Apparatus and method for detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed

Abstract

To provide an apparatus and method capable of detecting nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed without using a reference nucleic acid sequence.SOLUTION: Provided is an apparatus used for detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed. The apparatus comprises a first acquisition unit, a second acquisition unit, and a detection unit. The first acquisition unit acquires read data of the nucleic acid sequence to be analyzed. The second acquisition unit acquires data of the nucleic acid sequence to be detected. The detection unit compares a k-base-long nucleic acid sequence in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected with the nucleic acid sequence of the other data to detect the k-base-long nucleic acid sequence in the other data, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed is detected.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an apparatus and method for use in the detection of a nucleic acid sequence of interest in a nucleic acid sequence to be analysed. [Background technology]

[0002] Genetic engineering and genome editing techniques are used as methods for improving plant varieties. However, in the former case, plants containing foreign genes (GMOs) created using genetic engineering technology are subject to legal restrictions. For this reason, there are high barriers to selling GMOs as products. On the other hand, in the latter case, plants modified using genome editing technology are not subject to the legal restrictions regarding GMOs, so long as no foreign genes have been introduced into the genome. For this reason, compared to GMOs, plants modified using genome editing technology and that do not fall under GMOs have lower barriers to selling them as products, and as a result, attempts are being made to improve plants using genome editing technology.

[0003] In order to show that a plant modified using genome editing technology is not a GMO, it is necessary to show that no exogenous gene has been introduced into the genome of the modified plant. As a method for detecting the exogenous gene, there is a Southern blot method using a probe for a nucleic acid sequence introduced into the genome. However, when the length of the inserted exogenous gene is short (for example, 100 bases or less), it is difficult to detect the inserted exogenous gene by the Southern blot method. Therefore, a method using a next-generation sequencer is used as a more accurate method for detecting an exogenous gene. In the method using the next-generation sequencer, the genome to be analyzed is analyzed using the next-generation sequencer to obtain read data of the nucleic acid sequence of the genome to be analyzed. Next, in the method using the next-generation sequencer, the nucleic acid sequence of the entire length of the genome to be analyzed is reconstructed from the read data with reference to the nucleic acid sequence (also called the "reference nucleic acid sequence") of the target of analysis, and the obtained nucleic acid sequence of the entire length of the genome to be analyzed is compared with the reference nucleic acid sequence to identify the presence or absence of a nucleic acid sequence derived from the inserted exogenous gene or its nucleic acid sequence (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Stephan Pabinger et.al., “A survey of tools for variant analysis of next-generation genome sequencing data”, BRIEFINGS IN BIOINFORMATICS, 2014, VOL. 15, NO. 2, pages 256-278. Summary of the Invention [Problem to be solved by the invention]

[0005] However, when detecting an exogenous gene in the genome of the subject of analysis using a reference nucleic acid sequence, the nucleic acid sequence of the full-length genome of the subject of analysis must be decoded and the decoded nucleic acid sequence must have high analytical accuracy, that is, a full-length reference nucleic acid sequence with high analytical accuracy is required. Therefore, the method of detecting an exogenous gene using a next-generation sequencer has a problem that it cannot be applied to all subjects of analysis, including subjects with genome nucleic acid sequences with low analytical accuracy and subjects whose full-length genome nucleic acid sequences have not been decoded.

[0006] Therefore, an object of the present invention is to provide an apparatus and method capable of detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed without using a reference nucleic acid sequence. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention provides an apparatus for detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, the apparatus comprising: A first acquisition unit, a second acquisition unit, and a detection unit, The first acquisition unit acquires read data of a nucleic acid sequence to be analyzed, The second acquisition unit acquires data of a nucleic acid sequence to be detected, The detection unit detects the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a k-base-long nucleic acid sequence in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected with the nucleic acid sequence of the other data and detecting the k-base-long nucleic acid sequence in the other data.

[0008] The method of the present invention is a method used for detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising the steps of: The method includes a first acquiring step, a second acquiring step, and a detecting step, In the first obtaining step, read data of a nucleic acid sequence to be analyzed is obtained, In the second obtaining step, data on a nucleic acid sequence to be detected is obtained, In the detection step, a k-base-long nucleic acid sequence in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected is compared with the nucleic acid sequence of the other data, and the k-base-long nucleic acid sequence in the other data is detected, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed.

[0009] The program of the present invention causes a computer to execute a first acquisition process, a second acquisition process, and a detection process, In the first acquisition process, read data of a nucleic acid sequence to be analyzed is acquired, In the second acquisition process, data on a nucleic acid sequence to be detected is acquired, In the detection process, a nucleic acid sequence of k bases in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected is compared with the nucleic acid sequence of the other data, and the k bases in the other data are detected, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed. Effect of the Invention

[0010] According to the present invention, a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed can be detected without using a reference nucleic acid sequence. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a block diagram showing an example of a configuration of a detection device, a detection terminal, and a detection system including these in the first embodiment. [Diagram 2] FIG. 2 is a block diagram illustrating an example of a hardware configuration of the detection device according to the first embodiment. [Diagram 3] FIG. 3 is a block diagram illustrating an example of a hardware configuration of the detection terminal according to the first embodiment. [Figure 4] FIG. 4 is a flowchart showing an example of the detection method and program according to the first embodiment. [Diagram 5]FIG. 5 is a block diagram showing an example of a configuration of a detection device, a detection terminal, and a detection system including these in the second embodiment. [Figure 6] FIG. 6 is a flowchart showing an example of a detection method and a program according to the second embodiment. [Figure 7] FIG. 7 is a flowchart showing an example of a detection method and a program according to the second embodiment. [Figure 8] FIG. 8 is a diagram showing an outline of the detection method in Example 1. [Figure 9] FIG. 9 is a graph showing the results of detection of exogenous DNA in Example 1. [Figure 10] FIG. 10 is a schematic diagram showing the structure of the all-in-one binary vector in Example 4. [Figure 11] FIG. 11 is a photograph showing the detection results of the HPT gene fragment in Example 4. [Figure 12] FIG. 12 is a graph showing the results of detection of exogenous DNA (binary vector) in Example 4. [Figure 13] FIG. 13 is a graph showing the results of detection of foreign DNA (vector) in Example 5. [Figure 14] FIG. 14 is a graph showing the rate of identity of randomly generated nucleic acid sequences each having a length of k bases in Reference Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] <Definition> In the present invention, the term "analysis subject" refers to any subject for which a nucleic acid sequence derived from a detection subject is to be detected. The analysis subject may be any subject having a nucleic acid sequence, and specific examples include nucleic acid molecules or objects containing nucleic acid molecules. Specific examples of the analysis subject include animals, plants, or microorganisms, or the genomes of these organisms, and plasmids of microorganisms. The analysis subject is preferably a subject that may have undergone a nucleic acid sequence modification process, such as by mutation introduction or genome editing.

[0013] In the present invention, the "nucleic acid sequence derived from the detection target" refers to a nucleic acid sequence that is a nucleic acid sequence that is a part or all of a nucleic acid sequence of a nucleic acid molecule to be detected, and is presumed to be derived from the nucleic acid molecule to be detected. The "match" means that a certain nucleic acid sequence has 100% identity with another nucleic acid sequence. The nucleic acid molecule to be detected is not particularly limited and can be any nucleic acid molecule, and specific examples include genomes (DNA or RNA), chromosomes, genes, plasmids, gene exons or introns, etc. The nucleic acid sequence of the nucleic acid molecule to be detected can be, for example, a nucleic acid sequence registered in a database such as the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ). As a specific example, when the nucleic acid molecule to be analyzed is the genome of an organism, the nucleic acid molecule to be detected can be a nucleic acid such as a gene, a plasmid, or a gene exon of a gene other than the organism.

[0014] In the present invention, "nucleic acid sequence" means the base sequence of a nucleic acid molecule.

[0015] In the present invention, "nucleic acid sequence data" refers to data that provides information about the base sequence of a nucleic acid molecule. Specifically, the "nucleic acid sequence data" refers to data on the entire base sequence (sequence) of a certain nucleic acid sequence or a part of the base sequence (partial sequence).

[0016] In the present invention, the "reference nucleic acid sequence" refers to the full-length nucleic acid sequence of the nucleic acid molecule to be analyzed. The full-length nucleic acid sequence can be appropriately determined depending on the nucleic acid molecule to be analyzed, and specific examples include the nucleic acid sequence of the entire genome, the nucleic acid sequence of the full-length chromosome, the nucleic acid sequence of the full-length gene, the nucleic acid sequence of the full-length plasmid, the nucleic acid sequence of the full-length exon, and the nucleic acid sequence of the full-length intro. The full-length nucleic acid sequence can be, for example, a nucleic acid sequence registered in a database such as NCBI or DDBJ.

[0017] In the present invention, the term "subsequence" refers to any nucleic acid sequence contained in a nucleic acid sequence.

[0018] In the present invention, "next-generation sequencing" refers to a sequencing technique that parallelizes the process of determining the nucleic acid sequence of a nucleic acid molecule and generates tens to hundreds of millions of sequence data in a single run. In addition, in the present invention, "next-generation sequencer" refers to an instrument for performing next-generation sequencing.

[0019] In the present invention, "read data" refers to data that provides information obtained by fragmenting a nucleic acid molecule to be analyzed and performing a process of determining the nucleic acid sequence of the fragmented nucleic acid molecule. Examples of the fragmentation include mechanical fragmentation (shearing) and enzymatic fragmentation. As a specific example, the read data includes data that provides information obtained by performing a process of determining the nucleic acid sequence of a nucleic acid molecule using a next-generation sequencer. The read is also called, for example, a short read.

[0020] In the present invention, "eliminating coincidental identity" means reducing the expected value of occurrence of a sequence identical to a given sequence by chance to less than one.

[0021] In the present invention, "coverage" refers to the amount of data of a nucleic acid sequence that corresponds to how many times the full length of the nucleic acid sequence. The "coverage" may also be referred to as, for example, "coverage rate," "~ times reading," "coverage level," or "depth."

[0022] A detection system including the detection device of the present invention will be described in detail below with reference to the drawings. However, the present invention is not limited to the following description. In the following Figs. 1 to 14, the same parts are given the same reference numerals, and their description may be omitted. In addition, in the drawings, for convenience of description, the structure of each part may be appropriately simplified, and the dimensional ratio of each part may be shown diagrammatically, different from the actual one.

[0023] [Embodiment 1] This embodiment is an example of a detection system including a detection device and a detection terminal of the present invention. FIG. 1 is a block diagram showing a detection system 100 including a detection device 1, a detection terminal 2, and a sequence database (sequence DB) 3 of this embodiment. As shown in FIG. 1, the detection system 100 includes a detection device 1, a detection terminal 2, and a sequence DB 3. Also, as shown in FIG. 1, the detection device 1 includes a first acquisition unit 11, a second acquisition unit 12, and a detection unit 13. Also, as shown in FIG. 1, the detection terminal 2 includes an input unit 21 and an output unit 22. The sequence DB 3 stores read data (read data of the analysis target) 31 of one or more nucleic acid sequences to be analyzed and data (data of the detection target) 32 of nucleic acid sequences to be detected. As shown in FIG. 1, the detection device 1, the detection terminal 2, and the sequence DB 3 are connectable (communicable) in one direction or both directions via a communication line network 4 outside the detection system 100. The detection device 1, the detection terminal 2, and the sequence DB 3 of this embodiment may be incorporated into a personal computer (PC) in which the program of the present invention is installed, or into a server or the like as a system. The personal computer may also constitute a computer cluster. Although not shown, the detection device 1, the detection terminal 2, and the array DB 3 may be connected to an external terminal of a system administrator via a communication line network 4, and the system administrator may manage the detection device 1, the detection terminal 2, and the array DB 3 from the external terminal. In this embodiment, the detection system 100 includes one each of the detection device 1, the detection terminal 2, and the array DB 3, but each may be multiple.

