新型遗传标记微单倍型（MicroHaplotypes）的分型方案和群体遗传学特征及其法医学应用研究

Prof. Kidd originally defined microhaplotypes as a powerful new type of forensic marker at the 2013 25th ISFG conference in Melbourne. We also show the interesting of microhaps and began to study on them. The microhaps are the haplotypes based on two or more SNPs within a length of 300 bp and these SNPs have very rare recombination rates. The microhaps could be a multi-allelic locus with good heterozygosity and shows extremely low mutation rates. The amplicons of microhaps alleles are balance to each other and without any stutters. By these advantages of microhaps, it shows great value for analyzing the forensic applications, such as the degraded DNA typing, mixture stains analyzing, ancestor analysis, and relatedness characterizing. The typing of the microhaps must base on the single strand analyzing. Now the platform of the next generation sequencing can be used to typing the microhaps, such as Illumina MiSeq and Ion torrent of PGM. Our group has first reported the typing result of 15 microhaps at the 2015 26th ISFG conference in Krakow through oral presentation. It’s the first typing of microhaps in the world. The microhaps also could be typing by the variant analyzing method as SSCP, DGGE, and HRM etc. Our group has set up the microhaps typing method through SSCP. By the support of this project, we could select 50 microhaps with high heterozygosity from 350 microhaps candidates by the methods mentioned before. Also we planned to develop a system for typing the multiplex of microhaps. The study will improve the value of microhaps in forensic applications.

2013年墨尔本会议Kidd教授提出微单倍型，申请人同年开始微单倍型的前期探索工作。微单倍型是全新遗传标记，指在300bp范围多个高度连锁SNPs构成的单倍型。它无Stutter峰，扩增平衡，突变率极低，多态性较好。与传统遗传标记STR与SNP比较，它综合二者优势。在混合斑鉴定，种群分析鉴定，亲缘关系鉴定等方面有较高应用价值，是理想的法医学遗传标记。其精准分型必须依赖于单链分析，现阶段可通过基于单链测序原理的二代测序技术，如Illumina与PGM等。去年我们在克拉科夫会议上首次报告了微单倍型的二代测序分型结果。微单倍型亦可通过经典序列变异分析方法如SSCP，DGGE，HRM等分型，我们已建立了基于SSCP的分析方案。进一步拟通过上述方法在350个候选位点中筛选出50个具有较高法医学应用价值的微单倍型位点组成复合系统并建立二代测序分型方案，为其在法医学领域的应用提供基础信息与基本分型方案。

微单倍型遗传标记由2个以上SNPs构成，具有比STR标记突变率低比SNP标记多态性高的优点，且从近年来发表的文献可知，此种新型遗传标记具有补充传统法医学遗传标记体系的潜力。本研究将微单倍型遗传标记作为研究对象。探索建立了一套微单倍型遗传标记的检测分析方案。共选用SSCP，DGGE，HRM以及大规模并行测序四种方法进行比较，结果显示较多微单倍型位点不能通过前三种非大规模并行测序技术进行检测分析，得出大规模并行测序为最适用于微单倍型检测分析的实验技术的结论。进一步探索利用公共数据库中的SNPs数据构建筛选微单倍型候选位点的方案。开发筛选软件，建立微单倍型候选位点数据集，便于后续研究，并提出了此新型遗传标记的命名原则。成功构建了微单倍型位点的大规模并行测序分析方案，并完成测序实验。开发软件FLfinder用于解码Illumina公司大规模并行测序平台测序生成的FASTQ文件，并对微单倍型位点进行基因分型。进一步对测序数据进行分析，获得群体遗传学数据以及法医学应用参数，对微单倍型遗传标记的法医学应用价值进行评估。综合来看，微单倍型遗传标记具有个人识别，亲缘关系鉴定及族源推断等法医学重要研究领域方面的运用潜力。

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