Gene search device, gene search method, and gene search program
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- HU GROUP RESEARCH INSTITUTE G K
- Filing Date
- 2025-04-21
- Publication Date
- 2026-06-10
Smart Images
Figure 00000000_0000_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a gene search device, a gene search method, and a gene search program for assisting nucleic acid probe design.
Background Art
[0002] In microbiome research, comprehensive analysis using a next-generation sequencer (NGS: Next Generation Sequencer), which is a massively parallel sequencer, is the mainstream. In addition, such comprehensive analysis is a powerful screening method for microorganisms and genes.
[0003] On the other hand, after the target bacterial species to be detected is determined, it is conceivable that gene detection will be performed by another measurement method such as PCR (Polymerase chain reaction).
[0004] As prior art documents related to gene extraction, Non-Patent Document 1 and Non-Patent Document 2 can be cited. Non-Patent Document 1 discloses a technique for extracting genes characteristic of a virus and using the extracted genes for classifying unknown viruses. Non-Patent Document 2 discloses a technique for extracting only conserved sequences in the genome of a target species and designing primers for the extracted conserved sequences.
Prior Art Documents
Non-Patent Documents
[0005]
Non-Patent Document 1
Non-Patent Document 2
[0006] For example, when designing primers for a gene specific to a bacterium to be detected (such as a pathogenic gene), a deep prior knowledge and literature research for each bacterium are required when searching for the gene. Further, for example, when designing primers for a gene possessed by many bacteria (such as an essential growth gene), it is difficult to ensure specificity.
[0007] An object of the present invention is to provide a gene search device, a gene search method, and a gene search program that can search for gene information specific to a target only by setting information on the target microorganism species and the like, and can contribute to easily designing a nucleic acid probe optimal for microorganism detection. Means for Solving the Problems
[0008] In order to solve the above-described problems and achieve the object, a gene search device according to the present invention is a gene search device including a control unit for assisting nucleic acid probe design, and is accessible to possession gene information including the number of genes possessed by a plurality of predetermined genes in a plurality of predetermined microorganisms and the phylogenetic information of the plurality of predetermined microorganisms. The control unit has a search means for searching from the possession gene information for a gene having a lower possession rate in all microorganisms other than the microorganism that is commonly possessed by the microorganisms associated with the set phylogenetic information and is managed by the possession gene information.
[0009] Here, a nucleic acid probe is a nucleic acid molecule capable of hybridizing with a target nucleic acid. The target nucleic acid is a nucleic acid encoding a gene specific to a microorganism to be detected. Conceptually, nucleic acid probes include primers in nucleic acid amplification methods, guides in CRISPR (Clustered Regularly Interspaced Shot Palindromic Repeat)-CAS (CRISPR-associated protein) systems, probes in nucleic acid tests, and the like. The nucleic acid probe may be composed of a nucleic acid different from the target nucleic acid. That the nucleic acid probe is composed of a nucleic acid different from the target nucleic acid means that the nucleic acid probe has a backbone structure different from the backbone structure of the target nucleic acid (a structure composed of a sugar moiety and a phosphate moiety) as part or all of the backbone structure. For example, when the target nucleic acid is DNA, as the nucleic acid probe of a nucleic acid different from the target nucleic acid, a nucleic acid probe other than a DNA probe (for example, an RNA probe) can be used.
[0010] In addition, in the gene search device according to the present invention, the predetermined plurality of microorganisms and the strain information may be obtained from whole genome analysis results, and the predetermined plurality of genes may be determined by predicting gene region sequences on each whole genome sequence from the whole genome sequences of the predetermined plurality of microorganisms registered in the whole genome analysis results and performing functional annotation of the predicted gene region sequences.
[0011] Further, in the gene search device according to the present invention, the held gene information may further include the number of copies of the predetermined plurality of genes in a single or a plurality of contig sequences derived from a microbial community obtained by performing metagenome analysis, and group information of a group to which the single or a plurality of contig sequences belong, which corresponds to strain information. The search means may search the held gene information for genes with a lower retention rate in all contig sequences and all microorganisms other than the group commonly held by the group specified by the set group information and managed by the held gene information.
[0012] In the gene search device according to the present invention, the set group information may be the group information of the group to which the contig sequence having integrity belongs.
[0013] In the gene search device according to the present invention, the integrity may be defined by the retention rate of the single-copy gene set and the contamination rate.
[0014] In the gene search device according to the present invention, the group information may be the identification information of a bin obtained as a result of binning the plurality of contig sequences based on information regarding the base composition and data coverage.
[0015] In the gene search device according to the present invention, the search means may search for the gene having the lowest retention rate.
[0016] In the gene search device according to the present invention, the microorganism may be a bacterium, a virus, or a fungus.