[0024] The communication network 4 is not particularly limited and may be a known network, for example, a wired or wireless network. Examples of the communication network 4 include the Internet, WWW (World Wide Web), telephone lines, LAN (Local Area Network), and WiFi (Wireless Fidelity).

[0025] Examples of the detection terminal 2 include a PC, a mobile terminal such as a mobile phone, a smartphone, or a tablet terminal, a smart watch, smart glasses, a wearable terminal, etc. The detection terminal 2 may include, for example, an imaging means such as a camera or a scanner, an IC (integrated circuit) card reader, a voice input means such as a microphone, etc.

[0026] 2 illustrates a block diagram of a hardware configuration of the detection device 1. The detection device 1 includes, for example, a CPU (Central Processing Unit) 101, a memory 102, a bus 103, a storage device 104, an input device 106, a display 107, and a communication device 108. Each part of the detection device 1 is connected via the bus 103 by its respective interface (I / F).

[0027] The CPU 101 cooperates with other components, for example, as a controller (system controller, I / O controller, etc.), and controls the entire detection device 1. In the detection device 1, the CPU 101 executes, for example, the program 105 of the present invention and other programs, and also reads and writes various information stored in the memory 102 or the storage device 104. Specifically, for example, the CPU 101 functions as a first acquisition unit 11, a second acquisition unit 12, and a detection unit 13. The detection device 1 includes a CPU as a calculation device, but may also include other calculation devices such as a GPU (Graphics Processing Unit) and an APU (Accelerated Processing Unit), or may include a combination of the CPU and these. The CPU 101 functions, for example, as each unit in the detection device of the second embodiment described later.

[0028] The memory 102 includes, for example, a main memory. The main memory is also called a primary storage device. When the CPU 101 performs processing, the memory 102 reads various operation programs, such as a program 105 of the present invention, stored in, for example, a storage device 104 (auxiliary storage device) described below. The CPU 101 then reads data from the memory 102, decodes it, and executes the program. The main memory is, for example, a RAM (random access memory). The memory 102 further includes, for example, a ROM (read only memory).

[0029] The bus 103 can also be connected to, for example, an external device. Examples of the external device include an external storage device (external database, etc.) such as an array DB3, a printer, etc. The detection device 1 can be connected to a communication network 4, for example, by a communication device 108 connected to the bus, and can also be connected to an external device via the communication network 4. Therefore, the detection device 1 can also be connected to the detection terminal 2 and the array DB3 via the communication device 108 and the communication network 4.

[0030] The storage device 104 is also called an auxiliary storage device, for example, in contrast to the main memory (primary storage device). As described above, the storage device 104 stores an operation program including the program 105 of the present invention. The storage device 104 includes, for example, a storage medium and a drive for reading and writing data from and to the storage medium. The storage medium is not particularly limited, and may be either an internal or external type, and examples of the storage medium include a hard disk (HD), a floppy disk (FD), a CD-ROM, a CD-R, a CD-RW, an MO, a DVD, a flash memory, and a memory card, and the drive is not particularly limited. The storage device 104 may be, for example, a hard disk drive (HDD) in which the storage medium and the drive are integrated.

[0031] The detection device 1 further includes, for example, an input device 106 and a display 107. Examples of the input device 106 include a pointing device such as a touch panel, a track pad, or a mouse; a keyboard; an imaging means such as a camera or a scanner; a card reader such as an IC card reader or a magnetic card reader; and a voice input means such as a microphone. Examples of the display 107 include a display device such as an LED (light emitting diode) display or a liquid crystal display. In the first embodiment, the input device 106 and the display 107 are configured separately, but the input device 106 and the display 107 may be configured as an integrated device, such as a touch panel display.

[0032] In the detection device 1, the memory 102 and the storage device 104 can also store access information and log information from a user, as well as information acquired from an external database such as the array DB3.

[0033] 3 illustrates a block diagram of a hardware configuration of the detection terminal 2. The detection terminal 2 includes, for example, a CPU 201, a memory 202, a bus 203, a storage device 204, an input device (input unit) 21, a communication device 208, and a display (output unit) 22. Each component of the detection terminal 2 is connected via the bus 203 by its respective interface (I / F). The description of each component of the detection terminal 2 can be made by referring to the description of each component of the detection device 1.

[0034] In this embodiment, the array DB3 is a database server in which one or more analysis target read data 31 and one or more detection target data 32 are stored, as described later. The hardware configuration of the array DB3 can be explained by referring to the description of the hardware configuration of the detection device 1. In the array DB3, the analysis target read data 31 and the detection target data 32 are stored in a storage device. The number of analysis target read data 31 stored in the array DB3 is one or more. Also, the number of detection target data 32 stored in the array DB3 is one or more. In this embodiment, the analysis target read data 31 and the detection target data 32 are stored in the array DB3, but the analysis target read data 31 and / or the detection target data 32 may be stored in the storage device 104 of the detection device 1.

[0035] Next, an example of processing in the detection system 100 of this embodiment will be described with reference to the flowchart in Fig. 4, taking as an example a case where processing is performed based on the analysis target read data 31 and the detection target data 32 input from the detection terminal 2 and stored in the sequence DB 3. In Fig. 4, the detection device 1 performs steps S3 to S5. In Fig. 4, steps S1 and S2 are optional steps and may or may not be present.

[0036] Prior to processing by the detection device 1, first, the read data 31 to be analyzed and the data 32 to be detected are prepared. Specifically, a user inputs the read data 31 to be analyzed and the data 32 to be detected via the input unit 21 of the user's detection terminal 2 (S1).

[0037] The read data 31 of the analysis target can be obtained by fragmenting the nucleic acid molecule contained in the analysis target and decoding the nucleic acid sequence of each fragmented nucleic acid molecule. In the first embodiment, the read data 31 of the analysis target is read 1 (L T1 )~Lead n(L Tn) (n: positive integer). The read data 31 of the analysis target may be read data acquired by a user or read data registered in a database or the like. The read data 31 of the analysis target may be acquired, for example, by deciphering the nucleic acid sequence of the nucleic acid molecule contained in the analysis target using a next-generation sequencer. The next-generation sequencer may be a sequencer using pyrosequencing, a reversible terminator method, or ion semiconductor sequencing, and specific examples thereof include GS Junior and GS FLX+ platforms (manufactured by Roche), MiSeq, NextSeq and HiSeq platforms (manufactured by Illumina), Genetic Analyzer and Ion Proton System (manufactured by ThermoFisher Scientific), etc. The read data 31 of the analysis target is linked to, for example, identification information such as an identifier of the analysis target.

[0038] The length (base length) of each read included in the read data 31 to be analyzed is not particularly limited and varies depending on, for example, the method of decoding the nucleic acid sequence used to obtain the read data 31 to be analyzed. The length of the read is, for example, 50 to 2000 bps (base length), or 100 to 600 bps (base length).

[0039] The read data 31 to be analyzed may be composed of, for example, the nucleic acid sequence of each read decoded using the next-generation sequencer or the like, or may be composed of the nucleic acid sequence of each read decoded using the next-generation sequencer or the like, which has been pre-processed. The pre-processing may be, for example, trimming or masking of the nucleic acid sequence of each read. The trimming and masking may be performed using, for example, software (Trimmomatic, fastp (https: / / github.com / OpenGene / fastp), Cutadapt (https: / / cutadapt.readthedocs.io / en / stable / ), etc.). The trimming is, for example, a process of deleting a part or all of the nucleic acid sequence of an adapter (also called an index, tag, barcode, etc.) that is added to a fragment of a nucleic acid molecule derived from the nucleic acid molecule to be analyzed in the decoding of a nucleic acid sequence. The nucleic acid sequence of the adapter is usually added to both ends of the nucleic acid sequence derived from the nucleic acid molecule to be analyzed, and is present on both ends of each read. For this reason, in the trimming process, a part or all of the nucleic acid sequence corresponding to the adapters at both ends of the nucleic acid sequence of the read is deleted. If pre-processing has not been performed on the read data 31 to be analyzed, pre-processing may be performed on the read data 31 to be analyzed prior to detection in the detection step (S5) described below. By performing pre-processing on the read data 31 to be analyzed, the detection device 1 of the first embodiment can detect the nucleic acid sequence derived from the detection target with higher accuracy in the detection step (S5) described below.

[0040] The amount of the read data 31 of the analysis target is, for example, equal to or more than the amount of the nucleic acid sequence contained in one unit of the analysis target, and is preferably 10 times or more, more preferably 20 times or more, and even more preferably 30 times or more. In the detection device 1 of the first embodiment, if the amount of the read data 31 of the analysis target provided to the detection step (S5) described later is increased, the nucleic acid sequence derived from the detection target can be detected with better analysis accuracy (particularly, analysis sensitivity). For this reason, the upper limit of the amount of the read data 31 of the analysis target can be appropriately set according to, for example, the analysis accuracy required by the user and the detection processing time allowed by the user. As a specific example, the upper limit of the amount of the read data 31 of the analysis target can be, for example, 1000 times or less, 500 times or less, 250 times or less, or 100 times or less of one unit of the analysis target. The amount of the read data 31 to be analyzed is, for example, 10 to 1000 times, 20 to 1000 times, 30 to 1000 times, 10 to 500 times, 20 to 500 times, 30 to 500 times, 10 to 250 times, 20 to 250 times, and 30 to 250 times. The one unit means the minimum configuration of the analysis target. As a specific example, when the analysis target is a genome, a chromosome, and a gene, the one unit of the analysis target means the full length of the genome, the full length of the chromosome, and the full length of the gene. The amount of the read data 31 to be analyzed can be called coverage (coverage rate), or can also be called coverage rate, "~ times reading", coverage level, or depth. The amount of the read data 31 to be analyzed can be calculated by dividing the number of nucleic acid sequences (total number of bases) in the read data 31 to be analyzed by the number of nucleic acid sequences (total number of bases) contained in one unit of the analysis target. As a specific example, when the analysis target is a genome of an organism, the amount of read data 31 to be analyzed is read 1 (L T1 )~Lead n(L Tn It can be calculated by dividing the total number of bases in the entire genome by the total number of bases in the entire genome.

[0041] As described above, the detection target data 32 is data on the nucleic acid sequence of the detection target nucleic acid molecule. The detection target data 32 may be data acquired by a user or data registered in a database or the like. The number of detection target data 32 is not particularly limited and can be appropriately determined according to, for example, the number of detection targets considered by the user. The detection target data 32 is linked to, for example, identification information such as an identifier of the detection target.

[0042] Next, the read data 31 of the analysis target and the data 32 of the detection target input to the detection terminal 2 are output to the array DB 3 via the communication line network 4. Then, the array DB 3 stores the read data 31 of the analysis target and the data 32 of the detection target by linking them with the information (ID, identifier, etc.) of the analysis target and the information (ID, identifier, etc.) of the detection target, respectively (S2). Note that in the S2 step, the read data 31 of the analysis target and the data 32 of the detection target are stored simultaneously (in parallel), but may be processed separately. In this case, the order of storing the read data 31 of the analysis target and the data 32 of the detection target is not particularly limited, and the data 32 of the detection target may be stored after the read data 31 of the analysis target is stored, or the read data 31 of the analysis target may be stored after the data 32 of the detection target is stored.

[0043] Next, the detection device 1 starts processing using the read data 31 of the analysis target stored in the array DB3 and the data 32 of the detection target. First, the detection device 1 acquires, for example, the read data 31 of the analysis target and the data 32 of the detection target instructed by the user from the detection terminal 2 from the array DB3. Specifically, the first acquisition unit 11 of the detection device 1 requests the read data 31 of the analysis target stored in the array DB3 from the array DB3, and acquires the read data 31 of the analysis target output from the array DB3 (S3, first acquisition step). Next, the second acquisition unit 12 of the detection device 1 requests the data 32 of the detection target stored in the array DB3 from the array DB3, and acquires the data 32 of the detection target output from the array DB3 (S4, second acquisition step). Note that, in the first embodiment, S4 is processed after the processing of S3, but the processing order of S3 and S4 is not particularly limited, and S3 may be processed after the processing of S4, or S3 and S4 may be processed simultaneously.

