Guangzhou Key Laboratory of Forensic Multi‑Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou 510515, China.
Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases, College of Stomatology, Xi'an Jiaotong University, Xi'an 710049, China; Department of Forensic Medicine, Chongqing Medical University, Chongqing 400010, China.
Forensic Sci Int Genet. 2024 Nov;73:103126. doi: 10.1016/j.fsigen.2024.103126. Epub 2024 Aug 22.
Mitochondrial DNA (mtDNA) is an important genetic marker for degraded biological sample identification, maternal pedigree tracing, and population genetic structure study owing to its characteristics of high copy number, anti-degradable ring structure, and maternal inheritance. Whole mtDNA genome sequencing is an optimal method for the analysis of mtDNA polymorphism and heterogeneity because it allows for the comprehensive use of maternal genetic information. However, because of lacking quantitative evaluations for sequencing data, the scientific interpretation standards for mtDNA sequencing results of the previously used sequencing systems are often different, and false positive or false negative results are prone to occur when faced with the interference of nuclear genomic DNA, or the heterogeneities of mtDNA sequence and structure. In this study, we evaluated a novel mtDNA whole genome sequencing system using long fragment amplification strategy on the DNA nanoball (DNB) sequencing platform. This system demonstrated high sequencing quality and specific mtDNA sequencing efficiencies on positive control DNA and FTA bloodstain samples, as the average Q20 and Q30 values of the corresponding samples were 97.17 % and 91.93 %; 97.37 % and 92.48 %, respectively. The mean mapping percentages for the reference sequences of whole genome DNA (wgDNA), mtDNA, and nuclear genomic DNA (ngDNA) in the corresponding samples were 99.98 %, 99.97 %, 0.03 %, and 99.91 %, 99.40 %, 0.60 %; respectively. The average error calling rates for the bases A, C, G, and T of the whole mtDNA genome were 0.2519 %, 0.2550 %, 0.2906 %; and 0.2392 %, respectively. The efficacy of heteroplasmy identification was assessed using a set of theoretical sites with predetermined rates. These sites were created by combining the samples with known mtDNA haplotypes in certain proportions. The absolute errors between observed and theoretical heteroplasmy values were 89.59 %, 74.68 %, 50.20 %, 12.65 %, 8.31 %, and 4.85 %, while the theoretical heteroplasmy values were 5 %, 10 %, 20 %, 80 %, 90 %, and 95 %, respectively. The absolute error exhibited relative stability when the mtDNA sequencing depth exceeded 500×. Furthermore, the system sequencing efficiency was also confirmed among different kinds of samples, and these samples included natural samples (e.g., peripheral blood samples preserved on FTA cards for 2 and 11 years, and on filter paper for 6 and 9 years), degraded samples, sensitivity samples, samples derived from various bodily fluids, and maternal pedigree samples. In summary, the whole mtDNA genome sequencing system used for forensic identification demonstrated high performance in analyzing mtDNA sequence information, and showed significant prospects for forensic application and maternal genetic research.
线粒体 DNA（mtDNA）因其高拷贝数、抗降解的环状结构和母系遗传等特点，成为鉴定降解生物样本、追溯母系谱系和研究种群遗传结构的重要遗传标记。全 mtDNA 基因组测序是分析 mtDNA 多态性和异质性的最佳方法，因为它可以充分利用母系遗传信息。然而，由于缺乏对测序数据的定量评估，先前使用的测序系统的 mtDNA 测序结果的科学解释标准往往不同，并且当受到核基因组 DNA 的干扰或 mtDNA 序列和结构的异质性时，容易出现假阳性或假阴性结果。在本研究中，我们使用基于 DNA 纳米球（DNB）测序平台的长片段扩增策略，对一种新型 mtDNA 全基因组测序系统进行了评估。该系统在阳性对照 DNA 和 FTA 血斑样本上表现出了高测序质量和特异性 mtDNA 测序效率，相应样本的平均 Q20 和 Q30 值分别为 97.17%和 91.93%；97.37%和 92.48%。相应样本中全基因组 DNA（wgDNA）、mtDNA 和核基因组 DNA（ngDNA）参考序列的平均映射百分比分别为 99.98%、99.97%、0.03%和 99.91%、99.40%、0.60%。整个 mtDNA 基因组碱基 A、C、G 和 T 的平均错误呼叫率分别为 0.2519%、0.2550%、0.2906%和 0.2392%。使用一组具有预定突变率的理论位点来评估异质体识别的功效。这些位点是通过将具有已知 mtDNA 单倍型的样本以特定比例混合而创建的。观察到的和理论异质体值之间的绝对误差分别为 89.59%、74.68%、50.20%、12.65%、8.31%和 4.85%，而理论异质体值分别为 5%、10%、20%、80%、90%和 95%。当 mtDNA 测序深度超过 500×时，绝对误差表现出相对稳定性。此外，该系统的测序效率也在不同类型的样本中得到了证实，这些样本包括自然样本（例如，保存在 FTA 卡上 2 年和 11 年、保存在滤纸上 6 年和 9 年的外周血样本）、降解样本、灵敏度样本、来源于各种体液的样本和母系谱系样本。总之，用于法医鉴定的全 mtDNA 基因组测序系统在分析 mtDNA 序列信息方面表现出了高性能，并且在法医学应用和母系遗传研究方面具有广阔的前景。
