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.
Overall, the results demonstrate the ability to successfully
identify the body
fluid or tissue source of origin of forensic samples
using a targeted multiplex RNA NGS assay. The prototype multiplex
assay is currently being validated in terms of sensitivity and
specificity. The inclusion of body
fluid specific miRNA targets as
well as platform compatibility