Method for producing DNA molecules with adapter sequences added thereto and use thereof

Abstract

The present invention provides: a method for producing DNA molecules having a novel adaptor sequence added thereto; and a use thereof. One embodiment of the present invention provides a method for producing DNA molecules having an adaptor sequence added thereto, the method comprising: a preparation step for preparing double-strand DNA in which a first DNA strand and a second DNA strand are at least partially hybridized; and an annealing step for annealing a partial double-strand oligonucleotide adapter with the 3' end of the first DNA strand of the double-strand DNA. The partial double-strand oligonucleotide adapter is provided with a 3' overhang that is a protruding end including an oligonucleotide having at least eight continuous random or predetermined base sequences and that anneals with the 3' end of the first DNA strand.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a DNA molecule having an adapter sequence added thereto, and to uses thereof. [Background technology]

[0002] The recent widespread use of next-generation sequencers has made it easier to read the genetic information of living organisms. Currently, Illumina's platform is the most widely used next-generation sequencer. Sequencing using a next-generation sequencer requires the preparation of a DNA library sample by adding sequences called adapters to both ends of the genomic DNA fragments to be analyzed. A variety of kits for preparing such DNA library samples are commercially available. Well-known examples of these kits include the Illumina kit and the RThruPLEX® DNA-seq kit.

[0003] However, these kits require a step of adding an adapter using ligase, and are still expensive, starting at 6,000 yen per sample, which places a heavy burden on handling a large number of samples and is a major limitation on research.

[0004] Meanwhile, Patent Document 1 and Non-Patent Document 1 report a method for preparing a library by creating strand-specific cDNA from mRNA. In this method, cDNA is synthesized from mRNA, and an adapter sequence is inserted using a technique for inserting another sequence into the end of the formed RNA-DNA double strand. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2018-515081 (published June 14, 2018) [Non-patent literature]

[0006] [Non-Patent Document 1] Townsley, BT et al. Frontiers in plant science, 6 (2015): 366. [Non-patent document 2] Yasunori Ichihashi and Atsushi Fukushima, "The cutting edge of transcriptome analysis in plant science," Frontiers in Plant Science 7:110 (2016) Summary of the Invention [Problem to be solved by the invention]

[0007] However, the techniques described in Patent Document 1 and Non-Patent Document 1 relate to RNA, and no consideration has been given to their application to DNA.

[0008] Furthermore, existing commercially available kits are expensive per sample, which places a heavy burden on researchers when handling a large number of specimens, posing a major limitation to research.

[0009] Thus, further improvements in DNA library preparation are desired.

[0010] An object of the present invention is to provide a novel method for producing a DNA molecule having an adapter sequence added thereto, and uses thereof. [Means for solving the problem]

[0011] In order to solve the above problems, the present invention includes any one of the following aspects. <1> A method for producing a DNA molecule to which an adapter sequence has been added, comprising the steps of: a preparation step of preparing double-stranded DNA in which the first DNA strand and the second DNA strand are at least partially hybridized; an annealing step of annealing a partially double-stranded oligonucleotide adaptor to the 3' end of the first DNA strand of the double-stranded DNA; The method, wherein the partially double-stranded oligonucleotide adapter has a protruding end (3' overhang) comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences that anneals to the 3' end of the first DNA strand. <2> the 5'-end of the first DNA strand constituting the double-stranded DNA comprises a base sequence (second adapter sequence) different from each of the double-stranded portions (first adapter sequences) of the partially double-stranded oligonucleotide adapter; <1> The method described below. <3> The above preparation process is annealing an adaptor comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences and the second adaptor sequence located on the 5'-terminal side of the oligonucleotide to a single-stranded DNA fragment corresponding to the second DNA strand, followed by chain extension to prepare the double-stranded DNA; <2> The method described below. <4> annealing the adapter to the single-stranded DNA fragment corresponding to the second DNA strand at a temperature ranging from 30°C to 50°C; <3> The method described below. <5> The single-stranded DNA fragment corresponding to the second DNA strand is a collection of multiple DNA fragments obtained by fragmenting genomic DNA and denaturing it into single-stranded DNA. <1> ~ <4> A method according to any one of the above. <6> generating a third DNA strand complementary to the first DNA strand by extending the overhanging end of the partially double-stranded oligonucleotide adaptor; <1> ~ <5> A method according to any one of the above. <7> an amplification step of amplifying double-stranded DNA in which the first DNA strand and a third DNA strand complementary to the first DNA strand are hybridized; <1> ~ <6> A method according to any one of the above. <8> The size of the amplified fragment obtained is within the range of 300 bp or more and 1000 bp or less. <7> The method described below. <9> <7> or <8> A DNA library for next-generation sequencer analysis, obtained by the method described in the above, containing double-stranded DNA for analysis flanked by at least a portion of the second adapter sequence and its complementary sequence and at least a portion of the double-stranded portion (first adapter sequence) of the partially double-stranded oligonucleotide adapter. <10> <1> ~ <8> A kit for use in the method described above, comprising at least one of the following (A) to (C):

[0012] (A) A partially double-stranded oligonucleotide adapter with a protruding end (3' overhang) comprising an oligonucleotide consisting of at least eight consecutive random or predetermined base sequences that anneals to the 3' end of a DNA strand; (B) an adapter comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences and a second adapter sequence located on the 5'-terminal side of the oligonucleotide; and (C) a primer set consisting of a PCR primer that anneals to a complementary sequence of the second adapter sequence and a PCR primer that anneals to the strand (block strand) of the partially double-stranded oligonucleotide adapter that does not have a protruding end; [Effects of the Invention]

[0013] According to the present invention, it is possible to prepare a DNA library more easily and at lower cost. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a diagram illustrating an overview of a breath capture technique according to an embodiment of the present invention. [Figure 2] FIG. 1 shows the proportion of genomic regions sequenced in the reference genome for each sample obtained in Example 1 and Reference Example. [Figure 3] FIG. 1 shows the ratio of read bases to the reference chromosome for each sample obtained in Example 1 and Reference Example. [Figure 4] FIG. 1 shows the mapping efficiency to the reference genome for each sample obtained in Example 1 and Reference Example. [Figure 5] FIG. 1 shows the percentage of genomic regions sequenced in the reference genome for each sample obtained in Example 5 using 10 ng of Drosophila genomic DNA as input. DETAILED DESCRIPTION OF THE INVENTION

[0015] [Definitions of terms, etc.] As used herein, the term "polynucleotide" can be alternatively referred to as "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Furthermore, the term "base sequence" can be alternatively referred to as "nucleic acid sequence" or "nucleotide sequence," and refers to a sequence of deoxyribonucleotides or a sequence of ribonucleotides, unless otherwise specified. Furthermore, the term "polynucleotide" encompasses both single-stranded and double-stranded structures, and in the case of single strands, both sense and antisense strands.

