BR112018077489B1 - METHODS FOR ANALYZING GENOMIC DNA COMPRISING THE USE OF A DNA LIBRARY - Google Patents

Abstract

A DNA library with excellent reproducibility is easily produced. A nucleic acid amplification reaction is conducted in a reaction solution containing genomic DNA and a random primer at a high concentration to obtain a DNA fragment by the nucleic acid amplification reaction using the genomic DNA as a template.

Description

TECHNICAL FIELD

[001] The present invention relates to a method for producing a DNA library that can be used to analyze a DNA marker, for example, and a method for analyzing genomic DNA using such a DNA library.

FUNDAMENTALS OF THE TECHNIQUE

[002] In general, genomic analysis is performed to conduct comprehensive analysis of genetic information contained in the genome, such as nucleotide sequence information. However, an analysis aimed at determining the nucleotide sequence for the entire genome is disadvantageous in terms of the number of processes and the cost. In cases of organisms with large genomic sizes, furthermore, genomic analysis based on nucleotide sequence analysis has limitations because of the complexity of the genome.

[003] Patent Literature 1 discloses an amplified fragment length polymorphism (AFLP) marker technique in which a sample-specific index is incorporated into a restriction enzyme-treated fragment that has been ligated to an adapter and only a portion of the sequence of the restriction enzyme-treated fragment must be determined. According to the technique disclosed in Patent Literature 1, the complexity of genomic DNA is reduced by treating the genomic DNA with a restriction enzyme, the nucleotide sequence of a target part of the restriction enzyme treated fragment is determined, and the restriction enzyme treated target fragment is thus sufficiently determined. The technique disclosed in Patent Literature 1, however, requires processes such as treating genomic DNA with a restriction enzyme and ligation reaction using an adapter. Therefore, it is difficult to obtain a cost reduction.

[004] However, Patent Literature 2 discloses the following. That is, a DNA marker for identification that is highly correlated with taste assessment results was discovered among the DNA bands obtained through amplification of DNAs extracted from a rice sample through PCR in the presence of suitable primers using the RAPD technique (randomly amplified polymorphic DNA). The method disclosed in Patent Literature 2 involves the use of a plurality of tagged sequence sites (STSs, which are primers) identified by particular sequences. According to the method disclosed in Patent Literature 2, a DNA marker for identification amplified using an STS primer is detected by means of electrophoresis. However, the RAPD technique disclosed in Patent Literature 2 provides significantly poor reproducibility of PCR amplification and, consequently, such a technique cannot be generally adopted as a DNA marker technique.

[005] Patent Literature 3 discloses a method for producing a genomic library in which PCR is performed using a unique type of primer designed based on a sequence that appears relatively and frequently in the target genome, the total genomic region is substantially and uniformly amplified and a genomic library can be produced. Although Patent Literature 3 describes that a genomic library can be produced by conducting PCR using a random primer containing a random sequence, it does not describe any actual procedures or results of experimentation. Consequently, the method described in Patent Literature 3 is deduced to require genome nucleotide sequence information in order to identify the frequency of appearance of the genome, which would increase the number of procedures and the cost. According to the method described in Patent Literature 3, furthermore, the entire genome must be amplified and the complexity of the genomic DNA cannot be disadvantageously reduced.

QUOTE LIST PATENT LITERATURE Patent Literature 1: JP Patent No 5389638 Patent Literature 2: JP (Kokai) Patent Publication No 2003-79375 A Patent Literature 3: JP Patent No 3972106 SUMMARY OF THE INVENTION TECHNICAL PROBLEM

[006] For a technique for analyzing genome information, such as genetic linkage analysis conducted using a DNA marker, the production of a DNA library in a more convenient and highly reproducible manner is desired. As described above, a wide variety of techniques for producing a DNA library are known. To date, however, no techniques known as sufficient in terms of convenience and/or reproducibility have been presented. Under the above circumstances, it is an object of the present invention to provide a method for producing a DNA library with more convenience and greater reproducibility and it is another object to provide a method for analyzing genomic DNA with the use of such a DNA library.

SOLUTION TO THE PROBLEM

[007] The present inventors conducted concentrated studies in order to achieve the above objectives. As a result, they found that high reproducibility can be obtained by conducting PCR using a random primer by designating the concentration of such a random primer within a designated range in a reaction solution. This led to the completion of the present invention.

[008] The present invention includes the following. (1) A method for producing a DNA library, comprising conducting a nucleic acid amplification reaction in a reaction solution containing genomic DNA and a random primer at a high concentration using the genomic DNA as a template to obtain DNA fragments. (2) The method for producing a DNA library according to (1), wherein the reaction solution comprises the random primer at a concentration of 4 to 200 μM. (3) The method for producing a DNA library according to (1), wherein the reaction solution comprises the random primer at a concentration of 4 to 100 μM. (4) The method for producing a DNA library according to (1), wherein the random primer comprises 9 to 30 nucleotides. (5) The method for producing a DNA library according to (1), wherein the DNA fragments comprise 100 to 500 nucleotides. (6) A method for analyzing genomic DNA, comprising using a DNA library produced by the method to produce a DNA library according to any one of (1) to (5) as a DNA marker. (7) The method for analyzing genomic DNA according to (6), which comprises determining the nucleotide sequence of the DNA library produced by the method for producing a DNA library according to any one of (1) to (5) and confirm the presence or absence of the DNA marker based on the nucleotide sequence. (8) The method for analyzing genomic DNA according to (7), in which the presence or absence of the DNA marker is confirmed based on the number of nucleotide sequence reads from the DNA library in the step of confirming the presence or absence of the DNA marker. (9) The method for analyzing genomic DNA according to (7), in which the nucleotide sequence of the DNA library is compared with known sequence information or with the nucleotide sequence of a DNA library produced using the Genomic DNA from a different organism or tissue and the presence or absence of the DNA marker is confirmed based on differences in nucleotide sequences. (10) The method for analyzing genomic DNA according to (6), which comprises: a step of preparing a pair of primers to specifically amplify the DNA marker based on the nucleotide sequence of the DNA marker; a step of conducting a nucleic acid amplification reaction using genomic DNA extracted from a target organism as a template and the primer pair; and a step of confirming the presence or absence of the DNA marker in the genomic DNA based on the results of the nucleic acid amplification reaction. (11) A method for producing a DNA library, comprising: a step of conducting a nucleic acid amplification reaction in a first reaction solution comprising genomic DNA and a random primer at a high concentration to first obtain DNA fragments by the reaction nucleic acid amplification using genomic DNA as a template; and a step of conducting a nucleic acid amplification reaction in a second reaction solution comprising the first obtained DNA fragments and a nucleotide, such as a primer, having a 3' end nucleotide sequence having 70% identity to at least one nucleotide sequence from the 5' end of the random primer to link the nucleotides to the first DNA fragments, thereby obtaining second DNA fragments. (12) The method for producing a DNA library according to (11), wherein the first reaction solution comprises the random primer at a concentration of 4 to 100 μM. (13) The method for producing a DNA library according to (11), wherein the first reaction solution comprises the random primer at a concentration of 4 to 100 μM. (14) The method for producing a DNA library according to (11), wherein the random primer comprises 9 to 30 nucleotides. (15) The method for producing a DNA library according to (11), wherein the first DNA fragments comprise 100 to 500 nucleotides. (16) The method for producing a DNA library according to (11), wherein the primer for amplifying the second DNA fragments comprises a region used for a nucleotide sequencing reaction, or the primer used for a nucleotide sequencing reaction. nucleic acid amplification using the second DNA fragments as templates or a nucleic acid amplification reaction that will be conducted repeatedly comprises a region used for a nucleotide sequencing reaction. (17) A method for analyzing a DNA library, comprising a step of determining a nucleotide sequence for a second DNA fragment obtained by the method for producing a DNA library, according to any one of (11) to (15) , or a DNA fragment obtained using a primer comprising a region complementary to a sequencing primer that will be used in a nucleotide sequencing reaction in the method for producing a DNA library, according to (16). (18) A method for analyzing genomic DNA, comprising using a DNA library produced by the method to produce a DNA library, according to any one of (11) to (17), as a DNA marker. (19) The method for analyzing genomic DNA according to (18), which comprises determining the nucleotide sequence of the DNA library produced by the method for producing a DNA library according to any one of (11) to (17), and confirm the presence or absence of the DNA marker based on the nucleotide sequence. (20) The method for analyzing genomic DNA according to (19), in which the presence or absence of the DNA marker is confirmed based on the number of nucleotide sequence reads from the DNA library in the step of confirming the presence or absence of the DNA marker. (21) The method for analyzing genomic DNA according to (19), in which the nucleotide sequence of the DNA library is compared with the known sequence information or with the nucleotide sequence of a DNA library produced using the Genomic DNA from a different organism or tissue, and the presence or absence of the DNA marker is confirmed based on differences in nucleotide sequences. (22) The method for analyzing genomic DNA according to (18), which comprises: a step of preparing a pair of primers to specifically amplify the DNA marker based on the nucleotide sequence of the DNA marker; a step of conducting a nucleic acid amplification reaction using genomic DNA extracted from a target organism as a template and the primer pair; and a step of confirming the presence or absence of the DNA marker in the genomic DNA based on the results of the nucleic acid amplification reaction. (23) A DNA library, which is produced by the method for producing a DNA library, according to any one of (1) to (5) and (11) to (16).

[009] The present description includes part or all of the contents disclosed in the descriptions and/or drawings of Japanese Patent Application Nos. 2016-129048, 2016178528 and 2017-071020, which are priority documents of the present application.

ADVANTAGEOUS EFFECTS OF THE INVENTION

[010] A DNA library can be produced in a very convenient manner by the method for producing a DNA library, according to the present invention, by the fact that the method is based on a nucleic acid amplification method using random primers . Furthermore, the reproducibility of a nucleic acid fragment to be amplified is excellent in the method for producing a DNA library according to the present invention, even if the method is a nucleic acid amplification method using random primers. Therefore, according to the method for producing a DNA library of the present invention, the DNA library produced can be used as a DNA marker and thus can be used for genomic DNA analysis, such as genetic linkage analysis. .

[011] The method for analyzing genomic DNA using a DNA library, according to the present invention, involves the use of a DNA library produced in a simple manner with excellent reproducibility. Therefore, genomic DNA can be analyzed in a cost-effective manner with high precision.

BRIEF DESCRIPTION OF THE DRAWINGS

[012] Fig. 1 shows a flowchart demonstrating the method for producing a DNA library and the method for analyzing genomic DNA using the DNA library, in accordance with the present invention.

[013] Fig. 2 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library by means of PCR using the DNA of the NiF8 sugarcane variety as a template under general conditions.

[014] Fig. 3 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template at an annealing temperature of 45 °C.

[015] Fig. 4 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template at a girdling temperature of 40 °C.

[016] Fig. 5 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template at a girdling temperature of 37 °C.

[017] Fig. 6 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and 2.5 units of an enzyme.

[018] Fig. 7 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and 12.5 units of an enzyme.

[019] Fig. 8 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and MgCl2 at a concentration doubled from the original level.

[020] Fig. 9 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and MgCl2 at a concentration tripled from the original level.

[021] Fig. 10 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and MgCl2 at a concentration quadrupled from the original level.

[022] Fig. 11 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 8 nucleotides.

[023] Fig. 12 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 9 nucleotides.

[024] Fig. 13 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 11 nucleotides.

[025] Fig. 14 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 12 nucleotides.

[026] Fig. 15 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 14 nucleotides.

[027] Fig. 16 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 16 nucleotides.

[028] Fig. 17 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 18 nucleotides.

[029] Fig. 18 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 20 nucleotides.

[030] Fig. 19 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 2 μM.

[031] Fig. 20 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 4 μM.

[032] Fig. 21 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 6 μM.

[033] Fig. 22 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 6 μM.

[034] Fig. 23 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 8 μM.

[035] Fig. 24 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 8 μM.

[036] Fig. 25 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 10 μM.

[037] Fig. 26 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 10 μM.

[038] Fig. 27 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 20 μM.

[039] Fig. 28 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 20 μM.

[040] Fig. 29 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 40 μM.

[041] Fig. 30 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 40 μM.

[042] Fig. 31 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 60 μM.

[043] Fig. 32 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 60 μM.

[044] Fig. 33 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 100 μM.

[045] Fig. 34 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 100 μM.

[046] Fig. 35 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 200 μM.

[047] Fig. 36 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 200 μM.

[048] Fig. 37 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 300 μM.

[049] Fig. 38 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 300 μM.

[050] Fig. 39 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 400 μM.

[051] Fig. 40 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 400 μM.

[052] Fig. 41 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 500 μM.

[053] Fig. 42 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 500 μM.

[054] Fig. 43 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 600 μM.

[055] Fig. 44 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 700 μM.

[056] Fig. 45 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 800 μM.

[057] Fig. 46 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 900 μM.

[058] Fig. 47 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern of the amplified DNA library using DNA from the sugarcane variety NiF8 as a template and a random primer at a concentration of 1000 μM.

