Pushing the Boundaries: Forensic DNA Phenotyping Challenged by Single-Cell Sequencing

Abstract

Single-cell sequencing is a fast developing and very promising field; however, it is not commonly used in forensics. The main motivation behind introducing this technology into forensics is to improve mixture deconvolution, especially when a trace consists of the same cell type. Successful studies demonstrate the ability to analyze a mixture by separating single cells and obtaining CE-based STR profiles. This indicates a potential use of the method in other forensic investigations, like forensic DNA phenotyping, in which using mixed traces is not fully recommended. For this study, we collected single-source autopsy blood from which the white cells were first stained and later separated with the DEPArray™ N×T System. Groups of 20, 10, and 5 cells, as well as 20 single cells, were collected and submitted for DNA extraction. Libraries were prepared using the Ion AmpliSeq™ PhenoTrivium Panel, which includes both phenotype (HIrisPlex-S: eye, hair, and skin color) and ancestry-associated SNP-markers. Prior to sequencing, half of the single-cell-based libraries were additionally amplified and purified in order to improve the library concentrations. Ancestry and phenotype analysis resulted in nearly full consensus profiles resulting in correct predictions not only for the cells groups but also for the ten re-amplified single-cell libraries. Our results suggest that sequencing of single cells can be a promising tool used to deconvolute mixed traces submitted for forensic DNA phenotyping.

Forensic DNA Phenotyping: a review on SNP panels, genotyping techniques, and prediction models

“…However, this implies a longer preparation, sequencing, and analysis time [271]. As a result, the current focus is on testing SNaPshot™ panels using NGS instruments [98, 99,237,238,243,320], applying single cell sequencing and NGS to analyse mixtures and touch DNA samples [136,142,241,297], and automating analysis and result interpretation to reduce analysis time [23]. This last one would allow a better handling of the samples, increase simple size, and reduce costs and time.…”

Single cell genomics applications in forensic science: Current state and future directions

“…demonstrated the ability to use single cell semiconductor ion sequencing on white blood cells recovered from autopsy blood using digital cell sorting for ancestry (using bootstrapping admixture analysis across seven root populations) and DNA phenotyping (hair, skin and eye color) with both cell pools and single cells. 121 Single cell analysis with sufficient genotyping information for accurate ancestry and phenotyping inference was only achieved after library amplification prior to sequencing and the use of consensus profiling akin to that described earlier for STRs. In 2023, Kulhankova et al.…”

Cartilage Tissue in Forensic Science—State of the Art and Future Research Directions

“…Genetic phenotyping using next-generation sequencing (NGS) technology seems to be another interesting aspect of DNA isolation [86]. So far, DNA isolated from human remains from World War II allowed for determination of the eye and hair color in 100% of the studied cases [87].…”