[0044] Next, in the detection device 1, the detection unit 13 detects the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target using the read data 31 of the analysis target and the data 32 of the detection target (S5, detection step). Specifically, in S5, the detection unit 13 first extracts (generates) a nucleic acid sequence of k bases from the read data 31 of the analysis target to be used for comparison with the nucleic acid sequence of the data 32 of the detection target. The number of nucleic acid sequences of k bases to be extracted may be one or more. The nucleic acid sequence of k bases may be, for example, a nucleic acid sequence of k bases specified by a user, or a nucleic acid sequence of k bases may be extracted randomly or regularly from the nucleic acid sequence of the read data 31 of the analysis target. As a specific example, the nucleic acid sequence of k bases may be extracted from the read data 31 of the analysis target, read 1 (L T1 )~Lead n(L Tn ) may be used to extract one or more k-base-long nucleic acid sequences from any one read, one or more k-base-long nucleic acid sequences from multiple reads, or one or more k-base-long nucleic acid sequences from all reads.

[0045] In the k base length, the value of k can be appropriately set depending on the entire length of the nucleic acid sequence to be analyzed, and as a specific example, satisfies the following condition (1). In this case, the detection device 1 of embodiment 1 may be equipped with a setting unit that, prior to step S5, acquires information on the total base length of the nucleic acid sequence contained in one unit of the analysis subject, calculates the value of k based on the total base length of the nucleic acid sequence contained in one unit of the analysis subject, and sets the obtained value of k. In this case, the setting unit performs a setting step of acquiring information on the total base length of the nucleic acid sequence contained in one unit of the analysis subject, and calculating and setting the value of k. As a specific example, when the analysis subject is a rice genome, the total base length (N G ) is 400 Mb, and k is calculated to be 15, so it is preferable to set k in the range of 15 or more. The value of k may be a value input by the user. Condition (1): N G ≦4 k (In the above condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of analysis.)

[0046] The value of k is preferably a value that is unlikely to occur by chance, specifically 16, 17, 18, or 19, or any number or more, more preferably 20, 25, 30, 35, 40, 45, or 50, or any number or more. The upper limit of the value of k can be set, for example, according to the length of the full-length nucleic acid sequence of the detection target or the length of the full-length nucleic acid sequence of the analysis target. As a specific example, the upper limit of the value of k is the length of the full-length nucleic acid sequence of the detection target or the length of the full-length nucleic acid sequence of the analysis target. In the detection device 1 of embodiment 1, by setting the value of k to 20 or more, the possibility of occurrence of coincident identity can be further reduced, and the nucleic acid sequence derived from the detection target can be detected with higher accuracy (particularly, specificity of analysis).

[0047] Next, the detection unit 13 compares (collates) the extracted k-base-long nucleic acid sequence with the nucleic acid sequence of the data 32 to be detected, and detects a nucleic acid sequence that matches the k-base-long nucleic acid sequence in the nucleic acid sequence of the data 32 to be detected. The comparison can be performed, for example, using known software or processing used for aligning nucleic acid sequences or amino acid sequences. The detection unit 13 detects a sequence that matches the k-base-long nucleic acid sequence in the nucleic acid sequence of the data 32 to be detected, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed. In addition, when there are multiple k-base-long nucleic acid sequences, the detection unit 13 compares each k-base-long nucleic acid sequence with the nucleic acid sequence of the data 32 to be detected, and detects a nucleic acid sequence that matches the k-base-long nucleic acid sequence in the nucleic acid sequence of the data 32 to be detected. The detection unit 13 may link the detection result obtained for the k-base-long nucleic acid sequence with the data of the k-base-long nucleic acid sequence. Thereby, the detection device 1 can more accurately analyze whether or not a nucleic acid sequence derived from the detection target is present in the nucleic acid sequence of the analysis target by, for example, comparing the detection result obtained by the detection unit 13 with a threshold value, performing statistical processing, etc. Examples of the detection result include the count number (detection count) of the k-base-long nucleic acid sequence, the detection frequency of the k-base-long nucleic acid sequence, etc.

[0048] Then, the detection device 1 of the first embodiment ends the process.

[0049] The detection device 1 of the first embodiment may, for example, output the obtained detection result to the detection terminal 2. In this case, the detection terminal 2 outputs the detection result to the output unit 22.

[0050] According to the detection device 1, detection terminal 2, and detection system 100 of the first embodiment, a nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target can be detected from the read data 31 of the analysis target and the data 32 of the detection target. Therefore, the detection device 1 of the first embodiment can detect a nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target without using the above-mentioned reference nucleic acid sequence.

[0051] In the detection system 100 of the first embodiment, the lead data 31 of the analysis target and the data 32 of the detection target are stored in the sequence DB 3, but the present invention is not limited to this, and the lead data 31 of the analysis target and the data 32 of the detection target may be stored in the storage device 104 of the detection device 1. Furthermore, when the data 32 of the detection target is registered in a database outside the system, the detection device 1 may request the data 32 of the detection target from the database outside the system and acquire the transmitted data 32 of the detection target.

[0052] In the detection device 1 of the first embodiment, a k-base-long nucleic acid sequence is extracted from the read data 31 of the analysis target, but the present invention is not limited thereto, and a k-base-long nucleic acid sequence may be extracted from the data 32 of the detection target, or a k-base-long nucleic acid sequence may be extracted from both the read data 31 of the analysis target and the data 32 of the detection target. In this case, the detection unit 13 compares the extracted k-base-long nucleic acid sequence with the other nucleic acid sequence and detects a nucleic acid sequence that matches the k-base-long nucleic acid sequence, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target.

[0053] When the detection unit 13 links the detection result obtained for the k-base-long nucleic acid sequence with the data of the k-base-long nucleic acid sequence, the detection device 1 of the first embodiment may further include a determination unit, and the determination unit may determine whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the detection result of the k-base-long nucleic acid sequence in the other data. The determination in the determination unit may be, for example, a comparison of the count number with a first threshold value set in advance, a comparison with the detection result obtained using the control read data described later, or a statistical test. The first threshold value can be set, for example, by preparing a nucleic acid molecule to be analyzed that contains a nucleic acid molecule derived from the detection target and a nucleic acid molecule to be analyzed that does not contain a nucleic acid molecule derived from the detection target, and using the detection result obtained by the detection device 1 of the first embodiment.

[0054] [Embodiment 2] This embodiment is another example of a detection system including the detection device and detection terminal of the present invention. FIG. 5 is a block diagram showing a detection system 100A including the detection device 1A, the detection terminal 2, and the sequence DB3A of this embodiment. As shown in FIG. 5, the detection system 100A includes the detection device 1A, the detection terminal 2, and the sequence DB3. Also, as shown in FIG. 5, the detection device 1A includes a third acquisition unit 14, a comparison sequence generation unit 15, a determination unit 16, and a position identification unit 17 in addition to the configuration of the detection device 1 of the first embodiment. Also, the sequence DB3A stores the read data (control read data) 33 of the control nucleic acid sequence in addition to the data of the sequence DB3 of the first embodiment. The hardware configuration of the detection device 1A is the same as that of the detection device 1 of FIG. 2, except that the CPU 101 has the configuration of the detection device 1A of FIG. 5 instead of the configuration of the detection device 1 of FIG. 1. Except for these points, the configuration of the detection device 1A of the second embodiment is the same as the configuration of the detection device 1 of the first embodiment, and the description thereof can be used.

[0055] An example of the process in the detection system 100A of this embodiment will be described with reference to the flowcharts of FIG. 6 and FIG. 7. In FIG. 6 and FIG. 7, the detection device 1A performs steps S3 to S9. As shown in FIG. 6 and FIG. 7, the detection method of the second embodiment includes steps S1A and S2A instead of steps S1 and S2 of the detection method of the first embodiment. Moreover, the detection method of the second embodiment includes steps S51 to S54 instead of step S5 of the detection method of the first embodiment. The detection method of the second embodiment includes steps S6 to S9 in addition to the detection method of the first embodiment. Except for these points, the process of the detection system 100A of the second embodiment is similar to the process of the system 100 of the first embodiment, and the description thereof can be used.

[0056] First, similarly to step S1 in the first embodiment, prior to processing by the detection device 1A, the read data 31 of the analysis target, the data 32 of the detection target, and the read data 33 of the control are prepared. Specifically, the user inputs the read data 31 of the analysis target, the data 32 of the detection target, and the read data 33 of the control through the input unit 21 of the user's detection terminal 2 (S1A). In the second embodiment, the read data 31 of the analysis target is read 1 (L T1 )~Lead n(L Tn ) (n: positive integer). In the second embodiment, the control read data 33 is composed of read 1 (L R1 )~Lead m(L Rm ) (m: positive integer).

[0057] The control read data 33 can be obtained by fragmenting a nucleic acid molecule that is a control of the analysis target and deciphering the nucleic acid sequence of each fragmented nucleic acid molecule. The control read data 33 may be read data obtained by a user or read data registered in a database or the like. The control read data 33 can be obtained, for example, by deciphering the nucleic acid sequence of the nucleic acid molecule contained in the control using a next-generation sequencer, similar to the analysis target read data 31. The control read data 33 is preferably obtained under the same conditions as the analysis target read data 31. The control is any nucleic acid molecule used as a standard for detecting a difference from the analysis target, and can be set appropriately, for example, depending on the type of the analysis target. As a specific example, when the analysis target is a nucleic acid molecule modified by inserting, adding, substituting, deleting, modifying, etc. a nucleic acid sequence of a certain nucleic acid molecule, the control can be an unmodified nucleic acid molecule, i.e., a certain nucleic acid molecule. The control can also be called, for example, a control, a comparison target, etc. The control read data 33 is linked to identification information, such as the control identifier, for example.

[0058] The length of the control read data 33 can be determined, for example, from the description of the read data 31 to be analyzed. The control read data 33 can be, for example, preprocessed read data. The preprocessing can be determined from the description of the read data 31 to be analyzed. When the read data 31 to be analyzed is preprocessed, it is preferable that the control read data 33 is preprocessed in the same manner as the preprocessing performed on the read data 31 to be analyzed.

[0059] The amount of the control read data 33 can be, for example, the same as that of the analysis target read data 31. The amount of the control read data 33 and the amount of the analysis target read data 31, i.e., the coverage of the control read data 33 and the coverage of the analysis target read data 31, are, for example, approximately the same, preferably the same.

[0060] Next, the analysis target read data 31, the detection target data 32, and the control read data 33 input to the detection terminal 2 are output to the array DB 3A via the communication line network 4. Then, the array DB 3A stores the analysis target read data 31, the detection target data 32, and the control read data 33 by linking them to the analysis target information (ID, identifier, etc.), the detection target information (ID, identifier, etc.), and the control information (ID, identifier, etc.), respectively (S2A). In the S2A step, the analysis target read data 31, the detection target data 32, and the control read data 33 are stored simultaneously (in parallel), but may be processed separately. In this case, the order of storing the analysis target read data 31, the detection target data 32, and the control read data 33 is not particularly limited and may be any order.