[059] Fig. 48 shows a characteristic diagram demonstrating the results of MiSeq analysis of a DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer.

[060] Fig. 49 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the amplified DNA library using Nipponbare rice variety DNA as a template and a random primer.

[061] Fig. 50 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the amplified DNA library using Nipponbare rice variety DNA as a template and a random primer.

[062] Fig. 51 shows a characteristic diagram demonstrating the results of MiSeq analysis of a DNA library amplified using Nipponbare rice variety DNA as a template and a random primer.

[063] Fig. 52 shows a characteristic diagram demonstrating the positions of MiSeq reading patterns in the genome information of the Nipponbare rice variety.

[064] Fig. 53 shows a characteristic diagram that demonstrates the frequency distribution of the number of mismatched nucleotides between the random primer and the rice genome.

[065] Fig. 54 shows a characteristic diagram that demonstrates the number of reads of the sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at marker N80521152.

[066] Fig. 55 shows a photograph demonstrating the electrophoretic patterns of sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at PCR marker N80521152.

[067] Fig. 56 shows a characteristic diagram that demonstrates the number of reads of the sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at marker N80997192.

[068] Fig. 57 shows a photograph demonstrating the electrophoretic patterns of sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at PCR marker N80997192.

[069] Fig. 58 shows a characteristic diagram that demonstrates the number of reads of the sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at marker N80533142.

[070] Fig. 59 shows a photograph demonstrating the electrophoretic patterns of sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at PCR marker N80533142.

[071] Fig. 60 shows a characteristic diagram that demonstrates the number of reads of the sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at marker N91552391.

[072] Fig. 61 shows a photograph demonstrating the electrophoretic patterns of sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at PCR marker N91552391.

[073] Fig. 62 shows a characteristic diagram that demonstrates the number of reads of the sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at marker N91653962.

[074] Fig. 63 shows a photograph demonstrating the electrophoretic patterns of sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at PCR marker N91653962.

[075] Fig. 64 shows a characteristic diagram that demonstrates the number of reads of the sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at marker N91124801.

[076] Fig. 65 shows a photograph demonstrating the electrophoretic patterns of sugarcane varieties NiF8 and Ni9 and hybrid progeny lines thereof at PCR marker N91124801.

[077] Fig. 66 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 9 nucleotides.

[078] Fig. 67 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 9 nucleotides.

[079] Fig. 68 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 10 nucleotides.

[080] Fig. 69 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 10 nucleotides.

[081] Fig. 70 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 11 nucleotides.

[082] Fig. 71 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 11 nucleotides.

[083] Fig. 72 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 12 nucleotides.

[084] Fig. 73 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 12 nucleotides.

[085] Fig. 74 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 14 nucleotides.

[086] Fig. 75 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 14 nucleotides.

[087] Fig. 76 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 16 nucleotides.

[088] Fig. 77 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 16 nucleotides.

[089] Fig. 78 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 18 nucleotides.

[090] Fig. 79 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 18 nucleotides.

[091] Fig. 80 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 20 nucleotides.

[092] Fig. 81 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer comprising 20 nucleotides.

[093] Fig. 82 shows a characteristic diagram demonstrating the results of investigating the reproducibility of the DNA library amplified using DNA from the NiF8 sugarcane variety as a template and random primers comprising 8 to 35 nucleotides used in a concentration from 0.6 to 300 μM.

[094] Fig. 83 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 1 type of random primer.

[095] Fig. 84 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 1 type of random primer.

[096] Fig. 85 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 2 types of random primers.

[097] Fig. 86 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 2 types of random primers.

[098] Fig. 87 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 3 types of random primers.

[099] Fig. 88 shows a characteristic diagram that demonstrates a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 3 types of random primers.

[0100] Fig. 89 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 12 types of random primers.

[0101] Fig. 90 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 12 types of random primers.

[0102] Fig. 91 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 24 types of random primers.

[0103] Fig. 92 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 24 types of random primers.

[0104] Fig. 93 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 48 types of random primers.

[0105] Fig. 94 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and 48 types of random primers.

[0106] Fig. 95 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer B comprising 10 nucleotides.

[0107] Fig. 96 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer B comprising 10 nucleotides.

[0108] Fig. 97 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer C comprising 10 nucleotides.

[0109] Fig. 98 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer C comprising 10 nucleotides.

[0110] Fig. 99 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer D comprising 10 nucleotides.

[0111] Fig. 100 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer D comprising 10 nucleotides.

[0112] Fig. 101 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer E comprising 10 nucleotides.

[0113] Fig. 102 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer E comprising 10 nucleotides.

[0114] Fig. 103 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer F comprising 10 nucleotides.

[0115] Fig. 104 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer F comprising 10 nucleotides.

[0116] Fig. 105 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using human genomic DNA as a template and a random primer A comprising 10 nucleotides.

[0117] Fig. 106 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using human genomic DNA as a template and a random primer A comprising 10 nucleotides.

[0118] Fig. 107 schematically shows a characteristic diagram of a method for producing a DNA library applied to a next generation sequencer.

[0119] Fig. 108 schematically shows a characteristic diagram of a method for producing a DNA library applied to a next generation sequencer.

[0120] Fig. 109 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer G comprising 10 nucleotides.

[0121] Fig. 110 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer G comprising 10 nucleotides.

[0122] Fig. 111 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using a NiF8 sugarcane variety DNA library produced using a random primer G comprising 10 nucleotides as a template and a next generation sequencer.

[0123] Fig. 112 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using a NiF8 sugarcane variety DNA library produced using a random primer G comprising 10 nucleotides as a template and a next generation sequencer.

[0124] Fig. 113 shows a characteristic diagram demonstrating the results of MiSeq analysis of a DNA library amplified using DNA from the sugarcane variety NiF8 as a template and a random primer G comprising 10 nucleotides.

[0125] Fig. 114 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using Nipponbare rice variety DNA as a template and a random primer B comprising 12 nucleotides.

[0126] Fig. 115 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using Nipponbare rice variety DNA as a template and a random primer B comprising 12 nucleotides.

[0127] Fig. 116 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which first appeared time) from the DNA library amplified using a Nipponbare rice variety DNA library produced using a random primer B comprising 12 nucleotides as a template and a next generation sequencer.

[0128] Fig. 117 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a fluorescence unit (FU), in which the amplified fragment length is determined based on an electrophoretic pattern (which appeared for the second time) from the DNA library amplified using a Nipponbare rice variety DNA library produced using a random primer B comprising 12 nucleotides as a template and a next generation sequencer.

[0129] Fig. 118 shows a characteristic diagram demonstrating a distribution of the reading pattern obtained by MiSeq analysis of a DNA library amplified using DNA from the Nipponbare rice variety as a template and a random primer B comprising 12 nucleotides and the degree of consistency between the random primer sequence and the reference sequence of the Nipponbare rice variety.

[0130] Fig. 119 shows a characteristic diagram demonstrating the results of MiSeq analysis of a DNA library amplified using Nipponbare rice variety DNA as a template and a random primer B comprising 12 nucleotides.

DESCRIPTION OF EMBODIMENTS

[0131] Hereinafter, the present invention is described in detail.

[0132] According to the method for producing a DNA library of the present invention, a nucleic acid amplification reaction is conducted in a reaction solution, which is prepared to contain a primer having an arbitrary nucleotide sequence (hereinafter hereafter referred to as “random primer”) at a high concentration, and the amplified nucleic acid fragment is determined to be a DNA library. The term “high concentration” used in this report means that the concentration is higher than the primer concentration in a general nucleic acid amplification reaction. Specifically, the method for producing a DNA library of the present invention is characterized by the fact that a random primer is used in a higher concentration than a primer used in a general nucleic acid amplification reaction. As a template contained in a reaction solution, genomic DNA prepared from a target organism for which a DNA library is produced can be used.

[0133] In the method for producing a DNA library of the present invention, a target organism species is not particularly limited and a target organism species can be any species of organism, such as an animal, including a human, a plant, a microorganism, or a virus. In other words, according to the method for producing a DNA library of the present invention, a DNA library can be produced from any species of organism.

[0134] In the method for producing a DNA library of the present invention, the concentration of a random primer is specified, as described above. Thus, a nucleic acid fragment (or nucleic acid fragments) can be amplified with high reproducibility. The term “reproducibility” used in this report means a degree of agreement between nucleic acid fragments amplified by a plurality of nucleic acid amplification reactions carried out using the same template and the same random primer. That is, the term “high reproducibility (or the expression “reproducibility is high”)” means that the degree of concordance between nucleic acid fragments amplified by a plurality of nucleic acid amplification reactions carried out using the same template and from the same random initiator is high.

[0135] The degree of reproducibility can be assessed, for example, by conducting a plurality of nucleic acid amplification reactions using the same template and the same random primer, calculating the Spearman rank correlation coefficient for the fluorescence unit (FU) obtained as a result of electrophoresis of the resulting amplified fragments and assessment of the degree of reproducibility based on such coefficient. The Spearman rank correlation coefficient is generally represented by the symbol p. When p is greater than 0.9, for example, the reproducibility of the amplification reaction of interest can be assessed as sufficient.

RANDOM STARTER

[0136] A sequence that constitutes a random primer that can be used in the method for producing a DNA library, according to the present invention, is not particularly limited. For example, a random primer comprising nucleotides comprising 9 to 30 nucleotides can be used. In particular, a random primer may be composed of any nucleotide sequence comprising 9 to 30 nucleotides, a nucleotide type (i.e., a sequence type) is not particularly limited, and a random primer may be composed of 1 or more types of nucleotide sequences, preferably 1 to 10,000 types of nucleotide sequences, more preferably 1 to 1,000 types of nucleotide sequences, more preferably 1 to 100 types of nucleotide sequences, and most preferably 1 to 96 types of nucleotide sequences. By using nucleotides (or a group of nucleotides) within the range mentioned above for a random primer, an amplified nucleic acid fragment can be obtained with greater reproducibility. When a random primer comprises a plurality of nucleotide sequences, it is not necessary that all of the nucleotide sequences comprise the same number of nucleotides (9 to 30 nucleotides). A random primer may comprise a plurality of nucleotide sequences composed of a different number of nucleotides.

[0137] In general, in order to obtain a specific amplicon by a nucleic acid amplification reaction, the nucleotide sequence of a primer corresponding to the amplicon is designated. For example, a primer pair is designed such that the primers are placed between a site corresponding to an amplicon of a template DNA of genomic DNA or the like. In such a case, as the primers are designed to be hybridized to a specific region included in a template, they may be referred to as “specific primers”.

[0138] However, a random primer is different from a primer that is designed to obtain a specific amplicon and is designed to obtain a random amplicon, but not to be hybridized to a specific region of a template DNA. A random primer can have any nucleotide sequence and can contribute to random amplification of the amplicon when it is incidentally hybridized to a region included in the template DNA.

[0139] In other words, a random primer can be considered as nucleotides involved in the random amplification of the amplicon comprising an arbitrary sequence, as described above. In this report, such an arbitrary sequence is not particularly limited. However, it may be designated, for example, as a nucleotide sequence randomly selected from the group consisting of adenine, guanine, cytosine and thymine or a specific nucleotide sequence. Examples of a specific nucleotide sequence include a nucleotide sequence including a restriction enzyme recognition sequence or a nucleotide sequence that presents an adapter sequence used for a next generation sequencer.

[0140] When designating various types of nucleotides for random primers, it is possible to use a method for designating a plurality of nucleotide sequences having certain lengths by randomly selecting the group consisting of adenine, guanine, cytosine and thymine. Furthermore, when designating different types of nucleotides for random primers, it is also possible to use a method for designating a plurality of nucleotide sequences comprising a common part consisting of a specific nucleotide sequence and a non-common part consisting of a sequence of arbitrary nucleotides. In this report, the non-common part may consist of a nucleotide sequence randomly selected from the group consisting of adenine, guanine, cytosine and thymine or combinations of four types of nucleotides which are adenine, guanine, cytosine and thymine. The common part is not particularly limited and can consist of any nucleotide sequence. It may consist, for example, of a nucleotide sequence including a restriction enzyme recognition sequence, a nucleotide sequence that presents an adapter sequence used for a next generation sequencer, or a nucleotide sequence common in a specific family of genes .

[0141] When assigning various types of nucleotide sequences having certain lengths by randomly selecting nucleotides from four types of nucleotides to a plurality of random primers, 30% or more, preferably 50% or more, more preferably 70% or more, and more preferably 90% or more of the total, such sequences exhibit 70% or less, preferably 60% or less, more preferably 50% or less, and most preferably 40% or less. less identity. By designating different types of nucleotide sequences having certain lengths by randomly selecting nucleotides from different types of nucleotides to a plurality of random primers exhibiting identity within such a range, an amplified fragment can be obtained from total genomic DNA. of the target organism species. Thus, the uniformity of the amplified fragment can be enhanced.