[0017] The gene search method according to the present invention is a gene search method for assisting nucleic acid probe design, which is executed in an information processing apparatus including a control unit, and is executed in the control unit of the information processing apparatus that can access the retention gene information including the retention numbers of a plurality of predetermined genes in a plurality of predetermined microorganisms and the phylogenetic information of the plurality of predetermined microorganisms. The method includes a search step of searching the retention gene information for genes having a lower retention rate in all microorganisms other than the microorganisms commonly held by the microorganisms associated with the set phylogenetic information and managed by the retention gene information.
[0018] The gene search program according to the present invention is a gene search program for assisting nucleic acid probe design to be executed in an information processing apparatus including a control unit, and includes the number of copies of a plurality of predetermined genes in a plurality of predetermined microorganisms and phylogenetic information of the plurality of predetermined microorganisms. The control unit of the information processing apparatus capable of accessing the possession gene information including the above searches for genes having a lower possession rate in all microorganisms other than the microorganisms commonly possessed by the microorganisms associated with the set phylogenetic information and managed by the possession gene information from the possession gene information.
Advantages of the Invention
[0019] The present invention contributes to the effect that specific gene information for a target can be retrieved only by setting information such as the target microorganism species, and furthermore, an optimal nucleic acid probe for microorganism detection can be easily designed.
Brief Description of the Drawings
[0020]
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Embodiments for Carrying Out the Invention
[0021] Hereinafter, embodiments of the gene search device, gene search method, and gene search program according to the present invention will be described with reference to the drawings. Note that the present invention is not limited by this embodiment.
[0022] FIG. 1 is a block diagram showing an example of the configuration of the gene search device 100. The gene search device 100 includes a control unit 102 that comprehensively controls the device such as a CPU (Central Processing Unit), a communication interface unit 104 that communicably connects the device to a network 300 such as the Internet via a wired or wireless communication line, a storage unit 106 that can store various databases, tables, or files, and an input / output interface unit 108 that is connected to an input device 112 and an output device 114. Each unit included in the gene search device 100 is communicably connected via an arbitrary communication path.
[0023] As the input device 112, in addition to a keyboard, a mouse, or a microphone, a monitor that realizes a pointing device function in cooperation with the mouse, or a touch panel can be used. As the output device 114, in addition to a monitor, a speaker or a printer can be used.
[0024] The storage unit 106 is a storage means. As the storage unit 106, for example, a memory device such as a RAM (Random Access Memory) or a ROM (Read Only Memory), a fixed disk device such as a hard disk, a flexible disk, or an optical disk can be used. A computer program for giving commands to the CPU in cooperation with the OS (Operating System) to perform various processes may be recorded in the storage unit 106.
[0025] The storage unit 106 stores, for example, an owned gene data table 106a (corresponding to the owned gene information of the present invention). The owned gene data table 106a may be stored in an external DB (database) 200 accessible by the gene search device 100 via the network 300.
[0026] FIG. 2 is a diagram showing an example of the owned gene data table 106a. In the owned gene data table 106a, microorganism records corresponding to a plurality of predetermined microorganisms (strains) are stored. Each microorganism record stores microorganism identification information, the number of copies of each of a plurality of predetermined genes in the microorganism specified by the microorganism identification information, and phylogenetic information of the microorganism specified by the microorganism identification information. Each microorganism record stores gene identification information and the number of copies corresponding to each of a plurality of predetermined genes.
[0027] The microorganism is, for example, a bacterium, a virus, or a fungus. The microorganism identification information is, for example, a microorganism name or a microorganism ID. Specifically, the number of copies of each of a plurality of predetermined genes in the microorganism specified by the microorganism identification information is the number of times the entire genomic sequence of the microorganism specified by the microorganism identification information holds each of the plurality of predetermined gene region sequences. The gene search device 100 may have an information processing function for aggregating the number of copies. The gene identification information is, for example, a gene name or a gene ID. The phylogenetic information is, for example, a species name or a species ID.
[0028] The plurality of predetermined microorganisms and the phylogenetic information may be obtained from a public database (for example, a genomic database published by NCBI (National Center for Biotechnology Information) or a Taxonomy database published by NCBI) or from the results of whole-genome analysis. The gene search device 100 may have an information processing function for executing the acquisition.
[0029] The plurality of predetermined genes may be determined by predicting gene region sequences on each whole genome sequence from the whole genome sequences of a plurality of predetermined microorganisms registered in a public database (such as the genomic database publicly available from NCBI) or whole genome analysis results, and performing functional annotation on the predicted gene region sequences. The functional annotation of each predicted gene region sequence may be, for example, a functional annotation based on the result of a homology search between each predicted gene region sequence and gene region sequences registered in a public database (such as the KO (KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology) database). The gene search device 100 may have an information processing function for performing the prediction and the functional annotation.
[0030] The held gene data table 106a may further store contig sequence records corresponding to each of the single or multiple contig sequences derived from the microbial community obtained by performing metagenome analysis. Each contig sequence record stores contig sequence identification information, the number of copies of each of a plurality of predetermined genes in the contig sequence specified by the contig sequence identification information, and group information of the group to which the contig sequence specified by the contig sequence identification information belongs and which corresponds to phylogenetic information.
