WO2018147438A1 - Hla遺伝子のpcrプライマーセット及びそれを用いたシークエンス法 - Google Patents
Hla遺伝子のpcrプライマーセット及びそれを用いたシークエンス法 Download PDFInfo
- Publication number
- WO2018147438A1 WO2018147438A1 PCT/JP2018/004703 JP2018004703W WO2018147438A1 WO 2018147438 A1 WO2018147438 A1 WO 2018147438A1 JP 2018004703 W JP2018004703 W JP 2018004703W WO 2018147438 A1 WO2018147438 A1 WO 2018147438A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hla
- primer
- gene
- primer set
- base sequence
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Definitions
- the present invention relates to a PCR primer set for a Classical HLA gene and a highly efficient and highly uniform sequencing method using the PCR primer set.
- HLA Human leukocyte antigen
- MHC human major histocompatibility complex
- Non-patent Document 3 As another HLA gene typing approach, a 120-base probe corresponding to various sequences of the HLA allele is designed based on the database for the above six types of HLA genes, and the cDNA and probe are hybridized. A method of reading the obtained genomic fragment with NGS has been reported (Non-patent Document 3). However, in this method, in order to cover as much as possible the sequences of HLA alleles registered in the database, 10,000 types of probes are designed, and there is a problem that the uniformity of amplification and the collection efficiency deteriorate. . In addition, it is difficult to detect alleles other than the HLA alleles covered, and there is a high possibility that genomic fragments of unintended genes having the same sequence as the probe are mixed.
- the present inventors have come up with the idea that a primer set that satisfies the two conditions A and B can be developed by performing the following steps 1 to 4. Assemble sequence analysis data from the next generation sequence of 6 genes in 1.768 Japanese specimens, excluding reads from other genes. 2. Align the assembled results in all specimens per gene. In the result of 3.2, a region having a sequence common to all specimens is detected and set as a candidate primer. Among the candidates in 4.3, a sequence that does not share a sequence with other HLA genes and is suitable for the primer is selected. As a result of further studies based on the above idea, the present inventors have completed the present invention.
- a primer set for HLA-A gene amplification comprising a primer consisting of the base sequence shown in SEQ ID NO: 1 and a primer consisting of the base sequence shown in SEQ ID NO: 2.
- a primer set for HLA-B gene amplification comprising a primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 4.
- a primer set for HLA-C gene amplification comprising a primer consisting of the base sequence shown in SEQ ID NO: 5 and a primer consisting of the base sequence shown in SEQ ID NO: 6.
- Primer set for HLA-DRB1 gene amplification including primers.
- a primer set for amplifying the HLA-DQB1 gene comprising a primer consisting of the base sequence shown in SEQ ID NO: 11 and a primer consisting of the base sequence shown in SEQ ID NO: 12.
- Primer set for HLA-DPB1 gene amplification including primers.
- An HLA gene amplification primer set comprising all the primer sets according to [1] to [6].
- [9] A method for sequencing the HLA gene, which is performed using the primer set according to any one of [1] to [8].
- [10] A method for typing an HLA gene, which is performed using the nucleotide sequence information obtained by the method according to [9].
- [11] A kit for amplifying an HLA gene comprising the primer set according to any one of [1] to [8].
- the primer set of the present invention is designed based on a common sequence by assembling and aligning the results of a large-scale sequence, using the primer set of the present invention makes it difficult to amplify the HLA. Many alleles can be amplified without leaking.
- the primer set of the present invention since the primer set of the present invention has selected primers having no homology with other genes from a large number of sequence candidates by a computational approach, nonspecific amplification can be achieved by using the primer set of the present invention. It is possible to prevent. Accordingly, it is possible to reduce erroneous allele determinations due to contamination of non-target genes, contributing to highly accurate allele determination.
- a single primer is used for the target gene region, uniform PCR amplification is possible, the number of samples that can be performed at one time can be increased, and the HLA typing cost can be reduced.
- FIG. 1 shows a schematic diagram showing the assembly results of a large-scale sequence in the HLA-A gene of the present invention, the positions of primers for amplification, and an outline of the primers.
- FIG. 2 shows a schematic diagram showing the assembly results of a large-scale sequence in the HLA-B gene of the present invention, the positions of primers for amplification, and an outline of the primers.
- FIG. 3 shows a schematic diagram showing the assembly results of the large-scale sequence in the HLA-C gene of the present invention, the positions of the primers for amplification, and an outline of the primers.
- FIG. 1 shows a schematic diagram showing the assembly results of a large-scale sequence in the HLA-A gene of the present invention, the positions of primers for amplification, and an outline of the primers.
- FIG. 2 shows a schematic diagram showing the assembly results of a large-scale sequence in the HLA-B gene of the present invention, the positions of primers for amplification, and an outline of the primers.
- FIG. 4 shows a schematic diagram showing the assembly results of the large-scale sequence in the HLA-DRB1 gene of the present invention, the positions of the primers for amplification, and an outline of the primers.
- FIG. 5 shows a schematic diagram showing the assembly results of the HLA-DQB1 gene of the present invention, the positions of the primers for amplification, and an overview of the primers.
- FIG. 6 shows a schematic diagram showing the assembly results of the large-scale sequence in the HLA-DPB1 gene of the present invention, the positions of the primers for amplification, and an outline of the primers.
- FIG. 7 shows an analysis of the results of coverage average in the sequence results by MiSeq using the primer set of the present invention and the previously reported primer set.
- the present invention relates to HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 or HLA-DPB1 gene regions of HLA genes (translation region and intron region and 5 ′ and 3 ′ untranslated regions).
- a primer set (hereinafter abbreviated as “primer set of the present invention”) capable of uniformly amplifying a part of the region by PCR.
- the typing method of the HLA gene using the primer set of this invention is also provided.
- the “primer set” means a combination of two or more PCR primers capable of amplifying a predetermined region of each HLA gene, and includes a forward primer and a reverse primer corresponding to each region. Such a primer set is sometimes called a primer pair.
- the present invention provides at least one primer pair selected from the primer sets listed in Table 1 below.
- FIG. 1 shows a schematic diagram showing the assembly results of a large-scale sequence in the HLA-A gene of the present invention, the position of the amplification primer on the gene, and an outline of the primer.
- the primer set comprising the primer consisting of the base sequence shown in SEQ ID NO: 1 and the primer consisting of the base sequence shown in SEQ ID NO: 2 is a primer set capable of specifically amplifying the HLA-A gene. Yes (this primer set is abbreviated as “HLA-A gene amplification primer set”).
- the primer set for HLA-A gene amplification is designed to amplify a region from the upstream side of exon 1 to the downstream side of exon 8 of the HLA-A gene in the human genome base sequence (reference sequence: hg19).
- FIG. 2 shows a schematic diagram showing the assembly results of the large-scale sequence of the HLA-B gene of the present invention, the position of the primer for amplification on the gene, and an outline of the primer.
- the primer set comprising the primer consisting of the base sequence shown in SEQ ID NO: 3 and the primer consisting of the base sequence shown in SEQ ID NO: 4 is a primer set capable of specifically amplifying the HLA-B gene. Yes (this primer set is abbreviated as “HLA-B gene amplification primer set”).
- the primer set for amplifying HLA-B gene is designed to amplify a region from the upstream side of exon 1 to the downstream side of exon 7 of the HLA-B gene in the human genome base sequence (reference sequence: hg19).
