Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system

With the development and maturation of massively parallel sequencing (MPS) technology, the mitochondrial genome (mitogenome) sequencing is increasingly applied in the forensic field. In this study, we employed the strategy of short overlapping amplicons for the whole mitogenome, library preparation with tagmentation using the Nextera® XT DNA Library Preparation Kit, sequencing on the MiSeq FGx^TM Forensic Genomics System and analyzing data using the mitochondrial(mtDNA) MSR Plug-in and the mtDNA Variant Analyzer. A total of 27 libraries and 56 libraries were sequenced in a run using MiSeq Reagent Kit v2 and v3, respectively. Results showed more than 1800 × of averaged depth of coverage (DoC) at each position. Concordant haplotypes of 9947 A and 2800 M were obtained at 32 variants. Cross-reactivity was observed with 1 ng primate DNA and 10 ng non-primate DNA but could be easily distinguished. Full and accurate variants were obtained from at least 50 pg input DNA and from minor contributors between 19:1 and 1:19 mixed ratios with known reference profiles. More than 86% variants were detected from ≥200-bp degraded samples but its haplotype was assigned to more ancestral haplogroup. Further, a total of 3 962 variants were observed at 613 nucleotide positions from 103 Xibe mitogenomes with 25:1 ratio of transitions to transversions. Two new transversions (C13735A and A14755C) and two tri-alleles at nps 9824 and 16092 were identified. There were 103 unique mitogenome haplotypes from 103 Chinese Xibe that were assigned to 79 haplogroups. Haplogroup D was the preponderant top-level haplogroup in Xibe followed by F, B, M, A, N, G, C, Z, Y, HV and J. Random match probability (RMP) and haplotype diversity (HD) of the whole mitogenome was calculated as 0.0097 and 1.0000, respectively. Compared with HVS-I only, RMP decreased 33.56%, while the number of haplotypes and HD increased 15.73% and 0.49%, respectively. Principal component analysis (PCA) showed that Xibe was clustered to East and Southeast Asian. As a whole, this MPS strategy is suitable for the whole mitogenome sequencing especially for degraded samples and can facilitate generating mitogenome data to support the routine application in forensic sciences. EMP00726 is the first whole mitogenome dataset from Xibe contributed to the EMPOP. Supplemental data for this article are available online at.

随着大规模并行测序（massively parallel sequencing, MPS）技术的发展与成熟，线粒体基因组（mitogenome）测序在法医学领域的应用日益广泛。本研究针对全线粒体基因组采用短重叠扩增子策略，使用Nextera® XT DNA文库制备试剂盒通过转座酶片段化（tagmentation）完成文库制备，依托MiSeq FGx™法医基因组学系统进行测序，并借助线粒体DNA（mtDNA）MSR插件与mtDNA变异分析器开展数据分析。本研究分别使用MiSeq试剂试剂盒v2和v3，在单次运行中对总计27个和56个文库进行了测序。结果显示，每个位点的平均覆盖深度（depth of coverage, DoC）超过1800×。在32个变异位点处，成功获得了9947A和2800M的一致单倍型。实验发现，1ng灵长类DNA与10ng非灵长类DNA可产生交叉反应，但二者可轻易区分。当起始DNA量至少为50pg，且混合样本中次要贡献组分的比例介于19:1至1:19之间且配有已知参考图谱时，可获得完整且准确的变异位点信息。对≥200bp的降解样本，可检测到超过86%的变异位点，但所归属的单倍群更为原始。此外，对103例锡伯族个体的线粒体基因组进行分析后，在613个核苷酸位点上共检出3962个变异，其转换与颠换之比为25:1。本研究鉴定出2个新的颠换变异（C13735A和A14755C），以及位于nps 9824与16092位点的2个三等位基因位点。103例中国锡伯族个体共拥有103个独特的线粒体基因组单倍型，分别归属79个单倍群。锡伯族群体中占比最高的顶级单倍群为D，其后依次为F、B、M、A、N、G、C、Z、Y、HV与J。全线粒体基因组的随机匹配概率（random match probability, RMP）与单倍型多样性（haplotype diversity, HD）分别为0.0097和1.0000。与仅分析高变区I（HVS-I）相比，全线粒体基因组分析的RMP降低了33.56%，而单倍型数量与HD分别提升了15.73%和0.49%。主成分分析（principal component analysis, PCA）结果显示，锡伯族群体与东亚及东南亚人群聚为一类。总体而言，本研究采用的MPS策略适用于全线粒体基因组测序，尤其适用于降解样本，可为法医学常规应用提供有力的数据支持。EMP00726是首个提交至EMPOP（欧洲法医线粒体DNA人群数据库）的锡伯族全线粒体基因组数据集。本文的补充数据可在线获取。