred to as a covalently linked hybridization product intermediate.The covalently linked hybridization product intermediate is then linked to a second set of scaffold adaptors.The second set of scaffold adaptors may comprise an oligonucleotide component with a 5' end that can be phosphorylated as described herein.The second set of scaffold adaptors is hybridized to the covalently linked hybridization product intermediate, and the 5' phosphorylated end of the oligonucleotide component is covalently linked to the 3' end of the ssNA terminal region.

[0117] In some embodiments, the method involves stepwise ligation, including the use of scaffold adapters or components thereof having an adenylation modification (see FIG. 10). For example, a first set of scaffold adapters may include an adenylation modification (5'App) at the 5' end of an oligonucleotide component. The first set of scaffold adapters is hybridized to the ssNA, and the 5'App of the oligonucleotide component is covalently linked to the 3' end of the ssNA-terminal region. Covalent ligation may be performed in the absence of ATP. The product of this first round of hybridization and covalent ligation may be referred to as a covalently linked hybridization product intermediate. The covalently linked hybridization product intermediate is then ligated with a second set of scaffold adapters. The second set of scaffold adapters may include oligonucleotide components having a 3'OH terminus. The second set of scaffold adaptors is hybridized to the covalently linked hybridization product intermediate, and the 3'OH end of the oligonucleotide component is covalently linked (by adding ATP) to the 5' end (e.g., 5' phosphorylated end) of the ssNA-terminal region. In one variant, the first set of scaffold adaptors and the second set of scaffold adaptors are simultaneously linked to the ssNA in the absence of ATP. Ligation of the first set of scaffold adaptors can proceed in the absence of ATP, and ligation of the second set of scaffold adaptors can proceed only until ATP is added.

[0118] In some embodiments, the method involves stepwise ligation using an oligonucleotide (i.e., a single-stranded oligonucleotide) having a 3' phosphorylated end (see Figure 11). The oligonucleotide having a 3' phosphorylated end may include any of the subcomponents described herein for the oligonucleotide component of the scaffold adaptor (e.g., a primer binding site, an index, a UMI, a flow cell adaptor, etc.). The oligonucleotide having a 3' phosphorylated end is generally single-stranded and not hybridized to the scaffold polynucleotide. In one example, prior to the step of linking the scaffold adaptor or its component to the ssNA, the method may include the steps of: linking the ssNA to an oligonucleotide comprising a phosphate at its 3' end; and covalently linking the 3' phosphorylated end of the oligonucleotide to the 5' end (e.g., the 5' non-phosphorylated end) of the ssNA-terminal region. In some embodiments, prior to the step of covalently linking the oligonucleotide to the ssNA, the ssNA is contacted with an agent having phosphatase activity under conditions in which the ssNA is dephosphorylated, thereby generating a dephosphorylated ssNA. In some embodiments, the step of covalently linking the oligonucleotide to the ssNA comprises contacting the ssNA and the oligonucleotide with an agent having single-stranded ligase activity under conditions in which the 5' end of the ssNA is covalently linked to the 3' end of the oligonucleotide. In some embodiments, the agent having ligase activity is RtcB ligase. The product of such a covalent linking step may be referred to as a covalently linked product intermediate. The covalently linked product intermediate is then linked to a set of scaffold adaptors. The set of scaffold adaptors may include an oligonucleotide component having a 5' phosphorylated end. The set of scaffold adaptors is hybridized to the covalently linked product intermediate, and the 5' phosphorylated end of the oligonucleotide component is covalently linked to the 3' end of the ssNA terminal region.

[0119] In some embodiments, the methods include the use of oligonucleotides capable of hybridizing to oligonucleotide dimer products to reduce or eliminate adapter dimers (see Figure 20). The oligonucleotide dimer products are components of a scaffold adapter dimer and may contain an oligonucleotide component from a first scaffold adapter covalently linked to an oligonucleotide component from a second scaffold adapter. The methods herein may include a denaturation step that can release the oligonucleotide dimer product from the scaffold adapter dimer. The oligonucleotide dimer product can hybridize to an oligonucleotide having a sequence complementary to the oligonucleotide dimer product, or a portion thereof, thereby forming an oligonucleotide dimer hybridization product. In some embodiments, the oligonucleotide dimer hybridization product includes a cleavage site. In some embodiments, the cleavage site is a restriction enzyme recognition site. In some embodiments, the methods herein further include contacting the oligonucleotide dimer hybridization product with a cleavage agent (e.g., a restriction enzyme, a rare-cutter restriction enzyme).

[0120] In some embodiments, the method includes purifying or washing the nucleic acid products at various stages of library preparation to reduce or eliminate adapter dimers. In some cases, purifying or washing the nucleic acid products can reduce or eliminate adapter dimers. For example, covalently linked hybridization products (i.e., hybridized to a scaffold adapter and covalently linked to an oligonucleotide component, ssNA), single-stranded ligation products (i.e., denatured, covalently linked hybridization products; covalently linked to an oligonucleotide component and no longer able to hybridize to a scaffold polynucleotide, ssNA), or amplification products thereof can be purified or washed by any suitable purification or washing method. In some embodiments, the purifying or washing step includes the use of solid-phase reversible immobilization (SPRI). For example, SPRI beads can be suspended in a DNA binding buffer containing about 2.5 M to about 5 M NaCl, about 0.1 mM to about 1 M EDTA, about 10 mM Tris, about 0.01% to about 0.05% Tween®-20, and between about 8% and about 38% PEG-8000. For example, 1 ml of SPRI bead suspension can be combined with 2.5 M NaCl, 10 mM Tris, 1 mM EDTA, 0.05% Tween-20, and 20% PEG-8000. In some embodiments, SPRI includes sequential SPRI (sequential washes) and / or sequential SPRI (washes involving sequential addition and incubation of SPRI beads). Sequential SPRI can include multiple consecutive washes, which can include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more consecutive washes. Sequential SPRI can involve multiple sequential additions of SPRI beads (intervening incubations), which can include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sequential additions of SPRI beads. In some embodiments, the amount of SPRI beads used in SPRI purification can include between 0.1× and 3× SPRI beads (× is the ratio of beads to nucleic acid (e.g., bead volume to reaction volume)).For example, the amount of SPRI beads used in SPRI purification may include about 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x SPRI beads. In some embodiments, the amount of SPRI beads used in SPRI purification is 1.2x. In some embodiments, the amount of SPRI beads used in SPRI purification is 1.5x. In some embodiments, the purifying or washing step comprises column purification (e.g., column chromatography). In some embodiments, the purifying or washing step does not include column purification (e.g., column chromatography). In some embodiments, the covalently linked hybridization products, single-stranded ligation products, and / or their amplification products are not purified or washed.

[0121] SPRI purification is usually performed in the presence of a buffer. Any suitable buffer, such as Tris buffer or water of a similar pH, can be used. SPRI purification beads can be added directly to a sample solution (e.g., a sample solution containing covalently linked hybridization products (ligation products) or their amplified products). In certain cases, buffer can be added to increase the reaction volume, allowing additional beads to be added. In some embodiments, the SPRI bead solution is composed of carboxylated magnetic beads added to PEG 8000 dissolved in water, NaCl, Tris, and EDTA. The amount of PEG typically determines the PEG percentage of the SPRI bead solution. For example, adding 9 g of PEG 8000 to a 50 ml SPRI bead solution can be referred to as "18% SPRI." In another example, adding 19 g of PEG 8000 to a 50 ml SPRI solution can be referred to as "38% SPRI." Generally, the higher the PEG ratio, the smaller the size of the DNA fragments retained.

[0122] In some embodiments, the purification process includes contacting the covalently linked hybridization product (ligation product) with solid phase reversible immobilization (SPRI) beads and a buffer. In some embodiments, some or all of the SPRI buffer is replaced with isopropanol. In some embodiments, the SPRI buffer includes isopropanol. In some embodiments, the SPRI buffer is completely replaced with isopropanol. In some embodiments, the SPRI buffer includes about 5% volume / volume (v / v) isopropanol to about 50% v / v isopropanol. In some embodiments, the SPRI buffer includes about 10% v / v isopropanol to about 40% v / v isopropanol. For example, the SPRI buffer can contain about 10% v / v isopropanol, 15% v / v isopropanol, 20% v / v isopropanol, 25% v / v isopropanol, 30% v / v isopropanol, 35% v / v isopropanol, or 40% v / v isopropanol. In some embodiments, the SPRI buffer contains about 20% v / v isopropanol.

[0123] In some embodiments, the purification or washing step can enrich for nucleic acid fragments or their amplification products having a specific length or length range. In some embodiments, SPRI purification can enrich for nucleic acid fragments or their amplification products having a specific length or length range. In some embodiments, the amount of PEG 8000 in the SPRI bead solution used in SPRI purification can affect the length or length range of the enriched fragments. For example, SPRI purification at a 1.5x v / v ratio can recover more fragments in the <100 base range than SPRI purification at 1.2x because the final PEG 8000 concentration is higher at 1.5x than at 1.2x. In some embodiments, the methods herein include adjusting the SPRI ratio to enrich for a desired fragment length or length range. In some embodiments, the methods herein include adjusting the amount of isopropanol in SPRI purification to enrich for a desired fragment length or length range. In some embodiments, the methods herein include adjusting the amount of isopropanol in SPRI purification to enrich for a desired fragment length or length range while minimizing the amount of undesired artifacts (e.g., adapter dimers). For example, the methods herein include adjusting the amount of isopropanol in SPRI purification to enrich for a desired fragment length or length range, where the amount of adapter-dimer recovered is less than about 10% of the total nucleic acid recovered. In another example, the methods herein include adjusting the amount of isopropanol in SPRI purification to enrich for a desired fragment length or length range, where the amount of adapter-dimer recovered is less than about 5% of the total nucleic acid recovered.

[0124] In some embodiments, the methods herein (e.g., the steps of conjugating, hybridizing, and covalently linking ssNAs to scaffold adapters or components thereof) can be performed in a suitable reaction volume and / or using a suitable amount of ssNAs and / or a suitable ratio of ssNAs to scaffold adapters (or components thereof). A suitable reaction volume and / or a suitable amount of ssNAs and / or a suitable ratio of ssNAs to scaffold adapters (or components thereof) can include a reaction volume, amount of ssNAs, and / or ratio of ssNAs to scaffold adapters that reduces or prevents adapter-dimer formation. In some embodiments, a suitable amount of ssNAs can range from about 250 pg to about 5 ng of ssNAs. For example, a suitable amount of ssNAs can be about 250 pg, 500 pg, 750 pg, 1 ng, 1.5 ng, 2 ng, 2.5 ng, 3 ng, 3.5 ng, 4 ng, 4.5 ng, or 5 ng. In some embodiments, a suitable amount of ssNA can be about 1 ng of ssNA. In some embodiments, for a final reaction volume of 25 μl, 1 ng of ssNA can be combined with between about 1.0 and 2.0 pmoles of each scaffold adapter (i.e., about 1.0 to 2.0 pmoles of scaffold adapter hybridizing to the 5' end of the ssNA-terminal region (a pool of scaffold adapters containing multiple scaffold adapter species) and about 1.0 to 2.0 pmoles of scaffold adapter hybridizing to the 3' end of the ssNA-terminal region (a pool of scaffold adapters containing multiple scaffold adapter species)). For example, for a final reaction volume of 25 μl, 1 ng of ssNA can be combined with about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pmoles of each scaffold adapter. In some embodiments, for a final reaction volume of 25 μl, 1 ng of ssNA is combined with about 1.6 pmoles of each scaffold adaptor (i.e., about 1.6 pmoles of scaffold adaptor hybridizing with the 5' end of the ssNA-terminal region and about 1.6 pmoles of scaffold adaptor hybridizing with the 3' end of the ssNA-terminal region). For larger reaction volumes, the amounts of ssNA and scaffold adaptor can be scaled up, provided that the relative amounts are maintained.For smaller reaction volumes, the amounts of ssNA and scaffold adapter can be scaled down, provided that the relative amounts are preserved. In some embodiments, the scaffold adapter herein is combined with the ssNA at a molar ratio of between about 5:1 (scaffold adapter to ssNA) and about 50:1 (scaffold adapter to ssNA). For example, the scaffold adaptor can be conjugated to the ssNA at a molar ratio of about 5:1 (scaffold adaptor to ssNA), about 10:1 (scaffold adaptor to ssNA), about 15:1 (scaffold adaptor to ssNA), about 20:1 (scaffold adaptor to ssNA), about 25:1 (scaffold adaptor to ssNA), about 30:1 (scaffold adaptor to ssNA), about 35:1 (scaffold adaptor to ssNA), about 40:1 (scaffold adaptor to ssNA), about 45:1 (scaffold adaptor to ssNA), or about 50:1 (scaffold adaptor to ssNA). In some embodiments, the scaffold adaptor is conjugated to the ssNA at a molar ratio of about 15:1 (scaffold adaptor to ssNA). In some embodiments, the scaffold adaptor is conjugated to the ssNA at a molar ratio of about 30:1 (scaffold adaptor to ssNA).

[0125] In some embodiments, the methods herein include the use of a crowding agent. A suitable amount of crowding agent can be used to reduce or prevent adapter dimer formation. Examples of crowding agents include Ficoll 70, Dextran 70, polyethylene glycol (PEG) 2000, and polyethylene glycol (PEG) 8000. In some embodiments, the methods herein include the use of polyethylene glycol (PEG) 8000. PEG can be used, for example, in an amount between about 15% and about 20%, where this percentage refers to the final concentration of PEG in the ligation reaction. For example, PEG can be used at about 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20%. In some embodiments, 18.5% PEG is used. In some embodiments, 18% PEG is used.

[0126] During purification, SPRI bead solution can be added to the sample solution, and instructions for the v / v ratio are often provided. For example, 1.2 x 18% SPRI means adding 60 μl (50 x 1.2) of 18% SPRI beads to a 50 μl sample. This v / v ratio leads to a final PEG concentration of 9.8%, assuming no PEG is present in the sample solution. However, in many cases, there is already a certain amount of PEG present in the sample solution (i.e., the ligation product) after ligation. Therefore, users can adjust the amount of SPRI beads added to reach the desired final PEG concentration. The desired final PEG concentration can range from approximately 5% to approximately 15% final PEG. For example, the desired final PEG concentration can be approximately 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, the desired final PEG concentration is approximately 10% (e.g., for hair and cfDNA samples). In some embodiments, the desired final PEG concentration is about 12% (eg, for formalin-fixed, paraffin-embedded (FFPE) samples and samples with large template fragments).

[0127] Y adapter In some embodiments, a scaffold adaptor described herein comprises two strands, with a single-stranded scaffold region at a first end and two non-complementary strands at a second end. Such scaffold adaptors may be referred to as Y scaffold adaptors, Y adaptors, Y-shaped scaffold adaptors, Y-shaped adaptors, Y duplexes, Y-shaped duplexes, Y scaffold duplexes, Y scaffold duplexes, etc. Scaffold adaptors with a Y-shaped structure generally comprise a double-stranded duplex region, two single-stranded "arms" at one end, and a single-stranded scaffold region at the other end.

[0128] The Y scaffold adaptor may comprise multiple nucleic acid components and subcomponents. In some embodiments, the Y scaffold adaptor comprises a first nucleic acid strand and a second nucleic acid strand. In some embodiments, the first nucleic acid strand is complementary to the second nucleic acid strand. In some embodiments, a portion of the first nucleic acid strand is complementary to a portion of the second nucleic acid strand. In some embodiments, the first nucleic acid strand comprises a first region that is complementary to a first region in the second nucleic acid strand, and the first polynucleotide comprises a second region that is not complementary to a second region in the second polynucleotide. The complementary regions often form duplex regions of the Y scaffold adaptor, and the non-complementary regions often form arms or portions thereof of the Y scaffold adaptor. The first and second nucleic acid strands may comprise subcomponents (e.g., scaffold polynucleotide subcomponents, oligonucleotide subcomponents, and sequencing adaptor subcomponents described herein, such as amplification priming sites and / or individual sequencing adaptors (e.g., P5, P7 adaptors)). In some embodiments, the first and second nucleic acid strands do not include certain subcomponents of the sequencing adapters described herein, such as amplification priming sites and / or individual sequencing adapters (e.g., P5, P7 adapters).

[0129] In some embodiments, the Y scaffold adapter comprises a single-stranded scaffold region (ssNA hybridization region). The single-stranded scaffold region of the Y scaffold adapter is typically located adjacent to the double-stranded duplex portion, at the opposite end of the non-complementary strand (or "arm") portion. The single-stranded scaffold region of the Y scaffold adapter is typically complementary to a terminal region of the target nucleic acid (e.g., a terminal region of a single-stranded nucleic acid).

[0130] hairpin In some embodiments, the scaffold adaptor comprises one strand capable of forming a hairpin structure with a single-stranded loop. In some embodiments, the scaffold adaptor consists of one strand capable of forming a hairpin structure with a single-stranded loop. A scaffold adaptor having a hairpin structure generally comprises a double-stranded "stem" region and a single-stranded "loop" region. In some embodiments, the scaffold adaptor comprises one strand (i.e., one continuous strand) capable of adopting a hairpin structure. In some embodiments, the scaffold adaptor consists essentially of one strand (i.e., one continuous strand) capable of adopting a hairpin structure. Consisting essentially of one strand means that the scaffold adapter does not contain any additional strands of nucleic acid that are not part of a continuous strand (e.g., hybridized to the scaffold adapter). Thus, "consisting essentially of" here refers to the number of strands in the scaffold adapter, and the scaffold adapter may include other features that are not essential to the number of strands (e.g., it may include a detectable label; it may include other regions). A scaffold adapter that includes or consists essentially of one strand that can form a hairpin structure may be referred to herein as a hairpin, hairpin scaffold adapter, or hairpin adapter.

[0131] A hairpin scaffold adapter may comprise multiple nucleic acid components and subcomponents within a single strand. In some embodiments, a hairpin scaffold adapter comprises an oligonucleotide and a scaffold polynucleotide. In some embodiments, the oligonucleotide is complementary to an oligonucleotide hybridization region in the scaffold polynucleotide. In some embodiments, a portion of the oligonucleotide is complementary to a portion of the oligonucleotide hybridization region in the scaffold polynucleotide. In some embodiments, a hairpin scaffold adapter comprises a complementary region and a non-complementary region. The complementary region often forms the stem of the hairpin adapter, and the non-complementary region often forms the loop or a portion of the hairpin scaffold adapter. The oligonucleotide and scaffold polynucleotide may comprise subcomponents (e.g., scaffold polynucleotide subcomponents, oligonucleotide subcomponents, and sequencing adapter subcomponents described herein, such as amplification priming sites and / or individual sequencing adapters (e.g., P5, P7 adapters)). In some embodiments, the oligonucleotides and scaffold polynucleotides do not include certain subcomponents of the sequencing adapters described herein, such as amplification priming sites and individual sequencing adapters (e.g., P5, P7 adapters).