- FIG. 3 shows a schematic diagram showing the assembly results of the large-scale sequence of the HLA-C gene of the present invention, the position of the amplification primer on the gene, and an outline of the primer.
- a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 5 and a primer consisting of the base sequence shown in SEQ ID NO: 6 is a primer set that specifically amplifies the HLA-C gene (this primer The set is abbreviated as “HLA-C gene amplification primer set”.
- the primer set for amplifying the HLA-C gene is designed to amplify a region from the upstream side of exon 1 to the downstream side of exon 8 of the HLA-C gene in the human genome base sequence (reference sequence: hg19).
- FIG. 4 shows a schematic diagram showing the assembly results of the large-scale sequence in the HLA-DRB1 gene of the present invention, the position of the amplification primer on the gene, and the outline of the primer.
- a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 7 and a primer consisting of the base sequence shown in SEQ ID NO: 8 is a primer set that specifically amplifies a part of the HLA-DRB1 gene ( This primer set is abbreviated as “HLA-DRB1 gene amplification primer set 1”.
- HLA-DRB1 gene amplification primer set 1 is designed to amplify a region from the upstream side of exon 1 to the downstream side of exon 2 of the HLA-DRB1 gene in the human genome sequence (reference sequence: hg19).
- a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 9 and a primer consisting of the base sequence shown in SEQ ID NO: 10 is a primer set that specifically amplifies a part of the HLA-DRB1 gene (this primer The set is abbreviated as “HLA-DRB1 gene amplification primer set 2”.) HLA-DRB1 gene amplification primer set 2 amplifies the region from the intron region between exon 1 and exon 2 of the HLA-DRB1 gene to the downstream side of exon 6 in the human genome sequence (reference sequence: hg19). Designed to. Since the HLA-DRB1 gene has a long gene length, PCR is preferably performed using the two types of primer sets in order to have robustness against DNA degradation.
- FIG. 5 shows a schematic diagram showing the assembly results of the large-scale sequence of the HLA-DQB1 gene of the present invention, the position of the amplification primer on the gene, and an outline of the primer.
- a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 11 and a primer consisting of the base sequence shown in SEQ ID NO: 12 is a primer set that specifically amplifies the HLA-DQB1 gene (this primer The set is abbreviated as “HLA-DQB1 gene amplification primer set”).
- the primer set for amplifying the HLA-DQB1 gene is designed to amplify the region from the upstream side of exon 1 to the downstream side of exon 6 of the HLA-DQB1 gene in the human genome base sequence (reference sequence: hg19).
- FIG. 6 shows a schematic diagram showing the assembly results of the large-scale sequence of the HLA-DPB1 gene of the present invention, the position of the amplification primer on the gene, and the outline of the primer.
- a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 13 and a primer consisting of the base sequence shown in SEQ ID NO: 14 is a primer set that specifically amplifies a part of the HLA-DPB1 gene ( This primer set is abbreviated as “HLA-DPB1 gene amplification primer set 1”.
- HLA-DPB1 gene amplification primer set 1 is designed to amplify a region from the upstream side of exon 1 to the downstream side of exon 2 of the HLA-DPB1 gene in the human genome sequence (reference sequence: hg19).
- a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 15 and a primer consisting of the base sequence shown in SEQ ID NO: 16 is a primer set that specifically amplifies a part of the HLA-DPB1 gene (this primer The set is abbreviated as “HLA-DPB1 gene amplification primer set 2”.)
- HLA-DPB1 gene amplification primer set 2 is designed to amplify the region from the upstream side of exon 2 to the downstream side of exon 5 of the HLA-DPB1 gene in the human genome sequence (reference sequence: hg19). . Since the HLA-DPB1 gene has a long gene length, PCR is preferably performed using the two kinds of primer sets in order to have robustness against DNA degradation.
- the primer constituting the primer set of the present invention includes the nucleotide sequence represented by each sequence number in addition to the nucleic acid comprising the nucleotide sequence represented by each sequence number. And a variant thereof having a base sequence that hybridizes under stringent conditions with a complementary base sequence and also having a function as a primer.
- a variant thereof having a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and also having a function as a primer It has the same function as a primer as the oligonucleotide having the nucleotide sequence shown, that is, the ability to amplify a part of the HLA-A gene by combining with the primer having the nucleotide sequence shown by SEQ ID NO: 2.
- a variant thereof having a base sequence that hybridizes with a base sequence complementary to the base sequence shown in SEQ ID NO: 2 under stringent conditions and also having a function as a primer An oligonucleotide that hybridizes under stringent conditions with an oligonucleotide consisting of a base sequence complementary to the base sequence shown, and functions as a primer similar to the oligonucleotide consisting of the base sequence shown by SEQ ID NO: 2, By combining with a primer consisting of the base sequence shown in SEQ ID NO: 1, it has a function that allows PCR amplification of a part of the HLA-A gene.
- the stringency during hybridization is a function of temperature, salt concentration, primer chain length, GC content of primer nucleotide sequence, and concentration of chaotropic agent in the hybridization buffer.
- stringent conditions for example, the conditions described in Sambrook, J. et al. (1998) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York can be used. .
- Stringent temperature conditions are about 30 ° C. or higher, more preferably about 37 ° C. or higher, and most preferably about 42 ° C. or higher.
- Other conditions include the hybridization time, the concentration of the detergent (eg, SDS), the presence or absence of carrier DNA, and various stringencies can be set by combining these conditions.
- the primer of the present invention comprises a nucleic acid, but the nucleic acid is preferably a single-stranded nucleic acid.
- the nucleic acid means a molecule obtained by polymerizing nucleotides and molecules having functions equivalent to those of the nucleotides.
- DNA, RNA, and a polymer in which RNA and DNA are mixed are included, but when RNA is included, the base sequence is to read “T (thymine)” in the DNA sequence as “U (uracil)”.
- the primer of the present invention can be prepared by any chemical synthesis method known to those skilled in the art.
- the primer of the present invention has a labeling substance (for example, a fluorescent molecule, a dye molecule, a radioisotope, an organic compound such as digoxigenin or biotin) for facilitating detection and amplification of the primer at the 5 ′ end or 3 ′ end. Etc.) and / or an additional sequence (such as a loop primer portion used in the LAMP method).
- a labeling substance for example, a fluorescent molecule, a dye molecule, a radioisotope, an organic compound such as digoxigenin or biotin
- the primer of the present invention may be phosphorylated or aminated at the 5 ′ end.
- the primer of this invention may contain only a natural base and may contain a modified base. Examples of the modified base include, but are not limited to, deoxyinosine, deoxyuracil, S-modified base and the like.
- the primer of the present invention may contain any derivative oligonucleotide containing a phosphorothioate bond, phosphoramidate bond, or the like, or may contain a peptide-nucleic acid (PNA) containing a peptide nucleic acid bond. Good.
- PNA peptide-nucleic acid
- the primer of the present invention is preferably isolated or purified. “Isolated or purified” means that an operation for removing components other than the target component from a natural or synthesized state has been performed.
- the purity of the isolated or purified primer (ratio of the target primer contained in the total nucleic acid) is usually 50% or more, preferably 70% or more, more preferably 90% or more, most preferably 95% or more (for example, 100%).