[0031] The contig sequence identification information is, for example, a sequence name or sequence ID assigned to the contig sequence. The number of copies of each of the plurality of predetermined genes in the contig sequence specified by the contig sequence identification information is specifically the number of copies of each of the plurality of predetermined gene region sequences held by the contig sequence specified by the contig sequence identification information. The gene search device 100 may have an information processing function for aggregating the number of copies.
[0032] Group information is identification information for identifying a group (for example, a group name or a group ID), and is, for example, identification information (for example, a bin name or a bin ID, etc.) of a bin obtained as a result of binning a plurality of contig arrays based on information regarding base composition and data coverage. The gene search device 100 may include an information processing function for executing the binning.
[0033] The control unit 102 has an internal memory for storing a control program such as an OS, a program defining various processing procedures, etc., and required data, and executes various information processes based on these programs. The control unit 102 includes a search unit 102a.
[0034] The search unit 102a searches the held gene data table 106a for genes with a lower holding rate in "microorganisms associated with the strain information set by the operator" (detection target) and in "all microorganisms other than the said microorganisms managed in the held gene data table 106a" (non-detection target), and outputs information (for example, gene identification information and holding rate, etc.) regarding the searched genes to the output device 114.
[0035] The search unit 102a may search the held gene data table 106a for genes with a lower holding rate in "the group specified by the group information set by the operator" (detection target) and in "all contig arrays and all microorganisms other than the said group managed in the held gene data table 106a" (non-detection target).
[0036] The set group information may be, for example, the group information of a group to which a contig array having completeness belongs. Completeness may be defined, for example, by a holding rate of a single-copy gene set equal to or more than a predetermined value (for example, 95% etc.) or exceeding, and a contamination rate equal to or less than a predetermined value (for example, 5% etc.) or less. The gene search device 100 may include an information processing function for executing the confirmation of the completeness.
[0037] The search unit 102a may search for genes with a possession rate equal to or less than a predetermined value set by the operator. The predetermined value is preferably set to a small value such as, for example, 0.05, 0.01, 0.005, 0.001, etc., but is not limited to these exemplified values.
[0038] The search unit 102a may search for the gene with the lowest possession rate.
Example
[0039] In Example 1, a gene specific to the bacterium Akkermansia muciniphila (A. muciniphila) was searched for by the gene search apparatus 100, primers were designed for the searched gene, and it was verified whether the gene could actually be detected using the designed primers. The verification was performed in the following steps (Steps 11 to 13).
[0040] [Step 11. Step of creating a data table using a public database] The possessed gene data table 106a was created according to the following procedure (Procedures 111 to 117). Note that the possessed gene data table 106a shown in FIG. 2 was actually created by the following procedure.
[0041] [Procedure 111] A total of 11,596 strains of microbial whole genome sequences were downloaded from the genome database publicly available from NCBI.
[0042] [Procedure 112] Using the gene prediction software "Prokka", the gene region sequences on each of the downloaded whole genome sequences were predicted. As a result, a gene region was associated with each of the 11,596 whole genome sequences.
[0043] [Step 113] By performing a homology search of the sequences of each predicted gene region using the homology search software "DIAMOND" against the gene sequence database "KO (KEGG Orthology) database", annotation of each gene region was carried out. As a result, a functional classification was associated with each of all the predicted gene regions.
[0044] [Step 114] A gene ID was assigned to each functional classification of each gene region. As a result, a gene ID was associated with each of all the predicted gene regions.
[0045] [Step 115] For each of the downloaded whole genome sequences, the copy number of each of all the assigned gene IDs was tabulated.
[0046] [Step 116] Referring to the Taxonomy database publicly available from NCBI, taxonomic information (species name) was assigned to each of the downloaded whole genome sequences.
[0047] [Step 117] A gene possession data table 106a including the copy number of each tabulated gene ID and the assigned taxonomic information for each of the downloaded whole genome sequences was created.
[0048] [Step 12. Step of extracting gene regions specific to A. muciniphila] Gene regions specific to A. muciniphila were extracted by the following steps (Steps 121 to 125).
[0049] [Step 121] Using the gene search device 100 that stores the gene possession data table 106a created in Step 11, gene IDs were searched for that were commonly possessed by all strains of "A. muciniphila" to be detected and had a possession rate of less than 0.005 for all microorganisms other than A. muciniphila managed in the gene possession data table 106a, which were not targets for detection. The search results are shown in Table 1. Note that there were two strains of A. muciniphila in the NCBI genome database.
[0050]
Table 1
[0051] [Procedure 122] The gene region sequence corresponding to the gene ID "K10800" with the lowest retention rate in the non-detection target among the retrieved gene IDs was extracted from the whole genome sequence of A. muciniphila downloaded.
[0052] [Procedure 123] Using the consensus sequence creation program "cons", a consensus sequence was created for the extracted gene region sequence corresponding to the gene ID "K10800".
[0053] [Procedure 124] Using the primer design program "Primer3", a primer sequence set was created for the created consensus sequence. The created forward primer sequence is "5’-CTGCTTTCCCTCATCACCAT-3’" (SEQ ID NO: 1), and the reverse primer sequence is "5’-ATGCCCAGTTCCTTAAGCTG-3’" (SEQ ID NO: 2). The base sequence of SEQ ID NO: 1 represents the base sequence of the forward primer for detecting a gene specific to A. muciniphila (gene ID "K10800"). The base sequence of SEQ ID NO: 2 represents the base sequence of the reverse primer for detecting a gene specific to A. muciniphila (gene ID "K10800").
[0054] [Procedure 125] Using the homology search program "Blast+", a homology search of the designed primer sequences was performed using the 11,596 whole genome sequences downloaded in Step 11 as a database. As a result, it was confirmed that the designed primer sequences do not exist in the non-detection target.
[0055] [Step 13. Step of experimentally detecting a gene specific to A. muciniphila] The gene specific to A. muciniphila was experimentally detected by the following procedures (Procedures 131 to 134).
[0056] [Step 131] DNA was extracted from a bacterial culture solution (15 mL) of A. muciniphila, one human fecal sample (approx. 3 mg), and one human saliva sample (approx. 1 mL) using the "PureLink Microbiome DNA Purification kit" manufactured by Thermo Fisher Scientific. DNA was extracted from one human whole blood sample (approx. 200 μL) using the "QIAamp DNA Mini Kit" manufactured by Qiagen.
[0057] [Step 132] Using each of the extracted DNAs, 10 μL of PCR reaction solution was prepared. The components of the reaction solution are as follows. A total of 10 reaction solutions listed below were prepared. <Components of the reaction solution> · Extracted DNA: 1 μL · "NEBNext Q5 Hot Start HiFi 2X Master Mix" manufactured by New England Biolabs: 5 μL · Forward primer (SEQ ID NO: 1) prepared to 10 μM: 0.5 μL · Reverse primer (SEQ ID NO: 2) prepared to 10 μM: 0.5 μL · Purified water: 3 μL
[0058] <Prepared reaction solutions> · Reaction solution 1 using DNA derived from the bacterial culture solution of A. muciniphila · Reaction solution 2 using DNA derived from blood · Reaction solution 3 in which 0.01% of DNA derived from the bacterial culture solution of A. muciniphila is mixed into the reaction solution using DNA derived from blood · Reaction solution 4 in which 0.1% of DNA derived from the bacterial culture solution of A. muciniphila is mixed into the reaction solution using DNA derived from blood · Reaction solution 5 using DNA derived from feces · Reaction solution 6 in which 0.01% of DNA derived from the bacterial culture solution of A. muciniphila is mixed into the reaction solution using DNA derived from feces · Reaction solution 7 in which 0.1% of the DNA derived from the bacterial culture solution of A. muciniphila was mixed into the reaction solution using DNA derived from feces · Reaction solution 8 using DNA derived from saliva · Reaction solution 9 in which 0.01% of the DNA derived from the bacterial culture solution of A. muciniphila was mixed into the reaction solution using DNA derived from saliva · Reaction solution 10 in which 0.1% of the DNA derived from the bacterial culture solution of A. muciniphila was mixed into the reaction solution using DNA derived from saliva
[0059] [Procedure 133] PCR was performed using the prepared PCR reaction solution. For PCR, the thermal cycler "Veriti" manufactured by Thermo Fisher Scientific was used. The PCR reaction conditions were as follows: after incubation at 98°C for 30 seconds, 35 cycles of thermal denaturation at 98°C for 10 seconds, annealing at 65°C for 15 seconds, and extension reaction at 72°C for 30 seconds were performed.
[0060] [Procedure 134] The amplified products were analyzed using the "D1000 ScreenTape" of the fully automatic high-throughput electrophoresis system "Agilent 4200 TapeStation" manufactured by Agilent Technologies. The analysis results are shown in Figure 3. In Figure 3, "L" indicates the ladder, and "Am" indicates the detection result of the amplified product obtained from reaction solution 1. In Figure 3, "Bl" indicates the detection result of the amplified product obtained from reaction solution 2, "Bl + 0.01% Am" indicates the detection result of the amplified product obtained from reaction solution 3, and "Bl + 0.1% Am" indicates the detection result of the amplified product obtained from reaction solution 4. In Figure 3, "St" indicates the detection result of the amplified product obtained from reaction solution 5, "St + 0.01% Am" indicates the detection result of the amplified product obtained from reaction solution 6, and "St + 0.1% Am" indicates the detection result of the amplified product obtained from reaction solution 7. In Figure 3, "Sa" indicates the detection result of the amplified product obtained from reaction solution 8, "Sa + 0.01% Am" indicates the detection result of the amplified product obtained from reaction solution 9, and "Sa + 0.1% Am" indicates the detection result of the amplified product obtained from reaction solution 10.
[0061] According to Example 1, it was confirmed that the target bacterium can be actually detected by the primer designed for the gene searched by the gene search device 100, and that the target bacterium is not detected by the primer in a human genome sample or a metagenome sample in which various bacteria are present.
Example
[0062] In Example 2, for a metagenome sample, a gene specific to each individual bacterial species in the metagenome was searched by the gene search device 100, primers were designed for the searched gene, and it was verified whether the gene could be actually detected using the designed primers. The verification was performed in the following steps (Steps 21 to 24).
[0063] [Step 21. Step of creating a data table from metagenome analysis results] The held gene data table 106a was created according to the following procedure (Procedures 211 to 218).
[0064] [Procedure 211] DNA was extracted from 5 human fecal specimens (each about 3 mg) (Sample1 to Sample5) using the "PureLink Microbiome DNA Purification kit" manufactured by Thermo Fisher Scientific. DNA was extracted from 1 human whole blood specimen (about 200 μL) using the "QIAamp DNA Mini Kit" manufactured by Qiagen.
[0065] [Procedure 212] For the DNA of Sample1 and the DNA of Sample2 among the extracted DNA, a library for sequencing analysis was prepared using the "Nextera DNA Flex library prep kit" manufactured by Illumina.
[0066] [Procedure 213] Shotgun sequencing analysis of the prepared library was performed using NextSeq manufactured by Illumina.
[0067] [Step 214] Using the genome assembler "SPAdes", assembly analysis of the array information obtained in Step 213 was performed.
[0068] [Step 215] Binning was performed using the binning software "MetaBAT2". Specifically, based on information on nucleotide composition and data coverage, contig arrays considered to be derived from the same organism were grouped.
[0069] [Step 216] Using each group generated by binning as a unit, the same procedures as in Steps 112 to 115 were performed for each group.
[0070] [Step 217] For each contig array, an arbitrary ID (bin name) for specifying the corresponding group was assigned as lineage information.
[0071] [Step 218] Records including the count of each gene ID and the assigned lineage information for each group were joined to the held gene data table 106a created in Step 11.
[0072] [Step 22. Step of extracting gene regions specific to each individual bacterial species in the metagenome] The following procedures (Steps 221 to 225) were used to extract gene regions specific to each individual bacterial species in the metagenome.
[0073] [Step 221] Using the completeness evaluation software "CheckM", the completeness of all groups obtained in Step 21 was evaluated, and only contig arrays with a completeness of 95% or more and a contamination rate of 5% or less were selected as the targets for primer design. Among the selected groups, the bin names assigned to the groups derived from Sample1 were bin.33, bin.13, bin.43, and bin.18, and the bin names assigned to the groups derived from Sample2 were bin.14, bin.18, bin.52, and bin.11.
[0074] [Procedure 222] For each of the eight bin names obtained in Procedure 221, the following search process was executed to search for gene IDs for each bin name. <Search process> Using the gene search device 100 that stores the combined owned gene data table 106a, search for gene IDs that are owned by the "group specified by the bin name" to be detected and whose ownership rate of "all contig arrays and all microorganisms other than the group specified by the bin name, which are managed in the owned gene data table 106a" for non-detection targets is less than 0.05.
[0075] [Procedure 223] For each of the eight bin names, further select the gene ID with the lowest ownership rate for non-detection targets among the gene IDs searched in Procedure 222, and extract the gene region sequences corresponding to the selected gene IDs from the contig sequences associated with each bin name. Table 2 shows the gene IDs selected in Procedure 223 corresponding to each of the eight bin names.
[0076]
Table 2
[0077] [Procedure 224] Using the primer design program "Primer3", a primer sequence set was created for the gene region sequences extracted in Procedure 223 corresponding to each of the eight bin names. Table 3 shows the created primer sequence sets corresponding to each of the eight bin names.
[0078]
Table 3
[0079] The base sequences of SEQ ID NOs: 3 and 4 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K22330", which is specific to the bacterial species corresponding to the bin name "bin.13" in the metagenome. The base sequences of SEQ ID NOs: 5 and 6 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K15303", which is specific to the bacterium corresponding to the bin name "bin.18" in the metagenome. The base sequences of SEQ ID NOs: 7 and 8 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K05942", which is specific to the bacterium corresponding to the bin name "bin.33" in the metagenome. The base sequences of SEQ ID NOs: 9 and 10 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K05942", which is specific to the bacterium corresponding to the bin name "bin.43" in the metagenome.
[0080] The base sequences of SEQ ID NOs: 11 and 12 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K15022", which is specific to the bacterium corresponding to the bin name "bin.11" in the metagenome. The base sequences of SEQ ID NOs: 13 and 14 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K18205", which is specific to the bacterium corresponding to the bin name "bin.14" in the metagenome. The base sequences of SEQ ID NOs: 15 and 16 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K22340", which is specific to the bacterium corresponding to the bin name "bin.18" in the metagenome. The base sequences of SEQ ID NOs: 17 and 18 are the base sequences of the forward primer and the reverse primer for detecting the gene corresponding to the gene ID "K22607", which is specific to the bacterium corresponding to the bin name "bin.52" in the metagenome.
[0081] [Procedure 225] Using the homology search program "Blast+", a homology search of the designed primer sequences corresponding to each of the eight bin names was performed against all the contig sequences obtained in Step 21 and a total of 11,596 whole genome sequences downloaded in Step 11 as a database. As a result, it was confirmed that the designed primer sequences did not exist in the non-detection target.
[0082] [Step 23. Step of experimentally detecting genes specific to each individual bacterial species in the metagenome] In the following procedure (Procedures 231 to 233), genes specific to each individual bacterial species in the metagenome were experimentally detected.
[0083] [Procedure 231] Using the DNA extracted in Procedure 211, a 10 μL PCR reaction solution was prepared. The components of the reaction solution are as follows. A total of 48 reaction solutions listed below were prepared. <Components of the reaction solution> · Extracted DNA: 1 μL · "NEBNext Q5 Hot Start HiFi 2X Master Mix" manufactured by New England Biolabs: 5 μL · Forward primer prepared to 10 μM (sequence shown in Table 3): 0.5 μL · Reverse primer prepared to 10 μM (sequence shown in Table 3): 0.5 μL · Purified water: 3 μL
[0084] <Prepared reaction solution> · Reaction solution 1 using DNA derived from Sample 1 and primer set with primer set ID "bin.13.K22330" · Reaction solution 2 using DNA derived from Sample 2 and primer set with primer set ID "bin.13.K22330" · Reaction solution 3 using DNA derived from Sample 3 and primer set with primer set ID "bin.13.K22330" ·Reaction solution 4 using DNA from Sample4 and primer set with primer set ID "bin.13.K22330" ·Reaction solution 5 using DNA from Sample5 and primer set with primer set ID "bin.13.K22330" ·Reaction solution 6 using DNA from blood and primer set with primer set ID "bin.13.K22330"
[0085] ·Reaction solution 7 using DNA from Sample1 and primer set with primer set ID "bin.18.K15303" ·Reaction solution 8 using DNA from Sample2 and primer set with primer set ID "bin.18.K15303" ·Reaction solution 9 using DNA from Sample3 and primer set with primer set ID "bin.18.K15303" ·Reaction solution 10 using DNA from Sample4 and primer set with primer set ID "bin.18.K15303" ·Reaction solution 11 using DNA from Sample5 and primer set with primer set ID "bin.18.K15303" ·Reaction solution 12 using DNA from blood and primer set with primer set ID "bin.18.K15303"
[0086] ·Reaction solution 13 using DNA from Sample1 and primer set with primer set ID "bin.33.K05942" ·Reaction solution 14 using DNA from Sample2 and primer set with primer set ID "bin.33.K05942" ·Reaction solution 15 using DNA from Sample3 and primer set with primer set ID "bin.33.K05942" ·Reaction solution 16 using DNA from Sample4 and primer set with primer set ID "bin.33.K05942" ·Reaction solution 17 using DNA from Sample5 and primer set with primer set ID "bin.33.K05942" ·Reaction solution 18 using DNA from blood and primer set with primer set ID "bin.33.K05942"
[0087] ·Reaction solution 19 using DNA from Sample1 and primer set with primer set ID "bin.43.K05942" ·Reaction solution 20 using DNA from Sample2 and primer set with primer set ID "bin.43.K05942" ·Reaction solution 21 using DNA from Sample3 and primer set with primer set ID "bin.43.K05942" ·Reaction solution 22 using DNA from Sample4 and primer set with primer set ID "bin.43.K05942" ·Reaction solution 23 using DNA from Sample5 and primer set with primer set ID "bin.43.K05942" ·Reaction solution 24 using DNA from blood and primer set with primer set ID "bin.43.K05942"
[0088] ·Reaction solution 25 using DNA from Sample2 and primer set with primer set ID "bin.11.K15022" ·Reaction solution 26 using DNA from Sample3 and primer set with primer set ID "bin.11.K15022" ·Reaction solution 27 using DNA from Sample4 and primer set with primer set ID "bin.11.K15022" ·Reaction solution 28 using DNA from Sample1 and primer set with primer set ID "bin.11.K15022" ·Reaction solution 29 using DNA from Sample5 and primer set with primer set ID "bin.11.K15022" · Reaction solution 30 using DNA derived from blood and primer set with primer set ID "bin.11.K15022"
[0089] · Reaction solution 31 using DNA derived from Sample2 and primer set with primer set ID "bin.14.K18205" · Reaction solution 32 using DNA derived from Sample3 and primer set with primer set ID "bin.14.K18205" · Reaction solution 33 using DNA derived from Sample4 and primer set with primer set ID "bin.14.K18205" · Reaction solution 34 using DNA derived from Sample1 and primer set with primer set ID "bin.14.K18205" · Reaction solution 35 using DNA derived from Sample5 and primer set with primer set ID "bin.14.K18205" · Reaction solution 36 using DNA derived from blood and primer set with primer set ID "bin.14.K18205"
