JP2023542581A - Primer compositions, kits, methods and uses thereof for detecting Y-SNP haplogroups by next generation sequencing technology - Google Patents
Primer compositions, kits, methods and uses thereof for detecting Y-SNP haplogroups by next generation sequencing technology Download PDFInfo
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Abstract
本発明は、バイオ技術及び遺伝学技術分野に関し、次世代シーケンシング技術によりＹ－ＳＮＰハプログループを検出するプライマー組成物、キット、方法及び使用を提供する。本発明では、アジア人集団のハプログループ頻度に応じて、最新版の２０１９年ＩＳＯＧＧウェブサイト（Ｖｅｒｓｉｏｎ：１５．５８）に従って３８１個のＹ－ＳＮＰ部位をスクリーニングし、次世代シーケンスプラットフォーム及びＭｕｌｔｉｐＳｅｑ（登録商標）マルチプレックスＰＣＲアンプリコン捕捉技術によりアジア人集団ハプログループに対する検出方法を設計する。本方法は、感度が高く、特異性が良好で、事件検体に対する適用性が高く、分解性検体及び混合物検体に対しても良好な検出能力を示し、高解像度のハプログループ結果を得ることができ、ハプログループが細分化され、推定が正確である。前記方法は、家系解析及びサンプルの地理的起源が不明の父系種族的出身の推定に適用することができ、法医学の実践に感度が高く、解像度が高く、システムパフォーマンスが高い種族的出身推定技術を提供する。The present invention relates to the fields of biotechnology and genetics technology, and provides primer compositions, kits, methods and uses for detecting Y-SNP haplogroups by next generation sequencing technology. In the present invention, 381 Y-SNP sites were screened according to the haplogroup frequency of the Asian population according to the latest version of the 2019 ISOGG website (Version: 15.58), and a next-generation sequencing platform and MultipSeq (registered TM) Design a detection method for Asian population haplogroups using multiplex PCR amplicon capture technology. This method has high sensitivity, good specificity, high applicability to incident samples, good detection ability even for degradable samples and mixture samples, and can obtain high-resolution haplogroup results. , the haplogroups are subdivided and the estimation is accurate. The method can be applied to genealogy analysis and estimation of patrilineal ethnic origin where the geographical origin of samples is unknown, and provides an ethnic origin estimation technique with high sensitivity, high resolution and high system performance for forensic medicine practice. provide.
Description
本発明は、バイオ技術及び遺伝学技術分野に関し、具体的には、次世代シーケンシング技術により高解像度でＹ－ＳＮＰハプログループを検出するプライマー組成物、キット、方法及びその使用に関し、アジア人集団の主流ハプログループに対する３８１個のＹ－ＳＮＰ部位の増幅に使用される。 The present invention relates to the field of biotechnology and genetics technology, and specifically relates to primer compositions, kits, methods and their use for detecting Y-SNP haplogroups with high resolution by next-generation sequencing technology, and the use of the same in Asian populations. used to amplify 381 Y-SNP sites for the mainstream haplogroups.
Ｙ－ＳＴＲ検査は非常に成熟した法医学的ＤＮＡ分析技術であり、刑事事件の捜査を支援する上で非常に幅広い使用価値及び独特な使用価値を有する。しかし、Ｙ－ＳＴＲの変異率が高いため、同じ家系内でハプロタイプが一致しないという問題があり、これによって、家系調査が困難になり、サンプルの父系起源の推定が妨害される。 Y-STR testing is a very mature forensic DNA analysis technology and has very wide and unique use value in supporting criminal investigation. However, due to the high mutation rate of Y-STR, there is a problem of inconsistency of haplotypes within the same family, which makes genealogy research difficult and interferes with inferring the paternal origin of a sample.
Ｙ－ＳＮＰは、もう１つの遺伝子マーカーである。ＳＮＰは、変異率が低く、復帰変異が発生することがなく、遺伝的安定性が高いため、分解性検体に適用される。これらの特性により、Ｙ－ＳＮＰは進化と移動を研究するための理想的なマーカーとなっている。複数のＹ－ＳＮＰは１つのハプロタイプを構成し、ハプロタイプは突然変異の発生により複数の新しいハプロタイプを派生させる。これらの同じ祖先に由来するハプロタイプは、まとめてハプログループ（ｈａｐｌｏｇｒｏｕｐ）と呼ばれる。すべての男性が同じ父系祖先に由来する場合、その家族は理論的に１つのＹ－ＳＮＰハプログループに属する。ハプログループは互いに関連付けられ、マージされて共通祖先（最も近い共通祖先；ＴＭＲＣＡ）に由来するＹ染色体系統樹（Ｙ Ｃｈｒｏｍｏｓｏｍｅ Ｈａｐｌｏｇｒｏｕｐ Ｔｒｅｅ）を形成する。２００５年に遺伝子系譜学国際協会（ＩＳＯＧＧ）が設立され、２００６年にＹ－ＤＮＡ Ｈａｐｌｏｇｒｏｕｐ ＴｒｅｅがＩＳＯＧＧのＷｅｂサイトで公開された。２０１９年の時点で、ＩＳＯＧＧウェブサイト（バージョン：１４．１７７、日付：２０１９年１０月８日）に提供されたＹ染色体系統樹の構築に使用できるＹ－ＳＮＰ部位（Ｉｎｄｅｌ部位を含む）は、６９，２８０にも達した。細分化されたＹ染色体系統樹は、より短い世代数内の最も近い共通祖先までさかのぼることができるため、未知の男性サンプルの地理的、民族的、さらには家系の追跡にも適用できる。また、Ｙ－ＳＮＰハプログループによりＹ－ＳＴＲ遺伝子座の変異による不利な影響が回避され、即ち、遠い親族を含む同じ父系に由来する個体はすべて、完全に同じＹ－ＳＮＰハプログループタイプを有し、Ｙ染色体系統樹の同じ分岐に対応することができる。 Y-SNP is another genetic marker. SNPs are applied to degradable specimens because they have a low mutation rate, do not cause reversion, and have high genetic stability. These properties make Y-SNP an ideal marker for studying evolution and migration. Multiple Y-SNPs constitute one haplotype, and the haplotype derives multiple new haplotypes through the occurrence of mutations. These haplotypes derived from the same ancestor are collectively called a haplogroup. If all males are descended from the same paternal ancestor, the family theoretically belongs to one Y-SNP haplogroup. Haplogroups are associated with each other and merged to form a Y Chromosome Haplogroup Tree derived from a common ancestor (Temporal Common Ancestor; TMRCA). The International Society for Genetic Genealogy (ISOGG) was established in 2005, and the Y-DNA Haplogroup Tree was published on the ISOGG website in 2006. As of 2019, the Y-SNP sites (including Indel sites) that can be used to construct a Y chromosome phylogenetic tree provided on the ISOGG website (version: 14.177, date: October 8, 2019) are: It reached 69,280. A disaggregated Y-chromosome phylogenetic tree can be traced back to the nearest common ancestor within a shorter number of generations, making it applicable for geographic, ethnic, and even ancestry tracing of unknown male samples. Additionally, the Y-SNP haplogroup avoids the adverse effects of mutations at the Y-STR locus, i.e., all individuals descended from the same paternal line, including distant relatives, have exactly the same Y-SNP haplogroup type. , can correspond to the same branch of the Y chromosome phylogenetic tree.
近年、国内外の学者によるＹ－ＳＮＰハプログループの研究はますます盛んになっている。例えば、Ａｒｗｉｎ Ｒａｌらは、８５０個のＹ－ＳＮＰ部位を用いてワールドワイドなＹ染色体ハプログループツリーを構築した。このツリーは合計６４０個のＹハプログループを含み、主要なハプログループ（Ａ－Ｔ）及びそのサブグループをカバーしている。このツリーはワールドワイドであり、各ハプログループの数が制限されないため、特定の領域に対する解像度が低く、実際の使用において未知の男性検体の具体的な起源の推定が大きく制限されている。一部の学者は、アジア人集団のハプログループに対するＹ－ＳＮＰ複合増幅システムも開発したが、含まれるＹ－ＳＮＰの数が少ないため、集団に対する識別は不十分である。そのため、アジア人集団の主流Ｙ－ＳＮＰハプログループのより多い下流分岐を見つけ、ハイスループット検出システムを構築する必要がある。 In recent years, research on the Y-SNP haplogroup by domestic and international scholars has become increasingly active. For example, Arwin Ral et al. constructed a worldwide Y chromosome haplogroup tree using 850 Y-SNP sites. This tree contains a total of 640 Y haplogroups, covering the major haplogroups (AT) and their subgroups. Because this tree is worldwide and the number of each haplogroup is not limited, the resolution for specific regions is low, severely limiting the inference of the specific origin of unknown male specimens in practical use. Some scholars have also developed a Y-SNP complex amplification system for haplogroups in Asian populations, but the discrimination against the population is insufficient due to the small number of Y-SNPs involved. Therefore, it is necessary to find more downstream branches of the mainstream Y-SNP haplogroup in Asian populations and to build a high-throughput detection system.