[0142] When designating a plurality of nucleotide sequences comprising a common part consisting of a specific nucleotide sequence and a non-common part consisting of an arbitrary nucleotide sequence for a plurality of random primers, it is possible to designate, e.g. , a nucleotide sequence comprising a non-common part consisting of several nucleotides on the 3' end side and a common part consisting of the remaining nucleotides on the 5' end side. By allowing a non-common part to consist of n number of nucleotides on the 3' side, it is possible to designate 4n types of random primers. In this report, the expression “number n” may refer to 1 to 5, preferably 2 to 4, and most preferably 2 to 3.

[0143] For example, it is possible to designate, as a random primer comprising a common part and a non-common part, 16 types of random primers in total, each of which presents an adapter sequence (common part) used for a next sequencer. generation on the 5' end side and two nucleotides (non-common part) on the 3' end side in total. It is possible to designate 64 types of random primers in total by adjusting the number of nucleotides on the 3' end side to 3 nucleotides (non-common part). The more types of random primers, the more comprehensively amplified fragments can be obtained across the entire genomic DNA of the target organism species. Therefore, when designating a random primer consisting of a common part and a non-common part, it is preferable that 3 nucleotides exist on the 3' end side.

[0144] However, for example, after designating 64 types of nucleotide sequences comprising a common part and a non-common part consisting of 3 nucleotides, no more than 63 types of random primers selected from these 64 types of nucleotide sequences nucleotides can be used. In other words, compared to the case of using all 64 types of random primers, in the case of using no more than 63 types of random primers, excellent results can be obtained in a nucleic acid amplification reaction or analysis using a next generation sequencer. Specifically, when 64 types of random primers are used, the number of reads from a specific nucleic acid amplification fragment can become considerably large. In such a case, favorable analysis results can be obtained using the remaining 63 random primers, excluding one or more random primers involved in amplification of the specific nucleic acid amplification fragment from 64 types of random primers.

[0145] Similarly, in the case of designating 16 types of random primers comprising a common part and a non-common part of 2 nucleotides, when no more than 15 types of random primers selected from 16 types of random primers are used, results Favorable analysis results can be obtained in a nucleic acid amplification reaction or analysis using a next generation sequencer.

[0146] The nucleotides constituting a random primer are preferably designated such that the G-C content is from 5% to 95%, more preferably from 10% to 90%, more preferably from 15% to 80%, and, more preferably, from 20% to 70%. With the use of a group of nucleotides having a G-C content within the above range as a random primer, amplified nucleic acid fragments can be obtained with enhanced reproducibility. The G-C content is the percentage of guanine and cytosine contained in the total nucleotide chain.

[0147] Furthermore, the nucleotides that constitute a random primer are designated such that consecutive nucleotides account for, preferably, 80% or less, more preferably, 70% or less, more preferably, 60% or less, and, most preferably , 50% or less with respect to the total sequence length. Alternatively, the nucleotides constituting a random primer are designated such that the number of consecutive nucleotides is preferably 8 or less, more preferably 7 or less, more preferably 6 or less, and most preferably 5 or less. An amplified nucleic acid fragment can be obtained with enhanced reproducibility by using a group of nucleotides constituting a random primer, for which the number of consecutive nucleotides falls within the above range.

[0148] Furthermore, it is preferred that the nucleotides constituting a random primer are designed not to constitute a complementary region of 6 or more, more preferably, 5 or more, and, most preferably, 4 or more nucleotides in a molecule. When the nucleotides designated do not constitute a complementary region within the above range, double-strand formation occurring in a molecule can be prevented and amplified nucleic acid fragments can be obtained with enhanced reproducibility.

[0149] Furthermore, when multiple types of nucleotides are designed for a random primer, in particular, it is preferred that a plurality of nucleotides is designed not to constitute a complementary region of 6 or more, more preferably, 5 or more, and, more preferably, 4 or more nucleotides, while forming a plurality of nucleotide sequences. When different types of nucleotide sequences are designated, double-strand formation that occurs between nucleotide sequences can be prevented, and amplified nucleic acid fragments can be obtained with enhanced reproducibility.

[0150] When various types of nucleotides are designed for random primers, it is preferred that the nucleotides are designed not to constitute a complementary sequence of 6 or more, more preferably, 5 or more, and, most preferably, 4 or more nucleotides on the side from the 3' end. When they are designed not to form a complementary sequence within the above range on the 3' end side, the double-strand formation that occurs between the nucleotide sequences can be prevented, and amplified nucleic acid fragments can be obtained with enhanced reproducibility. .

[0151] The terms “complementary region” and “complementary sequence” refer, for example, to a region and a sequence that exhibit 80% to 100% identity (e.g., a region and a sequence comprising 5 nucleotides, wherein the 4 or 5 nucleotides are complementary to each other) or a region and a sequence that exhibit 90% to 100% identity (for example, a region and a sequence comprising 5 nucleotides, wherein the 5 nucleotides are complementary to each other).

[0152] Furthermore, nucleotides constituting a random primer are preferably designed to present a Tm value suitable for thermal cycling conditions (in particular, an annealing temperature) in a nucleic acid amplification reaction. A Tm value can be calculated by a conventional method, such as the nearest neighbor base pair method, the Wallace method, or the GC% method, although a calculation method is not particularly limited thereto. Specifically, the nucleotides used for a random primer are preferably designed to have a Tm value of 10°C to 85°C, more preferably 12°C to 75°C, more preferably 14°C to 70°C , and, more preferably, 16°C to 65°C. By assigning Tm values to nucleotides within the above range, amplified nucleic acid fragments can be obtained with enhanced reproducibility under given thermal cycling conditions (in particular, at a given annealing temperature) in a nucleic acid amplification reaction. .

[0153] Furthermore, when different types of nucleotides constituting a random primer are designated, in particular, a variation for Tm between a plurality of nucleotides is, preferably, 50 ° C or less, more preferably, 45 ° C or less, more preferably 40°C or less, and most preferably 35°C or less. When nucleotides are designed such that a variation for Tm among a plurality of nucleotides falls within the above range, amplified nucleic acid fragments can be obtained with enhanced reproducibility under given thermal cycling conditions (in particular, at a given temperature annealing) in a nucleic acid amplification reaction.

NUCLEIC ACID AMPLIFICATION REACTION

[0154] According to the method for producing a DNA library of the present invention, many amplification fragments are obtained through a nucleic acid amplification reaction conducted using the random primer and genomic DNA as a template described above. In particular, in such a nucleic acid amplification reaction, the concentration of a random primer in a reaction solution is set higher than the concentration of the primer in a usual nucleic acid amplification reaction. Thus, many amplification fragments can be obtained using genomic DNA as a template, while achieving high reproducibility. The many amplification fragments obtained can be used for a DNA library that can be applied for genotyping and the like.

[0155] A nucleic acid amplification reaction is a reaction to synthesize amplification fragments in a reaction solution containing genomic DNA as a template, the random primers mentioned above, DNA polymerase, deoxynucleoside triphosphate as a substrate (i.e., dNTP, which is a mixture of dATP, dCTP, dTTP and dGTP), and a buffer under given thermal cycling conditions. As it is necessary to add Mg2+ at a given concentration to a reaction solution in a nucleic acid amplification reaction, the buffer of the above composition contains MgCl2. When the buffer does not contain MgCl2, MgCl2 is still added to the above composition.

[0156] In particular, in a nucleic acid amplification reaction, it is preferable to appropriately adjust the concentration of a random primer according to the nucleotide length of the random primer. When different types of nucleotides constitute random primers with different nucleotide lengths, the average of the nucleotide lengths of the random primers can be adjusted as the nucleotide length (the average can be a simple average or the weighted average considering the number of nucleotides).

[0157] Specifically, a nucleic acid amplification reaction is conducted using a random primer comprising 9 to 30 nucleotides at a random primer concentration of 4 to 200 μM and, preferably, 4 to 100 μM. Under such conditions, many amplified fragments and, in particular, many amplified fragments comprising 100 to 500 nucleotides through a nucleic acid amplification reaction can be obtained, while achieving high reproducibility.

[0158] More specifically, when a random primer comprises 9 to 10 nucleotides, the concentration of the random primer is preferably 40 to 60 μM. When a random primer comprises 10 to 14 nucleotides, it is preferred that the concentration of the random primer is 100 μM or less and y > 3E + 08x-6.974, provided that the nucleotide length of the random primer is represented by “x” and the concentration of the random initiator is represented by “y”. When a random primer comprises 14 to 18 nucleotides, the concentration of the random primer is preferably 4 to 100 μM. When a random primer comprises 18 to 28 nucleotides, the concentration of the random primer is preferably 4 μM or more and y < 8E + 08x-5.533. When a random primer comprises 28 to 29 nucleotides, the concentration of the random primer is preferably 6 to 10 μM. By adjusting the random primer concentration according to the nucleotide length of a random primer as described above, many amplified fragments can be obtained with improved safety while achieving high reproducibility.

[0159] As described in the Examples below, the inequalities above (y > 3E + 08x-6.974 and y < 8E + 08x-5.533) are developed so that they are capable of representing the random primer concentration at which many DNA fragments comprising 100 to 500 nucleotides can be obtained with favorable reproducibility as a result of thorough inspection of the correlation between random primer length and random primer concentration.

[0160] The amount of genomic DNA as a template in a nucleic acid amplification reaction is not particularly limited. However, it is preferably 0.1 to 1000 ng, more preferably 1 to 500 ng, more preferably 5 to 200 ng, and most preferably 10 to 100 ng, when the amount of reaction solution is 50 μl. By adjusting the amount of genomic DNA as a template within the above range, many amplified fragments can be obtained without inhibiting the amplification reaction with a random primer, while achieving high reproducibility.

[0161] Genomic DNA can be prepared according to a conventional technique without particular limitations. With the use of a commercially available kit, genomic DNA can be easily prepared from a target organism species. Genomic DNA extracted from an organism according to a conventional technique or with the use of a commercially available kit may be used, genomic DNA extracted from an organism and purified may be used, or genomic DNA subjected to restriction enzyme treatment or ultrasonic treatment can be used.

[0162] The DNA polymerase used in a nucleic acid amplification reaction is not particularly limited and an enzyme that exhibits DNA polymerase activity under thermal cycling conditions for a nucleic acid amplification reaction can be used. Specifically, heat-stable DNA polymerase used for a general nucleic acid amplification reaction can be used. Examples of DNA polymerase include DNA polymerase derived from thermophilic bacteria, such as Taq DNA polymerase, and DNA polymerase derived from hyperthermophilic archaea, such as KOD DNA polymerase and Pfu DNA polymerase. In a nucleic acid amplification reaction, it is particularly preferable to use Pfu DNA polymerase as DNA polymerase in combination with the random primer described above. With the use of such DNA polymerases, many amplified fragments can be obtained with improved safety, while achieving high reproducibility.

[0163] In a nucleic acid amplification reaction, the concentration of deoxynucleoside triphosphate as a substrate (i.e., dNTP, which is a mixture of dATP, dCTP, dTTP, and dGTP) is not particularly limited and can be 5 μM to 0 6 mM, preferably 10 μM to 0.4 mM, and most preferably 20 μM to 0.2 mM. By adjusting the concentration of dNTP as a substrate within such a range, errors caused by incorrect incorporation by DNA polymerase can be prevented and many amplified fragments can be obtained, while achieving high reproducibility.

[0164] A buffer used in a nucleic acid amplification reaction is not particularly limited. For example, a solution comprising MgCl2 as described above, Tris-HCl (pH 8.3) and KCl can be used. The concentration of Mg2+ is not particularly limited. For example, it may be 0.1 to 4.0 mM, preferably 0.2 to 3.0 mM, more preferably 0.3 to 2.0 mM, and most preferably 0.5 to 1.5 mM. . By designating the concentration of Mg2+ in the reaction solution within such a range, many amplified fragments can be obtained, while achieving high reproducibility.

[0165] The thermal cyclization conditions of a nucleic acid amplification reaction are not particularly limited and a common thermal cycle can be adopted. A specific example of a thermal cycle comprises a first step of thermal denaturation in which genomic DNA as a template is dissociated into single strands, a cycle comprising thermal denaturation, annealing and extension repeated several times (e.g., 20 to 40 times), an extension stage during a given period of time according to the need and the final storage stage.

[0166] Thermal denaturation can be carried out, for example, between 93 °C to 99 °C, preferably, 95 °C to 98 °C, and, more preferably, 97 °C to 98 °C. Annealing can be carried out, for example, between 30°C to 70°C, preferably 35°C to 68°C, and more preferably 37°C to 65°C, although it may vary depending on the value of Tm from a random initiator. Extension may be carried out, for example, at 70°C to 76°C, preferably 71°C to 75°C, and more preferably 72°C to 74°C. Storage can be carried out, for example, at 4 °C.

