mRNA profiling and donor association of mock casework samples: Results of a 3rd and 4th EDNAP collaborative exercise

Simultaneous identification and association of body fluids to donors can serve as a powerful tool in the criminal investigation of mixed traces. Massively parallel sequencing of mRNA targets not only identifies the origin of the body fluids but may also provide additional contextual information about the body fluid donors of a (binary) mixture using coding region SNPs (cSNPs). Within the European DNA Profiling Group (EDNAP), two consecutive collaborative exercises (3rd and 4th EDNAP exercise) were organized, with the objective to evaluate the performance of two previously published high-resolution mRNA sequencing assays. In the 3rd EDNAP exercise, the BFID-cSNP-BSS assay (cSNPs for blood, saliva, and semen) was evaluated, while in the 4th EDNAP exercise, the BFID-cSNP-6F assay (cSNPs for six fluids/tissues, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin) was tested. Each RNA cSNP assay was accompanied by a genomic DNA assay for the genotyping of the cSNPs in the individual(s) or body fluid donor(s) of interest. A total of 11 laboratories participated in one or both collaborative exercises. In each exercise, the participating laboratories received a set of 16 standardized mock case stains for analysis and were encouraged to analyze additional, self-prepared stains and reference samples. Laboratories could participate using either the Ion Torrent S5™ or the Illumina MiSeq™ sequencing system. The results of the 16 mock case stains were very encouraging in both exercises, as body fluid components could be reliably identified for most of the stains. Since successful donor association depends on the number of body fluid markers covered in the sequencing results, we found that for stains consisting of blood, menstrual blood, vaginal secretion or a mixture thereof, the cSNPs provided substantial genetic discriminatory information for successful association of the respective body fluid to its donor. In mixtures, the difficulty in interpreting the cSNP genotypes might be attributed to the masking effect of the other body fluid(s) present. Body fluid identification and donor association of skin samples proved to be a significant challenge. In conclusion, body fluid identification and donor association using the BFID-cSNP-BSS and -6 F assays is a promising and effective method across laboratories and sequencing platforms.