JP2019523638A - 遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセット、及びその調製方法と応用 - Google Patents
遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセット、及びその調製方法と応用 Download PDFInfo
- Publication number
- JP2019523638A JP2019523638A JP2018558751A JP2018558751A JP2019523638A JP 2019523638 A JP2019523638 A JP 2019523638A JP 2018558751 A JP2018558751 A JP 2018558751A JP 2018558751 A JP2018558751 A JP 2018558751A JP 2019523638 A JP2019523638 A JP 2019523638A
- Authority
- JP
- Japan
- Prior art keywords
- adapter
- sequence
- seq
- double tag
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 45
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 20
- 238000012163 sequencing technique Methods 0.000 claims abstract description 66
- 230000035772 mutation Effects 0.000 claims abstract description 50
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 229960002685 biotin Drugs 0.000 claims abstract description 13
- 235000020958 biotin Nutrition 0.000 claims abstract description 13
- 239000011616 biotin Substances 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims description 54
- 102000004190 Enzymes Human genes 0.000 claims description 45
- 108090000790 Enzymes Proteins 0.000 claims description 45
- 238000003776 cleavage reaction Methods 0.000 claims description 35
- 230000007017 scission Effects 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 33
- 238000000137 annealing Methods 0.000 claims description 29
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- 239000012634 fragment Substances 0.000 claims description 14
- 238000001556 precipitation Methods 0.000 claims description 13
- 238000000746 purification Methods 0.000 claims description 13
- 239000011324 bead Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000013507 mapping Methods 0.000 claims description 8
- 238000012937 correction Methods 0.000 claims description 6
- 230000001681 protective effect Effects 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 230000002255 enzymatic effect Effects 0.000 claims description 4
- 230000001376 precipitating effect Effects 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 150000007523 nucleic acids Chemical group 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 24
- 239000000047 product Substances 0.000 description 19
- 239000000872 buffer Substances 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 13
- 239000000203 mixture Substances 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 11
- 238000003908 quality control method Methods 0.000 description 7
- 230000002441 reversible effect Effects 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000007847 digital PCR Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000011109 contamination Methods 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 230000007515 enzymatic degradation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 239000002096 quantum dot Substances 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009615 deamination Effects 0.000 description 2
