Developmental validation of an mRNA-cSNP profiling panel for body fluids identification and individualization.

Forensic analysis of body fluid stains is extremely important, which helps reconstruct crime scenes. It involves two main challenges: body fluid identification (BFID) and individualization. The former aims to ascertain the type/origin of the body fluid stain, while the latter tries to assign the stain to a specific donor. Generally, they are addressed through RNA and DNA analyses, respectively, but this can be time-consuming, laborious, and sometimes yield suboptimal results, especially mixed body fluid stains. To overcome these limitations, we developed an innovative mRNA-based sequencing panel with a dual function. This system comprises 34 body fluid-specific mRNA genes and 39 cSNP markers (amplicon length: 50 ~ 90 bp), enabling a direct link between specific body fluids and their donors. We thoroughly validated the integrated system according to the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM) on the MGI massively parallel sequencing (MPS) platform. As a result, this panel showed robust identification efficiency for body fluids at 20 ng RNA input, and the range of cumulative discrimination power (CDP) value was 0.802445047 ~ 0.999310789 in five types of body fluid. Furthermore, it had been employed in actual cases and provided the guidance. In conclusion, this system can efficiently, rapidly, and accurately perform body fluid identification and individualization for single-source and mixed samples, meeting the daily investigative demands in forensic genetics.