[0016] As used herein, the term "gene" is used interchangeably with "polynucleotide," "nucleic acid," or "nucleic acid molecule." A "polynucleotide" refers to a polymer of nucleotides. Therefore, the term "gene" as used herein encompasses not only double-stranded DNA, but also the single-stranded DNAs and RNAs (e.g., mRNA) that constitute it, such as the sense and antisense strands.

[0017] As used herein, "oligonucleotide" refers to a nucleotide polymer formed by polymerizing a predetermined number of nucleotides. The term "oligonucleotide" as used herein is not limited in length, but refers to a "polynucleotide" having a relatively short nucleotide chain.

[0018] As used herein, a "primer" refers to an oligonucleotide strand that hybridizes with a target or template nucleotide strand.

[0019] As used herein, "DNA" includes cDNA and genomic DNA obtained by, for example, cloning or chemical synthesis techniques, or a combination thereof. That is, DNA may be "genomic" DNA containing non-coding sequences such as introns, which is the form contained in the genome of an animal, or it may be cDNA obtained via mRNA using reverse transcriptase or polymerase, i.e., "transcribed" DNA not containing non-coding sequences such as introns.

[0020] As used herein, "RNA" refers to nucleic acids that have ribose sugars instead of deoxyribose sugars and generally have uracil instead of thymine as one of the pyrimidine bases.

[0021] Any of the nucleobases comprising the primers and oligonucleotides herein may contain one or more modifications known in the art, such as chemical modifications and substitutions, modified sugar moieties, and chemiluminescent or fluorescent labels.

[0022] [1. DNA molecule production method] In one embodiment, the present invention provides a method for producing a DNA molecule having an adapter sequence added thereto, the method comprising: a double-stranded DNA preparation step of preparing double-stranded DNA in which a first DNA strand and a second DNA strand are at least partially hybridized; and an annealing step of annealing a partially double-stranded oligonucleotide adapter to the 3' end of the first DNA strand of the double-stranded DNA, wherein the partially double-stranded oligonucleotide adapter has a protruding end (3' overhang) that anneals to the 3' end of the first DNA strand and includes an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences. Each step of this method will now be described in detail.

[0023] (1) Double-stranded DNA preparation process This step is a step of preparing double-stranded DNA in which a first DNA strand and a second DNA strand are at least partially hybridized. In FIG. 1 , this is the step of preparing double-stranded DNA shown in the third step from the top. In the following description, when referring to FIG. 1 , the lower strand of the double-stranded DNA shown in the third step from the top will be referred to as the first DNA strand, and the upper strand will be referred to as the second DNA strand. In one embodiment, the double-stranded DNA prepared in the double-stranded DNA preparation step has the following characteristics: 1) the first DNA strand and the second DNA strand are partially hybridized; 2) the 3'-end of the first DNA strand and the 5'-end of the second DNA strand are substantially blunt-ended; and 3) the 5'-end of the first DNA strand is not hybridized to the second DNA strand. Furthermore, in a preferred embodiment, the 5'-end of the first DNA strand contains a base sequence of known sequence (also referred to as a second adapter sequence, to be distinguished from the first adapter sequence described below).

[0024] As used herein, the term "adapter" or "adapter molecule" refers to an oligonucleotide having a specific sequence that can be annealed to a target polynucleotide.

[0025] (1-1) DNA fragmentation process In one embodiment, the double-stranded DNA preparation step includes a DNA fragmentation step in which a DNA sample is fragmented. Although not particularly limited, the DNA sample can be fragmented into fragments having a base length of preferably 300 bp to 1000 bp, more preferably 350 bp to 800 bp, and even more preferably 350 bp to 500 bp. In Figure 1, the double-stranded DNA fragment shown in the first step from the top is an example of a DNA fragment obtained in the DNA fragmentation step.

[0026] This fragmentation step can be carried out, for example, by heat-treating the genomic DNA. The conditions for the heat treatment are not particularly limited, but the solution containing the extracted genomic DNA can be heated, for example, at 95°C for about 45 minutes.

[0027] An example of a solution that dissolves the extracted genomic DNA during heating is 1 mM Tris (pH 7.5).

[0028] Other fragmentation techniques include enzyme digestion with restriction enzymes or the like, shearing, and ultrasonic treatment.

[0029] =DNA sample= The DNA sample to be fragmented is not particularly limited as long as it contains DNA. The DNA sample can be isolated from any living organism, such as an animal, plant, protist, yeast, fungus, bacterium, or virus (DNA sample isolation step). Examples of plants include plants from the grass family and the Brassicaceae family, and examples of animals include vertebrates such as mammals, birds, reptiles, and fish, as well as invertebrates such as insects, nematodes, and crustaceans. Known methods can be used to isolate DNA.

[0030] The DNA sample also includes samples derived from experimental plants such as Arabidopsis thaliana and experimental animals such as Drosophila. The DNA sample is not limited to samples derived from a single organism, but may be derived from multiple organisms. Examples of DNA samples derived from multiple organisms include, but are not limited to, samples for metagenomic analysis.

[0031] Examples of DNA contained in a DNA sample include genomic DNA and cDNA. The DNA includes wild-type DNA and DNA containing a single nucleotide polymorphism (SNP) or one or more mutations. While not particularly limited, the genomic DNA may be substantially the entire genomic DNA, or a portion of the genomic DNA recovered by a technique such as chromatin immunoprecipitation.

[0032] (1-2) Step of forming single strands of DNA obtained in the DNA fragmentation step (step of preparing second DNA strand) If the DNA fragments obtained in the DNA fragmentation step are double-stranded DNA fragments, these double-stranded DNA fragments are converted into single strands. The conversion of double-stranded DNA fragments into single strands can be performed by a known method such as heating at a predetermined temperature (this is the so-called process of converting double-stranded DNA into single strands by thermal denaturation). In Figure 1, this is the step of converting double-stranded DNA fragments into single strands, shown as the second step from the top.

