Methods for generating nucleic acid libraries and compositions and kits for practicing the methods

Abstract

A method for producing a nucleic acid library is provided. The method comprises combining a single-stranded nucleic acid (SSB-bound ssNA) bound by a single-stranded nucleic acid binding protein, an adapter oligonucleotide and a splint oligonucleotide to form a complex comprising the splint oligonucleotide and the terminal region of the SSB-bound ssNA and hybridizing with the adapter oligonucleotide. The end of the first adapter oligonucleotide is adjacent to the end of the first terminal region of the SSB-bound ssNA, and the method may further comprise covalently linking adjacent ends. Compositions and kits useful, for example, in practicing the methods disclosed herein are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 681,524, filed on June 6, 2018, which is incorporated herein by reference in its entirety. Background Art

[0003] Nucleic acid sequencing has become an increasingly important field of genetics research, for diagnosis and other applications. Usually, nucleic acid sequencing consists of determining nucleic acids, such as the nucleotide sequence of the fragment of RNA or DNA. Usually relatively short sequences will be analyzed, and the resulting sequence information can be used in various bioinformatics methods to compare the fragment with the reference sequence, or the fragment will be logically integrated together, so as to reliably determine the sequence of the much longer hereditary material derived from the fragment. Developed automated computer-based inspections of characteristic fragments, and recently used for genome mapping, genetic variation analysis between individuals, identification of genes and their functions, etc.

[0004] Several methods for high-throughput DNA sequencing rely on universal amplification reactions, whereby DNA samples are randomly fragmented and then processed so that the ends of different fragments all contain the same DNA sequence. Fragments with universal ends can be amplified with a single amplification primer in a single reaction. The addition of a universal priming sequence to the end of a target to be amplified by PCR can be achieved by a variety of methods. For example, a universal primer with a universal sequence at its 5' end and a degenerate sequence at its 3' end can be used to randomly amplify fragments from a complex target sequence or a complex mixture of target sequences. The degenerate 3' portion of the primer anneals at a random position on the DNA and can be extended to generate a copy of the target with a universal sequence at its 5' end.

[0005] Alternatively, an adapter containing a universal priming sequence can be connected to the end of the target sequence. One or more adapters can be used for the ligation reaction with the target sequence. A disadvantage associated with the current method for preparing a nucleic acid sequencing library by ligation of one or more adapter sequences for universal amplification is the time and expense required for such methods. Summary of the invention

[0006] A method for producing a nucleic acid library is provided. The method comprises combining a single-stranded nucleic acid (SSB-bound ssNA) bound by a single-stranded nucleic acid binding protein, an adapter oligonucleotide and a splint oligonucleotide to form a complex comprising the splint oligonucleotide and the terminal region of the SSB-bound ssNA and hybridizing with the adapter oligonucleotide. The end of the first adapter oligonucleotide is adjacent to the end of the first terminal region of the SSB-bound ssNA, and the method may further comprise covalently linking adjacent ends. Compositions and kits useful, for example, in practicing the methods disclosed herein are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 . Schematic illustration of a method for generating a nucleic acid library according to one embodiment of the present disclosure.

[0008] Figure 2 . Comparison of exemplary methods of the present disclosure with other methods for degraded DNA (upper panel) and modern DNA (lower panel).

[0009] Figure 3 Hair DNA length distribution. The left panel shows the observed lengths of template molecules generated from modern hair DNA by the Santa Cruz method (SRL3). As expected for DNA in the hair shaft, the length of the complete molecules is generally short. A similar length distribution is shown in the right panel (SRL4).

[0010] Figure 4 .Estimated library complexity (number of unique molecules) in SRL3 and SRL4.

[0011] Figure 5 . Comparison of library complexity. Sequencing libraries were prepared using the disclosed exemplary methods SS2.0, BEST, and forked adapter ligation. In three experiments, library complexity (the number of unique molecules in the library) was estimated based on millions of reads using Preseq (left) or by qPCR (right). The disclosed exemplary methods converted 2 to 3 times more extracted DNA into sequencing libraries than the next best option SS2.0. DETAILED DESCRIPTION

[0012] A method for producing a nucleic acid library is provided. The method comprises combining a single-stranded nucleic acid (SSB-bound ssNA) bound by a single-stranded nucleic acid binding protein, an adapter oligonucleotide and a splint oligonucleotide to form a complex comprising the splint oligonucleotide and the terminal region of the SSB-bound ssNA and hybridizing with the adapter oligonucleotide. The end of the first adapter oligonucleotide is adjacent to the end of the first terminal region of the SSB-bound ssNA, and the method may further comprise covalently linking adjacent ends. Compositions and kits useful, for example, in practicing the methods disclosed herein are also provided.

[0013] Before describing the methods, compositions and kits of the present disclosure in more detail, it should be understood that the methods, compositions and kits are not limited to the specific embodiments described, as such methods, compositions and kits may of course vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the methods, compositions and kits will be limited only by the appended claims.

[0014] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range (to one-tenth of the lower limit, unless the context clearly indicates otherwise) and any other stated or intervening value within the stated range are encompassed within the methods, compositions and kits. The upper and lower limits of these smaller ranges may independently be encompassed within the smaller ranges and are also encompassed within the methods, compositions and kits, subject to any explicitly excluded limits within the stated ranges. Where a stated range includes one or both of the limits, ranges excluding either or both of the included limits are also encompassed within the methods, compositions and kits.

[0015] Certain ranges are provided herein where the term "about" appears before a numerical value. The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or near the number that follows the term. In determining whether a number is close to or near a specifically recited number, the close or near unrecited number may be a number that, in the context in which it appears, provides a substantial equivalent to the specifically recited number.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the methods, compositions, and kits belong. Although any methods, compositions, and kits similar or equivalent to the methods, compositions, and kits described herein can also be used to practice or test the methods, compositions, and kits, representative illustrative methods, compositions, and kits are now described.

[0017] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference and are incorporated herein to disclose and describe the materials and / or methods in connection with the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods, compositions and kits are not entitled to antedate such publications, as the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0018] It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as antecedent basis for the use of such exclusive terminology (such as "solely", "only", etc.) in conjunction with the recitation of claim elements or the use of a "negative" limitation.

[0019] It should be understood that certain features of the methods, compositions, and kits described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the methods, compositions, and kits described in the context of separate embodiments for the sake of clarity may also be provided separately or in any suitable sub-combination. All combinations of embodiments are expressly contemplated in the present disclosure and disclosed herein, as if each combination were individually and expressly disclosed, provided that such combination includes operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing these variables are also expressly contemplated in the present methods, compositions, and kits and disclosed herein, as if each such sub-combination were individually and expressly disclosed herein.

[0020] When reading this disclosure, it will be apparent to those skilled in the art that each individual embodiment described and illustrated herein has separate components and features that can be easily separated or combined with the features of any other several embodiments without departing from the spirit or scope of the invention. Any described method can be carried out in the order of events described or in any other order that is logically possible.

[0021] method

[0022] As outlined above, the present disclosure provides a method for producing a nucleic acid library. The method comprises contacting a single-stranded nucleic acid (ssNA) with a single-stranded nucleic acid binding protein (SSB) to produce SSB-bound ssNA. The method further comprises combining the SSB-bound ssNA, a first adapter oligonucleotide, and a first splint oligonucleotide, wherein the first splint oligonucleotide comprises an SSB-bound ssNA hybridization region and a first adapter oligonucleotide hybridization region. The combination causes the formation of a complex comprising: the first splint oligonucleotide hybridizes with the terminal region of the SSB-bound ssNA through the SSB-bound ssNA hybridization region; and the first splint oligonucleotide hybridizes with the first adapter oligonucleotide through the first adapter oligonucleotide hybridization region, so that the end of the first adapter oligonucleotide is adjacent to the end of the first terminal region of the SSB-bound ssNA. In some embodiments, the combination further comprises combining the SSB-bound ssNA, a second adapter oligonucleotide and a second splint oligonucleotide, wherein the second splint oligonucleotide comprises a SSB-bound ssNA hybridization region and a second adapter oligonucleotide hybridization region, wherein the formed complex further comprises: the position at which the second splint oligonucleotide hybridizes to the terminal region of the SSB-bound ssNA through the SSB-bound ssNA hybridization region is opposite to the position of the terminal region hybridized to the first splint oligonucleotide; and the second splint oligonucleotide hybridizes to the second adapter oligonucleotide through the second adapter oligonucleotide hybridization region, so that the position at which the end of the second adapter oligonucleotide is adjacent to the end of the SSB-bound ssNA is opposite to the position of the end adjacent to the first adapter oligonucleotide.