[0090] · Reaction solution 37 using DNA derived from Sample2 and primer set with primer set ID "bin.18.K22340" · Reaction solution 38 using DNA derived from Sample3 and primer set with primer set ID "bin.18.K22340" · Reaction solution 39 using DNA derived from Sample4 and primer set with primer set ID "bin.18.K22340" · Reaction solution 40 using DNA derived from Sample1 and primer set with primer set ID "bin.18.K22340" · Reaction solution 41 using DNA derived from Sample5 and primer set with primer set ID "bin.18.K22340" · Reaction solution 42 using DNA derived from blood and primer set with primer set ID "bin.18.K22340"
[0091] ·Reaction solution 43 using DNA from Sample 2 and primer set with primer set ID "bin.52.K22607" ·Reaction solution 44 using DNA from Sample 3 and primer set with primer set ID "bin.52.K22607" ·Reaction solution 45 using DNA from Sample 4 and primer set with primer set ID "bin.52.K22607" ·Reaction solution 46 using DNA from Sample 1 and primer set with primer set ID "bin.52.K22607" ·Reaction solution 47 using DNA from Sample 5 and primer set with primer set ID "bin.52.K22607" ·Reaction solution 48 using DNA from blood and primer set with primer set ID "bin.52.K22607"
[0092] [Procedure 232] PCR was performed using the prepared PCR reaction solution. For PCR, a thermal cycler "Veriti" manufactured by Thermo Fisher Scientific was used. The PCR reaction conditions were incubation at 98°C for 30 seconds, followed by 35 cycles of thermal denaturation at 98°C for 10 seconds, annealing at 65°C for 15 seconds, and extension reaction at 72°C for 30 seconds.
[0093] [Procedure 233] The amplification products were analyzed using "D1000 ScreenTape" of the fully automatic high-throughput electrophoresis system "Agilent 4200 TapeStation" manufactured by Agilent Technologies. The analysis results are shown in Figure 4. In Figure 4, the detection results of the amplification products obtained from each of reaction solutions 1 to 24 are shown in order from the left in the upper row, and the detection results of the amplification products obtained from each of reaction solutions 25 to 48 are shown in order from the left in the lower row. In Figure 4, "L" indicates the ladder.
[0094] [Step 24: Step of performing quantitative analysis of specific genes in the metagenome] The quantitative analysis of specific genes in the metagenome was performed according to the following procedures (procedures 241 to 243).
[0095] [Procedure 241] Using the extracted DNA from Sample 1 and the DNA from Sample 2, a 20-μL quantitative PCR reaction solution was prepared. The components of the reaction solution are as follows. A total of 8 reaction solutions listed below were prepared. <Components of the reaction solution> · Template DNA: 1 μL · "2x KOD SYBR qPCR Mix" manufactured by Toyobo: 10 μL · Forward primer prepared to 10 μM (sequence shown in Table 3): 1 μL · Reverse primer prepared to 10 μM (sequence shown in Table 3): 1 μL · ROX: 0.4 μL · Purified water: 6.6 μL
[0096] <Prepared reaction solutions> · Reaction solution 1 using DNA from Sample 1 and the primer set with primer set ID "bin.13.K22330" · Reaction solution 2 using DNA from Sample 1 and the primer set with primer set ID "bin.18.K15303" · Reaction solution 3 using DNA from Sample 1 and the primer set with primer set ID "bin.33.K05942" · Reaction solution 4 using DNA from Sample 1 and the primer set with primer set ID "bin.43.K05942" · Reaction solution 5 using DNA from Sample 2 and the primer set with primer set ID "bin.11.K15022" · Reaction solution 6 using DNA from Sample 2 and the primer set with primer set ID "bin.14.K18205" · Reaction solution 7 using DNA from Sample 2 and the primer set with primer set ID "bin.18.K22340" ·Reaction solution 8 using DNA from Sample 2 and primer set with primer set ID "bin.52.K22607"
[0097] [Procedure 242] Quantitative PCR was performed using the prepared quantitative PCR reaction solution. For the quantitative PCR, "Mx3005P" manufactured by Agilent Technologies was used. The quantitative PCR reaction conditions were as follows: after incubation at 98°C for 2 minutes, 40 cycles of heat denaturation at 98°C for 10 seconds, annealing at 60°C for 10 seconds, and extension reaction at 68°C for 30 seconds were performed.
[0098] [Procedure 243] Relative quantitative values were calculated from the obtained data by the ΔCt method, and the calculation results were compared with the quantitative results obtained by shotgun sequencing. The comparison results are shown in Figure 5. The quantitative PCR was performed in 3 independent experiments. Within one experiment, it was performed with 2 technical replicates, and the average value was used for the analysis. The assumed quantitative value was calculated by normalizing the quantitative value of each sample with the quantitative value at the far left of the graph shown in Figure 5. In Figure 5, the white graph shows the quantitative value of qPCR, and the black graph shows the quantitative value of shotgun sequencing. In Figure 5, the dots in the white graph indicate the average value in one experiment.