[0033] That is, in one embodiment, the single-stranded DNA fragments (corresponding to the second DNA strand of the double-stranded DNA prepared in the double-stranded DNA preparation step) are a collection of multiple single-stranded DNA fragments obtained by fragmenting genomic DNA and denaturing it into single-stranded DNA.

[0034] (1-3) Step of preparing the first DNA strand using the second DNA strand This step is a step of preparing a double-stranded DNA consisting of a first DNA strand and a second DNA strand by preparing a first DNA strand using the second DNA strand obtained in (1-2) above. In Figure 1, this corresponds to the second and third steps from the top.

[0035] In one embodiment, a single-stranded adapter (3' adapter (or adapter comprising a second adapter sequence)) comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences and a second adapter sequence located 5'-terminally closer to the oligonucleotide is annealed to a single-stranded DNA fragment (which serves as a template DNA fragment) corresponding to the second DNA strand (corresponding to the second step from the top in Figure 1). Then, a first DNA strand complementary to the second DNA strand is extended by a primer extension reaction starting from the 3' end (containing an OH group) of the 3' adapter. As a result, a double-stranded DNA in which the first DNA strand and the second DNA strand are at least partially hybridized is prepared, as shown in the third step from the top in Figure 1. In this case, in the resulting double-stranded DNA, 1) the first DNA strand and the second DNA strand hybridize with the random oligonucleotide portion of the 3' adapter as the starting point, 2) the 3' end of the first DNA strand and the 5' end of the second DNA strand form substantially blunt ends, and 3) the 5' end of the first DNA strand (corresponding to the second adapter sequence) does not hybridize with the second DNA strand. Note that, as used herein, "forming a substantially blunt end" includes not only completely blunt ends, but also cases where a misalignment of one to several bases (e.g., 5, 4, 3, or 2 bases) occurs between the first DNA strand and the second DNA strand.

[0036] In this specification, the term "two DNA strands hybridized" does not necessarily mean that the respective base sequences are completely complementary to each other in the region where hybridization can occur, unless otherwise specified. In the region where hybridization can occur, for example, one DNA strand may be an oligonucleotide having a sequence identity of 80% or more with the other DNA strand, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0037] Similarly, in this specification, the term "two DNA strands are complementary" does not necessarily mean that the respective base sequences are completely complementary to each other in the region where hybridization can occur between the DNA strands, unless otherwise specified. In the region where hybridization can occur, for example, one DNA strand may be an oligonucleotide having a sequence identity of 80% or more with the other DNA strand, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0038] The oligonucleotide portion comprising a random or predetermined base sequence that constitutes the 3' adapter may be a sequence of at least 8 consecutive bases or a sequence of predetermined bases, but is preferably a sequence of 6 to 12 consecutive bases, and more preferably a sequence of 7 to 9 consecutive bases. In this specification, the term "random (base sequence)" is used in the same way as the general definition, and includes all possible types of base sequences (i.e., in the case of a sequence of n consecutive bases (n is an integer of 2 or more), 4 n (The term refers to a sequence containing multiple types of bases.)

[0039] Furthermore, the term "predetermined (base sequence)" refers to a specific base sequence designed to anneal to a desired region at the 3' end of the first DNA strand, for example. By using such a base sequence, it is possible to create a library containing only regions having the specific sequence.

[0040] The second adapter sequence constituting the 3' adapter can be selected to be compatible with a particular NGS platform. An example of the 3' adapter sequence is an oligonucleotide consisting of the base sequence set forth in SEQ ID NO:1.

[0041] This step can be carried out in the presence of a DNA polymerase, a template DNA fragment (second DNA strand), and a 3' adapter (functioning as a primer) under conditions similar to those for a general primer extension reaction using random primers. Also, please refer to the description of "Step (3) Extension Step" below.

[0042] However, the temperature at which the 3' adapter is annealed to the template DNA fragment affects the quality of the final library obtained. For the purpose of consistently obtaining a high-quality library, it is preferable to anneal the 3' adapter to the template DNA fragment within a temperature range of 30°C or higher and 50°C or lower. The annealing temperature is more preferably within a temperature range of 31°C or higher, or 35°C or higher, or 40°C or higher, or 42°C or higher, and 50°C or lower, or 49°C or lower, or 48°C or lower, or 47°C or lower.

[0043] The quantitative ratio between the 3' adaptor and the template DNA fragment (second DNA strand) is not particularly limited, but is preferably within the range of 1.4:1 to 69:1, for example.

[0044] For example, a DNA-dependent DNA polymerase (e.g., Klenow polymerase, Pol I DNA polymerase, etc.) can be used to extend the complementary strand of the template DNA fragment (i.e., to generate a strand). Strand extension is carried out, for example, in the presence of a suitable buffer, in the presence of DNA polymerase and deoxyribonucleotides (e.g., dNTPs), starting from a primer (here, a 3' adapter).

[0045] The DNA polymerase used for chain extension has polymerase activity and 3'-5' proofreading exonuclease activity, and may further contain 5'-3' exonuclease activity and / or terminal transferase activity. The DNA polymerase may be a thermophilic DNA polymerase such as Taq DNA polymerase, Pfu DNA polymerase, Bst DNA polymerase, Tli DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase, Vent DNA polymerase, SD DNA polymerase, or KOD DNA polymerase. Alternatively, the DNA polymerase may be a mesophilic DNA polymerase such as E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, phi29 DNA polymerase, T7 DNA polymerase, or T4 DNA polymerase. Examples are Ex Tag (Takara), KOD (Toyobo), Pfu (Agilent), PrimeSTAR HS (Takara), Q5 (NEB) Phusion High-Fidelity (NEB), Hifi (KAPA), Expand TM High Fidelity (Roche), etc. are used.

[0046] In the extension reaction using a 3' adapter, the temperature of the extension reaction is in the range of 60°C or higher and 95°C or lower, preferably 65°C or higher and 80°C or lower, for example, 72°C or 74°C.

[0047] The rate of the extension reaction is from 0.01 kb / min to 10 kb / min, preferably from 0.1 kb / min to 5 kb / min, for example, 1 kb / min, 1.5 kb / min, or 2 kb / min.

[0048] When MgCl 2 is used as an additive in the extension reaction solution, the concentration is 0.01 mM to 10 mM, preferably 0.1 mM to 5 mM, for example, 1 mM, 1.5 mM, or 2 mM.

[0049] When KCl is used as an additive in the extension reaction solution, the concentration is 0.1 mM to 1000 mM, preferably 1 mM to 100 mM, for example, 10 mM or 50 mM.