[0023] An example embodiment using a second adaptor oligonucleotide and a second splint oligonucleotide is schematically shown in Figure 1 In this example, ssNA is produced from dsNA by denaturing dsNA (e.g., ssDNA is produced from dsDNA). Figure 1 The top of the diagram shows dsNA 102. After denaturation of dsNA 102, the resulting ssNA is contacted with a single-stranded nucleic acid binding protein (SSB) to produce SSB-bound ssNA. Figure 11 is a SSB-bound ssNA 104 derived from a strand of dsNA 102. In this example, ssNA 104 is combined with a first adapter oligonucleotide 106 hybridized to a first splint oligonucleotide 108 and a second adapter oligonucleotide 110 hybridized to a second splint oligonucleotide 112. The hybridization of the first splint oligonucleotide 108 to the first adapter oligonucleotide 106 is through a first adapter oligonucleotide hybridization region 114 of the first splint oligonucleotide 108. The hybridization of the second splint oligonucleotide 112 to the second adapter oligonucleotide 110 is through a second adapter oligonucleotide hybridization region 116 of the second splint oligonucleotide 112. The hybridization of the first splint oligonucleotide 108 and the second splint oligonucleotide 112 to the SSB-bound NA 104, the first adapter region 106 and the second adapter region 110 forms a complex 122. The hybridization of the first splint oligonucleotide 108 to the 5' terminal region of the SSB-bound ssNA 104 is through the first SSB-bound ssNA hybridization region 118 of the first splint oligonucleotide 108. The hybridization of the second splint oligonucleotide 112 to the 3' terminal region of the SSB-bound ssNA 104 is through the second SSB-bound ssNA hybridization region 120 of the second splint oligonucleotide 112. The splint oligonucleotides are designed so that when the SSB-bound ssNA hybridization region of the splint oligonucleotide hybridizes with its corresponding terminal region of the SSB-bound ssNA, the end of the adapter oligonucleotide is adjacent to the end of the SSB-bound ssNA. The positions of the adjacent ends are indicated by asterisks. These adjacent ends can be covalently linked (e.g., by enzymatic linkage) to produce an adapted ssNA (e.g., Figure 1 The linked ssNA 124 shown in FIG. 1 ) can then be used in a downstream application of interest (e.g., PCR amplification, next generation sequencing, and / or the like) facilitated by one or more sequences in the linker portion of the linked ssNA. Figure 1 As shown in , after the adjacent ends are covalently linked, an optional cleanup step can be performed to separate the linked ssNA from one or more reagents or components of the complex formed, such as enzymes used for covalent linking, splint oligonucleotides, SSBs and / or the like. Suitable methods for such a cleanup step include, but are not limited to, solid phase reversible immobilization (SPRI - e.g., using magnetic beads) and nucleic acid column purification. Figure 1 In the example shown in , there is a blocking modification at each end of the splint oligonucleotide (black rectangle), and there is a further blocking modification at the end of each adapter oligonucleotide that is not adjacent to the ssNA (black rectangle) that binds to the SSB. The blocking modifications prevent the oligonucleotide and the ssNA from connecting to those ends.

[0024] As outlined above, the disadvantages associated with current methods for preparing nucleic acid sequencing libraries by ligation of one or more adapter sequences include the time and expense required for such methods. The methods disclosed herein constitute an improvement over prior art methods for preparing single-stranded libraries, such as those described by Gansauge et al. (2017) Nucleic Acids Research 45(10): e79 (referred to as "ssDNA 2.0"), in which it was surprisingly found that the methods were more efficient, required less time and reduced costs. Aspects of the methods disclosed herein will now be described in further detail.

[0025] The method comprises contacting a single-stranded nucleic acid (ssNA) with a single-stranded nucleic acid binding protein (SSB) to produce SSB-bound ssNA. "Single-stranded nucleic acid" or "ssNA" means a collection of polynucleotides that are single-stranded (i.e., not hybridized between or within molecules) for 70% or more of their length. In some embodiments, ssNA is single-stranded for 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, ssNA is single-stranded over the entire length of the polynucleotide.

[0026] ssNA can be any nucleic acid sample of interest (or can be prepared from any nucleic acid sample), including but not limited to nucleic acid samples isolated from single cells, multiple cells (e.g., cultured cells), tissues, organs, or organisms (e.g., bacteria, yeast, etc.). Exemplary sample types include but are not limited to blood, serum, saliva, sputum, urine, feces, vomitus, mucus, hair, nails (e.g., fingernails, toenails), swabs (e.g., cheek swabs, throat swabs, vaginal swabs), biopsies (e.g., puncture biopsies, fine needle biopsies, fine needle aspiration biopsies), cell cultures, environmental samples (e.g., water, soil, air, surfaces, contact DNA), and metagenomic samples. In certain aspects, nucleic acid samples are isolated from single cells, collections of cells, tissues, organs, and / or the like of an animal. In some cases, nucleic acid samples include cell-free nucleic acids (e.g., cell-free DNA (cfDNA)), such as but not limited to fetal cell-free nucleic acids (e.g., cell-free fetal DNA (cffDNA)) or circulating tumor nucleic acids (e.g., circulating tumor DNA (ctDNA)). In some embodiments, the animal is a mammal (e.g., a mammal from the genus Homo, a rodent (e.g., a mouse or rat), a dog, a cat, a horse, a cow, or any other mammal of interest). In other aspects, the nucleic acid sample is isolated / obtained from a source other than a mammal, such as bacteria, yeast, an insect (e.g., a fruit fly), an amphibian (e.g., a frog (e.g., Xenopus)), a virus, a plant, or any other non-mammalian nucleic acid sample source.

[0027] In some embodiments, ssNA is from a degraded nucleic acid sample. As used herein, a "degraded nucleic acid sample" is a sample of DNA that has been fragmented by an enzymatic, physical, chemical or other process. Examples of degraded nucleic acid samples are DNA fragments recovered from bone debris, hair, cell-free DNA from plasma, or environmental DNA recovered from soil or water. In some aspects, when ssNA is from a degraded nucleic acid sample, ssNA is from an ancient nucleic acid sample. "Ancient nucleic acid sample" means nucleic acid fragments recovered from biological debris. Non-limiting examples of ancient nucleic acid samples of interest are nucleic acid samples obtained (e.g., isolated) from extinct organisms or animals, such as extinct mammals. In some aspects, the extinct mammals are from the genus Homo. In some embodiments, ssNA is from a forensic nucleic acid sample. As used herein, a "forensic nucleic acid sample" is a nucleic acid sample related to a criminal investigation (e.g., obtained during its process).

[0028] In certain aspects, ssNA is from a tumor nucleic acid sample (that is, a nucleic acid sample isolated from a tumor). As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is generally characterized as unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include 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 cancer 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, etc.

[0029] In some embodiments, ssNA is from a cell-free nucleic acid sample, e.g., cell-free DNA, cell-free RNA, or both. In some aspects, the cell-free nucleic acid is obtained from a body fluid sample selected from the group consisting of: whole blood, plasma, serum, amniotic fluid, saliva, urine, pleural effusion, bronchial lavage, bronchial aspirate, breast milk, colostrum, tears, semen, peritoneal fluid, pleural effusion, and feces. In some embodiments, the cell-free nucleic acid is cell-free fetal DNA. In some aspects, the cell-free nucleic acid is circulating tumor DNA. In some embodiments, the cell-free nucleic acid includes infectious agent DNA. In some embodiments, the cell-free nucleic acid includes DNA from a transplant.

[0030] In certain aspects, ssNA is single-stranded deoxyribonucleic acid (ssDNA). The ssDNA of interest includes, but is not limited to, ssDNA derived from double-stranded DNA (dsDNA). For example, ssDNA can be derived from double-stranded DNA that is denatured (e.g., thermally and / or chemically denatured) to produce ssDNA. In some embodiments, the method comprises producing ssDNA by denaturing dsDNA prior to contacting the ssDNA with SSB.

