Quality Evaluation Method

Abstract

We provide a more accurate quality control method for genetic testing. [Solution] Quality control samples with different combinations of standard genes are prepared depending on the type of gene panel test used in the test, a sample for sequence analysis is prepared from the specimen, sequence information of the standard genes contained in the quality control sample and the genes contained in the sample for sequence analysis is obtained using a sequencer, and an index used to evaluate the quality of the gene panel test is output by a computer based on the sequence information, and the quality of the gene panel test is evaluated using the standard genes.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the quality of a genetic test. [Background technology]

[0002] With the recent advances in genetic testing technology, expectations are rising for personalized medicine, in which the genetic sequence of a subject is analyzed and appropriate treatments and drugs are selected according to the subject's characteristics. One known method of analyzing genetic sequences is panel testing, which uses next-generation sequencers to perform high-throughput analysis of abnormalities in specific genes associated with specific diseases or abnormalities in the exon regions that are translated into proteins.

[0003] Non-Patent Document 1 describes a quality control method for genetic testing using a next-generation sequencer. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lih et al., Analytical Validation of the Next-Generation Sequencing Assay for a Nationwide Signal-Finding Clinical Trial, The Journal of Molecular Diagnostics, Vol. 19, No. 2, March 2017 Summary of the Invention [Problem to be solved by the invention]

[0005] However, quality control in the field of genetic testing is still in the trial stage and has not yet been established as a standard quality control method for genetic testing. For example, if the technology described in Non-Patent Document 1 is used as a quality control method for panel testing that analyzes multiple genes, the accuracy of quality assessment may be low.

[0006] A primary object of the present invention is to provide a method for quality control of genetic testing with higher accuracy when multiple genes are the subject of analysis in panel testing and the like. [Means for solving the problem]

[0007] In order to solve the above problems, a quality evaluation method according to one aspect of the present invention includes: A gene that has a mutation relative to the sequence of the wild-type gene Standard genes At least three types A method for evaluating the quality of a gene panel test using the gene panel test is the first Type of If so, there are at least two types of The standard gene Contains Prepare quality control samples, If the gene panel test is of a second type, a quality control sample is prepared containing at least two types of the standard genes in a second combination different from the first combination; A sample for sequence analysis is prepared from the specimen, sequence information of the standard gene contained in the quality control sample and the gene contained in the sample for sequence analysis is obtained by a sequencer, and an index used for quality evaluation of the gene panel test is output by a computer based on the sequence information. Each of the standard genes used in the first type of genetic panel testing has a mutation that is a target of detection by the first type of genetic panel testing, and each of the standard genes used in the second type of genetic panel testing has a mutation that is a target of detection by the second type of genetic panel testing. . [Effects of the Invention]

[0008] According to one aspect of the present invention, a method for quality control of genetic testing with sufficient accuracy can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram showing a typical application example of a gene analysis system according to an embodiment of the present invention. [Figure 2] FIG. 2 is a sequence diagram showing an example of main processes performed in the gene analysis system. [Figure 3] FIG. 2 is a functional block diagram including an example of a software configuration of the quality evaluation device. [Figure 4] 1 is a flowchart showing an example of a process flow for analyzing the gene sequence of a sample. [Figure 5] 1 is a flowchart illustrating an example of a pre-processing procedure for analyzing the base sequence of a sample DNA using a sequencer. [Figure 6] FIG. 1 is a diagram illustrating an example of a quality control sample. [Figure 7] FIG. 1 is a diagram showing an example of the data structure of a gene panel-related information database. [Figure 8] FIG. 1 shows a specific example of a quality control sample. [Figure 9] FIG. 1 is a diagram illustrating an example of the step (a) of fragmenting a sample, and the step (b) of adding index sequences and adaptor sequences. [Figure 10] FIG. 1 is a diagram illustrating an example of a hybridization process. [Figure 11] FIG. 1 is a diagram illustrating an example of a process for recovering a DNA fragment to be analyzed. [Figure 12] 1 is a flowchart illustrating an example of a procedure for analyzing the base sequence of a sample DNA using a sequencer. [Figure 13] FIG. 1 is a diagram illustrating an example of a step of subjecting DNA fragments to a flow cell. [Figure 14] FIG. 1 is a diagram illustrating an example of a step of amplifying a DNA fragment to be analyzed. [Figure 15] FIG. 1 is a diagram illustrating an example of a sequencing process. [Figure 16] 10 is a flowchart illustrating an example of a flow of analysis by the quality evaluation device. [Figure 17] FIG. 10 is a diagram showing an example of a file format of read sequence information. [Figure 18] FIG. 10A is a diagram illustrating alignment by a data adjustment unit, and FIG. 10B is a diagram showing an example of a format of the alignment result by the data adjustment unit. [Figure 19] FIG. 1 is a diagram showing an example of the structure of a reference sequence database. [Figure 20] FIG. 1 shows examples of known mutations that are incorporated into reference sequences (not representing wild-type sequences) contained in a reference sequence database. [Figure 21] 10 is a flowchart illustrating an example of a detailed alignment process. [Figure 22] FIG. 10(a) is a diagram showing an example of score calculation, and FIG. 10(b) is a diagram showing another example of score calculation. [Figure 23] FIG. 10 is a diagram showing an example of the format of a result file generated by a mutation identification unit. [Figure 24] FIG. 1 is a diagram showing an example of the structure of a mutation database. [Figure 25] FIG. 10 is a diagram showing a detailed example of the structure of mutation information in a mutation database. [Figure 26] FIG. 10 is a diagram illustrating an example of a quality evaluation index. [Figure 27] FIG. 10 is a diagram illustrating an example of a quality evaluation index. [Figure 28] FIG. 10 is a diagram showing an example of a report to be created. [Figure 29] FIG. 1 shows an example of a reference gene containing a substitution mutation. [Figure 30] FIG. 1 shows an example of a reference gene containing a fusion mutation. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Embodiment 1] Hereinafter, one embodiment of the present invention will be described in detail.

[0011] (Application example of the gene analysis system 100) First, an overview of a genetic analysis system 100 according to one embodiment of the present invention will be described with reference to Fig. 1. Fig. 1 is a diagram showing a typical application example of the genetic analysis system 100 according to one embodiment of the present invention. The genetic analysis system 100 is a system that analyzes gene sequence information, and it is sufficient that it includes at least a quality evaluation device 1 and a management server 3.

[0012] The genetic analysis system 100 shown in Figure 1 is applied to an analysis system management institution 130 that manages all analyses performed at the testing institution 120, and to the testing institution 120 that analyzes provided samples in response to analysis requests from medical institutions 210 and provides the analysis results to the medical institutions 210.

[0013] The testing institution 120 is an institution that tests and analyzes samples provided by the medical institution 210, prepares a report based on the analysis results, and provides the report to the medical institution 210. The testing institution 120 is equipped with a sequencer 2, a quality evaluation device 1, and the like, but is not limited to these.

[0014] The analysis system management institution 130 is an institution that manages all analyses performed at each testing institution 120 that uses the genetic analysis system 100 .

[0015] The medical institution 210 is an institution where doctors, nurses, pharmacists, etc. provide medical services such as diagnosis, treatment, and prescriptions to patients, and examples thereof include hospitals, clinics, and pharmacies.

[0016] (Processing in an example where the genetic analysis system 100 is applied) Next, the processing flow in an application example of the genetic analysis system 100 shown in Fig. 1 will be explained in more detail with reference to Fig. 2. Fig. 2 is a sequence diagram showing an example of the main processing performed in the genetic analysis system 100. Note that the processing shown in Fig. 2 is only a part of the processing performed at each institution.

[0017] <Application for and start of use of the genetic analysis system> First, a testing institution 120 that wishes to use the gene analysis system 100 introduces the quality evaluation device 1. Then, the testing institution 120 applies to the analysis system management institution 130 for permission to use the gene analysis system 100 (step S101).

[0018] The testing institution 120 and the analysis system management institution 130 can conclude a desired contract in advance from among a plurality of contract types regarding the use of the genetic analysis system 100. For example, the contract may be selected from a plurality of contract types that differ in the content of the service provided by the analysis system management institution 130 to the testing institution 120, the method of determining the system usage fee that the analysis system management institution 130 will charge to the testing institution 120, and the method of payment of the system usage fee. The management server 3 of the analysis system management institution 130 identifies the content of the contract concluded with the testing institution 120 in response to an application from the testing institution 120 (step S102).

[0019] Next, the management server 3 managed by the analysis system management institution 130 assigns an inspection institution ID to the quality evaluation device 1 of the contracted inspection institution 120, and starts providing various services (step S103).

[0020] The quality evaluation device 1 receives various services from the management server 3. The various services include the provision of programs and information for controlling the analysis results of gene sequences that can be output from the quality evaluation device 1, and reports based on the analysis results. This allows the quality evaluation device 1 to output analysis results, reports, and the like that are compatible with the information about the input gene panel.

[0021] Gene panels often include a set of reagents, such as primers and probes. Gene panel analysis is not limited to this, and may also be used to analyze polymorphisms such as single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNVs). Gene panels may also be used to output information on the amount of mutations across the entire gene being analyzed (also known as tumor mutation burden) or to calculate methylation frequencies.

