Target RNA sequencing system for identification of body fluid contributors in mixtures

The cSNPs within body fluid-specific RNA molecules have emerged as a promising biomarker for body fluid identification and identifying body fluid donors in mixture stains. Unlike traditional DNA analysis, which is restricted to individualizing a forensic stain at a sub-source level, RNA-SNP profiling enables direct association of the body fluid with the suspect. However, the number of available RNA-SNPs is still not enough to achieve a satisfactory discriminatory power. To explore novel SNPs for RNA-based genotyping, we screened out a series of potential RNA markers and developed a target RNA sequencing assay comprising 135 RNA-SNPs across 42 body fluid genes to identify body fluid contributors in the mixture. The assessment of specificity was performed on single-source samples, and the results exhibited a clear separation between five body fluid types. The assay also showed complete genotype concordance between gDNA and cDNA, and had a capacity to analyze aged stains. In the artificial RNA mixtures consisting of 2-5 body fluid components and 2-component mock case mixtures, all body fluid components could be successfully detected, and most of the genotypes could be correctly associated with their body fluid contributors, indicating excellent discriminative capacity of our panel for mixture deconvolution. The panel presented a cumulative discriminatory power ranging from 0.987325116 (saliva) to 0.999993412 (venous blood), and could serve as an effective complementary tool to improve discriminatory power when combined with other currently available RNA-SNP panels.