[0050] The concentration of dNTP in the extension reaction solution is 0.01 mM or more and 10 mM or less, preferably 0.1 mM or more and 1 mM or less, for example, 0.2 mM, 0.25 mM, or 0.3 mM.

[0051] Furthermore, if necessary, for example, 6 mM or 10 mM (NH4)2SO4, 0.1% Triton X-100, and 0.001% or 0.1 mg / ml BSA are added.

[0052] (2) Annealing of partially double-stranded oligonucleotide adapters A method according to one embodiment of the present invention further includes an annealing step of annealing a partially double-stranded oligonucleotide adapter to the 3'-end of the first DNA strand of the double-stranded DNA obtained in step (1) (double-stranded DNA preparation step). In Figure 1, this step is shown as the fourth and fifth steps from the top. The first DNA strand is the strand depicted at the bottom of the double-stranded DNA.

[0053] Here, the "partially double-stranded oligonucleotide adaptor" has a protruding end (3' overhang) that anneals to the 3' end of the first DNA strand and includes an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences (e.g., corresponding to "NNNNNN" in the 5' adaptor in Figure 1). Hereinafter, the partially double-stranded oligonucleotide adaptor may also be referred to as the 5' adaptor.

[0054] The 5' adapter comprises a strand with an overhanging 3' region (the capture strand) and a shorter strand (the block strand). That is, the 5' adapter has a single-stranded portion and a double-stranded portion, and the block strand hybridizes to a portion of the capture strand. Herein, the double-stranded portion of the 5' adapter sequence is also referred to as the first adapter sequence. In some embodiments, the first adapter sequence (both strands thereof) has a different base sequence from the second adapter sequence. For example, the first adapter sequence may have 90% or less, 80% or less, 70% or less, or 60% or less sequence identity to the second adapter sequence. An example of the capture strand of the 5' adapter is an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 2. An example of the blocking strand of the 5' adapter is an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 3.

[0055] The hybridization of the block strand and the capture strand to each other does not necessarily mean that the respective base sequences are completely complementary to each other in the region where hybridization can occur. For example, the capture strand (excluding the 3' overhang) may be an oligonucleotide having a sequence identity with the block strand of 80% or more, preferably 85% or more, 86% or more, 87% or more, 88% or more, or 89% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0056] The first adapter sequence may be, for example, a sequence of eight contiguous known bases, preferably a sequence of six to twelve contiguous bases, and more preferably a sequence of seven to nine contiguous bases. For example, the first adapter sequence may be selected to be compatible with a specific NGS platform. The second adapter sequence may be similar, but specific NGS platforms include those commercialized by Illumina®, Roche Diagnostics®, Applied Biosystems®, Pacific Biosciences®, Thermo Fisher Scientific®, Bio-Rad®, and the like. The first adapter sequence may further include an index sequence or barcode sequence designed to label either the sample or the sequence of interest. In one example, these adapters may function as sequencing adaptors.

[0057] The portion of the oligonucleotide (which may be DNA or RNA) that comprises a random base sequence and that constitutes the 5' adapter may be a sequence of at least 8 consecutive or predetermined bases, but is preferably a sequence of 6 to 12 consecutive bases, and more preferably a sequence of 7 to 9 consecutive bases.

[0058] The 5' adapter can be prepared, for example, by hybridizing the above-mentioned block strand with the capture strand.

[0059] (2-1) Breathing process In one embodiment, the step of annealing the 5' adapter comprises a step of breathing the double-stranded DNA to be annealed. We have developed a technology for creating adapter directional sequencing (BrADseq) libraries (Non-patent Document 1 and Patent Document 1). This technology utilizes the fact that the double-stranded structure of DNA / RNA complexes exhibits partial opening and closing fluctuations (breathing), and specifically incorporates adapters into these complexes.

[0060] In one embodiment, the double-stranded DNA obtained in step (1) (double-stranded DNA preparation step) has a substantially blunt end at the 3' end of the first DNA strand (the 5' end of the second DNA strand), and the 5' end of the first DNA strand is not hybridized to the second DNA strand. In one embodiment, the breathing step is performed on double-stranded DNA having such different terminals.

[0061] In one embodiment, the breathing step can be carried out by leaving a solution containing the double-stranded DNA of interest and the 5' adaptor to stand at a temperature of, for example, 25°C or higher.

[0062] (2-2) Annealing of the 5' adapter to the breathing double-stranded DNA In this step, the double-stranded DNA that has been breathing in step (2-1) above (the fourth stage from the top in Figure 1) and the 5' adapter are allowed to coexist in solution, thereby selectively annealing the 3' overhang, which is the protruding end of the partially double-stranded oligonucleotide adapter, to the 3' end (the end that is breathing) of the first DNA strand of the double-stranded DNA.

[0063] This step can be carried out following or simultaneously with the above step (2-1). That is, breathing of the double-stranded DNA and annealing of the 5' adapter can be carried out simultaneously.

[0064] The conditions for annealing the 5' adapter to the double-stranded DNA are not particularly limited, but for example, the temperature is preferably within the range of 20° C. to 30° C. The quantitative ratio of the 5' adapter to the double-stranded DNA is also not particularly limited, but for example, it is preferably within the range of 14:1 to 713:1.

[0065] (3) Extension process In one embodiment, the method includes, following or simultaneously with the annealing step, extending the strand from the overhanging end (having an OH group) of the 5' adapter to generate a third DNA strand complementary to the first DNA strand.

[0066] In one embodiment, the double-stranded DNA obtained by this step comprises 1) a DNA duplex consisting of a first DNA strand and a third DNA strand complementary to the first strand, 2) one end consisting of the double-stranded portion of the 5' adaptor, and 3) the other end consisting of the 3' adaptor and its complementary sequence. The ends 2) and 3) are substantially blunt.

[0067] For example, a DNA-dependent DNA polymerase (e.g., Klenow polymerase, Pol I DNA polymerase, etc.) can be used for chain extension (i.e., production of the third DNA strand). Chain extension is carried out, for example, in the presence of a suitable buffer, in the presence of a DNA polymerase and deoxyribonucleotides (e.g., dNTPs), starting from a primer (here, the protruding end of the 5' adapter).

