Targeted S5 RNA sequencing assay for the identification and direct association of common body fluids with DNA donors in mixtures.

The evidentiary value of DNA profiles varies depending upon the context in which the DNA was found. Linking a DNA profile to a particular cellular phenotype in mixtures may aid in assessing its evidentiary relevance and value. We report the development of two dual-function high-resolution messenger RNA (mRNA) sequencing assays that can each identify the presence of 6 body fluids/tissues (blood, semen, saliva, vaginal secretions, menstrual blood, skin) and, via coding region SNPs (cSNPs) present in the body fluid-specific mRNA transcripts, directly associate particular body fluids with their specific DNA donors in mixtures. The original blood, semen, and saliva (BSS) assay contains 23 cSNPs for blood, semen, and saliva, while the expanded 6F (all 6 fluids/tissues) assay encompasses the BSS assay and also contains 23 additional cSNPs for vaginal secretions, menstrual blood, and skin. Software tools were developed to infer the identity of the body fluids present as well as providing the corresponding cSNP genotypes. Concomitant genomic DNA assays (BSS-d and 6F-d), required to genotype the same cSNPs from persons of interest/inferred contributors to the body fluid mixture, were also developed. Body fluid specificity was demonstrated by the ability to identify the body fluid origin of single-source and two-fluid admixtures. The discriminatory power (European Caucasians) for all body fluids is 0.957-0.997, with linkage disequilibrium considered. Reciprocal body fluid admixtures (mixture pairs with the same two donors but reversed body fluid types) were used to demonstrate the ability to identify the body fluid source of origin as well as associate the donor of each of the two fluids.