現在、Ｙ－ＳＮＰ検出に使用されている主流技術には、マルチプレックス一塩基伸長に基づくＳＮａＰｓｈｏｔ、ＭＡＬＤＩ－ＴＯＦ ＭＳ及び次世代シーケンシング（ｎｅｘｔ ｇｅｎｅｒａｔｉｏｎ ｓｅｑｕｅｎｃｉｎｇ，ＮＧＳ）などがある。ＳＮａＰｓｈｏｔは、感度が高く、キャピラリー電気泳動プラットフォームとの互換性が高いため、法医学分野では、ＳＮａＰｓｈｏｔに基づいて構築されたマルチプレックスＹ－ＳＮＰ複合検出システムがある。しかし、そのコストが高く、設計及びシステムの最適化が困難であり、操作手順が複雑で、時間がかかる。ＭＡＬＤＩ－ＴＯＦ ＭＳは、分子量の違いを直接検出するため、精度が高くてコストが低いが、技術的なスループットは限られており、検出できるＹ－ＳＮＰの数は５０以内であり、最大数百のＹ－ＳＮＰ部位を同時に検出することができないため、起源を正確に追跡することができない。そのため、スループットが高く、法医学分野での普及に適した検出方法を選択することは非常に重要である。ＮＧＳ技術は、精度及びスループットには大きな利点があり、複数のサンプル及び複数のＹ－ＳＮＰの検出に適している。この技術的手段を選択して高解像度のＹ染色体系統樹を構築する場合、検出マーカーの数によって制限されることがないため、検出マーカーの完全性が最大限に確保され、タイピングエラーが回避される。 Currently, mainstream technologies used for Y-SNP detection include SNaPshot based on multiplex single base extension, MALDI-TOF MS, and next generation sequencing (NGS). Due to SNaPshot's high sensitivity and high compatibility with capillary electrophoresis platforms, there are multiplex Y-SNP combined detection systems built on SNaPshot in the forensic field. However, its cost is high, design and system optimization are difficult, and operating procedures are complex and time-consuming. MALDI-TOF MS directly detects differences in molecular weight, so it has high accuracy and low cost, but the technical throughput is limited and the number of Y-SNPs that can be detected is within 50, up to several hundred. Y-SNP sites cannot be detected simultaneously, so the origin cannot be accurately traced. Therefore, it is very important to select a detection method that has high throughput and is suitable for widespread use in the forensic field. NGS technology has significant advantages in accuracy and throughput and is suitable for the detection of multiple samples and multiple Y-SNPs. When choosing this technical means to construct a high-resolution Y-chromosome phylogenetic tree, it is not limited by the number of detected markers, thus ensuring maximum integrity of detected markers and avoiding typing errors. Ru.
アジア人集団に適用され、解像度が高く、完全なＹ染色体系統樹を構築するために、本発明は、アジアの様々な地域及び集団のＹ染色体の遺伝構造の特徴に基づいて、次世代シーケンシング技術によりＹ－ＳＮＰハプログループの複合増幅検出に使用されるプライマー組成物、キット及び方法を提供する。
上記の目的を達成するために、本発明は、以下の技術的手段を採用する。
本発明の第１態様では、次世代シーケンシング技術によりＹ－ＳＮＰハプログループを検出するプライマー組成物が提供される。このプライマー組成物は、３８１個のＹ－ＳＮＰ部位を有する増幅プライマーを含み、
前記３８１個のＹ－ＳＮＰ部位は、ＡＦ６、Ｐ３０５、Ｐ１０８、Ｍ６０、Ｍ９１、Ｍ１３０、Ｆ３３９３、ＣＴＳ１１０４３、Ｍ８、Ｍ３５６、Ｍ３８、Ｍ２１７、Ｌ１３７３、Ｆ３４４７、Ｆ１６９９、Ｆ３９１８、ＦＧＣ２８８８１．２、Ｆ１７５６、Ｆ３８３０、Ｙ１０４２０、Ｍ４８、Ｍ８６、Ｂ９０、Ｍ５０４、Ｆ３７９６、Ｆ９９９２、Ｆ１０６７、Ｚ１３３８、Ｚ１３００、ＣＴＳ２６５７、ＣＴＳ１１９９０、ＣＴＳ８５７９、Ｍ４０７、Ｆ３８５０、Ｚ１２２０９、Ｆ１３１９、ＣＴＳ３３８５、ＦＧＣ４５５４８、Ｆ８４５、ＣＴＳ１０９２３、ＭＦ２０９１、Ｐ１４３、Ｍ１６８、Ｍ２９４、ＣＴＳ３９４６、ＪＳＴ０２１３５５、Ｍ１７４、ＣＴＳ１１５７７、Ｍ１５、Ｎ１、Ｚ２７２６９、ＰＨ４９７９、ＰＨ１９９１、Ｙ７８２０、ＳＫ５４１、Ｚ３１６２５、Ｚ３１５８４、Ｐ９９、Ｐ４７、Ｍ５３３、Ｚ３４３５９、ＰＨ９７、Ｚ４２５９９、Ｚ４２６００、Ｚ４２６０１、Ｚ４２６０２、Ｍ１１６．１、Ｍ１４５、Ｍ９６、Ｍ８９、Ｍ４２７、Ｍ２０１、Ｐ２８７、Ｐ１５、ＣＴＳ４３６７、Ｌ３０、Ｐ３０３、Ｌ１４０、Ｆ１３２９、Ｌ９０１、Ｍ６９、Ｍ５２、Ｍ８２、Ｍ５７８、Ｍ１７０、Ｍ４２９、Ｍ５２２、Ｍ３０４、Ｍ２６７、Ｍ１７２、Ｍ４１０、Ｌ２６、Ｍ４７、Ｍ６７、Ｍ９、Ｍ５２６、Ｍ２０、Ｍ２２、Ｍ２７、Ｍ３１７、Ｐ３２６、Ｐ２５６、Ｍ２３１、Ｆ３３７３、ＣＴＳ３７５０、ＣＴＳ１１４９９、Ｆ１２０６、Ｔａｔ、Ｍ１７８、Ｌ７０８、Ｐ２９８、Ｌ３９２、ＣＴＳ１０７６０、Ｚ１９３６、Ｂ１９７、Ｍ２０１９、Ｆ１００８、Ｍ１２８、Ｂ５２３、Ｐ６３、Ｂ５２５、Ｆ２９３０、ＣＴＳ５８２、Ｍ１８１９、Ｆ５４９、Ｍ２１４、Ｍ１７５、Ｆ２６５、Ｍ１１９、Ｂ３８４、Ｐ２０３．１、Ｆ４４６、Ｆ１４０、ＣＴＳ３２６５、Ｆ７８、Ｆ８１、Ｆ５３３、Ｆ４９２、Ｆ６５６、ＳＫ１５６８、Ｚ２３４０６、Ｍ１０１、Ｚ２３３９２、ＣＴＳ８４２３、ＣＴＳ５２、ＣＴＳ７０１、Ｚ２３２６６、ＣＴＳ７１６、ＣＴＳ３５２、Ｚ２３２７１、Ｍ５０、Ｆ１０３６、Ｐ３１、Ｍ２６８、Ｆ２３２０、Ｍ１４７０、ＰＫ４、Ｍ９５、Ｆ１２５２、Ｍ８８、Ｆ２７５８、Ｆ２８９０、ＣＴＳ５８５４、Ｚ２３８１０、Ｚ２３７８１、Ｆ７８９、Ｆ８３８、ＣＴＳ１４５６、Ｆ９９３、Ｆ４１７、ＣＴＳ９９９６、Ｍ１７６、Ｐ４９、Ｍ１２２、Ｍ３２４、Ｆ１１３、Ｌ１２７．１、ＫＬ１、Ｌ４６７、Ｆ１８７６、Ｆ２１５９、Ｆ１８６７、Ｆ８５２、Ｆ２２６６、Ｌ５９９、Ｚ４３９６１、Ｚ４３９６３、Ｆ８５４、Ｚ４３９６６、Ｆ９１５、ＩＭＳ－ＪＳＴ００２６１１、Ｆ２４０、Ｆ１８、Ｆ１１７、ＣＴＳ５８２０、Ｆ１３、Ｆ１１、Ｆ６３２、Ｆ１１０、Ｆ１７、Ｆ３７７、Ｆ８５６、Ｆ１０９５、Ｆ１４１８、Ｆ１４９５、ＣＴＳ７５０１、Ｆ７９３、Ｙ２０９５１、Ｙ２０９３２、Ｆ３８、Ｆ１２、Ｆ１９６、Ｆ９３０、Ｆ２６８５、Ｆ１３６５、Ｆ３２３２、Ｙ１５９７６、Ｙ２６３８３、ＦＧＣ５４４８６、ＣＴＳ１２８７７、Ｆ２５２７、Ｆ７２３、Ｆ１４２２、ＣＴＳ２１５４、ＳＫ１６９１、ＰＨ２０３、Ｆ４４９、Ｆ２３８、Ｆ１３４、Ｆ１２７３、Ｆ７２４、Ｆ１２６６、ＣＴＳ４９８、ＦＧＣ３７５０、Ｐ２０１、Ｍ１８８、Ｆ２５８８、ＣＴＳ４４５、ＣＴＳ２０１、Ｍ１５９、Ｍ７、Ｆ２６８０、Ｆ１２７６、ＣＴＳ６４８９、Ｆ１２７５、Ｍ１１３、Ｎ５、Ｚ２５４００、Ｆ１８６３、Ｆ１１３４、Ｆ１２６２、Ｙ２６４０３、Ｆ１８３７、Ｆ８６２、Ｆ８７９、Ｆ１２２６、Ｆ２８５９、Ｐ１６４、Ｍ１３４、Ｆ４５０、Ｍ１１７、Ｍ１３３、Ｆ４２、Ｆ４３８、Ｙ１７７２８、Ｆ１５５、Ｆ８１３、Ｙ２０９２８、Ｆ１７５４、Ｆ２１３７、Ｆ１４４２、Ｆ１１２３、Ｆ１３６９、Ｆ３１０、Ｆ４０２、Ｆ１５３１、Ｚ２５８５３、ＣＴＳ５４９２、ＣＴＳ６９８７、Ｚ４２６２０、ＣＴＳ４６５８、Ｚ２５９２８、ＳＫ１７３０、Ｚ２６０１０、Ａ９４６２、ＣＴＳ７６３４、Ｆ３１７、Ｆ３０３９、Ｙ２９８６１、ＣＴＳ５４８８、ＣＴＳ１０７３８、ＣＴＳ９６７８、Ｚ３９６６３、Ａ９４５７、ＹＰ４８６４、Ｚ４４０６８、Ｆ５５２４、Ｆ５５２５、Ｚ４４０７１、Ｚ４４０９１、ＣＴＳ４９６０、Ｆ１２２、Ｆ１１４、Ｆ４４４、Ｆ７９、Ｆ６２９、Ｆ４６、ＦＧＣ１６８４７、Ｆ４８、Ｆ２５０５、Ｆ２４２、ＣＴＳ４２６６、Ｚ２６１０８、Ｆ２８８７、Ｆ３６０７、Ｆ３５２５、ＣＴＳ３７６３、Ａ９４７２、ＦＧＣ１６８６３、Ｌ１３６０、ＦＧＣ１６８８９、ＳＫ１７６８、Ｆ４２４９、ＦＧＣ２３８６８、ＣＴＳ３３５、ＣＴＳ５３、ＣＴＳ６３７３、Ａ９４７３、Ｆ３３８６、Ｙ２９８２８、ＦＧＣ３４９７３、Ｆ１７３９、Ｆ７４３、Ｆ７４８、ＡＭ０１８２２、Ｎ６、ＡＭ０１８５６、Ｎ７、Ｆ４１１０、Ｆ４０６８、Ｆ４１２４、ＡＭ０１８４５、Ｆ７１７、Ａ１６４３３、Ｆ７４２、Ｆ１１５０、Ｆ８３７、Ｆ１０２５、Ｆ１０５５、Ｆ３０２１、Ｐ２９５、Ｍ４５、Ｍ２４２、Ｆ９０３、Ｍ３４６、Ｍ３、Ｍ２０７、Ｍ１７３、Ｍ４２０、ＳＲＹ１０８３１．２、Ｍ１７、Ｚ６４５、Ｍ４５８、Ｚ９１、Ｚ９３、Ｆ３１０５、Ｙ７、Ｚ２１２４、Ｚ２１２３、Ｓ４５７６、Ｆ１３４５、Ｍ３４３、Ｌ７５４、Ｌ３８９、Ｐ２９７、Ｍ２６９、Ｍ４７９、Ｍ１２４、ＦＧＣ２１７０６、Ｂ２５４、Ｍ１８４を含む。
In order to construct a high-resolution and complete Y-chromosome phylogenetic tree that is applied to Asian populations, the present invention uses next-generation sequencing based on the characteristics of the genetic structure of the Y-chromosome of various regions and populations in Asia. Primer compositions, kits, and methods for use in multiplex amplification detection of Y-SNP haplogroups by technology are provided.