[0068] The DNA polymerase used for strand extension and / or amplification has polymerase activity and 3'-5' proofreading exonuclease activity, and may further contain 5'-3' exonuclease activity and / or terminal transferase activity. Examples of DNA polymerases include Taq DNA polymerase, Pfu DNA polymerase, Bst DNA polymerase, Tli DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase, Vent DNA polymerase, SD The DNA polymerase may be a thermophilic DNA polymerase such as KOD DNA polymerase. Alternatively, the DNA polymerase may be a mesophilic DNA polymerase such as E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, phi29 DNA polymerase, T7 DNA polymerase, or T4 DNA polymerase. Examples of such polymerases include Ex Tag (Takara), KOD (Toyobo), Pfu (Agilent), PrimeSTAR HS (Takara), Q5 (NEB), Phusion High-Fidelity (NEB), Hifi (KAPA), and Expand TM High Fidelity (Roche), etc. are used.

[0069] (4) Amplification process The method according to one embodiment may include, following the step (3) described above, an amplification step of amplifying the double-stranded DNA obtained in step (3) (double-stranded DNA in which a first DNA strand is hybridized with a third DNA strand complementary to the first DNA strand).

[0070] Furthermore, this amplification step preferably enriches the target double-stranded DNA and also adds an adapter sequence.

[0071] As described above, in a typical example, the multiple types of double-stranded DNA to be amplified include 1) a DNA double-strand composed of a first DNA strand and a third DNA strand complementary to the first strand, 2) one end consisting of the double-stranded portion of the 5' adapter, and 3) the other end consisting of the 3' adapter and its complementary sequence. The ends of 2) and 3) are substantially blunt. That is, although the DNA double-strand of 1) above can contain a variety of sequences, the portions of 2) and 3) above are common to the multiple types of double-stranded DNA.

[0072] To generate multiple adaptor-linked amplified fragments, in one embodiment, the amplification step is performed by PCR using a PCR primer set containing sequences corresponding to the 5' and 3' adaptors (i.e., sequences that anneal to part or all of these adaptors). In a more specific example, the amplification step is performed using a primer set consisting of a PCR primer that anneals to the complementary strand of the 3' adaptor (second adaptor sequence) and a PCR primer that anneals to the blocked strand of the 5' adaptor (i.e., the strand without the overhanging end). This results in a collection of amplified DNA fragments with a common structure, as described below in Section 2. DNA Library, in which 1) double-stranded DNA derived from a DNA sample is sandwiched between 2) at least part (or possibly all) of the first adaptor sequence (the double-stranded portion of the partially double-stranded oligonucleotide adaptor) and 3) at least part (or possibly all) of the double-stranded portion consisting of the second adaptor sequence and its complementary sequence. This collection of amplified DNA fragments, both ends of which are sandwiched between adaptor sequences, can be used, for example, as a DNA library for next-generation sequence analysis.

[0073] The amplification step performed by PCR-based methods is described below. First, DNA polymerase (Pol1) and dNTPs are reacted in a suitable buffer in the presence of an appropriate PCR primer set. Each PCR cycle generally involves three steps: denaturation, annealing, and extension. The temperature in the denaturation step ranges, for example, from 90°C to 100°C, with 94°C being an example. The duration of the denaturation step is typically, for example, from 10 seconds to 10 minutes, with 30 seconds being an example. The total number of PCR cycles is, for example, 10 to 50 cycles, more preferably, from 16 to 21 cycles, but is not limited thereto. The temperature in the annealing step is determined according to the melting temperature of the amplification primers. For example, the temperature in the annealing step ranges, for example, from 50°C to 70°C, with 65°C being an example. The duration of the annealing step can be, for example, from 20 seconds to 4 minutes, with 30 seconds being an example. The temperature in the extension step may be in the range of 68°C to 75°C, and the duration of the extension step may be in the range of 10 seconds to 10 minutes, for example 30 seconds. The final extension step may be followed by a terminal extension step lasting for, for example, 5 to 10 minutes, for example 7 minutes.

[0074] The base length of the amplified fragment obtained through the above steps is not particularly limited, but is preferably 300 bp to 1000 bp, and more preferably 400 bp to 700 bp, for example.

[0075] When used for analysis by a next-generation sequencer, it is preferable that the sequence inserted in the next-generation sequencer (called an insert) be 300 bp or longer.

[0076] The amplification reaction is not limited to PCR-based amplification methods and may include any DNA amplification reaction, such as single primer isothermal amplification (SPIA), Ribo-SPIA, multiplex displacement amplification (FDA), transcription-modified amplifying amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), nicking enzyme amplification reaction (NEAR), and rolling circle amplification (RCA). Examples of PCR-based amplification methods include multiplex PCR, long-range PCR, routine PCR, high-speed PCR, hot-start PCR, touchdown PCR, and nested PCR.

[0077] The extended and amplified DNA may be size-selected and purified by size fractionation. Size fractionation may be performed using SPRI beads (Ampure XP beads, Agencourt, Sera-Mag beads, etc.). Column chromatography (spin column, etc.), polyacrylamide gel electrophoresis, agarose gel electrophoresis, etc. may also be used.

[0078] (5) Sequencing step In some embodiments, the methods provided herein further comprise a step of DNA sequencing the amplification products obtained in the above-mentioned steps. Examples of DNA sequencing methods include automated sequencing using the Sanger method and sequencing using a next-generation sequencing (NGS) platform. Next-generation sequencing includes, but is not limited to, pyrosequencing, ion semiconductor sequencing, sequencing-by-synthesis using reversible dye terminators, sequencing-by-ligation, and sequencing by oligonucleotide probe ligation or sequencing-by-synthesis using virtual terminators. Next-generation sequencing systems can be, for example, MiSeq (Illumina).

[0079] (6) Sequence analysis process In some embodiments, the quantitative genetic analysis further comprises a sequence analysis step in which the sequencing reads are analyzed.

[0080] Sequence analysis includes genome equivalent analysis, single nucleotide variation (SNV) analysis, gene copy number variation (CNV) analysis, genetic lesion detection and sequence alignment.In certain embodiments, this bioinformatics analysis is useful for quantifying the number of genome equivalents analyzed in DNA clone library, detecting gene mutations etc. at target gene loci and measuring copy number changes etc.

[0081] The methods described herein are useful for generating DNA libraries for a variety of purposes. The methods can be combined with well-known sequencing techniques, particularly high-throughput sequencing techniques.

[0082] [2. DNA Library] The present invention also encompasses a DNA library for next-generation sequence analysis obtained by carrying out the above-mentioned step (4) (amplification step). This DNA library is composed of multiple types of double-stranded DNA for analysis, but as described above, it has a common structure in which 1) the double-stranded DNA for analysis is sandwiched between 2) at least a portion (or may be the entirety) of a first adapter sequence (the double-stranded portion of a partially double-stranded oligonucleotide adapter) and 3) at least a portion (or may be the entirety) of a double-stranded portion composed of a second adapter sequence and its complementary sequence.

[0083] [3. Kit] The present invention also provides a kit for use in the method, the kit comprising at least one of the following (A) to (C):

[0084] (A) A partially double-stranded oligonucleotide adapter with a protruding end (3' overhang) comprising an oligonucleotide consisting of at least eight consecutive random or predetermined base sequences that anneals to the 3' end of a DNA strand; (B) an adapter comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences and a second adapter sequence located 5'-terminally of the oligonucleotide; and (C) A primer set consisting of a PCR primer that anneals to the complementary sequence of the second adapter sequence and a PCR primer that anneals to the blocked strand of the partially double-stranded oligonucleotide adapter (i.e., the strand without the overhanging end). The above (A) and (B) are materials for generating a sequence that serves as a template for subsequent PCR. The primer set (C) is used to amplify double-stranded DNA from the DNA sample generated as described above, including the adapter sequences (A and B) at both ends. The primer set (C) is based on the 5' adapter and its extended strand. In some embodiments, one of the primers in the (C) set is complementary to the blocking strand of the partially double-stranded oligonucleotide adapter, and the other is complementary to the 3' end of the extended strand.

[0085] The kit may include reagents necessary for generating a DNA library, such as a suitable buffer, a suitable polymerase, DTT, dNTPs, sterile water, MgCl, DNA amplification primers, and reagents for purifying the library. The kit may also include instructions for carrying out the methods according to the above-described embodiments.

[0086] Thus, the present invention demonstrates for the first time that breathing capture technology, which has been used conventionally in cDNA synthesis from mRNA, can be applied to DNA. The method of the present invention enables the simple and rapid preparation of a DNA library. For example, when using the above kit, preparation can be completed in approximately 1 to 2 hours.

[0087] As described above, the present invention proposes a low-cost and simple method for preparing a DNA library, as well as a DNA library prepared using this method. This method not only enables the preparation of a DNA library at low cost, but also produces a DNA library of higher quality than conventional products.

[0088] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0089] 1. Example 1. Preparation of a DNA library Experimental materials: 1 ng, 10 ng, or 50 ng of Arabidopsis thaliana genomic DNA (BioChain, D1634310-5) was used as input.

[0090] Experimental method: The experiment was carried out according to the following steps (a) to (d).

[0091] (Step (a) Obtaining fragmented dsDNA from genomic DNA) 10 μl of 100 ng / μl Arabidopsis thaliana genomic DNA (Cosmo Bio, D1634310-5) was taken and 90 μl of 10 mM Tris was added. 20 μl aliquots were heated at 95°C for 45 minutes to fragment the DNA. AMPureXP beads (Beckman Coulter, A63880) were added in an amount 1.5 times the volume of the solution, and the fragmented DNA was purified according to the manufacturer's instructions. The DNA was eluted with 20 μl of water.

[0092] (Step (b) Denaturation of dsDNA to ssDNA and Priming of the 3' End of ssDNA) The dsDNA obtained in step (a) above was denatured to ssDNA and the 3' end of the ssDNA was primed according to the following steps 1) to 8). For each concentration of fragmented DNA sample, two annealing procedures were performed, one at 35°C and the other at 45°C.

[0093] 1) The following materials were mixed: 5 μl of fragmented DNA (0.2 ng, 2 ng or 10 ng / μl) 3-prime priming adapter (SEQ ID NO: 1: 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN-3') (5 μM L-3ILL-N8.2) 1 μl 1.5 μl of 10x Buffer (500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl2, 10 mM DTT) (Takara Bio, Inc., RR006) dNTP 1.2 μl H2O 6.225 μl Ex taq (Takara Bio, RR006) 0.075 μl --Total 15μl 2) Incubated in a thermal cycler running the following program: 94°C for 2 minutes; 35°C or 45°C for 10 minutes; 42°C for 10 minutes; 72°C for 5 minutes; hold at 4°C. 3) 5 μl of 50 mM EDTA was added 4) 30 μl of Ampure XP beads (Beckman Caulter) were added, mixed, and size-selected. 5) Let stand for 5 minutes 6) The supernatant was discarded. 7) Wash twice with 200 μl of 80% EtOH 8) The beads were dried.

[0094] (Step (c) Breath capturing of the 5' end of ssDNA) Next, breathing capture of the 5' end of the ssDNA obtained in step (b) above was carried out according to the following steps 1) to 8).

[0095] 1) Add 4 μl of 10 μM 5-prime double-stranded adapter oligo (5pSense8n (SEQ ID NO: 2: 5'-CCTACACGACGCTCTTCCGATCTNNNNNNNN-3') and 5pAnti (SEQ ID NO: 3: 5'-AGATCGGAAGAGCGTCGTGTAGG-3') 2) The following mixture was added: 10xBuffer (500mM Tris-HCl (pH 7.5 at 25°C), 100mM MgCl2, 10mM DTT) 1μl 25 mM dNTPs 0.25 μl DNA Pol I (Thermo Fisher Scientific, EP0041) 0.25μl H2O 4.5 μl --Total 6 μl 3) Incubated in a thermal cycler running the following program: 15 min at 25°C 4) The following mixture was added: 50 mM EDTA 10 μl ABR 30 μl --Total 40μl 4) Let stand for 5 minutes 5) The supernatant was discarded. 6) Washed twice with 200 μl of 80% EtOH 7) The beads were dried 8) Eluted with 30 μl of 10 mM Tris.

[0096] (Step (d) Enrichment and Addition of Adapter Sequences) Subsequently, the DNA eluted in the above step (c) was concentrated, and an adapter sequence was added according to the following steps 1) to 5).