[0132] The hairpin scaffold adaptor may include one or more cleavage sites that can be cleaved under cleavage conditions. In some embodiments, the cleavage site is located between the oligonucleotide and the scaffold polynucleotide. Cleavage at the cleavage site often generates two separate strands from the hairpin scaffold adaptor. In some embodiments, cleavage at the cleavage site generates a partially double-stranded scaffold adaptor with two unpaired strands forming a "Y" structure. The cleavage site can include any suitable cleavage site, such as those described herein. In some embodiments, the cleavage site includes an RNA nucleotide and can be cleaved using, for example, an RNAse. In some embodiments, the cleavage site includes uracil and / or deoxyuridine and can be cleaved using, for example, a DNA glycosylase, an endonuclease, an RNAse, or the like, and combinations thereof. In some embodiments, the cleavage site does not include uracil and / or deoxyuridine. In some embodiments, the methods herein include, after binding the hairpin scaffold adaptor to the single-stranded nucleic acid, exposing the one or more cleavage sites to cleavage conditions, thereby cleaving the scaffold adaptor.

[0133] In some embodiments, the hairpin scaffold adapter comprises a single-stranded scaffold region (ssNA hybridization region). The single-stranded scaffold region of the hairpin scaffold adapter is typically located adjacent to the double-stranded stem portion and at the opposite end of the loop portion. The single-stranded scaffold region of the hairpin scaffold adapter is typically complementary to the terminal region of the target nucleic acid (e.g., the terminal region of the single-stranded nucleic acid).

[0134] In some embodiments, a hairpin scaffold adaptor comprises a scaffold polynucleotide comprising, in a 5' to 3' direction, an oligonucleotide; one or more cleavage sites; and an oligonucleotide hybridization region and a scaffold region (ssNA hybridization region). In some embodiments, a hairpin oligonucleotide comprises, in a 5' to 3' direction, a scaffold polynucleotide comprising a scaffold region (ssNA hybridization region) and an oligonucleotide hybridization region; one or more cleavage sites; and an oligonucleotide. In some embodiments, a plurality of hairpin scaffold adaptors, or a pool of hairpin scaffold adaptors, comprises a mixture of 1) a hairpin scaffold adaptor comprising, in a 5' to 3' direction, an oligonucleotide; one or more cleavage sites; and an oligonucleotide hybridization region and a scaffold region (ssNA hybridization region), and 2) a hairpin scaffold adaptor comprising, in a 5' to 3' direction, a scaffold polynucleotide comprising, in a 5' to 3' direction, a scaffold region (ssNA hybridization region) and an oligonucleotide hybridization region; one or more cleavage sites; and an oligonucleotide.

[0135] Modified Nucleotides In some embodiments, the scaffold adaptor, or a component thereof, comprises one or more modified nucleotides. Modified nucleotides may be referred to as modified bases and may include, for example, nucleotides conjugated to members of a binding pair, blocked nucleotides, non-natural nucleotides, nucleotide analogs, peptide nucleic acid (PNA) nucleotides, morpholino nucleotides, locked nucleic acid (LNA) nucleotides, bridged nucleic acid (BNA) nucleotides, glycol nucleic acid (GNA) nucleotides, threose nucleic acid (TNA) nucleotides, etc., and combinations thereof. In some embodiments, the scaffold adaptor, or a component thereof, comprises one or more modified nucleotides within the duplex region, within the scaffold region, at one end, or at both ends of the scaffold adaptor, or a component thereof. In some embodiments, the scaffold adaptor, or a component thereof, comprises one or more unpaired modified nucleotides. In some embodiments, the scaffold adaptor, or a component thereof, comprises one or more unpaired modified nucleotides at one end of the adaptor. In some embodiments, the scaffold adaptor, or a component thereof, comprises one or more unpaired modified nucleotides at the end of the adaptor opposite the end that hybridizes to the target nucleic acid (e.g., the end that comprises the single-stranded scaffold region). The modified nucleotide may be at the end of the strand having the 3' end or at the end of the strand having the 5' end.

[0136] In some embodiments, the oligonucleotide component comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides can block the covalent linkage of the oligonucleotide component to another oligonucleotide, polynucleotide, or nucleic acid molecule. In some embodiments, the oligonucleotide component comprises one or more modified nucleotides at the end not adjacent to the ssNA. In some embodiments, the scaffold polynucleotide comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides can block the covalent linkage of the scaffold polynucleotide to another oligonucleotide, polynucleotide, or nucleic acid molecule. The scaffold polynucleotide may comprise one or more modified nucleotides at one or both ends of the polynucleotide. In some embodiments, the one or more modified nucleotides comprise a modification that blocks ligation.

[0137] In some embodiments, the scaffold adapter, or a component thereof, comprises one or more blocked nucleotides. In one example, the scaffold adapter, or a component thereof, may comprise one or more modified nucleotides capable of blocking hybridization with nucleotides in another scaffold adapter, or a component thereof. In some cases, the one or more modified nucleotides may block ligation to nucleotides in another scaffold adapter, or a component thereof. In another example, the scaffold adapter, or a component thereof, may comprise one or more modified nucleotides capable of blocking hybridization with nucleotides in a target nucleic acid (e.g., ssNA). In some cases, the one or more modified nucleotides may block ligation to nucleotides in the target nucleic acid. In some embodiments, one or both ends of the scaffold polynucleotide comprise a blocking modification, and / or the end of the oligonucleotide component that is not adjacent to the ssNA fragment may comprise a blocking modification. A blocking modification refers to a modified end that cannot be ligated to the end of another nucleic acid component using the approach used to covalently link the adjacent ends of the oligonucleotide component and the ssNA fragment. In certain embodiments, the blocking modification is a ligation-blocking modification. Examples of blocking modifications that may be included on one or both ends of a scaffold polynucleotide and / or on the ends of oligonucleotide components not adjacent to the ssNA include the absence of a 3' OH and an inaccessible 3' OH. Non-limiting examples of blocking modifications with an inaccessible 3' OH at the end include amino modifiers, amino linkers, spacers, isodeoxy bases, dideoxy bases, inverted dideoxy bases, 3' phosphates, etc. In some embodiments, the scaffold adaptor, or a component thereof, contains one or more modified nucleotides that cannot bind to natural nucleotides.

[0138] In some embodiments, one or more modified nucleotides comprise an isodeoxy-base. In some embodiments, one or more modified nucleotides comprise isodeoxy-guanine (iso-dG). In some embodiments, one or more modified nucleotides comprise isodeoxy-cytosine (iso-dC). Iso-dC and iso-dG are chemical variants of cytosine and guanine, respectively. Iso-dC can hydrogen bond with iso-dG, but cannot with unmodified guanine (natural guanine). Iso-dG can base pair with iso-dC, but cannot with unmodified cytosine (natural cytosine). An iso-dC-containing backbone adaptor or a component thereof can be designed to hybridize with a complementary oligo containing iso-dG, but cannot hybridize with any naturally occurring nucleic acid sequence.

[0139] In some embodiments, the one or more modified nucleotides comprise an epigenetic modification, including, but not limited to, methylation, hydroxymethylation, and carboxylation. Examples of epigenetic modifications include carboxycytosine, 5-methylcytosine (5mC) and its oxidized derivatives (e.g., 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-arboxylcytosine (5caC)), N(6)-methyladenine (6mA), N4-methylcytosine (4mC), N(6)-methyladenosine (m(6)A), pseudouridine (Ψ), 5-methylcytidine (m(5)C), hydroxymethyluracil, 2'-O-methylation of the 3' end, tRNA modification, miRNA modification, and snRNA modification.

[0140] In some embodiments, one or more modified nucleotides comprise a dideoxy base. In some embodiments, one or more modified nucleotides comprise a dideoxy cytosine. In some embodiments, one or more modified nucleotides comprise an inverted dideoxy base. In some embodiments, one or more modified nucleotides comprise an inverted dideoxy thymine. For example, an inverted dideoxy thymine located at the 5' end of a sequence can prevent undesired 5' ligation.

[0141] In some embodiments, one or more modified nucleotides comprise a spacer. In some embodiments, one or more modified nucleotides comprise a C3 spacer. A C3 spacer phosphoramidite can be incorporated internally or at the 5' end of the oligonucleotide. Multiple C3 spacers can be added to either end of the backbone adaptor or its components to introduce a long hydrophilic spacer arm (e.g., for attachment of a fluorophore or other pendant group). Other spacers include, for example, a photocleavable (PC) spacer, hexanediol, spacer 9, spacer 18, 1',2'-dideoxyribose (dSpacer), and the like.

[0142] In some embodiments, the modified nucleotide comprises an amino linker or amino blocker. In some embodiments, the modified nucleotide comprises an amino linker C6 (e.g., a 5' amino linker C6 or a 3' amino linker C6). In one example, the amino linker C6 can be used to incorporate an activated primary amino group at the 5' end of an oligonucleotide, which can then be conjugated to a ligand. The amino group is then internalized into the 5'-terminal ligand. The amino group is separated from the 5'-terminal nucleotide base by a 6-carbon spacer arm to reduce steric interactions between the amino group and the oligonucleotide. In some embodiments, the modified nucleotide comprises an amino linker C12 (e.g., a 5' amino linker C12 or a 3' amino linker C12). In one example, the amino linker C12 can be used to incorporate an activated primary amino group at the 5' end of an oligonucleotide. The amino group is separated from the 5'-terminal nucleotide base by a 12-carbon spacer arm to minimize steric interactions between the amino group and the oligonucleotide.

[0143] In some embodiments, the modified nucleotide comprises a member of a binding pair. The binding pair may include, for example, antibody / antigen, antibody / antibody, antibody / antibody fragment, antibody / antibody receptor, antibody / protein A or protein G, hapten / anti-hapten, biotin / avidin, biotin / streptavidin, folate / folate binding protein, vitamin B12 / intrinsic factor, chemically reactive group / complementary chemically reactive group, digoxigenin moiety / anti-digoxigenin antibody, fluorescein moiety / anti-fluorescein antibody, steroid / steroid binding protein, operator / repressor, nuclease / nucleotide, lectin / polysaccharide, active compound / active compound receptor, hormone / hormone receptor, enzyme / substrate, oligonucleotide or polynucleotide / its corresponding complement, etc., or combinations thereof. In some embodiments, the modified nucleotide comprises biotin.

[0144] In some embodiments, the modified nucleotide comprises a first member of a binding pair (e.g., biotin), and the second member of the binding pair (e.g., streptavidin) is conjugated to a solid support or substrate. The solid support or substrate can be any physically separable solid that can directly or indirectly bind members of a binding pair, including, but not limited to, surfaces provided by microarrays and wells, and particles, such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. Solid supports can be, for example, chips, columns, optical fibers, wipes, filters (e.g., flat filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, semi-metals, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (acrylic resins, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethane, TEFLON®, polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride, (including PVDF, etc.), polysaccharides, nylon or nitrocellulose, resins, silica, or silica-based materials, including silicon, silica gel, and modified silicon, Sephadex®, Sepharose®, carbon, metals (e.g., steel, gold, silver, aluminum, silicon, and copper), inorganic glass, conductive polymers (including polymers such as polypyrrole and polyindole); micro- or nanostructured surfaces, e.g., nucleic acid tiling arrays, nanotube, nanowire, or nanoparticle-decorated surfaces; or porous surfaces, or gels, e.g., methacrylate, acrylamide, sugar polymers, cellulose, silicates, or other fibrous or chain-like polymers. In some embodiments, the solid support or substrate can be coated using a passivation or chemically derivatized coating with any number of materials, including polymers, such as dextran, acrylamide, gelatin, or agarose.The beads and / or particles can be discrete or connected to one another (e.g., sintered). In some embodiments, the solid support can be a collection of particles. In some embodiments, the particles can comprise silica, which can comprise silicon dioxide. In some embodiments, the silica can be porous, and in certain embodiments, the silica can be non-porous. In some embodiments, the particles further comprise an agent that imparts paramagnetic properties to the particles. In certain embodiments, the agent comprises a metal, and in certain embodiments, the agent is an iron oxide (e.g., iron or iron oxide, where the iron oxide contains a mixture of Fe2+ and Fe3+). Members of a binding pair can be linked to the solid support by covalent bonds or non-covalent interactions, and can be linked to the solid support directly or indirectly (e.g., by a spacer molecule or an intermediary such as biotin).

[0145] In some embodiments, the scaffold polynucleotide, the oligonucleotide component, or both, comprise one or more non-natural nucleotides, also referred to as nucleotide analogs. Non-limiting examples of non-natural nucleotides that may be included in the scaffold polynucleotide, the oligonucleotide component, or both, include LNA (locked nucleic acid), PNA (peptide nucleic acid), FANA (2'-deoxy-2'-fluoroarabinonucleotide), GNA (glycol nucleic acid), TNA (threose nucleic acid), 2'-O-Me RNA, 2'-fluoro RNA, morpholino nucleotides, and any combination thereof.

[0146] End Treatment In some embodiments, the methods herein include contacting a nucleic acid composition containing a single-stranded nucleic acid (ssNA) with an agent having end-processing activity under conditions in which the single-stranded nucleic acid (ssNA) molecule is processed, thereby producing an end-processed ssNA composition. End processing can include, but is not limited to, phosphorylation, dephosphorylation, methylation, demethylation, oxidation, deoxidation, base modification, extension, polymerization, and combinations thereof. End processing can be performed using enzymes, including, but not limited to, ligases, polynucleotide kinases (PNKs), terminal transferases, methyltransferases, methylases (e.g., 3' methylases, 5' methylases), polymerases (e.g., polyA polymerases), oxidases, and combinations thereof.

[0147] In some embodiments, the methods herein include contacting a nucleic acid composition comprising a single-stranded nucleic acid (ssNA) with an agent having phosphatase activity under conditions in which the single-stranded nucleic acid (ssNA) molecule is dephosphorylated, thereby producing a dephosphorylated ssNA composition. In some embodiments, the methods herein include contacting a scaffold adaptor, or a component thereof, with an agent having phosphatase activity under conditions in which the scaffold adaptor, or a component thereof, is dephosphorylated, thereby producing a dephosphorylated scaffold adaptor, or a component thereof (e.g., a dephosphorylated oligonucleotide; a dephosphorylated scaffold polynucleotide). Generally, the ssNA composition and / or the scaffold adaptor or a component thereof are dephosphorylated before the binding step (i.e., before hybridization). The ssNA may be dephosphorylated before the binding step (i.e., before hybridization), and then phosphorylated thereafter. The scaffold adaptor, or a component thereof, may be dephosphorylated before the binding step (i.e., before hybridization), and then phosphorylated thereafter. The scaffold adaptor, or a component thereof, may be dephosphorylated before the binding step (i.e., before hybridization), and then phosphorylated thereafter. The scaffold adaptor, or a component thereof, may be dephosphorylated and then unphosphorylated before the binding step (i.e., before hybridization). The scaffold adaptor, or a component thereof, may be dephosphorylated and unphosphorylated before the binding step (i.e., before hybridization), and then phosphorylated after the binding step (i.e., after hybridization) and before or during the ligation step. Reagents and kits for dephosphorylating nucleic acids are known and available. For example, the target nucleic acid (e.g., ssNA) and / or the scaffold adaptor, or a component thereof, may be treated with a phosphatase (i.e., an enzyme that uses water to cleave phosphate monoesters to phosphate ions and alcohols).

[0148] In some embodiments, the methods herein comprise contacting a nucleic acid composition comprising a single-stranded nucleic acid (ssNA) with an agent having phosphoryl transfer activity under conditions in which a 5' phosphate is added to the 5' end of the ssNA. In some embodiments, the methods herein comprise contacting a dephosphorylated ssNA composition with an agent having phosphoryl transfer activity under conditions in which a 5' phosphate is added to the 5' end of the ssNA. In some embodiments, the methods herein comprise contacting a scaffold adaptor, or a component thereof, with an agent having phosphoryl transfer activity under conditions in which a 5' phosphate is added to the 5' end of the scaffold adaptor, or a component thereof. In some embodiments, the methods herein comprise contacting a dephosphorylated scaffold adaptor, or a component thereof, with an agent having phosphoryl transfer activity under conditions in which a 5' phosphate is added to the 5' end of the scaffold adaptor, or a component thereof. In certain cases, the ssNA composition and / or the scaffold adaptor or a component thereof are phosphorylated prior to the binding step (i.e., prior to hybridization). 5' phosphorylation of nucleic acids can be achieved by various techniques. For example, ssNA compositions and / or scaffold adapters or components thereof can be treated with polynucleotide kinase (PNK) (e.g., T4 PNK), which catalyzes the transfer and exchange of Pi from the γ-position of ATP to the 5'-hydroxyl terminus and nucleoside 3'-monophosphate of polynucleotides (double- and single-stranded DNA and RNA). Suitable reaction conditions include, for example, incubation of the nucleic acid with PNK in 1x PNK reaction buffer (e.g., 70 mM Tris-HCl, 10 mM MgCl, 5 mM DTT, pH 7.6 at 25°C) for 30 minutes at 37°C; and incubation of the nucleic acid with PNK in T4 DNA ligase buffer (e.g., 50 mM Tris-HCl, 10 mM MgCl, 1 mM ATP, 10 mM DTT, pH 7.5 at 25°C) for 30 minutes at 37°C. If necessary, after the phosphorylation reaction, PNK may be heat inactivated, for example at 65° C. for 20 minutes.

[0149] In some embodiments, the method herein does not include the use of an agent with phosphoryl transfer activity. In some embodiments, the method does not include the step of generating 5'-phosphorylated ssNA by phosphorylating the 5'-end of ssNA from a nucleic acid sample. In certain cases, the nucleic acid sample contains ssNA with naturally phosphorylated 5'-ends. In some embodiments, the method does not include the step of generating 5'-phosphorylated scaffold adapters or their components by phosphorylating the 5'-end of the scaffold adapters or their components.