In order to achieve the above object, the present invention adopts the following technical means.
In a first aspect of the present invention, a primer composition for detecting Y-SNP haplogroups by next generation sequencing technology is provided. This primer composition includes an amplification primer having 381 Y-SNP sites,
The 381 Y-SNP sites are AF6, P305, P108, M60, M91, M130, F3393, CTS11043, M8, M356, M38, M217, L1373, F3447, F1699, F3918, FGC28881.2, F1756, F3830, Y10420, M48, M86, B90, M504, F3796, F9992, F1067, Z1338, Z1300, CTS2657, CTS11990, CTS8579, M407, F3850, Z12209, F1319, CTS3385, FGC4 5548, F845, CTS10923, MF2091, P143, M168, M294, CTS3946, JST021355, M174, CTS11577, M15, N1, Z27269, PH4979, PH1991, Y7820, SK541, Z31625, Z31584, P99, P47, M533, Z34359, PH97, Z42599, Z42600, Z42601, Z42602, M116.1, M145, M96, M89, M427, M201, P287, P15, CTS4367, L30, P303, L140, F1329, L901, M69, M52, M82, M578, M170, M429, M522, M304, M267, M172, M410, L26, M47, M67, M9, M526, M20, M22, M27, M317, P326, P256, M231, F3373, CTS3750, CTS11499, F1206, Tat, M178, L708, P298, L392, CTS10760, Z1936, B197, M2 019, F1008, M128, B523, P63, B525, F2930, CTS582, M1819, F549, M214, M175, F265, M119, B384, P203.1, F446, F140, CTS3265, F78, F81, F533, F492, F656, SK1568, Z 23406, M101, Z23392, CTS8423, CTS52, CTS701, Z23266, CTS716, CTS352, Z23271, M50, F1036, P31, M268, F2320, M1470, PK4, M95, F1252, M88, F2758, F289 0, CTS5854, Z23810, Z23781, F789, F838, CTS1456, F993, F417, CTS9996, M176, P49, M122, M324, F113, L127.1, KL1, L467, F1876, F2159, F1867, F852, F2266, L599, Z43961, Z43963, F85 4, Z43966, F915, IMS- JST002611, F240, F18, F117, CTS5820, F13, F11, F632, F110, F17, F377, F856, F1095, F1418, F1495, CTS7501, F793, Y20951, Y20932, F38, F12, F196 , F930, F2685, F1365, F3232, Y15976, Y26383, FGC54486, CTS12877, F2527, F723, F1422, CTS2154, SK1691, PH203, F449, F238, F134, F1273, F724, F1266, CTS498, FGC375 0, P201, M188, F2588, CTS445, CTS201, M159, M7, F2680, F1276, CTS6489, F1275, M113, N5, Z25400, F1863, F1134, F1262, Y26403, F1837, F862, F879, F1226, F2859, P164, M134, F450, M117, M1 33, F42, F438, Y17728, F155, F813, Y20928, F1754, F2137, F1442, F1123, F1369, F310, F402, F1531, Z25853, CTS5492, CTS6987, Z42620, CTS4658, Z25928, SK1730, Z26010, A9462, CTS7634, F317, F3039, Y29861, CTS5488, CTS10738, CTS9678, Z39663, A9457, YP4864, Z44068, F5524, F5525, Z44071, Z44091, CTS4960, F122, F114, F444, F79, F629, F46, FGC1684 7, F48, F2505, F242, CTS4266, Z26108, F2887, F3607, F3525, CTS3763, A9472, FGC16863, L1360, FGC16889, SK1768, F4249, FGC23868, CTS335, CTS53, CTS6373, A9473, F3386, Y29828, FGC34973, F1739, F743, F748, AM01822, N6, AM01856, N7, F4110, F4068, F4124, AM01845, F717, A16433, F742, F1150, F837, F1025, F1055, F3021, P295, M45, M242, F903, M346, M3, M207, M173, M420, SRY10831.2, M17, Z6 45, M458, Z91, Including Z93, F3105, Y7, Z2124, Z2123, S4576, F1345, M343, L754, L389, P297, M269, M479, M124, FGC21706, B254, M184.
さらに、前記増幅プライマー組成物は、ヌクレオチド配列が配列番号（SEQ ID No）１～７４６であるプライマーを含む。 Furthermore, the amplification primer composition includes primers whose nucleotide sequences are SEQ ID Nos. 1 to 746.
本発明の第２態様では、前記プライマー組成物を含む、次世代シーケンシング技術によりＹ－ＳＮＰハプログループを検出するキットが提供される。 A second aspect of the present invention provides a kit for detecting Y-SNP haplogroups by next-generation sequencing technology, which includes the primer composition.
さらに、前記キットは、マルチプレックスＰＣＲ反応に必要な他の試薬、リンカー配列ＰＣＲ反応に必要な試薬、及び磁気ビーズ精製に必要な試薬をさらに含む。 Furthermore, the kit further includes other reagents required for multiplex PCR reaction, reagents required for linker sequence PCR reaction, and reagents necessary for magnetic bead purification.
さらに、前記キットによって検出されるサンプルは、血液、精液、唾液、骨骼、毛髪、人体組織に由来し、血液（血痕）、精液（精液痕）、唾液（唾液痕）、骨骼、毛髪又は人体組織ゲノムＤＮＡを採用する。 Furthermore, the sample detected by the kit is derived from blood, semen, saliva, bone, hair, human tissue; Adopt genomic DNA.
本発明の第３態様では、前記キットを用いて次世代シーケンシング技術によりＹ－ＳＮＰハプログループを検出する方法が提供される。この方法は、以下のステップ１からステップ４を含む。
ステップ１において、マルチプレックスＰＣＲ反応を行い、具体的には、希釈したｇＤＮＡをテンプレートとし、増幅反応系を調製し、マルチプレックス断片増幅を行い、
ステップ２において、ＰＣＲ増幅産物を磁気ビーズ精製し、その後、リンカー配列ＰＣＲ反応を行い、
ステップ３において、ステップ２の増幅産物を磁気ビーズ精製してライブラリを構築し、その後、ライブラリについて濃度、断片の長さ及び純度を測定し、
ステップ４において、ライブラリを混合、変性した後、Ｉｌｌｕｍｉｎａ ＭｉＳｅｑシーケンスプラットフォームに入れてシーケンシングし、シーケンシング結果と参照ゲノム配列とを比較し、ｓａｍｔｏｏｌｓ、ＧＡＴＫなどのソフトウェアによりＳＮＰ及びＩｎｄｅｌを探し、次にＡＮＮＯＶＡＲソフトウェアによりＳＮＰ、Ｉｎｄｅｌ部位に注釈を付け、変異部位に対応する遺伝子情報などを確定する。
A third aspect of the present invention provides a method for detecting Y-SNP haplogroups by next generation sequencing technology using the kit. This method includes steps 1 to 4 below.