[0031] When ssNA is ssDNA derived from a dsDNA sample, the method may further include, after forming the complex, rehybridizing the ssDNA (which now contains one or more adapters (e.g., sequencing adapters) at one or both ends) to produce dsDNA. If desired, the produced dsDNA may be sequenced. In some embodiments, rehybridization is performed under sufficiently stringent hybridization conditions to produce dsDNA similar to the original dsDNA derived from the ssDNA. Sufficiently stringent hybridization conditions may include a selected hybridization temperature, a selected salt concentration, and / or any other convenient hybridization parameters selected to produce dsDNA similar to the original dsDNA derived from the ssDNA. One or both ends of at least one subset of such produced dsDNA will be similar to / copy the ends (e.g., overhangs) of the original dsDNA. Determining the end / overhang content (e.g., by sequencing) using the methods disclosed herein can provide a variety of useful information about nucleic acid samples derived from ssDNA. For example, knowing the overhang content is valuable in analyzing, for example, cell-free DNA (cfDNA) from plasma or another suitable source. It has been shown that cfDNA is derived from a variety of sources, including blood cells, fetal cells of pregnant women, tumor cells in individuals with cancer, transplanted organ tissue from organ transplant recipients, etc. The overhang content provided by embodiments of the disclosed methods can be used to classify sequencing reads, for example, by original source for diagnostic purposes.

[0032] In addition, the end / overhang content can be used to analyze mixed DNA from forensic samples. For example, DNA from semen, blood, or other sources of interest may have end characteristics that are diagnostic of the source, and the DNA sequence can be partitioned based on this information.

[0033] In addition, determining the overhang content in ancient DNA samples (e.g., samples from extinct organisms, plants, or animals) provides information for characterizing such samples and the organisms, plants, animals, etc. from which the samples were derived. For example, ancient DNA samples (e.g., DNA samples from extinct mammals) often contain contaminating DNA (e.g., contaminating bacterial DNA, etc.). In such cases, when these types of overhangs are associated with a particular source of DNA, the DNA sequences of interest can be partitioned from the contaminating DNA sequences based on the type of overhangs detected.

[0034] In certain embodiments, the methods of the present disclosure can be used to determine the rate and location of base damage in a DNA extract (e.g., an ancient DNA extract) based on the length and type of overhangs.

[0035] Thus, in some embodiments, methods are provided that include: combining an ssDNA derived from an SSB-bound dsDNA with an adapter oligonucleotide and a splint oligonucleotide to form a complex comprising an ssDNA derived from an SSB-bound dsDNA hybridized with an adapter oligonucleotide and a splint oligonucleotide as described herein; and after the formation of the complex, hybridizing the ssDNA to produce a dsDNA that is similar to the original dsDNA from which the ssDNA was derived (i.e., a "connected" dsDNA that now includes one or more adapters (e.g., sequencing adapters) at one or both ends). Such methods may further include sequencing the connected dsDNA. In certain aspects, sequencing is determining the end / overhang content of the ssDNA derived from the dsDNA. In any embodiments of the methods of the present disclosure involving sequencing, the method may include sequencing a subsample of the connected ssNA to reduce complexity during sequencing.

[0036] In some embodiments, ssNA is a single-stranded ribonucleic acid (ssRNA). The RNA of interest includes but is not limited to messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), trans-acting small interfering RNA (ta-siRNA), natural 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-interacting RNA (piRNA), siRNA prepared by endoribonuclease (esiRNA), small sequential RNA (stRNA), signal recognition RNA, telomeric RNA, ribozyme, or any combination of these RNA types or subtypes. In some embodiments, when ssNA is ssRNA, ssRNA is mRNA.

[0037] Methods, reagents, and kits for isolating, purifying, and / or concentrating DNA and RNA from a source of interest are known in the art and are commercially available. For example, kits for isolating DNA from a source of interest include the ELISA kit from Qiagen, Inc. (Germantown, Md.). and Nucleic acid isolation / purification kit; Life Technologies, Inc. (Carlsbad, CA) Nucleic acid isolation / purification kit; Clontech Laboratories, Inc. (Mountain View, CA) and Nucleic acid isolation / purification kits. In certain aspects, nucleic acids are isolated from fixed biological samples, such as formalin-fixed paraffin-embedded (FFPE) tissues. Commercially available kits can be used to isolate genomic DNA from FFPE tissues: for example, the ELISA kit from Qiagen (Germantown, MD). DNA / RNA FFPE Kit, Life Technologies Corporation (Carlsbad, CA) for FFPE Complete Nucleic Acid Isolation Kit and Cloning Technologies Laboratories, Inc. (Mountain View, CA) FFPE kit.

[0038] When the organism, plant, animal, etc. from which a nucleic acid sample was obtained (e.g., isolated) becomes extinct, suitable strategies for recovering such nucleic acids are known and include, for example, Green et al., (2010) Science 328(5979):710-722; Poinar et al., (2006) Science 311(5759):392-394; Stiller et al., 2006, Proc. Natl. Acad. Sci. 103(37):13578–13584; Miller et al., (2008) Nature 456(7220):387-90; Rasmussen et al., (2010) Nature 463(7282):757-762; and those described elsewhere.

[0039] As outlined above, the present method comprises contacting ssNA with a single-stranded nucleic acid binding protein (SSB) to produce ssNA bound by SSB. SSB binds to ssNA in a cooperative manner and does not bind well to double-stranded nucleic acid (dsNA). After binding to ssDNA, SSB destabilizes the helical duplex. SSBs that can be used in practicing the subject method include prokaryotic SSBs (e.g., bacterial or archaeal SSBs) and eukaryotic SSBs. Non-limiting examples of SSBs that can be used in practicing the subject method include Escherichia coli (E. coli) SSB, Escherichia coli RecA, extremely thermostable single-stranded DNA binding protein (ET SSB), Thermus thermophilus (Tth) RecA, T4 gene 32 protein, replication protein A (RPA-eukaryotic SSB), etc. ET SSB, Tth RecA, E. coli RecA, T4 gene 32 protein, and buffers and detailed protocols for preparing SSB-bound ssNA using such SSB can be obtained, for example, from New England Biolabs, Inc. (Ipswich, MA). The inventors have determined that, given equal molar concentration inputs, a larger input of SSB is beneficial for ssNAs with higher average fragment lengths. The "Experimental" section below provides detailed guidance on an exemplary method for contacting ssNA with SSB to produce ssNA bound to SSB.

[0040] As outlined above, the subject method comprises combining SSB-bound ssNA, adapter oligonucleotides and splint oligonucleotides to form a complex, wherein the splint oligonucleotide comprises an SSB-bound ssNA hybridization region and an adapter oligonucleotide hybridization region. As used herein, "oligonucleotide" is a single-stranded polymer of 5 to 500 nucleotides, for example 5 to 100 nucleotides. Oligonucleotides can be synthetic or can be prepared enzymatically, and in some embodiments, are 5 to 50 nucleotides in length. Oligonucleotides can contain ribonucleotide monomers (i.e., can be oligoribonucleotides or "RNA oligonucleotides"), deoxyribonucleotide monomers (i.e., can be oligodeoxyribonucleotides or "DNA oligonucleotides") or a combination thereof. For example, an oligonucleotide can be 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, or 150 to 200 or up to 500 nucleotides in length.

[0041] The "adapter oligonucleotide" of the present disclosure is an oligonucleotide comprising an adaptor or a portion thereof. "Adaptor" means a nucleotide sequence that can be used for one or more downstream applications (e.g., PCR amplification of the adapted ssNA or its derivatives, sequencing of the adapted ssNA or its derivatives, and / or the like). In certain aspects, the adaptor or a portion thereof present in an adaptor oligonucleotide is a sequencing adaptor. "Sequencing adaptor" means one or more nucleic acid domains comprising at least a portion of a nucleotide sequence (or its complement) utilized by a sequencing platform of interest as follows: Sequencing platforms provided (e.g., HiSeq TM 、MiSeq TM and / or Genome Analyzer TM Sequencing system); Oxford Nanopore TM Technology (e.g., MinION TM Sequencing System), Ion Torrent TM (For example, Ion PGM TM and / or Ion Proton TM Sequencing systems); Pacific Biosciences (e.g., Sequel or PacBio RS II Sequencing Systems); Life Technologies TM (For example, SOLiD TM Sequencing systems); Roche (e.g., 454GS FLX+ and / or GS Junior sequencing systems); or any other sequencing platform of interest.