[0150] Disconnect In some embodiments, the ssNA, scaffold adapter, and / or hybridization product (e.g., a scaffold adapter hybridized with an ssNA) is cleaved or sheared before, during, or after the methods described herein. In some embodiments, the ssNA, scaffold adapter, and / or hybridization product is cleaved or sheared at a cleavage site. In some embodiments, the scaffold adapter and / or hybridization product is cleaved or sheared at a cleavage site within a hairpin loop. In some embodiments, the scaffold adapter and / or hybridization product is cleaved or sheared at a cleavage site at an internal position of the scaffold adapter (e.g., within the duplex region of the scaffold adapter). In some embodiments, the scaffold adapter is cleaved at a cleavage site (e.g., uracil) that is present only on the scaffold polynucleotide and not on the complementary oligonucleotide component. Thus, in some embodiments, the scaffold polynucleotide comprises one or more uracil bases, and the oligonucleotide component does not comprise a uracil base. In some embodiments, the circular hybridization product is cleaved or sheared before, during, or after the methods described herein. In some embodiments, nucleic acids, e.g., circular nucleic acids and / or large fragments (e.g., longer than 500 base pairs in length), are cleaved or sheared before, during, or after the methods described herein. Large fragments may be referred to as high molecular weight (HMW) nucleic acids, HMW DNA, or HMW RNA. HMW nucleic acid fragments may include fragments greater than about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 2000 bp, about 3000 bp, about 4000 bp, about 5000 bp, about 10,000 bp, or longer. The terms "shearing" or "cleavage" generally refer to procedures or conditions that can separate a nucleic acid molecule into two (or more) smaller nucleic acid molecules. Such shearing or cleavage can be sequence-specific, base-specific, or non-specific, and can be accomplished by a variety of methods, reagents, or conditions, including, for example, chemical, enzymatic, and physical (e.g., physical fragmentation).The sheared or cleaved nucleic acids can have a nominal, representative, or average length of about 5 to about 10,000 base pairs, about 100 to about 1,000 base pairs, about 100 to about 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 base pairs.

[0151] Sheared or cleaved nucleic acids can be generated by suitable methods, non-limiting examples of which include physical methods (e.g., shearing, e.g., sonication, ultrasonication, French press, heat, UV irradiation, etc.), enzymatic processes (e.g., enzymatic cleaving agents (e.g., suitable nucleases, suitable restriction enzymes), chemical methods (e.g., alkylation, DMS, piperidine, acid hydrolysis, base hydrolysis, heat, etc., or combinations thereof), ultraviolet (UV) light (e.g., photocleavable moieties (e.g., including photocleavable spacers)), etc., or combinations thereof. The representative, average, or nominal length of the resulting nucleic acid fragments can be controlled by selecting an appropriate fragment generation method.

[0152] The term "cleaving agent" generally refers to an agent, sometimes a chemical or enzyme, that can cleave nucleic acids at one or more specific or non-specific sites. Specific cleaving agents often specifically cleave at specific sites according to a particular nucleotide sequence, which specific sites are sometimes referred to as cleavage sites. Cleavage agents can include enzymatic cleaving agents, chemical cleaving agents, and light (e.g., ultraviolet (UV) light).

[0153] Examples of enzymatic cleavage agents include, but are not limited to, endonucleases; deoxyribonucleases (DNases; e.g., DNase I, II); ribonucleases (RNases; e.g., RNAse A, RNAse E, RNAse F, RNAse H, RNAse III, RNAse L, RNAse P, RNAse PhyM, RNAse T1, RNAse T2, RNAse U2, and RNAse V); endonuclease VIII; CLEAVASE enzymes; TAQ DNA polymerase; E. coli DNA polymerase I; eukaryotic structure-specific endonucleases; mouse FEN-1 endonuclease; nicking enzymes; type I, II, or III restriction endonucleases (i.e., restriction enzymes), such as Acc I, Aci I, Afl III, Alu I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I, Bgl I.Bgl II, Bln I, Bsm I, BssH II, BstE II, BstUI, Cfo I, CIa I, Dde I, Dpn I, Dra I, EcIX I, EcoR I, EcoR I, EcoR II, EcoR V, Hae II, Hae II, HhaI, Hind II, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, MaeII, McrBC, Mlu I, MIuN I, Msp I, Nci I, Nco I, Nde I, Nde II, Nhe I, Not I, Nru I, Nsi I, Pst I, Pvu I, Pvu II, Rsa I, Sac I, Sal I, Sau3A I, Sca I, ScrF I, Sfi I, Sma I, Spe I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I, Xho I; glycosylases (e.g., uracil-DNA glycosylase (UDG), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA glycosylases (e.g., hypoxanthine-DNA glycosylase, uracil-DNA glycosylase (UDG), 5-hydroxymethyluracil-DNA glycosylase (HmUDG), 5-hydroxymethylcytosine-DNA glycosylase, or 1,N6-etheno- exonucleases (e.g., exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VIII); 5' to 3' exonucleases (e.g., exonuclease II); 3' to 5' exonucleases (e.g., exonuclease I); poly(A)-specific 3' to 5' exonucleases; ribozymes; DNAzymes; and the like, and combinations thereof.

[0154] In some embodiments, the cleavage site comprises a restriction enzyme recognition site. In some embodiments, the cleavage agent comprises a restriction enzyme. In some embodiments, the cleavage site comprises a rare-cutter restriction enzyme recognition site (e.g., a NotI recognition sequence). In some embodiments, the cleavage agent comprises a rare-cutter enzyme (e.g., a rare-cutter restriction enzyme). A rare-cutter enzyme generally refers to a restriction enzyme that has a recognition sequence that occurs extremely rarely in a genome (e.g., the human genome). One example is NotI, which cleaves after the first GC in the sequence 5'-GCGGCCGC-3'. Restriction enzymes with 7 and 8 base pair recognition sequences are often considered rare-cutter enzymes.

[0155] The procedure for selecting the cleavage method and restriction enzyme for cleaving DNA at specific sites is well known to those skilled in the art.For example, many restriction enzyme suppliers, including New England BioLabs, Pro-Mega Biochems, Boehringer-Mannheim, etc., provide information on the conditions and types of DNA sequences that are cleaved by specific restriction enzymes.Enzymes are often used under conditions that allow DNA to be cleaved with about 95% to 100% efficiency, preferably about 98% to 100% efficiency.

[0156] In some embodiments, the cleavage site comprises one or more ribonucleic acid (RNA) nucleotides. In some embodiments, the cleavage site comprises a single-stranded portion comprising one or more RNA nucleotides. In some embodiments, the single-stranded portion is adjacent to a double-stranded portion. In some embodiments, the single-stranded portion is a hairpin loop. In some embodiments, the cleavage site comprises one RNA nucleotide. In some embodiments, the cleavage site comprises two RNA nucleotides. In some embodiments, the cleavage site comprises three RNA nucleotides. In some embodiments, the cleavage site comprises four RNA nucleotides. In some embodiments, the cleavage site comprises five RNA nucleotides. In some embodiments, the cleavage site comprises more than five RNA nucleotides. In some embodiments, the cleavage site comprises one or more RNA nucleotides selected from adenine (A), cytosine (C), guanine (G), and uracil (U). In some embodiments, the cleavage site comprises one or more RNA nucleotides selected from adenine (A), cytosine (C), and guanine (G). In some embodiments, the cleavage site does not comprise uracil (U). In some embodiments, the cleavage site comprises one or more RNA nucleotides comprising guanine (G). In some embodiments, the cleavage site comprises one or more RNA nucleotides consisting of guanine (G). In some embodiments, the cleavage site comprises one or more RNA nucleotides comprising cytosine (C). In some embodiments, the cleavage site comprises one or more RNA nucleotides consisting of cytosine (C). In some embodiments, the cleavage site comprises one or more RNA nucleotides comprising adenine (A). In some embodiments, the cleavage site comprises one or more RNA nucleotides consisting of adenine (A). In some embodiments, the cleavage site comprises one or more RNA nucleotides consisting of adenine (A), cytosine (C), and guanine (G). In some embodiments, the cleavage site comprises one or more RNA nucleotides consisting of adenine (A) and cytosine (C). In some embodiments, the cleavage site comprises one or more RNA nucleotides consisting of adenine (A) and guanine (G).In some embodiments, the cleavage site comprises one or more RNA nucleotides consisting of cytosine (C) and guanine (G). In some embodiments, the cleavage agent comprises a ribonuclease (RNAse). In some embodiments, the RNAse is an endoribonuclease. The RNAse can be selected from one or more of RNAse A, RNAse E, RNAse F, RNAse H, RNAse III, RNAse L, RNAse P, RNAse PhyM, RNAse T1, RNAse T2, RNAse U2, and RNAse V.

[0157] In some embodiments, the cleavage site comprises a photocleavable spacer or photocleavable modification. Photocleavable modifications can include, for example, photolabile functional groups that are cleavable by ultraviolet (UV) light of a specific wavelength (e.g., 300-350 nm). An example of a photocleavable spacer (available from Integrated DNA Technologies; product number 1707) is a 10-atom linker arm that can be cleaved only upon exposure to UV light within the appropriate spectral range. Oligonucleotides containing a photocleavable spacer can have a 5' phosphate group available for subsequent ligase reactions. The photocleavable spacer can be positioned between DNA bases or between the oligo and a terminal modification (e.g., a fluorophore). In such embodiments, ultraviolet (UV) light can be considered the cleavage agent.

[0158] In some embodiments, the cleavage site comprises a diol. For example, the cleavage site may comprise vicinal diols incorporated at a 5'-to-5' linkage. A cleavage site comprising a diol can be chemically cleaved, for example, using periodate. In some embodiments, the cleavage site comprises a blunt-end restriction enzyme recognition site. A cleavage site comprising a blunt-end restriction enzyme recognition site can be cleaved by a blunt-end restriction enzyme.

[0159] Nick seal and fill-in In some embodiments, the methods herein include performing a nick-sealing reaction (e.g., using a DNA ligase or other suitable enzyme, and in certain cases, a kinase configured to 5' phosphorylate the nucleic acid (e.g., using polynucleotide kinase (PNK)). In some embodiments, the methods herein include performing a fill-in reaction. For example, if the scaffold adaptor is present as a duplex, some or all of the duplex may include an overhang at the end of the duplex opposite the end that hybridizes to the ssNA. If such a duplex overhang is present, the methods herein may further include, after the joining step, filling in the overhang formed by the duplex. In some embodiments, the fill-in reaction is performed to generate a blunt-ended hybridization product. Any suitable reagents can be used to perform the fill-in reaction. Suitable polymerases for performing the fill-in reaction include, for example, DNA polymerase I, large (Klenow) fragment, Bacillus stearothermophilus (Bst) DNA polymerase, etc. In some embodiments, a strand-displacing polymerase is used (e.g., Bst DNA polymerase).

[0160] Exonuclease treatment In some embodiments, nucleic acids (e.g., RNA-DNA duplexes, hybridization products, circularized hybridization products) are treated with an exonuclease. In some embodiments, RNA in an RNA-DNA duplex (e.g., an RNA-DNA duplex generated by first-strand cDNA synthesis) is treated with an exonuclease. An exonuclease is an enzyme that acts by cleaving nucleotides one by one from the end of a polynucleotide chain by a hydrolysis reaction that cleaves phosphodiester bonds at either the 3' or 5' end. Exonucleases include, for example, DNAses, RNAses (e.g., RNAse H), 5' to 3' exonucleases (e.g., exonuclease II), 3' to 5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3' to 5' exonucleases. In some embodiments, the exonuclease activity is provided by a reverse transcriptase (e.g., RNAse activity provided by an M-MLV reverse transcriptase having a fully functional RNAse H domain). In some embodiments, the hybridization product is treated with an exonuclease to remove contaminating nucleic acids, such as, for example, single-stranded oligonucleotides, nucleic acid fragments, or RNA from an RNA-DNA duplex. In some embodiments, the circularized hybridization product is treated with an exonuclease to remove any non-circularized hybridization product, unhybridized oligonucleotides, unhybridized target nucleic acids, oligonucleotide dimers, etc., and combinations thereof.

[0161] sample The present paper provides methods and compositions for processing and / or analyzing nucleic acid.The nucleic acid or nucleic acid mixture used in the methods and compositions described herein can be isolated from the sample obtained from subject (for example, test subject).Subject can be any living or non-living organism, including but not limited to human, non-human animal, plant, bacteria, fungus, protist or pathogen. Any human or non-human animal can be selected, including, for example, mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (e.g., cows), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), suids (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), plantigrade carnivores (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. The subject can be male or female (e.g., woman, or pregnant woman). The subject can be of any age (e.g., embryo, fetus, infant, child, adult). The subject may be a cancer patient, a patient suspected of having cancer, a patient in remission, a patient with a family history of cancer, and / or a subject undergoing cancer screening. The subject may be a patient suffering from an infectious disease or infectious disease or infected with a pathogen (e.g., bacteria, virus, fungus, protozoan, etc.), a patient suffering from an infectious disease or infectious disease or suspected of being infected by a pathogen, a patient who has recovered from an infectious disease, infectious disease, or pathogen infection, a patient with a history of an infectious disease, infectious disease, or pathogen infection, and / or a subject undergoing infectious disease or pathogen screening. The subject may be a transplant recipient. The subject may be a patient undergoing microbiome analysis. In some embodiments, the subject is female. In some embodiments, the subject is a female human. In some embodiments, the subject is male. In some embodiments, the subject is a male human.

[0162] Nucleic acid samples can be isolated or obtained from any type of suitable biological specimen or sample (e.g., test sample). Nucleic acid samples can be isolated or obtained from a single cell, multiple cells (e.g., cultured cells), cell culture medium, conditioned medium, tissue, organ, or organism (e.g., bacteria, yeast, etc.). In some embodiments, nucleic acid samples are isolated or obtained from cells, tissues, organs, and / or the like of an animal (e.g., an animal subject). In some embodiments, nucleic acid samples are isolated or obtained from sources such as bacteria, yeast, insects (e.g., Drosophila), mammals, amphibians (e.g., frogs (e.g., Xenopus)), viruses, plants, or any other mammalian or non-mammalian nucleic acid sample source.

[0163] The nucleic acid sample can be isolated or obtained from a living organism or animal. In some cases, the nucleic acid sample can be isolated or obtained from an extinct (or "ancient") organism or animal (e.g., an extinct mammal; an extinct mammal from the genus Homo). In some cases, the nucleic acid sample can be obtained as part of a diagnostic analysis.

[0164] In some cases, nucleic acid samples can be obtained as part of forensic analysis. In some embodiments, the single-stranded nucleic acid library preparation (ssPrep) method described herein is applied to forensic samples or specimens. Forensic samples or specimens can include any biological material containing nucleic acids. For example, forensic samples or specimens can include blood, semen, hair, skin, sweat, saliva, decomposed tissue, bone, fingernail scrapings, licked stamps / envelopes, exfoliation, touch DNA, razor residue, etc.

[0165] The sample or test sample can be any specimen isolated or obtained from a subject or a part thereof (e.g., a human subject, a pregnant female, a cancer patient, a patient suffering from an infectious or infectious disease, a transplant recipient, a fetus, a tumor, an infected organ or tissue, a transplanted organ or tissue, a microbiome). The sample can be from a pregnant female subject carrying a fetus at any stage of pregnancy (e.g., first, second, or third trimester for a human subject), or from a subject after birth. The sample can be from a pregnant subject carrying a fetus in which all chromosomes are euploid, or from a pregnant subject carrying a fetus with chromosomal aneuploidy (e.g., one, three (i.e., trisomy (e.g., T21, T18, T13)) or four copies of a chromosome) or other genetic mutation. Non-limiting examples of specimens include bodily fluids or tissues from a subject, including, but not limited to, blood or blood products (e.g., serum, plasma, etc.), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, stomach, peritoneal, ductal, ear, arthroscopic), biopsy samples (e.g., from preimplantation embryos; cancer biopsies), body cavity aspirate samples, cells (blood cells, placental cells, embryonic or fetal cells, nucleated fetal cells or fetal cell remnants, normal cells, abnormal cells (e.g., cancer cells)) or portions thereof (e.g., mitochondrial, nuclei, extracts, etc.), washings of the female reproductive tract, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage, semen, lymph, bile, tears, sweat, breast milk, milk, etc., or combinations thereof. In some embodiments, the biological sample is a cervical swab from a subject. The bodily fluid or tissue sample from which nucleic acids are extracted may be acellular (e.g., cell-free). In some embodiments, the bodily fluid or tissue sample may contain cellular elements or cellular debris. In some embodiments, fetal cells or cancer cells may be included in the sample.

[0166] The sample may be a liquid sample. The liquid sample may contain extracellular nucleic acids (e.g., circulating cell-free DNA). Examples of liquid samples include, but are not limited to, blood or blood products (e.g., serum, plasma, etc.), urine, cerebrospinal fluid, saliva, sputum, biopsy samples (e.g., liquid biopsy for cancer detection), the above liquid samples, etc., or combinations thereof. In certain embodiments, the sample is a liquid biopsy, which generally refers to the assessment of a liquid sample from a subject for the presence, absence, worsening, or remission of disease. Liquid biopsy can be used together with or as an alternative to solid biopsy (e.g., tumor biopsy). In certain cases, extracellular nucleic acids are analyzed in liquid biopsies.

[0167] In some embodiments, the biological sample may be blood, plasma, or serum. The term "blood" encompasses the conventional definition of whole blood, blood products, or any fraction of blood, such as serum, plasma, buffy coat, etc. Blood or fractions thereof often contain nucleosomes. Nucleosomes contain nucleic acids and may be acellular or intracellular. Blood also includes buffy coats. Buffy coats may be isolated using a Ficoll gradient. Buffy coats may contain white blood cells (e.g., white blood cells, T cells, B cells, platelets, etc.). Plasma refers to the fraction of whole blood obtained as a result of centrifugation of blood treated with an anticoagulant. Serum refers to the aqueous portion of the fluid remaining after a blood sample has clotted. Body fluid or tissue samples are often collected according to standard protocols commonly followed by hospitals or clinics. In the case of blood, an appropriate volume of peripheral blood (e.g., between 3 and 40 milliliters, between 5 and 50 milliliters) is often collected and may be stored according to standard procedures before or after preparation.

[0168] Analysis of nucleic acids found in a subject's blood can be performed, for example, using whole blood, serum, or plasma. For example, analysis of fetal DNA found in maternal blood can be performed, for example, using whole blood, serum, or plasma. For example, analysis of tumor or cancer DNA found in a patient's blood can be performed, for example, using whole blood, serum, or plasma. For example, analysis of pathogen DNA found in a patient's blood can be performed, for example, using whole blood, serum, or plasma. For example, analysis of transplant DNA found in a transplant recipient's blood can be performed, for example, using whole blood, serum, or plasma. Methods for preparing serum or plasma from blood obtained from a subject (e.g., maternal subject; patient; cancer patient) are known. For example, a subject's blood (e.g., a pregnant woman's blood; a patient's blood; or a cancer patient's blood) can be placed in tubes containing EDTA or specialized commercial products, such as Cell-Free DNA BCT (Streck, Omaha, NE) or Vacutainer SST (Becton Dickinson, Franklin Lakes, NJ), to prevent blood clotting. Plasma can then be obtained from the whole blood by centrifugation. Serum can be obtained with or without post-clotting centrifugation. When centrifugation is used, it is typically, but not exclusively, performed at an appropriate speed, e.g., 1,500-3,000 rpm. The plasma or serum may be subjected to an additional centrifugation step before being transferred to a fresh tube for nucleic acid extraction. In addition to the acellular portion of whole blood, nucleic acids, which are enriched in the buffy coat, can also be recovered from the cellular fraction, which can be obtained after centrifugation of a whole blood sample from a subject and removal of the plasma.