In step 1, a multiplex PCR reaction is performed, specifically, using diluted gDNA as a template, preparing an amplification reaction system, performing multiplex fragment amplification,
In step 2, the PCR amplification product is purified by magnetic beads, and then a linker sequence PCR reaction is performed,
In step 3, construct a library by magnetic bead purification of the amplification product of step 2, and then measure the concentration, fragment length and purity of the library,
In step 4, after mixing and denaturing the library, it is placed in the Illumina MiSeq sequencing platform and sequenced, the sequencing results are compared with the reference genome sequence, SNPs and Indels are searched by software such as samtools and GATK, and then SNP and Indel sites are annotated using ANNOVAR software, and genetic information corresponding to the mutation site is determined.
さらに、ステップ１のマルチプレックスＰＣＲ反応系は、１０μＬのｄｄＨ２Ｏ、４μＬのＥｎｈａｎｃｅｒ ｂｕｆｆｅｒ ＪＰ（２Ｎ）、４μＬのプライマー混合物（２μＭ）、１μＬのｇＤＮＡ及び６μＬのＩＧＴ－ＥＭ１０１ポリメラーゼ混合物を含み、合計２５μＬである。 Furthermore, the multiplex PCR reaction system in step 1 contained 10 μL ddH 2 O, 4 μL Enhancer buffer JP (2N), 4 μL primer mix (2 μM), 1 μL gDNA, and 6 μL IGT-EM101 polymerase mixture, in total. The volume is 25 μL.
さらに、ステップ１のマルチプレックスＰＣＲ反応条件は、１０５℃で予熱；９５℃で３分間３０秒；９８℃で２０秒、６０℃で３分間３０秒を１サイクルとして１４サイクル；７２℃で５分間である。 Furthermore, the multiplex PCR reaction conditions in step 1 were: preheating at 105°C; 3 minutes and 30 seconds at 95°C; 14 cycles of 20 seconds at 98°C and 3 minutes and 30 seconds at 60°C; 5 minutes at 72°C. It is.
さらに、前記リンカー配列ＰＣＲ反応系は、１７μＬのＰＣＲ磁気ビーズ精製産物、１μＬのＩＧＴ－Ｉ５ Ｉｎｄｅｘ（１０μＭ）、１μＬのＩＧＴ－Ｉ７ Ｉｎｄｅｘ（１０μＭ）及び６μＬのＩＧＴ－ＥＭ１０１ポリメラーゼ混合物を含み、合計２５μＬである。 Furthermore, the linker sequence PCR reaction system contained 17 μL of PCR magnetic bead purified product, 1 μL of IGT-I5 Index (10 μM), 1 μL of IGT-I7 Index (10 μM), and 6 μL of IGT-EM101 polymerase mixture, for a total of 25 μL. It is.
さらに、前記リンカー配列ＰＣＲ反応条件は、１０５℃で予熱；９５℃で３分間３０秒；９８℃で２０秒、５８℃で１分間、７２℃で３０秒を１サイクルとして９サイクル；７２℃で５分間である。 Furthermore, the linker sequence PCR reaction conditions were: preheating at 105°C; 95°C for 3 minutes and 30 seconds; 9 cycles each consisting of 98°C for 20 seconds, 58°C for 1 minute, and 72°C for 30 seconds; It is 5 minutes.
本発明の第４態様では、家系解析、及びサンプルの地理的起源が不明の父系種族的出身（ｅｔｈｎｉｃ ｏｒｉｇｉｎ）の推定における前記プライマー組成物又は前記キットの使用が提供される。 In a fourth aspect of the invention there is provided the use of said primer composition or said kit in genealogy analysis and estimation of paternal ethnic origin where the geographical origin of a sample is unknown.
本発明は、上記の技術的手段を採用することにより、従来技術に比べて以下の技術的効果を有する。
（１）本発明では、次世代シーケンシング技術により３８１個のＹ－ＳＮＰを有する増幅プライマーを含むプライマー組成物及びキットを構築する。部位を選択する際に、中国人集団のハプログループの頻度を参照し、中国人集団について高解像度のハプログループ結果を得ることができ、ハプログループが細分化され、推定が正確である。
（２）本発明では、ＭｕｌｔｉｐＳｅｑ（登録商標）マルチプレックスＰＣＲアンプリコン捕捉技術によりプライマー設計及びライブラリ構築を行い、熱力学的安定性のアルゴリズムにより特異的なプライマーを設計し、二段階ＰＣＲ増幅法により目標領域の増幅及びライブラリ構築を達成することにより、大量の時間及びコストが節約される。
（３）本発明では、様々なタイプのサンプル（例えば、口腔スワブ、指の爪、毛包のある毛髪及び精液斑などのゲノムＤＮＡ）を用いてライブラリ構築を行う。設計されたアンプリコン断片が短いため、このシステムは、分解性検体に対しても良好な検出及びタイピング効を得ることができる。
By employing the above technical means, the present invention has the following technical effects compared to the prior art.
(1) In the present invention, a primer composition and kit containing an amplification primer having 381 Y-SNPs are constructed using next-generation sequencing technology. When selecting sites, we refer to the frequency of haplogroups in the Chinese population, and can obtain high-resolution haplogroup results for the Chinese population, and the haplogroups are subdivided and the estimation is accurate.
(2) In the present invention, primer design and library construction are performed using MultipSeq (registered trademark) multiplex PCR amplicon capture technology, specific primers are designed using a thermodynamic stability algorithm, and two-step PCR amplification method is used to design specific primers. A large amount of time and cost is saved by achieving target region amplification and library construction.
(3) In the present invention, library construction is performed using various types of samples (eg, genomic DNA such as oral swabs, fingernails, hair with hair follicles, and semen spots). Because the designed amplicon fragments are short, this system can obtain good detection and typing efficacy even for degradable analytes.
本発明は、次世代シーケンシング技術により高解像度でＹ－ＳＮＰハプログループを検出するプライマー組成物に関し、この組成物は、３８１個のＹ－ＳＮＰ部位を有する増幅プライマーを含む。