[0042] In certain aspects, a sequencing adaptor is or comprises a nucleic acid domain selected from the group consisting of: a surface-attached sequencing platform oligonucleotide (e.g., A domain (e.g., a "capture site" or "capture sequence") that specifically binds to a P5 or P7 oligonucleotide attached to the surface of a flow cell in a sequencing system; a sequencing primer binding domain (e.g., A domain to which a read 1 or read 2 primer of a platform can bind); a unique identifier (e.g., a barcode or other domain that uniquely identifies the 3' region of an oligonucleotide probe, a probe complement oligonucleotide, or both, and / or uniquely identifies the sample source of the rRNA being sequenced to achieve sample multiplexing by labeling each molecule from a given sample with a specific barcode or "tag"); a barcode sequencing primer binding domain (a domain that binds to a primer used to sequence the barcode); a molecular identification domain (e.g., a molecular index tag, such as a randomized tag of 4, 6, or other number of nucleotides) that is used to uniquely label a molecule of interest, for example, to determine expression levels based on the number of instances in which a unique tag is sequenced; the complement of any such domain; or any combination thereof. In certain aspects, the barcode domain (e.g., sample index tag) and the molecular identification domain (e.g., molecular index tag) can be contained in the same nucleic acid.

[0043] When the adapter oligonucleotide comprises one or a portion of a sequencing adapter, one or more additional sequencing adapters and / or the remainder of the sequencing adapter can be added using a variety of methods. For example, the additional and / or remainder of the sequencing adapter can be added by ligation, reverse transcription, PCR amplification, and / or the like. In the case of PCR, an amplification primer pair comprising a first amplification primer and a second amplification primer can be used, wherein the first amplification primer comprises a 3' hybridization region (e.g., for hybridizing with the adapter region of the adapter oligonucleotide) and a 5' region comprising the additional and / or remainder of the sequencing adapter, and the second amplification primer comprises a 3' hybridization region (e.g., for hybridizing with the adapter region of the second adapter oligonucleotide added to the opposite end of the ssNA molecule) and optionally a 5' region comprising the additional and / or remainder of the sequencing adapter.

[0044] The "splint oligonucleotide" of the present disclosure is an oligonucleotide comprising an SSB-bound ssNA hybridization region and an adapter oligonucleotide hybridization region. The SSB-bound ssNA hybridization region is a region (nucleotide sequence) that hybridizes with the terminal region of the SSB-bound ssNA. The adapter oligonucleotide hybridization region is a region (nucleotide sequence) that hybridizes with all or a portion of the adapter oligonucleotide. The splint oligonucleotide is designed to hybridize with the SSB-bound ssNA and the adapter oligonucleotide at the same time, so that when the complex is formed, the end of the adapter oligonucleotide is adjacent to the end of the terminal region of the SSB-bound ssNA.

[0045] The SSB-bound ssNA hybridization region of the splint oligonucleotide can have any suitable length and sequence. In certain embodiments, the length of the SSB-bound ssNA hybridization region is 10 nucleotides or less. In certain aspects, the SSB-bound 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 certain embodiments, the SSB-bound ssNA hybridization region comprises (e.g., consisting of) a random nucleotide sequence, so that when a plurality of heterologous splint oligonucleotides with various random SSB-bound ssNA hybridization regions are employed, the collection can serve as a splint oligonucleotide for a heterologous population of ssNA bound by SSB, regardless of the sequence of the terminal region of the SSB-bound ssNA.

[0046] Thus, in certain aspects, the method comprises forming a complex by combining an SSB-bound ssNA, an adaptor oligonucleotide, and a plurality of heterologous splint oligonucleotides having various random SSB-bound ssNA hybridization regions capable of acting as splint oligonucleotides for a heterogeneous population of SSB-bound ssNAs having terminal regions of undefined sequence.

[0047] In some embodiments, the SSB-bound ssNA hybridization region comprises a known sequence designed to hybridize with an SSB-bound ssNA terminal region of a known sequence. In some aspects, two or more heterologous splint oligonucleotides having different SSB-bound ssNA hybridization regions of a known sequence are employed, and the known sequence is designed to hybridize with the corresponding SSB-bound ssNA terminal region of a known sequence. Embodiments in which the SSB-bound ssNA hybridization region has a known sequence are used, for example, when it is desired to produce a nucleic acid library from only a subset of SSB-bound ssNAs with terminal regions of a known sequence. Therefore, in some aspects, the method comprises forming a complex by combining SSB-bound ssNAs, adapter oligonucleotides, and one or more heterologous splint oligonucleotides, wherein the one or more heterologous splint oligonucleotides have one or more different SSB-bound ssNA hybridization regions of a known sequence that can serve as a splint oligonucleotide for one or more SSB-bound ssNAs with one or more terminal regions of a known sequence.

[0048] In certain aspects, the SSB-bound ssNA hybridization region comprises one or more universal bases. As used herein, 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 SSB-bound ssNA hybridization region include, but are not limited to, 2'-deoxyinosine (dI, dInosine) and 5-nitroindole.

[0049] The manner in which the SSB-bound ssNA, adapter oligonucleotide and splint oligonucleotide are combined can vary. In some embodiments, the combination comprises combining a complex comprising a splint oligonucleotide hybridized with an adapter oligonucleotide via an adapter oligonucleotide hybridization region with an SSB-bound ssNA. In other aspects, the combination comprises combining a complex comprising a splint oligonucleotide hybridized with an SSB-bound ssNA via an SSB-bound ssNA hybridization region with an adapter oligonucleotide. In still other aspects, the combination comprises combining an SSB-bound ssNA, an adapter oligonucleotide and a splint oligonucleotide, wherein prior to the combination, none of the components in the three components are pre-compounded (that is, hybridized with) another component.

[0050] The combination is performed under hybridization conditions such that the complex comprises the splint oligonucleotide hybridizing to the terminal region of the SSB-bound ssNA through the SSB-bound ssNA hybridization region, and the splint oligonucleotide hybridizing to the adapter oligonucleotide through the adapter oligonucleotide hybridization region. Whether specific hybridization occurs is determined by factors such as the degree of complementarity between the relevant (i.e., hybridization) regions of the splint oligonucleotide, the terminal region of the SSB-bound ssNA and the adapter oligonucleotide and their lengths, salt concentration, and the temperature at which hybridization occurs, which can be determined by the melting temperature (T) of the relevant region. M ). The melting temperature is the temperature at which half of the associated regions remain hybridized and half of the associated regions dissociate into single strands. The Tm of a duplex can be determined experimentally or predicted using the following formula: Tm = 81.5 + 16.6 (log10 [Na + ])+0.41(fraction G+C)–(60 / N), where N is the chain length and [Na + ] is less than 1 M. See Sambrook and Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor NY, Chapter 10). Other more advanced models that rely on various parameters can also be used to predict the Tm of the region of interest, depending on various hybridization conditions. Methods for achieving specific nucleic acid hybridization can be found, 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).

[0051] As used herein, the term "complementary" or "complementarity" refers to a nucleotide sequence that is paired with a region of a target nucleic acid by a non-covalent bond linking base, for example, a nucleotide sequence of a hybridization region of an SSB-bound ssNA that hybridizes to the terminal region of an SSB-bound ssNA, and a nucleotide sequence of a hybridization region of an adapter oligonucleotide that hybridizes to a probe complement oligonucleotide. In the classical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), just as guanine (G) forms a base pair with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Therefore, A is complementary to T, and G is complementary to C. In RNA, A is complementary to U, and vice versa. In general, "complementary" or "complementarity" refers to a nucleotide sequence that is at least partially complementary. These terms can also encompass fully complementary duplexes, such that each nucleotide in one chain is complementary to each nucleotide in the other chain in the corresponding position. In some cases, the nucleotide sequence may be partially complementary to the target, where not all nucleotides are complementary to every nucleotide in the target nucleic acid at all corresponding positions. For example, the hybridization region of the SSB-bound ssNA may be perfectly (i.e., 100%) complementary to the terminal region of the SSB-bound ssNA, or the hybridization region of the SSB-bound ssNA may share a certain degree of less than perfect complementarity (e.g., 70%, 75%, 85%, 90%, 95%, 99%). The percent identity of two nucleotide sequences can be determined by aligning the sequences for the purpose of optimal comparison (e.g., gaps can be introduced in the sequence of the first sequence for optimal alignment). The nucleotides at corresponding positions are then 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 = # of identical positions / total # of positions × 100). When a position in one sequence is occupied by the same nucleotide as the corresponding position in another sequence, the molecules at the position are identical. A non-limiting example of this mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., NBLAST) can be used. In one aspect, the parameters for sequence comparison can be set to score=100, word length=12, or can be varied (e.g., word length=5 or word length=20).