[0099] According to Example 2, it was confirmed that the target bacterium can be actually detected with primers designed for the genes searched by the gene search device 100, and that quantitative analysis almost equivalent to shotgun analysis can be performed with the designed primers.
Industrial Applicability
[0100] The present invention is useful, for example, in nucleic acid probe design.
Explanation of Signs
[0101] 100 Gene search device 102 Control unit 102a Search unit 104 Communication interface unit 106 Storage unit 106a Gene Data Table 108 Input / Output Interface Section
Claims
1. A gene search device equipped with a control unit for supporting nucleic acid probe design, It is possible to access gene information, including the number of genes containing genes with identified base sequences or genes with predicted functions in microorganisms whose genome sequences have been identified, and phylogenetic information for identifying the identified microorganisms. The control unit, The system information entered by the user is obtained, and the user-configured system information is obtained. By searching the possessed gene information using the user-defined lineage information as a query, microorganisms associated with the user-defined lineage information are detected. The common gene, which is a gene commonly possessed by microorganisms associated with the user-defined system information, is detected from the gene possessing information. Microorganisms other than those associated with the user-defined system information are detected from the possessed gene information. The possession rate of the common gene of microorganisms other than those associated with the user-defined system information is calculated using the gene possession information. The system includes a search means for outputting the commonly held genes whose possession rate is below a user-defined threshold. Genetic search device.
2. The identified microorganisms and the lineage information are as follows: This was obtained from whole genome sequencing results. The aforementioned functional prediction genes are This was determined by predicting the gene region sequences on each whole genome sequence from the whole genome sequences of the identified microorganisms registered in the whole genome analysis results, and then performing functional annotation on each of the predicted gene region sequences. The gene search device according to claim 1.
3. The aforementioned genetic information is Furthermore, it includes the number of genes present in the contig sequences derived from the microbiome obtained by metagenomic analysis, and group information of the group to which the contig sequences belong, which corresponds to the phylogenetic information. The search means is, The user-defined group information, which is the group information entered and set by the user, is obtained. By searching the possessed gene information using the user-defined group information as a query, the group identified by the user-defined group information is detected. The group-possessed genes, which are genes commonly possessed by the groups identified in the user-defined group information, are detected from the possessed gene information. The contig sequences and microorganisms other than those identified in the user-defined group information are detected from the gene information. The possession rate of contig sequences other than those identified in the user-defined group information and the group-possessed genes of microorganisms is calculated using the possessed gene information. The group outputs the genes whose possession rate is below the user-defined threshold. The gene search device according to claim 1 or 2.
4. The user-defined group information is, The gene search device according to claim 3, which is group information of the group to which the complete contig sequence belongs.
5. The aforementioned completeness is, The gene search device according to claim 4, defined by the possession rate and contamination rate of single-copy gene sets.
6. The aforementioned group information is, The gene search device according to claim 3, wherein the bin identification information is obtained as a result of binning the contig sequence based on the base composition and data coverage information.
7. The search means is, A gene search device according to claim 1 or 2, which searches for the gene with the lowest prevalence rate.
8. The aforementioned microorganisms A gene search device according to claim 1 or 2, wherein the gene is a bacterium, virus, or fungus.
9. A gene search method for supporting nucleic acid probe design, performed by an information processing device equipped with a control unit, The control unit of the information processing device, which has access to gene possession information including the number of genes possessed in a microorganism whose genome sequence has been identified, including genes with identified base sequences or genes with predicted functions, and phylogenetic information for identifying the identified microorganism, is executed. The system information entered by the user is obtained, and the user-configured system information is obtained. By searching the possessed gene information using the user-defined lineage information as a query, microorganisms associated with the user-defined lineage information are detected. The common gene, which is a gene commonly possessed by microorganisms associated with the user-defined system information, is detected from the gene possessing information. Microorganisms other than those associated with the user-defined system information are detected from the possessed gene information. The possession rate of the common gene of microorganisms other than those associated with the user-defined system information is calculated using the gene possession information. The search step includes outputting the commonly held genes whose possession rate is below a user-defined threshold, Genetic testing methods.
10. A gene search program for supporting nucleic acid probe design, to be executed in an information processing device equipped with a control unit, The control unit of the information processing device, which has access to gene possession information including the number of genes possessed in a microorganism whose genome sequence has been identified, including genes with identified base sequences or genes with predicted functions, and phylogenetic information for identifying the identified microorganism, is to be executed in the control unit of the information processing device. The system information entered by the user is obtained, and the user-configured system information is obtained. By searching the possessed gene information using the user-defined lineage information as a query, microorganisms associated with the user-defined lineage information are detected. The common gene, which is a gene commonly possessed by microorganisms associated with the user-defined system information, is detected from the gene possessing information. Microorganisms other than those associated with the user-defined system information are detected from the possessed gene information. The possession rate of the common gene of microorganisms other than those associated with the user-defined system information is calculated using the gene possession information. The search step includes outputting the commonly held genes whose possession rate is below a user-defined threshold, Genetic search program.