- the purity of the primer can be appropriately changed depending on the solvent and the solid / liquid state.
- the unit of purity may be (w / v)% or (v / v)%, and the desired purity should be calculated as appropriate in consideration of the definition of purity in the case of (w / w)%. Can do.
- These primers can be provided as a solid in a dry state or in an alcohol precipitation state, or can be provided in a state dissolved in water or an appropriate buffer (eg, TE buffer).
- an appropriate buffer eg, TE buffer
- the present invention provides a primer set comprising two or more primer sets selected from the primer sets of the present invention listed in Table 1. Furthermore, by using all the primers listed in Table 1, the above six important HLA genes (ie, HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 and HLA-DPB1) are used. Multiplex PCR) can be performed at a time, and high-efficiency, high-uniformity, and low-cost HLA gene typing can be realized.
- primer set 1 for HLA-DRB1 gene amplification and primer set 2 for HLA-DRB1 gene amplification have overlapping regions, it is preferable to perform multiplex PCR separately.
- HLA-DPB1 gene amplification primer set 1 and HLA-DPB1 gene amplification primer set 2 including the primer represented by SEQ ID NO: 15 or 16 are also preferably subjected to multiplex PCR separately. Accordingly, in a preferred embodiment, the present invention provides a primer set comprising all of the inventive primers listed in Table 1.
- PCR is performed by putting a plurality of primer sets for amplifying the target HLA gene in the same tube (container).
- the HLA-DRB1 gene amplification primer set 1 and the HLA-DRB1 gene amplification primer set 2 are preferably placed in separate containers because of overlapping regions.
- the HLA-DPB1 gene amplification primer set 1 and the HLA-DPB1 gene amplification primer set 2 are preferably placed in separate containers.
- the present invention provides an HLA-A gene amplification primer set, an HLA-C gene amplification primer set, an HLA-DRB1 gene amplification primer set 1, and an HLA-DPB1 gene amplification primer in one container.
- DNA used as a PCR template can be prepared from a test sample.
- a test sample for example, body fluid samples, such as blood and urine, cells, such as oral mucosa, body hairs, such as hair, etc. can be mentioned.
- DNA can be prepared using a known technique such as proteinase K / phenol extraction method, phenol / chloroform extraction method, alkali dissolution method, boiling method, etc., but using a commercially available DNA / RNA extraction kit, High-purity DNA can be prepared quickly and easily from a sample.
- PCR reaction conditions for HLA using the primer set of the present invention include the following conditions.
- Thermal denaturation step eg 92-98 ° C
- Annealing step eg 55-72 ° C
- Elongation step Example: 65-80 ° C
- the above PCR may be performed by using the annealing step and the extension step as one step (shuttle method), or by setting the annealing temperature higher and gradually lowering the temperature for each cycle (touch down method). May be. Or you may carry out combining them.
- the temperature of the annealing step and the extension step is typically 65 to 72 ° C.
- the base sequence of the obtained amplification product can be determined by a usual method, but it is preferable to determine the base sequence using a next-generation sequencer (NGS).
- NGS next-generation sequencer
- Illumina equipment eg MiSeq, HiSeq2500
- Life Technologies equipment eg Ion Proton, Ion PGM
- Roche Diagnostics examples of the device (eg, GS FLX +, GS Junior) manufactured by Roche Diagnostic may be mentioned, but the device is not limited thereto.
- the method of sequencing using NGS is, depends on the type of NGS, for example, company documentation (eg: Nextera R XT DNA Library Prep Reference Guide) can be carried out according to.
- paired end analysis is preferably used for sequencing of the obtained sample.
- the base sequence can be similarly determined by a method suitable for the apparatus.
- the amplification product of each sample is tagged with a kit (eg, Nextera XT DNA Sample Preparation Kit (Illumina)). 2. Using the kit (eg, Nextera XT v2 Index Kit Set A, B, C, D (Illumina)), a different index sequence is added to both sides of each sample, and PCR is performed. 3. Purify the amplification product. 4). The amplification product from each sample is used to confirm the library size. 5). Adjust the concentration between samples. 6). Sample amplification products are pooled, and quantitative PCR is performed on the pooled amplification products. 7). Sequence the pooled amplification products using MiSeq.
- the genotype of the HLA gene can be determined (typing) using a database.
- this database include an IPD-IMGT / HLA database in which more than 17,000 types of alleles are registered, and an HLA dictionary newly created by the present inventors.
- the HLA dictionary extracts all registered exon and intron base sequences of all HLA alleles from the IPD-IMGT / HLA database, groups them by base sequence pattern, and records these patterns and allele information to which they belong. It is a thing.
- OptiType As a typing method based on the nucleotide sequence information of the amplification product obtained by NGS, for example, mapping the reads to alleles of multiple HLA genes, applying the results to the linear programming problem, and finding the best allele pair Detection method (OptiType) (Szolek, A. et al., (2014) OptiType: Typeprecision HLA typing from next-generation sequencing data. Bioinformatics, 30, 3310-3316.) , Method of judging allele from the result (HLAreporter) (Huang, Y. et al., (2015) HLAreporter: a tool for HLA typing from next generation sequencing data.
- HLA-HD HLA-HD
- HLA typing may be performed without using NGS.
- methods include analysis methods using amplification products by long PCR, such as array-based PCR-SSOP, Sanger-based PCR-SBT, PCR-PHFA, and PCR-SSCP. Although not mentioned, it is not limited to these methods.
- the present invention also provides a kit that can be used in PCR using the primer set of the present invention.
- the kit of the present invention may contain other reagents necessary for PCR in addition to the primer set of the present invention.
- the reagent is a reagent that does not adversely affect the reaction when stored in the coexistence state with the primer set of the present invention, it can be mixed with the primer set and included in the kit.
- the reagent and the primer set of the present invention may be provided separately without mixing.
- the reagent include a DNA extraction reagent, a DNA polymerase enzyme, dNTP, a reaction buffer, a DNA molecule containing a target sequence serving as a PCR positive control, and instructions.
- the DNA polymerase enzyme may be a commercially available product.
- Examples of such a DNA polymerase enzyme include, for example, PrimeSTAR GXL DNA Polymerase, Tks Gflex DNA Polymerase, TaKaRa LA Taq, and Thermo SCIENTIFIC Long PCR Enzyme manufactured by TaKaRa. Examples include, but are not limited to, Mix.
- Example 1 Design of Multiplex PCR Primer Set for Classical HLA Genes
- HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 Describes the primer design procedure.
- bowtie2 (ver. 4.1.2) was used for all mapping of leads obtained from next-generation sequencers.
- mapping to the HLA dictionary The sequence results of MiSeq were mapped with bowtie2 to the exon / intron sequence information of all HLA alleles obtained from the IPD-IMGT / HLA database. Based on the map results, the weight of each gene in the lead was calculated. The weight calculation method will be briefly described below. Read the mapping result and check the correspondence of the lead that matches each exon. 50% or more of the leads overlap with the base sequence of any exon contained in the HLA dictionary, and both base sequences completely match within the overlapping range, or overlap with the base sequence of any intron contained in the HLA dictionary In addition, it is examined whether or not both base sequences match within 2 base mismatches in the overlapping range. The matched reads are weighted for each gene according to the number of alleles corresponding to the sequences matched by each exon and intron. The weight of the read for each gene is
- Steps 3 and 4 were performed for all complete alleles to obtain a set of classified leads.