- 238000006481 deamination reaction Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000036438 mutation frequency Effects 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101150039808 Egfr gene Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 238000011138 biotechnological process Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 108700021358 erbB-1 Genes Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Bioinformatics & Computational Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
5'-ACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3' SEQ ID NO:24
3'-CTGACCTCAAGTCTGCACACGAGAAGGCTAG-p-5' SEQ ID NO:25
汎用プライマー:
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT
CCGATC-s-T-3'(-s-はチオ(thio)を表す。以下同様) SEQ ID NO:26
Indexプライマー:SEQ ID NO:27がI7index配列を介してSEQ ID NO:28に連結されて得られる配列。
5'-CAAGCAGAAGACGGCATACGAGAT SEQ ID NO:27
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3' SEQ ID NO:28
このうち、I7Index配列はSEQ ID NO:12〜23から選択した。
PCR-P5: AATGATACGGCGACCACCG-s-A SEQ ID NO:29
PCR-P7: CAAGCAGAAGACGGCATACG-s-A SEQ ID NO:30
Claims (7)
- ダブルタグアダプターA、ダブルタグアダプターB及びダブルタグアダプターCを含み、ダブルタグアダプターA、ダブルタグアダプターB及びダブルタグアダプターCは、アダプタープライマーP5と、5’末端がビオチン修飾されたアダプタープライマーP7−A、アダプタープライマーP7−B及びアダプタープライマーP7−Cとが各々合成されることでそれぞれ得られ、
アダプタープライマーP5は、I5index配列を介してSEQ ID NO:01をSEQ ID NO:02に示す配列に連結して取得され、
アダプタープライマーP7−Aは、FFFFFEEEEEJJJJJNNNNNNNNNNNNを順にSEQ ID NO:03の5’末端に連結し、SEQ ID NO:03を更にI7index配列を介してSEQ ID NO:04に示す配列に連結して取得され、
アダプタープライマーP7−Bは、FFFFFEEEEEKKKKKNNNNNNNNNNNNを順にSEQ ID NO:03の5’末端に連結し、SEQ ID NO:03を更にI7index配列を介してSEQ ID NO:04に示す配列に連結して取得され、
アダプタープライマーP7−Cは、FFFFFEEEEELLLLLNNNNNNNNNNNNを順にSEQ ID NO:03の5’末端に連結し、SEQ ID NO:03を更にI7index配列を介してSEQ ID NO:04に示す配列に連結して取得され、
前記FFFFFは酵素切断点の保護塩基、EEEEEは酵素切断点、JJJJJ、KKKKK及びLLLLLはポジショニングタグ配列であり、且つ、JJJJJ、KKKKK及びLLLLLは互いに異なっており、NNNNNNNNNNNNはランダム分子タグ配列であり、FFFFF、JJJJJ、KKKKK、LLLLL及びEEEEEは5つの同一塩基を含むがこれに限らず、I7index配列は6〜8個の塩基であり、NNNNNNNNNNNNは4〜12のランダム塩基であり、且つ、4つの連続した同一塩基は存在しないことを特徴とする遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセット。 - 前記NNNNNNNNNNNNはBDHVBDHVで示され、Bは当該位置がA以外の塩基であることを、D位置は当該位置がC以外の塩基であることを、Hは当該位置がG以外の塩基であることを、Vは当該位置がT以外の塩基であることを示していることを特徴とする請求項1に記載の遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセット。
- 前記I5index配列はSEQ ID NO:05〜12から選択され、前記I7Index配列はSEQ ID NO:13〜23から選択され、前記JJJJJ、KKKKK及びLLLLLの配列は前記EEEEEの配列と部分的又は完全に重複してもよく、部分的又は完全に重複する場合、重複部分の塩基は1回のみ出現することを特徴とする請求項1に記載の遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセット。
- 請求項1〜3のいずれかに記載の遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセットの調製方法であって、
(1)アダプタープライマーP5、アダプタープライマーP7−A、アダプタープライマーP7−B、アダプタープライマーP7−C及び緩衝液を適量の脱イオン水と混合した後に、アニーリング処理を行ってアニーリングアダプターA、アニーリングアダプターB及びアニーリングアダプターCを取得するアニーリングステップと、
(2)取得したアニーリングアダプターA、アニーリングアダプターB及びアニーリングアダプターCをポリメラーゼで延伸させ、延伸アダプターA、延伸アダプターB及び延伸アダプターCを取得するアニーリングアダプター延伸ステップと、
(3)取得した延伸アダプターA、延伸アダプターB及び延伸アダプターCをそれぞれエタノール又はイソプロパノールで沈殿させて精製することで、精製後の延伸アダプターA、延伸アダプターB及び延伸アダプターCを取得する第1沈殿ステップと、
