Center for Computational and Integrative Biology, Rutgers University, Camden, NJ, 08102, USA.
Biomedical Forensic Sciences Program, Boston University School of Medicine, Boston, MA, 02118, USA.
Int J Legal Med. 2021 May;135(3):727-738. doi: 10.1007/s00414-021-02503-4. Epub 2021 Jan 23.
Current analysis of forensic DNA stains relies on the probabilistic interpretation of bulk-processed samples that represent mixed profiles consisting of an unknown number of potentially partial representations of each contributor. Single-cell methods, in contrast, offer a solution to the forensic DNA mixture problem by incorporating a step that separates cells before extraction. A forensically relevant single-cell pipeline relies on efficient direct-to-PCR extractions that are compatible with standard downstream forensic reagents. Here we demonstrate the feasibility of implementing single-cell pipelines into the forensic process by exploring four metrics of electropherogram (EPG) signal quality-i.e., allele detection rates, peak heights, peak height ratios, and peak height balance across low- to high-molecular-weight short tandem repeat (STR) markers-obtained with four direct-to-PCR extraction treatments and a common post-PCR laboratory procedure. Each treatment was used to extract DNA from 102 single buccal cells, whereupon the amplification reagents were immediately added to the tube and the DNA was amplified/injected using post-PCR conditions known to elicit a limit of detection (LoD) of one DNA molecule. The results show that most cells, regardless of extraction treatment, rendered EPGs with at least a 50% true positive allele detection rate and that allele drop-out was not cell independent. Statistical tests demonstrated that extraction treatments significantly impacted all metrics of EPG quality, where the Arcturus® PicoPure™ extraction method resulted in the lowest median allele drop-out rate, highest median average peak height, highest median average peak height ratio, and least negative median values of EPG sloping for GlobalFiler™ STR loci amplified at half volume. We, therefore, conclude the feasibility of implementing single-cell pipelines for casework purposes and demonstrate that inferential systems assuming cell independence will not be appropriate in the probabilistic interpretation of a collection of single-cell EPGs.
目前，法医 DNA 痕迹的分析依赖于对批量处理的样本进行概率解释，这些样本代表了由未知数量的每个贡献者的潜在部分表示组成的混合谱。相比之下，单细胞方法通过在提取前分离细胞来解决法医 DNA 混合物问题。一个与法医相关的单细胞管道依赖于高效的直接到 PCR 提取，这些提取与标准的下游法医试剂兼容。在这里，我们通过探索四种电泳图谱 (EPG) 信号质量指标——等位基因检测率、峰高、峰高比和低分子量到高分子量短串联重复 (STR) 标记的峰高平衡——来展示将单细胞管道纳入法医过程的可行性，这四种指标是通过四种直接到 PCR 提取处理和常见的 PCR 后实验室程序获得的。每种处理方法都用于从 102 个单个口腔细胞中提取 DNA，然后立即将扩增试剂添加到管中，并使用已知能够产生检测限 (LoD) 的 PCR 后条件来扩增/注射 DNA 一个 DNA 分子。结果表明，大多数细胞，无论提取处理如何，都产生了至少 50%的真阳性等位基因检测率的 EPG，并且等位基因缺失不是细胞独立的。统计检验表明，提取处理显著影响 EPG 质量的所有指标，其中 Arcturus® PicoPure™ 提取方法导致最低的中位等位基因缺失率、最高的中位平均峰高、最高的中位平均峰高比和 EPG 斜率的最小负中位值为 GlobalFiler™ STR 基因座的半体积扩增。因此，我们得出结论，实施单细胞管道用于案件工作是可行的，并表明在对一组单细胞 EPG 进行概率解释时，假设细胞独立性的推理系统将不适用。