- Arbitrary two complete alleles are A and B, C (A) and C (B) are A and B, respectively.
- a set of leads belonging to the consensus class of B D (A) and D (B) are A and A, respectively.
- C (A) ⁇ C (B), C (A) ⁇ D (B), D (A) ⁇ C (B), D (A) ⁇ D (B) The total number of leads after applying the weight of 2) was calculated.
- the number of reads was compared for all unions in all combinations of complete allele pairs, and the allele pair with the largest number of reads and the kind of union were selected.
- the pair exploration included combinations where A and B were the same complete allele.
- step 5 correcting different positions If the pair selected in step 5 is a different complete allele, the correspondence between the different positions of the two complete alleles is unknown, so use the mapped lead relationship. Different positions. First, at each different position of the complete allele, a set of leads mapped including this position was detected. This set of leads includes those belonging to both consensus and difference classes. Calculate the number of common leads for all combinations of sets of leads at different positions between two complete alleles. Corresponding to the same different positions. When there was no corresponding position on one complete allele side, the position was removed.
- step 7 Reclassification and determination of consensus sequence With respect to the allele pair determined in step 5 and the corresponding class and the difference position relationship between them, each lead is placed on which complete allele side by the method of step 3. Judged whether it belongs. A consensus sequence was created from each complete allele read set obtained from the judgment results.
- the sets of reads in the two consensus sequences finally obtained in the homo / hetero determination step 8 were compared. At this time, when the number of reads belonging only to the allele on one side was 10 times or more than the number of reads belonging only to the other side, alleles on the smaller side were deleted and output as homoalleles with the consensus sequence on the remaining side. Otherwise, the consensus sequence for both alleles was output.
- the output array is an assembly array.
- MUSCLE for a set of assembly sequence of each gene remaining in the execution step 1 of multiple alignment (ver 3.8.31) [Edgar, RC (2004) MUSCLE:.. Multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res, 32 : 1792-1797.] was used for multiple alignment.
- Primer candidate selection Primer3 [Schgasser, A. et al. (2012) Primer3--new capabilities and interfaces. Nucleic Acids Res., 40: e115.] was used to determine candidate primer pairs.
- Primers satisfy the following criteria. -The area masked in step 4 is not included. -A, B, C, DQB1 includes all translation regions in one pair. ⁇ Two pairs are prepared for DRB1 and DPB1, both pairs include exon 2 and include the translation region by two pairs before and after. • Each gene primer pair is specific to any other HLA gene. In addition, the unique position exists as much as possible inside (3 'side position for 5' primer, 5 'side for 3' primer).
- Example 2 Multiplex PCR and NGS Sequence I. Long PCR 1. Multiplex long PCR targeting 6 genes of HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 was performed on a total of 384 DNA samples. Amplification was performed for each of the following two sets. Set 1: HLA-A, -C, -DPB1 (FF, FR), -DRB1 (FF, FR) Set 2: HLA-B, -DQB1, -DPB1 (RF, RR),-DRB1 (RF, RR) Table 3 shows the primer sequences and final concentrations. The following were prepared for PCR.
- the amplification products were purified with Agencourt R AMPure R XP Beads. 4). Using 1 ⁇ L of the amplification product from each sample, the library size was confirmed by Agilent 2100 Bioanalyzer and High Sensitivity DNA chip (Agilent Technology). 5). Concentration adjustment between samples was performed using Library Normalization Beads in the kit. 6. Equal amounts of 384 amplification products were pooled. 7). Quantitative PCR was performed on the pooled amplification products using the KAPA Library Quantification Kit (for details, see the Technical Data Sheet on the KAPA Library Quantification Kit. Illumina R platforms). 8). 384 samples of HLA6 gene were sequenced using MiSeq (Illumina). 9. Confirmed output of data size (25M pair-end read) around 15GB.
- Fig. 7 shows the results of comparing the distribution of the average coverage of the six genes for each specimen using two primer sets. Since the number of samples performed is different, the average coverage is higher on the existing set side (because the output data size of MiSeq is the same). According to the results of the previously reported primer set, the average coverage of HLA-B and HLA-DRB1 was found to be lower than that of other genes. In particular, HLA-DRB1 has a median value of 0, which means that amplification products were not obtained for more than half of the samples. On the other hand, it was confirmed that the primer set of the present invention has low variation among genes, and the sequence of HLA-DRB1 can be read without any problem. Therefore, the use of the primer set of the present invention makes it possible to increase the number of samples for the next-generation sequencer, so that the primer set of the present invention has an excellent effect compared to the conventional primer set. Have
- the primer set of the present invention enables uniform PCR amplification, it is possible to increase the number of samples for one-time next-generation sequencer. Since the common sequence of large-scale Japanese specimens obtained at the Genomic Medicine Center is used, it is considered that the frequency of occurrence of unknown alleles that do not match the primers for the Japanese is extremely low. As a result, the re-execution rate is reduced, which is suitable for automation of HLA typing. It can be applied to organ transplantation and future safety tests using iPS cells, and can be applied to secure a large number of donors, and the number of primers can be reduced, making it easy to handle and commercialize.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
A.出来るだけ多種類のHLAアレルを包括できるようなプライマーの配列を選択する。
B.均一な増幅のため各増幅対象領域に対し、単一のプライマーを用意する。領域が重なる場合もオーバーラップ部は極力短くする。また目的外の遺伝子を増幅してはならない。
従って、上記2課題を解決する(即ち、上記A,Bの条件を充足する)プライマーセットの開発が望まれていた。Aの条件を上げるにはプライマーの数を増やすか、相同性の高い配列を選択する必要があるが、この場合にはBの条件達成が困難となる。本発明者らは、以下の1~4のステップを行うことで、条件Aと条件Bの2条件を充足するプライマーセットを開発できるのではないかとの着想を得た。
1.768人の日本人検体における6遺伝子の次世代シークエンスによる配列解析データを、他の遺伝子由来のリードを排除しつつ、アセンブリする。
2.アセンブリした結果を遺伝子毎の全検体においてアライメントする。
3.2の結果において全検体で共通した配列を持つ領域を検出し、プライマー候補とする。
4.3の候補のなかで配列が他のHLA遺伝子と共通せずかつプライマーに適した配列を選定する。
本発明者らは、上記着想に基づいてさらに研究を重ねた結果、本発明を完成するに至った。
[1]配列番号1に示される塩基配列からなるプライマー、及び配列番号2に示される塩基配列からなるプライマーを含む、HLA-A遺伝子増幅用プライマーセット。
[2]配列番号3に示される塩基配列からなるプライマー、及び配列番号4に示される塩基配列からなるプライマーを含む、HLA-B遺伝子増幅用プライマーセット。
[3]配列番号5に示される塩基配列からなるプライマー、及び配列番号6に示される塩基配列からなるプライマーを含む、HLA-C遺伝子増幅用プライマーセット。
[4]配列番号7に示される塩基配列からなるプライマー、配列番号8に示される塩基配列からなるプライマー、配列番号9に示される塩基配列からなるプライマー、及び配列番号10に示される塩基配列からなるプライマーを含む、HLA-DRB1遺伝子増幅用プライマーセット。
[5]配列番号11に示される塩基配列からなるプライマー、及び配列番号12に示される塩基配列からなるプライマーを含む、HLA-DQB1遺伝子増幅用プライマーセット。
[6]配列番号13に示される塩基配列からなるプライマー、配列番号14に示される塩基配列からなるプライマー、配列番号15に示される塩基配列からなるプライマー、及び配列番号16に示される塩基配列からなるプライマーを含む、HLA-DPB1遺伝子増幅用プライマーセット。
[7][1]に記載のプライマーセット、[2]に記載のプライマーセット、[3]に記載のプライマーセット、[4]に記載のプライマーセット、[5]に記載のプライマーセット及び[6]に記載のプライマーセットからなる群から2つ以上選択される、HLA遺伝子増幅用プライマーセット。
[8][1]~[6]に記載の全てのプライマーセットを含む、HLA遺伝子増幅用プライマーセット。
[9][1]~[8]のいずれかに記載のプライマーセットを用いて行う、HLA遺伝子のシークエンス方法。
[10][9]に記載の方法により得られた塩基配列情報を用いて行う、HLA遺伝子のタイピング方法。
[11][1]~[8]のいずれかに記載のプライマーセットを含む、HLA遺伝子の増幅用キット。
かかる変異体は、プライマーとしての機能が維持される範囲で、もととなるオリゴヌクレオチドから1個又は複数個のヌクレオチドが置換、欠失、挿入又は付加されている。
ハイブリダイゼーション時のストリンジェンシーは、温度、塩濃度、プライマーの鎖長、プライマーのヌクレオチド配列のGC含量及びハイブリダイゼーション緩衝液中のカオトロピック剤の濃度の関数であることが知られている。ストリンジェントな条件としては、例えば、Sambrook, J. et al. (1998) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New Yorkに記載された条件などを用いることができる。ストリンジェントな温度条件は、約30℃以上、より好ましくは約37℃以上、最も好ましくは約42℃以上である。その他の条件としては、ハイブリダイゼーション時間、洗浄剤(例えば、SDS)の濃度、及びキャリアDNAの存否等であり、これらの条件を組み合わせることによって、様々なストリンジェンシーを設定することができる。
熱変性ステップ(例:92~98℃)
アニーリングステップ(例:55~72℃)
伸長ステップ(例:65~80℃)
2.得られた断片配列に対し、キット(例:Nextera XT v2 Index Kit Set A、B、C、D(Illumina社))を用いて、サンプルそれぞれに異なるIndex配列を両側に付加し、PCRを行う。
3.増幅産物を精製する。
4.各サンプルからの増幅産物を用いて、ライブラリサイズを確認する。
5.サンプル間の濃度調整を行う。
6.サンプルの増幅産物をプールし、プールした増幅産物に対し、定量PCRを行う。
7.MiSeqを用いて、プールした増幅産物のシークエンスを行う。
6種のClassical HLA遺伝子(HLA-A, -B, -C, -DRB1, -DQB1, -DPB1)に対し、マルチプレックスPCRを行うためのプライマー設計手順について記述する。なお、次世代シークエンサーから得られたリードのマッピングには、全てbowtie2 (ver. 4.1.2)を用いた。
1.従来法によるHLA遺伝子のシークエンス
既存のプライマーセット[Hosomichi, K. et al. (2013) Phase-defined complete sequencing of the HLA genes by next-generation sequencing. BMC Genomics, 14:355.]を用いて各3遺伝子の2セットでのマルチプレックスPCRを768検体(未公開検体)に対して行った(表2参照)。ただしDRB1に関しては、配列を修正したプライマーを追加した。実験条件に関しては表3を参照のこと。得られた増幅産物をMiSeqによりシークエンスした。
IPD-IMGT/HLAデータベースから得られる全てのHLAアレルのエクソン・イントロンの配列情報に対し、MiSeqのシークエンス結果をbowtie2でマッピングした。マップ結果を基に、リードの各遺伝子における重みを計算した。重みの計算方法について以下簡潔に説明する。
マッピング結果を読み込み、各エクソンに一致したリードの対応関係を調べる。リードの50%以上がHLA辞書に含まれるいずれかのエクソンの塩基配列と重複しかつ該重複範囲において両塩基配列が完全に一致するもしくはHLA辞書に含まれるいずれかのイントロンの塩基配列と重複しかつ該重複範囲において両塩基配列が2塩基ミスマッチ以内で一致するかを調べる。マッチしたリードに対して各エクソン・イントロンでマッチした配列に対応するアレル数に応じて遺伝子毎に重み付けをする。各遺伝子に対する該リードの重みは以下の式1
遺伝子Gにおいて正(>0)の重みが得られたリード群を、IPD-IMGT/HLAにおいてイントロンを含めた完全長配列が登録されている遺伝子Gの全てのアレル(以下完全アレル)にマップした。各完全アレルに対し、5塩基のミスマッチ以内でマップされたリードを集め、マップされた各位置で各塩基のカバレッジを計算した。計算したカバレッジが各位置で最大となる塩基を選ぶことで得られる配列(以下「コンセンサス配列」と略記する。)を作成した。次に、ある位置でコンセンサス配列とは異なる塩基のカバレッジがコンセンサス配列に対応する塩基のカバレッジの10%以上ある場合、その位置を相異位置とし、その塩基配列を相異配列とした。ただし、10%以上のカバレッジをもつ塩基が複数存在する場合は、その中で最大のカバレッジを持つものを選択した。コンセンサスおよび相異をIDとする二つのクラスを用意し、相異位置を含む箇所にマップされたリードがコンセンサス配列・相異配列のどちらを多く含むか比較し、多い側のクラスにリードを所属させた。ただし同数若しくは相異位置を一つも含んでいないリードは両クラスに属するとした。