[0167] The first step of thermal denaturation can be carried out within the temperature range described above for a period, for example, from 5 seconds to 10 minutes, preferably, 10 seconds to 5 minutes, and, more preferably, 30 seconds to 2 minutes. In the cycle comprising “thermal denaturation, annealing and extension”, thermal denaturation may be carried out within the temperature range described above for a period of, for example, 2 seconds to 5 minutes, preferably 5 seconds to 2 minutes, and more preferably 10 seconds to 1 minute. In the cycle comprising "thermal denaturation, annealing and extension", annealing may be carried out within the temperature range described above for a period of, for example, 1 second to 3 minutes, preferably 3 seconds to 2 minutes, and most preferably , 5 seconds to 1 minute. In the cycle comprising "thermal denaturation, annealing and extension", extension may be carried out within the temperature range described above for a period of, for example, 1 second to 3 minutes, preferably 3 seconds to 2 minutes, and most preferably , 5 seconds to 1 minute.

[0168] According to the method for producing a DNA library of the present invention, amplified fragments can be obtained by a nucleic acid amplification reaction that uses a hot start method. The hot start method is intended to prevent erroneous initiation or non-specific amplification caused by primer-dimer formation prior to the cycle comprising “thermal denaturation, annealing and extension”. The hot start method involves the use of an enzyme in which the DNA polymerase activity has been suppressed by binding an anti-DNA polymerase antibody to it or chemically modifying it. Thus, DNA polymerase activity can be suppressed and a non-specific reaction before thermal cycling can be prevented. According to the hot start method, a temperature is set high in the first thermal cycle, DNA polymerase activity is thereby recovered, and the subsequent nucleic acid amplification reaction is allowed to proceed.

[0169] As described above, many amplified fragments can be obtained using genomic DNA as a template and a random primer by conducting a nucleic acid amplification reaction using a random primer comprising 9 to 30 nucleotides. and adjust its concentration between 4 and 200 μM in a reaction solution. By using the random primer comprising 9 to 30 nucleotides by adjusting its concentration between 4 to 200 μM in a reaction solution, a nucleic acid amplification reaction can be carried out with very high reproducibility. According to the nucleic acid amplification reaction specifically, many amplified fragments can be obtained, while achieving very high reproducibility. Therefore, the many amplified fragments obtained can be used for a DNA library in genetic analysis that targets genomic DNA.

[0170] By carrying out a nucleic acid amplification reaction using the random primer comprising 9 to 30 nucleotides and adjusting the concentration thereof in a reaction solution between 4 to 200 μM, in particular, many amplified fragments comprising about 100 to 500 nucleotides can be obtained using genomic DNA as a template. Such many amplified fragments comprising about 100 to 500 nucleotides are suitable for mass analysis of nucleotide sequences using, for example, a next generation sequencer, and highly accurate sequence information can be obtained. According to the present invention, a DNA library, including DNA fragments comprising about 100 to 500 nucleotides, can be produced.

[0171] By carrying out a nucleic acid amplification reaction using a random primer comprising 9 to 30 nucleotides and adjusting its concentration between 4 and 200 μM in a reaction solution, in particular, the amplified fragments can be obtained uniformly through genomic DNA. In other words, DNA fragments are amplified in a distributed manner throughout the genome, but not in a manner localized to a specific region of genomic DNA in a nucleic acid amplification reaction using such a random primer. That is, according to the present invention, a DNA library can be produced uniformly across the entire genome.

[0172] After carrying out the nucleic acid amplification reaction using the random primer mentioned above, restriction enzyme treatment, size selection treatment, sequence capture treatment and the like can be carried out on the obtained amplified fragments. By performing restriction enzyme treatment, size selection treatment, and sequence capture treatment on the amplified fragments, specific amplified fragments (a fragment having a specific restriction enzyme site, an amplified fragment with a size range) specific sequence and an amplified fragment that presents a specific sequence) can be obtained among the amplified fragments obtained. Then, the specific amplified fragments obtained by these treatments can be used for a DNA library.

GENOMIC DNA ANALYSIS METHOD

[0173] With the use of the DNA library produced in the manner described above, genomic DNA analysis, such as genotyping, can be performed. Such a DNA library has very high reproducibility, its size is suitable for a next generation sequencer and it presents uniformity across the entire genome. Consequently, the DNA library can be used as a DNA marker (also referred to as a “genetic marker” or “gene marker”). The term “DNA marker” refers to a broad range of characteristic nucleotide sequences present in genomic DNA. Furthermore, a DNA marker can especially be a nucleotide sequence in the genome that serves as a marker associated with genetic traits. A DNA marker can be used, for example, for genotype identification, linkage mapping, gene mapping, breeding comprising a selection step using a marker, backcrossing using a marker, quantitative trait locus mapping, bulk segregated analysis, variety identification or discontinuous disequilibrium mapping.

[0174] For example, the nucleotide sequence of a DNA library prepared as described above is determined using a next generation sequencer or the like and the presence or absence of a DNA marker can be confirmed based on the nucleotide sequence obtained.

[0175] As an example, the presence or absence of a DNA marker can be confirmed from the number of reads of the nucleotide sequence obtained. Although a next-generation sequencer is not particularly limited, such a sequencer is also referred to as a “second-generation sequencer”, and such a sequencer is an apparatus for sequencing nucleotides that allows the simultaneous determination of nucleotide sequences of several tens of millions of DNA fragments. The sequencing principle of a next-generation sequencer is not particularly limited. For example, sequencing can be performed, according to a method in which sequencing is performed, while amplifying and synthesizing target DNA in flow cells by the bridge PCR method and the sequencing-by-synthesis method, or, in other words, according to a method in which sequencing is performed by emulsion PCR and the Pyrosequencing method to analyze the amount of pyrophosphoric acids released after DNA synthesis. More specific examples of next-generation sequencers include the MiniSeq, MiSeq, NextSeq, HiSeq, and HiSeq X Series (Illumina, Inc.) and Roche 454 GS FLX sequencers (Roche).

[0176] In another example, the presence or absence of a DNA marker can be confirmed by comparing the nucleotide sequence obtained for the DNA library prepared, as described above, with the reference nucleotide sequence. In this report, reference nucleotide sequence means a sequence known as a reference, and may be, for example, a known sequence stored in a database. That is, a DNA library is prepared, as described above, for a given organism, its nucleotide sequence is determined, and the nucleotide sequence of the DNA library is compared with the reference nucleotide sequence. A nucleotide sequence that differs from the reference nucleotide sequence can be designated as a DNA marker (a characteristic nucleotide sequence existing in genomic DNA) related to the organism. For each specified DNA marker, relevance to the genetic trait (phenotype) can be determined by additional analysis according to a conventional method. In other words, a DNA marker related to a phenotype (sometimes referred to as a “selective marker”) can be identified among the identified DNA markers as described above.

[0177] Furthermore, in another example, the presence or absence of a DNA marker can be confirmed by comparing the nucleotide sequence obtained for the DNA library prepared, as described above, with the nucleotide sequence of a DNA library prepared as described above using genomic DNA from a different organism or tissue. In other words, a DNA library is prepared, as described above, for each of two or more organisms or two different tissues, the nucleotide sequences thereof are determined, and the nucleotide sequences of the DNA libraries are compared to each other. . Then, a nucleotide sequence that differs between DNA libraries can be designated as a DNA marker (a characteristic nucleotide sequence existing in genomic DNA) related to the experimented organism or tissue. For each specified DNA marker, relevance to the genetic trait (phenotype) can be determined by additional analysis according to a conventional method. In other words, a DNA marker related to a phenotype (sometimes referred to as a “selective marker”) can be identified among the identified DNA markers as described above.

[0178] As a aside, it is also possible to designate a pair of primers that specifically amplify the DNA marker based on the nucleotide sequence obtained. It is also possible to confirm the presence or absence of the DNA marker in the extracted genomic DNA by performing a nucleic acid amplification reaction using a designated primer pair and genomic DNA extracted from a target organism as a template.

[0179] Alternatively, DNA libraries prepared as described above can be used for metagenomic analysis to examine a wide variety of microorganisms and the like, mutation analysis of the genome of somatic cells from tumor tissue or the like, genotyping using microarrays, ploidy determination and analysis, calculation and analysis of chromosome number, analysis of increase and decrease of chromosomes, analysis of insertion/deletion/replication/partial translocation of chromosomes, analysis of contamination with foreign genome, analysis of kinship discrimination and cross-seed purity testing and analysis.

APPLICATION OF NEXT GENERATION SEQUENCING TECHNOLOGY

[0180] As described above, by conducting a nucleic acid amplification reaction with a random primer contained in a high concentration in a reaction solution, it is possible to obtain many amplified fragments with favorable reproducibility using genomic DNA as a template . Since each amplified fragment obtained has the nucleotide sequence at both ends of it that are the same as the random primer, it can be easily applied to next-generation sequencing technology using the nucleotide sequence.

[0181] Specifically, as described above, a nucleic acid amplification reaction is conducted in a reaction solution (first reaction solution) containing genomic DNA and a random primer at a high concentration to obtain many amplified fragments (first DNA fragments ) using genomic DNA as a template. Then, a nucleic acid amplification reaction is conducted in a reaction solution (second reaction solution) containing the many amplified fragments obtained (first DNA fragments) and a primer designed based on the nucleotide sequence of the random primer (referred to as as “next generation sequencer starter”). A next-generation sequencer primer that will be used in this report is a nucleotide sequence including a region used for a nucleotide sequencing reaction. More specifically, for example, the primer of the next generation sequencer may be a nucleotide sequence that presents a region necessary for a nucleotide sequencing reaction (sequence reaction) by a next generation sequencer, wherein the nucleotide sequence in the 3' end of the primer is a nucleotide sequence that shows 70% or more identity, preferably 80% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, more preferably, 97% or more identity, and more preferably, 100% identity to the nucleotide sequence on the 5' end side of the first DNA fragment.

[0182] In this report, the “region used for a nucleotide sequencing reaction” included in a next generation sequencer primer is not particularly limited, due to the fact that it may vary depending on the type of next generation sequencer. However, in the case of conducting a nucleotide sequencing reaction using a next generation sequencer with a sequencing primer, such a region may be, for example, a nucleotide sequence complementary to the nucleotide sequence of the sequencing primer. In a case where a sequencing reaction is conducted by a next-generation sequencer using capture beads linked to the supplied DNA, the “region used for a nucleotide sequencing reaction” refers to a nucleotide sequence complementary to the nucleotide sequence of the DNA bound to the capture beads. Furthermore, in a case where a next-generation sequencer reads a sequence based on a current change when a DNA strand featuring a terminal loop passes through a protein that features nanosized pores, the “region used for a sequencing reaction of nucleotides” may be a nucleotide sequence complementary to the nucleotide sequence that forms the loop.

[0183] By designating the nucleotide sequence at the 3' end of a next generation sequencer primer as described above, the next generation sequencer primer can be hybridized to the 3' end of the first DNA fragment under stringent conditions, and the second DNA fragment can be amplified using the first DNA fragment as a template. Stringent conditions mean conditions under which a specific hybrid is formed while a non-specific hybrid is not formed. For example, such conditions may be appropriately determined with reference to Molecular Cloning: A Laboratory Manual (Third Edition). Specifically, stringency can be determined by adjusting the temperature and salt concentration in a solution after Southern hybridization, and the temperature and salt concentration in a solution in the washing step of Southern hybridization. More specifically, for example, the sodium concentration is adjusted between 25 to 500 mM and preferably 25 to 300 mM, and the temperature is adjusted between 42 °C to 68 °C and preferably 42 °C to 65 °C , under stringent conditions. More specifically, the sodium concentration is 5 x SSC (83 mM NaCl, 83 mM sodium citrate) and the temperature is 42 °C.

[0184] In particular, when different types of random primers are used to obtain a first DNA fragment, next generation sequencer primers can be prepared to match all or some random primers.

[0185] For example, in a case where a group of different types of random primers (which have an arbitrary 3' end sequence of several nucleotides) comprising a common nucleotide sequence other than several nucleotides (e.g., about 1 to 3 nucleotides) at the 3' end is used, all the first DNA fragments obtained have a common 5' end sequence. Accordingly, the 3' end nucleotide sequence of a next generation sequencer primer is designed to be a nucleotide sequence that has 70% or more identity to the 5' end nucleotide sequence common to the first DNA fragments. By assigning next-generation sequencer primers as described above, it is possible to obtain next-generation sequencer primers corresponding to all random primers. Through the use of such next generation sequencer primers, it is possible to amplify the second DNA fragments using all of the first DNA fragments as templates.

[0186] Similarly, further in a case where a group of different types of random primers (which have an arbitrary 3' end sequence of several nucleotides) comprising a common nucleotide sequence other than several nucleotides (e.g., about 1 to 3 nucleotides) at the 3' end is used, it is also possible to obtain the second DNA fragments using some of the first DNA fragments obtained as templates. Specifically, the 3' end nucleotide sequence of a next generation sequencer primer is designed to be a nucleotide sequence that has 70% or more identity to the 5' end nucleotide sequence common to the first DNA fragments and the sequence comprising several nucleotides following the nucleotide sequence (corresponding to several nucleotides (arbitrary sequence) at the 3' end of the random primer), such that the second DNA fragments can be amplified using some of the first DNA fragments as templates.

