Huffman Kaitlin, Hanson Erin, Ballantyne Jack
Graduate Program in Chemistry, Department of Chemistry, University of Central Florida, PO Box 162366, Orlando, FL 32816-2366, USA.
Graduate Program in Chemistry, Department of Chemistry, University of Central Florida, PO Box 162366, Orlando, FL 32816-2366, USA; National Center for Forensic Science, PO Box 162367, Orlando, FL 32816-2367, USA; Department of Chemistry, University of Central Florida, PO Box 162366, Orlando, FL 32816-2366, USA.
Sci Justice. 2022 Mar;62(2):156-163. doi: 10.1016/j.scijus.2022.01.003. Epub 2022 Jan 20.
DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2 to 6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2-6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (≥10) from donors that had returned non-probative LRs (<10) by standard methods.
DNA混合物是犯罪现场证据的常见来源，并且通常是较难解释的生物证据来源之一。随着概率基因分型（PG）的实施，混合物分析发生了革命性变化，使得以前无法解析的混合图谱得以分析，并且能够从贡献者那里恢复 probative 基因型信息。然而，由于等位基因重叠、假象或低水平的次要贡献者，基因型信息丢失不可避免地会发生。为了减少复杂混合物中供体重要DNA信息的潜在损失，一种替代方法是在进行DNA分型之前从混合物中物理分离单个细胞，从而获得来自贡献者的单源图谱。在本研究中，一种简化的显微操作技术与增强的单细胞DNA分型相结合，用于收集一个或几个细胞，称为直接单细胞二次抽样（DSCS）。使用这种方法，从2至6人混合物中收集了单细胞和双细胞二次抽样。单细胞二次抽样产生了单源DNA图谱，而双细胞二次抽样则产生了单源DNA图谱或新的微型混合物，由于贡献者较少，这些微型混合物比原始混合物的复杂性更低。经过适当验证后，实施了PG（STRmix™）来分析原始大量混合物、单源细胞二次抽样以及来自原始2至6人混合物的双细胞微型混合物二次抽样。PG进一步允许进行重复分析，在许多情况下，这导致基因型信息显著增加，使得返回的供体似然比（LRs）与在其单源参考图谱中看到的相当（即其随机匹配概率的倒数）。在每种混合物中，与标准大量混合物分析相比，DSCS方法对每个供体都给出了更好的结果。对于5人和6人复杂混合物，DSCS从通过标准方法返回非 probative LRs（<10）的供体中恢复了高度 probative LRs（≥10）。