(4)精製後の延伸アダプターA、延伸アダプターB及び延伸アダプターCに、3’T突出末端を発生可能な制限酵素をそれぞれ導入し、酵素切断することにより酵素切断アダプターA、酵素切断アダプターB及び酵素切断アダプターCを取得する酵素切断ステップと、
(5)取得した酵素切断アダプターA、酵素切断アダプターB及び酵素切断アダプターCをエタノール又はイソプロパノールで沈殿させ、精製することで、ダブルタグアダプターA、ダブルタグアダプターB及びダブルタグアダプターCを取得する第2沈殿ステップと、
(6)ステップ(5)で取得したダブルタグアダプターA、ダブルタグアダプターB及びダブルタグアダプターCをビオチンと親和させて精製するビオチン精製ステップと、
(7)ステップ(6)で取得した生成物をエタノール又はイソプロパノールで沈殿させ、精製することで、前記マルチポジショニングダブルタグアダプターセットを取得する第3沈殿ステップと、を含むことを特徴とする方法。 - 10ng〜1μgの検出対象のDNAを200〜500bpのDNA断片に切断した後、DNA断片を末端修復酵素に加えて末端修復してからA尾部を付加し、請求項1〜3のいずれかに記載のマルチポジショニングダブルタグアダプターセットを付加してライゲーションし、ライゲーション完了後にAmpure磁性ビーズを用いるか、或いは切断により340〜660bpの断片を選択することを特徴とするライブラリー調製方法。
- (1)請求項5に記載のライブラリー調製方法でライブラリーを調製するステップと、
(2)前記シーケンスライブラリーをシーケンシングするステップ、を含むことを特徴とするシーケンシング方法。 - (1)請求項5に記載のライブラリー調製方法でライブラリーを調製するステップと、
(2)前記シーケンスライブラリーをシーケンシングするステップと、
(3)シーケンシング結果に基づいて結果を判定するステップと、を含み、
前記結果を判定する方法は、
a.設定パラメータに基づいて、アライメントされた塩基のQ値が30より大きいシーケンシングにおける唯一のマッピング配列を選択するステップと、
b.ランダムタグ配列に基づいて反復判定を実施することで塩基の再補正を実施するステップと、
c.SNP callingソフトを用いてSNP座位検出を実施し、SNP座位情報を統計して、最終的にSNP座位及び対応するMAF情報を取得するステップと、
d.検出したSNP座位及びMAF情報と、対照群の突然変異位置及び集団ゲノムの変異情報データベースとを比較して同一の突然変異位置をフィルタリングし、最後に残った突然変異位置情報を最終的に検出した突然変異位置情報とするステップ、を含むことを特徴とする核酸配列を特定する方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610754636.4A CN106367485B (zh) | 2016-08-29 | 2016-08-29 | 一种用于检测基因突变的多定位双标签接头组及其制备方法和应用 |
CN201610754636.4 | 2016-08-29 | ||
PCT/CN2017/099255 WO2018041062A1 (zh) | 2016-08-29 | 2017-08-28 | 一种用于检测基因突变的多定位双标签接头组及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2019523638A true JP2019523638A (ja) | 2019-08-29 |
JP6830496B2 JP6830496B2 (ja) | 2021-02-17 |
Family
ID=57900571
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2018558751A Active JP6830496B2 (ja) | 2016-08-29 | 2017-08-28 | 遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセット、及びその調製方法と応用 |
Country Status (5)
Country | Link |
---|---|
US (1) | US11286524B2 (ja) |
EP (1) | EP3505640A4 (ja) |
JP (1) | JP6830496B2 (ja) |
CN (1) | CN106367485B (ja) |
WO (1) | WO2018041062A1 (ja) |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106367485B (zh) | 2016-08-29 | 2019-04-26 | 厦门艾德生物医药科技股份有限公司 | 一种用于检测基因突变的多定位双标签接头组及其制备方法和应用 |
CN106906211B (zh) * | 2017-04-13 | 2020-11-20 | 苏州普瑞迈德医学检验所有限公司 | 一种分子接头及其应用 |
CN107190067B (zh) * | 2017-06-13 | 2019-12-13 | 厦门艾德生物医药科技股份有限公司 | 一种改进的二代测序用随机标签接头制作方法 |
CN107385030A (zh) * | 2017-07-14 | 2017-11-24 | 广州精科医学检验所有限公司 | 分子标签、接头及确定含有低频突变核酸序列的方法 |
US11739367B2 (en) | 2017-11-08 | 2023-08-29 | Twinstrand Biosciences, Inc. | Reagents and adapters for nucleic acid sequencing and methods for making such reagents and adapters |
CN107944223B (zh) * | 2017-11-10 | 2019-12-31 | 深圳裕策生物科技有限公司 | 基于二代测序的点突变检测过滤方法、装置和存储介质 |
CN108893466B (zh) * | 2018-06-04 | 2021-04-13 | 上海奥根诊断技术有限公司 | 测序接头、测序接头组和超低频突变的检测方法 |
CN110669823B (zh) * | 2018-07-03 | 2022-05-24 | 中国医学科学院肿瘤医院 | 一种同时检测多种肝癌常见突变的ctDNA文库构建和测序数据分析方法 |