[0052] The complex is formed so that the end of the adapter oligonucleotide is adjacent to the end of the terminal region of the ssNA that the SSB binds. "Adjacent to..." means that the terminal nucleotide at the end of the adapter oligonucleotide and the terminal nucleotide end of the terminal region of the ssNA that the SSB binds are close enough to each other so that the terminal nucleotide can be covalently linked, for example, by chemical connection, enzyme connection, etc. In certain embodiments, the end is adjacent to each other by hybridization of the terminal nucleotide end of the terminal region of the ssNA that the terminal nucleotide at the end of the adapter oligonucleotide and the terminal nucleotide end of the terminal region of the ssNA that the SSB binds with the adjacent nucleotides of the splint oligonucleotide. The splint oligonucleotide can be designed to ensure that the end of the adapter oligonucleotide is adjacent to the end of the terminal region of the ssNA that the SSB binds. Non-limiting examples of such splint oligonucleotides are provided in the experimental section of this article.

[0053] Any of the methods described herein may further comprise covalently linking the adjacent ends of the adapter oligonucleotide and the SSB-bound ssNA. The covalent linking may comprise linking the adjacent ends. Linking the adjacent ends may be performed using any suitable method. In some aspects, the linking is by chemical linking. In other aspects, the linking is by enzyme linking. Suitable reagents (e.g., ligases and corresponding buffers, etc.) and kits for performing enzyme ligation reactions are known and available, for example, the instant sticky-end ligase master mix (Instant Sticky-end Ligase Master Mix) available from New England Biolabs, Inc. (Ipswich, Massachusetts). The ligases that can be used include, for example, T4 DNA ligase (e.g., at low or high concentrations), T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Electro Etc. Conditions suitable for performing the ligation reaction will vary depending on the type of ligase used. Information about such conditions is readily available. If necessary, a phosphate group can be added to the 5' end of the adapter oligonucleotide or to the ssNA bound by the SSB using, for example, a suitable kinase, such as T4 polynucleotide kinase (PNK). Such kinases and guidance for using such kinases to phosphorylate the 5' end can be obtained, for example, from New England Biolabs, Inc. (Ipswich, MA).

[0054] In certain embodiments, the splint oligonucleotide, the adapter oligonucleotide or both include blocking modifications. For example, one or both ends of the splint oligonucleotide may include blocking modifications, and / or the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB may include blocking modifications. "Blocking modification" means that the end cannot be connected to the end of any other oligonucleotide component using a method for covalently connecting the adjacent ends of the ssNA bound to the adapter oligonucleotide and SSB. In some aspects, blocking modifications are connection blocking modifications. Examples of blocking modifications that may be included at one or both ends of the splint oligonucleotide and / or the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB include the absence of 3'OH at the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB, and the 3'OH at the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB is inaccessible. Non-limiting examples of blocking modifications where the end has an inaccessible 3'OH include: amino modifiers, spacers, dideoxy bases, reverse dideoxy bases and 3' phosphates, etc.

[0055] In certain aspects, the splint oligonucleotide, the adapter oligonucleotide, or both comprise one or more non-natural nucleotides (which may also be referred to as nucleotide analogs). Non-limiting examples of non-natural nucleotides that may be included in the splint oligonucleotide, the adapter oligonucleotide, or both are 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.

[0056] Covalently linking the adjacent ends of the adapter oligonucleotide and the SSB-bound ssNA produces an attached ssNA, where "attached" means that the ssNA now contains one or more adapter sequences or subregions thereof. The attached ssNA can be purified prior to use as input for a downstream application of interest. For example, the complex can be denatured (e.g., heat denatured) to separate the attached ssNA from the splint oligonucleotide, and the attached ssNA can be purified from the SSB and / or any other components or combinations thereof present during the contacting and / or combining steps (e.g., by solid phase reversible immobilization (SPRI), column purification, and / or the like).

[0057] In some embodiments, the one or more adapter sequences or subregions thereof are one or more sequencing adapters or subregions thereof, and the method further comprises sequencing at least a portion of the connected ssNA or any derivative thereof (e.g., an amplicon produced by PCR amplification using the connected ssNA as a template). Sequencing can be performed on any suitable sequencing platform, including a high-throughput sequencing (HTS) (or "next generation sequencing (NGS)") platform, etc. HTS / NGS sequencing platforms of interest include but are not limited to Sequencing platforms provided (e.g., HiSeq TM 、MiSeq TM and / or GenomeAnalyzer TM Sequencing system); Oxford Nanopore TM Technology (e.g., MinION TM GridIONx5 TM 、PromethION TM or SmidgION TM Nanopore-based sequencing systems), Ion Torrent TM (For example, Ion PGM TM and / or Ion Proton TM Sequencing systems); Pacific Biosciences (e.g., Sequel or PacBio RS II Sequencing Systems); Life Technologies TM (e.g., SOLiD sequencing system); Roche (e.g., 454GS FLX+ and / or GS Junior sequencing system); or any other sequencing platform of interest. Detailed protocols for direct sequencing (e.g., by nanopore-based sequencing) or preparing compatible nucleic acid molecules for sequencing on a particular platform (e.g., by amplification, e.g., solid phase amplification, etc.), sequencing compatible molecules, and analyzing sequencing data can be obtained from the manufacturer of the sequencing platform of interest.

[0058] As outlined above, the methods of the present disclosure constitute an improvement over prior art methods for single-stranded library preparation, such as the method described by Gansauge et al. (2017) Nucleic Acids Research 45(10):e79 (referred to as "ssDNA 2.0"), in which the present method was surprisingly found to be more efficient, require less time and reduce costs. In some embodiments, when the method comprises covalently linking the adjacent ends of the adapter oligonucleotide and the SSB-bound ssNA, the total duration of the combination and covalent linking steps is 4 hours or less, 3 hours or less, 2 hours or less or 1 hour or less. In certain embodiments, when the method comprises covalently linking the adjacent ends of the adapter oligonucleotide and the SSB-bound ssNA, the total duration of the contacting, combination and covalent linking steps is 4 hours or less, 3 hours or less, 2 hours or less or 1 hour or less. In some embodiments, the efficiency of the method is such that complexes are formed from 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more of the ssNA contacted with the SSB during the contacting step.

[0059] Composition

[0060] As outlined above, the present disclosure also provides compositions. The compositions can be used in a variety of applications, including, for example, any method in the method of practicing the present disclosure, including performing one or more steps in any of the steps described in the "method" chapters of the present disclosure. Thus, the compositions can include oligonucleotides (including multiple heterologous oligonucleotides / sets thereof), ssNA, SSB, other reagents, etc., described above in the method chapters of the present disclosure in any combination.

[0061] In certain aspects, compositions comprising SSB-bound ssNA, a first adaptor oligonucleotide, and a first splint oligonucleotide are provided, wherein the first splint oligonucleotide comprises a SSB-bound ssNA hybridization region and a first adaptor oligonucleotide hybridization region. Such compositions may further comprise a second adaptor oligonucleotide and a second splint oligonucleotide, wherein the second splint oligonucleotide comprises a SSB-bound ssNA hybridization region and a second adaptor oligonucleotide hybridization region.

[0062] In certain aspects, compositions comprising a complex are provided, the complex comprising a splint oligonucleotide hybridized to an adapter oligonucleotide via an adapter oligonucleotide hybridization region, present as a hybridized complex (e.g., in the absence of SSB-bound ssNA). In other aspects, compositions comprising a complex are provided, the complex comprising a splint oligonucleotide hybridized to an SSB-bound ssNA via an SSB-bound ssNA hybridization region, present as a hybridized complex.

[0063] ssNA can be ssDNA. When ssNA is ssDNA, ssDNA can be derived from dsDNA. In some embodiments, ssNA is ssRNA. In some embodiments, ssNA is from a degraded nucleic acid sample. In some aspects, when ssNA is from a degraded nucleic acid sample, ssNA is from an ancient nucleic acid sample, such as a nucleic acid sample obtained (e.g., isolated) from an extinct organism or animal, such as an extinct mammal. In some aspects, the extinct mammal is from the genus Homo. In some embodiments, ssNA is from a forensic nucleic acid sample.