[0061] Next, the detection device 1A starts processing using the analysis target read data 31, the detection target data 32, and the control read data 33 stored in the array DB3A. First, the detection device 1A acquires, for example, the analysis target read data 31, the detection target data 32, and the control read data 33 instructed by the user from the detection terminal 2 from the array DB3A. Specifically, the first acquisition unit 11 of the detection device 1A requests the analysis target read data 31 stored in the array DB3A from the array DB3A, and acquires the analysis target read data 31 output from the array DB3A (S3, first acquisition step). Next, the second acquisition unit 12 of the detection device 1A requests the detection target data 32 stored in the array DB3A from the array DB3A, and acquires the detection target data 32 output from the array DB3A (S4, second acquisition step). Furthermore, the third acquisition unit 14 of the detection device 1A requests the control read data 33 stored in the array DB3A from the array DB3A, and acquires the control read data 33 output from the array DB3A (S6, third acquisition step). Note that, in the second embodiment, S3, S4, and S6 are processed in this order, but the processing order of S3, S4, and S6 is not particularly limited, and they may be processed in any order, or S3, S4, and S6 may be processed simultaneously.

[0062] Next, in the detection device 1A, the comparison sequence generation unit 15 generates multiple k-base-long nucleic acid sequences to be used for comparison with the nucleic acid sequence of the detection target data 32 from the analysis target read data 31 and the control read data 33 (S7, comparison sequence generation step). The number of k-base-long nucleic acid sequences generated in the comparison sequence generation unit 15 may be multiple, and the upper limit is determined according to the number of reads of the analysis target read data 31 and the control read data 33 and the base length (length) of each read. If the number of the k-base-long nucleic acid sequences is relatively large, the detection device 1A can further improve the judgment accuracy in, for example, the judgment step (S8) described later. For this reason, it is preferable that the number of the k-base-long nucleic acid sequences is large.

[0063] The comparative sequence generating unit 15 may generate a k-base-long nucleic acid sequence from a nucleic acid sequence designated by a user in the analysis target read data 31 and the control read data 33, may randomly generate a k-base-long nucleic acid sequence, or may generate a k-base-long nucleic acid sequence with regularity (for example, at a predetermined interval). When the comparative sequence generating unit 15 generates a k-base-long nucleic acid sequence at a predetermined interval, the comparative sequence generating unit 15 generates a k-base-long nucleic acid sequence, for example, in the analysis target read data 31 and the control read data 33, using nucleic acids (bases) of one or more base lengths as a reference. Since the comparison sequence generation unit 15 can further improve the accuracy of judgment in the judgment step (S8) described below, for example, it is preferable to generate the multiple k-base-long nucleic acid sequences by generating a k-base-long nucleic acid sequence using one or more bases as a reference (end nucleic acid) from one end to the other end of the entire length of the nucleic acid sequence of each read for the read data 31 to be analyzed and the control read data 33, and it is particularly preferable to generate the multiple k-base-long nucleic acid sequences by generating a k-base-long nucleic acid sequence using one base as a reference (end nucleic acid).

[0064] As a specific example, when a nucleic acid sequence of k bases is generated for the read data 31 to be analyzed and the control read data 33, based on the nucleic acid of each base from one end to the other end of the entire length of the nucleic acid sequence of each read, the comparison sequence generation unit 15 performs the following process, for example. First, the comparison sequence generation unit 15 generates a nucleic acid sequence of k bases for the read data 31 to be analyzed, based on the nucleic acid of each base. T1 ) as a reference (starting point), a nucleic acid sequence having a length of k bases is extracted. Next, the comparison sequence generating unit 15 extracts a nucleic acid sequence having a length of k bases, using the nucleic acid of the second base as a reference. Furthermore, read 1 (L T1 ) has a length of l T1 In the case of T1 The reference is changed one base at a time toward the 3' side until the lead 2 (L−k)th nucleic acid becomes the reference, and a nucleic acid sequence of k bases in length is repeatedly extracted. T2 )~Lead n(LTn ) to extract a nucleic acid sequence of k bases in length. R1 )~Lead m(L Rm ) is subjected to a similar process to extract a nucleic acid sequence of k bases in length. Note that, although an example of extracting a nucleic acid sequence of k bases in length using the end of each read as a reference has been given, the comparison sequence generating unit 15 may extract the nucleic acid sequence using any nucleic acid of each read as a reference.

[0065] Next, in the detection device 1A, the detection unit 13 detects the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target using a plurality of k-base-long nucleic acid sequences generated from the read data 31 of the analysis target and a plurality of k-base-long nucleic acid sequences generated from the control read data 33 (S51 and S53). Then, the detection unit 13 links the obtained detection result to the data of the k-base-long nucleic acid sequence used to obtain the detection result or the data of the detection target corresponding to the k-base-long nucleic acid sequence (for example, the position of the k-base-long nucleic acid sequence in the nucleic acid molecule of the detection target) so that the result can be used in the determination step (S8) described later (S52 and S54, determination step). As a specific example, when the detection unit 13 detects that a certain k-base-long nucleic acid sequence matches the 20th to 39th nucleic acid sequence of the nucleic acid sequence derived from the detection target, the detection unit 13 links the detection result (for example, the count number) to the 20th to 39th nucleic acid sequence of the nucleic acid sequence derived from the detection target.

[0066] Specifically, first, in step S5, the detection unit 13 compares a certain k-base-long nucleic acid sequence generated from the read data 31 to be analyzed with the nucleic acid sequence of the data 32 to be detected, and detects the presence or absence of a nucleic acid sequence that matches the k-base-long nucleic acid sequence in the nucleic acid sequence of the data 32 to be detected (S51). Next, the detection unit 13 links the detection result of the k-base-long nucleic acid sequence (detection and / or non-detection) with the position information of the nucleic acid sequence to be detected corresponding to the k-base-long nucleic acid sequence in the data 32 to be detected, and generates detection data to be analyzed (S52). The linking can be performed, for example, by linking the number of detections and / or the number of non-detections (count number) with the position of the nucleic acid sequence. Then, the detection unit 13 performs the same process on all of the multiple k-base-long nucleic acid sequences generated by the comparison sequence generation unit 15 (S51). Next, the detection unit 13 links the detection results (detection and / or non-detection) of each k-base-long nucleic acid sequence with the position information of the nucleic acid sequence to be detected corresponding to each k-base-long nucleic acid sequence in the data 32 to generate detection data to be analyzed (S52). Note that, although the case where the detection unit 13 performs the process of step S51 on each k-base-long nucleic acid sequence and then performs the process of step S52 has been given as an example, the detection unit 13 may be configured to perform the processes of steps S51 and S52 on one k-base-long nucleic acid sequence and then perform the same process on the next k-base-long nucleic acid sequence. Also, although the detection unit 13 links the count number as the detection result, other information such as detection frequency may be linked as the detection result.

[0067] Next, the detection unit 13 compares a certain k-base-long nucleic acid sequence generated from the control read data 33 with the nucleic acid sequence of the detection target data 32, and detects the presence or absence of a nucleic acid sequence that matches the k-base-long nucleic acid sequence in the nucleic acid sequence of the detection target data 32 (S53). Next, the detection unit 13 links the detection result of the k-base-long nucleic acid sequence (detection and / or non-detection) with the position information of the detection target nucleic acid sequence corresponding to the k-base-long nucleic acid sequence in the detection target data 32, and generates control detection data (S54). The linking can be performed, for example, by linking the number of detections and / or the number of non-detections (count number) with the position of the nucleic acid sequence. Then, the detection unit 13 performs the same process on all of the multiple k-base-long nucleic acid sequences generated by the comparison sequence generation unit 15 (S53). Next, the detection unit 13 links the detection results of each k-base-long nucleic acid sequence with the position information of the nucleic acid sequence to be detected corresponding to each k-base-long nucleic acid sequence in the data 32 to generate control detection data (S54). Note that, although the case where the detection unit 13 performs the process of step S53 for each k-base-long nucleic acid sequence and then performs the process of step S54 has been given as an example, the detection unit 13 may be configured to perform the processes of steps S53 and S54 for one k-base-long nucleic acid sequence and then perform the same process for the next k-base-long nucleic acid sequence. Also, although the detection unit 13 links the count number as the detection result, other information such as detection frequency may be linked as the detection result.

[0068] Next, in the detection device 1A, the judgment unit 16 judges whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the results obtained in step S5, specifically, the detection result of the k-base-long nucleic acid sequence in the detection target data 32 obtained using the analysis target read data 31 and the detection result of the k-base-long nucleic acid sequence in the detection target data 32 obtained using the control read data 33 (S8).

[0069] Specifically, the determination unit 16 determines whether there is a difference in the detection results between the detection data of the analysis target obtained in step S52 and the detection data of the control obtained in step S54. The determination can be performed by comparing the detection data of the analysis target with the detection data of the control for each position of the nucleic acid sequence of the detection target. As a specific example, the determination unit 16 can perform the determination by comparing the detection results (the number of detections and / or the number of non-detections) linked to the position X in the detection data of the analysis target and the detection data of the control for the position X of the nucleic acid sequence of the detection target. Then, when there is a difference in the detection results of the detection data of the analysis target and the detection data of the control, for example, when the number of detections in the detection data of the analysis target is (significantly) greater than the number of detections in the detection data of the control, and / or when the number of non-detections in the detection data of the analysis target is (significantly) less than the number of non-detections in the detection data of the control, the determination unit 16 determines that the nucleic acid sequence of the analysis target contains a nucleic acid sequence derived from the detection target (Yes). Then, the detection device 1A proceeds to the process of step S9. On the other hand, if there is no difference between the detection results of the detection data of the analysis target and the detection data of the control, for example, if the number of detections in the detection data of the analysis target is equivalent to the number of detections in the detection data of the control (no significant difference), and / or if the number of non-detections in the detection data of the analysis target is equivalent to the number of non-detections in the detection data of the control (no significant difference), the judgment unit 16 judges that the nucleic acid sequence of the analysis target does not contain a nucleic acid sequence derived from the detection target (No). Then, the detection device 1A ends the process.

[0070] In the judgment by the judgment unit 16, for example, a statistical process capable of testing the independence of two groups of data can be used, and specific examples include chi-square test and G test. In this case, if the two groups of data are not independent, the judgment unit 16 judges that there is no difference between the two data, that is, the nucleic acid sequence of the analysis target does not contain the nucleic acid sequence derived from the detection target (No). Then, the detection device 1A ends the process. On the other hand, if the two groups of data are independent, the judgment unit 16 judges that there is a difference between the two data, that is, the nucleic acid sequence of the analysis target contains the nucleic acid sequence derived from the detection target (Yes). Then, the detection device 1A proceeds to the process of step S9.

[0071] Next, in the detection device 1A, the position determination unit 17 determines the position in the nucleic acid sequence of the detection target for the nucleic acid sequence detected in the nucleic acid sequence of the analysis target based on the detection data of the analysis target obtained in step S52 and the control detection data obtained in step S54 (S9, position determination step).

[0072] Specifically, the position identifying unit 17 compares the detection results at each position of the nucleic acid sequence in the data 32 of the detection target, for example, between the detection data of the analysis target obtained in step S52 and the detection data of the control obtained in step S54. The comparison can be performed in the same manner as the determination of each position in the description of the determination unit 16. Then, when there is a (significant) difference between the detection results in the detection data of the analysis target and the detection results in the detection data of the control at each position of the nucleic acid sequence of the detection target, the position identifying unit 17 determines the nucleic acid at the position where the comparison was performed as a nucleic acid sequence derived from the detection target, and identifies the position where the comparison was performed as a position in the nucleic acid sequence of the detection target. More specifically, when the count number of detections in the detection data of the analysis target obtained in step S52 is significantly larger than the count number of detections at each position of the nucleic acid sequence in the data 32 of the detection target, the position identifying unit 17 determines the nucleic acid at the position where the comparison was performed as a nucleic acid sequence derived from the detection target, and identifies the position where the comparison was performed as a position in the nucleic acid sequence of the detection target. In addition, when the determination unit 16 uses statistical processing, the position identification unit 17 may identify the position of the detection target in the nucleic acid sequence based on a value obtained by statistical processing of the detection results at each position. In this case, the position identification unit 17 may identify the position of the detection target in the nucleic acid sequence by comparing the value obtained by statistical processing of the detection results at each position with a second threshold value. The second threshold value can be set, for example, by preparing a nucleic acid molecule to be analyzed that contains a nucleic acid molecule derived from the detection target and a nucleic acid molecule to be analyzed that does not contain a nucleic acid molecule derived from the detection target, and using a value obtained by statistical processing of the detection results obtained by the detection device 1A of embodiment 2.