[0022] <Analysis request to testing organization 120> At the medical institution 210, a doctor or the like collects samples such as tissue and blood from the lesion site of the subject as needed. When requesting the testing institution 120 to analyze the collected samples, for example, an analysis request is transmitted from a communication terminal 5 provided at the medical institution 210 (step S105). When requesting the testing institution 120 to analyze the samples, the medical institution 210 transmits the analysis request and also provides the testing institution 120 with a sample ID assigned to each sample. The sample ID assigned to each sample associates each sample with information on the subject from whom the sample was collected, etc.

[0023] The following description will be given taking as an example a case where the medical institution 210 requests analysis of a panel test from the testing institution 120. Note that the panel test is not limited to clinical tests, but also includes tests for research purposes.

[0024] When a genetic panel test is requested from the medical institution 210, a desired genetic panel may be specified. Therefore, the analysis request sent from the medical institution 210 in step S105 of Fig. 2 may include information related to the genetic panel. Here, the information related to the genetic panel may be any information that can be used to identify the genetic panel, and may be, for example, the name of the genetic panel and the names of the genes to be analyzed in the panel test.

[0025] <Analysis at testing institution 120> The quality evaluation device 1 receives the analysis request from the medical institution 210 (S106). Furthermore, the quality evaluation device 1 receives the sample from the medical institution 210 that is the sender of the analysis request.

[0026] There are multiple gene panels that the testing institution 120 can use in the analysis requested by the medical institution 210, and the gene group to be analyzed is determined for each gene panel. The testing institution 120 can also use multiple gene panels appropriately depending on the purpose of the analysis. That is, a first gene panel can be used to analyze a first gene group to be analyzed for a first sample provided by the medical institution 210, and a second gene panel can be used to analyze a second gene group to be analyzed for a second sample.

[0027] In this embodiment, for example, the first type of genetic mutation is a "substitution," and the second type of genetic mutation is a "deletion." In this case, the presence and type of at least substitutions and deletions are tested in the genetic testing that is the subject of quality control. In this embodiment, the first reference gene contains a specific substitution mutation relative to the wild-type gene sequence, and the second reference gene contains a specific deletion mutation relative to the wild-type gene sequence.

[0028] The quality evaluation device 1 receives input of information about the gene panel to be used for analyzing the sample from the user (step S107).

[0029] At the testing institution 120, the received sample is pre-processed using a gene panel, and then sequenced using the sequencer 2 (step S108).

[0030] In addition, in addition to sequencing normal samples, the testing institution 120 performs quality control by pre-processing a specified quality control sample corresponding to the gene panel using a gene panel and then sequencing using a sequencer 2 (step S108).

[0031] The results of genetic testing, including pre-processing, sequencing, and sequence analysis, of quality control samples are used as indicators of the quality of panel testing.

[0032] One or more quality control samples may be associated with each gene panel, and for example, a corresponding quality control sample may be prepared in advance for each gene panel. The quality control sample may be measured independently or together with a sample provided by the medical institution 210.

[0033] Here, the quality control sample is a sample for quality control used in a genetic test for testing a first type of genetic mutation and a second type of genetic mutation different from the first type. The "quality control sample" is a preparation containing a first standard gene having the first type of genetic mutation and a second standard gene having the second type of genetic mutation.

[0034] Pre-processing may include fragmenting genes such as DNA contained in a sample and recovering the fragmented genes. Sequencing includes reading the sequences of one or more DNA fragments to be analyzed that were recovered in the pre-processing. Sequence information read by sequencing using the sequencer 2 is output to the quality assessment device 1 as read sequence information.

[0035] Pretreatment may also include a process of fragmenting genes such as DNA contained in the sample and quality control sample, and recovering the fragmented genes.

[0036] The read sequence refers to the polynucleotide sequence obtained by sequencing, and indicates the sequence output from the sequencer 2.

[0037] Sequencing involves the process of reading the sequences of one or more DNA fragments to be analyzed that were collected in the preprocessing. The sequence information read by sequencing using the sequencer 2 is output to the quality assessment device 1 as read sequence information.

[0038] The sequencer 2 may output read sequence information including a quality score, which is a quality evaluation index related to the gene sequence reading process, to the quality evaluation device 1. The sequencer 2 may also output a cluster concentration, which is a quality evaluation index related to the amplification process of the DNA fragment to be analyzed, to the quality evaluation device 1. The "quality score" and "cluster concentration" will be explained later.

[0039] There are multiple gene panels that the testing institution 120 can use in the analysis requested by the medical institution 210, and the gene group to be analyzed is determined for each gene panel. The testing institution 120 can also use multiple gene panels appropriately depending on the purpose of the analysis. That is, a first gene panel can be used to analyze a first gene group to be analyzed for a first sample provided by the medical institution 210, and a second gene panel can be used to analyze a second gene group to be analyzed for a second sample.

[0040] The quality evaluation device 1 acquires the read sequence information from the sequencer 2 and analyzes the gene sequence (step S109).

[0041] The quality control samples are also processed in the same manner as in the panel testing of samples from the medical institution 210, and the genetic sequence information of the quality control samples is analyzed. Based on the results of analyzing the quality control samples, a quality evaluation index for evaluating the quality of the panel testing is generated.

[0042] Next, the quality evaluation device 1 evaluates the quality of the panel inspection based on the generated quality evaluation index (step S110). Specifically, the quality evaluation device 1 can evaluate the quality of each panel inspection based on the comparison result obtained by comparing the generated quality evaluation index with the evaluation standard set for each quality evaluation index.

[0043] The quality evaluation device 1 creates a report based on the analysis results in step S109 and the indexes generated based on the results of analyzing the quality control sample (step S111), and transmits the created report to the communication terminal 5 (step S112). The report may include, for example, data on the alignment results of the read sequence information, data on the results of the analysis by the quality evaluation device 1 such as data on identified mutations, etc., and information on the quality of the panel test.

[0044] The created report may be printed by the testing institution 120, and for example, the testing institution 120 may send the created report to the medical institution 210 as a paper medium.

[0045] The quality evaluation device 1 of the testing institution 120 using the genetic analysis system 100 notifies the management server 3 of information regarding the genetic panel used in the analysis, information regarding the genes analyzed, analysis results, and quality evaluation indexes generated for the genetic tests performed (step S114).

[0046] The management server 3 acquires, for example, via the network 4, the testing institution ID, genetic panel ID, genetic ID, analysis results, etc. from the quality evaluation device 1 of each testing institution 120 that uses the genetic analysis system 100. Furthermore, the management server 3 associates and stores the acquired testing institution ID, genetic panel ID, genetic ID, analysis results, etc. (step S115).

[0047] The testing institution ID is information for identifying a user who performs gene sequence analysis, and may be a user ID, which is identification information given to each user who uses the quality evaluation device 1.

[0048] The gene panel ID is identification information assigned to identify a gene panel used in analyzing a target gene. The gene panel ID assigned to a gene panel is associated with the gene panel name, the name of the company providing the gene panel, etc.

[0049] The gene ID is identification information assigned to each gene to identify the gene to be analyzed.

[0050] (Configuration of Genetic Analysis System 100) The gene analysis system 100 is a system that analyzes gene sequence information, and includes at least a quality evaluation device 1 and a management server 3. The quality evaluation device 1 is connected to the management server 3 via a network 4 such as an intranet or the Internet.

[0051] (Sequencer 2) The sequencer 2 is a base sequence analyzer used to read the base sequence of the gene contained in the sample.

[0052] The sequencer 2 according to this embodiment is preferably a next-generation sequencer that performs sequencing using next-generation sequencing technology, or a third-generation sequencer. Next-generation sequencers are a group of base sequence analyzers that have been developed in recent years and have dramatically improved analytical capabilities by performing massive parallel processing of clonally amplified DNA templates or single DNA molecules in a flow cell.

[0053] Furthermore, the sequencing technology that can be used in this embodiment may be a sequencing technology that obtains multiple reads by reading the same region in duplicate (deep sequencing).

[0054] Examples of sequencing technologies that can be used in this embodiment include ion semiconductor sequencing, pyrosequencing, sequencing-by-synthesis using reversible dye terminators, sequencing-by-ligation, and sequencing by oligonucleotide probe ligation, which are based on sequencing principles other than the Sanger method and can obtain a large number of reads per run.

[0055] The sequencing primers used for sequencing are not particularly limited and are appropriately selected based on a sequence suitable for amplifying the target region. Furthermore, the reagents used for sequencing may be selected appropriately depending on the sequencing technology and sequencer 2 used. The steps from pretreatment to sequencing will be explained later with specific examples.

[0056] (Configuration of quality evaluation device 1) 3 shows an example of the configuration of the quality assessment device 1. The quality assessment device 1 is an apparatus including a control unit 11 that acquires lead sequence information read by a sequencer 2 and information about a gene panel including a plurality of genes to be analyzed, and an output unit 13 that outputs an analysis result of the lead sequence information based on the information about the gene panel acquired by the control unit 11. The quality assessment device 1 can be configured using a computer. For example, the control unit 11 is a processor such as a CPU, and the storage unit 12 is a hard disk drive.

[0057] The memory unit 12 also stores a program for sequence analysis, a program for generating a single reference sequence, etc. The output unit 13 includes a display, a printer, a speaker, etc. The input unit 17 includes a keyboard, a mouse, a touch sensor, etc. Alternatively, a device having both the functions of an input unit and an output unit, such as a touch panel in which a touch sensor and a display are integrated, may be used. The communication unit 14 is an interface that allows the control unit 11 to communicate with external devices.