[0064] The compositions of the present disclosure may further comprise a reagent for covalently linking the end of the adaptor oligonucleotide to the end of the SSB-bound ssNA. In some embodiments, the reagent is a chemical ligation reagent or an enzymatic ligation reagent, for example, a ligase.

[0065] The compositions of the present disclosure may include one or more components present in a container. Suitable containers include, but are not limited to, tubes, vials, and plates (eg, 96-well or other well plates).

[0066] In some aspects, the composition comprises one or more components in a liquid medium. The liquid medium can be an aqueous liquid medium, such as water, a buffer solution, etc. One or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), 2-(N-morpholino) ethanesulfonic acid sodium salt (MES), 3-(N-morpholino) propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS) etc.), solubilizing agents, detergents (e.g., nonionic detergents, such as tween (Tween)-20 etc.), nuclease inhibitors, glycerol, chelating agents etc. can be present in such compositions.

[0067] Reagent test kit

[0068] As outlined above, the present disclosure provides a kit. The kit can be used in a variety of applications, including, for example, practicing any of the methods of the present disclosure, including performing one or more of any of the steps described above in the methods section of the present disclosure. Thus, the kit can include oligonucleotides (including multiple heterologous oligonucleotides / sets thereof), ssNA, SSB, other reagents, etc., described above in the methods section of the present disclosure in any combination.

[0069] In some embodiments, the kit of the present disclosure comprises: a single-stranded nucleic acid binding protein (SSB, e.g., a single-stranded DNA binding protein, a single-stranded RNA binding protein, or both); a first adapter oligonucleotide; a first splint oligonucleotide, the first splint oligonucleotide comprising an SSB-bound ssNA hybridization region and a first adapter oligonucleotide hybridization region; and instructions for using the SSB, the first adapter oligonucleotide, and the first splint oligonucleotide to generate a nucleic acid library. In certain aspects, the kit further comprises a second adapter oligonucleotide and a second splint oligonucleotide, the second splint oligonucleotide comprising an SSB-bound ssNA hybridization region and a second adapter oligonucleotide hybridization region, wherein the instructions are for using the SSB, the first adapter oligonucleotide, the first splint oligonucleotide, the second adapter oligonucleotide, and the second splint oligonucleotide to generate a nucleic acid library.

[0070] The kits of the present disclosure may further comprise a reagent for covalently linking the end of the adaptor oligonucleotide to the end of the SSB-bound ssNA. In some embodiments, the reagent is a chemical ligation reagent or an enzymatic ligation reagent, for example, a ligase.

[0071] In some embodiments, the splint oligonucleotide, the adapter oligonucleotide or both include blocking modifications. For example, one or both ends of the splint oligonucleotide may include blocking modifications, and / or the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB may include blocking modifications. In some aspects, the blocking modification is a connection blocking modification. The example of blocking modifications that may be included at one or both ends of the splint oligonucleotide and / or the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB includes the absence of 3'OH at the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB, and the 3'OH at the end of the adapter oligonucleotide that is not adjacent to the ssNA bound to SSB is inaccessible. Non-limiting examples of blocking modifications in which the end has an inaccessible 3'OH include: amino modifiers, spacers, dideoxy bases, reverse dideoxy bases and 3' phosphates, etc.

[0072] In some embodiments, one or more splint oligonucleotides provided in the kits of the present disclosure comprise an SSB-bound ssNA hybridization region comprising (e.g., consisting of) a random nucleotide sequence, such that when the kit comprises a plurality of heterogeneous splint oligonucleotides having various random SSB-bound ssNA hybridization regions, the collection is capable of acting as a splint oligonucleotide for a heterogeneous population of SSB-bound ssNAs, regardless of the sequence of the terminal region of the SSB-bound ssNA of interest.

[0073] In certain aspects, the splint oligonucleotides provided in the kits of the present disclosure comprise a SSB-bound ssNA hybridization region comprising one or more universal bases. Universal bases that can be incorporated into the SSB-bound ssNA hybridization region include, but are not limited to, 2'-deoxyinosine (dI, dInosine) and 5-nitroindole.

[0074] In some embodiments, the length of the SSB-bound ssNA hybridization region of the splint oligonucleotide provided in the kit of the present disclosure is 10 nucleotides or less. In certain aspects, the SSB-bound 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.

[0075] The components of the subject kit can be present in separate containers, or multiple components can be present in a single container. Suitable containers include separate tubes (e.g., vials), one or more wells of plates (e.g., 96-well plates, 384-well plates, etc.), etc.

[0076] The instructions for using SSB, one or more adapter oligonucleotides and one or more splint oligonucleotides to produce a nucleic acid library can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Therefore, the instructions can be present in the test kit in the form of a package insert, in the label of the container of the test kit or its component (i.e., associated with the package or subpackage), etc. In other embodiments, the instructions exist in the form of an electronic storage data file present on a suitable computer-readable storage medium (e.g., a portable flash drive, DVD, CD-ROM, floppy disk, etc.). In yet other embodiments, there is no actual instruction in the test kit, but means for obtaining the instructions from a remote source, such as through the Internet, are provided. An example of this embodiment is a test kit comprising a website, in which the instructions can be viewed and / or can be downloaded from the website. Like the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0077] The following examples are offered by way of illustration and not by way of limitation.

[0078] experiment

[0079] Example 1

[0080] Disclosed herein is a method for fast, efficient and targeted connection of adapters to single-stranded DNA in one reaction. In the following examples, sequencing adapter oligonucleotides containing Illumina P7 or P5 adapter sequences are hybridized with splint oligonucleotides containing a series of 3'Ns for the P7 splint and a series of 5'Ns for the P5 splint. The splint creates an opportunity for the ligase that can only efficiently perform double-stranded connection to connect the adapter to the target single-stranded DNA. All oligonucleotide DNA ends that do not need to participate in the connection are blocked by oligonucleotide modifications (e.g., amino modifications) that prevent connection.

[0081] Adding single-strand binding protein (SSB) to the assay increases the efficiency of the reaction. Use the concentration and length of the target DNA to calculate the appropriate amount of SSB for optimal ligation efficiency. SSB prevents re-annealing of single-stranded DNA while preventing secondary structures.

[0082] This method can be favorably compared to the single-stranded library preparation referred to as SS2.0 described in Gansauge et al. (2017) Nucleic Acids Research 45(10):e79. Compared to SS2.0, this method requires significantly less time and exhibits significantly improved efficiency in converting DNA to suitable adapter-ligated DNA molecules that can be sequenced. In addition, compared to SS2.0, this method reduces reagent costs.

[0083] Adaptor and splint oligonucleotides are engineered to perform ligation blocking modifications on all ends that should not participate in the connection of appropriate adaptors. This includes blocking the 5' end of the P5 adaptor, the 3' end of the P7 adaptor, and all ends of the splint. These ligation blocking modifications can be any other suitable modification of amino modifiers, carbon spacers, dideoxy bases, or 5' phosphates that prevent ligase from approaching the 3' hydroxyl at the 3' end or the 5' end. Oligonucleotides can be synthesized, for example, by integrated DNA technology (IDT). Example oligonucleotides are shown in Table 1 below.

[0084] Table 1 - Example Oligonucleotides

[0085]

[0086] / 5AmMC12 / =5'amino modifier C12

[0087] / 5AmMC6 / =5'amino modifier C6

[0088] / 3AmMO / = 3' amino modifier

[0089] / 5Phos / = 5' phosphate

[0090] / 3ddC / = 3'dideoxycytidine

[0091] In the protocol used in this example, template DNA was combined with SSB and heat denatured. After denaturation, the reactions were placed on ice or in a PCR cooler at 4°C. After cooling, adapters were added to each reaction. Then, the reaction master mix was added, followed by mixing. Incubation at 37°C allowed ligation to begin immediately, with most ligation occurring before 45 minutes. The reactions can be cleaned up using established methods, and downstream applications such as amplification and sequencing remain unchanged.