[0073] Then, the detection device 1A ends the process.

[0074] The detection device 2 of the second embodiment may, for example, output the obtained detection result and / or the position in the nucleic acid sequence derived from the detection target to the detection terminal 2. In this case, the detection terminal 2 outputs the detection result and / or the position in the nucleic acid sequence derived from the detection target to the output unit 22.

[0075] The detection system 100A and the detection device 1A of the embodiment generate a plurality of nucleic acid sequences of k base length by the comparison sequence generation unit 15, and use them to detect the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed, so that the nucleic acid sequence derived from the detection target can be detected with higher accuracy (particularly, analysis sensitivity). Furthermore, the detection system 100A and the detection device 1A of the embodiment use the read data of the control nucleic acid sequence, so that the possibility of detecting a nucleic acid sequence not derived from the detection target as a nucleic acid sequence derived from the detection target can be suppressed, that is, the nucleic acid sequence derived from the detection target can be detected with higher accuracy (particularly, analysis specificity). Furthermore, the detection system 100A and the detection device 1A of the embodiment can specify the position in the nucleic acid sequence of the detection target for the nucleic acid sequence derived from the detection target introduced into the nucleic acid sequence to be analyzed by the position specifying unit 17, so that the nucleic acid sequence derived from the detection target can be confirmed using other means.

[0076] In the detection device 1A of the second embodiment, the comparison sequence generation unit 15 generates a k-base-long nucleic acid sequence in parallel from the analysis target read data 31 and the control read data 33, but the present invention is not limited to this, and the comparison sequence generation unit 15 may separately generate a k-base-long nucleic acid sequence from the analysis target read data 31 and the control read data 33. In this case, the order in which the comparison sequence generation unit 15 generates the k-base-long nucleic acid sequence from the analysis target read data 31 and the control read data 33 is not particularly limited, and can be any order.

[0077] In the detection device 1A of embodiment 2, the detection unit 13 performs steps S51 to S54 in this order in step S5, but in step S5, it is sufficient that step S52 is performed after step S51, and step S54 is performed after step S53, and the order of steps S51 and S52, and steps S53 and S54 can be any order.

[0078] [Embodiment 3] The program of this embodiment is a program that causes a computer to execute each step (also referred to as a process, procedure, command, or operation) of the above-mentioned detection method. The program of this embodiment may be recorded, for example, in a computer-readable recording medium. The recording medium is, for example, a non-transitory computer-readable storage medium. The recording medium is not particularly limited, and examples thereof include a random access memory (RAM), a read-only memory (ROM), a hard disk (HD), an optical disk, and a floppy disk (FD). EXAMPLES

[0079] The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents and kits were used according to their protocols.

[0080] [Example 1] A hypothetical genomic DNA was prepared and confirmed that the detection method of the present invention can detect foreign DNA inserted into genomic DNA. The detection method of Example 1 is outlined in FIG.

[0081] (1) Generation of virtual genome DNA First, the nucleic acid sequence of the genomic DNA into which the exogenous DNA was introduced was generated (FIG. 8(1)). Specifically, a 100-base-long nucleic acid sequence was randomly extracted from the nucleic acid sequence of the plasmid vector (ColE1 plasmid, International Nucleotide Sequence Database Collaboration (INSDC) accession number J015666), which is the nucleic acid molecule to be detected. Next, the extracted nucleic acid sequence was introduced into the nucleic acid sequence (IRGSP-1.0) of the genomic DNA of rice (Nipponbare). In this way, the nucleic acid sequence of the genomic DNA into which the exogenous DNA was introduced was generated. Note that, in the hypothetical genomic DNA used in Example 1, it was confirmed from the nucleic acid sequence that the nucleic acid sequence of the 5577th to 5676th bases of the vector was inserted.

[0082] (2) Generation of read data derived from virtual genome DNA Using the nucleic acid sequence of the virtual genome DNA obtained in Example 1(1), read data including reads derived from the virtual genome DNA of 100 base length (100 bps) was generated as read data of the nucleic acid sequence to be analyzed (FIGS. 8(2) and (3)). The amount of read data of the nucleic acid sequence to be analyzed was 50x coverage. When a nucleic acid sequence is deciphered for the entire genome using a next-generation sequencer, about 0.3% sequence errors occur. Therefore, when generating read data derived from the virtual genome DNA from the virtual genome DNA, about 0.3% artificial mutations were introduced into the obtained read data.

[0083] (3) Detection of nucleic acid sequences derived from plasmid vectors Next, for the read data derived from the virtual genome DNA, a 50-base-long nucleic acid sequence (k-base-long nucleic acid sequence, k-mer) was generated for each base from the 5' end to the 3' end of each read (Figure 8 (4)). Then, for each 50-base-long nucleic acid sequence, the nucleic acid sequence of the vector, which is the nucleic acid sequence to be detected, was compared to measure the number of times (count number) that the 50-base-long nucleic acid sequence was detected in the nucleic acid sequence of the vector. The obtained count number was linked to the position in the nucleic acid sequence of the vector where the 50-base-long nucleic acid sequence was detected and stored (detection data to be analyzed). Note that the ColE1 plasmid is a circular vector. For this reason, a nucleic acid sequence was generated by linearizing one revolution of the circular vector with a certain point in the ColE1 plasmid as a reference (starting point), and a nucleic acid sequence was generated in which the 50-base-long nucleic acid sequence on the 5' end side of the linearized plasmid vector was added to the 3' end of the nucleic acid sequence of the linearized plasmid vector, and this was used as the nucleic acid sequence of the vector to be compared (nucleic acid sequence to be detected).

[0084] In addition, except that the nucleic acid sequence of rice genomic DNA, into which no nucleic acid sequence derived from a plasmid vector was inserted, was used instead of the virtual genomic DNA, the number of detections (count number) was similarly linked to the position in the nucleic acid sequence of the vector where the 50-base-long nucleic acid sequence was detected and stored (control detection data).

[0085] Next, a test of independence was performed using the G test on the detection data of the analysis target and the detection data of the control. The test was performed assuming Model II described in Reference 1 below. The significance level was set to 1% (G=6.634). These results are shown in FIG. 9 and Table 1 below. Reference 1: Sokal, RR & Rohlf, FJ “Biometry: the principles and practice of statistics in biological research.”, WH Freeman and Co., New York, 1995 1995).

[0086] FIG. 9 is a graph showing the detection results of exogenous DNA. In FIG. 9, the upper row shows the results of the count number, and the lower row shows the results of the value obtained by the G test (G-statistic). In addition, the following Table 1 shows the detection results of all bases of 100 bases derived from the nucleic acid sequence of the vector. In FIG. 9, the horizontal axis shows the position in the nucleic acid sequence of the vector, and the vertical axis shows the count number or the value obtained by the G test. As shown in FIG. 9, according to the detection method of the present invention, the nucleic acid sequence of 5577th to 5676th of the vector was detected as having been inserted, which coincided with the above-mentioned condition. In addition, according to the detection method of the present invention, as shown in the following Table 1, all bases of 100 bases derived from the nucleic acid sequence of the vector were detected as nucleic acid sequences derived from the vector.

[0087] [Table 1]

[0088] From the above, it was found that the detection method of the present invention makes it possible to detect a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed without using a reference nucleic acid sequence.

[0089] [Example 2] In the detection method of the present invention, it was confirmed that the length of the foreign DNA inserted into the genomic DNA and the length of the k-base long nucleic acid sequence were changed, and the foreign DNA could be detected.

[0090] A hypothetical genomic DNA was generated in the same manner as in Example 1, except that a nucleic acid sequence of 15, 20, 30 or 50 bases was randomly extracted from the nucleic acid sequence of the plasmid vector (ColE1 plasmid), which was the nucleic acid molecule to be detected, and introduced into the nucleic acid sequence of the genomic DNA of rice (Nipponbare). Then, read data of the nucleic acid sequence to be analyzed was generated in the same manner as in Example 1(2), except that the generated hypothetical genomic DNA was used.

[0091] Next, the read data of the nucleic acid sequence to be analyzed in Example 2 was used as the read data of the nucleic acid sequence to be analyzed, and the nucleic acid sequence of k base length was a nucleic acid sequence of 10, 15, 20, 25, 30, 35, 40, 45, or 50 base length, except that the nucleic acid sequence of k base length was a nucleic acid sequence of 10, 15, 20, 25, 30, 35, 40, 45, or 50 base length, the nucleic acid sequence derived from the plasmid vector was detected in the same manner as in Example 1 (3). Furthermore, similarly, the generation of virtual genome DNA, the generation of read data of the nucleic acid sequence to be analyzed, the generation of the nucleic acid sequence of k base length, and the detection of the nucleic acid sequence derived from the plasmid vector were performed a total of 1000 times as one set, and the number of times that the entire length of the inserted foreign DNA could be detected was counted. In addition, the average value and standard deviation of the number of false positives in the 1000 detections were calculated. These results are shown in Tables 2 and 3 below.

[0092] [Table 2]

[0093] Table 2 shows the number of times that exogenous DNA was detected in 1000 detections. As shown in Table 2, when the length of the k-base-long nucleic acid sequence was 15 bases or more, the nucleic acid sequence of the exogenous DNA was detected with high accuracy. As shown in Reference Example 1 described later, when k≧15, there is almost no coincidence between the k-base-long nucleic acid sequence and the nucleic acid sequence of rice genomic DNA. For this reason, it was estimated that when the length of the k-base-long nucleic acid sequence is 15 bases or more, it can be detected with high accuracy.

[0094] [Table 3]

[0095] Next, Table 3 shows the average number of false positives and the standard deviation (Average ± SD) in 1000 detections. As shown in Table 3, as the length of the k-base-long nucleic acid sequence increases, the average number of false positives decreases and the standard deviation decreases. Also, as shown in Table 3, when the length of the k-base-long nucleic acid sequence is 20 bases or more, false positives can be significantly suppressed. Note that, as shown in Reference Example 1 described later, when k≧20, there is no chance of coincidence between the k-base-long nucleic acid sequence and the nucleic acid sequence of rice genomic DNA. Therefore, it was estimated that false positives can be significantly suppressed when the length of the k-base-long nucleic acid sequence is 20 bases or more.

[0096] From the above, it was found that the detection method of the present invention can detect exogenous DNA even if the length of the exogenous DNA inserted into the genomic DNA and the length of the nucleic acid sequence of k bases are changed.Furthermore, it was found that the detection method of the present invention can detect the nucleic acid sequence derived from the detection target with high accuracy by making the length of the nucleic acid sequence of k bases 15 bases or more, and can significantly suppress false positives by making the length of the nucleic acid sequence of k bases 20 bases or more.

[0097] [Example 3] In the detection method of the present invention, it was confirmed that exogenous DNA can be detected by changing the coverage of read data derived from virtual genomic DNA.

[0098] A virtual genome DNA was generated in the same manner as in Example 1, except that a 20-base-long nucleic acid sequence was randomly extracted from the nucleic acid sequence of a plasmid vector (ColE1 plasmid), which was a nucleic acid molecule to be detected, and introduced into the nucleic acid sequence of the genomic DNA of rice (Nipponbare).Then, using the generated virtual genome DNA, read data of the nucleic acid sequence to be analyzed was generated in the same manner as in Example 1(2), except that the amount of read data of the nucleic acid sequence to be analyzed was set to 10x, 20x, 30x, 40x, or 50x coverage.