[0187] However, in a case where the first DNA fragments are obtained using different types of random primers consisting of an arbitrary nucleotide sequence, it is possible to obtain the second DNA fragments using different types of next generation sequencer primers , such that the second DNA fragments correspond to all of the first DNA fragments, or it is also possible to obtain the second DNA fragments using different types of next generation sequencer primers, such that the second DNA fragments correspond to some of the first DNA fragments.

[0188] As described above, the second DNA fragments amplified using next-generation sequencer primers present a region necessary for a nucleotide sequencing reaction (sequence reaction) by a next-generation sequencer, which is included in the sequencer primers. next generation. The region required for a sequence reaction is not particularly limited and can vary depending on a next-generation sequencer. For example, when a next generation sequencer primer is used in a next generation sequencer based on the principle that sequencing is performed during amplification and synthesis of target DNA in flow cells by the bridge PCR method and the method In sequencing-by-synthesis, the next-generation sequencer primer must contain a region required for bridging PCR and a region required for the sequencing-by-synthesis method. The region required for bridging PCR is a region that is hybridized to an oligonucleotide immobilized on flow cells and is 9 nucleotides long, including the 5' end of the next generation sequencer primer. Furthermore, a region necessary for the sequencing by synthesis method is a region to which a sequencing primer used in a sequencing reaction is hybridized, and is a region in the middle of the primer of the next generation sequencer.

[0189] Additionally, a next generation sequencer may be an Ion Torrent sequencer. In the case of using the Ion Torrent sequencer, a next-generation sequencer primer presents an ion adapter on the 5' end side and binds to a particle to conduct emulsion PCR. Furthermore, in the Ion Torrent sequencer, particles coated with an emulsion PCR-amplified template are placed on an ion chip and subjected to a sequence reaction.

[0190] In this report, a nucleic acid amplification reaction using a next generation sequencer primer and a second reaction solution containing the first DNA is not particularly limited and conventional conditions for the nucleic acid amplification reaction can be applied. That is, the conditions in NUCLEIC ACID AMPLIFICATION REACTION described above can be used. For example, the second reaction solution contains the first DNA fragments as templates, the primer of the next generation sequencer described above, DNA polymerase, deoxynucleoside triphosphate as a substrate (i.e., dNTP, which is a mixture of dATP, dCTP, dTTP and dGTP) and a buffer.

[0191] In particular, the primer concentration of the next generation sequencer can be adjusted between 0.01 to 5.0 μM, preferably 0.1 to 2.5 μM, and most preferably 0.3 to 0. 7μM.

[0192] Although the amount of the first DNA fragments that serve as templates in a nucleic acid amplification reaction is not particularly limited, it is preferably 0.1 to 1000 ng, more preferably 1 to 500 ng, more preferably, 5 to 200 ng, and, more preferably, 10 to 100 ng when the amount of reaction solution is 50 μl.

[0193] A method for preparing the first DNA fragments as templates is not particularly limited. In the method, the reaction solution obtained after completion of the nucleic acid amplification reaction using the random primers described above can be used or the reaction solution can be used after purifying the first DNA fragments therefrom.

[0194] With respect to the type of DNA polymerase, the concentration of deoxynucleoside triphosphate as a substrate (dNTP, i.e., a mixture of dATP, dCTP, dTTP and dGTP), the composition of the buffer and the temperature cycling conditions used to the nucleic acid amplification reaction, the conditions in NUCLEIC ACID AMPLIFICATION REACTION described above can be used. Furthermore, in a nucleic acid amplification reaction using the next generation sequencer primers, a hot start method can be used or the amplified fragments can be obtained by a nucleic acid amplification reaction.

[0195] As described above, through the use of the first DNA fragments obtained using random primers as templates and using the second DNA fragments amplified using next generation sequencer primers, it is possible to easily prepare a DNA library that can be applied to a next generation sequencer.

[0196] In the above examples, a DNA library is prepared using the first DNA fragments obtained using random primers as templates and amplifying the second DNA fragments using the next generation sequencer primers. However, the scope of the present invention is not limited to such examples. For example, the DNA library according to the present invention can be prepared by amplifying the second DNA fragments using the first DNA fragments obtained using the random primers as templates and further obtaining the third DNA fragments using the second DNA fragments as next generation sequencer templates and primers, thereby obtaining a DNA library of third DNA fragments applicable to a next generation sequencer.

[0197] Similarly, in order to prepare a DNA library applicable to a next generation sequencer, after a nucleic acid amplification reaction using the second DNA fragments as templates, a nucleic acid amplification reaction is repeatedly conducted using DNA fragments obtained as templates and next-generation sequencer primers are used for the final nucleic acid amplification reaction. In such a case, the number of nucleic acid amplification reactions that will be repeated is not particularly limited, but is 2 to 10 times, preferably 2 to 5 times, and most preferably 2 to 3 times.

EXAMPLES

[0198] Hereinafter, the present invention is described in more detail with reference to the Examples below, although the scope of the present invention is not limited to these Examples.

1. Flowchart

[0199] In this example, a DNA library was prepared through PCR using genomic DNAs extracted from various types of organism species as templates and various groups of random primers, according to the flowchart shown in Fig. 1. Furthermore, using the prepared DNA library, sequence analysis was performed by a next generation sequencer and the genotype was analyzed based on the read data obtained.

2. Materials

[0200] In this example, genomic DNAs were extracted from the sugarcane varieties NiF8 and Ni9, 22 hybrid progeny lines thereof and the rice variety Nipponbare using the DNeasy Plant Mini Kit (QIAGEN) and the DNAs Extracted genomic genomes were purified. The purified genomic DNAs were used as NiF8-derived genomic DNA, Ni9-derived genomic DNA, genomic DNAs from 22 hybrid progeny lines, and Nipponbare-derived genomic DNA, respectively. In this example, Human Genomic DNA was purchased as human DNA from TakaraBio and used as human-derived genomic DNA.

3. Method 3.1. Correlation between PCR conditions and DNA fragment sizes 3.1.1. Random initiator designation

[0201] In order to designate the random primers, the GC content was adjusted between 20% and 70% and the number of consecutive nucleotides was adjusted to 5 or less. The nucleotide length was adjusted at 16 levels (i.e., 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 30, and 35 nucleotides). For each nucleotide length, 96 types of nucleotide sequences were designated and a set of 96 types of random primers were prepared for each nucleotide length. With respect to the 10-nucleotide primers, 6 groups (each comprising 96 types of random primers) were designated (these 6 groups are referred to as 10-nucleotide primer A and 10-nucleotide primer F). In this example specifically, 21 different groups of random primers were prepared.

[0202] Tables 1 to 21 show the nucleotide sequences of random primers contained in these 21 different groups of random primers.

3.1.2. Standard PCR

[0203] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers (final concentration: 0.6 μM; 10 nucleotide primer A), a mixture of 0.2 mM dNTP, MgCl2 1.0 mM and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. In this example, several nucleic acid fragments obtained through PCR using the random primers, including the standard PCR described above, are referred to as a DNA library.

3.1.3. DNA library purification and electrophoresis

[0204] The DNA library obtained in 3.1.2 above was purified using the MinElute PCR Purification Kit (QIAGEN) and subjected to electrophoresis using the Agilent 2100 bioanalyzer (Agilent Technologies) to obtain one unit of fluorescence ( FU).

3.1.4. Annealing temperature examination

[0205] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers (final concentration: 0.6 μM, 10 nucleotide primer A), a mixture of 0.2 mM dNTP, MgCl2 1.0 mM and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, different annealing temperatures for 15 seconds and 72°C for 20 seconds, followed by storage at 4°C. In this example, 37 °C, 40 °C and 45 °C were examined as annealing temperatures. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.1.5. Examination of the amount of enzyme

[0206] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers (final concentration: 0.6 μM, 10 nucleotide primer A), a mixture of 0.2 mM dNTP, MgCl21 .0 mM and 2.5 units or 12.5 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.1.6. Examination of MgCl2 concentration

[0207] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers (final concentration: 0.6 μM, 10 nucleotide primer A), a mixture of 0.2 mM dNTP, MgCl2 at a given concentration and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. In this example, concentrations of two, three and four times a usual concentration were examined as MgCl2 concentrations. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.1.7. Examination of random primer nucleotide length

[0208] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers (final concentration: 0.6 μM), a mixture of 0.2 mM dNTP, 1.0 mM MgCl2 and 1 .25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. In this example, primers that have 8 nucleotides (Table 7), 9 nucleotides (Table 8), 11 nucleotides (Table 9), 12 nucleotides (Table 10), 14 nucleotides (Table 11), 16 nucleotides (Table 12), 18 nucleotides (Table 13) and 20 nucleotides (Table 14) were examined as random primers. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.1.8. Random Initiator Concentration Examination

[0209] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers at a given concentration (primer A of 10 nucleotides), a mixture of 0.2 mM dNTP, 1.0 mM MgCl2 and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. In this example, 2, 4, 6, 8, 10, 20, 40, 60, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM were examined as random concentrations. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3. Furthermore, in this experiment, the reproducibility of repeated data was assessed based on Spearman rank correlation (p > 0.9).

3.2. Verification of reproducibility using MiSeq 3.2.2. DNA library preparation

[0210] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers (final concentration: 60 μM, 10 nucleotide primer A), a mixture of 0.2 mM dNTP, 1 MgCl2, 0 mM and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.2.3. Sequence library preparation

[0211] From the DNA library obtained in 3.2.1, a library sequence for MiSeq analysis was prepared using the KAPA Library Preparation Kit (Roche).

3.2.4. MiSeq Analysis

[0212] Using the MiSeq V2 500 Cycle Reagent Kit (Illumina), the sequence library for MiSeq analysis obtained in 3.2.2 was analyzed using 100 base paired terminal sequencing.

3.2.5. Analysis of reading data

[0213] Random primer sequence information was suppressed from the read data obtained in 3.2.3, and read patterns were identified. The number of reads was counted for each reading pattern, the number of reads for repeated analyzes and reproducibility were assessed using the correlational coefficient.

3.3. Nipponbare Rice Variety Analysis 3.3.1. DNA library preparation

[0214] To the genomic DNA described in 2. above (30 ng, Nipponbare-derived genomic DNA), random primers (final concentration: 60 μM, 10 nucleotide primer A), a mixture of 0.2 mM dNTP, 1 MgCl2, 0 mM and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.3.2. Sequence library preparation, MiSeq analysis, and read data analysis

[0215] Preparation of a sequence library using the DNA library prepared from Nipponbare-derived genomic DNA, MiSeq analysis and analysis of the read data were performed according to the methods described in 3.2.2, 3.2.3 and 3.2.4, respectively.

3.3.3. Assessment of genomic homogeneity

[0216] The reading patterns obtained in 3.3.2 were mapped to the genomic information of Nipponbare (NC_008394 to NC_008405) using bowtie2 and the genomic positions of the reading patterns were identified.

3.3.4. Non-specific amplification

[0217] Based on the positional information of the reading patterns identified in 3.3.3, the sequences of the random primers were compared to the genome sequences to which such random primers would be annealed, and the number of erroneous pairings was determined.

3.4. Polymorphism detection and genotype identification 3.4.1. DNA library preparation

[0218] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA, Ni9-derived genomic DNA, hybrid progeny-derived genomic DNA, or Nipponbare-derived genomic DNA), random primers (final concentration: 60 μM , 10-nucleotide primer A), a mixture of 0.2 mM dNTP, 1.0 mM MgCl2 and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared by adjusting the reaction level final in 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.4.2. HiSeq Analysis

[0219] Analysis of the DNA libraries prepared in 3.4.1 was assigned to TakaraBio under conditions where the number of samples was 16 per band through 100-base paired terminal sequencing and read data was obtained.

3.4.3. Analysis of reading data

[0220] Random primer sequence information was suppressed from the read data obtained in 3.4.2 and read patterns were identified. The number of reads was counted for each reading pattern.

3.4.4. Polymorphism detection and genotype identification

[0221] Based on the reading patterns and the number of readings obtained as a result of the analysis conducted in 3.4.3, polymorphisms peculiar to NiF8 and Ni9 were detected and the reading patterns thereof were designated as markers. Based on the number of reads, the genotypes of the 22 hybrid progeny lines were identified. Accuracy for genotype identification was assessed based on the reproducibility obtained by repeated data with respect to the 22 hybrid progeny lines.

3.5. Experiment for confirmation with PCR marker 3.5.1. Initiator designation

[0222] Primers were assigned to a total of 6 markers (i.e., 3 NiF8 markers and 3 Ni9 markers) among the markers identified in 3.4.4 based on marker sequence information obtained through paired-end sequencing (Table 22). Table 22. Marker Sequence Information and PCR Marker Primer Information *: Paired terminal sequence

3.5.2. PCR and electrophoresis

[0223] Using the TaKaRa Multiplex Ver.2 PCR Assay Kit (TAKARA) and the genomic DNA described in 2. above (15 ng, NiF8-derived genomic DNA, Ni9-derived genomic DNA, or progeny-derived genomic DNA hybrid) as a template, 1.25 μl of Multiplex PCR enzyme mix, 12.5 μl of 2x Multiplex PCR buffer and the primer designated 0.4 μM in 3.5.1 were added and a reaction solution was prepared, adjusting the final reaction level to 25 μl. PCR was performed under thermal cycling conditions comprising 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds and holding at 72°C for 10 minutes. followed by storage at 4°C. The amplified DNA fragment was subjected to electrophoresis using TapeStation (Agilent Technologies).