[0058] The quality evaluation device 1 includes a control unit 11 that controls all the units included in the quality evaluation device 1, a memory unit 12 that stores various data used by an analysis execution unit 110, an output unit 13, a communication unit 14, and an input unit 17. The control unit 11 includes the analysis execution unit 110 and a management unit 116. Furthermore, the analysis execution unit 110 includes a sequence data reading unit 111, an information selection unit 112, a data adjustment unit 113, a mutation identification unit 114, and a report creation unit 115. The memory unit 12 stores a gene panel related information database 121, a reference sequence database 122, a mutation database 123, and an analysis performance log 151.

[0059] The quality assessment device 1 creates a report including analysis results corresponding to the gene panel used, even if a different gene panel is used for each analysis. A user of the genetic analysis system 100 can analyze the results of panel testing using a common analysis program regardless of the type of gene panel and create a report. This eliminates the hassle of having to use different analysis programs for each gene panel or make special settings for the analysis program for each gene panel used when conducting a panel test, thereby improving user convenience.

[0060] When a user of the quality evaluation device 1 inputs information about a gene panel from the input unit 17, the information selection unit 112 refers to the gene panel related information database 121 and controls the algorithm of the analysis program so that the analysis program performs analysis of the genes to be analyzed according to the input information about the gene panel.

[0061] Here, the information regarding the gene panel may be any information that identifies the gene panel used for measurement by the sequencer 2, such as the gene panel name, the names of the genes being analyzed in the gene panel, and the gene panel ID.

[0062] Based on the information about the gene panel input from the input unit 17, the information selection unit 112 changes the analysis algorithm for performing an analysis corresponding to the genes to be analyzed in the gene panel indicated by the information about the gene panel.

[0063] The information selection unit 112 outputs instructions based on the information about the gene panel to at least one of the data adjustment unit 113, the mutation identification unit 114, and the report creation unit 115. By adopting this configuration, the quality assessment device 1 can output the analysis results of the read sequence information based on the input information about the gene panel.

[0064] In other words, the information selection unit 112 is a functional block that acquires information about a gene panel including multiple genes to be analyzed, and controls the output unit 13 to output the analysis results of the read sequence information based on the acquired information about the gene panel.

[0065] When genes contained in various samples are analyzed by a user who performs a panel test, various gene panels are used depending on the gene group to be analyzed for each sample.

[0066] That is, the quality evaluation device 1 can acquire first read sequence information read using a first gene panel for analyzing a first group of genes to be analyzed from a first sample, and second read sequence information read using a second gene panel for analyzing a second group of genes to be analyzed from a second sample.

[0067] The quality evaluation device 1 is equipped with an information selection unit 112, so that it can appropriately output the analysis results of the read sequence information, even when various combinations of target genes are analyzed using various gene panels.

[0068] In other words, the user can select information related to the gene panel, without having to set up an analysis program to be used to analyze the lead sequence information for each gene to be analyzed, or perform the analysis, and the analysis results for each lead sequence information can be output appropriately.

[0069] For example, when the information selection unit 112 outputs an instruction based on information about a gene panel to the data adjustment unit 113, the data adjustment unit 113 performs alignment processing that reflects the information about the gene panel.

[0070] Depending on the information regarding the gene panel, the information selection unit 112 instructs the data adjustment unit 113 to limit the reference sequences (reference sequences incorporating wild-type genomic sequences and mutant sequences) used for mapping the lead sequence information to only reference sequences related to genes corresponding to the information regarding the gene panel.

[0071] In this case, since the results of processing by the data adjustment unit 113 already reflect information about the gene panel, the information selection unit 112 does not need to output instructions based on information about the gene panel to the mutation identification unit 114, which performs processing after the processing by the data adjustment unit 113.

[0072] For example, when the information selection unit 112 outputs an instruction based on information about a gene panel to the mutation identification unit 114, the mutation identification unit 114 performs processing that reflects the information about the gene panel.

[0073] For example, the information selection unit 112 instructs the mutation identification unit 114 to limit the area of ​​the mutation database 123 referred to by the mutation identification unit 114 to only mutations related to the gene corresponding to the information on the gene panel, depending on the information on the gene panel. This allows the information on the gene panel to be reflected in the results of processing by the mutation identification unit 114.

[0074] (Process flow for analyzing the gene sequence of a sample) Here, the flow of processing for analyzing the gene sequences of samples and quality control samples will be described with reference to Fig. 4. Fig. 4 is a flowchart showing an example of the flow of processing for analyzing the gene sequence of a sample.

[0075] First, in step S31 of Fig. 4, preprocessing is performed to analyze the sequence of the gene to be analyzed. The preprocessing includes processes for fragmenting genes such as DNA contained in the sample and quality control sample and recovering the fragmented genes. Note that if the sample provided by the medical institution 210 is tissue or blood, the preprocessing also includes processes for extracting genes (e.g., DNA) from the tissue or blood.

[0076] Next, in step S32, the sequences of the genes contained in the sample that has undergone pretreatment and the quality control sample are read by the sequencer 2.

[0077] Specifically, step S32 is a step in which the sequences of one or more DNA fragments to be analyzed that have been recovered after pretreatment are read. The read sequence information includes the gene sequence read in this step. The one or more DNA fragments to be analyzed that have been recovered after pretreatment are sometimes referred to as a "library."

[0078] Subsequently, when a quality control sample is measured, in step S33, the quality evaluation device 1 analyzes the read gene sequence and identifies the presence or absence of a mutation in the sequence, the position of the mutation, the type of mutation, etc. By analyzing the read gene sequence, the detected mutation is identified.

[0079] Next, in step S34, the quality evaluation device 1 generates a quality evaluation index for evaluating the quality of the panel inspection. The quality evaluation device 1 may evaluate the quality of the panel inspection based on the generated quality evaluation index.

[0080] Finally, the quality evaluation device 1 creates a report including the analysis results such as information on the mutations identified in step S33 and information indicating the quality of the panel test such as the quality evaluation index generated in step S34. The created report is provided to the medical institution 210.

[0081] (Pretreatment) Next, the pre-processing procedure of step S31 in Fig. 4 will be described with reference to the flow shown in Fig. 5. Fig. 5 is a flowchart illustrating an example of the pre-processing procedure for analyzing the base sequence of a sample DNA by the sequencer 2.

[0082] When DNA is extracted from each of a sample and a quality control sample for sequence analysis, DNA is first extracted from the sample containing the gene to be analyzed and the quality control sample corresponding to the gene panel to be used (step 300 in (a) of Figure 5).

[0083] In this case, the DNA derived from the sample and the DNA derived from the quality control sample are each subjected to the processes from step S301 onwards.

[0084] By subjecting DNA extracted from quality control samples to the same treatment as DNA extracted from specimens, it is possible to generate quality assessment metrics that are useful for assessing the quality of sequence analysis in panel testing.

[0085] However, the method of using the quality control sample is not limited to this. For example, as shown in Figure 5(b), only DNA may be extracted from the quality control sample in step S300a, and the processes from step S301 onwards may be carried out.

[0086] Alternatively, as shown in FIG. 5(c), a quality control sample containing a mutation and a quality control sample not containing a mutation may be prepared as quality control samples, and DNA may be extracted from these samples (step 300b).

[0087] By comparing the analysis results of DNA from quality control samples containing mutations with the analysis results of DNA from quality control samples not containing mutations, a quality evaluation index useful for evaluating the quality of sequence analysis in panel testing can be generated.

[0088] Alternatively, as shown in Figure 5(d), DNA may be extracted from a sample containing the gene of interest, a quality control sample containing a mutation, and a quality control sample not containing a mutation (step 300c).The sample containing the gene of interest may be a combination of a blood sample and a tumor cell sample.

[0089] In the processes from step S301 onward, instead of processing the DNA derived from the sample and the DNA derived from the quality control sample separately, the DNA derived from the sample and the DNA derived from the quality control sample may be mixed and processed from step S301 onward. This ensures that the conditions for both are the same in all processes from step S301 onward, thereby generating a more accurate quality evaluation index. Furthermore, it is not necessary to use some of the lanes of the flow cell used in sequencer 2 exclusively for DNA fragments prepared from the quality control sample. This allows the limited number of lanes to be used effectively for DNA fragments derived from the sample containing the gene of interest.

[0090] In this case, it is desirable to use (1) a reagent for preparing a library by appropriately fragmenting the standard genes contained in the quality control sample and the genes to be analyzed in the panel test, and (2) a reagent containing an RNA bait for appropriately capturing each DNA fragment after fragmenting the standard genes contained in the quality control sample and the genes to be analyzed in the panel test.

[0091] (Quality control sample) In one embodiment, the quality control sample is a composition containing multiple standard genes. The quality control sample can be prepared by mixing multiple standard genes. These standard genes can be mixed and contained in a single container to form a reagent, which can be provided to the user as the quality control sample. Alternatively, the quality control sample can be provided to the user in the form of a kit in which multiple standard genes are contained in separate containers. The quality control sample can be in the form of a solution or a solid (powder). When provided as a solution, aqueous solvents known to those skilled in the art, such as water or TE buffer, can be used as the solvent.

[0092] The quality control sample will be described with reference to Fig. 6. Fig. 6 is a diagram for explaining an example of the quality control sample.

[0093] Figure 6(a) shows a list of genes that can be analyzed in a panel test using a gene panel. One or more genes in this list are associated as genes to be analyzed in the gene panel (see data 121B in Figure 7).