[0169] The sample may be a tumor nucleic acid sample (i.e., a nucleic acid sample isolated from a tumor). The term "tumor" generally refers to neoplastic cell growth and proliferation, whether malignant or benign, and may include precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" generally refer to the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, various types of head and neck cancer, and the like.

[0170] A sample may be heterogeneous. For example, a sample may contain more than one cell type and / or one or more nucleic acid species. In some cases, a sample may contain (i) fetal and maternal cells, (ii) cancer and non-cancerous cells, and / or (iii) pathogenic and host cells. In some cases, a sample may contain (i) cancer and non-cancer nucleic acids, (ii) pathogen and host nucleic acids, (iii) fetal and maternal nucleic acids, and / or more generally, (iv) mutated and wild-type nucleic acids. In some cases, a sample may contain minority and majority nucleic acid species, as described in more detail below. In some cases, a sample may contain cells and / or nucleic acids from a single subject or may contain cells and / or nucleic acids from multiple subjects.

[0171] nucleic acid Methods and compositions for processing and / or analyzing nucleic acids are provided herein. The terms nucleic acid, nucleic acid molecule, nucleic acid fragment, target nucleic acid, nucleic acid template, template nucleic acid, nucleic acid target, target nucleic acid, polynucleotide, polynucleotide fragment, target polynucleotide, polynucleotide target, etc. may be used interchangeably throughout this disclosure. These terms include DNA (e.g., complementary DNA (cDNA; synthesized from any RNA or DNA of interest), genomic DNA (gDNA), genomic DNA fragments, mitochondrial DNA (mtDNA), recombinant DNA (e.g., plasmid DNA), etc.), RNA (e.g., messenger RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA, trans-acting small interfering RNA (ta-siRNA), naturally occurring small interfering RNA (nat-siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), non-coding RNA (ncRNA), transfer messenger RNA (tmRNA), precursor mRNA (tmRNA), and so on. " refers to nucleic acids of any composition, such as messenger RNA (pre-mRNA), small Cajal body-specific RNA (scaRNA), piwi-binding RNA (piRNA), endoribonuclease-prepared siRNA (esiRNA), small RNA (stRNA), signal recognition RNA, telomeric RNA, RNA highly expressed by the fetus or placenta, and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs, and / or non-native backbones, etc.), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which can be in single- or double-stranded form and, unless otherwise limited, can include known analogs of natural nucleotides that can function in a similar manner to naturally occurring nucleotides.The nucleic acid may be or may be derived from a plasmid, phage, virus, bacterium, autonomously replicating sequence (ARS), mitochondrion, centromere, artificial chromosome, chromosome, or other nucleic acid that can be replicated or replicated in vitro or in a host cell, cell, cell nucleus, or cytoplasm in certain embodiments. In some embodiments, the template nucleic acid may be derived from a single chromosome (e.g., a nucleic acid sample may be derived from one chromosome of a sample obtained from a diploid organism). Unless otherwise specified, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid is used synonymously with locus, gene, cDNA, and mRNA encoded by a gene. The term may also include derivatives, variants, and analogs of RNA or DNA synthesized from nucleotide analogs, single-stranded ("sense" or "antisense," "plus" or "minus" strand, "forward" or "reverse" reading frame), and double-stranded polynucleotides. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it generally includes regions preceding and following the coding region (leader and trailer) involved in the transcription / translation of the gene product and in the regulation of transcription / translation, as well as intervening sequences (introns) between individual coding regions (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). For RNA, the base thymine is replaced by uracil.Nucleic acid length or size can be expressed in terms of the number of bases.

[0172] The target nucleic acid can be any nucleic acid of interest. The nucleic acid can be a polymer of any length composed of deoxyribonucleotides (i.e., DNA bases), ribonucleotides (i.e., RNA bases), or a combination thereof, for example, 10 bases or more, 20 bases or more, 50 bases or more, 100 bases or more, 200 bases or more, 300 bases or more, 400 bases or more, 500 bases or more, 1000 bases or more, 2000 bases or more, 3000 bases or more, 4000 bases or more, 5000 bases or more. In certain embodiments, a nucleic acid is a polymer made up of deoxyribonucleotides (i.e., DNA bases), ribonucleotides (i.e., RNA bases), or combinations thereof, e.g., 10 or fewer bases, 20 or fewer bases, 50 or fewer bases, 100 or fewer bases, 200 or fewer bases, 300 or fewer bases, 400 or fewer bases, 500 or fewer bases, 1000 or fewer bases, 2000 or fewer bases, 3000 or fewer bases, 4000 or fewer bases, or 5000 or fewer bases.

[0173] Nucleic acid can be single-stranded or double-stranded.For example, single-stranded DNA (ssDNA) can be generated by denaturing double-stranded DNA, for example, by heating or treating with alkali.Therefore, in some embodiments, ssDNA is derived from double-stranded DNA (dsDNA).In some embodiments, the method herein comprises a step of denaturing dsDNA before the step of combining the nucleic acid composition comprising dsDNA with the scaffold adaptor or its component herein, thereby generating ssDNA.

[0174] In certain embodiments, the nucleic acid is in a D-loop structure formed by strand invasion of a double-stranded DNA molecule by an oligonucleotide or DNA-like molecule, such as a peptide nucleic acid (PNA). For example, D-loop formation can be promoted by the addition of E. coli RecA protein and / or by altering salt concentration using methods known in the art.

[0175] Nucleic acids (e.g., nucleic acid targets, single-stranded nucleic acids (ssNAs), oligonucleotides, overhangs, scaffold polynucleotides, and their hybridization regions (e.g., ssNA hybridization regions, oligonucleotide hybridization regions)) may be described herein as being complementary to, having a region of complementarity with, being capable of hybridizing to, or having a hybridization region for, another nucleic acid. The terms "complementary" or "complementarity" or "hybridization" generally refer to nucleotide sequences that noncovalently base pair to a region of a nucleic acid (e.g., the nucleotide sequence of an ssNA hybridization region that hybridizes with a terminal region of an ssNA fragment and the nucleotide sequence of an oligonucleotide hybridization region that hybridizes with an oligonucleotide component of a scaffold adapter). In standard Watson-Crick base pairing, in DNA, adenine (A) base pairs with thymine (T), and guanine (G) base pairs with cytosine (C). In the case of RNA, thymine (T) is replaced by uracil (U). Thus, A is complementary to T and G is complementary to C. In the case of RNA, A is complementary to U, and vice versa. In the case of a DNA-RNA duplex, A (in the DNA strand) is complementary to U (in the RNA strand). In some embodiments, one or more thymine (T) bases are replaced by uracil (U) in the scaffold adapter or a component thereof, which is complementary to adenine (A). Typically, "complementary" or "complementarity" or "capable of hybridizing" refers to nucleotide sequences that are at least partially complementary. These terms can also encompass duplexes that are fully complementary, such that every nucleotide in one strand is complementary to or hybridizes with every nucleotide at the corresponding position in the other strand.

[0176] In certain cases, nucleotide sequences may be partially complementary to the target, and in this case, not all nucleotides are necessarily complementary to all nucleotides at all corresponding positions in the target nucleic acid.For example, ssNA hybridization region may be perfectly (i.e., 100%) complementary to the target ssNA terminal region, or the ssNA hybridization region may share a degree of complementarity that is less than perfect (e.g., 70%, 75%, 85%, 90%, 95%, 99%).In another example, oligonucleotide hybridization region may be perfectly (i.e., 100%) complementary to the oligonucleotide, or the oligonucleotide hybridization region may share a degree of complementarity that is less than perfect (e.g., 70%, 75%, 85%, 90%, 95%, 99%).

[0177] The percent identity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison (e.g., gaps can be introduced into the sequence of the first sequence for optimal alignment). In that case, nucleotides at corresponding positions are compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100). When a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, then the molecules are identical at that position.

[0178] In some embodiments, nucleic acids in a mixture of nucleic acids are analyzed. The mixture of nucleic acids can include two or more nucleic acid species with the same or different nucleotide sequences, different lengths, different origins (e.g., genomic origin, fetal vs. maternal origin, cell or tissue origin, cancer vs. non-cancer origin, tumor vs. non-tumor origin, host vs. pathogen, host vs. graft, host vs. microbiome, sample origin, subject origin, etc.), different overhang lengths, different overhang types (e.g., 5' overhang, 3' overhang, no overhang), or combinations thereof. In some embodiments, the mixture of nucleic acids includes single-stranded nucleic acids and double-stranded nucleic acids. In some embodiments, the mixture of nucleic acids includes DNA and RNA. In some embodiments, the mixture of nucleic acids includes ribosomal RNA (rRNA) and messenger RNA (mRNA). The nucleic acids subjected to the processes described herein may contain nucleic acids from one sample or from two or more samples (e.g., from one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more samples).

[0179] In some embodiments, the target nucleic acid (e.g., ssNA) comprises degraded DNA. Degraded DNA may be referred to as low-quality DNA or highly degraded DNA. Degraded DNA may be highly fragmented and may contain damage such as base analogs and abasic sites that are prone to miscoding and / or intermolecular crosslinking. For example, sequencing errors resulting from deamination of cytosine residues may be present in certain sequences obtained from degraded DNA (e.g., miscoding C to T and G to A). In some embodiments, the target nucleic acid (e.g., ssNA) is derived from a nicked double-stranded nucleic acid fragment. The nicked double-stranded nucleic acid fragment can be denatured (e.g., heat denatured) to generate ssNA fragments.

[0180] Nucleic acids can be obtained from one or more sources (e.g., biological samples, blood, cells, serum, plasma, buffy coat, urine, lymph, skin, hair, soil, etc.) by methods known in the art. Any suitable method can be used to isolate, extract, and / or purify DNA from a biological sample (e.g., from blood or blood products), non-limiting examples of which include DNA preparation methods (e.g., as described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001), various commercially available reagents or kits, such as DNeasy®, RNeasy®, QIAprep®, QIAquick®, and QIAamp® (e.g., QIAamp® Circulating Nucleic Acid Kit, QiaAmp® DNA Mini Kit, or QiaAmp® DNA Blood Mini Kit) nucleic acid isolation / purification kits by Qiagen, Inc. (Germantown, Md); GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wis.); GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, NJ); Life DNAzol®, ChargeSwitch®, Purelink®, GeneCatcher® nucleic acid isolation / purification kits from Biosciences Technologies, Inc. (Carlsbad, CA); NucleoMag®, NucleoSpin®, and NucleoBond® nucleic acid isolation / purification kits from Clontech Laboratories, Inc. (Mountain View, CA); etc., or combinations thereof. In certain embodiments, nucleic acids are isolated from fixed biological samples, such as formalin-fixed, paraffin-embedded (FFPE) tissue.Genomic DNA from FFPE tissue can be isolated using commercially available kits, such as the AllPrep® DNA / RNA FFPE kit from Qiagen, Inc. (Germantown, Md.), the RecoverAll® Total Nucleic Acid Isolation kit for FFPE from Life Technologies, Inc. (Carlsbad, Calif.), and the NucleoSpin® FFPE kit from Clontech Laboratories, Inc. (Mountain View, Calif.).

[0181] In some embodiments, nucleic acids are extracted from cells using a cell lysis procedure. Cell lysis procedures and reagents are known in the art and can generally be performed by chemical methods (e.g., detergents, hypotonic solutions, enzymatic procedures, etc., or a combination thereof), physical methods (e.g., French press, sonication, etc.), or electrolytic lysis. Any suitable lysis procedure can be used. For example, chemical methods generally utilize a lysis agent to disrupt cells and extract nucleic acids from the cells, followed by treatment with a chaotropic salt. Physical methods, such as freeze / thaw followed by crushing, the use of a cell press, etc., are also useful. In some cases, high-salt and / or alkaline lysis procedures can be used. In some cases, the lysis procedure can include a lysis step with EDTA / proteinase K, a binding buffer step using a large amount of salt (e.g., guanidine hydrochloride (GuHCl), sodium acetate) and isopropanol, and binding the DNA in this solution to a silica-based column. In some cases, the lysis protocol includes certain steps described in Dabney et al., Proceedings of the National Academy of Sciences 110, no. 39 (2013): 15758-15763.

[0182] Nucleic acids can include extracellular nucleic acids in certain embodiments. As used herein, the term "extracellular nucleic acid" can refer to nucleic acids isolated from a source that is substantially cell-free and can also be referred to as "cell-free" nucleic acids (cell-free DNA, cell-free RNA, or both), "circulating cell-free nucleic acids" (e.g., CCF fragments, ccf DNA), and / or "cell-free circulating vesicle nucleic acids." Extracellular nucleic acids can be present in blood, and extracellular nucleic acids can be obtained from blood (e.g., from the blood of a human subject). Extracellular nucleic acids often do not contain detectable cells and may contain cellular elements or cellular debris. Non-limiting examples of cell-free sources of extracellular nucleic acids are blood, plasma, serum, and urine. In certain aspects, cell-free nucleic acids are obtained from a bodily fluid sample selected from whole blood, plasma, serum, amniotic fluid, saliva, urine, pleural effusion, bronchial lavage, bronchial aspirate, breast milk, colostrum, tears, semen, peritoneal fluid, pleural effusion, and stool. As used herein, the term "obtaining cell-free circulating sample nucleic acid" includes obtaining a sample directly (e.g., collecting a sample, e.g., a test sample) or obtaining a sample from another person who has collected the sample. Extracellular nucleic acid can be a product of cellular secretion and / or nucleic acid release (e.g., DNA release). Extracellular nucleic acid can be, for example, a product of any form of dead cell. In some cases, extracellular nucleic acid is a product of any form of type I or type II cell death, including mitotic, swollen, toxic, ischemic, etc., and combinations thereof. Without being limited by theory, extracellular nucleic acid can be a product of cell apoptosis and cell destruction, which provides the basis for extracellular nucleic acid, which often has a range of lengths across a spectrum (e.g., a "ladder"). In some cases, extracellular nucleic acid is a product of cell necrosis, necroptosis, swollen death, entosis, pyroptosis, etc., and combinations thereof. In some embodiments, the sample nucleic acid from the test subject is circulating cell-free nucleic acid. In some embodiments, the circulating cell-free nucleic acid is from plasma or serum from a test subject. In some aspects, the cell-free nucleic acid is degraded. In some embodiments, the cell-free nucleic acid comprises cell-free fetal nucleic acid (e.g., cell-free fetal DNA).In certain aspects, the cell-free nucleic acid comprises circulating cancer nucleic acid (e.g., cancer DNA). In certain aspects, the cell-free nucleic acid comprises circulating tumor nucleic acid (e.g., tumor DNA). In some embodiments, the cell-free nucleic acid comprises infectious agent nucleic acid (e.g., pathogen DNA). In some embodiments, the cell-free nucleic acid comprises nucleic acid (e.g., DNA) from a transplant. In some embodiments, the cell-free nucleic acid comprises nucleic acid (e.g., DNA) from the microbiome (e.g., gut microbiome, blood microbiome, oral microbiome, spinal fluid microbiome, fecal microbiome).

[0183] Cell-free DNA (cfDNA) can be derived from degraded sources and often provides only small amounts of DNA when extracted. The method described herein for generating a single-stranded DNA (ssDNA) library can capture a large amount of short DNA fragments from cfDNA. For example, cfDNA from cancer samples tends to have a higher concentration of short fragments. In certain cases, the short fragments in cfDNA can be enriched for fragments generated by transcription factors rather than nucleosomes.

[0184] Extracellular nucleic acids may contain different nucleic acid species and are therefore referred to herein as "heterogeneous" in certain embodiments. For example, serum or plasma from a person suffering from a tumor or cancer may contain nucleic acids from tumor or cancer cells (e.g., neoplasms) and nucleic acids from non-tumor or non-cancer cells. In another example, serum or plasma from a pregnant female may contain maternal nucleic acids and fetal nucleic acids. In another example, serum or plasma from a patient suffering from an infectious or infectious disease may contain host nucleic acids and infectious agent or pathogen nucleic acids. In another example, a sample from a subject who has undergone a transplant may contain host nucleic acids and nucleic acids from a donor organ or tissue. In some cases, cancer, tumor, fetal, pathogen, or graft nucleic acids may represent about 5% to about 50% of the total nucleic acids (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49% of the total nucleic acids are cancer, tumor, fetal, pathogen, graft, or microbiome nucleic acids). In another example, heterogeneous nucleic acids may include nucleic acids from two or more subjects (e.g., a sample from a crime scene).

[0185] At least two different nucleic acid species may be present in different amounts in extracellular nucleic acids, and these nucleic acid species may be referred to as minor and major species. In certain cases, the minor nucleic acid species is from a diseased cell type (e.g., cancer cells, exhausted cells, cells attacked by the immune system). In certain embodiments, genetic mutations or genetic alterations (e.g., copy number alterations, copy number variations, single base alterations, single base variations, chromosomal alterations, and / or translocations) are determined for the minor nucleic acid species. In certain embodiments, genetic mutations or genetic alterations are determined for the majority nucleic acid species. In general, the terms "minority" and "majority" are not intended to be rigidly defined in any respect. In one aspect, for example, a nucleic acid considered "minor" can have an abundance of at least about 0.1% of the total nucleic acids in the sample to less than 50% of the total nucleic acids in the sample. In some embodiments, a minority nucleic acid can have an abundance of at least about 1% of the total nucleic acids in the sample to about 40% of the total nucleic acids in the sample. In some embodiments, the minority nucleic acid can have an abundance of at least about 2% of the total nucleic acids in the sample to about 30% of the total nucleic acids in the sample. In some embodiments, the minority nucleic acid can have an abundance of at least about 3% of the total nucleic acids in the sample to about 25% of the total nucleic acids in the sample. For example, the minority nucleic acid can have an abundance of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% of the total nucleic acids in the sample. In some cases, the minority species of extracellular nucleic acid may represent about 1% to about 40% of the total nucleic acid (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the nucleic acid is minor species nucleic acid). In some embodiments, the minority nucleic acid is extracellular DNA.In some embodiments, the minority nucleic acid is extracellular DNA from apoptotic tissue. In some embodiments, the minority nucleic acid is extracellular DNA from tissue in which some cells have undergone apoptosis. In some embodiments, the minority nucleic acid is extracellular DNA from necrotic tissue. In some embodiments, the minority nucleic acid is extracellular DNA from tissue in which some cells have undergone necrosis. Necrosis may, in certain cases, refer to a post-mortem process following cell death. In some embodiments, the minority nucleic acid is extracellular DNA from tissue affected by a cell proliferative disorder (e.g., cancer). In some embodiments, the minority nucleic acid is extracellular DNA from tumor cells. In some embodiments, the minority nucleic acid is extracellular DNA from a pathogen. In some embodiments, the minority nucleic acid is extracellular DNA from a transplant. In some embodiments, the minority nucleic acid is extracellular DNA from the microbiome.