3.5.3. Comparison of genotype data

[0224] Based on the electrophoresis results obtained in 3.5.2, the marker genotype was identified based on the presence or absence of a band and the results were compared to the number of marker reads.

3.6. Correlation between random primer density and length 3.6.1. Influence of random primer length at high concentration

[0225] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers having given lengths (final concentration: 10 μM), a mixture of 0.2 mM dNTP, 1.0 mM MgCl2 and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. In this experiment, 9 nucleotides (Table 8), 10 nucleotides (Table 1, 10-nucleotide primer A), 11 nucleotides (Table 9), 12 nucleotides (Table 10), 14 nucleotides (Table 11), 16 nucleotides (Table 12) , 18 nucleotides (Table 13) and 20 nucleotides (Table 14) were examined as random primer lengths. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. In the reaction system using random primers comprising 10 or more nucleotides, PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.6.2. Correlation between random primer density and length

[0226] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), random primers of a given length were added at a given concentration therein, a mixture of 0.2 mM dNTP, 1.0 MgCl2 mM and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added to them and a reaction solution was prepared by adjusting the final reaction level to 50 μl. In this experiment, the random primers comprising 8 to 35 nucleotides shown in Tables 1 to 21 were examined and the random primer concentration of 0.6 to 300 μM was examined.

[0227] In the reaction system using random primers comprising 8 nucleotides and 9 nucleotides, PCR was carried out under thermal cycling conditions comprising 98 °C for 2 minutes and 30 cycles of 98 °C for 10 seconds, 37 °C for 15 seconds and 72°C for 20 seconds, followed by storage at 4°C. In the reaction system using a random primer of 10 or more nucleotides, PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72° C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3. Furthermore, the reproducibility of repeated data was assessed based on Spearman rank correlation (p > 0.9).

3.7. Number of random primers

[0228] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), 1, 2, 3, 12, 24 or 48 types of random primers selected from the 96 types of random primers comprising 10 nucleotides ( primer A of 10 nucleotides) shown in Table 1 were added to the final concentration of 60 μM in them, a mixture of 0.2 mM dNTP, 1.0 mM MgCl2 and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added to them and a reaction solution was prepared, adjusting the final reaction level to 50 μl. In this experiment, as 1, 2, 3, 12, 24 or 48 types of random primers, the random primers were selected successively from No. 1 shown in Table 1, and the selected primers afterwards were examined. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3. Furthermore, the reproducibility of repeated data was assessed based on Spearman rank correlation (p > 0.9).

3.8. Random primer sequence

[0229] To the genomic DNA described in 2. above (30 ng, NiF8-derived genomic DNA), a group of primers selected from the 5 groups of random primers shown in Tables 2 to 6 were added at the final concentration of 60 μM in the same, a mixture of 0.2 mM dNTP, 1.0 mM MgCl2 and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added to them and a reaction solution was prepared, adjusting the final reaction level to 50 μl . PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3. Furthermore, the reproducibility of repeated data was assessed based on Spearman rank correlation (p > 0.9).

3.9. DNA library using human-derived genomic DNA

[0230] To the genomic DNA described in 2. above (30 ng, human-derived genomic DNA), random primers (final concentration: 60 μM, 10 nucleotide primer A), a mixture of 0.2 mM dNTP, 1 MgCl2 .0 mM and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same way as in 3.1.3. Furthermore, the reproducibility of repeated data was assessed based on Spearman rank correlation (p > 0.9).

4. Results and examination 4.1. Correlation between PCR conditions and DNA library size

[0231] When PCR was conducted using random primers, according to conventional PCR conditions (3.1.2 described above), the size of the amplified DNA library was 2 kbp or greater, but the amplification of the DNA library was DNA of a target size (i.e., 100 bp to 500 bp) was not observed (Fig. 2). A DNA library of 100 bp to 500 bp may not be obtainable because it is highly unlikely that a random primer will function as a primer in a region of 500 bp or smaller. In order to prepare a DNA library of the target size (i.e., 100 bp to 500 bp), it was considered necessary to induce nonspecific amplification with high reproducibility.

[0232] The correlation between the annealing temperature (3.1.4 above), the amount of enzyme (3.1.5 above), the MgCl2 concentration (3.1.6 above), the primer length (3.1.7 above) and Primer concentration (3.1.8 above), which are considered to affect PCR specificity, and DNA library size were examined.

[0233] Fig. 3 shows the results of the experiment described in 3.1.4 obtained at an annealing temperature of 45 °C, Fig. 4 shows the results obtained at an annealing temperature of 40 °C and Fig. 5 shows the results obtained at an annealing temperature of 37 °C. By reducing the annealing temperature from 45 °C, 40 °C to 37 °C, as shown in Figs. 3 to 5, increased amounts of amplified high molecular weight DNA library, although amplification of the low molecular weight DNA library is not observed.

[0234] Fig. 6 shows the results of the experiment described in 3.1.5 obtained when the amount of enzyme is increased by 2 times and Fig. 7 shows the results obtained when the amount of enzyme is increased by 10 times the original amount . By increasing the amount of enzyme by 2 times or 10 times a common amount, as shown in Figs. 6 and 7, the increased amounts of the amplified high molecular weight DNA library, although amplification of the low molecular weight DNA library is not observed.

[0235] Fig. 8 shows the results of the experiment described in 3.1.6 obtained when the MgCl2 concentration is increased by 2 times a common amount, Fig. 9 shows the results obtained when the MgCl2 concentration is increased by 3 times and Fig. 10 shows the results obtained when the MgCl2 concentration is increased by 4 times. By increasing the concentration of MgCl2 by 2 times, 3 times and 4 times the common amount, as shown in Figs. 8 to 10, the amounts of the amplified high molecular weight DNA library varied, although amplification of a low molecular weight DNA library was not observed.

[0236] Figs. 11 to 18 show the results of the experiment described in 3.1.7 obtained at random primer lengths of 8 nucleotides, 9 nucleotides, 11 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides and 20 nucleotides, respectively. Regardless of the length of a random primer, as shown in Figs. 11 to 18, no significant change was observed compared to the results shown in Fig. 2 (a random primer comprising 10 nucleotides).

[0237] The results of the experiment described in 3.1.8 are summarized in Table 23. Table 23

[0238] With the use of random primers comprising 10 nucleotides, as shown in Figs. 19 to 47, amplification was observed on a 1 kbp DNA fragment at a random primer concentration of 6 μM. As the concentration increased, the molecular weight of a DNA fragment decreased. Reproducibility at random primer concentration of 6 to 500 μM was examined. As a result, a relatively low p-value of 0.889 was obtained at the concentration of 6 μM, which is 10 times higher than the usual level. At a concentration of 8 μM, which is equivalent to 13.3 times higher than the usual level, and at 500 μM, which is 833.3 times higher than the usual level, a high p-value of 0.9 or more It was obtained. The results demonstrate that a DNA fragment of 1 kbp or less can be amplified while achieving high reproducibility by raising the random primer concentration to a level significantly greater than the concentration used under general PCR conditions. When the random primer concentration is excessively greater than 500 μM, amplification of a DNA fragment of a desired size cannot be observed. In order to amplify a low molecular weight DNA fragment with excellent reproducibility, it has consequently been found that the concentration of the random primer must be in an optimal range, which is greater than the concentration used in a general PCR procedure and equivalent to , or less than, a given level.

4.2. Confirmation of reproducibility using MiSeq

[0239] In order to confirm reproducibility for DNA library production, as described in 3.2 above, the DNA library amplified using genomic DNAs extracted from NiF8 as a template and random primers was analyzed using of a next generation sequencer (MiSeq), and the results are shown in Fig. 48. As a result of 3.2.4 above, 47,484 read patterns were obtained. As a result of comparing the number of readings obtained through the repeated measurements, a high correlation (i.e., a correlational coefficient “r” of 0.991) was obtained, just as with the electrophoresis results. Consequently, it was considered that a DNA library can be produced with satisfactory reproducibility using random primers.

4.3. Nipponbare Rice Variety Analysis

[0240] As described in 3.3 above, a DNA library was prepared using genomic DNAs extracted from the Nipponbare rice variety, the genomic information of which was disclosed, as a template, and random primers and subjected to electrophoresis, and the results are shown in Figs. 49 and 50. Based on the results shown in Figs. 49 and 50, the p value was 0.979. Furthermore, Fig. 51 shows the results of analyzing read data using MiSeq. Based on the results shown in Fig. 51, the correlational coefficient “r” was 0.992. These results demonstrate that a rice DNA library can be produced with very high reproducibility using random primers.

[0241] As described in 3.3.3, the obtained reading pattern was mapped to Nipponbare genomic information. As a result, DNA fragments were equally amplified throughout the genome at 6.2 kbp intervals (Fig. 52). As a result of comparing the sequence and genome information of random primers, 3.6 mismatches were discovered on average, and one or more mismatches were observed in 99.0% of primer pairs (Fig. 53). The results demonstrate that a DNA library involving the use of random primers is produced with satisfactory reproducibility through non-specific amplification uniformly throughout the genome.

4.4. Detection of polymorphism and identification of sugarcane genotype

[0242] As described in 3.4, DNA libraries from sugarcane varieties NiF8 and Ni9 and 22 hybrid progeny lines were produced using random primers, the resulting DNA libraries were analyzed with the next step sequencer generation (HiSeq), the polymorphisms of the parent varieties were detected and the genotypes of the hybrid progenies were identified based on the reading data. Table 24 shows the results. Table 24. Number of markers and genotyping accuracy of sugarcane varieties NiF8 and Ni9

[0243] As shown in Table 24, 8,683 markers for NiF8 and 11,655 markers for Ni9; that is, a total of 20,338 markers were produced. Furthermore, the reproducibility for identifying the genotype of hybrid progeny lines was 99.97%. This indicates that the accuracy for genotype identification is very high. In particular, sugarcane is polyploid (8x + n), the number of chromosomes is 100 to 130, and the genome size is 10 Gbp, which is at least 3 times larger than that of humans. Consequently, it is very difficult to identify the genotype across all genomic DNA. As described above, several markers can be produced using random primers and the sugarcane genotype can be identified with high precision.

4.5. Experiment for confirmation with the PCR marker

[0244] As described in 3.5 above, the sugarcane varieties NiF8 and Ni9 and 22 hybrid progeny lines were subjected to PCR using the primers shown in Table 22, the genotypes were identified through electrophoresis and the results were compared with the number of readings. Figs. 54 and 55 show the number of readings and the electrophoretic pattern of the NiF8 marker N80521152, respectively. Figs. 56 and 57 show the number of readings and the electrophoretic pattern of the NiF8 marker N80997192, respectively. Figs. 58 and 59 show the number of readings and the electrophoretic pattern of the NiF8 marker N80533142, respectively. Figs. 60 and 61 show the number of readings and the electrophoretic pattern of the Ni9 marker N91552391, respectively. Figs. 62 and 63 show the number of reads and the electrophoretic pattern of the Ni9 marker N91653962, respectively. Figs. 64 and 65 show the number of reads and the electrophoretic pattern of the Ni9 marker N91124801, respectively.

[0245] As shown in Figs. 54 to 65, the results for all PCR markers designated in 3.5 above were consistent with the results of analysis using a next generation sequencer. Thus, it was considered that genotype identification using a next generation sequencer would be applicable as a marker technique.

4.6. Correlation between random primer density and length

[0246] As described in 3.6.1, the results of DNA library production using random primers comprising 9 nucleotides (Table 8), 10 nucleotides (Table 1, 10 nucleotide primer A), 11 nucleotides (Table 9 ), 12 nucleotides (Table 10), 14 nucleotides (Table 11), 16 nucleotides (Table 12), 18 nucleotides (Table 13) and 20 nucleotides (Table 14) are shown in Figs. 66 to 81. The results are summarized in Table 25. Table 25

[0247] When random primers were used at a high concentration of 10.0 μM, which is 13.3 times higher than the usual level, as shown in Figs. 66 to 81, it was discovered that a low molecular weight fragment of DNA can be amplified using random primers comprising 9 to 20 nucleotides, while obtaining very high reproducibility. As the nucleotide length of a random primer increased (12 nucleotides or more, in particular), the molecular weight of the amplified fragment likely decreased. When random primers comprising 9 nucleotides were used, the amount of amplified DNA fragment was increased by adjusting the annealing temperature to 37 °C.