前記３８１個のＹ－ＳＮＰ部位は、いずれもアジア人集団の各主要なハプログループが占める割合に応じて、民族人口数と合わせて、Ｙ染色体の系統樹から選択した下流分岐Ｙ－ＳＮＰ遺伝子マーカーである。系統樹の構造の完全性を確保するために、多くの重要な分岐点及び主幹ハプログループを追加した。具体的には、ＡＦ６、Ｐ３０５、Ｐ１０８、Ｍ６０、Ｍ９１、Ｍ１３０、Ｆ３３９３、ＣＴＳ１１０４３、Ｍ８、Ｍ３５６、Ｍ３８、Ｍ２１７、Ｌ１３７３、Ｆ３４４７、Ｆ１６９９、Ｆ３９１８、ＦＧＣ２８８８１．２、Ｆ１７５６、Ｆ３８３０、Ｙ１０４２０、Ｍ４８、Ｍ８６、Ｂ９０、Ｍ５０４、Ｆ３７９６、Ｆ９９９２、Ｆ１０６７、Ｚ１３３８、Ｚ１３００、ＣＴＳ２６５７、ＣＴＳ１１９９０、ＣＴＳ８５７９、Ｍ４０７、Ｆ３８５０、Ｚ１２２０９、Ｆ１３１９、ＣＴＳ３３８５、ＦＧＣ４５５４８、Ｆ８４５、ＣＴＳ１０９２３、ＭＦ２０９１、Ｐ１４３、Ｍ１６８、Ｍ２９４、ＣＴＳ３９４６、ＪＳＴ０２１３５５、Ｍ１７４、ＣＴＳ１１５７７、Ｍ１５、Ｎ１、Ｚ２７２６９、ＰＨ４９７９、ＰＨ１９９１、Ｙ７８２０、ＳＫ５４１、Ｚ３１６２５、Ｚ３１５８４、Ｐ９９、Ｐ４７、Ｍ５３３、Ｚ３４３５９、ＰＨ９７、Ｚ４２５９９、Ｚ４２６００、Ｚ４２６０１、Ｚ４２６０２、Ｍ１１６．１、Ｍ１４５、Ｍ９６、Ｍ８９、Ｍ４２７、Ｍ２０１、Ｐ２８７、Ｐ１５、ＣＴＳ４３６７、Ｌ３０、Ｐ３０３、Ｌ１４０、Ｆ１３２９、Ｌ９０１、Ｍ６９、Ｍ５２、Ｍ８２、Ｍ５７８、Ｍ１７０、Ｍ４２９、Ｍ５２２、Ｍ３０４、Ｍ２６７、Ｍ１７２、Ｍ４１０、Ｌ２６、Ｍ４７、Ｍ６７、Ｍ９、Ｍ５２６、Ｍ２０、Ｍ２２、Ｍ２７、Ｍ３１７、Ｐ３２６、Ｐ２５６、Ｍ２３１、Ｆ３３７３、ＣＴＳ３７５０、ＣＴＳ１１４９９、Ｆ１２０６、Ｔａｔ、Ｍ１７８、Ｌ７０８、Ｐ２９８、Ｌ３９２、ＣＴＳ１０７６０、Ｚ１９３６、Ｂ１９７、Ｍ２０１９、Ｆ１００８、Ｍ１２８、Ｂ５２３、Ｐ６３、Ｂ５２５、Ｆ２９３０、ＣＴＳ５８２、Ｍ１８１９、Ｆ５４９、Ｍ２１４、Ｍ１７５、Ｆ２６５、Ｍ１１９、Ｂ３８４、Ｐ２０３．１、Ｆ４４６、Ｆ１４０、ＣＴＳ３２６５、Ｆ７８、Ｆ８１、Ｆ５３３、Ｆ４９２、Ｆ６５６、ＳＫ１５６８、Ｚ２３４０６、Ｍ１０１、Ｚ２３３９２、ＣＴＳ８４２３、ＣＴＳ５２、ＣＴＳ７０１、Ｚ２３２６６、ＣＴＳ７１６、ＣＴＳ３５２、Ｚ２３２７１、Ｍ５０、Ｆ１０３６、Ｐ３１、Ｍ２６８、Ｆ２３２０、Ｍ１４７０、ＰＫ４、Ｍ９５、Ｆ１２５２、Ｍ８８、Ｆ２７５８、Ｆ２８９０、ＣＴＳ５８５４、Ｚ２３８１０、Ｚ２３７８１、Ｆ７８９、Ｆ８３８、ＣＴＳ１４５６、Ｆ９９３、Ｆ４１７、ＣＴＳ９９９６、Ｍ１７６、Ｐ４９、Ｍ１２２、Ｍ３２４、Ｆ１１３、Ｌ１２７．１、ＫＬ１、Ｌ４６７、Ｆ１８７６、Ｆ２１５９、Ｆ１８６７、Ｆ８５２、Ｆ２２６６、Ｌ５９９、Ｚ４３９６１、Ｚ４３９６３、Ｆ８５４、Ｚ４３９６６、Ｆ９１５、ＩＭＳ－ＪＳＴ００２６１１、Ｆ２４０、Ｆ１８、Ｆ１１７、ＣＴＳ５８２０、Ｆ１３、Ｆ１１、Ｆ６３２、Ｆ１１０、Ｆ１７、Ｆ３７７、Ｆ８５６、Ｆ１０９５、Ｆ１４１８、Ｆ１４９５、ＣＴＳ７５０１、Ｆ７９３、Ｙ２０９５１、Ｙ２０９３２、Ｆ３８、Ｆ１２、Ｆ１９６、Ｆ９３０、Ｆ２６８５、Ｆ１３６５、Ｆ３２３２、Ｙ１５９７６、Ｙ２６３８３、ＦＧＣ５４４８６、ＣＴＳ１２８７７、Ｆ２５２７、Ｆ７２３、Ｆ１４２２、ＣＴＳ２１５４、ＳＫ１６９１、ＰＨ２０３、Ｆ４４９、Ｆ２３８、Ｆ１３４、Ｆ１２７３、Ｆ７２４、Ｆ１２６６、ＣＴＳ４９８、ＦＧＣ３７５０、Ｐ２０１、Ｍ１８８、Ｆ２５８８、ＣＴＳ４４５、ＣＴＳ２０１、Ｍ１５９、Ｍ７、Ｆ２６８０、Ｆ１２７６、ＣＴＳ６４８９、Ｆ１２７５、Ｍ１１３、Ｎ５、Ｚ２５４００、Ｆ１８６３、Ｆ１１３４、Ｆ１２６２、Ｙ２６４０３、Ｆ１８３７、Ｆ８６２、Ｆ８７９、Ｆ１２２６、Ｆ２８５９、Ｐ１６４、Ｍ１３４、Ｆ４５０、Ｍ１１７、Ｍ１３３、Ｆ４２、Ｆ４３８、Ｙ１７７２８、Ｆ１５５、Ｆ８１３、Ｙ２０９２８、Ｆ１７５４、Ｆ２１３７、Ｆ１４４２、Ｆ１１２３、Ｆ１３６９、Ｆ３１０、Ｆ４０２、Ｆ１５３１、Ｚ２５８５３、ＣＴＳ５４９２、ＣＴＳ６９８７、Ｚ４２６２０、ＣＴＳ４６５８、Ｚ２５９２８、ＳＫ１７３０、Ｚ２６０１０、Ａ９４６２、ＣＴＳ７６３４、Ｆ３１７、Ｆ３０３９、Ｙ２９８６１、ＣＴＳ５４８８、ＣＴＳ１０７３８、ＣＴＳ９６７８、Ｚ３９６６３、Ａ９４５７、ＹＰ４８６４、Ｚ４４０６８、Ｆ５５２４、Ｆ５５２５、Ｚ４４０７１、Ｚ４４０９１、ＣＴＳ４９６０、Ｆ１２２、Ｆ１１４、Ｆ４４４、Ｆ７９、Ｆ６２９、Ｆ４６、ＦＧＣ１６８４７、Ｆ４８、Ｆ２５０５、Ｆ２４２、ＣＴＳ４２６６、Ｚ２６１０８、Ｆ２８８７、Ｆ３６０７、Ｆ３５２５、ＣＴＳ３７６３、Ａ９４７２、ＦＧＣ１６８６３、Ｌ１３６０、ＦＧＣ１６８８９、ＳＫ１７６８、Ｆ４２４９、ＦＧＣ２３８６８、ＣＴＳ３３５、ＣＴＳ５３、ＣＴＳ６３７３、Ａ９４７３、Ｆ３３８６、Ｙ２９８２８、ＦＧＣ３４９７３、Ｆ１７３９、Ｆ７４３、Ｆ７４８、ＡＭ０１８２２、Ｎ６、ＡＭ０１８５６、Ｎ７、Ｆ４１１０、Ｆ４０６８、Ｆ４１２４、ＡＭ０１８４５、Ｆ７１７、Ａ１６４３３、Ｆ７４２、Ｆ１１５０、Ｆ８３７、Ｆ１０２５、Ｆ１０５５、Ｆ３０２１、Ｐ２９５、Ｍ４５、Ｍ２４２、Ｆ９０３、Ｍ３４６、Ｍ３、Ｍ２０７、Ｍ１７３、Ｍ４２０、ＳＲＹ１０８３１．２、Ｍ１７、Ｚ６４５、Ｍ４５８、Ｚ９１、Ｚ９３、Ｆ３１０５、Ｙ７、Ｚ２１２４、Ｚ２１２３、Ｓ４５７６、Ｆ１３４５、Ｍ３４３、Ｌ７５４、Ｌ３８９、Ｐ２９７、Ｍ２６９、Ｍ４７９、Ｍ１２４、ＦＧＣ２１７０６、Ｂ２５４、Ｍ１８４を含む。
The present invention relates to a primer composition for detecting Y-SNP haplogroups with high resolution by next-generation sequencing technology, and this composition includes an amplification primer having 381 Y-SNP sites.
The 381 Y-SNP sites are all downstream divergent Y-SNP gene markers selected from the phylogenetic tree of the Y chromosome according to the proportion of each major haplogroup in the Asian population, together with the number of ethnic populations. It is. To ensure the structural integrity of the phylogenetic tree, many important branch points and main haplogroups were added. Specifically, AF6, P305, P108, M60, M91, M130, F3393, CTS11043, M8, M356, M38, M217, L1373, F3447, F1699, F3918, FGC28881.2, F1756, F3830, Y10420, M48, M86 , B90, M504, F3796, F9992, F1067, Z1338, Z1300, CTS2657, CTS11990, CTS8579, M407, F3850, Z12209, F1319, CTS3385, FGC45548, F845, CTS1 0923, MF2091, P143, M168, M294, CTS3946, JST021355, M174 , CTS11577, M15, N1, Z27269, PH4979, PH1991, Y7820, SK541, Z31625, Z31584, P99, P47, M533, Z34359, PH97, Z42599, Z42600, Z426 01, Z42602, M116.1, M145, M96, M89, M427 , M201, P287, P15, CTS4367, L30, P303, L140, F1329, L901, M69, M52, M82, M578, M170, M429, M522, M304, M267, M172, M410, L26, M47, M67, M9, M526 , M20, M22, M27, M317, P326, P256, M231, F3373, CTS3750, CTS11499, F1206, Tat, M178, L708, P298, L392, CTS10760, Z1936, B197, M2019, F1008, M 128, B523, P63, B525 , F2930, CTS582, M1819, F549, M214, M175, F265, M119, B384, P203.1, F446, F140, CTS3265, F78, F81, F533, F492, F656, SK1568, Z23406, M101, Z23392, CTS8423, CTS52 , CTS701, Z23266, CTS716, CTS352, Z23271, M50, F1036, P31, M268, F2320, M1470, PK4, M95, F1252, M88, F2758, F2890, CTS5854, Z23810, Z 23781, F789, F838, CTS1456, F993, F417 , CTS9996, M176, P49, M122, M324, F113, L127.1, KL1, L467, F1876, F2159, F1867, F852, F2266, L599, Z43961, Z43963, F854, Z43966, F915, I MS-JST002611, F240, F18 , F117, CTS5820, F13, F11, F632, F110, F17, F377, F856, F1095, F1418, F1495, CTS7501, F793, Y20951, Y20932, F38, F12, F196, F930, F2685, F1365, F3232, Y15976, Y26383 , FGC54486, CTS12877, F2527, F723, F1422, CTS2154, SK1691, PH203, F449, F238, F134, F1273, F724, F1266, CTS498, FGC3750, P201, M188, F2588, C TS445, CTS201, M159, M7, F2680, F1276 , CTS6489, F1275, M113, N5, Z25400, F1863, F1134, F1262, Y26403, F1837, F862, F879, F1226, F2859, P164, M134, F450, M117, M133, F42, F438, Y1 7728, F155, F813, Y20928 , F1754, F2137, F1442, F1123, F1369, F310, F402, F1531, Z25853, CTS5492, CTS6987, Z42620, CTS4658, Z25928, SK1730, Z26010, A9462, C TS7634, F317, F3039, Y29861, CTS5488, CTS10738, CTS9678, Z39663 , A9457, YP4864, Z44068, F5524, F5525, Z44071, Z44091, CTS4960, F122, F114, F444, F79, F629, F46, FGC16847, F48, F2505, F242, CTS4266, Z26108, F2887, F3607, F3525, CTS3763, A9472 , FGC16863, L1360, FGC16889, SK1768, F4249, FGC23868, CTS335, CTS53, CTS6373, A9473, F3386, Y29828, FGC34973, F1739, F743, F748, AM01822, N 6, AM01856, N7, F4110, F4068, F4124, AM01845, F717 , A16433, F742, F1150, F837, F1025, F1055, F3021, P295, M45, M242, F903, M346, M3, M207, M173, M420, SRY10831.2, M17, Z645, M458, Z91, Z9 3, F3105, Y7 , Z2124, Z2123, S4576, F1345, M343, L754, L389, P297, M269, M479, M124, FGC21706, B254, M184.