[0186] In another aspect, for example, a nucleic acid considered to be the "majority" can have an abundance of greater than 50% of all nucleic acids in the sample to about 99.9% of all nucleic acids in the sample. In some embodiments, the majority nucleic acid can have an abundance of at least about 60% of all nucleic acids in the sample to about 99% of all nucleic acids in the sample. In some embodiments, the majority nucleic acid can have an abundance of at least about 70% of all nucleic acids in the sample to about 98% of all nucleic acids in the sample. In some embodiments, the majority nucleic acid can have an abundance of at least about 75% of all nucleic acids in the sample to about 97% of all nucleic acids in the sample. For example, the majority nucleic acid can have an abundance of at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total nucleic acids in the sample. In some embodiments, the majority nucleic acid is extracellular DNA. In some embodiments, the majority nucleic acid is extracellular maternal DNA. In some embodiments, the majority nucleic acid is DNA from healthy tissue. In some embodiments, the majority nucleic acid is DNA from non-tumor cells. In some embodiments, the majority nucleic acid is DNA from host cells.

[0187] In some embodiments, the minority species of extracellular nucleic acids are about 500 base pairs or less in length (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the minority species nucleic acids are about 500 base pairs or less in length), In some embodiments, the minority species of extracellular nucleic acids are about 300 base pairs or less in length (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the minority species nucleic acids are about 300 base pairs or less in length). In some embodiments, the minority species of extracellular nucleic acids are about 250 base pairs or less in length (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the minority species nucleic acids are about 250 base pairs or less in length), In some embodiments, the minority species of extracellular nucleic acids are about 200 base pairs or less in length (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the minority species nucleic acids are about 200 base pairs or less in length). In some embodiments, the minority species of extracellular nucleic acids are about 150 base pairs or less in length (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the minority species nucleic acids are about 150 base pairs or less in length), In some embodiments, the minority species of extracellular nucleic acids are about 100 base pairs or less in length (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the minority species nucleic acids are about 100 base pairs or less in length). In some embodiments, the minority species of extracellular nucleic acids are about 50 base pairs or less in length (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the minority species nucleic acids are about 50 base pairs or less in length).

[0188] Nucleic acids can be subjected to the methods described herein with or without processing of the sample containing the nucleic acid. In some embodiments, nucleic acids are subjected to the methods described herein after processing of the sample containing the nucleic acid. For example, nucleic acids can be extracted, isolated, purified, partially purified, or amplified from the sample. The term "isolated," as used herein, refers to nucleic acids that have been removed from their original environment (e.g., the natural environment if it naturally occurs, or a host cell if exogenously expressed) and thus have been altered from their original environment by human intervention (e.g., "by the hand of man"). The term "isolated nucleic acid," as used herein, can refer to nucleic acids that have been removed from a subject (e.g., a human subject). Isolated nucleic acids can be provided that have less non-nucleic acid components (e.g., proteins, lipids) than the amount of components present in the source sample. A composition containing isolated nucleic acids can be about 50% to greater than 99% free of non-nucleic acid components. A composition comprising an isolated nucleic acid can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of non-nucleic acid components. The term "purified," as used herein, can refer to a provided nucleic acid that contains fewer non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. A composition comprising a purified nucleic acid can be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of other non-nucleic acid components. The term "purified," as used herein, can refer to a provided nucleic acid that contains fewer nucleic acid species than in the sample source from which the nucleic acid is derived. A composition containing purified nucleic acid can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of other nucleic acid species. For example, fetal nucleic acid can be purified from a mixture containing maternal and fetal nucleic acid. In certain instances, small fragments of nucleic acid (e.g., 30-500 bp fragments) can be purified or partially purified from a mixture containing nucleic acid fragments of different lengths.In certain examples, nucleosomes containing smaller fragments of nucleic acid can be purified from a mixture of larger nucleosome complexes containing larger fragments of nucleic acid. In certain examples, larger nucleosome complexes containing larger fragments of nucleic acid can be purified from nucleosomes containing smaller fragments of nucleic acid. In certain examples, small fragments of fetal nucleic acid (e.g., 30-500 bp fragments) can be purified or partially purified from a mixture containing both fetal and maternal nucleic acid fragments. In certain examples, nucleosomes containing smaller fragments of fetal nucleic acid can be purified from a mixture of larger nucleosome complexes containing larger fragments of maternal nucleic acid. In certain examples, cancer cell nucleic acid can be purified from a mixture containing cancer cell nucleic acid and non-cancer cell nucleic acid. In certain examples, nucleosomes containing smaller fragments of cancer cell nucleic acid can be purified from a mixture of larger nucleosome complexes containing larger fragments of non-cancer nucleic acid. In some embodiments, nucleic acids are subjected to the methods described herein without prior processing of the nucleic acid-containing sample. For example, nucleic acids can be analyzed directly from a sample without prior extraction, purification, partial purification, and / or amplification.

[0189] Nucleic acids can be amplified under amplification conditions. The terms "amplified" or "amplification" or "amplification conditions," as used herein, refer to subjecting a target nucleic acid (e.g., ssNA) in a sample or a nucleic acid product produced by a method herein to a process that linearly or exponentially produces amplicon nucleic acids having the same or substantially the same nucleotide sequence as the target nucleic acid (e.g., ssNA) or a portion thereof. In certain embodiments, the terms "amplified" or "amplification" or "amplification conditions" refer to a method involving polymerase chain reaction (PCR). In certain cases, the amplification product can contain one or more more nucleotides than the amplified nucleotide region of the nucleic acid template sequence (e.g., a primer can contain "extra" nucleotides, such as a transcription initiation sequence, in addition to nucleotides complementary to the nucleic acid template gene molecule, such that the amplification product contains "extra" nucleotides or nucleotides that do not correspond to the amplified nucleotide region of the nucleic acid template gene molecule).

[0190] Before nucleic acid is subjected to the method described herein, nucleic acid can be exposed to a process that modifies certain nucleotides in nucleic acid.For example, nucleic acid can be subjected to a process that selectively modifies nucleic acid based on the methylation state of internal nucleotides.In addition, conditions such as high temperature, ultraviolet irradiation, and X-ray irradiation can induce changes in the sequence of nucleic acid molecules.Nucleic acid can be provided in any suitable form that is useful for sequence analysis.

[0191] In some embodiments, the target nucleic acid (e.g., ssNA) is not modified before binding to a scaffold adaptor or a component thereof herein. In some embodiments, the target nucleic acid (e.g., ssNA) is not modified in length before binding to a scaffold adaptor or a component thereof herein. "Unmodified" in this context means that the target nucleic acid is isolated from a sample and then bound to a scaffold adaptor or a component thereof without modifying the length or composition of the target nucleic acid. For example, the target nucleic acid (e.g., ssNA) may not be shortened (e.g., they are not contacted with a restriction enzyme, a nuclease, or physical conditions that reduce the length (e.g., shearing conditions, cleavage conditions)), or the length may not be increased by one or more nucleotides (e.g., the end is not filled in at an overhanging position; nucleotides are not added to the end). The addition of a phosphate group or a chemically reactive group to one or both ends of a target nucleic acid (e.g., ssNA) is generally not considered a modification of the nucleic acid or a modification of the length of the nucleic acid. The denaturation of double-stranded nucleic acid (dsNA) fragments to produce ssNA fragments is generally not considered a modification of the nucleic acid or a modification of the length of the nucleic acid.

[0192] In some embodiments, one or both native ends of a target nucleic acid (e.g., ssNA) are present when the ssNA is bound to a scaffold adaptor or a component thereof herein. Native ends generally refer to unmodified fragments of nucleic acid fragments. In some embodiments, the native ends of a target nucleic acid (e.g., ssNA) are not modified in length before the step of binding to a scaffold adaptor or a component thereof herein. "Unmodified" in this context means that the target nucleic acid is isolated from a sample and then bound to a scaffold adaptor or a component thereof without modifying the length of the native end of the target nucleic acid. For example, the target nucleic acid (e.g., ssNA) is not shortened by one or more nucleotides (e.g., it is not contacted with a restriction enzyme, a nuclease, or a physical condition that reduces the length (e.g., shearing conditions, cleavage conditions) to generate a non-native end) and is not increased in length (e.g., the native end is not filled in with an overhang; nucleotides are not added to the native end). The addition of phosphate groups or chemically reactive groups to one or both of the native termini of a target nucleic acid is generally not considered a modification of the length of the nucleic acid.

[0193] In some embodiments, the target nucleic acid (e.g., ssNA) is not contacted with a cleavage agent (e.g., endonuclease, exonuclease, restriction enzyme) and / or polymerase prior to the step of binding to a scaffold adapter or a component thereof herein. In some embodiments, the target nucleic acid is not subjected to mechanical shearing (e.g., sonication (e.g., the Adaptive Focused Acoustics™ (AFA) process by Covaris)) prior to the step of binding to a scaffold adapter or a component thereof herein. In some embodiments, the target nucleic acid is not contacted with an exonuclease (e.g., DNAse) prior to the step of binding to a scaffold adapter or a component thereof herein. In some embodiments, the target nucleic acid is not amplified prior to the step of binding to a scaffold adapter or a component thereof herein. In some embodiments, the target nucleic acid is not bound to a solid support prior to the step of binding to a scaffold adapter or a component thereof herein. In some embodiments, the target nucleic acid is not conjugated to another molecule prior to the step of binding to a scaffold adapter or a component thereof herein. In some embodiments, the target nucleic acid is not cloned into a vector prior to the step of binding to a scaffold adaptor or a component thereof herein. In some embodiments, the target nucleic acid may be subjected to dephosphorylation prior to the step of binding to a scaffold adaptor or a component thereof herein. In some embodiments, the target nucleic acid may be subjected to phosphorylation prior to the step of binding to a scaffold adaptor or a component thereof herein.

[0194] In some embodiments, the step of binding a target nucleic acid (e.g., ssNA) to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid and binding the isolated target nucleic acid to a scaffold adapter herein or a component thereof. In some embodiments, the step of binding a target nucleic acid to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid, phosphorylating the isolated target nucleic acid, and binding the phosphorylated target nucleic acid to a scaffold adapter herein or a component thereof. In some embodiments, the step of binding a target nucleic acid to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid, dephosphorylating the scaffold adapter herein or a component thereof, and binding the isolated target nucleic acid to a dephosphorylated scaffold adapter herein or a dephosphorylated component thereof. In some embodiments, the step of binding a target nucleic acid to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid, dephosphorylating the isolated target nucleic acid, phosphorylating the dephosphorylated target nucleic acid, and binding the phosphorylated target nucleic acid to a scaffold adapter herein or a component thereof. In some embodiments, the step of binding the target nucleic acid to a scaffold adaptor herein or a component thereof comprises isolating the target nucleic acid, dephosphorylating the isolated target nucleic acid, phosphorylating the dephosphorylated target nucleic acid, dephosphorylating the scaffold adaptor or a component thereof, and binding the phosphorylated target nucleic acid to the dephosphorylated scaffold adaptor herein or a dephosphorylated component thereof.

[0195] In some embodiments, the step of binding a target nucleic acid (e.g., ssNA) to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid and binding the isolated target nucleic acid to a scaffold adapter herein or a component thereof. In some embodiments, the step of binding a target nucleic acid to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid, phosphorylating the isolated target nucleic acid, and binding the phosphorylated target nucleic acid to a scaffold adapter herein or a component thereof. In some embodiments, the step of binding a target nucleic acid to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid, dephosphorylating the scaffold adapter herein or a component thereof, and binding the isolated target nucleic acid to a dephosphorylated scaffold adapter herein or a dephosphorylated component thereof. In some embodiments, the step of binding a target nucleic acid to a scaffold adapter herein or a component thereof comprises isolating the target nucleic acid, dephosphorylating the isolated target nucleic acid, phosphorylating the dephosphorylated target nucleic acid, and binding the phosphorylated target nucleic acid to a scaffold adapter herein or a component thereof. In some embodiments, the step of binding the target nucleic acid to a scaffold adaptor herein or a component thereof comprises isolating the target nucleic acid, dephosphorylating the isolated target nucleic acid, phosphorylating the dephosphorylated target nucleic acid, dephosphorylating the scaffold adaptor or a component thereof, and binding the phosphorylated target nucleic acid to the dephosphorylated scaffold adaptor herein or a dephosphorylated component thereof.

[0196] single stranded nucleic acid Provided herein are methods and compositions for capturing single-stranded nucleic acids (ssNAs) using dedicated adapters (e.g., for generating sequencing libraries).Single-stranded nucleic acids or ssNAs generally refer to a group of polynucleotides that are single-stranded (i.e., have no intermolecular or intramolecular hybridization) over 70% or more of their length.In some embodiments, ssNAs are single-stranded over 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more of the length of the polynucleotide.In certain aspects, ssNAs are single-stranded over the entire length of the polynucleotide.Single-stranded nucleic acids may be referred to herein as target nucleic acids.

[0197] ssNA may include single-stranded deoxyribonucleic acid (ssDNA). In some embodiments, ssDNA includes, but is not limited to, ssDNA derived from double-stranded DNA (dsDNA). For example, ssDNA is obtained from double-stranded DNA that is denatured (e.g., thermally denatured and / or chemically denatured) to generate ssDNA. In some embodiments, the method herein includes a step of generating ssDNA by denaturing dsDNA before the step of binding the ssDNA to the scaffold adaptor or a component thereof described herein.

[0198] In some embodiments, the ssNA comprises a single-stranded ribonucleic acid (ssRNA). The RNA may include, for example, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), trans-acting small interfering RNA (ta-siRNA), naturally occurring small interfering RNA (nat-siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), non-coding RNA (ncRNA), transfer messenger RNA (tmRNA), precursor messenger RNA (pre-mRNA), small Cajal body-specific RNA (scaRNA), piwi-binding RNA (piRNA), endoribonuclease-prepared siRNA (esiRNA), small RNA (stRNA), signal recognition RNA, telomeric RNA, ribozyme, or a combination thereof. In some embodiments, when the ssNA is ssRNA, the ssRNA is mRNA. In some embodiments, the ssNA comprises single-stranded complementary DNA (cDNA).

[0199] In some embodiments, the methods herein include contacting ssNA with a single-stranded nucleic acid-binding protein (SSB) to generate SSB-binding ssNA. SSBs generally bind cooperatively to ssNA and typically bind poorly to double-stranded nucleic acids (dsNA). Upon binding to ssDNA, SSBs destabilize the helical duplex. The SSB may be a prokaryotic SSB (e.g., a bacterial or archaeal SSB) or a eukaryotic SSB. Examples of SSBs include E. coli SSB, E. coli RecA, highly thermostable single-stranded DNA-binding protein (ET SSB), Thermus thermophilus (Tth) RecA, T4 gene 32 protein, and replication protein A (RPA—eukaryotic SSB). ET SSB, Tth RecA, E. coli RecA, T4 gene 32 protein, as well as buffers and detailed protocols for preparing SSB-conjugated ssNAs using such SSBs, are commercially available (New England Biolabs, Inc., Ipswich, MA).

[0200] In some embodiments, the methods herein do not include a step of contacting ssNAs with a single-stranded nucleic acid-binding protein (SSB) to generate SSB-bound ssNAs. Thus, the methods herein can omit the step of generating SSB-bound ssNAs. For example, the methods herein may include a step of binding ssNAs to a scaffold adaptor described herein or a component thereof without contacting the ssNAs with SSB. In such cases, the methods herein may be referred to as "SSB-free" methods for generating nucleic acid libraries. Certain SSB-free methods described herein can generate libraries with parameters similar to those of libraries prepared using SSBs, as shown in the figures and discussed in the examples. In some embodiments, the methods herein include a step of contacting ssNAs with a single-stranded nucleic acid binder other than SSB. Such a single-stranded nucleic acid binder can stably bind to single-stranded nucleic acids, prevent or reduce the formation of nucleic acid duplexes, still allow ligation or otherwise end-modification of the bound nucleic acids, and be thermostable. Examples of single-stranded nucleic acid binding agents include, but are not limited to, topoisomerases, helicases, domains thereof, and fusion proteins containing these domains.

[0201] In some embodiments, the methods herein comprise combining a nucleic acid composition comprising a single-stranded nucleic acid (ssNA) with a scaffold adaptor described herein or a component thereof. In some embodiments, the methods herein comprise combining a nucleic acid composition consisting of a single-stranded nucleic acid (ssNA) with a scaffold adaptor described herein or a component thereof. In some embodiments, the methods herein comprise combining a nucleic acid composition consisting essentially of a single-stranded nucleic acid (ssNA) with a scaffold adaptor described herein or a component thereof. A nucleic acid composition "essentially consisting of" a single-stranded nucleic acid (ssNA) generally comprises ssNA and no additional protein or nucleic acid component. For example, a nucleic acid composition "essentially consisting of" a single-stranded nucleic acid (ssNA) may not comprise double-stranded nucleic acid (dsNA) or may comprise a low percentage of dsNA (e.g., less than 10% dsNA, less than 5% dsNA, less than 1% dsNA). A nucleic acid composition "essentially consisting of" a single-stranded nucleic acid (ssNA) may not comprise protein. For example, a nucleic acid composition "consisting essentially of" a single-stranded nucleic acid (ssNA) may not contain single-stranded binding proteins (SSBs) or other proteins useful for stabilizing ssNAs. A nucleic acid composition "consisting essentially of" a single-stranded nucleic acid (ssNA) may contain chemical components typically present in nucleic acid compositions, such as buffers, salts, alcohols, crowding agents (e.g., PEG), and may also contain residual components (e.g., nucleic acids, proteins, cell membrane components) from the nucleic acid source (e.g., sample) or nucleic acid extraction. A nucleic acid composition "consisting essentially of" a single-stranded nucleic acid (ssNA) may contain ssNA fragments having one or more phosphates (e.g., terminal phosphate, 5'-terminal phosphate). A nucleic acid composition "consisting essentially of" a single-stranded nucleic acid (ssNA) may also contain ssNA fragments containing one or more modified nucleotides.