[0099] Next, the read data of the nucleic acid sequence to be analyzed in Example 3 was used as the read data of the nucleic acid sequence to be analyzed, and the nucleic acid sequence of k bases was changed to a nucleic acid sequence of 20 bases, but the nucleic acid sequence derived from the plasmid vector was detected in the same manner as in Example 1 (3). Similarly, the generation of virtual genome DNA, the generation of read data of the nucleic acid sequence to be analyzed, the generation of the nucleic acid sequence of k bases, and the detection of the nucleic acid sequence derived from the plasmid vector were performed a total of 1000 times as one set, and the number of times (detection count) that the entire length of the inserted foreign DNA was detected was counted, and the detection rate (detection count / 1000×100(%)) was calculated. These results are shown in Table 4 below.

[0100] [Table 4]

[0101] The detection rate of the full-length exogenous DNA is shown in Table 4. As shown in Table 4, the detection rate improved with increasing coverage, and the detection rate was 100% at a coverage of 30× or more.

[0102] From the above, it was found that the detection method of the present invention can detect exogenous DNA even if the coverage of the read data derived from the virtual genome DNA is changed.Furthermore, it was found that the detection method of the present invention can detect the nucleic acid sequence derived from the detection target with extremely high accuracy by setting the amount of read data of the nucleic acid sequence to be analyzed to 20x coverage or more, particularly 30x coverage or more.

[0103] [Example 4] It was confirmed that the detection method of the present invention can detect foreign DNA in genome-edited rice plants.

[0104] (1) Preparation of genome-edited rice plants With reference to Reference 2 below, an all-in-one binary vector (pZH_gALS-2_Cas9) containing nucleic acid sequences encoding HPT, SpCas9, and sgRNA targeting the OsALS2 gene, as shown in Figure 10, was prepared. Reference 2: Mikami, M et.al., “Comparison of CRISPR / Cas9 expression constructs for efficient targeted mutagenesis in rice.”, Plant Mol. Biol., 2015, vol. 88, pages 561-572

[0105] Next, the binary vector was introduced into callus derived from the scutellum of a rice plant (Nipponbare) using Agrobacterium according to the protocol described in Reference 3 below. Specifically, a rice plant callus cultured for one month was infected with Agrobacterium (EHA10548 strain) containing the binary vector. After the start of the infection, Agrobacterium and callus were co-cultured for three days. Next, the co-cultured callus was transferred to a callus induction medium containing 50 mg / ml hygromycin B (manufactured by Wako Pure Chemical Industries, Ltd.) and 25 mg / ml meropenem (manufactured by Wako Pure Chemical Industries, Ltd.). Furthermore, hygromycin-resistant callus was selected by culturing for four weeks in the callus induction medium. The selected callus was then transferred to a regeneration medium to obtain a regenerated plant (T0 generation). The regenerated plants were then pollinated to obtain null segregants (T1 generation) from the self-fertilized progeny lines. Reference 3: Hood, EE et.al., “New Agrobacterium helper plasmids for gene transfer to plants.”, Transgenic Res., 1993, vol. 2, pages 208-218

[0106] (2) Confirmation of foreign DNA Whether a nucleic acid sequence derived from a foreign DNA (binary vector) was introduced into the nucleic acid sequence of the genomic DNA of the T0 generation was determined by Southern blotting with reference to Reference 4 below. Specifically, wild-type (Nipponbare) and T0 generation genomic DNA were isolated by the CTAB method of Reference 5 below. 10 μg of the isolated genomic DNA was digested with a restriction enzyme (SpeI). Next, a part of the HPT gene was amplified by PCR using the obtained fragment of genomic DNA and the following HPT primer set to obtain an amplified fragment. The PCR amplification conditions were 94° C. for 5 minutes, followed by 30 cycles of 98° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 50 seconds, and then further treatment at 68° C. for 5 minutes. Then, Southern blotting was performed using the obtained amplified fragment of the HPT gene and a probe capable of hybridizing to the amplified fragment of the predicted HPT gene (SEQ ID NO: 3) below. Reference 4: Abe, K. et al. “Development and characterization of transgenic dominant male sterile rice toward an outcross-based breeding system.”, Breed. Sci., 2018, vol. 68, pages 248-257 Reference 5: Murray, MG & Thompson, WF, “Rapid isolation of high molecular weight plant DNA.”, Nucleic Acids Res., 1980, vol. 8, pages 4321-4326

[0107] ·HPT primer set Forward primer (HPT-F01, SEQ ID NO:1) 5'-CAAAGATCGTTATGTTTATCGGCACTTTG-3' Reverse primer (HPT-R01, SEQ ID NO:2) 5'-GAAGAAGATGTTGGCGACCTCGTATTG-3' Amplified fragment of putative HPT gene (SEQ ID NO:3) 5'--3'

[0108] Next, PCR was used to confirm that the nucleic acid sequence of the genomic DNA of the T1 generation had not been introduced with a nucleic acid sequence derived from the foreign DNA (binary vector). Specifically, after isolating genomic DNA in the same manner as in the T0 generation, the HPT gene fragment was amplified under the PCR amplification conditions using the obtained genomic DNA and the HPT primer set. The obtained amplification product was then electrophoresed using agarose gel to detect the HPT gene fragment. The HPT gene fragment was also detected in the same manner, except that the wild type, the T0 generation, or the binary vector was used instead of the T1 generation. These results are shown in FIG. 11.

[0109] FIG. 11 is a photograph showing the detection results of the HPT gene fragment. In FIG. 11, (A) shows the results of Southern blotting, and (B) shows the results of electrophoresis using an agarose gel. In FIG. 11(A), the lanes show, from the left, the results of the wild type (Npb) and the T0 generation (2, 8, 20, 21, 44, 46). In FIG. 11(B), the lanes show, from the left, the marker (M), the wild type (Npb), the binary vector (Vector), the T0 generation (T0 #3-8), and the T1 generation (T1 #3-8-7). As shown in FIG. 11(A), the insertion of the HPT gene was confirmed in all six individuals in the T0 generation, but the insertion of the HPT gene was not confirmed in the wild type. As shown in FIG. 11(B), the insertion of the HPT gene was confirmed in the binary vector and the T0 generation, whereas the insertion of the HPT gene was not confirmed in the wild type. From these results, it was confirmed that a nucleic acid sequence derived from the nucleic acid sequence of the binary vector was inserted in the T0 generation, and that a nucleic acid sequence derived from the nucleic acid sequence of the binary vector was not inserted in the T1 generation, which is a null segregating individual.

[0110] (3) Detection of exogenous DNA Using the T0 generation obtained in Example 4(1), it was confirmed whether the detection method of the present invention can detect a nucleic acid sequence derived from a binary vector, which is a nucleic acid molecule to be detected. First, read data (read data to be analyzed) was obtained for the genomic DNA of the T0 generation. Specifically, a DNA library was prepared using the genomic DNA of the T0 generation and a DNA library preparation kit (TruSeq DNA PCR-Free Library Preparation Kit (manufactured by Illumina)), and then read data (read data to be analyzed) consisting of reads of 151 bases was obtained using the obtained DNA library and Illumina HiSeq X platform. The read data derived from the T0 generation was composed of a nucleic acid sequence of 69,170,747,118 bases. Moreover, read data was obtained in the same manner, except that the wild type (n=2) and the T1 generation were used instead of the T0 generation. One of the two wild-type read data is the read data to be analyzed (wild-type 1), the other is the control read data (wild-type 2), and the T1 generation read data is the reference read data. The wild-type read data was composed of nucleic acid sequences of 63,610,009,038 bases (wild-type 1) and 71,368,754,156 bases (wild-type 2), and the T1 generation read data was composed of a nucleic acid sequence of 63,340,672,150 bases.

[0111] The T0 generation read data, the wild type read data, and the T1 generation read data were trimmed using software (Trimmomatic ver. 0.36, options: ILLUMINACLIP: TruSeq_custom.fa: 2: 30: 10 LEADING: 10 TRAILING: 10 SLIDINGWINDOW: 4: 20 MINLEN: 20) to trim the nucleic acid sequences corresponding to the adapters present at both ends of each read. As a result, the T0 generation read data, the wild type read data, and the T1 generation read data were composed of 60,730,824,843 bases (T0 generation), 52,292,878,177 bases (wild type 1), 58,863,113,535 bases (wild type 2), and 54,313,317,595 bases (T1 generation).

[0112] Then, the read data from the T0 generation, the read data from the wild type (wild type 1), and the read data from the T1 generation were used as the read data to be analyzed, the nucleic acid sequence of k bases was changed to a nucleic acid sequence of 20 bases, and the nucleic acid sequence of the binary vector was used as the nucleic acid sequence to be detected, and the detection data to be analyzed was obtained in the same manner as in Example 1 (3). In addition, except that the read data from the wild type (wild type 2) was used as the control read data, the detection data of the control was measured by analyzing in the same manner as the detection data to be analyzed. Then, a test of independence was performed using a G test for the detection data to be analyzed and the detection data of the control. These results are shown in FIG. 12. When the read data from the T0 generation and the read data from the T1 generation were used, the count threshold was set to 20, and when the read data from the wild type (wild type 1) was used, the count threshold was set to 250.

[0113] FIG. 12 is a graph showing the detection results of exogenous DNA (binary vector). In FIG. 12(A) to (C), the upper row shows the results of the count number, and the lower row shows the results of the value obtained by the G test (G-statistic). In FIG. 12, (A) shows the results using the read data derived from the wild type, (B) shows the results using the read data derived from the T0 generation, and (C) shows the results using the read data derived from the T1 generation. In FIG. 12(A) to (C), the horizontal axis shows the position in the nucleic acid sequence of the binary vector, and the vertical axis shows the count number or the value obtained by the G test. As shown in FIG. 12(A), the nucleic acid sequence derived from the binary vector was detected in some regions even in the read data derived from the wild type. Further investigation of these revealed that the region indicated by the arrow Y was the region in the binary vector where the rice-derived nucleic acid sequence was inserted. In addition, the region indicated by the arrow X was because the nucleic acid sequence derived from the kit used for constructing the DNA library was detected. It was confirmed that the count of the region was not performed when a different DNA library preparation kit (KAPA Hyper Prep Kit / PCR Free) was used. Furthermore, the positions 16002 to 16021 and 17122 to 17141 were detected as coincidentally identical, and were presumed to have arisen due to data fluctuations (measurement errors) during read data acquisition. Next, as shown in FIG. 12(B), in the T0 generation, a nucleic acid sequence derived from a wide region of the binary vector was detected, which was consistent with the results of the Southern blot method shown in FIG. 11. On the other hand, as shown in FIG. 12(C), in the T1 generation, a nucleic acid sequence derived from the binary vector was not detected. The same results were obtained when the nucleic acid sequence of k bases was changed to a nucleic acid sequence of 50 bases and the nucleic acid sequence derived from the binary vector was detected.

[0114] It was found that the detection method of the present invention can detect foreign DNA in genome-edited rice plants.

[0115] [Example 5] It was confirmed that the detection method of the present invention can detect foreign DNA in genome-edited wheat plants.

[0116] The nucleic acid sequence data of the genomic DNA of wheat plants that had undergone genome editing (the nucleic acid sequence to be analyzed) and the nucleic acid sequence data of the genomic DNA of wheat plants that had not undergone genome editing (the control nucleic acid sequence) (BioProject accession number PRJDB7455, available for download from DDBJ Sequence Read Archive31) were taken from the T0 generation and T1 generation (null segregants) described in Reference 6 below. Reference 6: Abe, F. et al. “Genome-edited triple-recessive mutation alters seed dormancy in wheat.”, Cell Rep., 2019, vol. 28, pages 1362-1369.e4

[0117] The read data of the nucleic acid sequence to be analyzed was generated in the same manner as in Example 1(2), except that the nucleic acid sequence of the T0 generation genomic DNA and the nucleic acid sequence of the T1 generation genomic DNA were used instead of the nucleic acid sequence of the virtual genomic DNA obtained in Example 1(1). The amount of read data of the nucleic acid sequence to be analyzed was 50x coverage. In addition, the read data of the control nucleic acid sequence was generated in the same manner using the nucleic acid sequence of the genomic DNA of a wheat plant that had not been subjected to genome editing.