[0092] To prepare the adapter, 1X final concentration of T4 RNA ligase buffer (Catalog No. B0216L) was combined with the P5 adapter to a final concentration of 10 uM and with the P5 splint oligonucleotide to a final concentration of 20 μM. Similar separate mixtures were prepared using the P7 adapter oligonucleotide and the P7 splint oligonucleotide. The adapter was hybridized by heating to 95°C for 10 seconds and then ramping down to 10°C at a rate of 0.1°C / s.

[0093] An example scenario is provided below.

[0094] 1. Sample input (36uL)

[0095] a. Combine sheared DNA and ET SSB (Catalog No. M2401S) to a volume of 36uL

[0096] i.1uL ET SSB facilitates connection without inhibition for most sample types tested

[0097] ii. Fill the remaining volume with buffer EBT (10 mM Tris-HCl, pH 8.0 and 0.05% Tween 20)

[0098] 2. Deform the sample

[0099] a. Incubate samples in a thermal cycler with lid preheated to 95°C for 3 minutes.

[0100] b. Immediately place the denatured samples on ice or in a PCR cooler for 30 seconds.

[0101] 3. Add 2uL of pooled adapter mixture (equal volumes of P5 and P7 adapters)

[0102] a. The adaptor input will depend on the molarity of the input. A molar ratio of 6 to 10:1 adaptor to template is preferred.

[0103] 4. Add reaction master mix and mix thoroughly by pipetting

[0104] a. 8uL of T4 DNA Ligase Buffer (Catalog #M0202M)

[0105] b. 32uL of 50% PEG 8000 (Catalog No. B0216L)

[0106] c. 1uL of T4 Polynucleotide Kinase – 10,000U / mL (Catalog No. M0201L)

[0107] d. 1uL of T4 DNA Ligase – 2,000,000U / mL (Catalog No. M0202M)

[0108] 5. Incubate at 37°C for up to 60 minutes

[0109] a. Most connections occur in the first 15 minutes, but do not plateau until around 45 minutes.

[0110] 6. Clearing reaction

[0111] a. Column cleanup (eg, for degraded DNA) or SPRI (eg, for modern samples).

[0112] Comparison and optimization of assays

[0113] After cleaning, qPCR was performed on dilutions of the pre-amplified library to determine the ligation efficiency. A lower CT value indicates a higher ligation efficiency relative to another sample with a higher CT value in the same run. The difference in its value is approximately equal to a two-fold difference in library efficiency. An aliquot of the pre-amplified library was also amplified using an index PCR reaction. Post-indexing. The library was cleaned up with SPRI and visualized on the Agilent TapeStation 2200 system to estimate the proportion of adapter artifacts in each library.

[0114] Single-chain binding protein

[0115] It has been observed that single-strand binding proteins (SSBs) such as the ET SSB protein provided by NEB enhance the ligation efficiency of single-strand ligations. Given equal molar inputs, samples with higher average fragment lengths require greater SSB inputs to achieve peak ligation. The molar concentration of DNA in the reaction also affects the amount of SSB required. In large excesses of ET, SSB has the potential to inhibit ligation.

[0116] Comparison of plans

[0117] The efficiency of this protocol was compared with that of the NEB Ultra 2 kit (dsDNA), SS2.0 (Gansauge et al. (2017) Nucleic Acids Res. 45(10):e79), and the blunt-end single tube (BEST) method described in Caroe et al. (2017) Methods in Ecology and Evolution 9(2):410-419 and Mak et al. (2017) GigaScience 6:1-13. Comparative results were obtained using modern human DNA with an average fragment length of approximately 350 bp and ancient bison samples with a severely degraded average fragment length of approximately 35-40 bp.

[0118] The NEB Ultra 2 kit is identified as an efficient library preparation method for modern samples, while SS2.0 is identified as an efficient library preparation method for degraded samples. The BEST protocol involves blunt-end repair using T4 DNA polymerase and T4 PNK to blunt-end the DNA (without tailing) and phosphorylate the 5' end. Next, the blunt-end dsDNA adapter is ligated to the blunt end using T4 DNA ligase, followed by a fill-in reaction using Bst 2.0 Warmstart polymerase and cleanup using SPRI beads or columns.

[0119] The comparison results are Figure 2 . From left to right on the upper figure: the method described in this example (asterisk), ss2.0, and BEST. From left to right on the lower figure: the method described in this example (asterisk), NEB Ultra 2 kit, ss2.0, and BEST. The method described in this example showed higher ligation efficiency for ancient samples compared to ss2.0. For modern samples, the method described in this example was between 0.3 and 0.5 qPCR cycles, after the NEB Ultra 2 kit.

[0120] Example 2 – Hair DNA

[0121] DNA was collected from hair using standard proteinase K treatment at high temperature. 6 nanograms of DNA was used as template for library preparation. The protocol described above was followed. Two sequencing libraries were generated from the adaptor-ligated products. One used 1 μL (SRL3) of the 50 μL total ligated product. The other used 2.5 μL of this product (SRL4). Both libraries were sequenced on the Illumina MiSeq sequencing platform to assess library characteristics and complexity (number of unique library molecules).

[0122] After 2×75 paired-end sequencing, the forward and reverse read pairs that overlap each other are combined using the SeqPrep program. This occurs when the original DNA template is short enough that the forward and reverse reads cover some of the same sequence (referred to herein as "merged reads"). After merging, the merged and unmerged read maps are mapped to the reference human genome sequence. Figure 3 Shown in is the observed raw template length distribution for the pooled and mapped, pooled and unmapped, and pooled and unmapped reads for both the SRL3 and SRL4 libraries. Note that for the unpooled and unmapped, it is not possible to infer the length of the template DNA.

[0123] The number of unique library molecules in the two libraries was estimated using the Preseq software program. This program counts the number of observed duplicate molecules to model the complexity of the nucleic acid library based on a large sample of observed reads as generated here. This program shows an estimate of the fraction of observed reads predicted to be unique at various depths of library sequencing. Figure 4 As shown in , both libraries were predicted to have a complexity of over 250,000,000 unique molecules. SRL4, made from 2.5 μL of adapter-ligated template, had more unique molecules than SRL3.

[0124] Example 3 – Ancient DNA from bison bones

[0125] The efficiency of converting template DNA molecules to sequencing libraries was compared using DNA extracted from ancient bison bones. Libraries were generated from the same amount of DNA from the same extract using four different protocols, including the protocol described above.

[0126] The complexity of the libraries was measured using two methods: qPCR and direct sequencing of adapter-ligated products. qPCR was performed in triplicate. Figure 5 The two methods shown in demonstrate that the methods described herein are more efficient in converting DNA into sequencing libraries.

[0127] Therefore, the foregoing only shows the principles of the present disclosure.It should be understood that those skilled in the art can design various arrangements, although not explicitly described or shown herein, but the various arrangements embody the principles of the present invention and are included in the spirit and scope of the present invention.In addition, all examples and conditional language described here are mainly intended to help readers understand the principles and concepts of the present invention contributed by the inventors to promote the development of the art, and will be regarded as without being limited to these particularly described examples and conditions.In addition, all statements of the principles, aspects and embodiments of the present invention and its specific examples are described here to cover both their structural and functional equivalents.In addition, it is expected that such equivalents include both currently known equivalents and equivalents developed one day, that is, any element of the development that performs the same function regardless of structure.Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Sequence Listing <110> The Regents of the University of California California R. Green J. Karp <120> Methods for generating nucleic acid libraries and compositions and kits for practicing the methods <130> UCSC-376WO <150> 62 / 681,524 <151> 2018-06-06 <160> 4 <170> PatentIn Version 3.5 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 1 acactctttc cctacacgac gctcttccga tct 33 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (1)..(7) <223> n is a, c, g or t <400> 2 nnnnnnnaga tcggaagagc gtcgtgtagg gaaagagtgt 40 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 3 agatcggaag agcacacgtc tgaactccag tcac 34 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (35)..(41) <223> n is a, c, g or t <400> 4 gtgactggag ttcagacgtg tgctcttccg atctnnnnnn n 41

