法医学杂志

法医学杂志 ›› 2017, Vol. 33 ›› Issue (4): 368-373.DOI: 10.3969/j.issn.1004-5619.2017.04.007

• 论著 • 上一篇    下一篇

基于Ion Torrent PGMTM测序系统的人mtDNA全测序分析

曹  禹1，2，邹凯南1，黄江平1，马  克3，平  原1   

  1. （1. 上海市公安局物证鉴定中心 法医物证学现场应用技术公安部重点实验室 上海市现场物证重点实验室，上海 200083； 2. 复旦大学生命科学学院，上海 200438； 3. 上海市刑事科学技术研究院，上海 200083）
  • 发布日期:2017-08-25 出版日期:2017-08-28
  • 通讯作者: 平原，男，主任法医师，主要从事法医DNA分析技术的应用和研究；E-mail：pingyuan803@gmail.com
  • 作者简介:曹禹（1984—），男，硕士研究生，主检法医师，主要从事法医物证检验鉴定；E-mail：e1195850@163.com
  • 基金资助:

    法医现场物证快速性别判断及DNA信息综合获取资助项目（14JG0500400）

Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGMTM Platform

CAO YU1,2, ZOU KAI-NAN1, HUANG JIANG-PING1, MA KE3, PING YUAN1   

  1. （1. Shanghai Key Laboratory of Crime Scene Evidence, Key Laboratory of Forensic Evidence and Science Technology, Ministry of Public Security, Institute of Forensic Science, Shanghai Public Security Bureau, Shanghai 200083, China; 2. School of Life Sciences, Fudan University, Shanghai 200438, China; 3. Shanghai Research Institute of Criminal Science and Technology, Shanghai 200083, China）
  • Online:2017-08-25 Published:2017-08-28

摘要： 目的 应用Ion Torrent PGMTM测序系统对人线粒体DNA（mitochondria DNA，mtDNA）全序列进行分析检测，研究不同组织间mtDNA序列差异情况。 方法 通过法医尸体检验采集6名无关个体的组织样本，包括胸腔血液、头发、肋软骨、指甲、骨骼肌和口腔上皮。使用4对引物对线粒体全序列进行扩增，应用Ion ShearTM Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库，并在Ion Torrent PGMTM测序系统上进行线粒体基因组全序列测序，并针对异质性位点和在HVⅠ区域突变位点，进行Sanger测序验证。 结果 所有样本的全基因组mtDNA都扩增成功，6名无关个体分属于6种不同的单倍型，同一个体不同组织之间mtDNA存在异质性差异。异质性位点和HVⅠ区域突变位点采用Sanger测序结果均得到验证。通过Kappa统计方法进行一致性检验后发现，相同个体不同组织的mtDNA序列检验结果仍具有较好的一致性。 结论 本研究所采用的人线粒体基因组全序列的测序检验方法，可以检测出同一个体不同组织间mtDNA的异质性差异，该差异具有较高的一致性，该结果对mtDNA在法庭科学中的应用具有指导作用。

关键词: 法医遗传学；DNA, 线粒体；遗传异质性；Ion Torrent PGMTM测序系统；高通量核苷酸序列分析

Abstract: Objective To analyze and detect the whole genome sequence of human mitochondrial DNA （mtDNA） by Ion Torrent PGMTM platform and to study the differences of mtDNA sequence in different tissues. Methods Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion ShearTM Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGMTM platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. Results The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. Conclusion The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science.

Key words: forensic genetics, DNA, mitochondrial, genetic heterogeneity, Ion Torrent PGMTM sequencing system, high-throughput nucleotide sequencing

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