コンセンサスのクラスに属するリードの集合だけでカバレッジを再計算し、相異位置のコンセンサス配列のカバレッジが相異配列のカバレッジを下回る場合は相異位置でのコンセンサス配列・相異配列に対応する塩基を入れ替えた。この後、相異側のクラスに属するリードの集合だけでカバレッジを計算し、相異位置のコンセンサス配列のカバレッジが相異配列のカバレッジを上回る場合は、この相異位置を除去した。反転および除去された情報をもとに、再び全リードに対してステップ3のリードのクラス分けを行い、相異位置でのコンセンサス配列・相異配列の反転および相異位置の除去を行った。この作業を反転および相異位置の除去が発生しなくなるまで繰り返した。
全ての完全アレルにおいてステップ3、4を行い、クラス分けされたリードの集合を得た。任意の二つの完全アレルをそれぞれA、Bとし、C(A)、C(B)をそれぞれA、Bのコンセンサスのクラスに属するリードの集合、D(A)、D(B)をそれぞれA、Bの相異のクラスに属するリードの集合とした。C(A)∪C(B)、C(A)∪D(B)、D(A)∪C(B)、D(A)∪D(B)のそれぞれの和集合において、式2(数2)の重みをかけた上でのリード数合計をそれぞれ計算した。完全アレルペアの全ての組み合わせにおける全ての和集合でリード数を比較して、最もリード数が多くなるアレルペアとその和集合の種類を選択した。ペアの探査にはAとBが同じ完全アレルとする組み合わせを含めた。
ステップ5で選ばれたペアが異なる完全アレルであった場合、二つの完全アレルが持つ相異位置の互いの対応関係が不明なため、マップされたリードの関係を用いて相異位置を対応させた。まず完全アレルの各相異位置において、この位置を含んでマップされたリードの集合を検出した。このリードの集合には、コンセンサス・相異の両クラスに属するものを含む。二つの完全アレル間の相異位置のリードの集合同士における全ての組み合わせで共通リード数を計算し、共通リード数が多い集合の組みの順番に、集合に対応する相異位置同士を完全アレル間における同じ相異位置として対応させた。片方の完全アレル側に対応する相異位置が存在しなかった場合は、その相異位置を除去した。
ステップ5で決定したアレルペアと対応するクラスおよびそれらの間で対応させた相異位置関係に対して、各リードをステップ3の方法によってどちらの完全アレル側に属するか判定した。判定結果で得られた各完全アレルのリードの集合からコンセンサス配列をそれぞれ作成した。
1)ステップ7で得られた二つのコンセンサス配列に対し、全リードを再マップした。2)各リードを二つのマップ結果のミスマッチ数を比較して少ない方に所属させた。同ミスマッチ数の場合は両方に属するとした。それぞれの所属したリードの集合を用いて再びコンセンサス配列を作成した。このときコンセンサスのカバレッジが5リード数に達しない位置は“N”とした。3)得られた二つの配列に対し、再びステップ1)、2)を行った。これを両配列にどちらにおいても更新がなくなるまで繰り返した。
ステップ8で最終的に得られた二つのコンセンサス配列におけるリードの集合を比較した。この時、片側のアレルにのみ属するリードの数が相手側にのみ属するリードの数に対し10倍以上あった場合、少ない側のアレルを消去し、残った側のコンセンサス配列によるホモアレルとして出力した。そうでない場合は、両アレルのコンセンサス配列を出力した。出力した配列をアセンブリ配列とする。
事前に京都大学医学研究科付属ゲノム医学センター(以下「ゲノム医学センター」と略記する。)で保管する768検体のDNAサンプルそれぞれにおいて、Iの方法を用いて6種類のHLA遺伝子のアセンブリ配列を作成した。
得られたアセンブリ配列から塩基配列数が以下の長さに達しないものを除去した。この際、“N”の部分は配列数から引いた。
HLA-A: 2,800bp
HLA-B: 2,800bp
HLA-C: 2,800bp
HLA-DRB1: 10,000bp
HLA-DQB1: 6,500bp
HLA-DPB1: 9,500bp
ただし、“N”が配列全体の5%以上含まれる場合は長さの如何を問わずそのアセンブリ配列を除去した。
ステップ1で残った各遺伝子のアセンブリ配列の集合に対しMUSCLE (ver 3.8.31) [Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res., 32:1792-1797.]を用いてマルチプルアラインメントを行った。
アラインメント結果から得られたコンセンサス配列のある区間が1つのアセンブリ配列によって20塩基以上の挿入が生じていた場合(=カバレッジが1である区間が20塩基以上続く)、その区間を除去しアライメント位置を詰めた。
ステップ3で得られたコンセンサス配列のカバレッジがアセンブリ配列数の95%以下になっている位置をマスクした。またコンセンサス配列上でエクソンに該当する位置を、類似する配列を検索するソフトウェアであるBLASTにより既知のHLA遺伝子の配列データから検索し、一致したエクソン区間をマスクした。
Primer3 [Untergasser, A. et al. (2012) Primer3--new capabilities and interfaces. Nucleic Acids Res., 40:e115.] を用いてプライマーペアの候補を決定した。プライマーは以下の基準を満たすものとした。
・ステップ4でマスクした領域を含まない。
・A,B,C,DQB1は一つのペアで全ての翻訳領域を包括する。
・DRB1, DPB1に対しては2ペア用意し、両ペアともエクソン2を含みかつ前後2ペアによって翻訳領域を包括する。
・各遺伝子のプライマーペアは、他のどのHLA遺伝子に対しても特異的である。また特異な位置はできる限り内側 (5’側のプライマーなら3’側の位置、3’側のプライマーなら5’側)に存在している。
PRIMER_OPT_SIZE=30
PRIMER_MIN_SIZE=20
PRIMER_MAX_SIZE=35
PRIMER_MIN_TM=40
PRIMER_MAX_TM=90
PRIMER_MIN_GC=10
PRIMER_MAX_GC=90
PRIMER_MAX_NS_ACCEPTED=1
P3_FILE_FLAG=1
PRIMER_MAX_SELF_END=3
PRIMER_PAIR_MAX_COMPL_END=3
PRIMER_MAX_HAIRPIN_TH=60
I.ロングPCR
1.計384検体の各DNAサンプルに対し、HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 の6遺伝子を標的としたマルチプレックスロングPCRを行った。増幅は以下の2セットをそれぞれまとめて行った。
セット1: HLA-A,-C,-DPB1(FF,FR),-DRB1(FF,FR)
セット2: HLA-B,-DQB1,-DPB1(RF,RR),-DRB1(RF,RR)
プライマー配列と最終濃度は表3の通りである。またPCRに向けて以下のものを準備した。
・DNA 25 ng
・0.25 μL of Tks GflexTM DNA Polymerase
・6.25 μL 2 × GflexTM PCR Buffer
最終容量が12.5 μLになるように調製した後Cool StartTM法(TaKaRa社)を適用し、表5における温度条件、サイクル数でPCR反応を行った。PCR装置にはGeneAmpR PCR System 9700 (Thermo Fisher Scientific inc., Waltham, MA, USA) を用いた。
2.増幅産物をAgencourtR AMPureR XP Beads(Beckman Coulter, Brea, CA, USA)で精製した。
3.各サンプル1μLをアガロースゲルで泳動し、目的バンドの有無を確認した。想定されるバンドサイズは表3に記載した通りである。
4.二つのセットを等モル混合し、サンプル毎の増幅産物の最終濃度が0.2ng/μL(2.5μL)になるように調製した。
これより以下で特に記載のないプロトコルは、Illumina社のマニュアルに準拠した(Nextera XT DNA Library Prep Reference Guide (15031942 Rev. D))。
1.各サンプルの増幅産物の 0.5 ng を Nextera XT DNA Sample Preparation Kit (Illumina)適用してTagmentationを行った(300bp×2のペアエンドリード)。
2.得られた断片配列に対し、Nextera XT v2 Index Kit Set A、B、C、Dを用いて(各行、列で2.5 μL)384検体それぞれに異なるIndex配列を両側に付加し、PCRを行った。
3.AgencourtR AMPureR XP Beadsで増幅産物を精製した。
4.各サンプルから増幅産物を1 μL用いて、Agilent 2100 Bioanalyzer及びHigh Sensitivity DNA chip (Agilent Technology)によりライブラリサイズを確認した。
5.Kit中のLibrary Normalization Beadsによるサンプル間の濃度調整を行った。
6.384検体の増幅産物を等量プールした。
7.プールした増幅産物に対し、 KAPA Library Quantification Kit を用いて定量PCRを行った(詳細はKAPA Library Quantification Kit. IlluminaR platformsのTechnical Data Sheetを参照)。
8.MiSeq (Illumina) を用いて384検体、HLA6遺伝子のシークエンスを行った。
9.15GB前後のデータサイズ(25Mのペアエンドリード)の出力が得られているか確認した。
ゲノム医学センターで保管されている384検体を用いて、デザインしたHLA遺伝子用のプライマーセット及び上記の実験方法を用いて予備実験を行った。比較のため、既報のプライマーセット及び実験方法を用いた96検体によるシークエンスも同時に行った[Hosomichi, K. et al. (2013) Phase-defined complete sequencing of the HLA genes by next-generation sequencing. BMC Genomics, 14:355.]。なお、比較用の検体は、予備実験とは重複していない。これらの検体のMiSeqによるシークエンス結果に対し、IMGT/HLAデータベースの完全長配列が既知のHLAアレル全てにマップした。いずれかのアレルと一致したリードを集め各遺伝子での平均カバレッジを計算した。平均カバレッジの分母となる遺伝子長はHLAアレルによって異なるため、各遺伝子のアレルセットでの平均長を用いた。