以下、本発明をより良く理解するために、具体的な実施例及び図面により本発明を詳しく具体的に説明するが、以下の実施例は、本発明の範囲を制限するものではない。 Hereinafter, in order to better understand the present invention, the present invention will be specifically explained in detail with reference to specific examples and drawings, but the following examples are not intended to limit the scope of the present invention.
実施例において、特記のない場合、常法を用い、通常の市販試薬又は常法により調製された試薬を使用する。 In the Examples, unless otherwise specified, conventional methods are used, and conventional commercially available reagents or reagents prepared by conventional methods are used.
実施例１
本実施例によれば、次世代シーケンシング技術により高解像度でＹ－ＳＮＰハプログループを検出するプライマー組成物及び方法が提供される。具体的なＹ－ＳＮＰ部位スクリーニング、プライマー設計及び検出方法の手順は、以下の通りである。
１．Ｙ－ＳＮＰ部位スクリーニング及びプライマー設計
アジア人集団の各主要なハプログループが占める割合に応じて、民族人口数と合わせて、Ｙ染色体の系統樹から異なる数の下流分岐Ｙ－ＳＮＰ遺伝子マーカーを選択した。系統樹の構造の完全性を確保するために、多くの重要な分岐点及び主幹ハプログループを追加した。中国人集団に対して合計３８１個のＹ－ＳＮＰ部位がスクリーニングされ、３５９個のハプログループが構成された（２０１９年、ＩＳＯＧＧウェブサイト（Ｖｅｒｓｉｏｎ：１５．５８）（ｈｔｔｐｓ：／／ｉｓｏｇｇ．ｏｒｇ／ｔｒｅｅ／ＨａｐｌｏｇｒｏｕｐＴｒ２０１９．ｈｔｍｌ））。選択された３８１個のＹ－ＳＮＰ部位のヒト参照ゲノム（ＧＲＣｈ３７／ｈｇ１９）の染色体位置などの詳細情報をプライマー設計ウェブサイト（ｈｔｔｐｓ：／／ｄｅｓｉｇｎ．ｉｇｅｎｅｔｅｃｈ．ｃｏｍ／）にアップロードして多重プライマー設計を行った。より良好なカバレッジ（Ｃｏｖｅｒａｇｅ）を得るために、プライマーに対して２ラウンドの設計及び最適化を行った。
Example 1
According to this example, a primer composition and method for detecting Y-SNP haplogroups with high resolution using next-generation sequencing technology are provided. The specific steps for Y-SNP site screening, primer design, and detection method are as follows.
1. Y-SNP site screening and primer design Different numbers of downstream divergent Y-SNP gene markers were selected from the Y-chromosome phylogenetic tree according to the proportion of each major haplogroup in the Asian population, together with the number of ethnic populations. . To ensure the structural integrity of the phylogenetic tree, many important branch points and main haplogroups were added. A total of 381 Y-SNP sites were screened for the Chinese population, and 359 haplogroups were constructed (2019, ISOGG website (Version: 15.58) (https://isogg.org/ tree/HaplogroupTr2019.html)). Detailed information such as the chromosomal location of the human reference genome (GRCh37/hg19) of the selected 381 Y-SNP sites was uploaded to the primer design website (https://design.igenetech.com/) for multiplex primer design. I did it. Two rounds of design and optimization were performed on the primers to obtain better coverage.
本実施例に記載の増幅プライマーのＹ染色体における位置及びプライマー配列を表１に示す。 Table 1 shows the positions of the amplification primers described in this example on the Y chromosome and the primer sequences.
２．ライブラリの構築
ＭｕｌｔｉｐＳｅｑマルチプレックスＰＣＲアンプリコン捕捉技術を選択し、二段階ＰＣＲ増幅法により目標領域の増幅及びライブラリの構築を行った。ライブラリ構築の手順は、マルチプレックスＰＣＲ反応、磁気ビーズによる結合した産物の精製、リンカー配列ＰＣＲ反応、磁気ビーズ精製、ライブラリ濃度の測定及び品質検査を含む。具体的な過程は、以下の通りである。
（１）マルチプレックスＰＣＲ：希釈したｇＤＮＡをテンプレートとし、ＭｕｌｔｉｐＳｅｑ ＡＩ－Ｍｉｔｏ－Ｍｕｌｔｉ Ｐａｎｅｌキットを用いてマルチプレックス断片増幅を行い、増幅反応系の総体積は２５μＬであり、組成を表２に示す。
2. Library Construction MultipSeq multiplex PCR amplicon capture technology was chosen to amplify the target region and construct the library using a two-step PCR amplification method. The library construction procedure includes multiplex PCR reactions, purification of bound products by magnetic beads, linker sequence PCR reactions, magnetic bead purification, library concentration measurements, and quality checks. The specific process is as follows.
(1) Multiplex PCR: Using diluted gDNA as a template, multiplex fragment amplification was performed using the MultipSeq AI-Mito-Multi Panel kit. The total volume of the amplification reaction system was 25 μL, and the composition is shown in Table 2.
ＰＣＲ反応条件：１０５℃で予熱；９５℃で３分間３０秒；９８℃で２０秒、６０℃で３分間３０秒を１サイクルとして１４サイクル；７２℃で５分間 PCR reaction conditions: Preheat at 105°C; 95°C for 3 minutes and 30 seconds; 14 cycles of 98°C for 20 seconds and 60°C for 3 minutes and 30 seconds; 72°C for 5 minutes
（２）ＡＭＰｕｒｅ ＸＰ磁気ビーズによるＰＣＲ産物の精製 (2) Purification of PCR products using AMPure XP magnetic beads
（３）リンカー配列ＰＣＲ反応：反応系を表３に示す。 (3) Linker sequence PCR reaction: The reaction system is shown in Table 3.
ＰＣＲ反応条件：１０５℃で予熱；９５℃で３分間３０秒；９８℃で２０秒、５８℃で１分間、７２℃で３０秒を１サイクルとして９サイクル；７２℃で５分間 PCR reaction conditions: Preheat at 105°C; 95°C for 3 minutes and 30 seconds; 9 cycles of 98°C for 20 seconds, 58°C for 1 minute, and 72°C for 30 seconds; 72°C for 5 minutes
（４）第２ラウンドの磁気ビーズ精製を行い、その後、２μＬライブラリを取り、Ｑｕｂｉｔ（登録商標）３．０フルオロメーターを用いて濃度を測定し、ライブラリ濃度を記録し、１μＬライブラリサンプルを取り、Ａｇｉｌｅｎｔ ２１００バイオアナライザシステム（高感度ＤＮＡキット）を用いてライブラリ断片の長さ及び純度を測定した。 (4) Perform a second round of magnetic bead purification, then take 2 μL library, measure the concentration using a Qubit® 3.0 fluorometer, record the library concentration, and take 1 μL library sample; The length and purity of the library fragments were measured using the Agilent 2100 Bioanalyzer System (High Sensitivity DNA Kit).
（５）ライブラリを混合、変性した後、Ｉｌｌｕｍｉｎａ ＭｉＳｅｑシーケンスプラットフォームに入れてシーケンシングを行った。シーケンシング結果を参照ゲノム配列と比較し、ｓａｍｔｏｏｌｓ、ＧＡＴＫなどのソフトウェアによりＳＮＰ及びＩｎｄｅｌを探し、その後、ＡＮＮＯＶＡＲソフトウェアによりＳＮＰ、Ｉｎｄｅｌ部位に注釈を付け、変異部位に対応する遺伝子情報などを確定した。 (5) After mixing and denaturing the library, it was placed in an Illumina MiSeq sequencing platform and sequenced. The sequencing results were compared with the reference genome sequence, SNPs and Indels were searched for using software such as samtools and GATK, and then SNPs and Indel sites were annotated using ANNOVAR software to determine genetic information corresponding to the mutation site.
前記複合増幅系に係るＹ－ＤＮＡハプログループ基本樹（２０１９年）及び選択されたＹ－ＳＮＰの数を図１に示す。 FIG. 1 shows the Y-DNA haplogroup basic tree (2019) and the number of selected Y-SNPs related to the composite amplification system.
実施例２
本実施例は、実施例１で提供される次世代シーケンシング技術により高解像度でＹ－ＳＮＰハプログループを検出する方法の法医学的検証作業である。
Example 2
This example is a forensic verification of the method for detecting Y-SNP haplogroups with high resolution using the next-generation sequencing technology provided in Example 1.
ＤＮＡ分析方法に関する技術作業部会（Ｓｃｉｅｎｔｉｆｉｃ Ｗｏｒｋｉｎｇ Ｇｒｏｕｐ ｆｏｒ ＤＮＡ Ａｎａｌｙｓｉｓ Ｍｅｔｈｏｄｓ，ＳＷＧＤＡＭ）の要件に従って実施例１で構築された方法のシーケンシング品質、感度、再現性、精度、事件検体適用性を評価し、法医学的パラメータを計算した。 We evaluated the sequencing quality, sensitivity, reproducibility, precision, and case specimen applicability of the method constructed in Example 1 in accordance with the requirements of the Scientific Working Group for DNA Analysis Methods (SWGDAM), and The target parameters were calculated.
１００個のサンプルに基づいて計算されたそれぞれのＹ－ＳＮＰ部位の平均シーケンス深度を図２に示す。ライブラリ構築はスムーズであり、シーケンシング品質は高かった。 The average sequencing depth of each Y-SNP site calculated based on 100 samples is shown in Figure 2. Library construction was smooth and sequencing quality was high.
