The potential of mRNA markers in body fluids and personal source analysis based on the QNome nanopore sequencing

In forensic casework, unraveling the criminal nature of an event requires not only identifying the individual source of specific biological stains but also confirming their cellular origin. Recent studies have demonstrated the feasibility of identifying body fluids using specific mRNA markers and linking body fluids to their donors via coding region SNPs (cSNPs) within these mRNAs. Nanopore sequencing enables the detection of more cSNPs within longer mRNA amplicons due to its long-read capability. In this proof-of-principle study, we developed a targeted mRNA nanopore sequencing panel to simultaneously infer body fluid sources and identify individuals. This panel includes 12 body fluid-specific mRNAs and two reference genes (RGs), with a total of 41 cSNPs included in these 14 mRNA transcripts. Sequencing data showed that specific mRNAs were highly expressed in peripheral blood, semen, and menstrual blood, with an average read proportion exceeding 95% (excluding RGs reads). Cross-reactivity was observed in saliva and vaginal secretions, but all body fluid samples could still be accurately clustered. Alternative alleles were detected for 16 cSNPs, and genotyping results for randomly selected samples were validated for consistency with Sanger sequencing. The system demonstrated discriminatory power (DP) ranging from 0.5645 to 0.9017, providing information about the body fluid donor. However, further research is needed to identify more specific mRNAs, introduce additional highly polymorphic cSNPs, and perform evaluations on larger populations.