[0094] Figure 6(b) shows examples of mutation types that can be detected in panel testing, including "SNV (single nucleotide polymorphism)," "insertion" and "deletion" (denoted as "InDel" in the figure), "CNV (copy number variation)," and "fusion."

[0095] Quality control sample A1 corresponding to gene panel A contains at least two of the following standard genes: SNV-containing standard genes, insertion-containing standard genes, deletion-containing standard genes, CNV-containing standard genes, and fusion-containing standard genes. For example, quality control sample A1 contains, as standard genes, a partial sequence of gene A containing "SNV" relative to the wild type and a partial sequence of gene B containing "insertion" relative to the wild type.

[0096] Figure 6(d) shows an example output of the analysis results of a genetic test using gene panel A and the analysis results of a quality control sample. In this example, the analysis results of gene panel A detected GNA11, AKT1, and PIK3CA SNVs, EGFR long insertions and long deletions, the SLC34A2-ROS1 fusion gene, the CCDC6-RET fusion gene, MET gene amplification, MYC-N gene amplification, and MYC-C gene amplification. The quality control sample for gene panel A contains a standard gene containing a GNA11 SNV, a standard gene containing an AKT1 SNV, a standard gene containing a PIK3CA SNV, a standard gene containing an EGFR long insertion, a standard gene containing an EGFR long deletion, a standard gene containing an SLC34A2-ROS1 fusion sequence, a standard gene containing a CCDC6-RET fusion sequence, a standard gene containing a MET gene amplification, a standard gene containing a MYC-N gene amplification, and a standard gene containing a MYC-C gene amplification. Here, an example is shown in which the quality control sample contains 10 standard genes, but the present invention is not limited to this.

[0097] The first and second standard genes contained in the quality control sample may be different DNA molecules or may be linked together. When the first and second standard genes are linked together, the sequences of the first and second standard genes may be directly linked, or a spacer sequence may be interposed between the sequences of the first and second standard genes.

[0098] The spacer sequence is preferably a sequence that is unlikely to be contained in a sample to be subjected to genetic testing, for example, a sequence of multiple (e.g., 100) consecutive adenine bases.

[0099] The reference gene may be a gene included in the gene panel to be analyzed, or may not be a gene included in the gene panel. It may be a gene of the organism species to be genetically tested, or a gene of a different organism species. For example, if the subject of genetic testing is humans, the reference gene may be a gene of a non-human animal, plant, bacterium, etc.

[0100] The method for synthesizing a standard gene is not particularly limited. For example, it can be synthesized using a known DNA synthesizer. Alternatively, it may be obtained by amplifying a gene derived from an organism as a template by PCR and purifying it. It may also be obtained by amplifying a standard gene synthesized by a DNA synthesizer as a template by PCR and purifying it.

[0101] The length of a standard gene is not particularly limited. For example, the length of a standard gene can be 50 nucleotides or more. When amplifying by PCR, a gene of up to 2000 nucleotides can be easily amplified and is suitable. When synthesizing using a DNA synthesizer, up to several kbp can be synthesized.

[0102] The concentration of the standard gene in the quality control sample is not particularly limited. For example, The degree of the ion beam can be made equal to that of the ion beam.

[0103] The standard gene in the quality control sample may be single-stranded or double-stranded, and may be linear or circular.

[0104] An example of the preparation of a quality control sample will now be described in detail.

[0105] (1) Preparation of a standard gene containing substitution mutations A standard gene having the sequence of SEQ ID NO: 1 is synthesized using a known DNA synthesizer. The synthesized DNA is amplified by PCR using commercially available reagents including DNA polymerase, dNTPs, and buffer. The sequence of SEQ ID NO: 1 is shown in Figure 29. This sequence is the sequence of exon 20 of the PIK3CA gene and contains a substitution of A to G (A3140G) at position 3140 of the wild-type PIK3CA gene (see U.S. Patent Publication No. 8,026,053 for A3140G, which is incorporated herein by reference). The sequence of SEQ ID NO: 1 is 476 mer in length, and the A3140G substitution mutation is located at position 204. The coding sequence of the wild-type PIK3CA gene is shown in SEQ ID NO: 2.

[0106] The amplified product is subjected to agarose gel electrophoresis, and a band around 500 bp is excised. The excised gel is purified by standard methods. After purification, the DNA is quantified and diluted to the desired concentration with TE buffer to obtain the standard gene having the sequence of SEQ ID NO: 1.

[0107] (2) Preparation of a standard gene containing a fusion mutation A standard gene having the sequence of SEQ ID NO: 3 is synthesized using a known DNA synthesizer. The synthesized DNA is amplified by PCR using commercially available reagents including DNA polymerase, dNTPs, and a buffer solution. The sequence of SEQ ID NO: 3 is shown in Figure 30. The sequence of SEQ ID NO: 3 is a partial sequence of a fusion gene of the EML4 gene and the ALK gene. In the sequence of SEQ ID NO: 3, the sequence from positions 1 to 500 is derived from the EML4 gene, and the sequence from positions 501 to 1000 is derived from the ALK gene (see Figure 30). The EML4-ALK fusion gene is registered in GenBank Accession No. AB663645.1. The sequence of SEQ ID NO: 3 is the sequence from positions 1158 to 2157 of GenBank Accession No. AB663645.1.

[0108] A standard gene having the sequence of SEQ ID NO: 3 is obtained in the same manner as in (1) above.

[0109] (3) Preparation of quality control samples containing standard genes A quality control sample is prepared by mixing a standard DNA molecule having the sequence of SEQ ID NO: 1 and a standard DNA molecule having the sequence of SEQ ID NO: 3 at an arbitrary concentration. This quality control sample is mixed with a specimen to prepare a sample for sequence analysis.

[0110] (4) Analysis The quality of gene panel testing is evaluated using a next-generation sequencer (e.g., Illumina NextSeq500) using the prepared sequencing sample. This gene panel targets multiple genes, including the PIK3CA gene and the EML4-ALK fusion gene. Genomic DNA from the specimen and standard genes in the sequencing sample are subjected to preprocessing (fragmentation, DNA enrichment, PCR amplification using tag primers, etc.) and sequence analysis to obtain sequence information for the target genes. Quality control indicators are obtained during sequencing, and the quality of the analysis results for the target genes is evaluated based on the indicators from the sequence analysis of the standard DNA molecules. Based on the results of this quality assessment, users can determine the reliability of the analysis results for the genes being analyzed.

[0111] In the above example, the quality control sample and the specimen are mixed in (3), but the quality control sample and the specimen may be subjected to sequence analysis separately without being mixed.

[0112] Furthermore, when panel testing using the same gene panel is performed repeatedly, the same quality control sample may be used repeatedly. As shown in data 121D in Figure 7, multiple types of quality control samples containing different standard genes and mutation types may be prepared as quality control samples corresponding to each gene panel.

[0113] By using multiple quality control samples with different combinations of standard genes for each panel test, week, or month, a quality evaluation index for evaluating the quality of the mutation detection process in the panel test can be generated by detecting mutations in a wider variety of standard genes, thereby improving the comprehensiveness of the quality control of the panel test.

[0114] For example, Figure 8 shows quality control samples A and B, which are quality control samples corresponding to gene panel A. The standard genes a1, a2, and a3 contained in quality control sample A have been changed to standard genes b1, b2, and b3 in quality control sample B, respectively.

[0115] Next, as shown in FIG. 9(a), the sample (genomic DNA derived from the specimen and / or the standard gene) is fragmented to a length that allows the sequence to be read by the sequencer 2 (step S301 in FIG. 5). The sample DNA can be fragmented by known methods, such as ultrasonic treatment or treatment with a reagent that fragments nucleic acids. The resulting DNA fragments (nucleic acid fragments) can be, for example, several tens to several hundreds of base pairs in length.

[0116] Next, as shown in Figure 9(b), adapter sequences corresponding to the type of sequencer 2 to be used and the sequencing protocol are added to both ends (3' end and 5' end) of the DNA fragment obtained in step S301 (step S302 in Figure 5). Note that this step is essential when the sequencer 2 is an Illumina sequencer or a device that employs a system similar to that of an Illumina sequencer, but may be omitted when using another type of sequencer 2.

[0117] The adapter sequence is a sequence used to perform sequencing in a later step, and in one embodiment, can be a sequence for hybridizing to an oligo DNA immobilized on a flow cell in a Bridge PCR method.

[0118] In one embodiment, as shown in the upper part of Figure 9(b), adapter sequences (e.g., adapter 1 sequence and adapter 2 sequence in Figure 9) may be added directly to both ends of a DNA fragment. Addition of adapter sequences to a DNA fragment can be achieved by techniques known in the art. For example, the DNA sequence may be blunted and then adapter sequences may be ligated.

[0119] Adapter sequences can be added to DNA fragments by techniques known in the art. For example, a DNA fragment may be blunted, an index sequence may be ligated, and then an adapter sequence may be ligated.

[0120] Next, as shown in FIG. 10, a biotinylated RNA bait library is hybridized to the DNA fragments to which the adapter sequences have been added (step S303 in FIG. 5).

[0121] The biotinylated RNA bait library is composed of biotinylated RNA (hereinafter referred to as RNA bait) that hybridizes with the gene to be analyzed. The length of the RNA bait is arbitrary, but for example, a long oligo RNA bait of about 120 bp may be used to increase specificity.

