Single-cell analysis has gained increasing interest in forensic genetics, primarily for complex mixture deconvolution based on nuclear DNA in cases where the minor component is severely underrepresented. In mitochondrial DNA (mtDNA) analysis, single-cell sequencing becomes relevant for understanding single-base sequence discrepancies in inconclusive interpretation results. Conventional bulk-cell sequencing reflects an averaged mtDNA signal across multiple cells and may obscure low-frequency variants or cellular heterogeneity, which can occur in casework samples where only a limited number of cells contribute, such as hair shafts. Investigating mtDNA variation at single-cell level may allow the detection of cellular heterogeneity and provide additional insights into mtDNA heteroplasmy, potentially improving the interpretation of challenging mtDNA profiles.

This study explores the feasibility of single-cell mtDNA sequencing in forensically relevant cell types using the semi-automated DEPArray Plus system in forensic application mode for isolating single cells. Individual epithelia cells derived from buccal-mucosa and saliva, as well as white blood cells were isolated and subjected to massively parallel sequencing using the Precision ID mtDNA Whole Genome Kit on the Ion Torrent S5 Prime System. To evaluate potential effects of the DEPArray sample preparation, modified protocols with respect to fixation and permeabilization were tested. 

Sequencing performance was assessed across different cell types and preparation conditions, focusing on metrics such as base-level retention, sequence quality, and the number of single cells meeting predefined quality criteria. Differences between cell types and preliminary trends suggesting a potential influence of sample preparation were observed.

MtDNA sequences derived from single cells passing these criteria were compared within and between cell types from the same donor and with corresponding bulk reference samples to explore patterns of mtDNA variation. Preliminary observations indicate, that point heteroplasmy variants appearing manifest in bulk samples may only be present in a subset of individual cells, while other cells display either the reference base or are homoplasmic for the variant. These findings suggest that cellular composition may contribute to (heteroplasmic) variation observed in bulk mtDNA data and highlight the importance of considering cellular heterogeneity when interpreting mtDNA variation in forensic samples, particularly in inconclusive scenarios.

Authors

• Lena Ewers (Institute of Legal Medicine, Medical University of Innsbruck, Austria)
• Arthur Brommer (Forensic Science Institute, Bavarian State Criminal Police Office (BLKA), Germany)
• Arthur Brommer (Faculty of Biology, LMU Munich, Germany)
• Thorsten Hadrys (Forensic Science Institute, Bavarian State Criminal Police Office (BLKA), Germany)
• Walther Parson (Institute of Legal Medicine, Medical University of Innsbruck, Austria)