Claims (11)
- 配列番号1に示される塩基配列からなるプライマー、及び配列番号2に示される塩基配列からなるプライマーを含む、HLA-A遺伝子増幅用プライマーセット。
- 配列番号3に示される塩基配列からなるプライマー、及び配列番号4に示される塩基配列からなるプライマーを含む、HLA-B遺伝子増幅用プライマーセット。
- 配列番号5に示される塩基配列からなるプライマー、及び配列番号6に示される塩基配列からなるプライマーを含む、HLA-C遺伝子増幅用プライマーセット。
- 配列番号7に示される塩基配列からなるプライマー、配列番号8に示される塩基配列からなるプライマー、配列番号9に示される塩基配列からなるプライマー、及び配列番号10に示される塩基配列からなるプライマーを含む、HLA-DRB1遺伝子増幅用プライマーセット。
- 配列番号11に示される塩基配列からなるプライマー、及び配列番号12に示される塩基配列からなるプライマーを含む、HLA-DQB1遺伝子増幅用プライマーセット。
- 配列番号13に示される塩基配列からなるプライマー、配列番号14に示される塩基配列からなるプライマー、配列番号15に示される塩基配列からなるプライマー、及び配列番号16に示される塩基配列からなるプライマーを含む、HLA-DPB1遺伝子増幅用プライマーセット。
- 請求項1に記載のプライマーセット、請求項2に記載のプライマーセット、請求項3に記載のプライマーセット、請求項4に記載のプライマーセット、請求項5に記載のプライマーセット及び請求項6に記載のプライマーセットからなる群から2つ以上選択される、HLA遺伝子増幅用プライマーセット。
- 請求項1~6に記載の全てのプライマーセットを含む、HLA遺伝子増幅用プライマーセット。
- 請求項1~8のいずれか1項に記載のプライマーセットを用いて行う、HLA遺伝子のシークエンス方法。
- 請求項9に記載の方法により得られた塩基配列情報を用いて行う、HLA遺伝子のタイピング方法。
- 請求項1~8のいずれか1項に記載のプライマーセットを含む、HLA遺伝子の増幅用キット。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020197025629A KR20190116989A (ko) | 2017-02-13 | 2018-02-09 | Hla 유전자를 위한 pcr 프라이머 세트, 및 이를 이용한 서열 분석 방법 |
CN201880011698.3A CN110494562A (zh) | 2017-02-13 | 2018-02-09 | 用于hla基因的pcr引物组和使用其的测序方法 |
US16/485,187 US20190367983A1 (en) | 2017-02-13 | 2018-02-09 | Pcr primer set for hla gene, and sequencing method using same |
EP18750659.7A EP3581652A4 (en) | 2017-02-13 | 2018-02-09 | HLA GENE PCR PRIMER SET, AND SEQUENCING PROCESS USING THIS PCR PRIMER SET |
CA3053302A CA3053302A1 (en) | 2017-02-13 | 2018-02-09 | Pcr primer set for hla gene, and sequencing method using same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2017-024397 | 2017-02-13 | ||
JP2017024397A JP6798697B2 (ja) | 2017-02-13 | 2017-02-13 | Hla遺伝子のpcrプライマーセット及びそれを用いたシークエンス法 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018147438A1 true WO2018147438A1 (ja) | 2018-08-16 |
Family
ID=63107030
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2018/004703 WO2018147438A1 (ja) | 2017-02-13 | 2018-02-09 | Hla遺伝子のpcrプライマーセット及びそれを用いたシークエンス法 |
Country Status (7)
Country | Link |
---|---|
US (1) | US20190367983A1 (ja) |
EP (1) | EP3581652A4 (ja) |
JP (1) | JP6798697B2 (ja) |
KR (1) | KR20190116989A (ja) |
CN (1) | CN110494562A (ja) |
CA (1) | CA3053302A1 (ja) |
WO (1) | WO2018147438A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3626835A1 (en) * | 2018-09-18 | 2020-03-25 | Sistemas Genómicos, S.L. | Method for genotypically identifying both alleles of at least one locus of a subject's hla gene |
CN111057706A (zh) * | 2019-10-09 | 2020-04-24 | 西安市中心血站(陕西省血液中心) | 一组引物以及hla-dpb1基因测序分型的检测方法 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102475292B1 (ko) * | 2020-09-03 | 2022-12-08 | 주식회사 엔젠바이오 | Hla 유전자 증폭용 조성물 및 이의 용도 |
CN114032295A (zh) * | 2021-12-20 | 2022-02-11 | 德必碁生物科技(厦门)有限公司 | 一种人类白细胞抗原abcdrb1dqb1基因分型检测方法及试剂盒 |
CN114854737B (zh) * | 2022-03-11 | 2023-07-14 | 郑州大学 | 基于三代测序平台的i类hla基因扩增引物、试剂盒及分型方法 |
CN117512085B (zh) * | 2023-11-21 | 2024-06-04 | 江苏伟禾生物科技有限公司 | 一种用于检测hla-dpb1基因分型的引物组及试剂盒 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011500041A (ja) * | 2007-10-16 | 2011-01-06 | エフ.ホフマン−ラ ロシュ アーゲー | クローナルシークエンシングによる高分解能かつ高効率のhla遺伝子型決定法 |
US20120122719A1 (en) * | 2009-11-16 | 2012-05-17 | Hogan Michael E | Methods for PCR and HLA typing using unpurified samples |
WO2014065410A1 (ja) * | 2012-10-26 | 2014-05-01 | ジェノダイブファーマ株式会社 | Hla遺伝子のdnaタイピング方法及びキット |
US20150005179A1 (en) * | 2013-06-27 | 2015-01-01 | Instant Chemistry Inc. | Hla primers for personal compatibility |
JP2017024397A (ja) | 2016-05-10 | 2017-02-02 | 株式会社Dnpファインケミカル | インクジェット記録用受理溶液、この受理溶液を含むインクジェット記録用インクセット及びこのインクセットを用いた印刷物の製造方法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100086914A1 (en) * | 2008-10-03 | 2010-04-08 | Roche Molecular Systems, Inc. | High resolution, high throughput hla genotyping by clonal sequencing |
US20140141436A1 (en) * | 2008-10-03 | 2014-05-22 | Roche Molecular Systems, Inc. | Methods and Compositions for Very High Resolution Genotyping of HLA |
JPWO2013011734A1 (ja) * | 2011-07-21 | 2015-02-23 | ジェノダイブファーマ株式会社 | Hla遺伝子のdnaタイピング方法及びキット |
JP6643902B2 (ja) * | 2013-11-27 | 2020-02-12 | ジェノダイブファーマ株式会社 | 超並列高速シークエンサーによる簡便なhla遺伝子のdnaタイピング方法およびキット |
-
2017
- 2017-02-13 JP JP2017024397A patent/JP6798697B2/ja active Active
-
2018
- 2018-02-09 CN CN201880011698.3A patent/CN110494562A/zh active Pending
- 2018-02-09 WO PCT/JP2018/004703 patent/WO2018147438A1/ja active Application Filing
- 2018-02-09 CA CA3053302A patent/CA3053302A1/en active Pending
- 2018-02-09 US US16/485,187 patent/US20190367983A1/en not_active Abandoned