[0122] In the panel test using the sequencer 2 in this embodiment, a large number of genes (for example, 100 or more) are the genes to be analyzed.

[0123] The reagents used in panel testing contain a set of RNA baits corresponding to each of the multiple genes. Since different panels have different numbers and types of genes to be tested, the sets of RNA baits contained in the reagents used in panel testing also differ. When a gene different from the gene to be analyzed is used as a standard gene, a bait that binds to the standard gene must also be prepared.

[0124] Then, as shown in Fig. 11, the DNA fragments to be analyzed are collected (step S304 in Fig. 5). In detail, as shown in the upper part of Fig. 11, the DNA fragments hybridized with the biotinylated RNA bait library are mixed with streptavidin magnetic beads in which streptavidin is bound to magnetic beads.

[0125] This causes the streptavidin portion of the streptavidin magnetic beads to bind to the biotin portion of the RNA bait, as shown in the middle of Figure 11. Then, as shown in the bottom of Figure 11, the streptavidin magnetic beads are attracted with a magnet, and fragments that have not hybridized with the RNA bait (i.e., DNA fragments that are not to be analyzed) are removed by washing.

[0126] This allows the selection and enrichment of DNA fragments that hybridize with the RNA bait, i.e., DNA fragments to be analyzed. Sequencer 2 obtains multiple read sequences by reading the nucleic acid sequences of the DNA fragments selected using multiple RNA baits in this way.

[0127] (Read sequence by sequencer 2) Next, the procedure of step S32 in Fig. 4 will be described along the flow shown in Fig. 12, with appropriate reference to Fig. 13 to Fig. 15. Fig. 12 is a flowchart illustrating an example of the procedure for analyzing the base sequence of a sample DNA by the sequencer 2.

[0128] As shown in the left to center columns of FIG. 13, the streptavidin magnetic beads and RNA bait are removed from the concentrated DNA fragments, and the fragments are amplified by PCR to complete the pretreatment.

[0129] First, as shown in the right column of FIG. 13, the sequence of the amplified DNA fragment is applied to a flow cell (step S305 in FIG. 12).

[0130] Subsequently, as shown in FIG. 14, the DNA fragment to be analyzed is amplified on a flow cell by Bridge PCR (step S306 in FIG. 12).

[0131] That is, the DNA fragment to be analyzed (e.g., Template DNA in Figure 14) has two different adapter sequences (e.g., Adapter 1 sequence and Adapter 2 sequence in Figure 14) attached to both ends by the above-mentioned pretreatment ("1" in Figure 14), and this DNA fragment is made single-stranded, and the Adapter 1 sequence on the 5' end is immobilized on the flow cell ("2" in Figure 14).

[0132] The adapter 2 sequence at the 5' end is fixed on the flow cell in advance, and the adapter 2 sequence at the 3' end of the DNA fragment binds to the adapter 2 sequence at the 5' end on the flow cell, forming a bridge ("3" in Figure 14).

[0133] In this state, a DNA extension reaction is carried out using DNA polymerase ("4" in Figure 14), and when denatured, two single-stranded DNA fragments are obtained ("5" in Figure 14).

[0134] By repeating this process of bridge formation, DNA extension reaction, and denaturation in this order, a large number of single-stranded DNA fragments can be locally amplified and fixed, forming clusters ("6" to "10" in Figure 14).

[0135] Then, as shown in FIG. 15, the single-stranded DNA forming the cluster is used as a template to read the sequence by sequencing-by-synthesis (step S307 in FIG. 12).

[0136] First, DNA polymerase and fluorescently labeled dNTPs with blocked 3' ends are added to single-stranded DNA immobilized on a flow cell (top left column of Figure 15) (top center column of Figure 15), and then a sequencing primer is added (top right column of Figure 15).

[0137] The sequence primer may be designed to hybridize to a portion of the adapter sequence, i.e., to amplify a DNA fragment derived from the sample DNA, and, if an index sequence is added, to amplify the index sequence.

[0138] After adding a sequencing primer, a DNA polymerase is used to extend the 3'-blocked fluorescent dNTP by one base. Because a 3'-blocked dNTP is used, the polymerase reaction stops after one base has been extended. The DNA polymerase is then removed (middle right column of Figure 15). The single-stranded DNA that has been extended by one base (bottom right column of Figure 15) is then exposed to laser light to excite the fluorescent substance attached to the base, and the resulting light emission is photographed and recorded (bottom left column of Figure 15).

[0139] To determine the four bases, photographs are taken using a fluorescence microscope, with changing wavelength filters to capture each fluorescent color corresponding to A, C, G, and T. After all the photographs are imported, the bases are determined from the photographic data. The fluorescent substance and the protecting group blocking the 3' end are then removed, and the next step, the polymerase reaction, begins. This process constitutes one cycle, and by repeating the second and third cycles, the entire length can be sequenced.

[0140] Using the above method, the chain length that can be analyzed can reach 150 bases x 2, which allows analysis in units much smaller than a picotiter plate. Therefore, by increasing the density, it is possible to obtain enormous amounts of sequence information, 40 to 200 Gb, in a single analysis.

[0141] (c. Gene panel) As described above, the gene panel used to read the lead sequence using sequencer 2 refers to an analysis kit for analyzing multiple analysis targets in a single run, and in one embodiment, it may be an analysis kit for analyzing multiple gene sequences related to a specific disease.

[0142] As used herein, the term "kit" refers to a package containing containers (e.g., bottles, plates, tubes, dishes, etc.) containing specific materials. Preferably, instructions for using each material are provided. When used in the context of a kit, "containing" refers to a state in which the material is contained in any of the individual containers that make up the kit. The kit may also be a package containing multiple different compositions together, where the composition may be in the form described above, and in the case of a solution form, may be contained in a container.

[0143] The kit may contain substance A and substance B mixed in the same container or in separate containers. The "instructions" indicate procedures for applying each component of the kit to therapy and / or diagnosis. The "instructions" may be written or printed on paper or other media, or may be attached to electronic media such as magnetic tape, computer-readable disk or tape, CD-ROM, etc. The kit may also contain containers containing diluents, solvents, washing solutions, or other reagents. Furthermore, the kit may also include tools necessary for therapeutic and / or diagnostic application.

[0144] In one embodiment, the gene panel may include one or more of the above-mentioned reagents, such as quality control samples, reagents for fragmenting nucleic acids, reagents for ligation, wash solutions, PCR reagents (dNTPs, DNA polymerase, etc.), and magnetic beads. The gene panel may also include one or more of oligonucleotides for adding adapter sequences to fragmented DNA, oligonucleotides for adding index sequences to fragmented DNA, RNA bait libraries, etc.

[0145] In particular, the index sequence of each gene panel can be a sequence specific to the gene panel for identifying the gene panel, and the RNA bait library of each gene panel can be a library specific to the gene panel, containing RNA baits corresponding to each test gene of the gene panel.

[0146] (Sequence data reading unit 111, data adjustment unit 113, mutation identification unit 114) Next, the sequence data reading unit 111, data adjusting unit 113, and mutation identifying unit 114 of the analysis execution unit 110 will be described along the processing flow shown in Fig. 16, with appropriate reference to Figs. 17 to 25. Fig. 16 is a flowchart illustrating an example of the flow of analysis by the quality evaluation device 1. The processing shown in Fig. 16 corresponds to step S109 shown in Fig. 2 and step S33 shown in Fig. 4.

[0147] <Sequence data reading unit 111> First, in step S11 of FIG. 16, the sequence data reading unit 111 reads the read sequence information provided by the sequencer 2.

[0148] The read sequence information is data indicating the base sequence read by the sequencer 2. The sequencer 2 sequences a large number of nucleic acid fragments obtained using a specific gene panel, reads their sequence information, and provides it to the quality assessment device 1 as read sequence information.

[0149] In one embodiment, the read sequence information may include the read sequence as well as the quality score of each base in the sequence. Furthermore, both the read sequence information obtained by subjecting an FFPE sample collected from a lesion site of a subject to the sequencer 2 and the read sequence information obtained by subjecting a blood sample of the same subject to the sequencer 2 are input to the quality assessment device 1.

[0150] FIG. 17 is a diagram showing an example of a file format of read sequence information. In the example shown in FIG. 22, the read sequence information includes a sequence name, a sequence, and a quality score. The sequence name may be a sequence ID or the like assigned to the read sequence information output by the sequencer 2. The sequence indicates the base sequence read by the sequencer 2. The quality score indicates the probability that base assignment by the sequencer 2 will be incorrect. The sequence quality score (Q) of any base is expressed by the following formula:

[0151] Q=-10log10E In this formula, E represents an estimate of the probability that a base assignment will be incorrect. A higher Q value means a lower probability of error. A lower Q value means a larger portion of the read is unusable.

[0152] It may also increase the number of false positive mutation assignments, reducing the accuracy of the results. Note that a "false positive" refers to a read sequence being determined to have a mutation even though it does not have the true mutation of interest.

[0153] "Positive" means that the read sequence has the true mutation of interest, and "negative" means that the read sequence does not have the mutation of interest. For example, a quality score of 20 means that the probability of error is 1 in 100, and therefore the accuracy of each base in the read gene sequence (also called "base call accuracy") is 99%.

