Abstract:
Conventional methods for the identification of biological fluids in a forensic setting are largely focused on
presumptive testing for protein products associated with the relevant fluid type. Recent confirmatory tests
have focused on the differential expression of mRNA in target tissues to identify blood, menstrual fluid,
vaginal material, seminal fluid and saliva. The next development in mRNA based methods for the
identification of biological fluids is the use of massively parallel sequencing techniques to target body fluid
specific biomarkers for the identification of typical forensically relevant body fluids, with the addition of
skin and urine.
This project compared the results obtained by processing the same samples in parallel through an mRNA
based reverse transcription polymerase chain reaction multiplex called CellTyper 2, and an MPS panel
with primers designed by Integrated DNA Technologies. Both single source and mixed samples were
analysed. This project aimed to not only compare the two processes, but to evaluate the performance of
the selected biomarkers in the MPS panel and establish some initial parameters for the use and analysis
of MPS data.
CellTyper 2 detected body fluid specific markers in most samples. There is some contention over whether
one of its menstrual fluid specific markers, MMP10 is specific enough, while the seminal fluid marker
TNP1 is amplified in the presence of residual DNA. The MPS panel produced promising results. Samples
with higher inputs of RNA and cDNA produced higher total read counts which was a predictor of the
quality of the MPS results. Body fluid specific biomarkers were more abundant in their target body fluid,
except in the cases of samples with low read counts (for example urine and skin). Some biomarkers were
identified as more informative than others. Overall, it appears the MPS panel of biomarkers should be
viewed holistically rather than placing too much value on any single biomarker. Future research might
involve testing more samples from a wider range of donors, identifying more specific biomarkers for the
identification of skin and urine, reviewing primer design, and replacing or removing some of the less
specific biomarkers.