BR112021000409A2 (pt) | 2018-07-12 | 2021-04-06 | Twinstrand Biosciences, Inc. | Métodos e reagentes para caracterizar edição genômica, expansão clonal e aplicações associadas |
CN113166796A (zh) * | 2018-10-04 | 2021-07-23 | 蓝星基因组股份有限公司 | 来自单个生物样品的蛋白质、核糖体和无细胞核酸的同时基于测序的分析 |
CN109735900A (zh) * | 2019-03-20 | 2019-05-10 | 嘉兴菲沙基因信息有限公司 | 一种适用于Hi-C的小片段DNA文库构建方法 |
CN111748613A (zh) * | 2019-03-27 | 2020-10-09 | 华大数极生物科技(深圳)有限公司 | 一种双标签接头设计方法及制备方法 |
CN110129415B (zh) * | 2019-05-17 | 2023-08-18 | 迈杰转化医学研究(苏州)有限公司 | 一种ngs建库分子接头及其制备方法和用途 |
CN110257480A (zh) * | 2019-07-04 | 2019-09-20 | 北京京诺玛特科技有限公司 | 核酸序列测序接头及其构建测序文库的方法 |
CN110409001B (zh) * | 2019-07-25 | 2022-11-15 | 北京贝瑞和康生物技术有限公司 | 一种构建捕获文库的方法和试剂盒 |
CN110331187A (zh) * | 2019-08-12 | 2019-10-15 | 天津华大医学检验所有限公司 | 组合标签、组合标签接头及其应用 |
CN113257351A (zh) * | 2020-02-12 | 2021-08-13 | 赛纳生物科技(北京)有限公司 | 一种用于多碱基基因测序的基因文库及其构建方法 |
CN111471746A (zh) * | 2020-04-14 | 2020-07-31 | 深圳市新合生物医疗科技有限公司 | 检测低突变丰度样本的ngs文库制备接头及其制备方法 |
CN112226514B (zh) * | 2020-11-23 | 2021-08-03 | 苏州京脉生物科技有限公司 | 用于早期胃癌检测的标志物组合、试剂盒及其应用 |
CN113005188A (zh) * | 2020-12-29 | 2021-06-22 | 阅尔基因技术(苏州)有限公司 | 用一代测序评估样本dna中碱基损伤、错配和变异的方法 |
CN113981043B (zh) * | 2021-11-22 | 2024-04-16 | 广州迈景基因医学科技有限公司 | 一种制备二代测序接头的方法 |
Family Cites Families (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10027218A1 (de) * | 2000-05-31 | 2001-12-06 | Hubert Bernauer | Artifizielle genetische Markierung mit synthetischer DNA |
GB0522310D0 (en) * | 2005-11-01 | 2005-12-07 | Solexa Ltd | Methods of preparing libraries of template polynucleotides |
CN101967476B (zh) * | 2010-09-21 | 2012-11-14 | 深圳华大基因科技有限公司 | 一种基于接头连接的DNA PCR-Free标签文库构建方法 |
CN102978205B (zh) * | 2012-11-19 | 2014-08-20 | 北京诺禾致源生物信息科技有限公司 | 一种应用于标记开发的高通量测序的接头及其运用方法 |
CN103667273B (zh) * | 2013-12-05 | 2016-01-20 | 北京诺禾致源生物信息科技有限公司 | 双链接头、其应用及构建末端配对dna文库的方法 |
CA2940764C (en) * | 2014-02-26 | 2019-10-29 | Nelson Alexander | Photo-selective method for biological sample analysis field |
CN103882530B (zh) * | 2014-03-26 | 2016-02-24 | 清华大学 | 用随机序列标记质粒对dna片段进行高通量两端测序的方法 |
CN104263726A (zh) | 2014-09-25 | 2015-01-07 | 天津诺禾致源生物信息科技有限公司 | 适用于扩增子测序文库构建的引物及扩增子测序文库的构建方法 |
CN104313699A (zh) | 2014-10-31 | 2015-01-28 | 天津诺禾致源生物信息科技有限公司 | 测序文库的构建方法及用于测序文库构建的试剂盒 |
CN104561294B (zh) * | 2014-12-26 | 2018-03-30 | 北京诺禾致源科技股份有限公司 | 基因分型测序文库的构建方法和测序方法 |
KR101651817B1 (ko) | 2015-10-28 | 2016-08-29 | 대한민국 | Ngs 라이브러리 제작용 프라이머 세트 및 이를 이용한 ngs 라이브러리 제작방법 및 키트 |
CN106086162B (zh) | 2015-11-09 | 2020-02-21 | 厦门艾德生物医药科技股份有限公司 | 一种用于检测肿瘤突变的双标签接头序列及检测方法 |
CN105586427B (zh) | 2016-03-10 | 2020-06-19 | 厦门艾德生物医药科技股份有限公司 | 检测人类brca1和brca2基因突变的引物、试剂盒及方法 |
CN106282177A (zh) | 2016-08-23 | 2017-01-04 | 厦门基源医疗科技有限公司 | 一种16S rDNA高通量测序文库的构建方法 |
CN106367485B (zh) | 2016-08-29 | 2019-04-26 | 厦门艾德生物医药科技股份有限公司 | 一种用于检测基因突变的多定位双标签接头组及其制备方法和应用 |
CN106893774A (zh) | 2017-01-22 | 2017-06-27 | 苏州首度基因科技有限责任公司 | 用多分子标签检测dna变异水平的方法 |
-
2016
- 2016-08-29 CN CN201610754636.4A patent/CN106367485B/zh active Active
-
2017