[0154] <Data Adjustment Unit 113> Subsequently, in step S12 of FIG. 16, the data adjusting unit 113 aligns the read sequences of the nucleic acid fragments included in the read sequence information based on the read sequence information read by the sequence data reading unit 111.

[0155] 18(a) is a diagram illustrating alignment by the data adjusting unit 113. The data adjusting unit 113 performs alignment by referring to reference sequences (reference sequence information) stored in the reference sequence database 122 and mapping the read sequence of each nucleic acid fragment to the reference sequence to be compared with the read sequence information. In one embodiment, the reference sequence database 122 stores multiple types of reference sequences corresponding to each gene to be analyzed.

[0156] In addition, the data adjustment unit 113 performs alignment on both the lead sequence information obtained by subjecting an FFPE sample collected from the lesion site of the subject to the sequencer 2 and the lead sequence information obtained by subjecting a blood sample from the same subject to the sequencer 2.

[0157] 18(b) is a diagram showing an example of the format of the alignment result of the data adjusting unit 113. The format of the alignment result is not particularly limited as long as it can identify the lead sequence, the reference sequence, and the mapping position, but may include reference sequence information, the name of the lead sequence, position information, map quality, and sequence, as shown in FIG. 18(b).

[0158] The reference sequence information is information indicating the reference sequence name (reference sequence ID) in the reference sequence database 122, the sequence length of the reference sequence, etc. The read sequence name is information indicating the name (read sequence ID) of each read sequence to be aligned. The position information is information indicating the position on the reference sequence to which the leftmost base of the read sequence is mapped (leftmost mapping position). The map quality is information indicating the mapping quality corresponding to the read sequence. The sequence is information indicating the base sequence corresponding to each read sequence (e.g., ...GTAAGGCACGTCATA...).

[0159] Fig. 19 is a diagram showing an example of the structure of the reference sequence database 122. As shown in Fig. 19, the reference sequence database 122 stores reference sequences indicating wild-type sequences (for example, genomic sequences of chromosomes #1 to #23) and reference sequences in which known mutations have been incorporated into the wild-type sequences.

[0160] Furthermore, metadata indicating information about a gene panel is attached to each reference sequence in the reference sequence database 122. The information about the gene panel attached to each reference sequence may, for example, directly or indirectly indicate the gene to be analyzed that corresponds to each reference sequence.

[0161] In one embodiment, the information selection unit 112 may control the data adjustment unit 113 to select a reference sequence corresponding to the information about the input gene panel by referring to the information about the gene panel and the metadata of each reference sequence when the data adjustment unit 113 obtains a reference sequence from the reference sequence database 122.

[0162] For example, in one embodiment, the information selection unit 112 may control the data adjustment unit 113 to select reference sequences corresponding to genes to be analyzed that are identified by the input information about the gene panel, thereby improving the efficiency of analysis because the data adjustment unit 113 only needs to perform mapping to reference sequences related to the used gene panel.

[0163] In another embodiment, the information selection unit 112 does not need to perform the above control. In that case, the information selection unit 112 may control the mutation identification unit 114 or the report creation unit 115, as will be described later.

[0164] FIG. 20 is a diagram showing examples of known mutations incorporated into reference sequences (not representing wild-type sequences) included in the reference sequence database 122. Known mutations are mutations registered in external databases (e.g., COSMIC, ClinVar, etc.), and as shown in FIG. 20, the chromosomal location, gene name, and mutation are specified. In the example of FIG. 25, an amino acid mutation is specified, but a nucleic acid mutation may also be specified. The type of mutation is not particularly limited, and may be various mutations such as substitution, insertion, or deletion, or may be a mutation in which a partial sequence of another chromosome or a reverse complementary sequence is attached.

[0165] Fig. 21 is a flowchart illustrating an example of detailed steps of the alignment in step S12 in Fig. 16. In one aspect, the alignment in step S12 in Fig. 16 is performed by steps S401 to S205 shown in Fig. 21.

[0166] 21, the data adjusting unit 113 selects an unaligned read sequence from among the read sequences of each nucleic acid fragment included in the read sequence information acquired by the sequence data reading unit 111, and compares it with a reference sequence acquired from the reference sequence database 122. Then, in step S402, the data adjusting unit 113 identifies a position on the reference sequence where the degree of match with the read sequence satisfies a predetermined criterion. Here, the degree of match is a value indicating the degree of match between the acquired read sequence information and the reference sequence, and examples include the number or percentage of matching bases.

[0167] In one embodiment, the data adjusting unit 113 calculates a score indicating the degree of identity between the read sequence and the reference sequence. The score indicating the degree of identity can be, for example, the percentage identity between the two sequences. The data adjusting unit 113, for example, identifies the number of positions where the bases in the read sequence and the bases in the reference sequence are identical, calculates the number of matching positions, and calculates the percentage by dividing the number of matching positions by the number of bases in the read sequence compared to the reference sequence (the number of bases in the comparison window).

[0168] Figure 22(a) is a diagram showing an example of score calculation. In one embodiment, at the position shown in Figure 22(a), the score of the degree of identity between read sequence R1 and the reference sequence is 100% because 13 of the 13 bases in the read sequence match, and the score of the degree of identity between read sequence R2 and the reference sequence is 92.3% because 12 of the 13 bases in the read sequence match.

[0169] In addition, when calculating a score indicating the degree of identity between a lead sequence and a reference sequence, the data adjustment unit 113 may calculate a score that is lower than the normal calculation if the lead sequence contains a predetermined mutation (e.g., an insertion / deletion (InDel)) relative to the reference sequence.

[0170] In one embodiment, the data adjusting unit 113 may correct the score of a read sequence containing at least one of an insertion and a deletion relative to a reference sequence by multiplying the score calculated by the normal calculation as described above by a weighting factor corresponding to the number of bases corresponding to the insertion or deletion. The weighting factor W may be calculated, for example, as W = {1 - (1 / 100) × (number of bases corresponding to the insertion or deletion)}.

[0171] Figure 22(b) shows another example of score calculation. In one embodiment, at the position shown in Figure 22(b), the score of the degree of identity between read sequence R3 and the reference sequence is 88% by normal calculation because 15 of the 17 bases in the read sequence (* indicating a deletion is counted as one base) match, and the corrected score is 88% x 0.98 = 86%. Furthermore, the score of the degree of identity between read sequence R4 and the reference sequence is 81% by normal calculation because 17 of the 21 bases in the read sequence match, and the corrected score is 81% x 0.96 = 77.8%.

[0172] The data adjusting unit 113 calculates the score of the degree of match while changing the mapping position of the read sequence relative to each reference sequence, thereby identifying the position on the reference sequence where the degree of match with the read sequence satisfies a predetermined standard. In this case, algorithms known in the art, such as dynamic programming, FASTA, and BLAST, may be used.

[0173] Returning to Figure 21, next, if there is a single position on the reference sequence whose degree of match with the lead sequence satisfies a predetermined criterion (NO in step S203), the data adjustment unit 113 aligns the lead sequence to that position, and if there are multiple positions on the reference sequence whose degree of match with the lead sequence satisfies a predetermined criterion (YES in step S403), the data adjustment unit 113 aligns the lead sequence to the position with the highest degree of match (step S404).

[0174] Then, if the data adjustment unit 113 has not aligned all of the read sequences included in the read sequence information acquired by the sequence data reading unit 111 (NO in step S405), it returns to step S401, and if it has aligned all of the read sequences included in the read sequence information (YES in step S405), it completes the processing of step S12.

[0175] <Mutation Identification Division 114> Next, returning to FIG. 16, in step S13, the mutation identification unit 114 compares the sequence of a reference sequence (alignment sequence) obtained by aligning the lead sequence obtained by providing a sample collected from the lesion site of the subject with the sequence of a reference sequence (so-called alignment sequence) obtained by aligning the lead sequence obtained by providing a blood sample from the same subject.

[0176] 16, differences between the two alignment sequences are extracted as mutations. For example, if the alignment sequence derived from a blood sample for the same position in the same gene to be analyzed is ATCGA and the alignment sequence derived from a tumor tissue is ATCCA, the mutation identification unit 114 extracts the difference between G and C as a mutation.

[0177] In one embodiment, the mutation identification unit 114 generates a result file based on the extracted mutations. Figure 23 is a diagram showing an example of the format of the result file generated by the mutation identification unit 114. The format may be based on, for example, Variant Call Format (VCF).

[0178] As shown in Figure 23, the result file contains position information, a reference base, and a mutated base for each extracted mutation. The position information indicates a position on the reference genome, and includes, for example, a chromosome number and a position on the chromosome. The reference base indicates a reference base (A, T, C, G, etc.) at the position indicated by the position information. The mutated base indicates a base after mutation of the reference base. The reference base is a base on an alignment sequence derived from a blood sample, and the mutated base is a base on an alignment sequence derived from tumor tissue.

[0179] In Figure 23, a mutation in which the reference base is C and the mutated base is G is an example of a substitution mutation, a mutation in which the reference base is C and the mutated base is CTAG is an example of an insertion mutation, and a mutation in which the reference base is TCG and the mutated base is T is an example of a deletion mutation. Also, a mutation in which the mutated base is G]17:198982], ]13:123456]T, C[2:321682[, or [17:198983[A] is an example of a mutation in which a partial sequence or reverse complementary sequence of another chromosome is linked.