[0202] Nucleic acid concentration In some embodiments, nucleic acids (e.g., extracellular nucleic acids) are enriched or relatively enriched for a subpopulation or species of nucleic acids. Nucleic acid subpopulations can include, for example, fetal nucleic acids, maternal nucleic acids, cancer nucleic acids, tumor nucleic acids, patient nucleic acids, host nucleic acids, pathogen nucleic acids, graft nucleic acids, microbiome nucleic acids, nucleic acids comprising fragments of a particular length or length range, or nucleic acids from a particular genomic region (e.g., a single chromosome, a set of chromosomes, and / or a particular chromosomal region). Such enriched samples can be used with the methods provided herein. Thus, in certain embodiments, the methods of the present technology include a further step of enriching for a subpopulation of nucleic acids in the sample. In certain embodiments, nucleic acids from normal tissues (e.g., non-cancer cells, host cells) are selectively (partially, substantially, almost completely, or completely) removed from the sample. In certain embodiments, maternal nucleic acids are selectively (partially, substantially, almost completely, or completely) removed from the sample. In certain embodiments, enrichment for specific low copy number nucleic acids (e.g., cancer, tumor, fetal, pathogen, graft, microbiome nucleic acids) can improve quantitative sensitivity. Methods for enriching a sample for a particular species of nucleic acid are described, for example, in U.S. Pat. No. 6,927,028, International Patent Application Publication No. WO2007 / 140417, International Patent Application Publication No. WO2007 / 147063, International Patent Application Publication No. WO2009 / 032779, International Patent Application Publication No. WO2009 / 032781, International Patent Application Publication No. WO2010 / 033639, International Patent Application Publication No. WO2011 / 034631, International Patent Application Publication No. WO2006 / 056480, and International Patent Application Publication No. WO2011 / 143659, the entire contents of each of which are incorporated herein by reference, including all text, tables, formulas, and figures.

[0203] In some embodiments, nucleic acids are enriched for a specific target fragment type and / or reference fragment type.In certain embodiments, nucleic acids are enriched for a specific nucleic acid fragment length or fragment length range using one or more length-based separation methods described below.In certain embodiments, nucleic acids are enriched for fragments from selected genomic regions (e.g., chromosomes) using one or more sequence-based separation methods described herein and / or known in the art.

[0204] Non-limiting examples of methods for enriching for nucleic acid subpopulations in a sample include methods that utilize epigenetic differences between nucleic acid species (e.g., the methylation-based fetal nucleic acid enrichment method described in U.S. Patent Application Publication No. 2010 / 0105049, incorporated herein by reference); approaches that enhance polymorphic sequences with restriction endonucleases (e.g., such as the method described in U.S. Patent Application Publication No. 2009 / 0317818, incorporated herein by reference); selective enzymatic digestion approaches; massively parallel signature sequencing (MPSS) approaches; amplification (e.g., PCR)-based approaches (e.g., locus-specific amplification, multiplex SNP allele PCR approaches; universal amplification); pull-down approaches (e.g., biotinylated ultramer pull-down); extension and ligation-based methods (e.g., molecular inversion probe (MIP) extension and ligation); and combinations thereof.

[0205] In some embodiments, modified nucleic acids can be enriched. Nucleic acid modifications include, but are not limited to, carboxycytosine, 5-methylcytosine (5mC) and its oxidized derivatives (e.g., 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-arboxylcytosine (5caC)), N(6)-methyladenine (6mA), N4-methylcytosine (4mC), N(6)-methyladenosine (m(6)A), pseudouridine (Ψ), 5-methylcytidine (m(5)C), hydroxymethyluracil, 2'-O-methylation of the 3' end, tRNA modifications, miRNA modifications, and snRNA modifications. Nucleic acids containing one or more modifications can be enriched by various methods, including, but not limited to, antibody-based pull-down. Enrichment of modified nucleic acids can be performed before (Figures 52A-B) or after (Figures 52C-D) denaturation of dsDNA. Enrichment before denaturation may also result in enrichment of complementary strands that may lack the modification, while enrichment after denaturation does not enrich for complementary strands that lack the modification.

[0206] In some embodiments, nucleic acids are enriched for fragments from selected genomic regions (e.g., chromosomes) using one or more sequence-based separation methods described herein. Sequence-based separation is generally based on nucleotide sequences that are present in the fragment of interest (e.g., target and / or reference fragments) but are substantially absent or present in only trace amounts (e.g., 5% or less) in other fragments of the sample. In some embodiments, sequence-based separation can produce separated target fragments and / or separated reference fragments. The separated target fragments and / or separated reference fragments are often isolated from fragments remaining in the nucleic acid sample. In certain embodiments, the separated target fragments and the separated reference fragments are also isolated from each other (e.g., in separate assay compartments). In certain embodiments, the separated target fragments and the separated reference fragments are isolated together (e.g., in the same assay compartment). In some embodiments, unbound fragments can be removed, degraded, or digested separately.

[0207] In some embodiments, scaffold adapters are used to enrich target nucleic acids. For example, as shown in Figure 51, scaffold adapters can be designed so that some or all of the bases in the ssNA hybridization region are defined or known bases. These scaffold adapters can preferentially hybridize to target nucleic acids having sequences complementary to the defined or known bases in the scaffold adapter ssNA hybridization region, thereby enriching the target nucleic acids in the resulting library. For example, as shown in Figure 51, incorporation of GC dinucleotides into the ssNA hybridization region can be used to enrich target nucleic acids with terminal CG (also known as CpG) dinucleotides. Part or all of the length of the scaffold adapter ssNA hybridization region can be used to similarly target any other defined sequence, including, but not limited to, nuclease cleavage sites, gene promoter regions, pathogen sequences, tumor-associated sequences, and other motifs. In one example, a library was prepared using non-enriching scaffold adapters and CG dinucleotide-enriching scaffold adapters. For libraries prepared without enrichment, 1.7% of reads started with CG and 1.1% of reads ended with CG. For libraries prepared with enrichment, 5.2% of reads started with CG and 19.6% of reads ended with CG. In another example, shown in Figure 55, a sample containing RNA (e.g., host and pathogen RNA) is reverse transcribed using primers specific to the pathogen RNA of interest to generate cDNA. The cDNA is then purified and prepared using single-stranded library preparation methods as discussed herein, using either standard scaffold adapters or scaffold adapters with ssNA hybridization regions targeted to the enriched region by the reverse transcription primer. Pathogen DNA can be similarly enriched.

[0208] In some cases, the target nucleic acid sequence at the 5' or 3' nucleic acid end is defined or known. In other cases, scaffold adapters can be used to identify novel targets of interest at the 5' or 3' nucleic acid end. Nucleic acid sequences or patterns of interest can be characterized from the scaffold adapter library output with or without enrichment. In some cases, specific sequences or sequence patterns at the 5', 3', or both nucleic acid ends can be associated with specific conditions. Such conditions include, but are not limited to, disease states, methylation states, and expression states. Scaffold adapters can be used to quantify the presence or relative abundance of known or novel target sequences at nucleic acid ends between samples and controls, for example, between cell-free DNA from cancer patients and healthy controls. These data can be used to learn the relationship between sequence information at the DNA end and a given condition. By training with well-characterized datasets of patient and healthy samples, in one example, analytical methods or algorithms can be used to predict conditions or transitions through conditions. For example, the present inventors have found that cfDNA from patients with acute myeloid leukemia (AML) has an increased AT dinucleotide and a decreased CpG dinucleotide at the 5' and 3' DNA ends compared to non-AML patient samples. In this example, analytical tools can be used with cfDNA end sequence information to predict the patient's risk of developing AML.

[0209] In some embodiments, selective nucleic acid capture process is used to separate target and / or reference fragments from nucleic acid samples.Commercially available nucleic acid capture systems include, for example, Nimblegen sequence capture system (Roche NimbleGen, Madison, WI); ILLUMINA BEADARRAY platform (Illumina, San Diego, CA); Affymetrix GENECHIP platform (Affymetrix, Santa Clara, CA); Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA); and related platforms.Such methods usually involve hybridization of capture oligonucleotides with part or all of the nucleotide sequence of target or reference fragments, and may include the use of solid-phase (e.g., solid-phase array) and / or solution-based platforms.Capture oligonucleotides (sometimes referred to as "bait") can be selected or designed so that they preferentially hybridize with nucleic acid fragments from selected genomic regions or loci, or with specific sequences in nucleic acid targets. In certain embodiments, hybridization-based methods (e.g., those using oligonucleotide arrays) can be used to enrich for fragments containing certain nucleic acid sequences. Thus, in some embodiments, a nucleic acid sample is optionally enriched, for example, by capturing a subset of fragments using capture oligonucleotides complementary to selected sequences in the sample nucleic acid. In certain cases, the captured fragments are amplified. For example, captured fragments containing adapters can be amplified using primers complementary to the adapter sequences to form a collection of amplified fragments indexed according to the adapter sequences. In some embodiments, nucleic acids are enriched for fragments from selected genomic regions (e.g., chromosomes, genes) by amplifying one or more regions of interest using oligonucleotides (e.g., PCR primers) complementary to the sequences of fragments containing the regions of interest or portions thereof.

[0210] In some embodiments, nucleic acids are enriched for a particular nucleic acid fragment length, length range, or length(s) below or above a particular threshold or cutoff using one or more length-based separation methods. Nucleic acid fragment length typically refers to the number of nucleotides in a fragment. Nucleic acid fragment length may also be referred to as nucleic acid fragment size. In some embodiments, length-based separation methods are performed without measuring the length of individual fragments. In some embodiments, length-based separation methods are performed in conjunction with methods that determine the length of individual fragments. In some embodiments, length-based separation refers to a size fractionation procedure, in which all or a portion of the fractionated pool can be isolated (e.g., retained) and / or analyzed. Size fractionation procedures are known in the art (e.g., array separation, molecular sieve separation, gel electrophoresis separation, column chromatography (e.g., size exclusion column) separation, and microfluidics-based approaches). In certain cases, length-based separation approaches may include, for example, selective sequence tagging approaches, fragment circularization, chemical treatment (e.g., formaldehyde, polyethylene glycol (PEG) precipitation), mass spectrometry, and / or size-specific nucleic acid amplification.

[0211] In some embodiments, nucleic acids are enriched for fragments associated with one or more nucleic acid binding proteins. Exemplary enrichment methods include chromatin immunoprecipitation (ChIP), crosslinking ChIP (XCHIP), native ChIP (NChIP), bead-free ChIP, carrier ChIP (CChIP), fast ChIP (qChIP), and rapid and quantitative ChIP (QChIP). 2 These include, but are not limited to, microchip-ChIP, microchip (µChIP), matrix-ChIP, pathogen-ChIP (PAT-ChIP), ChIP-exo, ChIP-on-chip, RIP-ChIP, HiChIP, ChIA-PET, and HiChIRP.

[0212] In some embodiments, the methods herein include enriching an RNA species in a mixture of RNA species. For example, the methods herein may include enriching messenger RNA (mRNA) present in a mixture of mRNA and ribosomal RNA (rRNA). Any suitable mRNA enrichment method can be used, including rRNA depletion and / or mRNA enrichment methods, such as magnetic bead rRNA depletion (e.g., Ribo-zero™, Ribominus™, and MICROBExpress™, which use rRNA depletion probes in combination with magnetic beads to deplete rRNA from a sample, thereby enriching mRNA), oligo(dT)-based poly(A) enrichment (e.g., BioMag® Oligo(dT)20), nuclease-based rRNA depletion (e.g., Terminator™ 5'-phosphate-dependent exonuclease digestion), and combinations thereof.

[0213] The enrichment strategy can increase the relative abundance of the target nucleic acid (e.g., as assessed by percent of sequencing reads) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, or more.

[0214] Length-based separation In some embodiments, the methods herein include separating target nucleic acids (e.g., ssNAs) according to fragment length. For example, target nucleic acids (e.g., ssNAs) can be enriched for a particular nucleic acid fragment length, length range, or length(s) below or above a particular threshold or cutoff using one or more length-based separation methods. Nucleic acid fragment length typically refers to the number of nucleotides in a fragment. Nucleic acid fragment length may also be referred to as nucleic acid fragment size. In some embodiments, length-based separation methods are performed without measuring the length of individual fragments. In some embodiments, length-based separation methods are performed in conjunction with a method for determining the length of individual fragments. In some embodiments, length-based separation refers to a size fractionation procedure in which all or a portion of the fractionated pool can be isolated (e.g., retained) and / or analyzed. Size fractionation procedures are known in the art (e.g., array separation, molecular sieve separation, gel electrophoresis separation, column chromatography (e.g., size exclusion column) separation, and microfluidics-based approaches). In some embodiments, length-based separation approaches may include, for example, fragment circularization, chemical treatment (e.g., formaldehyde, polyethylene glycol (PEG)), mass spectrometry, and / or size-specific nucleic acid amplification. In some embodiments, length-based separation is performed using solid-phase reversible immobilization (SPRI) beads.

[0215] In some embodiments, nucleic acid fragments of a certain length, length range, or length(s) below or above a certain threshold or cutoff are separated from a sample. In some embodiments, fragments having lengths below a certain threshold or cutoff (e.g., 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp) are referred to as "short" fragments, and fragments having lengths above a certain threshold or cutoff (e.g., 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp) are referred to as "long," large, and / or high molecular weight (HMW) fragments. In some embodiments, fragments of a certain length, length range, or length(s) below or above a certain threshold or cutoff are retained for analysis, while fragments of a different length, length range, or length(s) above or below the threshold or cutoff are not retained for analysis. In some embodiments, fragments that are less than about 500 bp are retained. In some embodiments, fragments less than about 400 bp are retained. In some embodiments, fragments less than about 300 bp are retained. In some embodiments, fragments less than about 200 bp are retained. In some embodiments, fragments less than about 150 bp are retained. For example, fragments less than about 190 bp, 180 bp, 170 bp, 160 bp, 150 bp, 140 bp, 130 bp, 120 bp, 110 bp, or 100 bp are retained. In some embodiments, fragments between about 100 bp and about 200 bp are retained. For example, fragments between about 190 bp, 180 bp, 170 bp, 160 bp, 150 bp, 140 bp, 130 bp, 120 bp, or 110 bp are retained. In some embodiments, fragments within the range of about 100 bp to about 200 bp are retained. For example, fragments within the range of about 110 bp to about 190 bp, 130 bp to about 180 bp, 140 bp to about 170 bp, 140 bp to about 150 bp, 150 bp to about 160 bp, or 145 bp to about 155 bp are retained.

[0216] In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of less than about 1000 bp are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of less than about 500 bp are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of less than about 400 bp are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of less than about 300 bp are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of less than about 200 bp are combined with a plurality of scaffold adaptor species, or a pool of scaffold adaptor species, or a component of a scaffold adaptor species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of less than about 100 bp are combined with a plurality of scaffold adaptor species, or a pool of scaffold adaptor species, or a component of a scaffold adaptor species, as described herein.

[0217] In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of about 100 bp or greater are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of about 200 bp or greater are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of about 300 bp or greater are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of about 400 bp or greater are bound to a plurality of scaffold adapter species, or a pool of scaffold adapter species, or a component of a scaffold adapter species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of about 500 bp or greater are combined with a plurality of scaffold adaptor species, or a pool of scaffold adaptor species, or components of a scaffold adaptor species, as described herein. In some embodiments, target nucleic acids (e.g., ssNAs) having a fragment length of about 1000 bp or greater are combined with a plurality of scaffold adaptor species, or a pool of scaffold adaptor species, or components of a scaffold adaptor species, as described herein.

[0218] In some embodiments, target nucleic acids (e.g., ssNAs) having any fragment length or combination of fragment lengths are combined with a plurality of scaffold adaptor species, or a pool of scaffold adaptor species, or a component of a scaffold adaptor species, as described herein. For example, target nucleic acids (e.g., ssNAs) having fragment lengths of less than 500 bp and fragment lengths of 500 bp or longer can be combined with a plurality of scaffold adaptor species, or a pool of scaffold adaptor species, or a component of a scaffold adaptor species, as described herein.

[0219] Certain length-based separation methods that can be used with the methods described herein utilize, for example, a selective sequence tagging approach. Such methods can selectively tag fragment-size species (e.g., short fragments) of nucleic acids in a sample containing both long and short nucleic acids. Such methods typically involve performing a nucleic acid amplification reaction using a set of nested primers, including an internal primer and an external primer. In some embodiments, one or both of the internal primers can be tagged, thereby introducing a tag onto the target amplification product. External primers generally do not anneal to short fragments containing the (internal) target sequence. Internal primers can anneal to short fragments to generate amplification products containing tags and target sequences. Typically, tagging of long fragments is inhibited by a combination of mechanisms, including, for example, blocking extension of internal primers by prior annealing and extension of external primers. Enrichment of tagged fragments can be achieved by any of a variety of methods, including, for example, exonuclease digestion of single-stranded nucleic acids and amplification of tagged fragments using at least one tag-specific amplification primer.

[0220] Another length-based separation method that can be used with the methods described herein includes subjecting a nucleic acid sample to polyethylene glycol (PEG) precipitation. Exemplary methods include those described in International Patent Application Publication Nos. WO2007 / 140417 and WO2010 / 115016. This method generally requires contacting a nucleic acid sample with PEG in the presence of one or more monovalent salts under conditions sufficient to substantially precipitate large nucleic acids without substantially precipitating small (e.g., less than 300 nucleotides) nucleic acids.

[0221] Another length-based enrichment method that can be used with the method described herein includes ligation circularization, for example, using circligase.Usually, short nucleic acid fragments can be circularized more efficiently than long fragments.Non-circularized sequences can be separated from circularized sequences, and enriched short fragments can be used for further analysis.

[0222] Nucleic Acid Library The methods herein may include preparing a nucleic acid library and / or modifying nucleic acids for the nucleic acid library. In some embodiments, the ends of nucleic acid fragments are modified so that the fragments or their amplification products can be incorporated into a nucleic acid library. Generally, a nucleic acid library refers to a plurality of polynucleotide molecules (e.g., a sample of nucleic acids) that are prepared, assembled, and / or modified for a specific process, non-limiting examples of which include immobilization on a solid phase (e.g., a solid support, a flow cell, a bead), enrichment, amplification, cloning, and detection, and / or for nucleic acid sequencing. In certain embodiments, the nucleic acid library is prepared before or during the sequencing process. The nucleic acid library (e.g., a sequencing library) can be prepared by a suitable method known in the art. The nucleic acid library can be prepared by a targeted or non-targeted preparation process.