法医学的検証の結果から明らかなように、本方法の推奨ＤＮＡ投入量は少なくとも１．２５ｎｇであるが、法医学の実践に適用される場合、ＤＮＡ濃度が極めて低い微量サンプルでも有意なハプログループ結果が得られる可能性がある。感度試験の結果を図３に示す。再現性が良好である。種特異性が良好であり、動物サンプル及び女性サンプルに本方法を適用した場合、いずれも効果的な増幅が得られなかった。正確性が高い。様々なタイプの検体のＤＮＡタイピングに適用される。２００例の漢民族集団において、合計７つの主幹ハプログループが見つかり、主なハプログループは依然としてＯ、Ｃ、Ｎ及びＤであり、中国人集団の頻度分布と一致する。細分化すると、ハプログループには９３個の末端ハプログループもあり、ＨＤ値は０．９８８３と高い。 As is clear from the results of forensic validation, the recommended DNA input amount for this method is at least 1.25 ng, but when applied to forensic practice, significant haplogroup results can be obtained even in trace amounts of samples with extremely low DNA concentrations. There is a possibility that you can get it. The results of the sensitivity test are shown in Figure 3. Good reproducibility. Species specificity was good, and when this method was applied to animal samples and female samples, no effective amplification was obtained in either case. High accuracy. Applicable to DNA typing of various types of specimens. In the Han population of 200 cases, a total of 7 main haplogroups were found, and the main haplogroups are still O, C, N and D, which is consistent with the frequency distribution of the Chinese population. When subdivided, there are 93 terminal haplogroups in the haplogroup, and the HD value is as high as 0.9883.
上記の実施例から分かるように、本発明に記載の次世代シーケンシング技術により高解像度でＹ－ＳＮＰハプログループを検出する方法は、家系解析及びサンプルの地理的起源が不明の父系種族的出身の推定に適用することができ、法医学の実践に感度が高く、解像度が高く、システムパフォーマンスが高い種族的出身推定技術を提供する。 As can be seen from the above examples, the method of detecting Y-SNP haplogroups at high resolution by next-generation sequencing technology described in the present invention is useful for genealogical analysis and for cases of paternal ethnic origin where the geographical origin of the sample is unknown. To provide an ethnic origin estimation technique that can be applied to estimation and has high sensitivity, high resolution, and high system performance in forensic medicine practice.
以上、本発明の具体的な実施例を詳しく説明したが、これらは例示に過ぎず、本発明は上記の具体的な実施例に限定されるものではない。当業者にとって、本発明に対するいかなる同等修正及び置換も本発明の範囲内に含まれる。したがって、本発明の思想及び範囲から逸脱することなくなされた同等変更及び修正は、全て本発明の範囲内に含まれるべきである。 Although specific embodiments of the present invention have been described in detail above, these are merely illustrative, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention will fall within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the invention are to be included within the scope of the invention.
Claims (10)
３８１個のＹ－ＳＮＰ部位を有する増幅プライマーを含み、
前記３８１個のＹ－ＳＮＰは、ＡＦ６、Ｐ３０５、Ｐ１０８、Ｍ６０、Ｍ９１、Ｍ１３０、Ｆ３３９３、ＣＴＳ１１０４３、Ｍ８、Ｍ３５６、Ｍ３８、Ｍ２１７、Ｌ１３７３、Ｆ３４４７、Ｆ１６９９、Ｆ３９１８、ＦＧＣ２８８８１．２、Ｆ１７５６、Ｆ３８３０、Ｙ１０４２０、Ｍ４８、Ｍ８６、Ｂ９０、Ｍ５０４、Ｆ３７９６、Ｆ９９９２、Ｆ１０６７、Ｚ１３３８、Ｚ１３００、ＣＴＳ２６５７、ＣＴＳ１１９９０、ＣＴＳ８５７９、Ｍ４０７、Ｆ３８５０、Ｚ１２２０９、Ｆ１３１９、ＣＴＳ３３８５、ＦＧＣ４５５４８、Ｆ８４５、ＣＴＳ１０９２３、ＭＦ２０９１、Ｐ１４３、Ｍ１６８、Ｍ２９４、ＣＴＳ３９４６、ＪＳＴ０２１３５５、Ｍ１７４、ＣＴＳ１１５７７、Ｍ１５、Ｎ１、Ｚ２７２６９、ＰＨ４９７９、ＰＨ１９９１、Ｙ７８２０、ＳＫ５４１、Ｚ３１６２５、Ｚ３１５８４、Ｐ９９、Ｐ４７、Ｍ５３３、Ｚ３４３５９、ＰＨ９７、Ｚ４２５９９、Ｚ４２６００、Ｚ４２６０１、Ｚ４２６０２、Ｍ１１６．１、Ｍ１４５、Ｍ９６、Ｍ８９、Ｍ４２７、Ｍ２０１、Ｐ２８７、Ｐ１５、ＣＴＳ４３６７、Ｌ３０、Ｐ３０３、Ｌ１４０、Ｆ１３２９、Ｌ９０１、Ｍ６９、Ｍ５２、Ｍ８２、Ｍ５７８、Ｍ１７０、Ｍ４２９、Ｍ５２２、Ｍ３０４、Ｍ２６７、Ｍ１７２、Ｍ４１０、Ｌ２６、Ｍ４７、Ｍ６７、Ｍ９、Ｍ５２６、Ｍ２０、Ｍ２２、Ｍ２７、Ｍ３１７、Ｐ３２６、Ｐ２５６、Ｍ２３１、Ｆ３３７３、ＣＴＳ３７５０、ＣＴＳ１１４９９、Ｆ１２０６、Ｔａｔ、Ｍ１７８、Ｌ７０８、Ｐ２９８、Ｌ３９２、ＣＴＳ１０７６０、Ｚ１９３６、Ｂ１９７、Ｍ２０１９、Ｆ１００８、Ｍ１２８、Ｂ５２３、Ｐ６３、Ｂ５２５、Ｆ２９３０、ＣＴＳ５８２、Ｍ１８１９、Ｆ５４９、Ｍ２１４、Ｍ１７５、Ｆ２６５、Ｍ１１９、Ｂ３８４、Ｐ２０３．１、Ｆ４４６、Ｆ１４０、ＣＴＳ３２６５、Ｆ７８、Ｆ８１、Ｆ５３３、Ｆ４９２、Ｆ６５６、ＳＫ１５６８、Ｚ２３４０６、Ｍ１０１、Ｚ２３３９２、ＣＴＳ８４２３、ＣＴＳ５２、ＣＴＳ７０１、Ｚ２３２６６、ＣＴＳ７１６、ＣＴＳ３５２、Ｚ２３２７１、Ｍ５０、Ｆ１０３６、Ｐ３１、Ｍ２６８、Ｆ２３２０、Ｍ１４７０、ＰＫ４、Ｍ９５、Ｆ１２５２、Ｍ８８、Ｆ２７５８、Ｆ２８９０、ＣＴＳ５８５４、Ｚ２３８１０、Ｚ２３７８１、Ｆ７８９、Ｆ８３８、ＣＴＳ１４５６、Ｆ９９３、Ｆ４１７、ＣＴＳ９９９６、Ｍ１７６、Ｐ４９、Ｍ１２２、Ｍ３２４、Ｆ１１３、Ｌ１２７．１、ＫＬ１、Ｌ４６７、Ｆ１８７６、Ｆ２１５９、Ｆ１８６７、Ｆ８５２、Ｆ２２６６、Ｌ５９９、Ｚ４３９６１、Ｚ４３９６３、Ｆ８５４、Ｚ４３９６６、Ｆ９１５、ＩＭＳ－ＪＳＴ００２６１１、Ｆ２４０、Ｆ１８、Ｆ１１７、ＣＴＳ５８２０、Ｆ１３、Ｆ１１、Ｆ６３２、Ｆ１１０、Ｆ１７、Ｆ３７７、Ｆ８５６、Ｆ１０９５、Ｆ１４１８、Ｆ１４９５、ＣＴＳ７５０１、Ｆ７９３、Ｙ２０９５１、Ｙ２０９３２、Ｆ３８、Ｆ１２、Ｆ１９６、Ｆ９３０、Ｆ２６８５、Ｆ１３６５、Ｆ３２３２、Ｙ１５９７６、Ｙ２６３８３、ＦＧＣ５４４８６、ＣＴＳ１２８７７、Ｆ２５２７、Ｆ７２３、Ｆ１４２２、ＣＴＳ２１５４、ＳＫ１６９１、ＰＨ２０３、Ｆ４４９、Ｆ２３８、Ｆ１３４、Ｆ１２７３、Ｆ７２４、Ｆ１２６６、ＣＴＳ４９８、ＦＧＣ３７５０、Ｐ２０１、Ｍ１８８、Ｆ２５８８、ＣＴＳ４４５、ＣＴＳ２０１、Ｍ１５９、Ｍ７、Ｆ２６８０、Ｆ１２７６、ＣＴＳ６４８９、Ｆ１２７５、Ｍ１１３、Ｎ５、Ｚ２５４００、Ｆ１８６３、Ｆ１１３４、Ｆ１２６２、Ｙ２６４０３、Ｆ１８３７、Ｆ８６２、Ｆ８７９、Ｆ１２２６、Ｆ２８５９、Ｐ１６４、Ｍ１３４、Ｆ４５０、Ｍ１１７、Ｍ１３３、Ｆ４２、Ｆ４３８、Ｙ１７７２８、Ｆ１５５、Ｆ８１３、Ｙ２０９２８、Ｆ１７５４、Ｆ２１３７、Ｆ１４４２、Ｆ１１２３、Ｆ１３６９、Ｆ３１０、Ｆ４０２、Ｆ１５３１、Ｚ２５８５３、ＣＴＳ５４９２、ＣＴＳ６９８７、Ｚ４２６２０、ＣＴＳ４６５８、Ｚ２５９２８、ＳＫ１７３０、Ｚ２６０１０、Ａ９４６２、ＣＴＳ７６３４、Ｆ３１７、Ｆ３０３９、Ｙ２９８６１、ＣＴＳ５４８８、ＣＴＳ１０７３８、ＣＴＳ９６７８、Ｚ３９６６３、Ａ９４５７、ＹＰ４８６４、Ｚ４４０６８、Ｆ５５２４、Ｆ５５２５、Ｚ４４０７１、Ｚ４４０９１、ＣＴＳ４９６０、Ｆ１２２、Ｆ１１４、Ｆ４４４、Ｆ７９、Ｆ６２９、Ｆ４６、ＦＧＣ１６８４７、Ｆ４８、Ｆ２５０５、Ｆ２４２、ＣＴＳ４２６６、Ｚ２６１０８、Ｆ２８８７、Ｆ３６０７、Ｆ３５２５、ＣＴＳ３７６３、Ａ９４７２、ＦＧＣ１６８６３、Ｌ１３６０、ＦＧＣ１６８８９、ＳＫ１７６８、Ｆ４２４９、ＦＧＣ２３８６８、ＣＴＳ３３５、ＣＴＳ５３、ＣＴＳ６３７３、Ａ９４７３、Ｆ３３８６、Ｙ２９８２８、ＦＧＣ３４９７３、Ｆ１７３９、Ｆ７４３、Ｆ７４８、ＡＭ０１８２２、Ｎ６、ＡＭ０１８５６、Ｎ７、Ｆ４１１０、Ｆ４０６８、Ｆ４１２４、ＡＭ０１８４５、Ｆ７１７、Ａ１６４３３、Ｆ７４２、Ｆ１１５０、Ｆ８３７、Ｆ１０２５、Ｆ１０５５、Ｆ３０２１、Ｐ２９５、Ｍ４５、Ｍ２４２、Ｆ９０３、Ｍ３４６、Ｍ３、Ｍ２０７、Ｍ１７３、Ｍ４２０、ＳＲＹ１０８３１．２、Ｍ１７、Ｚ６４５、Ｍ４５８、Ｚ９１、Ｚ９３、Ｆ３１０５、Ｙ７、Ｚ２１２４、Ｚ２１２３、Ｓ４５７６、Ｆ１３４５、Ｍ３４３、Ｌ７５４、Ｌ３８９、Ｐ２９７、Ｍ２６９、Ｍ４７９、Ｍ１２４、ＦＧＣ２１７０６、Ｂ２５４及びＭ１８４を含むことを特徴とする、組成物。 A primer composition for detecting Y-SNP haplogroup by next generation sequencing technology, comprising:
Contains an amplification primer with 381 Y-SNP sites,