[0097] 1) The following PCR mixture was made: 10xBuffer (500mM Tris-HCl (pH 7.5 at 25°C), 100mM MgCl2, 10mM DTT) 2μl dNTP 1.6 μl 2 μM PE1 (SEQ ID NO: 4: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') 1 μl 2 μM PE2 (SEQ ID NO: 5: 5'-CAAGCAGAAGACGGCATACGAGAT-index 8nt-GTGACTGGAGTTCAGACGTGTGCTCTTCCGAT-3') (Use a different index for each sample) 1 μl H2O 4.3 μl Ex Taq 0.1 μl Breathing captured DNA 10 μl Total 20 μl 2) Incubated in a thermal cycler running the following program: 94°C for 2 minutes; (21 cycles for 1 ng of input genomic DNA, 18 cycles for 10 ng, and 16 cycles for 50 ng) x (94°C for 30 seconds; 65°C for 30 seconds; 72°C for 30 seconds); 72°C for 7 minutes; hold at 4°C 3) AmpureXP bead purification was performed using 0.8x beads (washed twice). 4) Eluted with 10 μl of 10 mM Tris 5) Sequencing was performed using MiSeq (Illumina, model number).

[0098] [Reference Example: Creation of a DNA library using existing technology] Furthermore, as conventional techniques, Illumina (TruSeq ChIP Sample Preparation Kit v2 - Set A, IP-202-1012) and Takara (SMARTer® ThruPLEX® DNA-seq 6S(12) Kit, R400523) Samples were prepared using 10 ng and 50 ng of input genomic DNA, respectively, according to the standard protocol.

[0099] As the experimental material, the same genomic DNA as in Example 1 was used.

[0100] [2. Example 2. Quality Consideration 1] The quality of the DNA library obtained by the method of Example 1 was examined. First, to examine the bias against genomic regions that occurs during library preparation, a comparison was made with a DNA library prepared by conventional techniques using fragmented DNA as the target, as described in the above-mentioned Reference Example.

[0101] (Analysis method) Data of 850K reads / sample was acquired and analyzed on a personal computer running Linux®. Specifically, mapping to genome data was performed using bowtie2. Next, the depth function of samtools was used to calculate the coverage of each sample relative to the reference genome.

[0102] (result) The results are shown in Figure 2. Figure 2 shows the proportion of genomic regions sequenced in the reference genome for each sample. In the figure, the samples obtained in Example 1 are listed as BrAD-Seq, and the samples obtained using Takara and Illumina kits are listed as Takara and Illumina, respectively. The same applies to all figures below.

[0103] At 45°C, higher values ​​were obtained compared to other companies' kits, which means that the genome was more widely mapped, resulting in less bias.

[0104] The method of the present invention was able to obtain results similar to those of other companies' kits in terms of the number of reads obtained from sequencing, mapping rate, and GC content.

[0105] [3. Example 3. Quality Consideration 2] To further examine the quality of the DNA library obtained by the method of Example 1, the ratio of read bases to the reference chromosomes (chromosomes 1 to 5) was examined for each sample obtained in the Example and Reference Examples.

[0106] (Analysis method) The number of reads mapped to each chromosome was calculated using the idxstats function of samtools from the results of mapping in Example 2. Quality Review 1 above. This value was multiplied by each read length to calculate the total number of bases mapped to each chromosome. Finally, the coverage was calculated by dividing the total number of bases by the total length of each chromosome.

[0107] (result) The results are shown in Figure 3. Figure 3 shows the ratio of read bases to the reference chromosome for each sample.

[0108] In the present invention, more uniform values ​​were obtained for each chromosome than with existing kits, which means that sequence information for each chromosome was obtained equally, resulting in less bias.

[0109] These results demonstrate that the DNA library obtained by the method of the present invention reflects the original genome length better than that obtained by existing techniques, and is superior to conventional methods at both 35°C and 45°C.

[0110] [4. Example 4. Quality Consideration 3] Furthermore, the mapping efficiency to the reference genome was examined for each sample obtained in the Examples and Reference Examples.

[0111] (Analysis method) The results of mapping in Example 2, Quality Review 1 were converted to bed using the bamtobed, makewindows, and coverage functions of bedtools. Furthermore, bedtools was used to calculate mapping coverage per 1000 bp.

[0112] (result) The results are shown in Figure 4. Figure 4 shows the mapping efficiency for each sample relative to the reference genome. Figure 4A shows the results for BrAD-seq at 35°C, Figure 4B shows the results for BrAD-seq at 45°C, Figure 4C shows the results for Takara, and Figure 4D shows the results for Illumina. Figure 4A and Figure 4B show the data for samples with input genomic DNA of 1 ng, 10 ng, and 50 ng, respectively, from left to right, and Figure 4C and Figure 4D show the data for samples with input genomic DNA of 10 ng and 50 ng, respectively, from left to right.

[0113] When the existing kit was used, the baseline was low and a high peak was observed in a certain region, whereas in the present invention, the baseline was high, wide, and evenly mapped.

[0114] 5. Example 5. Preparation of DNA Library Experimental materials: 10 ng of Drosophila melanogaster genomic DNA was used as input.

[0115] Experimental method: The experiment was carried out according to the following steps (a) to (d).

[0116] (Step (a) Obtaining fragmented dsDNA from genomic DNA) Fragmented dsDNA was obtained from genomic DNA under the following conditions.

[0117] Genomic DNA fragmentation conditions: Duty factor 10% Peak Incident power(w) 140 cycle / Burst 200 Time 80 seconds Covaris model used: S220 (Step (b) Denaturation of dsDNA to ssDNA and Priming of the 3' End of ssDNA) The dsDNA obtained in step (a) above was denatured to ssDNA and the 3' end of the ssDNA was primed according to the following steps 1) to 8). Three annealing temperatures of 40°C, 45°C, and 50°C were used for each concentration of fragmented DNA sample.

[0118] 1) The following materials were mixed: Fragmented DNA (2ng / μl) 5μl 3-prime priming adapter (SEQ ID NO: 1: 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN-3') (5 μM L-3ILL-N8.2) 1 μl 1.5 μl of 10x Buffer (500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl2, 10 mM DTT) (Takara Bio, Inc., RR006) dNTP 1.2 μl H2O 6.225 μl Ex taqcc 0.075μl --Total 15μl 2) Incubated in a thermal cycler running the following program: 94°C for 2 minutes; 40°C, 45°C, or 50°C for 1 minute, 5 minutes, 10 minutes, and 15 minutes, respectively; 42°C for 10 minutes; 72°C for 5 minutes; and hold at 4°C. 3) 5 μl of 50 mM EDTA was added 4) 30 μl of Ampure XP beads (Beckman Caulter) were added, mixed, and size-selected. 5) Let stand for 5 minutes 6) The supernatant was discarded. 7) Wash twice with 200 μl of 80% EtOH 8) The beads were dried.

[0119] (Step (c) Breath capturing of the 5' end of ssDNA) Next, breathing capture of the 5' end of the ssDNA obtained in step (b) above was carried out according to the following steps 1) to 8).