[0118] Next, instead of the read data derived from the virtual genome DNA, the read data derived from the T0 generation, the read data derived from the T1 generation, and the read data of the control nucleic acid sequence were used, the nucleic acid sequence of k bases was changed to a nucleic acid sequence of 20 bases, and the nucleic acid sequence of the vector (ColE1 plasmid) was changed to the nucleic acid sequence of the vector (pZH_OsU6gRNA_PubiMMCas9-TaQsd1_t1) of Reference 6. Except for this, the detection data of the analysis target and the detection data of the control were obtained in the same manner as in Example 1 (3). Then, a test of independence was performed using the G test for the detection data of the analysis target and the detection data of the control. These results are shown in FIG. 13.

[0119] FIG. 13 is a graph showing the detection results of exogenous DNA (vector). In FIG. 13(A)-(B), the upper row shows the results of the count number, and the lower row shows the results of the value obtained by the G test (G-statistic). In FIG. 13, (A) shows the results using the read data derived from the T0 generation, and (B) shows the results using the read data derived from the T1 generation. In FIG. 13(A)-(B), the horizontal axis shows the position in the nucleic acid sequence of the vector, and the vertical axis shows the count number or the value obtained by the G test. As shown in FIG. 13(A), in the T0 generation, a nucleic acid sequence derived from a wide region of the vector was detected, and this result was consistent with the result of Reference 6. On the other hand, as shown in FIG. 13(B), in the T1 generation, a nucleic acid sequence derived from the vector was not detected, and it was confirmed that it was a null segregant.

[0120] From the above, it was found that the detection method of the present invention can detect foreign DNA in genome-edited wheat plants.

[0121] [Reference example 1] It was confirmed that the value of k in a nucleic acid sequence of k bases in length can be set by referring to the total length of the nucleic acid sequence to be analyzed.

[0122] A randomly generated nucleic acid sequence of k bases in length (4 kFor all of the above (as listed below), the percentage of identity with the nucleic acid sequence of wheat genomic DNA, the nucleic acid sequence of corn genomic DNA, the nucleic acid sequence of pig genomic DNA, the nucleic acid sequence of silkworm genomic DNA, the nucleic acid sequence of Bacillus subtilis genomic DNA, the nucleic acid sequence of rice genomic DNA, and the nucleic acid sequence of ColE1 plasmid (ColE1) was examined. Note that k was set to 1 to 30. The nucleic acid sequences of wheat genomic DNA, rice genomic DNA, and ColE1 plasmid were the same as those described above. The nucleic acid sequences of corn genomic DNA, pig genomic DNA, and silkworm genomic DNA were registered in the following database. These results are shown in Figure 14 and Table 5 below. Pig genomic DNA: swine (Sscrofa11.1) in the database (http: / / www.ensembl.org / ) Maize plant genomic DNA: maize (Zm-B73-REFERENCE-GRAMENE-4.0) in the database (https: / / www.maizegdb.org / ) Silkworm genomic DNA: Silkworm (as of December 12, 2017) in the database (http: / / sgp.dna.affrc.go.jp / ) Bacillus subtilis genomic DNA: AL009126.3 in the database (https: / / www.ncbi.nlm.nih.gov / nuccore / ) Wheat plant genomic DNA (before genome editing): Wheat (TGACv1) in the database (EnsemblPlants: http: / / plants.ensembl.org / )

[0123] [Table 5]

[0124] FIG. 14 is a graph showing the rate of match between randomly generated nucleic acid sequences of k bases in length. In FIG. 14, the horizontal axis shows the value of k, and the vertical axis shows the rate of match. Moreover, Table 5 shows the rate of match between randomly generated nucleic acid sequences of k bases in length. As shown in FIG. 14 and Table 5, as the value of k is increased, the rate of match relatively decreases, and when the combination of nucleic acid sequences of k bases in length exceeds the full length of each DNA, the rate of match decreases significantly and becomes higher than the total number of bases in the genomic DNA (N G ) is 4 k To be smaller (N G ≦4 k ), it was believed that by setting k, the match rate was suppressed to less than 1%, enabling detection with high accuracy and specificity. Furthermore, when the value of k was set to 20, the match rate for all nucleic acid sequences was suppressed to less than 1%. From these results, it was confirmed that the value of k for a nucleic acid sequence of k bases in length can be set by referring to the total length of the nucleic acid sequence to be analyzed.

[0125] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-mentioned embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0126] <Additional Notes> Some or all of the above-described embodiments and examples may be described as follows, but are not limited to the following: <Detection device> (Appendix 1) 1. An apparatus for use in detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising: A first acquisition unit, a second acquisition unit, and a detection unit, The first acquisition unit acquires read data of a nucleic acid sequence to be analyzed, The second acquisition unit acquires data of a nucleic acid sequence to be detected, The detection unit detects a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a k-base-long nucleic acid sequence in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected with the nucleic acid sequence of the other data and detecting the k-base-long nucleic acid sequence in the other data. (Appendix 2) A comparison sequence generating unit is provided, the comparison sequence generation unit generates a plurality of nucleic acid sequences each having a length of k bases from at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected; The device described in Appendix 1, wherein the detection unit compares each k-base-long nucleic acid sequence with the nucleic acid sequence of the other data and detects the k-base-long nucleic acid sequence in the other data, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target. (Appendix 3) The apparatus of Appendix 2, wherein the comparison sequence generation unit generates the multiple k-base-long nucleic acid sequences by generating a k-base-long nucleic acid sequence based on nucleic acids of one or more bases from one end to the other end of the entire length of each nucleic acid sequence for at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected. (Appendix 4) A determination unit is provided, The device according to any one of appendices 1 to 3, wherein the determination unit determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in length in the other data. (Appendix 5) A third acquisition unit and a determination unit are provided, The third acquisition unit acquires read data of a control nucleic acid sequence, the detection unit compares a k-base-long nucleic acid sequence in at least one of the read data of the control nucleic acid sequence and the data of the nucleic acid sequence to be detected with a nucleic acid sequence in another data, and detects the k-base-long nucleic acid sequence in the other data; The device described in any of Appendices 1 to 3, wherein the determination unit determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in other data obtained using the read data of the nucleic acid sequence to be analyzed and the detection result of a nucleic acid sequence of k bases in other data obtained using the read data of the control nucleic acid sequence. (Appendix 6) 6. The device according to claim 4 or 5, wherein the detection result is a count of the k-base long nucleic acid sequence. (Appendix 7) A position identifying unit is provided, The device described in any of Appendices 4 to 6, wherein the position identification unit identifies a position in the nucleic acid sequence derived from the detection target for a nucleic acid sequence detected in the nucleic acid sequence to be analyzed based on the detection result of a nucleic acid sequence of k bases in length in the other data. (Appendix 8) The device described in any of Appendices 1 to 7, wherein the detection unit detects the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a k-base-long nucleic acid sequence in the read data of the nucleic acid sequence to be analyzed with the data of the nucleic acid sequence to be detected and detecting the k-base-long nucleic acid sequence in the data of the nucleic acid sequence to be detected. (Appendix 9) The device according to any one of appendix 1 to 8, wherein k satisfies the following condition (1): Condition (1): N G <=4 k (In the above condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of the analysis target. (Appendix 10) 10. The apparatus of any one of claims 1 to 9, wherein k is 20 or greater. (Appendix 11) An apparatus described in any one of appendix 1 to 10, wherein the read data of the nucleic acid sequence to be analyzed includes data of nucleic acid sequence that is 10 times or more the amount of nucleic acid sequence contained in one unit of the analysis target. (Appendix 12) the analysis target is a genome of an organism, 12. The device according to any one of claims 1 to 11, wherein the detection target is a nucleic acid other than that of the organism. (Appendix 13) 13. The apparatus of any of claims 1 to 12, wherein the apparatus is a server. <Detection method> (Appendix 14) A method for detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising the steps of: The method includes a first acquiring step, a second acquiring step, and a detecting step, In the first obtaining step, read data of a nucleic acid sequence to be analyzed is obtained, In the second obtaining step, data on a nucleic acid sequence to be detected is obtained, In the detection step, a nucleic acid sequence of k bases in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected is compared with the nucleic acid sequence of the other data, and the k bases in the other data are detected, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed. (Appendix 15) A comparison sequence generating step is included, In the comparative sequence generating step, a plurality of nucleic acid sequences each having a length of k bases are generated from at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected; The method described in Appendix 14, wherein the detection step compares each k-base-long nucleic acid sequence with the nucleic acid sequence of the other data, and detects the k-base-long nucleic acid sequence in the other data, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target. (Appendix 16) The method described in Appendix 15, wherein the comparison sequence generation step generates a k-base-long nucleic acid sequence based on one or more bases of nucleic acid from one end to the other end of the entire length of each nucleic acid sequence for at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected, thereby generating the multiple k-base-long nucleic acid sequences. (Appendix 17) A determination step is included, A method according to any one of Appendices 14 to 16, wherein the determination step determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in the other data. (Appendix 18) A third acquisition step and a determination step are included, In the third obtaining step, read data of a control nucleic acid sequence is obtained; The detection step includes collating a k-base-long nucleic acid sequence in at least one of the read data of the control nucleic acid sequence and the data of the nucleic acid sequence to be detected with a nucleic acid sequence in another data, and detecting the k-base-long nucleic acid sequence in the other data; A method according to any one of Appendices 14 to 16, in which the determination step determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in other data obtained using the read data of the nucleic acid sequence to be analyzed and the detection result of a nucleic acid sequence of k bases in other data obtained using the read data of the control nucleic acid sequence. (Appendix 19) The method according to claim 17 or 18, wherein the detection result is a count of the k-base-long nucleic acid sequence. (Appendix 20) A location determination step is included, A method according to any one of Appendices 14 to 19, wherein the position identification step identifies the position of a nucleic acid sequence detected in the nucleic acid sequence to be analyzed in the nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in length in the other data. (Appendix 21) A method according to any one of Appendices 14 to 20, wherein the detection step detects the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a k-base-long nucleic acid sequence in the read data of the nucleic acid sequence to be analyzed with the data of the nucleic acid sequence to be detected and detecting the k-base-long nucleic acid sequence in the data of the nucleic acid sequence to be detected. (Appendix 22) 22. The method according to any one of Appendices 14 to 21, wherein k satisfies the following condition (1): Condition (1): N G <=4 k (In the above condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of the analysis target. (Appendix 23) 23. The method of any one of claims 14 to 22, wherein k is 20 or more. (Appendix 24) A method according to any one of Appendices 14 to 23, wherein the read data of the nucleic acid sequence to be analyzed contains data of nucleic acid sequence that is 10 times or more the amount of nucleic acid sequence contained in one unit of the analysis target. (Appendix 25) the analysis target is a genome of an organism, 25. The method according to any one of claims 14 to 24, wherein the detection target is a nucleic acid other than that of the organism. <Program> (Appendix 26) A program used for detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising: The program causes a computer to execute a first acquisition process, a second acquisition process, and a detection process; In the first acquisition process, read data of a nucleic acid sequence to be analyzed is acquired, In the second acquisition process, data on a nucleic acid sequence to be detected is acquired, The detection process detects a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a nucleic acid sequence of k bases in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected with the nucleic acid sequence of the other data and detecting the nucleic acid sequence of k bases in the other data. (Appendix 27) A comparative sequence generation process is included. In the comparative sequence generation process, a plurality of nucleic acid sequences each having a length of k bases are generated from at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected; The program described in Appendix 26, wherein the detection process compares each k-base-long nucleic acid sequence with the nucleic acid sequence of the other data, and detects the k-base-long nucleic acid sequence in the other data, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target. (Appendix 28) The program described in Appendix 27, in which the comparison sequence generation process generates a k-base-long nucleic acid sequence based on nucleic acids of one or more bases from one end to the other end of the entire length of each nucleic acid sequence for at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected, thereby generating the multiple k-base-long nucleic acid sequences. (Appendix 29) Including a judgment process, A program described in any of Appendices 26 to 28, in which the determination process determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in length in the other data. (Appendix 30) A third acquisition process and a determination process are included, In the third acquisition process, read data of a control nucleic acid sequence is acquired; The detection process includes: comparing a k-base-long nucleic acid sequence in at least one of the read data of the control nucleic acid sequence and the data of the nucleic acid sequence to be detected with a nucleic acid sequence in other data; and detecting the k-base-long nucleic acid sequence in the other data; A program described in any of Appendices 26 to 28, in which the determination process determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in other data obtained using the read data of the nucleic acid sequence to be analyzed and the detection result of a nucleic acid sequence of k bases in other data obtained using the read data of the control nucleic acid sequence. (Appendix 31) 31. The program according to claim 29 or 30, wherein the detection result is a count of the k-base-long nucleic acid sequence. (Appendix 32) Including location identification processing, A program described in any of Appendices 26 to 31, in which the position identification process identifies the position of a nucleic acid sequence detected in the nucleic acid sequence to be analyzed in the nucleic acid sequence derived from the detection target based on the detection result of a nucleic acid sequence of k bases in length in the other data. (Appendix 33) A program described in any of Appendices 26 to 32, in which the detection process detects a nucleic acid sequence derived from the target nucleic acid sequence in the target nucleic acid sequence by comparing a k-base-long nucleic acid sequence in the read data of the target nucleic acid sequence with the data of the target nucleic acid sequence and detecting the k-base-long nucleic acid sequence in the data of the target nucleic acid sequence. (Appendix 34) The program according to any one of Appendices 26 to 33, wherein k satisfies the following condition (1): Condition (1): N G ≦4 k (In the above condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of the analysis target. (Appendix 35) 35. The program of any one of Appendices 26 to 34, wherein k is 20 or more. (Appendix 36) A program described in any one of Appendices 26 to 35, wherein the read data of the nucleic acid sequence to be analyzed includes data of nucleic acid sequence that is 10 times or more the amount of nucleic acid sequence contained in one unit of the analysis target. (Appendix 37) the analysis target is a genome of an organism, 37. The program according to any one of appendix 26 to 36, wherein the detection target is a nucleic acid other than that of the organism. <Recording Media> (Appendix 38) A computer-readable recording medium having a program according to any one of appendices 26 to 37 recorded thereon. <Detection System> (Appendix 39) A system for use in detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising: A terminal and a server are provided. The terminal and the server can be connected via a communication line network outside the system, The terminal and the server as a whole A first acquisition unit, a second acquisition unit, and a detection unit, The first acquisition unit acquires read data of a nucleic acid sequence to be analyzed, The second acquisition unit acquires data of a nucleic acid sequence to be detected, The detection unit detects a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a k-base-long nucleic acid sequence in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected with the nucleic acid sequence of the other data and detecting the k-base-long nucleic acid sequence in the other data. (Appendix 40) A system for use in detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising: The system comprises one or more computers, The one or more computers: A first acquisition unit, a second acquisition unit, and a detection unit, The first acquisition unit acquires read data of a nucleic acid sequence to be analyzed, The second acquisition unit acquires data of a nucleic acid sequence to be detected, The detection unit detects a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a k-base-long nucleic acid sequence in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected with the nucleic acid sequence of the other data and detecting the k-base-long nucleic acid sequence in the other data. (Appendix 41) The system is composed of a plurality of computers, 41. The system of claim 40, wherein the plurality of computers are connectable via a communications network external to the system. (Appendix 42) 42. The system of claim 40 or 41, wherein the computer is a server and / or a terminal. [Industrial Applicability]

