Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory (AFMES-AFDIL), Dover Air Force Base, DE 19002, USA.
SNA International, LLC; Alexandria, VA 22314, USA.
Genes (Basel). 2020 Oct 29;11(11):1290. doi: 10.3390/genes11111290.
A total of 1327 platinum-quality mitochondrial DNA haplotypes from United States (U.S.) populations were generated using a robust, semi-automated next-generation sequencing (NGS) workflow with rigorous quality control (QC). The laboratory workflow involved long-range PCR to minimize the co-amplification of nuclear mitochondrial DNA segments (NUMTs), PCR-free library preparation to reduce amplification bias, and high-coverage Illumina MiSeq sequencing to produce an average per-sample read depth of 1000 × for low-frequency (5%) variant detection. Point heteroplasmies below 10% frequency were confirmed through replicate amplification, and length heteroplasmy was quantitatively assessed using a custom read count analysis tool. Data analysis involved a redundant, dual-analyst review to minimize errors in haplotype reporting with additional QC checks performed by EMPOP. Applying these methods, eight sample sets were processed from five U.S. metapopulations (African American, Caucasian, Hispanic, Asian American, and Native American) corresponding to self-reported identity at the time of sample collection. Population analyses (e.g., haplotype frequencies, random match probabilities, and genetic distance estimates) were performed to evaluate the eight datasets, with over 95% of haplotypes unique per dataset. The platinum-quality mitogenome haplotypes presented in this study will enable forensic statistical calculations and thereby support the usage of mitogenome sequencing in forensic laboratories.
共生成了 1327 个来自美国（U.S.）人群的铂级线粒体 DNA 单倍型，使用了一种强大的、半自动的下一代测序（NGS）工作流程，并进行了严格的质量控制（QC）。实验室工作流程涉及长距离 PCR，以最小化核线粒体 DNA 片段（NUMTs）的共扩增；PCR 免费文库制备，以减少扩增偏差；高覆盖率 Illumina MiSeq 测序，以产生平均每个样本 1000×的读取深度，用于低频（5%）变体检测。通过重复扩增来确认频率低于 10%的点异质性，使用自定义读取计数分析工具定量评估长度异质性。数据分析涉及冗余的双分析师审查，以最大程度地减少单倍型报告中的错误，并由 EMPOP 进行额外的 QC 检查。应用这些方法，从五个美国混合人群（非裔美国人、白种人、西班牙裔、亚裔美国人和美洲原住民）中处理了八个样本集，这些人群在样本采集时报告了自己的身份。进行了种群分析（例如，单倍型频率、随机匹配概率和遗传距离估计）来评估这八个数据集，每个数据集的单倍型超过 95%都是独特的。本研究中呈现的铂级线粒体基因组单倍型将能够进行法医统计计算，从而支持在法医实验室中使用线粒体基因组测序。