[0120] 1) Add 4 μl of 10 μM 5-prime double-stranded adapter oligo (5pSense8n (SEQ ID NO: 2: 5'-CCTACACGACGCTCTTCCGATCTNNNNNNNN-3') and 5pAnti (SEQ ID NO: 3: 5'-AGATCGGAAGAGCGTCGTGTAGG-3') 2) The following mixture was added: 10xBuffer (500mM Tris-HCl (pH 7.5 at 25°C), 100mM MgCl2, 10mM DTT) 1μl 25 mM dNTPs 0.25 μl DNA Pol I (Thermo Fisher Scientific, EP0041) 0.25μl H2O 4.5 μl --Total 6 μl 3) Incubated in a thermal cycler running the following program: 15 min at 25°C 4) The following mixture was added: 50 mM EDTA 10 μl ABR 30 μl --Total 40μl 4) Let stand for 5 minutes 5) The supernatant was discarded. 6) Washed twice with 200 μl of 80% EtOH 7) The beads were dried 8) Eluted with 30 μl of 10 mM Tris.

[0121] (Step (d) Enrichment and Addition of Adapter Sequences) Subsequently, the DNA eluted in the above step (c) was concentrated, and an adapter sequence was added according to the following steps 1) to 5).

[0122] 1) The following PCR mixture was made: 10xBuffer (500mM Tris-HCl (pH 7.5 at 25°C), 100mM MgCl2, 10mM DTT) 2μl dNTP 1.6 μl 2 μM PE1 (SEQ ID NO: 4: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') 1 μl 2 μM PE2 (SEQ ID NO: 5: 5'-CAAGCAGAAGACGGCATACGAGAT-index 8nt-GTGACTGGAGTTCAGACGTGTGCTCTTCCGAT-3') (Use a different index for each sample) 1 μl H2O 4.3 μl Ex Taq 0.1 μl Breathing captured DNA 10 μl Total 20 μl 2) Incubated in a thermal cycler running the following program: 94°C for 2 min; 18 cycles (94°C for 30 sec; 65°C for 30 sec; 72°C for 30 sec); 72°C for 7 min; hold at 4°C 3) AmpureXP bead purification was performed using 0.8x beads (washed twice). 4) Eluted with 10 μl of 10 mM Tris 5) Sequencing was performed using NovaSeq (Illumina).

[0123] 6. Example 6. Quality Consideration The quality of the DNA library obtained by the method of Example 4 was examined. To verify the effect of annealing temperature, coverage relative to the genome was calculated.

[0124] (Analysis method) Data of 9M reads / sample was acquired and analyzed on a personal computer running Linux®. Specifically, mapping to genome data was performed using bowtie2. Next, the depth function of samtools was used to calculate the coverage of each sample relative to the reference genome.

[0125] (result) The results are shown in Figure 5. Figure 5 shows the proportion of genomic regions sequenced in the reference genome for each sample. In the figure, the annealing temperature is written at the bottom.

[0126] The highest values ​​were obtained under the reaction conditions of 45°C for 5 or 10 minutes, which indicates a broader mapping to the genome and less bias.

[0127] The method of the present invention provided satisfactory results for genomic DNA derived from experimental animals in terms of the number of reads obtained from sequencing, mapping rate, and GC content. [Industrial Applicability]

[0128] The present invention can be used for preparing DNA libraries used in next-generation genome sequencing (NGS) technology and the like.

Claims

1. A method for producing a DNA molecule having an adapter sequence added thereto, comprising: preparing a double-stranded DNA in which the first DNA strand and the second DNA strand are at least partially hybridized; an annealing step of annealing a partially double-stranded oligonucleotide adaptor to the 3' end of the first DNA strand of the double-stranded DNA; the partially double-stranded oligonucleotide adaptor comprises a 3' overhang, which is a protruding end comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences, and which anneals to the 3' end of the first DNA strand; The method, wherein the single-stranded DNA fragments corresponding to the second DNA strand are a collection of multiple DNA fragments obtained by fragmenting genomic DNA and denaturing it into single-stranded DNA.

2. The method of claim 1, wherein the 5' end of the first DNA strand constituting the double-stranded DNA comprises a second adapter sequence having a base sequence different from each of the first adapter sequences that are the double-stranded portion of the partially double-stranded oligonucleotide adapter.

3. The above preparation process is The method of claim 2, comprising annealing an adapter comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences and the second adapter sequence located 5'-terminally of the oligonucleotide to a single-stranded DNA fragment corresponding to the second DNA strand, followed by strand extension to prepare the double-stranded DNA.

4. 4. The method of claim 3, wherein the adapter is annealed to the single-stranded DNA fragment corresponding to the second DNA strand within a temperature range of 30°C or higher and 50°C or lower.

5. 5. The method of claim 1, further comprising generating a third DNA strand complementary to the first DNA strand by extending the third DNA strand from the overhanging end of the partially double-stranded oligonucleotide adaptor.

6. The method according to any one of claims 1 to 5, comprising an amplification step of amplifying double-stranded DNA in which the first DNA strand and a third DNA strand complementary to the first DNA strand are hybridized.

7. The method according to claim 6, wherein the size of the amplified fragment obtained is in the range of 300 bp or more and 1000 bp or less.

8. 8. A DNA library for next-generation sequencer analysis obtained by the method of claim 6 or 7, comprising double-stranded DNA for analysis sandwiched between at least a portion of the second adapter sequence and its complementary sequence and at least a portion of the first adapter sequence, which is the double-stranded portion of the partially double-stranded oligonucleotide adapter, and the double-stranded DNA for analysis has been amplified using fragmented genomic DNA as a template.

9. A kit for analyzing genomic DNA, which is used in the method according to any one of claims 1 to 7, and comprises all of the following (A) to (C): (A) a partially double-stranded oligonucleotide adapter having a 3' overhang, which is a protruding end comprising an oligonucleotide of at least 8 consecutive random or predetermined base sequences that anneals to the 3' end of a DNA strand; (B) an adapter comprising an oligonucleotide consisting of at least 8 consecutive random or predetermined base sequences and a second adapter sequence located on the 5'-terminal side of the oligonucleotide; and (C) a primer set consisting of a PCR primer that anneals to a complementary sequence of the second adapter sequence and a PCR primer that anneals to a block strand, which is the strand without a protruding end, of the partially double-stranded oligonucleotide adapter.

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