- 2017-08-28 JP JP2018558751A patent/JP6830496B2/ja active Active
- 2017-08-28 EP EP17845364.3A patent/EP3505640A4/en not_active Ceased
- 2017-08-28 US US16/322,340 patent/US11286524B2/en active Active
- 2017-08-28 WO PCT/CN2017/099255 patent/WO2018041062A1/zh unknown
Also Published As
Publication number | Publication date |
---|---|
CN106367485A (zh) | 2017-02-01 |
CN106367485B (zh) | 2019-04-26 |
WO2018041062A1 (zh) | 2018-03-08 |
US11286524B2 (en) | 2022-03-29 |
US20200010892A1 (en) | 2020-01-09 |
EP3505640A1 (en) | 2019-07-03 |
JP6830496B2 (ja) | 2021-02-17 |
EP3505640A4 (en) | 2020-04-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6830496B2 (ja) | 遺伝子突然変異を検出するマルチポジショニングダブルタグアダプターセット、及びその調製方法と応用 | |
CN106086162B (zh) | 一种用于检测肿瘤突变的双标签接头序列及检测方法 | |
US11555220B2 (en) | Methods of lowering the error rate of massively parallel DNA sequencing using duplex consensus sequencing | |
KR102028375B1 (ko) | 희귀 돌연변이 및 카피수 변이를 검출하기 위한 시스템 및 방법 | |
DK2631336T3 (en) | DNA library and the method for producing the same as well as method and apparatus for detecting the SNP | |
CN110033829B (zh) | 基于差异snp标记物的同源基因的融合检测方法 | |
JP6664575B2 (ja) | 核酸分子数計測法 | |
US20170321270A1 (en) | Noninvasive prenatal diagnostic methods | |
CN113373524A (zh) | 一种ctDNA测序标签接头、文库、检测方法和试剂盒 | |
CN113564266B (zh) | Snp分型遗传标记组合、检测试剂盒及用途 | |
CN108359723B (zh) | 一种降低深度测序错误的方法 | |
CN111575349A (zh) | 一种接头序列及其应用 | |
EP3474168B1 (en) | Method for measuring mutation rate | |
US20230416812A1 (en) | Method capable of making one cluster by connecting information of strands generated during pcr process and tracking generation order of generated strands | |
US20220364080A1 (en) | Methods for dna library generation to facilitate the detection and reporting of low frequency variants | |
CN114774517A (zh) | 一种人免疫组库测序的方法及试剂盒 | |
CN108304693B (zh) | 利用高通量测序数据分析基因融合的方法 | |
US20200216888A1 (en) | Method for increasing accuracy of analysis by removing primer sequence in amplicon-based next-generation sequencing | |
WO2018219581A1 (en) | Method and system for nucleic acid sequencing | |
KR101967879B1 (ko) | 핵산 서열분석에서 uid 핵산 서열의 순결도를 측정하는 방법 | |
CN118406760A (zh) | 一种针对人软组织肿瘤融合基因进行检测的探针、试剂盒及方法 | |
CN118460707A (zh) | 检测寻常鱼鳞病相关flg基因突变的引物组、试剂盒及方法 | |
CN108647494A (zh) | 一种评估组装基因序列的准确性的方法 | |
Di Pierro | Exome sequencing in Mendelian diseases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20181213 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190412 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200218 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20200515 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20200717 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200817 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20210112 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210126 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6830496 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |