JP6417465B2 - Methods for evaluating the genotoxicity of substances - Google Patents

Description

  The present invention relates to a method for analyzing mutations or evaluating the genotoxicity of substances.

  Genotoxicity is a general term for toxicity to intracellular genetic material, mainly DNA, and in a narrower sense, it has the property (mutagenicity) that causes DNA damage or mutation and changes its genetic information. Say. Genetic information of DNA is held in a base sequence composed of four types of bases A (adenine), T (thymine), G (guanine), and C (cytosine). A genotoxic substance acts on DNA directly or indirectly, changes its base sequence qualitatively or quantitatively, and changes genetic information. Changes in genetic information due to genotoxic substances are known to cause carcinogenesis and reproductive and developmental toxicity. Evaluating genotoxicity of pharmaceuticals, cosmetics, various chemicals, etc. is important for public safety. is important.

  The mechanisms of genotoxicity are diverse and can be broadly divided into base pair substitution mutations that change DNA base pair information to other base pairs, and short insertions that cause short base sequences to be inserted or deleted in DNA sequences. There are genomic structural changes that change the genomic structure, causing deletion mutations and insertions, deletions, translocations, inversions, etc. of relatively long base sequences throughout the genome sequence. In particular, short insertion / deletion mutations are also called frameshift mutations when the reading frame of a protein encoded by a gene is changed. In genotoxicity caused by chemical substances, it is said that many cause base pair substitution mutations or short insertion / deletion mutations.

  Until now, various genotoxicity tests using in vitro and in vivo models have been developed as methods for evaluating the genotoxicity of substances. For example, as a test for detecting the aforementioned base pair substitution mutation or frame shift mutation, Bruce N. et al. There is an Ames test developed by Dr. Ames (Non-Patent Document 1). In the Ames test, a Salmonella strain that has a mutation in the histidine biosynthesis gene and cannot grow in a medium not containing histidine is used. When the gene is mutated by substance exposure and histidine can be synthesized, colonies can be formed on a medium not containing histidine. The mutagenicity of the substance is confirmed by counting the resulting colonies. In addition, a micronucleus test using mammalian cells is used as a test for detecting the presence or absence of genomic structural changes (Non-patent Document 2). Furthermore, by combining multiple genotoxicity tests, the presence or absence of the genotoxicity of the substance can be confirmed with high sensitivity.

  However, in conventional genotoxicity tests as described above, genotoxicity detection relies on indirect indicators that do not directly reflect the amount or quality of the mutation. For this reason, in the conventional test, qualitative and quantitative information on the mutation such as what mutation has occurred and how many mutations have occurred at one base rate cannot be obtained in detail. In addition, since there is no uniform index between various tests, it is difficult to compare results between different tests. Thus, conventional genotoxicity tests do not provide sufficient information to advance a systematic understanding of genotoxicity, such as comparing strengths among multiple mutagens and classifying genotoxicity by mechanism.

  Application of high-throughput sequencing technology using next-generation sequencers to genotoxicity evaluation has been proposed. Maslov et al. (Non-Patent Document 3) disclose a methodology for applying high-throughput sequencing to genotoxicity assessment. One of the methods is to identify a mutation site by exposing a cell to a substance, creating a uniform population of genome information derived from a single cell, and acquiring the genome information using a next-generation sequencer. is there. As a practical example of this methodology, Matsuda et al. (Non-Patent Document 4) isolated a single colony of a strain TA100 derived from Salmonella Typhimurium exposed to a mutagen, and obtained its entire genome sequence with a next-generation sequencer. By comparing the sequence between the target sequence and the lead sequence, and detecting the site where the same base change occurred in multiple lead sequences at a certain frequency as the mutation site, the individual mutation and its position were identified. Has been reported. Furthermore, Matsuda et al. (Non-Patent Document 5) detect mutations in the same manner as Non-Patent Document 4 using a small amount of diluted strain culture solution collected instead of isolating a single colony. Reporting method. As another method, there has been reported a method for evaluating accumulation of DNA mutation due to radiation or the like using a next-generation sequencer. Specifically, attention is paid to a sequence (tag sequence) specific to a restriction enzyme site and the like, and evaluation is performed to estimate the mutation frequency in the genome based on the appearance frequency (Patent Document 1). In addition, a unique tag sequence is added to each molecule of cell-free (cf) DNA to obtain a consensus sequence of multiple read sequences obtained from the same molecule, and then align multiple read sequences at the same location on the genome. Mutation detection methods to be compared are disclosed (Non-Patent Documents 6 and 7).

International Publication No. 2014/175427

Mortelmans et al., Mutation Research, 2000, 455: 29-60 Matsushima et al., Mutagenesis, 1999, 14: 569-580 Maslov et al., Mutation Research 2015, 776: 136-143 Matsuda, Genes and Environment, 2013, 35: 53-56 Matsuda et al., Genes and Environment, 2015, 37: 15-24 Nucleic Acids Research, 2016, 44 (11): e105 Clinical Oncology, 2016, 28: 735-738

  The present inventor has determined that a site where a change in the base sequence at a specific site occurs at a certain frequency in a plurality of lead sequences as a mutation site by comparison between a plurality of read sequences including a specific site of the reference sequence. Instead of the conventional method of detection, each lead sequence is compared with a reference sequence, base mutations are detected from each lead sequence, and the results are analyzed to calculate the mutation pattern and its frequency. The analysis method was found. This analysis method makes it possible to detect mutations based on a large amount of nucleotide sequence information, or to detect mutations with high speed and sensitivity more efficiently than conventional methods, and as a result, quantitative and qualitative mutations in the entire cell population can be detected. Data that reflects trends can be provided.

In one embodiment, the present invention is a method for evaluating the genotoxicity of a test substance, comprising:
(1) The cell population exposed to the test substance is taken as a test group, and its DNA is obtained;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5),
Providing a method.

In another embodiment, the present invention provides a method for evaluating the genotoxicity of a test substance, comprising:
(1 ′) taking a cell population exposed to the test substance as a test group and obtaining the DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′),
Providing a method.

In yet another embodiment, the present invention provides a method for evaluating the genotoxicity of a test substance, comprising:
(1 ″) A cell population exposed to a test substance is taken as a test group, and the DNA is obtained;
(2 ″) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ″) comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ″) obtaining the site detected in (3 ″) as a mutation site having an insertion or deletion mutation;
(5 ″) determining the length of the inserted or deleted base and / or the type of the inserted base for each acquired mutation;
(6 ″) determining the base length of the insertion or deletion site determined in (5 ″) and / or the mutation frequency for each type of inserted base;
Providing a method.

In yet another embodiment, the present invention is a method for evaluating a mutation in a cancer cell, comprising:
(1) Acquiring DNA of a cancer cell population as a test group;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5),
Providing a method.

In yet another embodiment, the present invention is a method for evaluating genetic information of cultured cells, comprising:
(1) The cultured cell population is used as a test group, and the DNA is obtained;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5),
Providing a method.

  According to the method of the present invention, quantitative and qualitative information about mutations in a cell population can be obtained by a single analysis. Therefore, according to the method of the present invention, mutation analysis at the cell population level can be performed at a much simpler and lower cost than the conventional method. The method of the present invention is particularly effective when it is desired to grasp the tendency of mutation in a cell population having heterogeneous genetic information, such as genotoxicity evaluation of a substance or cancer evaluation.

Mutation call ratio of base pair mutation pattern in synthetic DNA sample. A: GC base pair mutation pattern, B: AT base pair mutation pattern. The amount of increase in the mutation frequency of the base pair mutation pattern in the synthetic DNA sample. A: GC base pair, B: AT base pair. Increase in mutation frequency of insertion mutation in a synthetic DNA sample. A: Number of inserted bases, B: Type of inserted base. Increase in mutation frequency of base pair mutation pattern in mutagen-exposed samples. A: GC base pair, B: AT base pair. *: P <0.05, **: p <0.01, ***: p <0.001 (Dunnett's test). Difference in mutation frequency increase in mutation pattern of base pair in mutagen-exposed samples depending on the type of original base. Spectral analysis results of base pair mutation patterns in mutagen-exposed samples. Results of sequence context analysis of base pair mutations in mutagen-exposed samples. Signature 11: Pattern of mutation signature by known alkylating agent, ENU: Mutation pattern by treatment with Ethylnitrosoura in Example 2. Sequence context analysis result of base pair mutation in mutagen exposed sample (continuation of FIG. 7). Signature 11: Pattern of mutation signature by known alkylating agent, ENU: Mutation pattern by treatment with Ethylnitrosoura in Example 2.

(1. Definition)
As used herein, “mutation” refers to a mutation that occurs in DNA, for example, base or sequence deletions, insertions, substitutions, additions, inversions, and translocations in DNA. Is mentioned. Mutations herein include single base deletions, insertions, substitutions, additions, and deletions, insertions, substitutions, additions, inversions, and translocations of sequences of two or more bases. The mutation in the present specification includes a mutation in a coding region and a non-coding region, and also includes a mutation accompanied by a change in the expressed amino acid and a mutation not accompanied (silent mutation).

  The “genotoxicity” of a substance evaluated in the present invention refers to a property that the substance causes mutation (so-called mutagenicity).

  In the present specification, the “original fragment” is a fragment of DNA to be analyzed, and refers to a single-stranded DNA fragment whose sequence is read by a sequencing reaction. In the present specification, the “original base” related to the base at the mutation site refers to the base before mutation at the mutation site of the original fragment.

  All patent documents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

(2. Methods for analyzing mutations in cell populations)
The genotoxicity assessment method using high-throughput sequencing is expected to directly assess the amount and quality of mutations caused by mutagens. In addition, such a method is basically applicable to all living species as long as genome sequences are available. On the other hand, in the conventional genotoxicity evaluation method using high-throughput sequencing as described in Non-Patent Document 4, in order to detect a base mutation at a partial position, all read sequences including the site are all aligned. Therefore, a large amount of sequence information is required, and much time, labor, and cost are required for obtaining sequence information and detecting a mutation site. In addition, since mutation due to substance exposure is generally very infrequent and does not occur evenly in individual cells within the population, in the analysis of single cells as in Non-Patent Document 4, the cell population It is considered difficult to accurately assess the frequency of mutations within the cell and the effect of mutagens on the cell population. Even in the method described in Non-Patent Document 5, since the genetic information of the sample subjected to the analysis was still homogeneous, it cannot be said that the result reflected the information of the cell population. In the evaluation of genotoxicity, it is an important point how to detect low-frequency mutations in individual cells and how to evaluate genotoxicity to a population based on it.

  If the analysis as described in Non-Patent Document 4 or 5 is repeated for a plurality of samples derived from a single cell, information reflecting the events in the cell population can be obtained, but the time, labor and cost for that are It is enormous. This method is not practical, especially when evaluating multiple substances and when evaluating dose-response relationships. On the other hand, the method of Patent Document 1 is a relatively low cost method, but it only estimates the mutation frequency using a specific sequence such as a restriction enzyme site. It is not a feasible method for genotoxicity assessment based on accurate mutation information.

  In addition, conventional genotoxicity assessment methods using high-throughput sequencing are used to evaluate multiple substances when considering technological hurdles in mammalian cell isolation and culture and the cost of mutation analysis due to genome size. Application to is extremely difficult. Development of a method for detecting quantitative and qualitative information on mutations caused by mutagens in a cell population retaining various mutation information simply and at low cost is desired.

  The present inventor has determined that a site where a change in the base sequence at a specific site occurs at a certain frequency in a plurality of lead sequences as a mutation site by comparison between a plurality of read sequences including a specific site of the reference sequence. Instead of the conventional method of detection, each lead sequence is compared with a reference sequence, base mutations are detected from each lead sequence, and the results are analyzed to calculate the mutation pattern and its frequency. The analysis method was found. This analysis method makes it possible to detect mutations based on a large amount of nucleotide sequence information, or to detect mutations with high speed and sensitivity more efficiently than conventional methods, and as a result, quantitative and qualitative mutations in the entire cell population can be detected. Data that reflects trends can be provided.

  According to the method of the present invention, quantitative and qualitative information about mutations in a cell population can be obtained by a single analysis. Therefore, according to the method of the present invention, mutation analysis at the cell population level can be performed at a much simpler and lower cost than the conventional method. The method of the present invention is particularly effective when it is desired to grasp the tendency of mutation in a cell population having heterogeneous genetic information, such as genotoxicity evaluation of a substance or cancer evaluation.

Accordingly, in one embodiment, the present invention provides a method for analyzing mutations in a cell population. The basic procedure of the method of the present invention is as follows:
(A) obtaining DNA from a cell population;
(B) sequencing fragments of the DNA (ie, the original fragment) to obtain one or more read sequences for each fragment;
(C) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence;
(D) obtaining a site detected for the one or more lead sequences as a mutation site;
(E) Information on the mutation at the mutation site is acquired, and the tendency of the mutation is analyzed based on the information.

  The cell population used in the method of the present invention may be a homo population (for example, a cell population derived from a single colony or the like with a uniform genetic information) or a hetero population (a population with non-uniform genetic information). A population in which the uniformity of genetic information is unknown may be used, but a hetero population or a cell population presumed to be a hetero population is preferable. Examples of cell populations used in the method of the present invention include specimens collected from animals or plants, and populations of cultured cells derived from animals, plants or microorganisms, preferably from animal, plant or microorganism strains. A population of cultured cells can be mentioned. Examples of animals are preferably mammals such as humans, silkworms, nematodes and the like, and examples of microorganisms are preferably Escherichia coli, Salmonella, yeast and the like. As another example of the cell population used in the method of the present invention, a specimen collected from a living body or a culture thereof, a cultured cell exposed to a mutagen or a candidate substance thereof, or a cultured cell administered with a drug or a candidate substance thereof, etc. Can be mentioned.

  The DNA derived from the cell population used in the present invention can be obtained by extraction or isolation from the cell population using a conventional method in the art. For the extraction or isolation, for example, a commercially available DNA extraction kit can be used. Alternatively, DNA derived from the cell population preserved after extraction or isolation may be obtained and used in the method of the present invention. Examples of DNA derived from the cell population used in the present invention include genomic DNA, mitochondrial genomic DNA, chloroplast genomic DNA, plasmid DNA, and viral genomic DNA. Of these, genomic DNA is preferred.

  Alternatively, when analyzing RNA viruses in a cell population, RNA may be obtained and analyzed instead of DNA. RNA in cells can be extracted or isolated by a conventional method in the art, such as a commercially available RNA extraction kit. Alternatively, RNA derived from the cell population preserved after extraction or isolation may be obtained and used in the method of the present invention. When RNA is obtained and analyzed in the method of the present invention, “DNA” in the present specification is read as “RNA”, and base T is read as base U.

  The reference sequence used in the method of the present invention is a known sequence contained in DNA to be subjected to mutation analysis. Here, as a known sequence, it is preferable to use a sequence registered in a public database or the like. However, in the analysis target DNA sequenced in advance by a sequencer or the like prior to (C) of the method of the present invention described above, The arrangement of The region, length and number of the reference sequence are not particularly limited, and can be appropriately selected from DNA according to the purpose of mutation analysis. For example, when the purpose of the mutation analysis according to the present invention is genotoxicity evaluation of a substance, the length of the reference sequence is not particularly limited, but is preferably 1,000 bp or more in total, more preferably 10,000 bp or more, 000 bp or more is more preferable, and 1,000,000 bp or more is still more preferable.

  Fragmentation of DNA can be performed using a usual method in this field, such as ultrasonic treatment and enzyme treatment. The length of the fragment to be prepared can be appropriately selected according to the length that can be accurately read by the sequencer. In general, 100 to 10,000 bp can be selected, but as long as the sequencer can read accurately, a fragment having a length of 10,000 bp or more may be prepared, and a more appropriate range depending on the type of the sequencer. Can be selected. For example, when applied to a sequencing reaction sequencer that amplifies fragments, the average length of the fragments is preferably 100 to 500 bp, more preferably 150 to 200 bp. For example, in order to apply to a sequencer that performs single molecule real-time sequencing, the length of the fragment is preferably 150 to 10,000 bp in average length, more preferably 200 to 1,000 bp in average length, and more preferably 200 to 500 bp in average length. preferable.

  The resulting fragments are then sequenced. Fragment sequencing only needs to be performed on a portion to be used for sequence comparison with a reference sequence described later. For example, at least a part, preferably the whole of the sequence, may be sequenced with a fragment corresponding to the DNA region of the reference sequence. In the case of mammalian cells and the like, exon regions and the like may be selectively sequenced. Kits such as SureSelect (manufactured by Agilent Technologies) are marketed for selection of areas.

  In the sequencing reaction for amplification of fragments, the fragments are amplified by PCR or the like, and the sequence of each amplified fragment is determined. In a single molecule real-time sequencing reaction, the sequence is determined without amplifying the fragment. A known sequencer may be used for fragment sequencing, but a high-throughput sequencer (so-called next-generation sequencer) is preferably used. HiSeq (manufactured by Illumina), MiSeq (manufactured by Illumina) and the like are marketed as high-throughput sequencers for amplifying fragments. In addition, as high-throughput sequencers for performing single-molecule real-time sequencing, Pac Bio RSII (Pacific Biosciences), Pac Bio Sequence System (Pacific Biosciences), and the like are on the market.

  Although the detailed procedure of the fragment sequencing method is not particularly limited, it is preferably a more accurate sequencing method. As one of such methods, in sequencing in which fragment amplification is performed, there is a method in which sequences are obtained from both sides of a fragment and a common part is utilized as described later. In addition, the comparison accuracy can be further improved by comparing sequences obtained from two original fragments complementary to each other between complementary strands. As such a technique, when preparing a HiSeq or MiSeq library, an adapter specific to each fragment molecule is added, and after sequencing, the base sequence between complementary strands is referred based on the adapter sequence information (Proc Natl Acad Sci USA, 2012, 109 (36): 14508-14513). In single molecule real-time sequencing, a single hairpin sequence adapter is added to both ends of a double-stranded fragment to form a single circle, and the single molecule circular fragment is sequenced several times in succession to integrate its sequence information. (Nucleic Acids Res, 2010, 38 (15): e159) and the like.

  As a result of the above sequencing, a reading result (read sequence) is obtained for each of the original fragments obtained by the above-described DNA fragmentation. The number of read sequences to be acquired for each original fragment may be one or more, but from the viewpoint of improving accuracy in the analysis of mutation tendency described later, a plurality of read sequences are preferably acquired for each fragment. The number of lead sequences obtained from one fragment is preferably 2 or more, more preferably 10 or more. On the other hand, from the viewpoint of analysis efficiency, the number of lead sequences is preferably 5 or less, more preferably 2 or less.

  The obtained one or more read sequences can be directly used for comparison with the subsequent reference sequences. From the viewpoint of improving the accuracy of the mutation analysis, it is preferable to extract one having high reading reliability by sequencing from the bases of the obtained read sequence. Preferably, a highly reliable base is extracted from the corresponding bases of two or more lead sequences obtained from the same fragment. The extracted highly reliable base is referred to herein as a “consensus base”. The extraction of “consensus base” can be performed using a program attached to the next-generation sequencer. As a more detailed procedure, for example, when two kinds of read sequences obtained from the same fragment have the same base type at the corresponding position, or when the two read sequences are complementary strands, A method of extracting bases that are complementary as “consensus bases”; comparing bases between two or more read sequences obtained from the same fragment and including a complementary strand at each position on the sequence In this case, the base that appears at the highest frequency, including complementary bases, is determined and extracted as a “consensus base”; the reading accuracy (quality value) of the base sequence at the corresponding position between the read sequences ) Method of adopting the highest base as “consensus base”; Method of probabilistically determining “consensus base” based on quality value, base appearance frequency, etc .; How the corresponding base of all lead sequence obtained from a single fragment to be "consensus nucleotide" the base if they match Flip; method combining or these, and the like. In the method of the present invention, the extraction of the “consensus base” may be performed before the mapping of the lead sequence described later to the reference sequence, or after the mapping, as necessary.

  The one or more read sequences for each original fragment obtained by sequencing are then mapped to reference sequences, respectively, and the sequences are compared. As a result of the comparison, a site where the base does not match between each lead sequence and the reference sequence is detected. Preferably, the comparison can be performed only for “consensus bases” on the lead sequence described above. Examples of the type of “non-matching site” include a site where the type of base on the lead sequence differs from the reference sequence (substitution site), and a site where the base on the lead sequence is deleted from the reference sequence. (Deletion site), a site where the base is inserted on the lead sequence relative to the reference sequence (insertion site), and the like.

  The detected “unmatched site” is acquired as a mutation site in the DNA to be analyzed. More specifically, information on the mutation at the mutation site is acquired. The information to be acquired includes, for example, the type of the non-matching site (mutation site) (for example, whether it is a substitution site, a deletion site, or an insertion site), the type of base at the site, and the base before mutation (or base) Type (for example, the type of base at the position corresponding to the site on the reference sequence), the type of base on both sides of the site (for example, the base on the side of the position corresponding to the site on the reference sequence) Type), but is not limited thereto. In a preferred embodiment, if the unmatched site is a substitution site, information on the type of base at the site and the type of base before mutation is obtained; Information on the type of base and the type of base on both sides of the deletion site; if the unmatched site is an insertion site, the type of base on the site and the type of base on both sides of the insertion site Information is acquired.

  From the comparison between the lead sequence and the reference sequence, a site where the base matches between the lead sequence and the reference sequence can also be detected. These “matched sites” can be obtained as sites without mutation in the DNA to be analyzed. Information on sites without these mutations can be obtained. Examples of the information to be acquired include the type of base at the site and the types of bases on both sides of the site.

  For each of the one or more lead sequences, information on the mutation at the above-described mutation site or information on a site without mutation is obtained. The acquired information can be collected to create a mutation analysis database. For example, a database may be created in which all the information about mutation sites obtained from each lead sequence is collected; a database in which mutation information obtained from each lead sequence is classified for each type of mutation site may be created; A database may be created in which the mutation information obtained from each lead sequence is classified according to the type of base before mutation (for example, reference sequence) or after mutation at the mutation site; mutation information obtained from each lead sequence is mutated You may create the database classified according to the base length (for example, the length of an insertion, deletion, or substitution site); you may create the database which combined these classification. Or you may create the database which integrated the information regarding a variation | mutation site | part, and the information regarding the site | part which does not have a variation | mutation. For example, you may create the database which put together the information of the site | part of a variation | mutation, and the site | part which does not have variation | mutation for every kind (A, T, G, and C) of the base before a variation | mutation (for example, reference sequence). Alternatively, the position of the mutation site on the genome is specified, and it corresponds to the coding region or non-coding region of the gene, and in the case of the coding region of the gene, it is an intron or an exon, or the strand that is transcribed into RNA A database may be created with information on whether or not.

The above-described detection of “non-matching sites” and acquisition of information on mutation sites or sites without mutations may be performed for all read sequences obtained by sequencing, but for some lead sequences. You may go. The total amount of the lead sequence used for the detection (the total length of the lead sequence to be used) is not particularly limited as long as it is an amount that allows analysis of the tendency of the subsequent mutation, but it may be a base length that is not less than the reciprocal of the mutation frequency. Preferably, the base length is 100 times or more the reciprocal of the mutation frequency. For example, since the frequency of mutation in Example 2 described later is on the order of about 1/10 ⁵ bp, the total length of the read sequences used is preferably 1 × 10 ⁵ bp or more, preferably 1 × 10 5 It is more preferably ⁷ bp or more, and further preferably 1 × 10 ⁹ bp or more. In addition, in order to analyze the tendency of mutation having a mutation frequency on the order of 1/10 ⁶ bp, the total amount of the read sequence used for detection is preferably 10 times the above amount, and the mutation frequency is 1 In order to analyze the tendency of mutations on the order of / 10 ⁴ bp, the total amount of read sequences used for detection is preferably 1/10 times the above amount. On the other hand, from the viewpoint of analysis efficiency, the total amount of read sequences used for detection is preferably 10,000 times or less of the reciprocal of the mutation frequency, more preferably 1000 times or less, and more preferably 100 times. More preferably, the base length is twice or less. For example, the total amount of lead sequence used for detection is preferably 1 × is 10 ¹⁰ bp or less, more preferably 1 × is 10 ⁹ bp or less, more preferably at most 1 × 10 ⁸ bp, 1 more more preferably × at 10 ⁷ bp or less, and still preferably not more than 1 × 10 ⁶ bp. In addition, the mutation analysis database may be created based on information about all of the acquired mutation sites or sites without mutation, but as long as it enables analysis of subsequent mutation trends, It may be created based only on information.