[0223] In some embodiments, a library of nucleic acids is modified to include chemical moieties (e.g., functional groups) configured for immobilization of the nucleic acids to a solid support. In some embodiments, a library of nucleic acids is modified to include biomolecules (e.g., functional groups) and / or members of binding pairs configured for immobilization of the library to a solid support, non-limiting examples of biomolecules and binding pairs include thyroxine-binding globulin, steroid-binding proteins, antibodies, antigens, haptens, enzymes, lectins, nucleic acids, repressors, protein A, protein G, avidin, streptavidin, biotin, complement component C1q, nucleic acid-binding proteins, receptors, carbohydrates, oligonucleotides, polynucleotides, complementary nucleic acid sequences, etc., and combinations thereof. Some examples of specific binding pairs include, but are not limited to, an avidin moiety and a biotin moiety; an antigen epitope and an antibody or immunoreactive fragment thereof; an antibody and a hapten; a digoxigenin moiety and an anti-digoxigenin antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; an oligonucleotide or polynucleotide and its corresponding complement; and the like, or combinations thereof.

[0224] In some embodiments, a nucleic acid library is modified to include one or more polynucleotides of known composition, including, but not limited to, an identifier (e.g., a tag, an indexing tag), a capture sequence, a label, an adapter, a restriction enzyme site, a promoter, an enhancer, an origin of replication, a stem-loop, a complementary sequence (e.g., a primer binding site, an annealing site), a suitable integration site (e.g., a transposon, a viral integration site), a modified nucleotide, a unique molecular identifier (UMI) as described herein, a palindromic sequence as described herein, or a combination thereof. The polynucleotides of known sequence can be added to a suitable position, for example, at the 5' end, the 3' end, or within the nucleic acid sequence. The polynucleotides of known sequence can be the same or different sequences. In some embodiments, the polynucleotides of known sequence are configured to hybridize with one or more oligonucleotides immobilized on a surface (e.g., the surface of a flow cell). For example, a nucleic acid molecule containing a 5' known sequence can hybridize with a first plurality of oligonucleotides, while a 3' known sequence can hybridize with a second plurality of oligonucleotides. In some embodiments, the library of nucleic acids can include chromosome-specific tags, capture sequences, labels, and / or adapters (e.g., oligonucleotide adapters described herein). In some embodiments, the library of nucleic acids includes one or more detectable labels. In some embodiments, one or more detectable labels can be incorporated at the 5' end, the 3' end, and / or any nucleotide position within the nucleic acids in the library. In some embodiments, the library of nucleic acids includes hybridized oligonucleotides. In certain embodiments, the hybridized oligonucleotides are labeled probes. In some embodiments, the library of nucleic acids includes hybridized oligonucleotide probes prior to immobilization to a solid phase.

[0225] In some embodiments, the polynucleotide of known sequence comprises a universal sequence. A universal sequence is a specific nucleotide sequence incorporated into two or more nucleic acid molecules or into two or more subsets of nucleic acid molecules, and the universal sequence is the same for all molecules or subsets of molecules into which it is incorporated. Universal sequences are often designed to hybridize with and / or amplify multiple different sequences using a single universal primer that is complementary to the universal sequence. In some embodiments, two (e.g., a pair) or more universal sequences and / or universal primers are used. The universal primer often comprises a universal sequence. In some embodiments, an adapter (e.g., a universal adapter) comprises a universal sequence. In some embodiments, one or more universal sequences are used to capture, identify, and / or detect multiple species or subsets of nucleic acids.

[0226] In certain embodiments of nucleic acid library preparation (e.g., in certain single-base synthesis procedures), nucleic acids are size-selected and / or fragmented (e.g., in preparation for library generation) to lengths of a few hundred base pairs or less. In some embodiments, library preparation is performed without fragmentation (e.g., when using cell-free DNA).

[0227] In certain embodiments, ligation-based library preparation methods are used (e.g., ILLUMINA TRUSEQ, Illumina, San Diego, CA). Ligation-based library preparation methods can incorporate index sequences (e.g., sample index sequences for identifying the sample origin of a nucleic acid sequence) in the first ligation step, and often use adapter (e.g., methylated adapter) designs that can be used to prepare samples for single-read sequencing, paired-end sequencing, and multiplexed sequencing. For example, nucleic acids (e.g., fragmented nucleic acids or cell-free DNA) can be end-repaired by a fill-in reaction, an exonuclease reaction, or a combination thereof. In some embodiments, the resulting blunt-end repaired nucleic acid can then be extended with a single nucleotide that is complementary to the single nucleotide overhang at the 3' end of the adapter / primer. Any nucleotide can be used for the extension / overhang nucleotide. In some embodiments, end repair is omitted and scaffold adaptors (e.g., scaffold adaptors described herein) are ligated directly to the native ends of nucleic acids (e.g., single-stranded nucleic acids, fragmented nucleic acids, and / or cell-free DNA).

[0228] In some embodiments, nucleic acid library preparation includes ligating a scaffold adapter, such as a scaffold adapter described herein, or a component thereof (e.g., to a sample nucleic acid, a sample nucleic acid fragment, a template nucleic acid, a target nucleic acid, or an ssNA). The scaffold adapter, or a component thereof, can include a sequence complementary to a flow cell anchor and may be utilized to immobilize the nucleic acid library on a solid support, such as the interior surface of a flow cell. In some embodiments, the scaffold adapter, or a component thereof, includes an identifier, one or more sequencing primer hybridization sites (e.g., a sequence complementary to a universal sequencing primer, a single-end sequencing primer, a paired-end sequencing primer, a multiplexed sequencing primer, etc.), or a combination thereof (e.g., adapter / sequencing, adapter / identifier, adapter / identifier / sequencing). In some embodiments, the scaffold adapter, or a component thereof, comprises one or more of: a primer-annealing polynucleotide (e.g., for annealing to a flow cell-bound oligonucleotide and / or to a free amplification primer), also referred to herein as a priming sequence or primer-binding domain; an index polynucleotide (e.g., a sample index sequence for tracking nucleic acids from different samples; also referred to as a sample ID); a barcode polynucleotide (e.g., a single molecule barcode (SMB) for tracking individual molecules of a sample nucleic acid to be amplified prior to sequencing; also referred to as a molecular barcode or unique molecular identifier (UMI)). In some embodiments, the primer-annealing component (or priming sequence or primer-binding domain) of the scaffold adapter, or a component thereof, comprises one or more universal sequences (e.g., sequences complementary to one or more amplification primers). In some embodiments, the index polynucleotide (e.g., a sample index; sample ID) is a component of the scaffold adapter, or a component thereof. In some embodiments, the index polynucleotide (e.g., a sample index; sample ID) is a component of a universal amplification primer sequence.

[0229] In some embodiments, the scaffold adapter, or components thereof, when used in combination with an amplification primer (e.g., a universal amplification primer), is designed to generate a library construct that includes one or more of a universal sequence, a molecular barcode, a sample ID sequence, a spacer sequence, and a sample nucleic acid sequence (e.g., an ssNA sequence). In some embodiments, the scaffold adapter, or components thereof, when used in combination with a universal amplification primer, is designed to generate a library construct that includes an ordered combination of one or more of a universal sequence, a molecular barcode, a sample ID sequence, a spacer sequence, and a sample nucleic acid sequence (e.g., an ssNA sequence). For example, a library construct may include a first universal sequence, followed by a second universal sequence, followed by a first molecular barcode, followed by a spacer sequence, followed by a template sequence (e.g., a sample nucleic acid sequence; an ssNA sequence), followed by a spacer sequence, followed by a second molecular barcode, followed by a third universal sequence, followed by a sample ID, followed by a fourth universal sequence. In some embodiments, scaffold adapters, or components thereof, when used in combination with amplification primers (e.g., universal amplification primers), are designed to generate library constructs for each strand of a template molecule (e.g., a sample nucleic acid molecule; ssNA molecule). In some embodiments, the scaffold adapter is a double-stranded adapter.

[0230] The identifier may be a suitable detectable label incorporated into or attached to a nucleic acid (e.g., a polynucleotide) that allows for detection and / or identification of the nucleic acid containing the identifier. In some embodiments, the identifier is incorporated into or attached to the nucleic acid during the sequencing method (e.g., by a polymerase). In some embodiments, the identifier is incorporated into or attached to the nucleic acid before the sequencing method (e.g., by an extension reaction, an amplification reaction, or a ligation reaction). Non-limiting examples of identifiers include nucleic acid tags, nucleic acid indexes, or barcodes, radiolabels (e.g., isotopes), metal labels, fluorescent labels, chemiluminescent labels, phosphorescent labels, fluorophore quenchers, dyes, proteins (e.g., enzymes, antibodies or portions thereof, linkers, members of binding pairs), etc., or combinations thereof. In some embodiments, the identifier (e.g., nucleic acid index or barcode) is a unique, known, and / or identifiable sequence of nucleotides or nucleotide analogs. In some embodiments, the identifier is six or more consecutive nucleotides. Numerous fluorophores with a variety of different excitation and emission spectra are available. Any suitable type and / or number of fluorophores can be used as identifiers.In some embodiments, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, thirty or more, or fifty or more different identifiers are utilized in the methods described herein (for example, nucleic acid detection and / or sequencing methods).In some embodiments, one or two types of identifiers (for example, fluorescent labels) are linked to each nucleic acid in library.Detection and / or quantification of the identifiers may be performed by any suitable method, device or machine, non-limiting examples of which include flow cytometry, quantitative polymerase chain reaction (qPCR), gel electrophoresis, a luminometer, a fluorometer, a spectrophotometer, suitable gene chip or microarray analysis, Western blot, mass spectrometry, chromatography, cytofluorometry, fluorescence microscopy, suitable fluorescence or digital imaging methods, confocal laser scanning microscopy, laser scanning cytometry, affinity chromatography, manual batch mode separation, electric field suspension, suitable nucleic acid sequencing methods and / or nucleic acid sequencing devices, and the like, and combinations thereof.

[0231] In some embodiments, the identifier, sequencing-specific index / barcode, and sequencer-specific flow cell-bound primer site are incorporated into the nucleic acid library by single primer extension (e.g., by a strand-displacing polymerase).

[0232] In some embodiments, the nucleic acid library, or a portion thereof, is amplified under amplification conditions (e.g., amplified by a PCR-based method). In some embodiments, the sequencing method includes amplification of the nucleic acid library. The nucleic acid library can be amplified before or after immobilization to a solid support (e.g., a solid support in a flow cell). Nucleic acid amplification includes a process of amplifying or increasing the number of nucleic acid templates and / or their complements present (e.g., in a nucleic acid library) by generating one or more copies of the templates and / or their complements. Amplification can be performed by a suitable method. The nucleic acid library can be amplified by thermocycling or isothermal amplification. In some embodiments, rolling circle amplification is used. In some embodiments, amplification is performed on a solid support (e.g., in a flow cell) to which the nucleic acid library, or a portion thereof, is immobilized. In certain sequencing methods, the nucleic acid library is added to a flow cell and immobilized by hybridization to anchors under suitable conditions. This type of nucleic acid amplification is often referred to as solid-phase amplification. In some embodiments of solid-phase amplification, all or a portion of the amplification products are synthesized by extension initiated from immobilized primers. Solid-phase amplification reactions are similar to standard liquid-phase amplification, except that at least one of the amplification oligonucleotides (e.g., primers) is immobilized to a solid support. In some embodiments, modified nucleic acids (e.g., nucleic acids modified by the addition of adapters) are amplified.

[0233] In some embodiments, solid-phase amplification involves a nucleic acid amplification reaction that includes only one type of oligonucleotide primer immobilized on a surface. In certain embodiments, solid-phase amplification involves multiple different immobilized oligonucleotide primer species. In some embodiments, solid-phase amplification can involve a nucleic acid amplification reaction that includes one type of oligonucleotide primer immobilized on a solid surface and a second, different oligonucleotide primer species in solution. Multiple different types of immobilized or solution-based primers can be used. Non-limiting examples of solid-phase nucleic acid amplification reactions include interface amplification, bridge amplification, emulsion PCR, WildFire amplification (e.g., U.S. Patent Application Publication No. 2013 / 0012399), etc., or combinations thereof.

[0234] Nucleic Acid Sequencing In some embodiments, nucleic acids (e.g., nucleic acid fragments, sample nucleic acids, cell-free nucleic acids, single-stranded nucleic acids, single-stranded DNA, single-stranded RNA) are sequenced. In some embodiments, ssNAs hybridized with scaffold adapters provided herein ("hybridization products") are sequenced by a sequencing process. In some embodiments, ssNAs hybridized with oligonucleotide components provided herein ("single-stranded ligation products") are sequenced by a sequencing process. In some embodiments, hybridization products and / or single-stranded ligation products are amplified by an amplification process, and the amplified products are sequenced by a sequencing process. In some embodiments, hybridization products and / or single-stranded ligation products are not amplified by an amplification process, and the hybridization products and / or single-stranded ligation products are sequenced by a sequencing process without prior amplification. In some embodiments, the sequencing process generates sequence reads (or sequencing reads). In some embodiments, the methods herein include determining the sequence of the single-stranded nucleic acid molecule based on the sequence reads.

[0235] For certain sequencing platforms (e.g., paired-end sequencing), generating sequence reads can include generating forward sequence reads and generating reverse sequence reads. For example, sequencing using certain paired-end sequencing platforms sequences each nucleic acid fragment from both directions, generally resulting in two reads for each nucleic acid fragment, including a first read in the forward direction (forward read) and a second read in the reverse complementary direction (reverse read). For certain platforms, the forward read is generated from a specific primer in the sequencing adapter (e.g., ILLUMINA adapter, P5 primer), and the reverse read is generated from a different primer in the sequencing adapter (e.g., ILLUMINA adapter, P7 primer).

[0236] Nucleic acids may be sequenced using a Sanger sequencing platform, a high-throughput or massively parallel sequencing (next-generation sequencing (NGS)) platform, such as a sequencing platform provided by Illumina® (e.g., HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), a sequencing platform provided by Oxford Nanopore™ Technologies (e.g., MinION sequencing system), a sequencing platform provided by Ion Torrent™ (e.g., Ion PGM™ and / or Ion Proton™ sequencing systems), a sequencing platform provided by Pacific Biosciences (e.g., PACBIO RS II sequencing system), a sequencing platform provided by Life Technologies™ (e.g., SOLiD sequencing system), a sequencing platform provided by Roche (e.g., 454 GS FLX+ and / or GS Sequencing can be performed using any suitable sequencing platform, including a nucleotide sequencer (e.g., a nucleotide sequencer ...In some embodiments, the sequencing process generates short sequencing reads or "short reads." In some embodiments, the nominal, representative, average, or absolute length of the short reads can be from about 10 consecutive nucleotides to about 250 or more consecutive nucleotides. In some embodiments, the nominal, representative, average, or absolute length of the short reads can be from about 50 consecutive nucleotides to about 150 or more consecutive nucleotides.

[0237] The length of a sequence read is often related to the particular sequencing technology employed. For example, high-throughput methods produce sequence reads that can range in size from tens to hundreds of base pairs (bp). For example, nanopore sequencing can produce sequence reads that can range in size from tens to hundreds to thousands of base pairs. In some embodiments, sequence reads have a mean, median, typical, or absolute length of about 15 bp to about 900 bp. In certain embodiments, sequence reads have a mean, median, typical, or absolute length of about 1000 bp or more. In some embodiments, sequence reads have a mean, median, typical, or absolute length of about 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 bp or more. In some embodiments, sequence reads have a mean, median, typical, or absolute length of about 100 bp to about 200 bp.

[0238] In some embodiments, the nominal, representative, average, or absolute length of a single-end read can be from about 10 contiguous nucleotides to about 250 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 200 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 150 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 125 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 100 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 75 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 60 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 50 or more contiguous nucleotides, from about 15 contiguous nucleotides to about 40 or more contiguous nucleotides, or can be about 15 contiguous nucleotides, or about 36 or more contiguous nucleotides. In certain embodiments, the nominal, typical, average, or absolute length of a single-end read is about 20 to about 30 bases in length, or about 24 to about 28 bases in length. In certain embodiments, the nominal, typical, average, or absolute length of a single-end read is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, or about 29 bases in length or more. In certain embodiments, the nominal, typical, average, or absolute length of a single-end read is about 20 to about 200 bases in length, about 100 to about 200 bases in length, or about 140 to about 160 bases in length. In certain embodiments, the nominal, typical, average, or absolute length of a single-end read is about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or about 200 or more bases in length.In certain embodiments, the nominal, representative, average, or absolute length of paired-end reads is from about 10 contiguous nucleotides to about 25 or more contiguous nucleotides (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides in length), sometimes from about 15 contiguous nucleotides to about 20 or more contiguous nucleotides, sometimes about 17 contiguous nucleotides, or sometimes about 18 contiguous nucleotides. In certain embodiments, the nominal, representative, average, or absolute length of paired-end reads ranges from about 25 contiguous nucleotides to about 400 or more contiguous nucleotides (e.g., about 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 or more nucleotides in length), from about 50 contiguous nucleotides to about 350 contiguous nucleotides. The length of the paired-end read may be up to about 150 or more contiguous nucleotides, from about 100 to about 325 contiguous nucleotides, from about 150 to about 325 contiguous nucleotides, from about 200 to about 325 contiguous nucleotides, from about 275 to about 310 contiguous nucleotides, from about 100 to about 200 contiguous nucleotides, from about 100 to about 175 contiguous nucleotides, from about 125 to about 175 contiguous nucleotides, or from about 140 to about 160 contiguous nucleotides. In certain embodiments, the nominal, representative, average, or absolute length of the paired-end read is about 150 contiguous nucleotides, and may be 150 contiguous nucleotides.

[0239] A read is generally a representation of a nucleotide sequence in a physical nucleic acid. For example, for a read containing an ATGC representation of a sequence, "A" represents an adenine nucleotide, "T" represents a thymine nucleotide, "G" represents a guanine nucleotide, and "C" represents a cytosine nucleotide in the physical nucleic acid. Sequence reads obtained from a sample from a subject may be reads from a mixture of minority and majority nucleic acids. For example, sequence reads obtained from the blood of a cancer patient may be reads from a mixture of cancer and non-cancer nucleic acids. In another example, sequence reads obtained from the blood of a pregnant female may be reads from a mixture of fetal and maternal nucleic acids. In another example, sequence reads obtained from the blood of a patient suffering from an infectious or infectious disease may be reads from a mixture of host and pathogen nucleic acids. In another example, sequence reads obtained from the blood of a transplant recipient may be reads from a mixture of host and graft nucleic acids. In another example, the sequence reads obtained from a sample may be reads from a mixture of nucleic acids from microorganisms that collectively comprise a subject's microbiome (e.g., gut microbiome, blood microbiome, oral microbiome, spinal fluid microbiome, fecal microbiome). In another example, the sequence reads obtained from a sample may be reads from a mixture of nucleic acids from microorganisms that collectively comprise a microbiome (e.g., gut microbiome, blood microbiome, oral microbiome, spinal fluid microbiome, fecal microbiome) and nucleic acids from the host subject. A mixture of relatively short reads can be converted by the processes described herein into a representation of genomic nucleic acids present in the subject and / or into a representation of genomic nucleic acids present in a tumor, fetus, pathogen, graft, or microbiome.