[0180] 16 , subsequently, in step S15, the mutation identification unit 114 searches the mutation database 123. Then, in step S16, the mutation identification unit 114 refers to the mutation information in the mutation database 123 and identifies the mutations by adding annotations to the mutations included in the result file.

[0181] Figure 24 is a diagram showing an example of the structure of the mutation database 123. The mutation database 123 is constructed based on an external database such as COSMIC or ClinVar. In one embodiment, each piece of mutation information in the database is provided with metadata related to information on the gene panel. In the example shown in Figure 24, each piece of mutation information in the database is provided with the gene ID of the gene to be analyzed as metadata.

[0182] Fig. 25 is a diagram showing a detailed example of the structure of mutation information in the mutation database 123. As shown in Fig. 25, in one embodiment, the mutation information included in the mutation database 123 may include a mutation ID, position information of the mutation (e.g., "CHROM" and "POS"), "REF", "ALT", and "Annotation". The mutation ID is an identifier for identifying a mutation.

[0183] In the mutation location information, "CHROM" indicates the chromosome number, and "POS" indicates the position on the chromosome. "REF" indicates the base in the wild type, and "ALT" indicates the base after mutation. "Annotation" indicates information about the mutation. "Annotation" may be information indicating an amino acid mutation, such as "EGFR C2573G" or "EGFR L858R." For example, "EGFR C2573G" indicates a mutation in which the cysteine ​​at residue 2573 of the protein "EGFR" has been replaced with glycine.

[0184] As in the above example, the "Annotation" of the mutation information may be information for converting a mutation based on base information into a mutation based on amino acid information. In this case, the mutation identification unit 114 can convert a mutation based on base information into a mutation based on amino acid information based on the referenced "Annotation" information.

[0185] The mutation identification unit 114 searches the mutation database 123 using information identifying the mutation included in the result file (e.g., position information of the mutation and base information corresponding to the mutation) as a key. For example, the mutation identification unit 114 may search the mutation database 123 using any of the information "CHROM," "POS," "REF," and "ALT" as a key. If a mutation extracted by comparing an alignment sequence derived from a blood sample with an alignment sequence derived from a lesion site is registered in the mutation database 123, the mutation identification unit 114 identifies the mutation as one present in the sample and annotates the mutation included in the result file (e.g., "EGFR L858R," "BRAF V600E," etc.).

[0186] (Report Writing Department 115) The report creation unit 115 creates a report based on the information output by the mutation identification unit 114 and the information on the gene panel provided by the information selection unit 112 (corresponding to step S110 in FIG. 2 and step S35 in FIG. 4). The information included in the created report includes information on the gene panel and information on the identified mutations.

[0187] The report creation unit 115 selects and discards information to be included in the report based on the information on the gene panel from the information selection unit 112, and deletes unselected information from the report. Alternatively, the information selection unit 112 may be configured to control the report creation unit 115 so that the information selection unit 112 selects information related to genes corresponding to the information on the gene panel input via the input unit 17 as information to be included in the report, and deletes unselected information from the report.

[0188] (Output section 13) The report created by the report creation unit 115 may be transmitted as a result of the analysis of the lead sequence information from the output unit 13 to the terminal device 5 installed in the medical institution 210 (corresponding to step S111 in FIG. 2). Alternatively, the report may be transmitted to a printer (not shown) connected to the quality evaluation device 1, printed by the printer, and then sent as a paper medium from the testing institution 120 to the medical institution 210.

[0189] (Quality evaluation indicators) Examples of quality evaluation indexes obtained by measuring the quality evaluation sample include the following: · Index (i): A quality evaluation index that indicates the quality of the read sequence information by sequencer 2. Indicator (ii): A quality evaluation indicator that indicates the proportion of bases read by Sequencer 2 among the bases contained in multiple genes to be analyzed. · Metric (iii): A quality evaluation metric that indicates the depth of read sequence information. · Index (iv): A quality evaluation index that indicates the variability in the depth of read sequence information. Index (v): A quality evaluation index that indicates whether all mutations in each standard gene contained in the quality control sample were detected.

[0190] And, indicator (i) is Indicator (i-1): Quality score, and Indicator (i-2): Cluster concentration may be included.

[0191] Here, the above quality evaluation index will be explained with reference to FIGS. 26 to 28. FIG.

[0192] Index (i-1): Quality score The quality score is an index indicating the accuracy of each base in the gene sequence read by sequencer 2.

[0193] For example, when the read sequence information is output from the sequencer 2 as a FASTQ file, the quality score is also included in the read sequence information (see Figure 17). Details of the quality score have already been explained, so they will not be explained here.

[0194] Indicator (i-2): Cluster concentration Sequencer 2 locally amplifies and fixes a large number of single-stranded DNA fragments on the flow cell to form clusters (see 9 in Figure 14). The clusters on the flow cell are then imaged using a fluorescence microscope, and the sequences are read by detecting the fluorescent colors (i.e., different fluorescent wavelengths) corresponding to A, C, G, and T. Cluster density is an index that indicates how closely the clusters of each gene formed on the flow cell are located when sequencing.

[0195] For example, if the cluster density becomes too high, causing the clusters to be too close together or overlapping, the contrast of the image captured from the flow cell (i.e., the signal-to-noise ratio) will be low, making it difficult to focus the fluorescence microscope, which will result in the inability to detect the fluorescence correctly and, as a result, the sequence will not be read accurately.

[0196] Indicator (ii): A quality evaluation indicator indicating the proportion of bases in the target region read by sequencer 2 out of the bases read by sequencer 2 This index indicates how many bases in the target region have been read out of the bases read by sequencer 2 (including bases outside the target region), and can be calculated as the ratio of the total number of bases read to the total number of bases in the target region.

[0197] Metric (iii) is a quality evaluation metric that indicates the depth of read sequence information.

[0198] This index is based on the total number of read sequence information read for each base contained in the gene being analyzed, and can be calculated as the ratio of the total number of bases read whose depth is equal to or greater than a predetermined value to the total number of bases read.

[0199] The depth refers to the total number of read sequence information read for the same base.

[0200] FIG. 26 shows a graph indicating the depth of each base read when the entire length of the gene to be analyzed is T bases and the bases in the read region are t1 bases. In this graph, the horizontal axis represents the position of the base, and the vertical axis represents the depth of each base. In the example shown in FIG. 26, of the t1 bases in the read region, the total number of bases in the region whose depth is equal to or greater than a predetermined value (e.g., 100) is (t2+t3) bases. In this case, index (iii) is generated as the value of (t2+t3) / t1.

[0201] Indicator (iv): A quality assessment indicator that indicates the variability in the depth of read sequence information.

[0202] This index indicates the uniformity of depth. When there is an extremely large amount of read sequence information from only a certain portion of the read region, the uniformity of depth is low, whereas when the read sequence information is distributed relatively evenly across the read region, the uniformity of depth is high. The uniformity of depth can be quantified, for example, using the interquartile range (IQR), although this is not limited to this. A higher IQR indicates lower uniformity, and a lower IQR indicates higher uniformity.

[0203] Index (v): A quality evaluation index that indicates whether all mutations in each standard gene contained in the quality control sample were detected.

[0204] This index indicates that mutations in each standard gene contained in the quality control sample were detected and correctly identified. The mutations in each standard gene contained in quality control sample A shown in Figure 27 (see the "Variant" column) are known mutations. Whether or not the positions and types of these mutations were correctly identified is used as a quality evaluation index.

[0205] 28 is a diagram showing an example of a report created by the report creation unit 115. The upper left part of the report shown in this example lists a "patient ID" indicating the subject ID, "patient's sex," "patient's disease name," "attending physician's name" which is the name of the physician in charge of the subject at the medical institution 210, and "institution name" indicating the name of the medical institution.

[0206] Below that, information about the gene panel is included, including the name of the gene panel, "Panel A." Additionally, the report also includes quality assessment indicators, or "QC indicators," obtained from the processing and analysis results using quality control samples.

[0207] In this report, a section entitled "Detected Gene Mutations and Associated Drugs" contains information about the mutations identified by the mutation identification unit 114 and a list of drugs.

[0208] If the quality assessment index falls below a predetermined standard, the detected gene mutation can be marked with an asterisk (*). In addition, a comment indicating low reliability can be added.

[0209] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0210] A quality assessment method according to one embodiment of the present invention is a quality assessment method for genetic testing in which genes in a sample collected from a subject are tested for multiple types of genetic mutations, including a first type of genetic mutation and a second type of genetic mutation different from the first type. The method includes the steps of: preparing a quality control sample containing a first standard gene, which is a nucleic acid molecule having the first type of genetic mutation but not the second type of genetic mutation; and a second standard gene, which is a nucleic acid molecule having the second type of genetic mutation but not the first type of genetic mutation; preparing a sequence analysis sample containing multiple fragmented genes contained in the sample collected from the subject and the first and second standard genes contained in the quality control sample; preparing a sequence analysis sample containing the multiple fragmented genes contained in the sequence analysis sample and the first and second standard genes contained in the quality control material; obtaining sequence information for the multiple fragmented genes contained in the sequence analysis sample and the first and second standard genes using a next-generation sequencer; and using the obtained sequence information, a computer (quality assessment device 1) outputs an index for evaluating the quality of the genetic testing.

[0211] According to the above configuration, an index for evaluating the quality of a genetic test is output by analyzing a quality control sample. The quality control sample contains at least one standard gene having a mutation of the type of mutation to be detected in the genetic test, so that an index corresponding to various mutation types can be output when evaluating the quality of the genetic test. This enables quality control of genetic tests with sufficient accuracy.