[0248] In order to clarify the correlation between the density and length of random primers, as described in 3.6.2 above, PCR was performed using random primers comprising 8 to 35 nucleotides at a concentration of 0.6 to 300 μM, in order to produce a DNA library. The results are shown in Table 26. Table 26. The correlation between the concentration and length of the Random Primer for the DNA library O: DNA library covering 100 to 500 bases can be amplified with satisfactory reproducibility (P>0.9) x: DNA library not covering 100 to 500 bases or reproducibility was low (p<=0.9) -: Not performed

[0249] As shown in Table 26, it has been discovered that a low molecular weight DNA fragment (100 to 500 nucleotides) can be amplified with high reproducibility with the use of random primers comprising 9 to 30 nucleotides between 4.0 to 200 μM . In particular, it was confirmed that low molecular weight DNA fragments (100 to 500 nucleotides) can be safely amplified with high reproducibility using random primers comprising 9 to 30 nucleotides at 4.0 to 100 μM.

[0250] The results shown in Table 26 are examined in more detail. As a result, the correlation between length and random primer concentration preferably lies within a range surrounded by a structure, as shown in Fig. 82. More specifically, the random primer concentration is preferably 40 to 60 μM, when random primers comprise 9 to 10 nucleotides. It is preferred that a concentration of the random primer satisfies the condition represented by an inequality: y > 3E + 08x-6.974, as long as the nucleotide length of the random primer is represented by x and the concentration of the random primer is represented by y, and 100 μM or smaller, when the random primer comprises 10 to 14 nucleotides. The concentration of the random primer is preferably 4 to 100 mM when the random primer comprises 14 to 18 nucleotides. When a random primer comprises 18 to 28 nucleotides, the concentration of the random primer is preferably 4 μM or greater, and satisfies the condition represented by an inequality: y < 8E + 08x-5.533. When a random primer comprises 28 to 29 nucleotides, the concentration of the random primer is preferably 4 to 10 μM. The inequalities y > 3E + 08x-6.974 and y < 8E + 08x-5.533 are determined based on the Microsoft Excel power approximation.

[0251] By prescribing the number of nucleotides and the concentration of random primers within the ranges provided, as described above, it was discovered that low molecular weight DNA fragments (100 to 500 nucleotides) can be amplified with high reproducibility. For example, the accuracy of data obtained through the analysis of high molecular weight DNA fragments using a next generation sequencer is known to deteriorate to a significant extent. As described in this example, the number of nucleotides and concentration of random primers can be prescribed within provided ranges so that a DNA library with a molecular size suitable for analysis with a next generation sequencer can be produced with satisfactory reproducibility. , and such a DNA library may be suitable for marker analysis using a next generation sequencer.

4.7. Number of random primers

[0252] As described in 3.7 above, 1, 2, 3, 12, 24 or 48 types of random primers (concentration: 60 μM) were used to produce a DNA library and the results are shown in Figs. 83 to 94. The results are summarized in Table 27. Table 27

[0253] As shown in Figs. 83 to 94, it was discovered that low molecular weight DNA fragments can be amplified using any of 1, 2, 3, 12, 24 or 48 types of random primers, while achieving very high reproducibility. In particular, it should be understood that as the number of random primer types increases, a peak in the electrophoretic pattern decreases, and a shift probably disappears. 4.8. Random primer sequence

[0254] As described in 3.8 above, DNA libraries were produced using the random primer groups shown in Tables 2 to 6 (i.e., 10-nucleotide primer B, 10-nucleotide primer C, 10-nucleotide primer D , 10-nucleotide primer E and 10-nucleotide primer F), and the results are shown in Figs. 95 to 104. The results are summarized in Table 28. Table 28

[0255] As shown in Figs. 95 to 104, it was discovered that low molecular weight DNA fragments can be amplified using any groups of 10-nucleotide primer B, 10-nucleotide primer C, 10-nucleotide primer D, 10-nucleotide primer E, or primer F. of 10 nucleotides, while achieving very high reproducibility.

4.9. Human DNA library production

[0256] As described in 3.9 above, a DNA library was produced using human-derived genomic DNA and random primers at a final concentration of 60 μM (10 nucleotide primer A), and the results are shown in Figs. . 105 and 106. Fig. 105 shows the results of the first repeated experiment and Fig. 106 shows the results of the second repeated experiment. As shown in Figs. 105 and 106, it was discovered that low molecular weight DNA fragments can be amplified while achieving very high reproducibility even if human-derived genomic DNA is used.

Example 2 1. Flowchart

[0257] In this example, the first DNA fragments were prepared by PCR using genomic DNA as a template and random primers, according to the schematic diagrams shown in Figs. 107 and 108. Subsequently, second DNA fragments were prepared by PCR using the first DNA fragments as templates and next generation sequencer primers. The prepared second DNA fragments were used as a sequencer library to conduct sequence analysis using a next-generation sequencer. The genotype was analyzed based on the reading data obtained.

2. Materials

[0258] In this example, genomic DNAs were extracted from the sugarcane variety NiF8 and the rice variety Nipponbare using the DNeasy Plant Mini Kit (QIAGEN) and the extracted genomic DNAs were purified. The purified genomic DNAs were used as NiF8-derived genomic DNA and Nipponbare-derived genomic DNA, respectively.

3. Method 3.1. Examination of NiF8 sugarcane variety 3.1.1. Designation of random primers and next generation sequencer primers

[0259] In this example, random primers were designed based on the 10 nucleotides 3' end of the next generation sequencer adapter (Nextera adapter, Illumina, Inc.). Specifically, in this example, GTTACACACG (SEQ ID NO: 2041, G of 10 nucleotides) was used as a random primer. Furthermore, next generation sequencer primers were designed based on the sequence information in the Nextera adapter from Illumina, Inc. in the manner mentioned above (Table 29). Table 29

3.1.2. DNA library preparation

[0260] A mixture of dNTP at a final concentration of 0.2 mM, MgCl2 at a final concentration of 1.0 mM and DNA Polymerase (TAKARA, PrimeSTAR) at a final concentration of 1.25 units and a random primer (G of 10 nucleotides) at a final concentration of 60 μM were added to the NiF8-derived genomic DNA (30 ng) described in 2. above. A DNA library (first DNA fragments) was prepared by PCR (treatment at 98 °C for 2 minutes, reaction for 30 cycles of 98 °C for 10 seconds, 50 °C for 15 seconds and 72 °C for 20 seconds and storage at 4 °C) in a final reaction volume of 50 μl.

3.1.3. Purification and electrophoresis

[0261] The DNA library obtained in 3.1.2 above was purified using the MinElute PCR Purification Kit (QIAGEN) and subjected to electrophoresis using the Agilent 2100 bioanalyzer (Technologies) to obtain one fluorescence unit (FU ). Furthermore, the reproducibility of repeated data was assessed based on Spearman rank correlation (p > 0.9).

3.1.4. Next Generation Sequencer DNA Library Preparation

[0262] A mixture of dNTP at a final concentration of 0.2 mM, MgCl2 at a final concentration of 1.0 mM, DNA Polymerase (TAKARA, PrimeSTAR) at a final concentration of 1.25 units and a DNA sequencer primer next generation at a final concentration of 0.5 μM the first DNA fragment (100 ng) purified in 3.1.3 above was added. A next generation sequencer DNA library (second DNA fragments) was prepared by PCR (treatment at 95°C for 2 minutes, reaction for 25 cycles of 98°C for 15 seconds, 55°C for 15 seconds, 72° C for 20 seconds, treatment at 72 °C for 1 minute and storage at 4 °C) in a final reaction volume of 50 μl. The DNA library for a next generation sequencer was subjected to purification and electrophoresis in the same way as in 3.1.3.

3.1.5. MiSeq Analysis

[0263] The DNA library from the next generation sequencer (a second DNA fragment) in 3.1.4 above was analyzed by MiSeq via 100 base paired end sequencing using MiSeq V2 500 Cycle Reagent Kit (Illumina).

3.1.6. Analysis of reading data

[0264] Reading patterns were identified from reading data obtained in 3.1.5. The number of reads was counted for each reading pattern, for the number of reads from the repeated analyses, and reproducibility was assessed using the correlational coefficient.

3.2. Examination of Nipponbare Rice Variety 3.2.1. Designation of random primers and next generation sequencer primers

[0265] In this example, the random primers were designed based on 10 nucleotides from the 3' end of the Nextera next generation sequencer adapter from Illumina, Inc. That is, in this example, a sequence of 10 nucleotides positioned at the 3' end of the Nextera adapter and 16 types of nucleotide sequences prepared by adding an arbitrary 2-nucleotide nucleotide sequence to the 3' end of the 10-nucleotide sequence for results in a total length of 12 nucleotides were designated as random primers (Table 30 , B of 12 nucleotides). Table 30

[0266] Furthermore, in this example, a next generation sequencer primer was designated based on the sequence information in the Nextera adapter from Illumina, Inc. in the same manner as in 3.1.1.

3.2.2. DNA library preparation

[0267] A mixture of dNTP at a final concentration of 0.2 mM, MgCl2 at a final concentration of 1.0 mM and DNA Polymerase (TAKARA, PrimeSTAR) at a final concentration of 1.25 units and a random primer (B of 12 nucleotides) at a concentration of 40 μM were added to the Nipponbare-derived genomic DNA (30 ng) described in 2. above. A DNA library (first DNA fragments) was prepared by PCR (treatment at 98 °C for 2 minutes, reaction for 30 cycles of 98 °C for 10 seconds, 50 °C for 15 seconds, 72 °C for 20 seconds and storage at 4 °C) in a final reaction volume of 50 μl.

3.2.3. Purification and electrophoresis

[0268] The DNA library obtained in 3.2.2 above was purified using the MinElute PCR Purification Kit (QIAGEN) and subjected to electrophoresis using the Agilent 2100 bioanalyzer (Technologies) to obtain one fluorescence unit (FU ). Furthermore, the reproducibility of repeated data was assessed based on Spearman rank correlation (p > 0.9).

3.2.4. Next Generation Sequencer DNA Library Preparation

[0269] A mixture of dNTP at a final concentration of 0.2 mM, MgCl2 at a final concentration of 1.0 mM, DNA Polymerase (TAKARA, PrimeSTAR) at a final concentration of 1.25 units and a DNA sequencer primer next generation at a concentration of 0.5 μM were added to the first DNA fragment (100 ng) purified in 3.2.3 above. A next generation sequencer DNA library (second DNA fragments) was prepared by PCR (treatment at 95°C for 2 minutes, reaction for 25 cycles of 98°C for 15 seconds, 55°C for 15 seconds, 72° C for 20 seconds, treatment at 72 °C for 1 minute and storage at 4 °C) in a final reaction volume of 50 μl. DNA library purification for next generation sequencers and electrophoresis were conducted in the same manner as in 3.1.3.

3.2.5. MiSeq Analysis

[0270] The DNA library from the next generation sequencer (second DNA fragment) in 3.2.4 above was analyzed by MiSeq via 100 base paired end sequencing using MiSeq V2 500 Cycle Reagent Kit (Illumina).

3.2.6. Analysis of reading data

[0271] The reading patterns in 3.2.5 were mapped to the genomic information of Nipponbare (NC_008394 to NC_008405) using bowtie2, the degree of consistency between the random primer sequence and the genomic DNA was confirmed. Reading patterns were identified from reading data obtained in 3.2.5. The number of reads was counted for each reading pattern, for the number of reads from the repeated analyses, and reproducibility was assessed using the correlational coefficient.

4. Results and Exam 4.1. NiF8 sugarcane variety examination results

[0272] Figs. 109 and 110 show the results of electrophoresis after conducting PCR using a random primer consisting of 10 nucleotides (G of 10 nucleotides) from the 3' end of the next generation sequencer adapter (Nextera adapter, Illumina, Inc.) in a high concentration of 60 μl. As shown in Figs. 109 and 110, amplification was observed in a broad region ranging from 100 bp to 500 bp (the first DNA fragment). It was considered that amplification can be observed over a wide region, due to the fact that amplification was also observed in a region, except the region of genomic DNA corresponding to the random primer. Furthermore, since the rank correlation coefficient between the repeated data was 0.957 (>0.9), reproducibility was confirmed in the amplification pattern.

[0273] Next, Figures 111 and 112 show the results of electrophoresis after conducting PCR using the next generation sequencer primer in the same way as in 3.1.4. That is, in order to prepare a DNA library (second DNA fragments) linked to a next generation sequencer adapter (Nextera adapter), PCR was conducted using a next generation sequencer primer comprising the Nextera adapter sequence from Illumina, Inc. and the first DNA fragment as a template. The accuracy of analysis using the Illumina, Inc. next generation sequencer is significantly reduced in a case where the DNA library includes short fragments that are 100 bp in length or less or long fragments that are 1 kbp in length or less. larger. Since the next-generation sequencer DNA library (second DNA fragments) prepared in this example was mainly distributed in a range of 150 bp to 1 kbp with a peak around 500 bp, as illustrated in Figs. 111 and 112, the DNA library was considered as an appropriate next generation sequencer DNA library. Furthermore, since the rank correlation coefficient between the repeated data was 0.989 (>0.9), reproducibility was confirmed in the amplification pattern.