- 2018-02-09 EP EP18750659.7A patent/EP3581652A4/en not_active Withdrawn
- 2018-02-09 KR KR1020197025629A patent/KR20190116989A/ko not_active Application Discontinuation
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011500041A (ja) * | 2007-10-16 | 2011-01-06 | エフ.ホフマン−ラ ロシュ アーゲー | クローナルシークエンシングによる高分解能かつ高効率のhla遺伝子型決定法 |
US20120122719A1 (en) * | 2009-11-16 | 2012-05-17 | Hogan Michael E | Methods for PCR and HLA typing using unpurified samples |
WO2014065410A1 (ja) * | 2012-10-26 | 2014-05-01 | ジェノダイブファーマ株式会社 | Hla遺伝子のdnaタイピング方法及びキット |
JP2016010318A (ja) | 2012-10-26 | 2016-01-21 | ジェノダイブファーマ株式会社 | Hla遺伝子のdnaタイピング方法及びキット |
US20150005179A1 (en) * | 2013-06-27 | 2015-01-01 | Instant Chemistry Inc. | Hla primers for personal compatibility |
JP2017024397A (ja) | 2016-05-10 | 2017-02-02 | 株式会社Dnpファインケミカル | インクジェット記録用受理溶液、この受理溶液を含むインクジェット記録用インクセット及びこのインクセットを用いた印刷物の製造方法 |
Non-Patent Citations (11)
Title |
---|
EDGAR, R.C.: "MUSCLE: multiple sequence alignment with high accuracy and high throughput", NUCLEIC ACIDS RES., vol. 32, 2004, pages 1792 - 1797, XP008137003, doi:10.1093/nar/gkh340 |
EHRENBERG, P.K. ET AL., BMC GENOMICS, vol. 15, 2014, pages 864 |
HOSOMICHI, K. ET AL.: "Phase-defined complete sequencing of the HLA genes by next-generation sequencing", BMC GENOMICS, vol. 14, 2013, pages 355, XP055536363, doi:10.1186/1471-2164-14-355 |
HOSOMICHI, K. ET AL.: "Phase-defined complete sequencing of the HLA genes by next-generation sequencing", BMC GENOMICS, vol. 14, no. 1, 2013, pages 355, XP055536363 * |
HUANG, Y. ET AL.: "HLA reporter: a tool for HLA typing from next generation sequencing data", GENOME MED, vol. 7, 2015, pages 25, XP021215655, doi:10.1186/s13073-015-0145-3 |
KAWAGUCHI ET AL.: "HLA-HD: An accurate HLA typing algorithm for next-generation sequencing data", HUMAN MUTATION, vol. 38, 2017, pages 788 - 797, XP055597991, doi:10.1002/humu.23230 |
SAMBROOK, J. ET AL.: "Molecular Cloning: A Laboratory Manual", 1998, COLD SPRING HARBOR LABORATORY PRESS |
See also references of EP3581652A4 |
SZOLEK, A. ET AL.: "OptiType: precision HLA typing from next-generation sequencing data", BIOINFORMATICS, vol. 30, 2014, pages 3310 - 3316, XP055346451, doi:10.1093/bioinformatics/btu548 |
UNTERGASSER, A. ET AL.: "Primer3--new capabilities and interfaces", NUCLEIC ACIDS RES., vol. 40, 2012, pages ell5 |
WITTIG, M. ET AL., NUCLEIC ACIDS RES., vol. 43, no. 11, 2015, pages e70 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3626835A1 (en) * | 2018-09-18 | 2020-03-25 | Sistemas Genómicos, S.L. | Method for genotypically identifying both alleles of at least one locus of a subject's hla gene |
CN111057706A (zh) * | 2019-10-09 | 2020-04-24 | 西安市中心血站(陕西省血液中心) | 一组引物以及hla-dpb1基因测序分型的检测方法 |
Also Published As
Publication number | Publication date |
---|---|
JP6798697B2 (ja) | 2020-12-09 |
CN110494562A (zh) | 2019-11-22 |
CA3053302A1 (en) | 2018-08-16 |
EP3581652A1 (en) | 2019-12-18 |
US20190367983A1 (en) | 2019-12-05 |
EP3581652A4 (en) | 2021-03-10 |
KR20190116989A (ko) | 2019-10-15 |
JP2018130036A (ja) | 2018-08-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2018147438A1 (ja) | Hla遺伝子のpcrプライマーセット及びそれを用いたシークエンス法 | |
US9920370B2 (en) | Haplotying of HLA loci with ultra-deep shotgun sequencing | |
DK2366031T3 (en) | METHODS FOR SEQUENCING In PRENATAL DIAGNOSTICS | |
EP3006571B1 (en) | Hla gene multiplex dna typing method and kit | |
Liu et al. | Extended blood group molecular typing and next-generation sequencing | |
CZ293278B6 (cs) | Způsob přípravy komplexních DNA methylačních peptidových map | |
CN110863056A (zh) | 一种人类dna精准分型的方法、试剂和应用 | |
US20220228212A1 (en) | Major histocompatibility complex single nucleotide polymorphisms | |
CN112592981B (zh) | 用于dna档案建库的引物组、试剂盒和方法 | |
BR112012014466B1 (pt) | método para detectar uma mutação usando um microarranjo de dna apresentando uma pluralidade de sondas de polinucleotídeo imobilizadas no mesmo | |
CN108441547B (zh) | 一种hla基因扩增、基因分型的引物组、试剂盒和方法 | |
CN113840923A (zh) | 用于核酸检测的方法、***和设备 | |
CN112266963B (zh) | 一种联合检测慢性粒细胞白血病检测试剂盒 | |
KR101724130B1 (ko) | 장 베체트병 진단용 바이오마커 및 이의 용도 | |
JP2001137000A (ja) | 遺伝子変異の判別方法 | |
Wu et al. | Next-generation sequencing for clinical HLA typing | |
CN116179671A (zh) | 一种用于hla基因分型的扩增引物组、试剂盒及方法 | |
CN116377084A (zh) | 一种高效能常染色体微单倍型遗传标记体系及其检测引物和试剂盒 | |
Kaub et al. | Genetic and Epigenetic Basis of Development and Disease | |
Lareu et al. | B Mitochondrial DNA in forensic genetics | |
KR20190054243A (ko) | 노란목도리담비의 개체식별을 위한 마이크로새틀라이트 마커 및 이를 이용한 개체 식별 방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18750659 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3053302 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20197025629 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2018750659 Country of ref document: EP |