[0212] "Subject" refers to human subjects as well as non-human subjects, such as mammals, invertebrates, vertebrates, fungi, yeast, bacteria, viruses, and plants. Although the examples herein relate to human subjects, the concepts of the present invention can be applied to genomes from any organism other than humans, such as animals or plants, and are useful in fields such as medicine, veterinary medicine, and animal science.

[0213] The term "sample" can be alternatively referred to as specimen or sample, and is used synonymously with specimen or preparation in the art, and refers to any preparation obtained from a biological material source (e.g., an individual, body fluid, cell line, tissue culture, or tissue section).

[0214] The term "quality control sample" refers to a preparation that has been prepared for example to undergo pre-processing for analyzing the gene sequence and processing for reading sequence information by the sequencer 2.

[0215] "Mutation" includes nucleotide substitution, deletion, or insertion in a gene, gene fusion, or copy number variation. "Substitution" refers to a phenomenon in which at least one base in a gene sequence is different. "Substitution" includes point mutations and single-nucleotide polymorphisms. "Deletion" and "insertion" are also referred to as "InDel (Insertion and / or Deletion)." InDel is a phenomenon in which at least one base in a gene sequence is inserted and / or deleted. "Gene fusion" refers to a phenomenon in which the 5' sequence of one gene is joined to the 3' sequence of another gene due to chromosomal translocation or the like. "Copy number variation" refers to differences in the number of copies of a gene on the genome per cell between individuals. Specific examples include VNTR (Variable Nucleotide Tandem Repeat, repeat polymorphism), STRP (Short Tandem Repeat Polymorphism, microsatellite polymorphism), and gene amplification.

[0216] The first type of genetic variation is a nucleotide substitution, deletion, or insertion, a polymorphism, a gene copy number abnormality, or a gene fusion, and the second type of genetic variation is a nucleotide substitution, deletion, or insertion, a polymorphism, a gene copy number abnormality, or a gene fusion, and may be different from the first type of genetic variation.

[0217] The quality control sample may contain a standard gene having at least one gene mutation for each of a plurality of mutation types to be detected in the genetic test.

[0218] The quality of a genetic test may be evaluated based on the indicators.

[0219] Along with the sequence information of the first and second standard genes contained in the quality control sample, sequence information of genes derived from a sample collected from a subject contained in the sample for sequence analysis may also be obtained.

[0220] The quality control sample may further include genes that do not have mutations.

[0221] The quality of a genetic test may be evaluated based on whether the indicator meets predetermined criteria.

[0222] The predetermined criteria may be different between a first gene panel used in a genetic test and a second gene panel different from the first gene panel.

[0223] The sequence information of the gene contained in the quality control sample may be obtained by a sequencer, and the indicator may be the quality of the sequence information read by the sequencer.

[0224] The indicator may indicate the accuracy of each base in the gene sequence read by the sequencer.

[0225] The sequencer amplifies genes on the flow cell to form clusters, and the indicator may indicate the proximity of each gene cluster on the flow cell.

[0226] Sequence information of the first and second standard genes contained in the quality control sample may be obtained by a sequencer, and the indicator may indicate the proportion of bases in the target region among the bases read by the sequencer.

[0227] The index may indicate the depth of sequence information of the first and second standard genes contained in the acquired quality control sample.

[0228] The index may indicate the variation in depth of sequence information of the first and second standard genes contained in the acquired quality control sample.

[0229] A plurality of quality control samples containing different genes may be prepared, and a quality control sample to be prepared may be selected from the plurality of quality control samples.

[0230] A quality control sample may be prepared that contains at least one standard gene that is not a target gene for analysis in the genetic test and has a first or second type of gene mutation.

[0231] The computer (quality evaluation device 1) may transmit the index to a management server (3) connected to multiple testing facilities.

[0232] The computer (quality evaluation device 1) may receive the quality evaluation results from the management server (3).

[0233] A quality evaluation device (1) according to one embodiment of the present invention is a quality evaluation device (1) that evaluates the quality of a genetic test that tests at least a first type of genetic mutation and a second type of genetic mutation different from the first type for genes in a sample collected from a subject, and is equipped with: a first standard gene that is a nucleic acid molecule having a first type of genetic mutation but not having a second type of genetic mutation; a second standard gene that is a nucleic acid molecule having a second type of genetic mutation but not having the first type of genetic mutation and different from the first standard gene; sequence information of the fragmented genes and the sequence information of the first and second standard genes obtained by a next-generation sequencer from a sequence analysis sample containing the fragmented genes contained in the sample collected from the subject; and a quality control unit (117) that generates an index for evaluating the quality of the genetic test based on the sequence information.

[0234] According to the above configuration, a quality control sample is analyzed to generate an index for evaluating the quality of a genetic test. The quality control sample contains at least one standard gene having a mutation of a mutation type to be detected in the genetic test, so that indices corresponding to various mutation types can be generated for evaluating the quality of the genetic test. Therefore, by providing the index to the user in the quality evaluation device, it is possible to perform quality control of the genetic test with sufficient accuracy.

[0235] A program according to one embodiment of the present invention is a quality assessment program for genetic testing in which genes in a sample collected from a subject are tested for at least a first type of genetic mutation and a second type of genetic mutation different from the first type, and the program causes a computer to execute the following steps: acquiring, using a next-generation sequencer, sequence information of the fragmented genes and the sequence information of the first and second standard genes from a sequence analysis sample containing a first standard gene that is a nucleic acid molecule having a first type of genetic mutation but not having a second type of genetic mutation; a second standard gene that is a nucleic acid molecule having a second type of genetic mutation but not having the first type of genetic mutation and different from the first standard gene; and a plurality of fragmented genes contained in the sample collected from the subject; and generating an index for evaluating the quality of the genetic testing based on the acquired sequence information.

[0236] According to the above configuration, the same effects as those of the quality evaluation method according to one aspect of the present invention can be achieved.

[0237] A recording medium according to one aspect of the present invention is a computer-readable recording medium on which a program according to one aspect of the present invention is recorded.

[0238] A quality control sample according to one embodiment of the present invention is a quality control sample for genetic testing in which genes in a sample collected from a subject are tested for at least a first type of genetic mutation and a second type of genetic mutation different from the first type, and includes a first standard gene having the first type of genetic mutation and a second standard gene having the second type of genetic mutation.

[0239] According to the above configuration, at least one standard gene having a mutation of a mutation type to be detected in the genetic test is included, and therefore, when evaluating the quality of the genetic test, it is possible to generate indicators corresponding to various mutation types by applying the quality control sample. [Explanation of symbols]

[0240] 1. Quality evaluation equipment 2. Sequencer 3 Management Server 4 Network 11 Control section 13 Output section 100 Genetic Analysis System 117 Quality Control Department 121 Gene Panel Related Information Database 122 Reference Sequence Database 124 Drug Database 125 Reference Database 126 Quality Evaluation Criteria

Claims

1. A quality assessment method for evaluating the quality of a gene panel test using at least three types of standard genes, which are genes having mutations relative to the sequence of a wild-type gene, comprising: If the genetic panel test used in the test is of a first type, a quality control sample containing at least two types of the standard genes in a first combination is prepared; if the genetic panel test is of a second type, a quality control sample containing at least two types of the standard genes in a second combination different from the first combination is prepared; preparing a sample for sequence analysis from the specimen; obtaining sequence information of the standard gene contained in the quality control sample and the gene contained in the sample for sequence analysis using a sequencer; outputting, by a computer, an index used for evaluating the quality of the gene panel testing based on the sequence information; A quality assessment method, characterized in that each of the standard genes used in the first type of genetic panel testing has a mutation that is a target of detection by the first type of genetic panel testing, and each of the standard genes used in the second type of genetic panel testing has a mutation that is a target of detection by the second type of genetic panel testing.

2. The quality assessment method according to claim 1 , wherein the type of mutation in the standard gene contained in the quality control sample varies depending on the type of the gene panel test.

3. 3. The quality assessment method according to claim 1, wherein the quality control sample is prepared by combining a first standard gene having a nucleotide substitution, deletion, or insertion relative to the sequence of a wild-type gene with a second standard gene having a gene copy number abnormality or gene fusion.

4. The quality assessment method according to claim 1 , wherein the quality control sample contains at least one standard gene for each of a plurality of mutation types to be detected in the gene panel test.

5. The quality assessment method according to claim 1 , wherein the sample for sequence analysis contains a fragmented gene.

6. The quality assessment method according to claim 1 , wherein the quality control sample further contains a gene that does not have a mutation in the sequence of the wild-type gene.

7. The quality assessment method according to claim 1 , wherein the quality of the genetic panel test is assessed based on whether the index satisfies a predetermined standard.

8. The quality evaluation method according to claim 1 , wherein the index includes a quality of reading the sequence information by the sequencer.

9. The quality assessment method according to claim 1 , wherein the indicator includes the accuracy of bases in the gene sequence read by the sequencer.

10. The sequencer amplifies genes on a flow cell to form clusters; The quality assessment method according to claim 1 , wherein the index includes a degree of proximity of clusters of genes in the flow cell.

11. The quality assessment method according to claim 1 , wherein the index includes a depth of the sequence information.

12. The quality evaluation method according to claim 1 , wherein the index includes a depth variation of the sequence information.

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