Single-cell forensic genetics is emerging as a valuable approach for the resolution of complex forensic DNA mixtures, especially where probabilistic models are limited or minor components are insufficiently captured, as it enables deconvolution of the biological material prior to downstream genetic analysis. This technology is particularly promising in sexual assault cases involving multiple perpetrators. Furthermore, other genetic applications, such as forensic DNA phenotyping, are difficult to implement on DNA mixtures.

The semi-automated DEPArray technology enables the isolation of single white blood, epithelial and sperm cells for subsequent DNA profiling. Single cells can be recovered from various sources, including single-source samples as well as complex tissue mixtures from different donors. Despite its strong potential for resolving complex mixtures, single-cell forensic genetics remains challenged by the downstream analysis of extremely low-template DNA. In the framework of the SCORE (Single Cell Forensic Genetics) consortium we aim to evaluate and optimize a workflow for single-cell analysis using the DEPArray technology.

In this study, we evaluated the performance of STR analysis on single cells using Massively Parallel Sequencing (MPS). We showed that its sensitivity is comparable to that of conventional capillary electrophoresis analysis, with respect to platform-specific differences in profile quality measures, such as stutter and allelic imbalance. In addition, MPS provides further information, including sequence-based allele discrimination and the detection of isoalleles. We further investigated whether whole genome amplification (WGA) can enhance the efficiency of single-cell analysis without increasing the risk for drop-ins or other artefacts. For this purpose, single-cell STR analyses were performed with and without WGA, and sensitivity and additional quality parameters were compared. Employing a WGA protocol prior to genetic analysis may furthermore enable multiple downstream applications from a single cell, including forensic DNA phenotyping.

Authors

• Arthur Brommer (Forensic Science Institute, Bavarian State Criminal Police Office (BLKA), Germany)
• Arthur Brommer (Faculty of Biology, Ludwig-Maximilians-Universität München (LMU Munich), Germany)
• Lisa Dierig (Institute of Legal Medicine, University Hospital of Ulm, Germany)
• Angelika Fürst (Forensic Science Institute, Bavarian State Criminal Police Office (BLKA), Germany)
• Michael Kluge (Forensic Science Institute, Bavarian State Criminal Police Office (BLKA), Germany)
• Bernhard Polzer (Fraunhofer Insitute for Toxicology and Experimental Medicine, Germany)
• Walther Parson (Institute of Legal Medicine, Medical University of Innsbruck, Austria)
• Walther Parson (Forensic Science Program, The Pennsylvania State University, United States of America)
• Thorsten Hadrys (Forensic Science Institute, Bavarian State Criminal Police Office (BLKA), Germany)