Single-cell sequencing offers new opportunities in forensic genetics, particularly for resolving complex mixtures and distinguishing contributions from multiple body fluids. The application has been limited in the past by challenging sample quality conditions, which are often caused by environmental factors and low input quantities. Recent advances in single-cell sequencing, such as reduced cell loss and lower required input quantities, now enable the highly sensitive analysis of over 100,000 cells simultaneously. However, the conditions of cells in real forensic samples and the quality of the cells extracted for single-cell transcriptomics remain unclear. 

To address this issue, we conducted the first systematic evaluation of the feasibility of single-cell whole transcriptome analysis across forensically relevant body fluids. Key requirements for single-cell sequencing were assessed, including recovery of at least 1,000 intact cells per loading and sample viability ideally above 80%. We established an optimized workflow to extract and resuspend cells while preserving cellular integrity. Using this workflow, we evaluated cell quantity and viability across multiple body fluids and storage conditions. Single-cell sequencing was subsequently performed on four forensic sample types on the BD Rhapsody™ single-cell platform (Waters Biosciences), a microwell-based approach designed for high cell recovery and effective profiling of difficult samples. Samples included a blood–blood mixture, touch DNA, a saliva- blood mixture, and a vaginal–sperm mixture.  

In our cell counting study, more than 1,000 cells were recovered from each of the cotton swab sample tested. Sufficient intact cells and viability for single-cell sequencing were retained in blood and buccal samples stored for more than 18 months. This finding supports the compatibility of the cell extraction method with standard forensic collection procedures. Notably, even challenging forensic samples such as touch DNA or mixtures yielded informative single-cell data with robust cell counts and quality metrics. We have successfully begun conducting a whole-transcriptome analysis using a specialized bioinformatics pipeline that links transcriptional profiles to individual contributors. Our findings indicate potential applications in mixture deconvolution, phenotyping, and ancestry inference, demonstrating the technical feasibility of using single-cell transcriptomics on forensic samples. 

Authors

• Vanessa van Ackeren (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland) 
• Peter Resutik (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland) 
• Nikolai J.L. Püllen (Functional Genomics Center Zurich, University of Zurich, Switzerland) 
• Paul Gueguen (Functional Genomics Center Zurich, University of Zurich, Switzerland) 
• Natasha Arora (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland) 
• Cordula Haas (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)