[0274] Furthermore, as a result of analysis of the DNA library (second DNA fragment) by the MiSeq next generation sequencer, 3.5 Gbp read data and 3.6 Gbp read data were obtained. The values indicating accuracy of MiSeq data (>= Q30) were 93.3% and 93.1%. Since the manufacturer's recommended values were 3.0 Gbp or more for read data and 85.0 % or more for >= Q30, the next generation sequencer DNA library (second DNA fragments) prepared in this example was considered Applicable to next generation sequencer analysis. In order to confirm reproducibility, the number of reads from repeated analyzes was compared to 34,613 read patterns obtained by MiSeq. Fig. 113 shows the results. As shown in Fig. 113, there was a high correlation of r = 0.996 in terms of the number of readings from the repeated analyses, as well as with the electrophoresis results.

[0275] As described above, a DNA library (first DNA fragments) was obtained by conducting PCR using a random primer comprising 10 nucleotides at the 3' end of a next generation sequencer adapter (Nextera Adapter, Illumina, Inc.) at a high concentration and then PCR was conducted using a next generation sequencer primer comprising the Nextera Adapter sequence. Consequently, it was possible to conveniently produce a next-generation sequencer DNA library (second DNA fragments) comprising many fragments with favorable reproducibility.

4.2. Nipponbare rice variety examination results

[0276] Figs. 114 and 115 show the results of electrophoresis after conducting PCR using 10 nucleotides positioned at the 3' end of the next generation sequencer adapter (Nextera adapter, Illumina, Inc.) and 16 types of random primers (B of 12 nucleotides) that have a total length of 12 nucleotides obtained by adding an arbitrary sequence of 2 nucleotides to the sequence of 10 nucleotides at the 3' end in a high concentration of 40 μl. As shown in Figs. 114 and 115, amplification was observed in a broad region ranging from 100 bp to 500 bp (the first DNA fragment). It was considered that the amplification can be observed in a wide region, due to the fact that the amplification was also observed in a region, except the region of the genomic DNA corresponding to the random primer as in the case of 4.1. Furthermore, since the rank correlation coefficient was 0.950 (>0.9), reproducibility was confirmed in the amplification pattern.

[0277] Next, Figures 116 and 117 show the results of electrophoresis after conducting PCR using the next generation sequencer primer in the same way as in 3.2.4. That is, in order to prepare a DNA library (second DNA fragments) linked to a next generation sequencer adapter (Nextera adapter), PCR was conducted using a next generation sequencer primer comprising the Nextera adapter sequence from Illumina, Inc. and the first DNA fragment as a template. As a result, since the next-generation sequencer DNA library (the second DNA fragment) prepared in this example was mainly distributed in a range of 150 bp to 1 kbp with a peak around 300 bp, as illustrated in Figs. . 116 and 117, the DNA library was considered an appropriate next generation sequencer DNA library. Furthermore, since the rank correlation coefficient between the repeated data was 0.992 (>0.9), reproducibility was confirmed in the amplification pattern.

[0278] Furthermore, as a result of analysis of the obtained DNA library (second DNA fragments) by MiSeq next generation sequencer, 4.0 Gbp read data and 3.8 Gbp read data were obtained. The values indicating accuracy of MiSeq data (>= Q30) were 94.0% and 95.3%. As in the case of 4.1.1, in view of the above results, the next generation sequencer DNA library (second DNA fragments) prepared in this example was considered applicable to next generation sequencer analysis. Fig. 118 shows the results obtained by comparing the random primer sequence and the reference sequence of the Nipponbare rice variety, in order to evaluate the degree of consistency between the random primer sequence of 19,849 reading patterns obtained by MiSeq and the genome. As shown in Fig. 118, the average degree of consistency between the random primer sequence and the reference sequence of the Nipponbare rice variety was 34.5%. In particular, since there was no identical reading pattern between the random primer sequences and the Nipponbare rice variety reference sequence, it was considered that any reading pattern indicated that a random primer was ligated to a sequence not corresponding to the random primer. and the resulting sequence was amplified. The above results were considered to correspond to the results obtained by the bioanalyzer. In order to confirm the reproducibility of the reading pattern, the numbers of readings from the repeated analyzes were compared. Fig. 119 shows the results. As shown in Fig. 119, there was a high correlation of r = 0.999 in terms of the numbers of readings from the repeated analyzes as with the electrophoresis results.

[0279] As described above, a DNA library (first DNA fragments) was obtained by conducting PCR using 16 types of random primers that have a total length of 12 nucleotides obtained by adding an arbitrary sequence of 2 nucleotides to the 3' end of 10 nucleotides at high concentrations, where the 10 nucleotide positions at the 3' end of a next generation sequencer adapter (Nextera Adapter, Illumina, Inc.) and then PCR was conducted using a primer comprising the sequel to the Nextera Adapter. Consequently, it was possible to conveniently produce a next-generation sequencer DNA library (second DNA fragments) comprising many fragments with favorable reproducibility.

Example 3 1. Materials and Method 1.1. Materials

[0280] In this example, genomic DNA was extracted from the Nipponbare rice variety using the DNeasy Plant Mini kit (QIAGEN) and the extracted genomic DNAs were purified. Purified genomic DNA was used as Nipponbare-derived genomic DNA.

1.2. DNA library preparation

[0281] To the genomic DNA described in 1.1 above (30 ng, Nipponbare-derived genomic DNA), random primers (final concentration: 60 μM, 10 nucleotide primer A), a mixture of 0.2 mM dNTP, 1.0 MgCl2 mM and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added and a reaction solution was prepared, adjusting the final reaction level to 50 μl. PCR was performed under thermal cycling conditions comprising 98°C for 2 minutes and 30 cycles of 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 20 seconds, followed by storage at 4°C. The DNA library obtained in this experiment was purified by the MinElute PCR Purification Kit (QIAGEN).

1.3. Sequence library preparation

[0282] From the DNA library obtained in 1.2, a sequence library for MiSeq analysis was prepared using the KAPA Library Preparation Kit (Roche).

1.4. MiSeq Analysis

[0283] Using the MiSeq V2 500 Cycle Reagent Kit (Illumina), the sequence library for MiSeq analysis obtained in 1.3 was analyzed through 100 base paired end sequencing.

1.5. Analysis of nucleotide sequence information

[0284] Random primer sequence information was suppressed from the read data obtained in 1.4, and nucleotide sequence information from each read was identified. Mapping of nucleotide sequence information of each read onto Kasalath rice genomic information (kasalath_genome) was conducted by bowtie2 and single nucleotide polymorphism (SNP) and insertion or deletion mutation (InDel) were identified as markers for each chromosome.

2. Results and examination

[0285] Table 31 shows the results of mapping the nucleotide sequence information of the DNA library prepared using the random primers based on genomic DNA from the Nipponbare rice variety onto the genomic information of Kasalath rice. Table 31

[0286] As shown in Table 31, it was possible to identify 2,694 to 5,579 SNPs (3,812.6 SNPs on average, 45,751 SNPs in total) for each chromosome. As shown in Table 31, it was also possible to identify the insertion/deletion (InDel) of 227 to 569 SNPs (349.3 SNPs on average, 4,191 SNPs in total) for each chromosome. The above results revealed that it is possible to identify a DNA marker as a characteristic nucleotide sequence present in the genome of a test organism by comparing the nucleotide sequence information in a DNA library prepared using random primers and known sequence information. of nucleotides in the manner shown in this example.

[0287] All publications, patents and patent applications cited in this description are fully incorporated into this report by reference.

Claims (11)

1. Method for analyzing genomic DNA, CHARACTERIZED by the fact that it comprises the steps of preparing a DNA library by the method for producing a DNA library comprising conducting a nucleic acid amplification reaction in a reaction solution containing genomic DNA and a random primer comprising 9 to 30 nucleotides using genomic DNA as a template to obtain DNA fragments by the nucleic acid amplification reaction, wherein when the random primer contains 9 to 10 nucleotides, the concentration of the random primer is adjusted to 40 to 60 μM; when the random primer contains 10 to 14 nucleotides, the concentration of the random primer is adjusted to 100 μM or less, and said concentration is defined by the formula y > 3E + 08x-6.974, provided that the nucleotide length of the random primer is represented by “x” and the concentration of the random primer is represented by “y”; when the random primer contains 14 to 18 nucleotides, the random primer concentration is adjusted to 4 to 100 μM; when the random primer contains 18 to 28 nucleotides, the concentration of the random primer is adjusted to 4 μM or more, and said concentration is defined by the formula y < 8E + 08x-5.533; when the random primer contains 28 to 29 nucleotides, the random primer concentration is adjusted to 6 to 10 μM; and when the random primer contains 30 nucleotides, the random primer concentration is adjusted to 6 μM; determine the nucleotide sequence of the DNA library produced; and confirming the presence or absence of a characteristic nucleotide sequence based on the nucleotide sequence. 2. Method for analyzing genomic DNA, according to claim 1, CHARACTERIZED by the fact that the presence or absence of the characteristic nucleotide sequence is confirmed based on the number of nucleotide sequence readings from the DNA library. 3. Method for analyzing genomic DNA according to claim 1, CHARACTERIZED by the fact that the nucleotide sequence of the DNA library is compared with known sequence information or with the nucleotide sequence of a DNA library produced using the genomic DNA of a different organism or tissue, and the presence or absence of the characteristic nucleotide sequence is confirmed based on differences in the nucleotide sequences. 4. Method for analyzing genomic DNA, according to claim 1, CHARACTERIZED by the fact that it further comprises the steps of: preparing a pair of primers to specifically amplify the characteristic nucleotide sequence; conducting a nucleic acid amplification reaction using genomic DNA extracted from a target organism as a template and the primer pair; and confirm the presence or absence of the characteristic nucleotide sequence in the genomic DNA based on the results of the nucleic acid amplification reaction. 5. Method for analyzing genomic DNA, according to claim 1, CHARACTERIZED by the fact that each of the DNA fragments comprises 100 to 500 nucleotides. 6. Method for analyzing genomic DNA, CHARACTERIZED by the fact that it comprises the steps of: conducting a nucleic acid amplification reaction in a first reaction solution comprising genomic DNA and a random primer comprising 9 to 30 nucleotides in a high concentration to obtaining the first DNA fragments by the nucleic acid amplification reaction using genomic DNA as a template, wherein when the random primer contains 9 to 10 nucleotides, the concentration of the random primer is adjusted to 40 to 60 μM; when the random primer contains 10 to 14 nucleotides, the concentration of the random primer is adjusted to 100 μM or less, and said concentration is defined by the formula y > 3E + 08x-6.974, provided that the nucleotide length of the random primer is represented by “x” and the concentration of the random primer is represented by “y”; when the random primer contains 14 to 18 nucleotides, the random primer concentration is adjusted to 4 to 100 μM; when the random primer contains 18 to 28 nucleotides, the concentration of the random primer is adjusted to 4 μM or more, and said concentration is defined by the formula y < 8E + 08x-5.533; when the random primer contains 28 to 29 nucleotides, the random primer concentration is adjusted to 6 to 10 μM; and when the random primer contains 30 nucleotides, the random primer concentration is adjusted to 6 μM; conduct a nucleic acid amplification reaction in a second reaction solution comprising the first DNA fragments obtained and a primer, which has a nucleotide sequence at the 3' end capable of hybridizing with a nucleotide sequence at the 5' end of the first fragments of DNA to obtain the second DNA fragments; preparing a DNA library with the second DNA fragments or DNA fragments obtained by a nucleic acid amplification reaction using the second DNA fragments as templates or a nucleic acid amplification reaction to be conducted repeatedly; determine the nucleotide sequence of the prepared DNA library; and confirming the presence or absence of a characteristic nucleotide sequence based on the nucleotide sequence. 7. Method for analyzing genomic DNA, according to claim 6, CHARACTERIZED by the fact that the primer for amplifying the second DNA fragments comprises a region used for a nucleotide sequencing reaction, or the primer used for a nucleotide sequencing reaction. nucleic acid amplification using the second DNA fragments as templates or a nucleic acid amplification reaction that will be conducted repeatedly comprises a region used for a nucleotide sequencing reaction. 8. Method for analyzing genomic DNA, according to claim 6, CHARACTERIZED by the fact that the presence or absence of the characteristic nucleotide sequence is confirmed based on the number of nucleotide sequence readings from the DNA library. 9. Method for analyzing genomic DNA, according to claim 6, CHARACTERIZED by the fact that the nucleotide sequence of the DNA library is compared with the known sequence information or with the nucleotide sequence of a DNA library produced using the genomic DNA of a different organism or tissue, and the presence or absence of the characteristic nucleotide sequence is confirmed based on differences in the nucleotide sequences. 10. Method for analyzing genomic DNA, according to claim 6, CHARACTERIZED by the fact that it further comprises the steps of: preparing a pair of primers to specifically amplify the characteristic nucleotide sequence; conducting a nucleic acid amplification reaction using genomic DNA extracted from a target organism as a template and the primer pair; and confirm the presence or absence of the characteristic nucleotide sequence in the genomic DNA based on the results of the nucleic acid amplification reaction. 11. Method for analyzing genomic DNA, according to claim 6, CHARACTERIZED by the fact that each of the first DNA fragments comprises 100 to 500 nucleotides.