[0127] As described above, according to the present invention, a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed can be detected without using a reference nucleic acid sequence. Therefore, the present invention can be suitably used, for example, for detecting an exogenous gene in the genome of an organism to be analyzed. Therefore, the present invention is extremely useful, for example, in the medical field.

Claims

1. 1. An apparatus for use in detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising: The apparatus includes a first acquisition unit, a second acquisition unit, a third acquisition unit, a detection unit, and a determination unit, The first acquisition unit acquires read data of a nucleic acid sequence to be analyzed, The second acquisition unit acquires data of a nucleic acid sequence to be detected, The third acquisition unit acquires read data of a control nucleic acid sequence, The detection unit is a nucleic acid sequence of k bases in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence of k bases in the other data is detected, thereby detecting a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed; A nucleic acid sequence having a length of k bases in at least one of the read data of the control nucleic acid sequence and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence having a length of k bases in the other data is detected; the determination unit determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the nucleic acid sequence to be analyzed and a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the control nucleic acid sequence; the analysis target is a genome of an organism, the detection target is a nucleic acid other than that of the organism, An apparatus, wherein k is 20 or more or satisfies the following condition (1): Condition (1): N G ≦4k (In the condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of the analysis target.)

2. A comparison sequence generating unit is provided, the comparison sequence generation unit generates a plurality of nucleic acid sequences each having a length of k bases from at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected; The device described in claim 1, wherein the detection unit detects the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the target to be analyzed by comparing each k-base-long nucleic acid sequence with the nucleic acid sequence of the other data and detecting the k-base-long nucleic acid sequence in the other data.

3. The apparatus of claim 2, wherein the comparison sequence generation unit generates the multiple k-base-long nucleic acid sequences by generating a k-base-long nucleic acid sequence based on nucleic acids of one or more bases from one end to the other end of the entire length of each nucleic acid sequence for at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected.

4. The apparatus according to claim 1 , wherein the detection result is a count number of the k-base long nucleic acid sequence.

5. A position identifying unit is provided, The device according to claim 1 , wherein the position identification unit identifies a position in the nucleic acid sequence to be detected for a nucleic acid sequence detected in the nucleic acid sequence to be analyzed based on the detection result of a nucleic acid sequence of k bases in the other data.

6. The device described in any one of claims 1 to 5, wherein the detection unit detects the nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed by comparing a k-base-long nucleic acid sequence in the read data of the nucleic acid sequence to be analyzed with the data of the nucleic acid sequence to be detected and detecting the k-base-long nucleic acid sequence in the data of the nucleic acid sequence to be detected.

7. The apparatus according to claim 1 , wherein the read data of the nucleic acid sequence to be analyzed includes data of nucleic acid sequence that is 10 times or more the amount of nucleic acid sequence contained in one unit of the analysis target.

8. The device according to claim 1 , wherein the device is a server.

9. A computer-implemented method for detecting a nucleic acid sequence of interest in a nucleic acid sequence of interest, comprising: The method includes a first acquisition step, a second acquisition step, a third acquisition step, a detection step, and a determination step, In the first obtaining step, read data of a nucleic acid sequence to be analyzed is obtained, In the second obtaining step, data on a nucleic acid sequence to be detected is obtained, The third obtaining step includes obtaining read data of a control nucleic acid sequence; In the detection step, a nucleic acid sequence of k bases in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence of k bases in the other data is detected, thereby detecting a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed; A nucleic acid sequence having a length of k bases in at least one of the read data of the control nucleic acid sequence and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence having a length of k bases in the other data is detected; The determination step determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target, based on a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the nucleic acid sequence to be analyzed and a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the control nucleic acid sequence, the analysis target is a genome of an organism, the detection target is a nucleic acid other than that of the organism, k is 20 or more or satisfies the following condition (1): Condition (1): N G ≦4k (In the condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of the analysis target.)

10. A comparative sequence generating step, In the comparative sequence generating step, a plurality of nucleic acid sequences each having a length of k bases are generated from at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected; The method according to claim 9, wherein in the detection step, each k-base-long nucleic acid sequence is compared with the nucleic acid sequence of the other data, and the k-base-long nucleic acid sequence in the other data is detected, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target.

11. A position determining step is included, The method according to claim 9 or 10, in the position identification step, a position in the nucleic acid sequence to be detected is identified for a nucleic acid sequence detected in the nucleic acid sequence to be analyzed based on the detection result of a nucleic acid sequence of k bases in length in the other data.

12. A program used for detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising: The program causes a computer to execute a first acquisition process, a second acquisition process, a third acquisition process, a detection process, and a determination process; In the first acquisition process, read data of a nucleic acid sequence to be analyzed is acquired, In the second acquisition process, data on a nucleic acid sequence to be detected is acquired, The third acquisition process includes acquiring read data of a control nucleic acid sequence, In the detection process, a nucleic acid sequence of k bases in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence of k bases in the other data is detected, thereby detecting a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed; A nucleic acid sequence having a length of k bases in at least one of the read data of the control nucleic acid sequence and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence having a length of k bases in the other data is detected; The determination process determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the nucleic acid sequence to be analyzed and a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the control nucleic acid sequence, the analysis target is a genome of an organism, the detection target is a nucleic acid other than that of the organism, A program in which k is 20 or greater or satisfies the following condition (1). Condition (1): N G ≦4k (In the condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of the analysis target.)

13. A comparative sequence generation process is included, In the comparison sequence generation process, a plurality of nucleic acid sequences each having a length of k bases are generated from at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected; The program of claim 12, wherein the detection process compares each k-base-long nucleic acid sequence with the nucleic acid sequence of the other data, and detects the k-base-long nucleic acid sequence in the other data, thereby detecting the nucleic acid sequence derived from the detection target in the nucleic acid sequence of the analysis target.

14. Including a location identification process, The program according to claim 12 or 13, wherein the position identification process identifies the position of a nucleic acid sequence detected in the nucleic acid sequence to be analyzed in the nucleic acid sequence to be detected based on the detection result of a nucleic acid sequence of k bases in the other data.

15. A computer-readable recording medium having the program according to any one of claims 12 to 14 recorded thereon.

16. A system for use in detecting a nucleic acid sequence derived from a detection target in a nucleic acid sequence to be analyzed, comprising: A terminal and a server are provided. The terminal and the server can be connected via a communication line network outside the system, The terminal and the server as a whole The apparatus includes a first acquisition unit, a second acquisition unit, a third acquisition unit, a detection unit, and a determination unit, The first acquisition unit acquires read data of a nucleic acid sequence to be analyzed, The second acquisition unit acquires data of a nucleic acid sequence to be detected, The third acquisition unit acquires read data of a control nucleic acid sequence, The detection unit is a nucleic acid sequence of k bases in at least one of the read data of the nucleic acid sequence to be analyzed and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence of k bases in the other data is detected, thereby detecting a nucleic acid sequence derived from the detection target in the nucleic acid sequence to be analyzed; A nucleic acid sequence having a length of k bases in at least one of the read data of the control nucleic acid sequence and the data of the nucleic acid sequence to be detected is compared with a nucleic acid sequence in the other data, and the nucleic acid sequence having a length of k bases in the other data is detected; the determination unit determines whether the nucleic acid sequence to be analyzed contains a nucleic acid sequence derived from the detection target based on a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the nucleic acid sequence to be analyzed and a detection result of a k-base-long nucleic acid sequence in the other data obtained using the read data of the control nucleic acid sequence; the analysis target is a genome of an organism, the detection target is a nucleic acid other than that of the organism, A system in which k is 20 or greater or satisfies the following condition (1). Condition (1): N G ≦4k (In the condition (1), N G is the total base length of the nucleic acid sequence contained in one unit of the analysis target.)

17. A position identification unit is provided, The system according to claim 16 , wherein the position identifying unit identifies a position in the nucleic acid sequence to be detected for a nucleic acid sequence detected in the nucleic acid sequence to be analyzed based on a detection result of a nucleic acid sequence of k bases in the other data.

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