Deconvoluting multi-person biological mixtures and accurate characterization and identification of separated contributors using non-targeted single-cell DNA sequencing.

The genetic characterization and identification of individuals who contributed to biological mixtures are complex and mostly unresolved tasks. These tasks are relevant in various fields, particularly in forensic investigations, which frequently encounters crime scene stains generated by more than one person. Currently, forensic mixture deconvolution is mostly performed subsequent to forensic DNA profiling at the level of the mixed DNA profiles, which comes with several limitations. Some previous studies attempted at separating single cells prior to forensic DNA profiling. However, these approaches are biased at selection of the cells and, due to their targeted DNA analysis on low template DNA, provide incomplete and unreliable forensic DNA profiles. We recently demonstrated the feasibility of performing mixture deconvolution prior to forensic DNA profiling through the utilization of a non-targeted single-cell transcriptome sequencing (scRNA-seq). In addition to individual-specific mixture deconvolution, this approach also allowed accurate characterisation of biological sex, biogeographic ancestry and individual identification of the separated mixture contributors. However, RNA has the forensic disadvantage of being prone to degradation, and sequencing RNA - focussing on coding regions - limits the number of single nucleotide polymorphisms (SNPs) utilized for genetic mixture deconvolution, characterization, and identification. These limitations can be overcome by performing single-cell sequencing on the level of DNA instead of RNA. Here, for the first time, we applied non-targeted single-cell DNA sequencing (scDNA-seq) by applying the scATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) technique to address the challenges of mixture deconvolution in the forensic context. We demonstrated that scATAC-seq, together with our recently developed De-goulash data analysis pipeline, is capable of deconvoluting complex blood mixtures of five individuals from both sexes with varying biogeographic ancestries. We further showed that our approach achieved correct genetic characterization of the biological sex and the biogeographic ancestry of each of the separated mixture contributors and established their identity. Furthermore, by analysing in-silico generated scATAC-seq data mixtures, we demonstrated successful individual-specific mixture deconvolution of i) highly complex mixtures of 11 individuals, ii) balanced mixtures containing as few as 20 cells (10 per each individual), and iii) imbalanced mixtures with a ratio as low as 1:80. Overall, our proof-of-principle study demonstrates the general feasibility of scDNA-seq in general, and scATAC-seq in particular, for mixture deconvolution, genetic characterization and individual identification of the separated mixture contributors. Furthermore, it shows that compared to scRNA-seq, scDNA-seq detects more SNPs from fewer cells, providing higher sensitivity, that is valuable in forensic genetics.

Single-cell transcriptome sequencing allows genetic separation, characterization and identification of individuals in multi-person biological mixtures.

Identifying individuals from biological mixtures to which they contributed is highly relevant in crime scene investigation and various biomedical research fields, but despite previous attempts, remains nearly impossible. Here we investigated the potential of using single-cell transcriptome sequencing (scRNA-seq), coupled with a dedicated bioinformatics pipeline (De-goulash), to solve this long-standing problem. We developed a novel approach and tested it with scRNA-seq data that we de-novo generated from multi-person blood mixtures, and also in-silico mixtures we assembled from public single individual scRNA-seq datasets, involving different numbers, ratios, and bio-geographic ancestries of contributors. For all 2 up to 9-person balanced and imbalanced blood mixtures with ratios up to 1:60, we achieved a clear single-cell separation according to the contributing individuals. For all separated mixture contributors, sex and bio-geographic ancestry (maternal, paternal, and bi-parental) were correctly determined. All separated contributors were correctly individually identified with court-acceptable statistical certainty using de-novo generated whole exome sequencing reference data. In this proof-of-concept study, we demonstrate the feasibility of single-cell approaches to deconvolute biological mixtures and subsequently genetically characterise, and individually identify the separated mixture contributors. With further optimisation and implementation, this approach may eventually allow moving to challenging biological mixtures, including those found at crime scenes.