The 381 Y-SNPs are AF6, P305, P108, M60, M91, M130, F3393, CTS11043, M8, M356, M38, M217, L1373, F3447, F1699, F3918, FGC28881.2, F1756, F3830, Y1 0420 , M48, M86, B90, M504, F3796, F9992, F1067, Z1338, Z1300, CTS2657, CTS11990, CTS8579, M407, F3850, Z12209, F1319, CTS3385, FGC45548, F 845, CTS10923, MF2091, P143, M168, M294, CTS3946 , JST021355, M174, CTS11577, M15, N1, Z27269, PH4979, PH1991, Y7820, SK541, Z31625, Z31584, P99, P47, M533, Z34359, PH97, Z42599, Z42600, Z42601, Z42602, M116.1, M145, M96 , M89, M427, M201, P287, P15, CTS4367, L30, P303, L140, F1329, L901, M69, M52, M82, M578, M170, M429, M522, M304, M267, M172, M410, L26, M47, M67 , M9, M526, M20, M22, M27, M317, P326, P256, M231, F3373, CTS3750, CTS11499, F1206, Tat, M178, L708, P298, L392, CTS10760, Z1936, B197, M2019 , F1008, M128, B523 , P63, B525, F2930, CTS582, M1819, F549, M214, M175, F265, M119, B384, P203.1, F446, F140, CTS3265, F78, F81, F533, F492, F656, SK1568, Z2340 6, M101, Z23392 , CTS8423, CTS52, CTS701, Z23266, CTS716, CTS352, Z23271, M50, F1036, P31, M268, F2320, M1470, PK4, M95, F1252, M88, F2758, F2890, CTS585 4, Z23810, Z23781, F789, F838, CTS1456 , F993, F417, CTS9996, M176, P49, M122, M324, F113, L127.1, KL1, L467, F1876, F2159, F1867, F852, F2266, L599, Z43961, Z43963, F854, Z43 966, F915, IMS-JST002611 , F240, F18, F117, CTS5820, F13, F11, F632, F110, F17, F377, F856, F1095, F1418, F1495, CTS7501, F793, Y20951, Y20932, F38, F12, F196, F930, F26 85, F1365, F3232 , Y15976, Y26383, FGC54486, CTS12877, F2527, F723, F1422, CTS2154, SK1691, PH203, F449, F238, F134, F1273, F724, F1266, CTS498, FGC3750, P20 1, M188, F2588, CTS445, CTS201, M159, M7 , F2680, F1276, CTS6489, F1275, M113, N5, Z25400, F1863, F1134, F1262, Y26403, F1837, F862, F879, F1226, F2859, P164, M134, F450, M117, M133 , F42, F438, Y17728, F155 , F813, Y20928, F1754, F2137, F1442, F1123, F1369, F310, F402, F1531, Z25853, CTS5492, CTS6987, Z42620, CTS4658, Z25928, SK1730, Z2 6010, A9462, CTS7634, F317, F3039, Y29861, CTS5488, CTS10738 , CTS9678, Z39663, A9457, YP4864, Z44068, F5524, F5525, Z44071, Z44091, CTS4960, F122, F114, F444, F79, F629, F46, FGC16847, F48, F2 505, F242, CTS4266, Z26108, F2887, F3607, F3525 , CTS3763, A9472, FGC16863, L1360, FGC16889, SK1768, F4249, FGC23868, CTS335, CTS53, CTS6373, A9473, F3386, Y29828, FGC34973, F1739, F743, F748, AM01822, N6, AM01856, N7, F4110, F4068, F4124 , AM01845, F717, A16433, F742, F1150, F837, F1025, F1055, F3021, P295, M45, M242, F903, M346, M3, M207, M173, M420, SRY10831.2, M17, Z645, M4 58, Z91, Z93 , F3105, Y7, Z2124, Z2123, S4576, F1345, M343, L754, L389, P297, M269, M479, M124, FGC21706, B254 and M184.
マルチプレックスＰＣＲ反応に必要な他の試薬、リンカー配列ＰＣＲ反応に必要な試薬、及び磁気ビーズ精製に必要な試薬をさらに含むことを特徴とする、キット。 A kit for detecting Y-SNP haplogroups by next generation sequencing technology, comprising the primer composition according to claim 1 or 2,
A kit further comprising other reagents necessary for multiplex PCR reaction, reagents necessary for linker sequence PCR reaction, and reagents necessary for magnetic bead purification.
以下のステップ１からステップ４を含み、
ステップ１において、マルチプレックスＰＣＲ反応を行い、具体的には、希釈したｇＤＮＡをテンプレートとし、増幅反応系を調製し、マルチプレックス断片増幅を行い、
ステップ２において、ＰＣＲ増幅産物を磁気ビーズ精製し、その後、リンカー配列ＰＣＲ反応を行い、
ステップ３において、ステップ２の増幅産物を磁気ビーズ精製してライブラリを構築し、その後、ライブラリについて濃度、断片の長さ及び純度を測定し、
ステップ４において、ライブラリを混合、変性した後、Ｉｌｌｕｍｉｎａ ＭｉＳｅｑシーケンスプラットフォームに入れてシーケンシングし、シーケンシング結果と参照ゲノム配列とを比較し、ＳＮＰ及びＩｎｄｅｌを探し、次にＡＮＮＯＶＡＲソフトウェアによりＳＮＰ、Ｉｎｄｅｌ部位に注釈を付け、変異部位に対応する遺伝子情報を確定することを特徴とする、方法。 A method for detecting a Y-SNP haplogroup by next generation sequencing technology using the kit according to claim 3 or 4, comprising:
Including steps 1 to 4 below,
In step 1, a multiplex PCR reaction is performed, specifically, using diluted gDNA as a template, preparing an amplification reaction system, performing multiplex fragment amplification,
In step 2, the PCR amplification product is purified by magnetic beads, and then a linker sequence PCR reaction is performed,
In step 3, construct a library by magnetic bead purification of the amplification product of step 2, and then measure the concentration, fragment length and purity of the library,
In step 4, after mixing and denaturing the library, it is sequenced in the Illumina MiSeq sequencing platform, and the sequencing results are compared with the reference genome sequence to search for SNPs and Indels, and then the SNP and Indel sites are determined by ANNOVAR software. 1. A method, characterized in that the genetic information corresponding to the mutation site is determined by annotating the mutation site.
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| PCT/CN2022/115542 WO2023030260A1 (en) | 2021-08-30 | 2022-08-29 | Primer composition, kit and method for detecting y-snp haplogroups based on next-generation sequencing technology and application thereof |
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| RU2539733C2 (en) * | 2012-10-24 | 2015-01-27 | Общество с ограниченной ответственностью "БИОЧИП-ИМБ" | Set of differentiating nucleotides and biochip applicable in method for genetic typing of human y-chromosomes haplogroup markers: m130 (c), m145 (de) |
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