  In conventional methods (for example, Non-Patent Documents 4 and 5), after obtaining a plurality of lead sequences corresponding to a specific part of a reference sequence, the same type of non-matching bases are present at the same part among the plurality of lead sequences. When observed at a certain frequency, the site was determined as a mutation site in the DNA to be analyzed. This method can miss low frequency mutations. In this method, the DNA region for mutation detection is limited to a limited length region where the plurality of read sequences overlap, and a large amount of data is required to overlap the leads. It takes a lot of time and labor to analyze over a wide area and to grasp the tendency of mutation in the whole.

  On the other hand, in the method of the present invention, the mutation site is not determined based on the appearance frequency of such mismatched bases between the read sequences. In the method of the present invention, basically, mutation information based on comparison with a reference sequence is obtained for each of one or more lead sequences corresponding to a specific site of a reference sequence, and the information is stored as necessary. Categorize and create a database. Based on the database, the tendency of mutation in the DNA to be analyzed is analyzed. For example, statistical analysis (for example, mutation frequency analysis, mutation pattern analysis, etc.) using any element included in the database as a sample population can be performed.

  In the method of the present invention, mutation information is acquired for each lead sequence, so that detection can be performed without missing a low-frequency mutation. In addition, the method of the present invention enables detection and analysis of mutations in a wide region on DNA corresponding to any of the lead sequences used for mutation detection. Therefore, according to the method of the present invention, mutations can be detected at a higher speed and with higher sensitivity than in the conventional method, so that more efficient and more accurate mutation analysis can be performed.

(2-1. Detection of mutation in each fragment using next-generation sequencer)
As a preferred embodiment of the method of the present invention, the sequence of DNA fragment sequencing using a next-generation sequencer that performs PCR and the analysis of mutations in the DNA to be analyzed by comparison with a reference sequence will be described in detail below.

  A fragment derived from the DNA to be analyzed to be sequenced is added with an adapter sequence for sequencing at both ends. The fragment to which the adapter has been added is amplified to an amount detectable by the PCR method upon sequencing in the next-generation sequencer. The amplified fragment is read from the sequence, and the read sequence is output as a read sequence. In a preferred embodiment of the invention, two read sequences (Lead 1, Lead 2) are obtained for each amplified fragment. At this time, the lead 1 corresponds to the sequence side of the original fragment read by sequencing, and the lead 2 corresponds to the complementary strand side thereof. When a fragment is prepared with a size less than twice the read length of the sequencer, lead 1 and lead 2 of each amplified fragment include at least a part of the fragment as a common region, and further, an upstream region or a downstream region, respectively. Includes area. By superimposing the read lead 1 and the read 2 in the common region, one synthetic lead sequence is constructed for each amplified fragment. The construction of the synthetic lead sequence from the two lead sequences is PEAR (Bioinformatics, 2014, 30 (5): 614-620), FLASH (Bioinformatics, 2011, 27 (21): 2957-2963), PANDAseq (BMC Bioinformatics , 2012, 13:31).

  Each lead sequence is then mapped onto a reference sequence and compared. In a preferred embodiment, the comparison can be performed only for the above-mentioned “consensus base” on the lead sequence for improved comparison accuracy. In another preferred embodiment, a synthetic lead sequence is used as a lead sequence for comparison in order to improve comparison accuracy. More preferably, the region to be compared with the reference sequence in the synthetic lead sequence is limited to the overlapping portion of lead 1 and lead 2, and the base on the synthetic lead sequence used for comparison is the base between lead 1 and lead 2. Limited to those that are complementary (ie, “consensus bases”). This can reduce the adverse effect of sequencing errors on sequence comparison. The limitation to the “consensus base” may be performed before mapping to the reference sequence, or may be performed after mapping.

  By mapping and comparison to the reference sequence, a site (mutation site) where the base sequence does not match the reference sequence in each lead sequence can be detected. Furthermore, the mutation information such as the type of the mutation site (substitution site, deletion site or insertion site), the type of base at the site, the type of base before mutation, the type of bases on both sides of the site, etc. Can be acquired. By performing these procedures on the read sequences derived from each original fragment obtained by sequencing, mutation information can be collected and a database of mutation information from each read sequence can be created. The above procedure can be performed sequentially for each lead array, but may be performed for a plurality of lead arrays in parallel.

  For example, the mapping of the lead sequence to the reference sequence, the comparison of the comparison region, the display of the site where the base sequence does not match the reference sequence, and the acquisition of the mutation information at the site, for example, the mapping is Bowtie 2 software (Nature Methods, 2012). , 9 (4): 357-359), BWA software (Bioinformatics, 2009, 25 (14): 1754-1760), comparison region narrowing, and display of bases that do not match the reference sequence are shown in Samtools software (Bioinformatics, 2009, 25 (16): 2078-2079) and acquisition of mutation information at the site can be executed by a program for detecting a base different from a reference sequence created using a programming language such as Python. However, the software or programming language for executing the procedure of the method of the present invention is not limited to these.

(2-2. Analysis of mutation in cell population)
Furthermore, the tendency of mutation in the cell population can be examined based on the acquired database of mutation information. Preferably, the mutation analyzed by the present invention includes a base pair substitution mutation that changes the base pair of DNA to another base pair, and a short insertion that causes insertion or deletion of a short base sequence in the DNA sequence. Deletion mutations. Examples of the base pair substitution mutation include a single base pair substitution mutation and a multi-base pair substitution mutation in which 2 or 3 or more base pairs are substituted. Of these, in the present invention, a single base pair substitution mutation is preferably analyzed. According to the present invention, the mutation pattern and mutation frequency of these mutations can be determined. Hereinafter, the analysis procedure will be described in detail.

(2-2-1. Analysis of base pair substitution mutation)
In one embodiment, single base pair substitution mutations are analyzed. In the present embodiment, as described above, one or more lead sequences from each original fragment are compared with a reference sequence, and a site where the base sequence does not match the reference sequence in each lead sequence is detected. These sites are obtained as mutation sites having base pair substitution mutations with respect to the reference sequence. Next, each mutation is classified according to the mutation pattern of the base based on the detected base type of the mutation site and the base before the mutation. Next, the appearance frequency is determined for each mutation pattern of the base. These procedures can be performed using a program or the like created using the programming language such as Python described above.

In a more detailed example, each base contained in the lead sequence is divided into the following (i) to (iv).
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) a base present at a position where the base on the reference sequence is C The above (i) and (ii) are bases present at a site where the base pair of the reference sequence was AT, (iv) is a base present at a site where the base pair of the reference sequence was GC. Among these bases, those in which the base does not match the reference sequence (that is, base pair substitution mutation) are detected. Next, for each of the detected mutated bases, based on the classifications (i) to (iv) above, obtain the base pair before mutation existing in the mutation site from the base information of the reference sequence, and each Based on the base information of the lead sequence, the base pair after mutation is determined. From these data, for each mutation, when the base pair before mutation was AT [AT → TA, AT → CG, and AT → GC], when the base pair before mutation was GC [GC → TA, GC → CG, and GC → AT] can be classified into 6 base pair mutation patterns in total. Furthermore, the appearance frequency of each mutation pattern can be determined based on the total number of mutations belonging to each mutation pattern and the total number of analyzed bases. For example, based on the total number of bases analyzed for each of AT and GC base pairs, the appearance frequency of three types of mutation patterns can be calculated for each base pair.

  Furthermore, the mutation pattern of each mutation obtained above can be further classified according to the original base. The original base of the mutation at the site where the base pair before mutation is AT is A or T, and the original base of the mutation at the site where the base pair before mutation is GC is G or C. Therefore, each of the six base pair mutation patterns can be further divided into two according to the original base. Such a classification is useful for eliminating sequencing read errors caused by base modifications that occur during the extraction or isolation of DNA from cells. In particular, it is known that the G base is subject to chemical modification by oxidation during the DNA preparation process, and is likely to cause an error in reading G as T. Normally, since both bases constituting the base pair change due to the base pair mutation, the base pair mutation pattern divided into two according to the original base should show the same mutation frequency. If the mutation frequency is biased towards either original base, it suggests a sequencing error due to base modification.

  In another embodiment of the invention, polybase pair substitution mutations are analyzed. Examples of the multi-base pair substitution mutation include a 2-base pair substitution mutation and a 3-base pair substitution mutation. In the case of analysis of multiple base pair substitution mutations, for example, mutation patterns are classified according to the base sequence before mutation (for example, 4 × 4 = 16 patterns in the two base pair substitution type), and then each mutation pattern The frequency of appearance of each mutation pattern can be determined based on the total number of mutations belonging to, and the total number of analyzed mutations.

(2-2-2. Sequence context analysis)
In recent years, approaches have been proposed in which mutation elements (mutation signatures) caused by mutagens are extracted from mutation information accumulated on the genome of cancer cells using mathematical techniques. Various mutation signatures have been identified from mutation information accumulated in various human cancer genomes (Cell Rep, 2013, 3: 246-259). In the theory of mutation signatures, mutation patterns of base pair substitution mutations are classified based on the sequence context before and after the base pair is located. By performing sequence context analysis, more detailed analysis of base pair substitution mutations becomes possible.

Therefore, as another embodiment, a sequence context analysis procedure for a single base pair substitution mutation is shown. In this embodiment, first, each base sequence is compared with a reference sequence as described above, thereby detecting a 1 base pair substitution mutation in each lead sequence. Next, for each detected mutation, a sequence (so-called context) including a base before the mutation and bases adjacent to the upstream and downstream of the base before the mutation is determined based on the reference sequence. Subsequently, each mutation is typed according to the mutation pattern of the base pair and the context. That is, the detected mutations are converted into six base pair mutation patterns [AT → TA, AT → CG, AT → GC, GC → TA, GC → CG in the same manner as (2-2-1.) Described above. And GC → AT]. On the other hand, each detected mutation is classified according to the context. For example, the context of 3 base length including one base on both sides of the mutation site is 4 × 4 16 groups [For example, in the case of mutation from C, ACA, ACC, ACG, ACT, CCA, CCC, CCG, CCT, GCA, GCC, GCG, GCT, TCA, TCC, TCG, and TCT]. As a result, each mutation is classified into 96 (4 × 6 × 4) types in total according to the mutation pattern and context of the base pair. The context sequence used for this analysis consists of a base before mutation, one or more bases adjacent to the upstream of the base before mutation, and one or more bases adjacent to the downstream of the base before mutation. I just need it. The length of the context may be 3 bases or more, but is not limited thereto, and a longer context can be analyzed as necessary. For example, according to the context of 5 base length including 2 bases on both sides of the mutation site, each mutation is classified into 256 groups (4 × 4 × 4 × 4). Mutations are finally classified into a total of 1536 (4 × 4 × 6 × 4 × 4) types. Furthermore, according to the 2n + 1 base length context including n bases on both sides of the mutation site, each mutation is classified into 4 ²ⁿ group, and according to this classification and 6 base pair patterns, each mutation finally becomes 4 ²ⁿ in total. X Classified into 6 types. Next, based on the total number of mutations belonging to each mutation type and the total number of analyzed bases, the mutation frequency of each of the mutation types can be determined.

(2-2-3. Analysis of short insertion / deletion mutation)
In yet another embodiment, short insertion / deletion mutations are analyzed. In the present embodiment, as described above, each lead sequence is compared with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in each lead sequence. These sites are obtained as mutation sites having insertion or deletion mutations relative to the reference sequence. Further, for each obtained mutation, the type of mutation (insertion mutation or deletion mutation), the base length of the insertion or deletion site, and / or the type of inserted or deleted base are determined. The insertion or deletion site detected in this embodiment is preferably a site where the length of the inserted or deleted base is preferably 10 bp or less, more preferably 1 to 5 bp, but is not limited thereto. The procedure for detecting the insertion or deletion site of a specific base length can be performed using a program created using a programming language such as Python described above. Furthermore, the type of inserted or deleted base can be identified by comparing each lead sequence with a reference sequence. By these, the base length of the insertion or deletion site in each lead sequence and / or the type of base at the insertion or deletion site can be determined. Next, the frequency of insertion or deletion is determined for each base length and / or base type. For example, the insertion or deletion mutation obtained for each lead sequence can be classified by base length, and the frequency of each can be determined. In addition, for example, inserted or deleted bases can be classified according to their types (A, T, G, and C), and the respective frequencies can be determined. Furthermore, the classification of finer mutations combining the classification based on the base length and the type of base can be performed, and the frequency of each can be determined.

(2-3. Analysis of increase in mutation frequency)
Mutations detected by comparison of the lead sequence with a reference sequence according to the procedure described above can include base reading errors in sequencing. For more accurate mutation analysis, it is preferable to remove this error component. The error component can be removed by subtracting the mutation frequency of the control cell population from the mutation frequency of the cell population to be analyzed. Furthermore, when the cell population to be analyzed is a cell population that has been exposed to specific conditions, the effect of the specific conditions on the mutation frequency can be analyzed by determining the difference in mutation frequency between the analysis target and the control. become. For example, if the cell population to be analyzed is a cell population exposed to specific conditions, such as a cell population exposed to a mutagen, a cell population administered with a drug, etc., it is exposed to these conditions. The same non-cell population is taken as the control cell population. This control cell population is analyzed for base pair substitution mutation, sequence context analysis, or insertion / deletion mutation in the same procedure as described above to determine the mutation frequency. The mutation frequency of the obtained control cell population is subtracted from the mutation frequency of the cell population to be analyzed. This removes the component of the sequencing error from the mutation frequency of the cell population to be analyzed, and examines whether there is an increase in the mutation frequency due to the specific condition, or the amount of increase in the mutation frequency relative to the control under the specific condition be able to. Furthermore, it is preferable to analyze the mutation frequency based on the classification according to the above-mentioned original base because a sequencing error due to the modification of the base can be detected.

(3. Application)
By the mutation analysis method according to the present invention described above, mutations occurring in a cell population can be analyzed quantitatively and qualitatively. The analysis method of the present invention can be applied to various analyzes or evaluations related to mutations. Typical application examples include evaluation of the genotoxicity of a substance, evaluation method of mutation associated with tumor development (for example, evaluation of mutation in cancer cells and evaluation method of mutation in cfDNA), and quality control of cultured cells ( For example, the evaluation of genetic information such as the presence or absence of mutation or the evaluation of mutation type).

  Accordingly, a method for evaluating the genotoxicity of a test substance is provided as a preferred embodiment of the present invention. The specific procedure of this method will be described below.

(3-1. Method for evaluating genotoxicity of substances)
(3-1-1. Evaluation based on analysis of base pair substitution mutation)
One embodiment of the method for evaluating the genotoxicity of a test substance according to the present invention is based on the analysis of the base pair substitution mutation described above. The method is
(1) The cell population exposed to the test substance is taken as a test group, and its DNA is obtained;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5),
including.

  The steps (1) to (6) are the same as those described in the above (2.), particularly (2-2-1.). More specifically, in the case of analyzing a single base pair substitution mutation, in step (5), each mutation is converted into [AT → TA, AT → CG, and AT → for the site where the base pair of the reference sequence is AT. GC] is classified into three mutation patterns, and the site where the reference sequence base pair is GC is classified into three patterns [GC → TA, GC → CG, and GC → AT]. Preferably, these are combined and classified into a total of 6 base pair mutation patterns. Further, these six base pair mutation patterns are further divided into 2 based on the type of original base (A or T for AT, G or C for GC), respectively. They may be divided into groups and classified into 12 mutation patterns in total. Next, in step (6), the mutation frequency is determined for each of the mutation patterns determined in step (5). Thereby, the mutation frequency of the mutation pattern of each base pair can be determined.

In a preferred embodiment, the method further comprises:
(7) Using the cell population not exposed to the test substance as a control group, the mutation frequency of the mutation pattern of each base pair in the control group is determined by the same procedure as in (1) to (6). ;
(8) Subtract the mutation frequency of each mutation pattern in the control group obtained in (7) from the mutation frequency of each mutation pattern in the test group obtained in (6).
Thereby, the variation | mutation frequency increase amount in the test group which removed the influence of the sequencing error can be calculated | required.

(3-1-2. Evaluation Based on Sequence Context Analysis)
A further embodiment of the test substance genotoxicity assessment method according to the invention is based on the sequence context analysis described above. The method is
(1 ′) taking a cell population exposed to the test substance as a test group and obtaining the DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′),
including.

  The steps (1 ') to (7') are as described in the above (2.), particularly (2-2-2.). More specifically, in step (6 ′), each mutation is converted into the above-described six mutation patterns of base pairs [AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT. And 16 groups according to the types of bases adjacent to the mutation site [for example, in the case of mutation from C, ACA, ACC, ACG, ACT, CCA, CCC, CCG, CCT, GCA, GCC, GCG, GCT, TCA, Based on TCC, TCG, and TCT], it is classified into 96 types in total. Next, in step (7 '), the mutation frequency is determined for each of the mutation types determined in step (6'). Thereby, the type of mutation and the mutation frequency can be determined.

In a preferred embodiment, the method further comprises:
(8 ′) The cell population not exposed to the test substance is used as a control group, and the mutation frequency of each mutation type in the control group is determined by the same procedure as in (1 ′) to (7 ′);
(9 ′) Subtract the mutation frequency of each mutation type in the control group obtained in (8 ′) from the mutation frequency of each mutation type in the test group obtained in (7 ′).
Thereby, the variation | mutation frequency increase amount in the test group which removed the influence of the sequencing error can be calculated | required.

(3-1-3. Evaluation based on analysis of short insertion or deletion mutation)
A further embodiment of the method for assessing the genotoxicity of a test substance according to the invention is based on the analysis of short insertion or deletion mutations described above. The method is
(1 ″) A cell population exposed to a test substance is taken as a test group, and the DNA is obtained;
(2 ″) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ″) comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ″) obtaining the site detected in (3 ″) as a mutation site having an insertion or deletion mutation;
(5 ″) determining the length of the inserted or deleted base and / or the type of the inserted base for each acquired mutation;
(6 ″) determining the base length of the insertion or deletion site determined in (5 ″) and / or the mutation frequency for each type of inserted base;
including.

  The steps (1 ″) to (6 ″) are as described in the above (2.), particularly (2-2-3.). More specifically, in the step (3 ″), the length of the inserted or deleted base in each lead sequence is preferably 10 bp or less, more preferably, by comparison with the reference sequence. In addition, when an insertion or deletion is detected, the type of base inserted or deleted can be identified by comparing the sequence of each lead sequence with a reference sequence. Thereby, the base length of the insertion or deletion site in each lead sequence and / or the type of the inserted or deleted base can be determined (step (5 ″)). Then, in step (6 ″), the mutation frequency is determined for each of the base length of the insertion or deletion site determined in step (5 ″) and / or the type of base inserted or deleted. Thereby, a mutation pattern and a mutation frequency can be determined.

In a preferred embodiment, the method further comprises:
(7 ″) The cell population not exposed to the test substance is used as a control group, and the base length of the insertion or deletion site in the control group and / or the same procedure as in (1 ″) to (6 ″) Determining the mutation frequency for each type of inserted or deleted base;
(8 ″) Obtained from (7 ″) from the base length of the insertion or deletion site in the test group obtained in (6 ″) and / or the mutation frequency for each type of inserted or deleted base. Subtract the mutation frequency in the control group.
Thereby, the variation | mutation frequency increase amount in the test group which removed the influence of the sequencing error can be calculated | required.

  According to the method for evaluating the genotoxicity of a test substance according to the present invention, analyzing the tendency of mutation at the cell population level for a population of cells that may have different mutations due to exposure to the test substance. Can do. Therefore, according to the present invention, information closer to the actual influence of the test substance in vivo can be obtained as compared with the conventional analysis method based on a single cell (for example, Non-Patent Documents 4 and 5). Further, according to the present invention, the influence of a test substance on a cell population can be analyzed quantitatively and qualitatively at the level of individual bases on DNA. Detailed information can be obtained.

(3-2. Method for evaluating mutation associated with tumor development)
(3-2-1. Method for evaluating mutations in cancer cells)
As another preferred embodiment of the present invention, a method for evaluating mutations in cancer cells is provided. The specific procedure of this method is basically the same as the method for evaluating the genotoxicity of the test substance described above. However, as a test group, a population of cancer cells is used. Alternatively, instead of a cancer cell population, a cell suspected of cancer or a population of cells for which the risk of canceration is to be evaluated may be used as a test group. As a control group, a group of non-cancer cells (for example, normal cells) or a group of cells having a low cancer risk is used. This method is useful for identifying the tendency of mutations specific to cancer types, evaluating canceration risk, and confirming the degree of progression and malignancy of cancer. Regarding the evaluation of mutations in cancer cells, a more detailed analysis is possible by sequence context analysis as shown in (2-2-2.). So far, in human cancer genome analysis, 96 mutations (4 × 6 × 4) based on a 3 base context, or 1536 variations (4 × 4 × 6 ×) based on a 5 base context. 4 × 4) has been reported (Cell Rep, 2013, 3: 246-259).

  According to the method for evaluating a mutation in a cancer cell according to the present invention, a conventional method for analyzing a mutation at a specific site on the genome, that is, a method for aligning and comparing a plurality of read sequences for the same site on the genome (for example, Unlike the method of extracting only mutations present in cells in cancer tissue at a certain ratio as a result of selection in cancer tissue as in Non-Patent Documents 4 and 5), The tendency of the mutation specific to the cancer type that is occurring can be identified. It has been reported that the amount and quality of mutations in cancer cells vary depending on the type of cancer (Nature, 2013, 500 (7463): 415-421). According to the method of the present invention, the tendency of mutation in a cancer cell population can be analyzed quantitatively and qualitatively. Therefore, the method may be useful for diagnosis of cancer progression and type. Conceivable.

  Furthermore, in the analysis of mutation signatures so far, a conventional method (for example, non-patent literature) is obtained by decoding the genomic DNA obtained from each person in the group for which the tumor is induced by a specific cause. After extracting mutations that exist in a certain percentage of cancer tissue according to 4, 5), perform sequence context analysis on the extracted mutations, and then aggregate the mutation information obtained from each person, Identify the mutated signature of. However, when mutation signature analysis is performed in an individual diagnosis, since the number of identified mutations is small, it may be difficult to determine the similarity between the obtained sequence context and a known mutation signature. On the other hand, according to the present invention, as shown in FIGS. 7 and 8, it is possible to obtain sequence context data having a quality capable of confirming similarity with a mutation signature by one-time next generation sequencing analysis. . This is because, unlike the conventional method, by adding mutations that do not occupy a certain ratio or more to the analysis target, a sufficient number of mutations can be secured for the analysis of the sequence context. Therefore, it is considered that the present invention can be utilized for high-throughput analysis of sequence contexts in individual mutations.

(3-2-2. Method for evaluating mutations in cfDNA)
As another preferred embodiment of the present invention, analysis of blood cell-free DNA (cfDNA) is provided. cfDNA has attracted attention as a minimally invasive diagnostic method for human tumorigenesis. cfDNA is obtained from liquid biopsies such as plasma, serum, and urine. The specific procedure relating to the application of the method to cfDNA analysis is basically the same as the method for evaluating the genotoxicity of the test substance described above. However, as the DNA of the test group, cfDNA in a liquid biopsy collected from a human or the like of a cancer patient is used instead of DNA obtained from a cancer cell population. As a control group, for example, cfDNA derived from a healthy human, or the same human cfDNA collected in advance before suffering from cancer is used. This method is useful for identifying the tendency of mutation in cfDNA unique to cancer patients and for confirming the degree of progression and malignancy of cancer.

  According to the method for evaluating mutations in cfDNA according to the present invention, a unique tag sequence is added to each molecule of cfDNA, and a consensus sequence of a plurality of read sequences obtained from the same molecule is obtained. , Such as a method in which a plurality of read sequences are aligned and compared at the same location on the genome (see Non-Patent Documents 6 and 7), ocDNA occupies a certain ratio, and is constant in cells in cancer tissue. Unlike the method of extracting only mutations that are estimated to exist in a proportion, the tendency of mutations specific to the cancer type that occurs in the entire cancer cell population can be identified. Regarding the evaluation of mutations in cfDNA, more detailed analysis can be performed by sequence context analysis as shown in (2-2-2.). The method of the present invention is minimally invasive, and is useful for identifying the tendency of mutations peculiar to cancer cells, confirming the degree of progression and malignancy of cancer, detecting a microtumor having a low degree of progression, and the like. Therefore, it can be considered that the method of the present invention is applied to regular checkups and health checkups for cancer.

(3-3. Method for evaluating genetic information of cultured cells)
As another preferred embodiment of the present invention, a method for evaluating genetic information of cultured cells is provided. The specific procedure of this method is basically the same as the method for evaluating the genotoxicity of the test substance described above. However, as a test group, use a population of cultured cells to be examined for the presence of mutations. For example, the test group includes cells that have been passaged for a certain period of time and that want to confirm their mutation tendency. As a control group, a group of cultured cells of the same type and having known genetic information (for example, the presence or absence of mutation and its mutation type are confirmed) is used. For example, the control group includes cells before being subjected to passage. By this method, the presence or absence of a mutation in cultured cells or the mutation type can be evaluated. This method is useful for quality control of cultured cells.

  According to the method for evaluating genetic information of cultured cells according to the present invention, it is possible to identify the tendency of mutations unique to the cultured cells occurring in the cultured cell population, not mutations occurring in individual cells. According to this method, it is possible to evaluate whether or not genetic quality is maintained in cultured cells such as iPS cells (whether or not mutation has occurred). For example, when human-derived iPS cells are prepared, it is extremely important to perform genetic quality control for clinical application. In the genome of iPS cells, it has been reported that various mutations occur during the establishment process. These may lead to carcinogenesis after transplantation into a patient, and management of their genetic quality is essential (Nature, 2011, 471 (7336): 63-67). By using the method of the present invention, it is possible to easily grasp the tendency of mutation occurring in a population of iPS cells. Furthermore, the method of the present invention is a more comprehensive method than the conventional general iPS cell quality evaluation method using PCR, and is another conventional method of tumor formation using SCID mice (PLoS One). , 2012, 7 (5): e37342). Therefore, the method of the present invention will be useful as a simple and inexpensive screening technique for genetic quality control of iPS cells.

(3.4. Various conditions)
In any of the above embodiments, a known sequence in the DNA of the cell population of the test group can be used as the reference sequence. The reference sequence is preferably a sequence registered in a public database or the like, but is a sequence in the genomic DNA of the cell population previously sequenced by a sequencer or the like prior to the method of the present invention. Also good.

  Examples of the test substance used in the method for evaluating genotoxicity of a test substance according to the present invention are not particularly limited as long as the substance is desired to be evaluated for genotoxicity. For example, a substance suspected of having genotoxicity, a substance for which the presence / absence of genotoxicity is to be confirmed, a substance for which a mutation is to be induced, and the like are included. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, etc., or may be a compound, a composition or a mixture. Good. Alternatively, the test substance may be ultraviolet light or radiation.

  The means for exposing the cell population to the test substance may be appropriately selected according to the type of the test substance, and is not particularly limited. For example, a method of adding a test substance to a medium containing the cell population, a method of placing the cell population in an atmosphere containing the test substance, and the like can be mentioned.

  Examples of cell populations used in the method of the present invention include specimens collected from animals or plants, and populations of cultured cells derived from animals, plants or microorganisms, preferably animal, plant or microorganism strains. A population of cultured cells derived from it. Examples of animals are preferably mammals such as humans, silkworms, nematodes and the like, and examples of microorganisms are preferably Escherichia coli, Salmonella, yeast and the like.

  Of the cell populations listed above, the method for evaluating the genotoxicity of a test substance preferably uses a population of cultured cells derived from a microbial strain, more preferably from a group consisting of a population of E. coli cells and a population of Salmonella cells. At least one selected is used. Preferred examples of Salmonella include S. cerevisiae. Typhimurium LT-2 strain and S. typhimurium used for Ames test. Examples include Typhimurium TA100 strain, TA98 strain, TA1535 strain, TA1538 strain, TA1537 strain and the like. Preferable examples of E. coli include WP2 strain and WP2 uvrA strain which are also used for Ames test.

  The types of cancer cells that can be used in the present invention are not particularly limited. For example, lung cancer, breast cancer, prostate cancer, tongue cancer, laryngeal or pharyngeal cancer, digestive organ cancer (for example, esophageal cancer, Stomach cancer, duodenal cancer, colon cancer, colon or rectal cancer, etc.), liver cancer, pancreatic cancer, cervical cancer, endometrial cancer, renal cell cancer, renal pelvis cancer, bladder cancer, brain tumor Bone tumor, leukemia, lymphoma, myeloma, skin cancer, malignant melanoma and the like. These cancer cells may be derived from specimens collected from animals, or may be cultured cancer cell lines.

  As exemplary embodiments of the present invention, the following substances, production methods, uses, methods and the like are further disclosed herein. However, the present invention is not limited to these embodiments.

[1] A method for evaluating the genotoxicity of a test substance,
(1) The cell population exposed to the test substance is taken as a test group, and its DNA is obtained;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5),
Including a method.

[2] Preferably, the method further comprises extracting a base having a high reliability of reading by sequencing from the base of the read sequence obtained in (2), and in (3), the extracted read Compare bases on the sequence with bases on the reference sequence,
[1] The method according to [1].

[3] Preferably, the method further includes extracting a base having a high reliability of reading by sequencing among the bases of the site detected by comparing the lead sequence and the reference sequence in the above (3). [1] The method described.

[4] Preferably, (3) to (5) are
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
For each detected non-matching base, obtain a base pair before and after the mutation at the mutation site, and a base pair substitution mutation at the mutation site, the base pair before the mutation and the base pair after the mutation Classifying into 6 base pair mutation patterns of AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT, according to the type of
The method according to any one of [1] to [3].

[5] The method according to [4], preferably further comprising classifying each of the six base pair mutation patterns into two groups according to the original base.

[6] Preferably,
(7) Using the cell population not exposed to the test substance as a control group, and determining the mutation frequency of the mutation pattern of each base pair in the control group by the same procedure as in the above (1) to (6) ;
(8) subtracting the mutation frequency of each mutation pattern in the control group obtained in (7) from the mutation frequency of each mutation pattern in the test group obtained in (6),
The method according to any one of [1] to [5], comprising:

[7] A method for evaluating the genotoxicity of a test substance,
(1 ′) taking a cell population exposed to the test substance as a test group and obtaining the DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′),
Including a method.

[8] Preferably, the method further comprises extracting a base having a high reliability of reading by sequencing from the base of the read sequence obtained in (2 ′), and the extraction in (3 ′). Compare the base on the lead sequence with the base of the reference sequence,
[7] The method according to [7].

[9] Preferably, the method further includes extracting a base having a high reliability of reading by sequencing among the bases of the site detected by comparing the lead sequence and the reference sequence in (3 ′) above. ] The method of description.

[10] Preferably, (3 ′) to (6 ′) are
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
Obtaining a base pair before and after mutation at the mutation site for each non-matching detected base;
According to the type of base pair before mutation and base pair after mutation, AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT Classifying into 6 base pair mutation patterns
Determining a context sequence comprising a base before mutation at the mutation site, one or more bases adjacent to the upstream of the base before mutation, and one or more bases adjacent to the downstream of the base before mutation; And typing the base pair substitution mutations according to the mutation pattern of the six base pairs and the context sequence;
The method according to any one of [7] to [9], comprising:

[11] Preferably, the context sequence is a 3-base long sequence including a base before mutation at the mutation site and one base on both sides thereof, and the mutation pattern of the six base pairs and the three bases [10] The method according to [10], wherein the base pair substitution mutation is typed into 96 according to a long context sequence.

[12] Preferably,
(8 ′) determining the mutation frequency of each mutation type in the control group by the same procedure as in the above (1 ′) to (7 ′) using the cell population not exposed to the test substance as a control group;
(9 ′) subtracting the mutation frequency of each mutation type in the control group obtained in (8 ′) from the mutation frequency of each mutation type in the test group obtained in (7 ′),
The method according to any one of [7] to [11], comprising:

[13] A method for evaluating the genotoxicity of a test substance,
(1 ″) A cell population exposed to a test substance is taken as a test group, and the DNA is obtained;
(2 ″) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ″) comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ″) obtaining the site detected in (3 ″) as a mutation site having an insertion or deletion mutation;
(5 ″) determining the length of the inserted or deleted base and / or the type of the inserted base for each acquired mutation;
(6 ″) determining the base length of the insertion or deletion site determined in (5 ″) and / or the mutation frequency for each type of inserted base;
Including a method.

[14] Preferably, the method further comprises extracting a base having a high reliability of reading by sequencing from the base of the read sequence obtained in (2 ″), and the extraction in (3 ″). Compare the base on the lead sequence with the base of the reference sequence,
[13] The method described.

[15] Preferably, the method further includes extracting a base having a high reliability of reading by sequencing from the bases of the site detected by comparing the lead sequence and the reference sequence in (3 ″). ] The method of description.

[16] The method according to any one of [13] to [15], wherein the detected base length of the inserted or deleted site is preferably 10 bp or less, more preferably 1 to 5 bp.

[17] Preferably, furthermore,
(7 ″) The cell population not exposed to the test substance is used as a control group, and the base length of the insertion or deletion site in the control group and / or in the same procedure as in the above (1 ″) to (6 ″) Determining the mutation frequency for each type of inserted base;
(8 ″) From the base length of the insertion or deletion site in the test group obtained in (6 ″) and / or the mutation frequency for each type of inserted base, the control group obtained in (7 ″) Subtracting the mutation frequency in
The method according to any one of [13] to [16], comprising:

[18] Preferably, any one of [1] to [12], wherein the base pair substitution mutation is a one base pair substitution mutation, a two base pair substitution mutation, or a three base pair substitution mutation. The method described.

[19] The method according to any one of [1] to [18], wherein the cell population is preferably at least one selected from the group consisting of a Salmonella cell population and an E. coli cell population.

[20] Preferably, the Salmonella is S. cerevisiae. [19] The method according to [19], wherein the strain is Typhimurium LT-2, TA100, TA98, TA1535, TA1538, or TA1537.

[21] A method for evaluating mutations in cancer cells,
(1) Acquiring DNA of a cancer cell population as a test group;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5),
Including a method.

[22] A method for evaluating genetic information of cultured cells,
(1) The cultured cell population is used as a test group, and the DNA is obtained;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5),
Including a method.

[23] Preferably, the method further comprises extracting a base having a high reliability of reading by sequencing from the base of the read sequence obtained in (2), and in the above (4), the extracted read Compare bases on the sequence with bases on the reference sequence,
[21] or [22] The method.

[24] Preferably, the method further includes extracting a base having a high reliability of reading by sequencing among the bases of the site detected by the comparison between the read sequence and the reference sequence in (3). Or the method of [22] description.

[25] Preferably, (3) to (5) are
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
For each detected non-matching base, obtain a base pair before and after the mutation at the mutation site, and a base pair substitution mutation at the mutation site, the base pair before the mutation and the base pair after the mutation Classifying into 6 base pair mutation patterns of AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT, according to the type of
The method according to any one of [21] to [24], comprising:

[26] The method of [25], preferably further comprising classifying each of the six base pair mutation patterns into two groups according to the original base.

[27] Preferably,
(7) Determining the mutation frequency of the mutation pattern of each base pair in the control group in the same procedure as the above (1) to (6);
(8) subtracting the mutation frequency of each mutation pattern in the control group obtained in (7) from the mutation frequency of each mutation pattern in the test group obtained in (6),
The method according to any one of [21] to [26], comprising:

[28] Preferably,
The method is a method for evaluating mutations in cancer cells, and the control group is a non-cancer cell population, or
The test group is a population of cells suspected of being cancerous or cells to be evaluated for canceration risk, the control group is a non-cancer cell population or a population of cells having a low canceration risk, and The cancer risk of the cells is evaluated by the method,
[27] The method described.

[29] Preferably, the method is a method for evaluating genetic information of cultured cells, and the control group is a cultured cell of the same type as the test group, and is a population of cells with known genetic information. 27] The method described.

[30] A method for evaluating mutations in cancer cells,
(1 ′) taking a cancer cell population as a test group and obtaining its DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′),
Including a method.

[31] A method for evaluating genetic information of cultured cells,
(1 ′) using the cultured cell population as a test group and obtaining the DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′),
Including a method.

[32] Preferably, the method further comprises extracting a base having high reliability of reading by sequencing from the base of the read sequence obtained in (2 ′), and the extraction in (3 ′). Compare the base on the lead sequence with the base of the reference sequence,
[30] or [31] The method.

[33] Preferably, the method further includes extracting a base having a high reliability of reading by sequencing among the bases of the site detected by comparing the lead sequence and the reference sequence in (3 ′) above [30] ] Or the method according to [31].

[34] Preferably, (3 ′) to (6 ′) are
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
Obtaining a base pair before and after mutation at the mutation site for each non-matching detected base;
According to the type of base pair before mutation and base pair after mutation, AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT Classifying into 6 base pair mutation patterns
Determining a context sequence comprising a base before mutation at the mutation site, one or more bases adjacent to the upstream of the base before mutation, and one or more bases adjacent to the downstream of the base before mutation; And typing the base pair substitution mutations according to the mutation pattern of the six base pairs and the context sequence;
The method according to any one of [30] to [33].

[35] Preferably, the context sequence is a 3-base long sequence including a base before mutation at the mutation site and one base on both sides thereof, and the mutation pattern of the six base pairs and the three bases [34] The method according to [34], wherein the base pair substitution mutation is typed into 96 according to a long context sequence.

[36] Preferably, furthermore,
(8 ′) determining the mutation frequency of each mutation type in the control group by the same procedure as in the above (1 ′) to (7 ′);
(9 ′) subtracting the mutation frequency of each mutation type in the control group obtained in (8 ′) from the mutation frequency of each mutation type in the test group obtained in (7 ′),
The method according to any one of [30] to [35], comprising:

[37] Preferably,
The method is a method for evaluating mutations in cancer cells, and the control group is a non-cancer cell population, or
The test group is a population of cells suspected of being cancerous or cells to be evaluated for canceration risk, the control group is a non-cancer cell population or a population of cells having a low canceration risk, and The cancer risk of the cells is evaluated by the method,
[36] The method described.

[38] Preferably, the method is a method for evaluating genetic information of cultured cells, and the control group is a cultured cell of the same type as the test group, and is a population of cells with known genetic information. 36] The method described.

[39] A method for evaluating mutations in cancer cells,
(1 ″) Acquiring DNA of a cancer cell population as a test group;
(2 ″) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ″) comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ″) obtaining the site detected in (3 ″) as a mutation site having an insertion or deletion mutation;
(5 ″) determining the length of the inserted or deleted base and / or the type of the inserted base for each acquired mutation;
(6 ″) determining the base length of the insertion or deletion site determined in (5 ″) and / or the mutation frequency for each type of inserted base;
Including a method.

[40] A method for evaluating genetic information of cultured cells,
(1 ″) using the cultured cell population as a test group and obtaining the DNA;
(2 ″) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ″) comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ″) obtaining the site detected in (3 ″) as a mutation site having an insertion or deletion mutation;
(5 ″) determining the length of the inserted or deleted base and / or the type of the inserted base for each acquired mutation;
(6 ″) determining the base length of the insertion or deletion site determined in (5 ″) and / or the mutation frequency for each type of inserted base;
Including a method.

[41] Preferably, the method further comprises extracting a base having a high reliability of reading by sequencing from the base of the read sequence obtained in (2 ″), and the extraction in (3 ″). Compare the base on the lead sequence with the base of the reference sequence,
[39] The method according to [40].

[42] Preferably, the method further includes extracting a base having a high reliability of reading by sequencing among the bases of the site detected by comparing the read sequence with the reference sequence in (3 ″) above [39] ] Or the method according to [40].

[43] The method according to any one of [39] to [42], wherein the detected base length of the inserted or deleted site is preferably 10 bp or less, more preferably 1 to 5 bp.

[44] Preferably,
(7 ″) determining the base length of the insertion or deletion site in the control group and / or the mutation frequency for each type of inserted base in the same procedure as in the above (1 ″) to (6 ″);
(8 ″) From the base length of the insertion or deletion site in the test group obtained in (6 ″) and / or the mutation frequency for each type of inserted base, the control group obtained in (7 ″) Subtracting the mutation frequency in
The method according to any one of [39] to [43].

[45] Preferably,
The method is a method for evaluating mutations in cancer cells, and the control group is a non-cancer cell population, or
The test group is a population of cells suspected of being cancerous or cells to be evaluated for canceration risk, the control group is a non-cancer cell population or a population of cells having a low canceration risk, and The cancer risk of the cells is evaluated by the method,
[44] The method described.

[46] Preferably, the method is a method for evaluating genetic information of cultured cells, and the control group is a cultured cell of the same type as the test group, and is a population of cells with known genetic information. 44].

[47] Preferably, the base pair substitution mutation is one base pair substitution mutation, two base pair substitution mutation, or three base pair substitution mutation, any one of [21] to [38] the method of.

[48] The total amount of lead sequences used for the detection is preferably 1 × 10 ¹⁰ bp or less, more preferably 1 × 10 ⁹ bp or less, further preferably 1 × 10 ⁸ bp or less, and even more preferably 1 × 10 ⁷ The method according to any one of [1] to [47], which is not more than bp, more preferably not more than 1 × 10 ⁶ bp.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Example 1 Validation of Analysis Method In this example, the effectiveness of the analysis method of the present invention was verified by analyzing a synthetic DNA sample with a known mutation frequency using the test method of the present invention and qualitatively and quantitatively evaluating the mutation. .

1. Preparation of DNA samples Synthetic DNA samples containing various mutation patterns in known amounts were prepared. Schematic diagram 1 shows a conceptual diagram of a DNA sample preparation procedure. A synthetic DNA sequence (SEQ ID NO: 1; hereinafter referred to as a random DNA sequence) having a random sequence of 1000 bp was synthesized. In this random DNA sequence, about 50% of GC and AT base pairs were present. Based on this random DNA sequence, a DNA sequence (hereinafter referred to as a mutant DNA sequence) into which a mutation (base pair substitution mutation or short insertion / deletion mutation) was introduced was prepared. Details will be described below.

For mutant DNA sequences containing base pair substitution mutations, the GC base pair (501st) located at the center of the random DNA sequence was replaced with another base pair (GC → TA, CG or AT, see Table 1) 3 Various types of mutant sequences were prepared and each incorporated into a pTAKN-2 vector. The obtained vector was dissolved in TE buffer (pH 8.0, manufactured by Wako Pure Chemical Industries, Ltd.), adjusted to a concentration of 100 ng / μL, and equal amounts of solutions containing each mutant DNA sequence were mixed. Similarly, three types of mutant sequences were prepared by substituting the AT base pair (the 502nd position) with other base pairs (AT → TA, CG or GC, see Table 1), and each was incorporated into the pTAKN-2 vector. A TE buffer solution (100 ng / μL) was prepared, and equal amounts of the three types of solutions obtained were mixed. Each equivalent mixed solution was used as a mutant DNA solution. Further, 10 μL of the mutant DNA solution was mixed with 90 μL of TE buffer to prepare a 10-fold diluted mutant DNA solution. Further, 10 μL of the 10-fold diluted mutant DNA solution was mixed with 90 μL of TE buffer to prepare a 100-fold diluted mutant DNA solution. Separately, a random DNA sequence was incorporated into the pTAKN-2 vector to prepare a TE buffer solution (100 ng / μL) of the vector (random DNA solution). A random DNA solution is mixed with a mutant DNA solution, a 10-fold diluted mutant DNA solution, or a 100-fold diluted mutant DNA solution, and each base pair substitution is observed at an equal frequency, and the total mutation frequency is 1/10 ³ , 1 DNA samples of / 10 ⁴ , 1/10 ⁵ , and 1/10 ⁶ bp were prepared (see Table 2).

As for the mutant DNA sequence containing a short insertion / deletion mutation, a mutant sequence in which one base (A, ie, AT base pair) was inserted before the 501st base pair was prepared (see Table 1). This was incorporated into the pTAKN-2 vector in the same manner as described above, and a TE buffer solution (100 ng / μL) of the vector was prepared to obtain a mutant DNA solution. Moreover, 10-fold and 100-fold diluted mutant DNA solutions of the mutant sample solution were prepared. A random DNA solution is mixed with a mutant DNA solution, a 10-fold diluted mutant DNA solution, or a 100-fold diluted mutant DNA solution, and the total mutation frequency is 1/10 ³ , 1/10 ⁴ , 1/10 ⁵ , 1/10. ^{A 6} bp DNA sample was prepared (see Table 2). The following sequencing was performed using a DNA sample containing each mutant DNA solution as a mutant sample and a DNA sample not containing the mutant DNA solution (only the random DNA solution) as a control sample.

2. High-throughput sequencing For each mutant sample and control sample prepared in step 1, the base sequence was decoded using a next-generation sequencer HiSeq 2500 (manufactured by Illumina, hereinafter also referred to as HiSeq) according to a standard protocol. At that time, the DNA was fragmented to an average length of about 150 bp by sonication, adapters were added to both ends of each fragment, and sequencing was performed with a read length of 2 × 125 bp. The average base sequence information of 1.9 Gbp was obtained per sample.

3. Editing of Read Sequence and Production of Mutation Analysis Format A schematic diagram of the read sequence editing and analysis flow obtained by sequencing is shown in FIG. First, using the Cutadapt software (Martin, 2011), the adapter sequence at each lead end was removed and the base with low quality was removed.
i) In HiSeq, for each DNA fragment (original fragment) subjected to sequencing, the read 1 sequence is read from one side, and then the read 2 sequence forming the pair is obtained from the opposite side. Therefore, for the read sequences of each pair that passed the selection of Cutadapt, PEAR software was used to superimpose the portions where the sequences of both leads form a pair, thereby constructing a single combined read. All bases in the synthetic lead sequence were matched to lead 1. The quality value of each base in the synthetic lead is the sum of the quality values of both bases when the bases of lead 1 and lead 2 are complementary, and the higher quality value when the bases of both leads are not complementary. A value obtained by subtracting the smaller quality value from the value was adopted. Thereby, according to the difference in quality value, among the bases in the synthetic lead, those in which the bases of lead 1 and 2 are paired can be selected.
ii) The synthetic reads for each original fragment prepared were mapped to a reference sequence (sequence of pTAKN-2 vector into which a random DNA sequence was inserted) using Bowtie 2 software, and a file in Sam format was created.
iii) The obtained Sam file was converted into a pileup format using Samtools software. At this time, based on the base quality value, the base information to be analyzed was limited to a range in which the bases of both leads were paired in a complementary manner in the overlapping region of the pair of leads.
iv) The resulting pileup format was subjected to mutation analysis using a program created using the programming language Python.

4). Mutation Analysis 1) Detection of Base Pair Substitution Mutation A schematic diagram of a mutation analysis algorithm for a single base pair substitution mutation is shown in FIG. Using the program created using the programming language Python from the pileup format subjected to the analysis, all the bases to be analyzed in the lead sequence are grouped with a group whose base of the corresponding reference sequence is A, a group with T, and G The group was classified into 4 groups, one group and C group. Next, the total number of bases assigned to each group and the mutated bases were detected. From the obtained data, the mutation call ratio of each mutation pattern (AT → TA, AT → CG, AT → GC) of the AT base pair occupying per 10 ⁶ bp of the AT base pair before mutation, and the GC base pair before mutation The mutation call ratio of each mutation pattern (GC → TA, GC → CG, GC → AT) of GC base pairs occupying around 10 ⁶ bp was calculated.

  FIG. 1 shows the ratio of mutation calls for each mutation pattern of GC and AT base pairs in a mutation sample containing a base pair substitution mutation. For any mutation pattern, the mutation call rate increased depending on the mutation frequency in the sample. A mutation was also detected in the control sample, which represents a background error (an error that occurred during the sequencing process from sample preparation including sequencing errors). Furthermore, the mutation call ratio in the control sample also varied with the mutation pattern, and the GC base pair tended to have a higher mutation call ratio than the AT base pair. This is considered to be because GC base pairs are easily affected by chemical modification such as oxidation during the library preparation process such as DNA extraction.

2) Calculation of mutation frequency increase amount Next, by subtracting the mutation call ratio of the control sample from the mutation call ratio of the mutation sample, background errors including sequence errors are excluded, and the mutation frequency of the mutation sample relative to the control sample is calculated. The increase amount (hereinafter referred to as the mutation frequency increase amount) was calculated. The calculated mutation frequency increase is shown in FIG. In any mutation pattern, the amount of increase in mutation frequency and the introduced mutation frequency were almost the same, indicating that this method was able to detect a single base pair substitution mutation at about 10 ⁵ bp. .

3) Detection of short insertion / deletion mutations For short insertion / deletion mutations, a base inserted or deleted at a length of 10 bp or less from a random DNA sequence using a program created using the programming language Python Were detected, and the insertion or deletion length (bp), and the frequency of occurrence for each type of insertion or deletion base were counted. Further, similarly to the analysis of the base pair substitution mutation described above, the mutation frequency of the control sample was subtracted to exclude the background error, and the mutation frequency increase was calculated. The result of the mutation frequency increase amount of the insertion mutation is shown in FIG. 3A shows the length (bp) of the inserted base, and FIG. 3B shows the amount of increase in mutation frequency for the type of inserted base. By this method, a short insertion mutation with a frequency of about 10 ⁵ bp could be detected. In addition, single nucleotide deletion was also examined by the same method as insertion mutation, and one deletion mutation with a short frequency of about 10 ⁵ bp could be detected (data not shown).

5. Conclusion In this example, with regard to base pair substitution mutations and short insertion / deletion mutations in DNA containing various mutation information, comprehensive mutation information including quantity (frequency) and quality (mutation pattern) is highly sensitive. I was able to get it. From these results, it was shown that low-frequency mutations present in DNA can be analyzed qualitatively and quantitatively by the analysis method of the present invention.

Example 2 Analysis of genotoxicity by mutagen In this example, genotoxicity of the mutagen was determined by qualitative and quantitative analysis of the mutation pattern generated in the genome of the organism by exposure to the mutagen by the analysis method of the present invention. Analyzed. Ethylnitrosourea (ENU, CAS No. 759-73-9) was used as a mutagen. As an organism exposed to the mutagen, Salmonella TA100 strain, which is widely used for the Ames test and capable of detecting a base pair substitution mutation, was used. In the experiment, three independent operations were performed to prepare samples (n = 3).

1. TA100 strain exposure to mutagens Exposure to mutagens is determined by the pre-incubation method of the Ames test (K. Mortelmans et al., Mutat. Res.-Fundam. Mol. Mech. Mutagen., 2000, 455, 29-60). ). TA100 strain was added to a 2 mL Nutrient bouillon no. 2 (manufactured by Oxoid) and cultured with shaking at 37 ° C. and 180 rpm for 4 hours. D. A preculture solution having a 660 value of 1.0 or more was obtained. ENU (54%; manufactured by Sigma-Aldrich) was diluted with dimethyl sulfoxide (DMSO; manufactured by Wako Pure Chemical Industries, Ltd.). In a test tube, add 100 μL of ENU solution diluted to an appropriate concentration, 500 μL of 0.1 M phosphate buffer, and 100 μL of preculture (ENU concentrations: 67.5, 135, 270, 405, 540, 810, and 1080 μg). / Tube, shaking culture at 100 rpm for 20 minutes in a water bath at 37 ° C. As a control group, 100 μL of solvent (DMSO) was added instead of the ENU solution. Remove the test tube from the water bath, add 50 μL of the culture solution to 2 mL of the Nutrient Broth solution previously dispensed, perform additional culture at 37 ° C. and 180 rpm for 14 hours, collect 1 mL of the bacterial suspension, and then add 7500 rpm. Was centrifuged for 5 minutes, the supernatant was removed, and the cells were collected.

  In addition, for the Ames test, a bacterial suspension exposed to ENU under the same conditions as described above was prepared, and 2 mL of top agar (1% NaCl, 1% agar, 0.05 mM Histidine and 0%) heated to 45 ° C. (Containing 0.05 mM Biotine), suspended by vortexing, and overlaid on a minimal glucose agar medium (Tesmedia (registered trademark) AN; manufactured by Oriental Yeast Co., Ltd.). The obtained plate was cultured at 37 ° C. for 48 hours, and the observed colonies were counted.

2. Total DNA recovery Total DNA was recovered from the bacterial cells obtained in 1 above using DNeasy Blood & Tissue Kit (Qiagen) according to the recommended protocol.

3. High-throughput sequence Using the total DNA solution from the control group and the ENU-treated group collected in (1), the base sequence was decoded by the next-generation sequencer HiSeq 2500 (manufactured by Illumina) according to the standard protocol. At that time, the DNA was fragmented to an average length of about 150 bp by sonication, adapters were added to both ends of each fragment, and sequencing was performed with a read length of 2 × 125 bp. On average, 5.0 Gbp of base sequence information was obtained per sample.

4). Editing of lead sequence and analysis of mutation The editing and mutation analysis of the read sequence obtained by sequencing are performed in the same manner as in Example 1, according to the conceptual diagram of the analysis flow shown in FIG. This was done using Samtools software and programs created using the programming language Python. In this example, PCR duplicates were removed using Picard tools (broadinstitute.github.io/picard/) after mapping with Bowtie 2 software.

  The reference sequence to be mapped by the Bowtie 2 software is S. cerevisiae. It was constructed based on the genomic sequence of Typhimurium TA100 strain. First, DNA was extracted from the TA100 strain, and the nucleotide sequence was decoded with a next-generation sequencer HiSeq 2500 (manufactured by Illumina) according to a standard protocol. At that time, the DNA was fragmented to an average length of about 300 bp by sonication, and adapters were added to both ends of each fragment, followed by sequencing with a read length of 2 × 125 bp. The resulting lead sequence was designated as S. After mapping to the genomic sequence of the Typhimurium LT-2 strain, the pSLT plasmid sequence (GCA0000006945.2), and the R46 plasmid sequence (NC — 003292.1), mutation detection was performed using Samtool software. A reference sequence based on the genome sequence of the TA100 strain reflecting the obtained mutation information was prepared and used for mutation analysis. The genome sequence of the TA100 strain is shown in SEQ ID NO: 2.

5. Calculation of increase amount of mutation frequency In the same procedure as in Example 1, using a program using the programming language Python, all the bases to be analyzed in all the read sequences mapped to the reference sequence are converted to the corresponding reference sequence. The group was divided into 4 groups by base, and then the total number of bases in each group and the bases mutated with respect to the reference sequence were detected. By comparing the mutated base with the base of the reference sequence, each mutation pattern (AT → TA, AT in each 10 ⁶ bp of the AT base pair and the GC base pair in the base to be analyzed for each of the ENU treatment group and the control group. → CG, AT → GC, and GC → TA, GC → CG, GC → AT), and the mutation call ratio of each mutation pattern was calculated. A statistical test for the frequency of each mutation pattern between the control group and the ENU-treated group was performed by Dunnett's multiple comparison test for each mutation pattern.

  Next, the amount of increase in mutation frequency due to exposure to ENU is calculated by subtracting the mutation call ratio of the control group from the mutation call ratio of the ENU treatment group for each base pair mutation pattern in the same procedure as 2) of Example 1. did.

6). Sequence context analysis In this study, the mutation call ratio was analyzed based on the sequence context. That is, 5. Based on the mutation call information and the reference sequence information obtained in step 1, the mutation type of the base pair at each mutation call site was detected. Further, based on the information of the reference sequence, information of 3 bases including each mutation call site and the bases on both sides thereof was collected. The mutations at each mutation call site were classified into 96 types obtained by multiplying 6 types of base pair mutation types and the base information (4 × 4 = 16 types) on both sides. The mutation call ratio (/ 10 ⁶ bp) was calculated for each of 96 types of mutations based on this sequence context. For each mutation type, the amount of increase in mutation frequency due to ENU exposure was calculated by subtracting the mutation call ratio in the control group from the mutation call ratio in the ENU treatment group.

7). Results 1) Number of revertants in Ames test Table 3 shows the number of revertant colonies after exposure to ENU. The data shows the average and standard deviation of the measurements from the three plates. An increase in the number of back mutation mutants was observed with ENU exposure, indicating that the mutation was introduced into the genome of TA100 strain by ENU exposure.

2) Calculation of change in mutation call ratio due to ENU exposure Regarding ENU treatment groups (ENU concentrations 135, 270, 405 and 540 μg / tube), The amount of increase in mutation frequency calculated in is shown in FIG. An increase in the frequency of multiple base pair mutation patterns was observed with ENU exposure. In GC base pairs, an increase in the frequency of GC> AT mutation was observed (FIG. 4A). On the other hand, in the AT base pair, an increase in the frequency of AT> TA and AT> GC mutation was mainly observed (FIG. 4B).

3) Classification of each mutation pattern by original base In HiSeq, the sequence of lead 1 corresponds to the original DNA fragment (original fragment) subjected to the sequencing reaction. Therefore, the base before mutation (that is, the original base) at the mutation site of the original fragment can be confirmed by examining which base of A, T, G and C of the reference sequence is mapped to the base of the lead 1 sequence. .
Since the background error frequency may vary depending on the original base, mutation patterns were further classified according to the original base, and the amount of increase in mutation frequency for each classification was determined. That is, the above 5. Each of the 6 base pair mutation patterns in the control sample and the mutation sample obtained in (1) was further classified into two according to the type of original base, and the mutation frequency of each classification was obtained. Subsequently, the mutation frequency increase amount was calculated by subtracting the mutation frequency of the corresponding control sample from the mutation frequency of the mutation sample.

  FIG. 5 shows the amount of increase in the mutation frequency of the base pair mutation pattern divided for each type of original base in the ENU-exposed sample. Usually, in the case of a mutation fixed in the genome by exposure to ENU, both bases of the base pair change, and therefore, the increase in the mutation frequency is observed at the same frequency in both bases forming the base pair. However, in the GC> TA mutation in FIG. 5, when the original base is G, the tendency that the mutation frequency tends to be higher than when the original base is C was clearly observed.

  The above bias strongly suggests that the majority of GC> TA mutations were caused by chemically modified G. That is, it was considered that the increase in GC> TA mutation observed in this study was not a mutation fixed in the genome but reflected a reading error of a chemically modified G base in sequencing. It is known that the G base is susceptible to chemical modification by oxidation during the preparation of a DNA sample, and is prone to error in reading G as T in sequencing. Therefore, the increase in GC> TA mutation frequency observed in some samples in this study was considered to be an artificial effect caused by oxidation of G during the DNA preparation process. Therefore, it was suggested that this analysis method can eliminate sequencing errors caused by errors generated in the process of preparing DNA samples from cells.

4) Mutation spectrum analysis Next, a mutation spectrum was analyzed based on the mutation frequency increase amount of each mutation pattern in which an increase was observed in the ENU540 μg / tube group. Specifically, the amount of increase in mutation frequency at 10 ⁶ bp of each mutation pattern was summed, and the ratio of each mutation pattern to the whole was calculated. The results are shown in FIG. The mutation pattern with the highest frequency increase was GC> AT mutation, and the next highest mutation pattern was AT> GC mutation. This is the same result as the ENU mutation pattern observed in YG7108, which is one of the Ames test strains in Non-Patent Document 5, and thus the mutation pattern by ENU could be accurately detected by this method. It has been suggested.

5) Sequence context analysis FIGS. 7 and 8 show the amount of increase in the mutation frequency calculated by the sequence context analysis. The notation of the mutation pattern in the figure is the pyrimidine base mutation pattern (C> A, C> G, C> T, T> A, T> C and T> G) of the mutated base pair, and the pyrimidine. It is represented by a three-base sequence containing a base and its adjacent bases (for example, when C> T, when the C base is sandwiched between A and T, it is expressed as ACT). In the C> T mutation, which showed the largest increase in mutation frequency in this analysis, a tendency was found that the increase in mutation frequency increased in the context where the pyrimidine base (C or T) is located on the 3 ′ base side of C. . This is similar to the pattern of mutation signatures with alkylating agents (see Signature 11, Nature, 2013, 500 (7463): 415-421 in FIGS. 7-8), where ENU is the alkylating agent. The result was consistent with that. From these results, it was considered that sequence context analysis is a useful method that can easily estimate the genotoxic mechanism of mutagen and its role in human carcinogenesis.

Claims (28)

A method for evaluating the genotoxicity of a test substance,
(1) The cell population exposed to the test substance is taken as a test group, and its DNA is obtained;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5);
(7) Using the cell population not exposed to the test substance as a control group, the mutation frequency of the mutation pattern of each base pair in the control group is determined by the same procedure as in (1) to (6). ;
(8) subtracting the mutation frequency of each mutation pattern in the control group obtained in (7) from the mutation frequency of each mutation pattern in the test group obtained in (6),
Including a method. The method further comprises extracting a base having high reliability of reading by sequencing from the base of the read sequence obtained in (2), and referring to the base on the extracted lead sequence in (3) Compare with the base of the sequence,
The method of claim 1. Said (3)-(5)
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
For each detected non-matching base, obtain a base pair before and after the mutation at the mutation site, and a base pair substitution mutation at the mutation site, the base pair before the mutation and the base pair after the mutation Classifying into 6 base pair mutation patterns of AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT, according to the type of
The method according to claim 1 or 2, comprising: A method for evaluating the genotoxicity of a test substance,
(1 ′) taking a cell population exposed to the test substance as a test group and obtaining the DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′);
(8 ′) The cell population not exposed to the test substance is used as a control group, and the mutation frequency of each mutation type in the control group is determined by the same procedure as in (1 ′) to (7 ′);
(9 ′) subtracting the mutation frequency of each mutation type in the control group obtained in (8 ′) from the mutation frequency of each mutation type in the test group obtained in (7 ′),
Including a method. Further comprising extracting a base having a high reliability of reading by sequencing from the base of the lead sequence obtained in (2 ′), and in the step (3 ′), a base on the extracted lead sequence To the base of the reference sequence,
The method of claim 4. Said (3 ')-(6') are
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
Obtaining a base pair before and after mutation at the mutation site for each non-matching detected base;
According to the type of base pair before mutation and base pair after mutation, AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT Classifying into 6 base pair mutation patterns
Determining a context sequence comprising a base before mutation at the mutation site, one or more bases adjacent to the upstream of the base before mutation, and one or more bases adjacent to the downstream of the base before mutation; And typing the base pair substitution mutations according to the mutation pattern of the six base pairs and the context sequence;
The method according to claim 4 or 5, comprising: A method for evaluating the genotoxicity of a test substance,
(1 ") taking the cell population exposed to the test substance as a test group and obtaining its DNA;
(2 ") sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ") comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ") obtaining the site detected in (3") as a mutation site having an insertion or deletion mutation;
(5 ") for each of the obtained mutated, the base length of the insertion or deletion, and / or insertion or to determine the type of the deleted bases;
(6 ") determining the base length of the insertion or deletion site determined in (5") and / or the mutation frequency for each type of inserted or deleted base;
(7 ") The cell population not exposed to the test substance as a control group, and the base length of the insertion or deletion site in the control group and / or Determining the mutation frequency for each type of inserted or deleted base;
(8 ") Obtained from (7") from the base length of the insertion or deletion site in the test group obtained in (6 ") and / or the mutation frequency for each type of inserted or deleted base. Subtracting the mutation frequency in the control group,
Including a method. Further comprising extracting a base having a high reliability of reading by sequencing from the base of the read sequence obtained in (2 "), and in the base of the extracted lead sequence in (3") To the base of the reference sequence,
The method of claim 7. The base pair substitution is performed without performing the step of determining the site as a mutation site based on the frequency of appearance of the base between the lead sequences in the site where the base sequence that does not match the reference sequence is detected. The method according to claim 1, which is obtained as a mutation site having a type mutation. Without performing the step of determining a site on the lead sequence into which the base has been inserted or deleted from the reference sequence as a mutation site based on the frequency of appearance of the inserted or deleted site between the lead sequences The method according to claim 7 or 8, which is obtained as a mutation site having an insertion or deletion mutation. The method according to any one of claims 1 to 6 and 9 , wherein the base pair substitution mutation is a one base pair substitution mutation, a two base pair substitution mutation, or a three base pair substitution mutation. The method according to any one of claims 1 to 11 , wherein the cell population is at least one selected from the group consisting of a Salmonella cell population and an E. coli cell population. The Salmonella is S. The method according to claim 12 , which is Typhimurium LT-2 strain, TA100 strain, TA98 strain, TA1535 strain, TA1538 strain or TA1537 strain. A method for evaluating mutations in cancer cells,
(1) Acquiring DNA of a cancer cell population as a test group;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5);
(7) Determining the mutation frequency of the mutation pattern of each base pair in the control group by the same procedure as in (1) to (6);
(8) subtracting the mutation frequency of each mutation pattern in the control group obtained in (7) from the mutation frequency of each mutation pattern in the test group obtained in (6),
Including a method. A method for evaluating genetic information of cultured cells,
(1) The cultured cell population is used as a test group, and the DNA is obtained;
(2) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3) comparing each of the one or more lead sequences with a reference sequence and detecting a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is known in the DNA An array;
(4) obtaining the site detected in (3) as a mutation site having a base pair substitution mutation;
(5) classifying each acquired mutation according to the mutation pattern of the base pair;
(6) determining the mutation frequency of each of the mutation patterns obtained in (5);
(7) Determining the mutation frequency of the mutation pattern of each base pair in the control group by the same procedure as in (1) to (6);
(8) subtracting the mutation frequency of each mutation pattern in the control group obtained in (7) from the mutation frequency of each mutation pattern in the test group obtained in (6),
Including a method. Further comprising extracting a base having a high reliability of reading by sequencing from the base of the lead sequence obtained in (2), and referring to the base on the extracted lead sequence in ( 3 ) 16. A method according to claim 14 or 15 , wherein the method is compared with the base of the sequence. Said (3)-(5)
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
For each detected non-matching base, obtain a base pair before and after the mutation at the mutation site, and a base pair substitution mutation at the mutation site, the base pair before the mutation and the base pair after the mutation Classifying into 6 base pair mutation patterns of AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT, according to the type of
Including, any one method according to claim 14-16. A method for evaluating mutations in cancer cells,
(1 ′) taking a cancer cell population as a test group and obtaining its DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′);
(8 ′) determining the mutation frequency of each mutation type in the control group by the same procedure as in (1 ′) to (7 ′);
(9 ′) subtracting the mutation frequency of each mutation type in the control group obtained in (8 ′) from the mutation frequency of each mutation type in the test group obtained in (7 ′),
Including a method. A method for evaluating genetic information of cultured cells,
(1 ′) using the cultured cell population as a test group and obtaining the DNA;
(2 ′) sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ′) comparing each of the one or more lead sequences with a reference sequence to detect a site where the base does not match between the lead sequence and the reference sequence, wherein the reference sequence is Is a known sequence;
(4 ′) obtaining the site detected in (3 ′) as a mutation site having a base pair substitution mutation;
(5 ′) for each obtained mutation, based on the reference sequence, determining a context sequence including a base before mutation and bases adjacent to the upstream and downstream of the base before mutation;
(6 ′) typing each mutation obtained in (4 ′) according to the context sequence determined in (5 ′) and the type of base after mutation;
(7 ′) determining the mutation frequency of each of the mutation types obtained in (6 ′);
(8 ′) determining the mutation frequency of each mutation type in the control group by the same procedure as in (1 ′) to (7 ′);
(9 ′) subtracting the mutation frequency of each mutation type in the control group obtained in (8 ′) from the mutation frequency of each mutation type in the test group obtained in (7 ′),
Including a method. Further comprising extracting a base having a high reliability of reading by sequencing from the base of the lead sequence obtained in (2 ′), and in the step (3 ′), a base on the extracted lead sequence To the base of the reference sequence,
20. A method according to claim 18 or 19 . Said (3 ')-(6') are
Dividing the base contained in the lead sequence into the following (i) to (iv):
(i) a base present at a position where the base on the reference sequence is A
(ii) a base present at a position where the base on the reference sequence is T
(iii) a base present at a position where the base on the reference sequence is G
(iv) A base present at a position where the base on the reference sequence is C Among the bases contained in the lead sequence, those that do not match the base with the reference sequence are detected, and the site where the base is present is determined as a base pair. Obtaining as a mutation site having a substitution mutation,
Obtaining a base pair before and after mutation at the mutation site for each non-matching detected base;
According to the type of base pair before mutation and base pair after mutation, AT → TA, AT → CG, AT → GC, GC → TA, GC → CG, and GC → AT Classifying into 6 base pair mutation patterns
Determining a context sequence comprising a base before mutation at the mutation site, one or more bases adjacent to the upstream of the base before mutation, and one or more bases adjacent to the downstream of the base before mutation; And typing the base pair substitution mutations according to the mutation pattern of the six base pairs and the context sequence;
21. The method according to any one of claims 18 to 20 , comprising: A method for evaluating mutations in cancer cells,
(1 ") taking a cancer cell population as a test group and obtaining its DNA;
(2 ") sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ") comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ") obtaining the site detected in (3") as a mutation site having an insertion or deletion mutation;
(5 ") for each of the obtained mutated, the base length of the insertion or deletion, and / or insertion or to determine the type of the deleted bases;
(6 ") determining the base length of the insertion or deletion site determined in (5") and / or the mutation frequency for each type of inserted or deleted base;
(7 ") the (1") - the same procedure as in (6 "), determining the mutation frequency of each type of bases deleted bases in length and / or insertion or deletion of the insertion or deletion site in the control group ;
(8 ") Obtained from (7") from the base length of the insertion or deletion site in the test group obtained in (6 ") and / or the mutation frequency for each type of inserted or deleted base. Subtracting the mutation frequency in the control group,
Including a method. A method for evaluating genetic information of cultured cells,
(1 ") using the cultured cell population as a test group and obtaining the DNA;
(2 ") sequencing the DNA fragments to obtain one or more read sequences for each fragment;
(3 ") comparing each of the one or more lead sequences with a reference sequence to detect a site where a base is inserted or deleted from the reference sequence in the lead sequence, wherein the reference sequence is A known sequence in the DNA;
(4 ") obtaining the site detected in (3") as a mutation site having an insertion or deletion mutation;
(5 ") for each of the obtained mutated, the base length of the insertion or deletion, and / or insertion or to determine the type of the deleted bases;
(6 ") determining the base length of the insertion or deletion site determined in (5") and / or the mutation frequency for each type of inserted or deleted base;
(7 ") the (1") - the same procedure as in (6 "), determining the mutation frequency of each type of bases deleted bases in length and / or insertion or deletion of the insertion or deletion site in the control group ;
(8 ") Obtained from (7") from the base length of the insertion or deletion site in the test group obtained in (6 ") and / or the mutation frequency for each type of inserted or deleted base. Subtracting the mutation frequency in the control group,
Including a method. Further comprising extracting a base having a high reliability of reading by sequencing from the base of the read sequence obtained in (2 "), and in the base of the extracted lead sequence in (3") To the base of the reference sequence,
24. A method according to claim 22 or 23 . The base pair substitution is performed without performing the step of determining the site as a mutation site based on the frequency of appearance of the base between the lead sequences in the site where the base sequence that does not match the reference sequence is detected. The method according to any one of claims 14 to 21, which is obtained as a mutation site having a type mutation. Without performing the step of determining a site on the lead sequence into which the base has been inserted or deleted from the reference sequence as a mutation site based on the frequency of appearance of the inserted or deleted site between the lead sequences The method according to any one of claims 22 to 24, which is obtained as a mutation site having an insertion or deletion mutation. The method according to any one of claims 14 to 21 and 25 , wherein the base pair substitution mutation is a one base pair substitution mutation, a two base pair substitution mutation, or a three base pair substitution mutation. 28. The method according to any one of claims 1 to 27 , wherein the total amount of the lead sequence used for the detection is 1 × 10 ¹⁰ bp or less.