[0240] In certain embodiments, "obtaining" nucleic acid sequence reads of a sample from a subject and / or "obtaining" nucleic acid sequence reads of a biological specimen from one or more reference individuals can include directly sequencing the nucleic acid to obtain the sequence information. In some embodiments, "obtaining" can include receiving sequence information obtained directly from the nucleic acid by another party.

[0241] In some embodiments, some or all of the nucleic acids in the sample are enriched and / or amplified (e.g., non-specifically, for example, by PCR-based methods) before or during sequencing. In certain embodiments, specific nucleic acid species or subsets in the sample are enriched and / or amplified before or during sequencing. In some embodiments, species or subsets of a preselected pool of nucleic acids are randomly sequenced. In some embodiments, nucleic acids in the sample are not enriched and / or amplified before or during sequencing.

[0242] In some embodiments, a representative fraction of the genome is sequenced, which may be referred to as "coverage" or "coverage fold." For example, 1x coverage indicates that approximately 100% of the nucleotide sequence of the genome is represented by the reads. In some cases, coverage fold is referred to as (and is directly proportional to) "sequencing depth." In some embodiments, "coverage fold" is a relative term that refers to a previous sequencing experiment as a reference. For example, a second sequencing experiment may have half the coverage of the first sequencing experiment. In some embodiments, the genome is sequenced with redundancy, where a given region of the genome may be covered by two or more reads or overlapping reads (e.g., a "coverage fold" greater than 1, e.g., 2x coverage). In some embodiments, a genome (e.g., a whole genome) is sequenced at about 0.01x to about 100x coverage, about 0.1x to about 20x coverage, or about 0.1x to about 1x coverage (e.g., about 0.015x, 0.02x, 0.03x, 0.04x, 0.05x, 0.06x, 0.07x, 0.08x, 0.09x, 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x or more coverage). In some embodiments, a specific portion of the genome (e.g., a genome portion from a targeted method) is sequenced, and the coverage fold value generally refers to the fraction of the specific genome portion that is sequenced (i.e., the coverage fold value does not refer to the entire genome). In some cases, the specific genome portion is sequenced at a coverage of 1000x or more. For example, the specific genome portion can be sequenced at a coverage of 2000x, 5,000x, 10,000x, 20,000x, 30,000x, 40,000x, or 50,000x. In some embodiments, the sequencing is at a coverage of about 1,000x to about 100,000x.In some embodiments, the sequencing is at about 10,000-fold to about 70,000-fold coverage. In some embodiments, the sequencing is at about 20,000-fold to about 60,000-fold coverage. In some embodiments, the sequencing is at about 30,000-fold to about 50,000-fold coverage.

[0243] In some embodiments, one nucleic acid sample from one individual is sequenced.In certain embodiments, the nucleic acid from each of two or more samples, and the samples from one individual or from different individuals, is sequenced.In certain embodiments, the nucleic acid samples from two or more biological samples, each biological sample from one individual or two or more individuals, are pooled, and this pool is sequenced.In the latter embodiment, the nucleic acid sample from each biological sample is often identified by one or more unique identifiers.

[0244] In some embodiments, sequencing method utilizes identification, which allows multiplexing of sequence reaction in sequencing process.The more unique identifiers there are, for example, the more samples and / or chromosomes that can be multiplexed in sequencing process for detection.The sequencing process can be carried out using any suitable number of unique identifiers (for example, 4, 8, 12, 24, 48, 96 or more).

[0245] The sequencing process may use a solid phase, which may include a flow cell, a flow chamber capable of binding nucleic acids from a library and allowing reagents to flow and contact the bound nucleic acids. The flow cell may include flow cell lanes, and the use of identifiers can facilitate the analysis of several samples in each lane. Flow cells are often solid supports that can be configured to hold bound analytes and / or allow reagent solutions to pass over them in an ordered manner. Flow cells are often planar, optically transparent, generally on the millimeter or submillimeter scale, and often have channels or lanes where analyte / reagent interactions occur. In some embodiments, the number of samples analyzed in a given flow cell lane depends on the number of unique identifiers utilized during library preparation and / or probe design. For example, multiplexing using 12 identifiers allows for the simultaneous analysis of 96 samples (e.g., equivalent to the number of wells in a 96-well microwell plate) in an 8-lane flow cell. Similarly, multiplexing using, for example, 48 identifiers allows for simultaneous analysis of 384 samples in an 8-lane flow cell (e.g., equivalent to the number of wells in a 384-well microwell plate). Non-limiting examples of commercially available multiplex sequencing kits include Illumina's multiplexing sample preparation oligonucleotide kit and multiplexing sequencing primers and PhiX control kit (e.g., Illumina catalog numbers PE-400-1001 and PE-400-1002, respectively).

[0246] Any suitable nucleic acid sequencing method can be used, including, but not limited to, Maxim & Gilbert, chain termination, single-base synthesis, sequencing by ligation, mass spectrometry sequencing, microscopy-based techniques, or combinations thereof. In some embodiments, first-generation techniques, such as Sanger sequencing, including automated Sanger sequencing, including microfluidic Sanger sequencing, can be used in the methods provided herein. In some embodiments, sequencing techniques involving the use of nucleic acid imaging techniques (e.g., transmission electron microscopy (TEM) and atomic force microscopy (AFM)) can be used. In some embodiments, high-throughput sequencing methods are used. High-throughput sequencing methods generally involve clonally amplified DNA templates or single DNA molecules being sequenced in a massively parallel manner, sometimes within a flow cell. Next-generation (e.g., second and third generation) sequencing methods that can sequence DNA in a massively parallel manner can be used in the methods described herein, and these methods are collectively referred to herein as " massively parallel sequencing" (MPS).In some embodiments, MPS sequencing methods utilize a targeted approach, where specific chromosomes, genes or regions of interest are sequenced.In certain embodiments, a non-targeted approach is used, where most or all nucleic acids in a sample are randomly sequenced, amplified and / or captured.

[0247] In some embodiments, targeted enrichment, amplification, and / or sequencing approaches are used. Targeted approaches often isolate, select, and / or enrich a subset of nucleic acids in a sample for further processing by using sequence-specific oligonucleotides. In some embodiments, a library of sequence-specific oligonucleotides is utilized to target (e.g., hybridize with) one or more sets of nucleic acids in a sample. The sequence-specific oligonucleotides and / or primers are often selective for specific sequences (e.g., unique nucleic acid sequences) present in one or more chromosomes, genes, exons, introns, and / or regulatory regions of interest. Any suitable method or combination of methods can be used to enrich, amplify, and / or sequence one or more subsets of target nucleic acids. In some embodiments, target sequences are isolated and / or enriched by capture onto a solid phase (e.g., flow cell, bead) using one or more sequence-specific anchors. In some embodiments, target sequences are enriched and / or amplified by polymerase-based methods (e.g., PCR-based methods by any suitable polymerase-based extension) using sequence-specific primers and / or primer sets. The sequence-specific anchor can often be used as a sequence-specific primer.

[0248] MPS sequencing may also use single-base synthesis and certain imaging processes.The nucleic acid sequencing technology that can be used in the method described herein is single-base synthesis and reversible terminator-based sequencing (for example, Illumina's Genome Analyzer; Genome Analyzer II; HISEQ 2000; HISEQ 2500 (Illumina, San Diego CA)).This technology can be used to sequence millions of nucleic acid (e.g., DNA) fragments in parallel.One example of this type of sequencing technology uses a flow cell that contains an optically transparent slide, and oligonucleotide anchors (e.g., adapter primers) are attached to eight individual lanes on its surface.

[0249] Single-base synthesis methods are generally performed by iteratively adding nucleotides (via covalent addition) to a primer or an existing nucleic acid strand in a template-directed manner. Each iterative addition of nucleotides is detected, and the process is repeated multiple times until the sequence of the nucleic acid strand is obtained. The length of the resulting sequence depends, in part, on the number of addition and detection steps performed. In some embodiments of single-base synthesis methods, one, two, three, or more nucleotides of the same type (e.g., A, G, C, or T) are added and detected in a single round of nucleotide addition. Nucleotides can be added by any suitable method (e.g., enzymatically or chemically). For example, in some embodiments, a polymerase or ligase adds nucleotides to a primer or an existing nucleic acid strand in a template-directed manner. In some embodiments of single-base synthesis methods, different types of nucleotides, nucleotide analogs, and / or identifiers are used. In some embodiments, reversible terminators and / or removable (e.g., cleavable) identifiers are used. In some embodiments, fluorescently labeled nucleotides and / or nucleotide analogs are used. In certain embodiments, the single base synthesis method includes a cleavage (e.g., cleavage and removal of the identifier) ​​and / or a washing step. In some embodiments, the addition of one or more nucleotides is detected by a suitable method described herein or known in the art, non-limiting examples of which include any suitable imaging device, suitable camera, digital camera, CCD (charge-coupled device)-based imaging device (e.g., CCD camera), CMOS (complementary metal oxide silicon)-based imaging device (e.g., CMOS camera), photodiode (e.g., photomultiplier tube), electron microscopy, field effect transistor (e.g., DNA field effect transistor), ISFET ion sensor (e.g., CHEMFET sensor), etc., or a combination thereof.

[0250] Any suitable MPS method, system, or technology platform for performing the methods described herein can be used to obtain nucleic acid sequence reads. Non-limiting examples of MPS platforms include ILLUMINA / SOLEX / HISEQ (e.g., Illumina's Genome Analyzer; Genome Analyzer II; HISEQ 2000; HISEQ), SOLiD, Roche / 454, PACBIO and / or SMRT, Helicos True Single Molecule Sequencing, Ion Torrent and Ion semiconductor-based sequencing (e.g., as developed by Life Technologies), WildFire, 5500, 5500xl W, and / or 5500xl W Genetic Analyzer-based technologies (e.g., Life Technologies). Technologies and sold; US Patent Application Publication No. 2013 / 0012399; Polony sequencing, pyrosequencing, massively parallel signature sequencing (MPSS), RNA polymerase (RNAP) sequencing, LaserGen system and method, nanopore-based platform, chemical-sensitive field-effect transistor (CHEMFET) array, electron microscopy-based sequencing (such as those developed by ZS Genetics, Halcyon Molecular), nanoball sequencing, etc., or combinations thereof. Other sequencing methods that can be used to carry out the methods herein include digital PCR, hybridization sequencing, nanopore sequencing, chromosome-specific sequencing (such as DANSR (digital analysis of selected regions)) technology.

[0251] In some embodiments, nucleic acid is sequenced, and sequencing product (for example, a group of sequence reads) is processed before or simultaneously with the analysis of sequenced nucleic acid.For example, sequence reads can be processed according to one or more of the following, and combinations thereof: alignment, mapping, filtering, counting, normalization, weighting, profile generation, etc. Certain processing steps can be performed in any order, and certain processing steps can be repeated.

[0252] The disclosed methods can be used to reduce sequencing error rates. In some embodiments, double-stranded molecules can be labeled with barcodes before the initial denaturation step, so that sequences from originally paired nucleic acid molecules can be associated after subsequent denaturation, single-strand library preparation, and sequencing. In some embodiments, after the initial ligation of the scaffold adapter, a pool of index primers is used to perform index PCR, resulting in copies of both the original sample nucleic acid molecule and the nucleic acid from the initial PCR first-strand synthesis, both of which contain the same barcode or UMI (or its complement). These or other means of associating originally hybridized (and therefore complementary) strands allow the sequencing read information for both strands to be compared, which can be used to reduce sequencing error rates.

[0253] Lead Mapping Sequence reads can be mapped, and the number of reads that map to a specified nucleic acid region (e.g., a chromosome or portion thereof) is referred to as a count. Any suitable mapping method (e.g., a process, algorithm, program, software, module, etc., or a combination thereof) can be used. Certain aspects of the mapping process are described below.

[0254] Mapping of nucleotide sequence reads (i.e., sequence information from fragments whose physical genomic location is unknown) can be performed in several ways, and often involves aligning the obtained sequence reads with matching sequences in a reference...

Claims

1. 1. A method for generating a nucleic acid library, comprising: I. Combining (i) a nucleic acid composition comprising a single stranded nucleic acid (ssNA), (ii) a first oligonucleotide, (iii) a plurality of first scaffold polynucleotide species, (iv) a second oligonucleotide, and (v) a plurality of second scaffold polynucleotide species, (a) each polynucleotide in said plurality of first scaffold polynucleotide species comprises a ssNA hybridization region and a first oligonucleotide hybridization region; (b) each polynucleotide in said plurality of second scaffold polynucleotide species comprises a ssNA hybridization region and a second oligonucleotide hybridization region; (c) the nucleic acid composition, the first oligonucleotide, the plurality of first scaffold polynucleotide species, the second oligonucleotide, and the plurality of second scaffold polynucleotide species are A. A molecule of the first scaffold polynucleotide species is hybridized with (i) a first ssNA terminal region and (ii) a molecule of the first oligonucleotide, thereby forming a hybridization product in which the end of the molecule of the first oligonucleotide is adjacent to the end of the first ssNA terminal region; and B. A molecule of the second scaffold polynucleotide species is hybridized with (i) a second ssNA terminal region and (ii) a molecule of the second oligonucleotide, thereby forming a hybridization product in which the end of the molecule of the second oligonucleotide is adjacent to the end of the second ssNA terminal region. bound under conditions; (d) the ssNA is not bound to a single stranded nucleic acid binding protein (SSB) prior to or during the binding step, and neither the ssNA nor the nucleic acid containing the ssNA is modified after obtaining the biological sample and prior to the binding step; and II. Covalently linking adjacent ends of the first oligonucleotide and the first ssNA-terminal region, and covalently linking adjacent ends of the second oligonucleotide and the second ssNA-terminal region, thereby generating a covalently linked hybridization product. A method comprising:

2. 2. The method of claim 1, further comprising, prior to said binding step, contacting said first oligonucleotide and / or said plurality of first scaffold polynucleotide species with an agent having phosphatase activity under conditions in which said first oligonucleotide and / or said plurality of first scaffold polynucleotide species are dephosphorylated, thereby generating dephosphorylated first oligonucleotides and / or dephosphorylated first scaffold polynucleotide species.

3. 3. The method of claim 1 or 2, further comprising, prior to said binding step, contacting said second oligonucleotide and / or said plurality of second scaffold polynucleotide species with an agent having phosphatase activity under conditions in which said second oligonucleotide and / or said plurality of second scaffold polynucleotide species are dephosphorylated, thereby generating dephosphorylated second oligonucleotides and / or dephosphorylated second scaffold polynucleotide species.

4. 4. The method of claim 1, wherein prior to the binding step, each of the first scaffold polynucleotide species is hybridized to a first oligonucleotide to form a plurality of first scaffold duplex species, and each of the second scaffold polynucleotide species is hybridized to a second oligonucleotide to form a plurality of second scaffold duplex species.

5. 5. The method of claim 1, wherein the covalent linking step comprises contacting the hybridization product with an agent having ligase activity under conditions in which an end of the first ssNA terminal region is covalently linked to an end of the first oligonucleotide and an end of the second ssNA terminal region is covalently linked to an end of the second oligonucleotide.

6. 5. The method of claim 4, wherein some or all of the duplexes in said plurality of first scaffold duplex species comprise an adenylation modification at the 5' end of said first oligonucleotide, and said plurality of first scaffold duplex species are combined with said ssNA in the absence of ATP and covalently linked to said ssNA, thereby forming a covalently linked hybridization product intermediate.

7. 7. The method of claim 6, wherein the covalently linked hybridization product intermediate is combined with the plurality of second scaffold duplex species and ATP and covalently linked to the plurality of second scaffold duplex species and ATP, thereby forming a covalently linked hybridization product.

8. the ssNA hybridization region for each of the first scaffold polynucleotide species is distinct from the ssNA hybridization regions in other first scaffold polynucleotide species among the plurality of first scaffold polynucleotide species; and 8. The method of claim 1, wherein the ssNA hybridization region for each of the second scaffold polynucleotide species is different from the ssNA hybridization regions in other second scaffold polynucleotide species among the plurality of second scaffold polynucleotide species.

9. The method of claim 1 , wherein the ssNA hybridization region comprises a random sequence.

10. The method of any one of claims 1 to 9, wherein the ssNA hybridization region comprises one or more universal bases.

11. (1) the first oligonucleotide comprises one or more modified nucleotides, or some or all of the first scaffold polynucleotide species comprises one or more modified nucleotides, or the first oligonucleotide comprises one or more modified nucleotides and some or all of the first scaffold polynucleotide species comprises one or more modified nucleotides; and / or (2) the second oligonucleotide comprises one or more modified nucleotides, or some or all of the second scaffold polynucleotide species comprises one or more modified nucleotides, or the second oligonucleotide comprises one or more modified nucleotides and some or all of the second scaffold polynucleotide species comprises one or more modified nucleotides, wherein: the one or more modified nucleotides are capable of blocking the covalent linkage of the oligonucleotide to another oligonucleotide, polynucleotide or nucleic acid molecule, and the oligonucleotide comprises the one or more modified nucleotides at an end not adjacent to the ssNA; and / or 11. The method of any one of claims 1 to 10, wherein the one or more modified nucleotides are capable of blocking covalent linkage of the scaffold polynucleotide species to another oligonucleotide, polynucleotide or nucleic acid molecule, and the scaffold polynucleotide species comprises the one or more modified nucleotides at one or both ends of the polynucleotide.

12. 12. The method of claim 11 , wherein the one or more modified nucleotides comprises a modification that blocks ligation.

13. 12. The method of any one of claims 1 to 11, further comprising the step of denaturing the covalently linked hybridization product, thereby generating a single-stranded ligation product.

14. (1) combining the single-stranded ligation product with a third oligonucleotide under conditions in which the third oligonucleotide hybridizes to a dimer of the first oligonucleotide and the second oligonucleotide, thereby forming an oligonucleotide dimer hybridization product, wherein the oligonucleotide dimer hybridization product contains a cleavage site; (2) contacting the oligonucleotide dimer hybridization product with a cleavage agent; The method of claim 13 further comprising:

15. 15. The method of any one of claims 1 to 14, wherein the nucleic acid composition comprises single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), or ssDNA and ssRNA.

16. 16. The method of any one of claims 1 to 15, wherein one or both native ends of the ssNA are present when the ssNA is combined with the first oligonucleotide and the plurality of first scaffold polynucleotide species.

17. 17. The method of any one of claims 1 to 16, wherein the ssNA is from cell-free nucleic acid.

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