3. Results and discussion
We developed a targeted multiplex next generation RNA sequencing assay for the tissue source determination of forensic samples using the Illumina MiSeq platform. Custom targeted oligonucleotide pools were designed to amplify thirty to fifty-five gene targets in a single reaction. Overall, sixty-six body fluid- and tissue-specific gene targets were evaluated in a number of iterative combinations, including three housekeeping genes.
The specificity of each biomarker within each of the iterative multiplex systems was evaluated using four to seven donors of each target body fluid or tissue. The ‘target hit’ or count values for each biomarker was obtained. A biomarker was determined to be suitable candidate when it demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. An example of the expression data obtained for a single source blood sample is shown in Fig. 1. As can be seen, high count values are observed for each of the eight included blood biomarkers with little to no expression of the other thirty non-blood biomarkers in the multiplex. Therefore, using this multiplex assay a blood sample could easily be distinguished due to the high specificity of the included blood-specific biomarkers. Modifications were made to each subsequent multiplex system in order to remove biomarkers with poor specificity or sensitivity (i.e. abundance). Additionally, biomarkers with extremely high expression levels, such as HBB and HBA, were also removed due to the reduction in overall efficiency of the multiplex. After initial specificity testing, a prototype 34plex system was developed. A high degree of specificity was obtained for each body fluid. For example, the percentage of reads attributable to saliva biomarkers in saliva samples ranges from 83 to 98%, for blood 96–100%, semen 98–100%, vaginal secretions 87–100% and skin 93–96%. The percentage of reads from menstrual blood, as expected, included contributions from menstrual blood biomarkers (29–96%), vaginal secretions and blood. Using the developed multiplex assay, both components in two-fluid admixed samples (blood-saliva and vaginal secretions-semen) were correctly identified.