Development of DNA methylation markers for sperm, saliva and blood identification using pyrosequencing and qPCR/HRM

Discrimination of body fluids can provide important information in the investigation of crime scenes. The goal of this project was to identify new sets of tissue specific differentially methylated regions (tDMRs) and develop assays that can be utilized for forensic discrimination of body fluids, in particular sperm, saliva and blood. In this study, a sample set containing semen with sperm, semen without sperm, buccal swabs, saliva (oral fluids), venous blood, menstrual blood, vaginal secretions, and sweat/skin samples were used to develop four assays. Two methods for the analysis of DNA methylation biomarkers were developed in this paper: pyrosequencing and quantitative PCR/high resolution melt (HRM) analysis. Using an epigenome wide association study, two markers, NMUR2 and UBE2U, were found to be specific for sperm, based on the fact that mean DNA methylation levels for semen (containing sperm cells) were significantly lower than mean DNA methylation levels of other body fluids. In addition, one marker (SA-6) was hypermethylated in saliva when compared to other body fluids. The assays developed for NMUR2, UBE2U and SA-6 markers can be applied in forensic tissue identification using both pyrosequencing and HRM analysis. Additionally, a set of CpG sites in the AHRR locus were hypomethylated in blood when compared to other tissues using pyrosequencing. However, this locus was not amenable to HRM analysis. Overall, this work demonstrates the discovery and application of tDMRs for forensic applications.