Claims

1. A method for producing a nucleic acid library, the method comprising: contacting a single-stranded nucleic acid (ssNA) with a single-stranded nucleic acid binding protein (SSB) to produce SSB-bound ssNA; Combine the following: The SSB binds to the ssNA, a first adaptor oligonucleotide, and a first splint oligonucleotide comprising a SSB-binding ssNA hybridization region and a first adaptor oligonucleotide hybridization region, to form a complex comprising: the first splint oligonucleotide hybridizes to the terminal region of the SSB-bound ssNA through the SSB-bound ssNA hybridization region; and the first splint oligonucleotide hybridizes to the first adapter oligonucleotide through the first adapter oligonucleotide hybridization region, such that an end of the first adapter oligonucleotide is adjacent to an end of the terminal region of the SSB-bound ssNA; and Combine the following: The SSB binds to the ssNA, a second adaptor oligonucleotide; and a second splint oligonucleotide comprising a SSB-binding ssNA hybridizing region and a second adaptor oligonucleotide hybridizing region, wherein the formed complex further comprises: the position at which the second splint oligonucleotide hybridizes to the terminal region of the SSB-bound ssNA through the SSB-bound ssNA hybridization region is opposite to the position of the terminal region hybridized to the first splint oligonucleotide; and the second splint oligonucleotide hybridizes to the second adapter oligonucleotide through the second adapter oligonucleotide hybridization region, such that the position at which the end of the second adapter oligonucleotide is adjacent to the end of the SSB-bound ssNA is opposite to the position of the end adjacent to the first adapter oligonucleotide, wherein the ends of the first adapter oligonucleotide and the second adapter oligonucleotide that are not adjacent to the SSB-bound ssNA include blocking modifications, and The terminus comprising a blocking modification means that the terminus is incapable of ligating to the terminus of any other oligonucleotide component using methods used to covalently ligate adjacent termini of an adaptor oligonucleotide and an SSB-bound ssNA; The method further comprises covalently linking adjacent ends of the first adaptor oligonucleotide and the SSB-bound ssNA, and The second adaptor oligonucleotide and adjacent ends of the SSB-bound ssNA are covalently linked.

2. The method of claim 1, wherein the total duration of the contacting, combining and covalently linking steps is less than or equal to 3 hours.

3. The method of claim 1, wherein the complex is formed by greater than or equal to 80% of the ssNA being in contact with the SSB.

4. The method of claim 1, wherein the ssNA is from a degraded nucleic acid sample. The method of claim 4 , wherein the ssNA is derived from an ancient nucleic acid sample.

6. The method of claim 1, wherein the ssNA is from a forensic nucleic acid sample.

7. The method of claim 1, wherein the ssNA is single-stranded DNA (ssDNA).

8. The method of claim 7, wherein the ssDNA is derived from double-stranded DNA (dsDNA).

9. The method of claim 8, further comprising generating the ssDNA by denaturing the dsDNA prior to contacting the ssDNA with SSB.

10. The method of claim 8, further comprising re-hybridizing the ssDNA to produce dsDNA after forming the complex.

11. The method of claim 10, further comprising sequencing the generated dsDNA.

12. The method of claim 11, wherein a subsample of the generated dsDNA is sequenced.

13. The method of claim 1, wherein the ssNA is single-stranded RNA (ssRNA). The method of claim 1 , wherein the covalent linkage comprises chemical linkage or enzymatic linkage.

15. The method of claim 1, wherein the blocking modification is a ligation blocking modification.

16. The method of claim 1, wherein the blocking modification is selected from the group consisting of: the absence of a 3' OH at the end of the adaptor oligonucleotide that is not adjacent to the ssNA that binds to the SSB; and the presence of a modified 3' OH at the end of the adaptor oligonucleotide that is not adjacent to the ssNA that binds to the SSB.

17. The method of claim 16, wherein the blocking modification is the presence of a modified 3' OH at the end of the adaptor oligonucleotide that is not adjacent to the ssNA to which the SSB binds, and wherein the blocking modification is selected from the group consisting of: an amino modifier, a spacer, a dideoxy base, an inverted dideoxy base, and a 3' phosphate.

18. The method of claim 1, wherein the SSB-bound ssNA hybridization region comprises a random sequence.

19. The method of claim 1, wherein the SSB-bound ssNA hybridization region comprises a universal base.

20. The method of claim 1, wherein the SSB-bound ssNA hybridization region is less than or equal to 10 nucleotides in length.

21. The method of claim 1, wherein the adaptor oligonucleotide comprises an adaptor or its complement for PCR amplification.

22. The method of claim 1, wherein the adapter oligonucleotide comprises a partial or complete sequencing adapter or its complement.

23. The method of any one of claims 1 to 22, further comprising sequencing at least a portion of the ssNA.

24. A composition comprising: single-stranded nucleic acid bound by single-stranded nucleic acid binding protein (SSB-bound ssNA); a first adaptor oligonucleotide; a first splint oligonucleotide comprising a SSB-binding ssNA hybridization region and a first adaptor oligonucleotide hybridization region; a second adaptor oligonucleotide; and a second splint oligonucleotide comprising a SSB-bound ssNA hybridization region and a second adaptor oligonucleotide hybridization region, wherein the ends of the first adaptor oligonucleotide and the second adaptor oligonucleotide not adjacent to the SSB-bound ssNA comprise a blocking modification, and The terminus comprising a blocking modification means that the terminus is incapable of ligating to the terminus of any other oligonucleotide component using methods used to covalently ligate adjacent termini of an adaptor oligonucleotide and an SSB-bound ssNA; The composition further comprises: Reagents for covalently linking the ends of adaptor oligonucleotides to the ends of the SSB-bound ssNA.

25. The composition of claim 24, wherein the ssNA is from a degraded nucleic acid sample.

26. The composition of claim 25, wherein the ssNA is from an ancient nucleic acid sample.

27. The composition of claim 24, wherein the ssNA is from a forensic nucleic acid sample.

28. The composition of claim 24, wherein the ssNA is single-stranded DNA (ssDNA).

29. The composition of claim 28, wherein the ssDNA is derived from double-stranded DNA (dsDNA).

30. The composition of claim 24, wherein the ssNA is single-stranded RNA (ssRNA).

31. The composition of claim 24, wherein the agent is a ligase.

32. A kit comprising: Single-stranded nucleic acid binding protein (SSB); a first adaptor oligonucleotide; a first splint oligonucleotide comprising a SSB-binding ssNA hybridization region and a first adaptor oligonucleotide hybridization region, instructions for generating a nucleic acid library using the SSB, the first adaptor oligonucleotide, and the first splint oligonucleotide; a second adaptor oligonucleotide; as well as a second splint oligonucleotide comprising a SSB-bound ssNA hybridization region and a second adaptor oligonucleotide hybridization region, wherein the ends of the first adaptor oligonucleotide and the second adaptor oligonucleotide not adjacent to the SSB-bound ssNA comprise a blocking modification, and The termini include a blocking modification meaning that the termini are incapable of ligating to the termini of any other oligonucleotide component using methods used to covalently ligate an adapter oligonucleotide and an adjacent termini of an SSB-bound ssNA, and wherein the instructions are for generating a nucleic acid library using the SSB, the first adaptor oligonucleotide, the first splint oligonucleotide, the second adaptor oligonucleotide, and the second splint oligonucleotide; The kit further comprises: Reagents used to ligate the end of an adaptor oligonucleotide to the end of a single-stranded nucleic acid bound by a single-stranded nucleic acid binding protein (SSB-bound ssNA).

33. The kit of claim 32, wherein the SSB is a single-stranded DNA binding protein.

34. The kit of claim 32, wherein the SSB is a single-stranded RNA binding protein.

35. The kit of claim 32, wherein the reagent is a ligase.

36. The kit of claim 32, wherein the blocking modification is a ligation blocking modification.

37. A kit according to claim 32, wherein the blocking modification is selected from the group consisting of: the absence of a 3' OH at the end of the adaptor oligonucleotide that is not adjacent to the ssNA that binds to the SSB; and the presence of a modified 3' OH at the end of the adaptor oligonucleotide that is not adjacent to the ssNA that binds to the SSB.

38. A kit according to claim 32, wherein the blocking modification is the presence of a modified 3' OH at the end of the adaptor oligonucleotide that is not adjacent to the ssNA to which the SSB binds, and wherein the blocking modification is selected from the group consisting of: an amino modifier, a spacer, a dideoxy base, an inverted dideoxy base, and a 3' phosphate.

39. The kit of claim 32, wherein the SSB-binding ssNA hybridization region comprises a random sequence.

40. The kit of claim 32, wherein the SSB-bound ssNA hybridization region comprises a universal base.

41. The kit of any one of claims 32 to 40, wherein the SSB-bound ssNA hybridization region is less than or equal to 10 nucleotides in length.

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