Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Department of Public Health, Experimental and Forensic Medicine, Section of Legal Medicine and Forensic Sciences, University of Pavia, Pavia, Italy.
Electrophoresis. 2022 Jul;43(13-14):1521-1530. doi: 10.1002/elps.202100143. Epub 2022 May 11.
The recent introduction of polymerase chain reaction (PCR)-massively parallel sequencing (MPS) technologies in forensics has changed the approach to allelic short tandem repeat (STR) typing because sequencing cloned PCR fragments enables alleles with identical molecular weights to be distinguished based on their nucleotide sequences. Therefore, because PCR fidelity mainly depends on template integrity, new technical issues could arise in the interpretation of the results obtained from the degraded samples. In this work, a set of DNA samples degraded in vitro was used to investigate whether PCR-MPS could generate "isometric drop-ins" (IDIs; i.e., molecular products having the same length as the original allele but with a different nucleotide sequence within the repeated units). The Precision ID GlobalFiler NGS STR panel kit was used to analyze 0.5 and 1 ng of mock samples in duplicate tests (for a total of 16 PCR-MPS analyses). As expected, several well-known PCR artifacts (such as allelic dropout, stutters above the threshold) were scored; 95 IDIs with an average occurrence of 5.9 IDIs per test (min: 1, max: 11) were scored as well. In total, IDIs represented one of the most frequent artifacts. The coverage of these IDIs reached up to 981 reads (median: 239 reads), and the ratios with the coverage of the original allele ranged from 0.069 to 7.285 (median: 0.221). In addition, approximately 5.2% of the IDIs showed coverage higher than that of the original allele. Molecular analysis of these artifacts showed that they were generated in 96.8% of cases through a single nucleotide change event, with the C > T transition being the most frequent (85.7%). Thus, in a forensic evaluation of evidence, IDIs may represent an actual issue, particularly when DNA mixtures need to be interpreted because they could mislead the operator regarding the number of contributors. Overall, the molecular features of the IDIs described in this work, as well as the performance of duplicate tests, may be useful tools for managing this new class of artifacts otherwise not detected by capillary electrophoresis technology.
最近在法医学中引入聚合酶链反应（PCR）-大规模平行测序（MPS）技术改变了等位基因短串联重复序列（STR）分型的方法，因为对克隆 PCR 片段进行测序可以根据其核苷酸序列区分具有相同分子量的等位基因。因此，由于 PCR 保真度主要取决于模板完整性，因此在解释从降解样本中获得的结果时可能会出现新的技术问题。在这项工作中，使用一组体外降解的 DNA 样本来研究 PCR-MPS 是否可以产生“等体积插入物”（IDIs；即，具有与原始等位基因相同长度但在重复单元内具有不同核苷酸序列的分子产物）。使用 Precision ID GlobalFiler NGS STR 面板试剂盒分析了 0.5 和 1ng 的模拟样本，进行了两次重复测试（总共进行了 16 次 PCR-MPS 分析）。正如预期的那样，评分了几个众所周知的 PCR 伪影（例如等位基因缺失，阈值以上的停顿）；评分了 95 个 IDI，每个测试的平均 IDI 发生率为 5.9 个（最小值：1，最大值：11）。总的来说，IDIs 是最常见的伪影之一。这些 IDI 的覆盖率高达 981 个读数（中位数：239 个读数），与原始等位基因覆盖率的比值范围为 0.069 至 7.285（中位数：0.221）。此外，大约 5.2%的 IDI 显示的覆盖率高于原始等位基因。对这些伪影的分子分析表明，在 96.8%的情况下，它们是通过单个核苷酸变化事件产生的，其中 C>T 转换最为常见（85.7%）。因此，在对证据进行法医评估时，IDIs 可能是一个实际问题，尤其是在需要解释 DNA 混合物时，因为它们可能会使操作人员对贡献者的数量产生误解。总的来说，本工作中描述的 IDI 的分子特征以及重复测试的性能可能是管理这种新型未被毛细管电泳技术检测到的伪影的有用工具。
