Linking STRs/SNPs and DNA methylation using massively parallel sequencing for potential forensic applications.

DNA methylation is an important target for crime trace analysis. Amongst others, the combined analysis of tissue-specific differentially methylated DNA positions (tDMP) and person-identifying single nucleotide polymorphisms (iSNP) has demonstrated potential to link known mixture contributors to their respective body fluid. As STRs are the gold standard for individualization, we aimed to implement a pipeline to perform STR analysis of bisulfite converted DNA and to characterize DNA methylation sites in their close proximity, potentially identifying body fluid-varying methylation. We analyzed forensically used STR regions in blood, semen, saliva, buccal mucosa, nasal secretion, menstrual blood and vaginal fluid using amplicon-based bisulfite massively parallel sequencing. Furthermore, we evaluated the DNA methylation of eight known tDMPs for blood, semen, saliva and vaginal fluid and ten surrounding iSNPs. Successful STR analysis and allele structure reporting from bisulfite converted DNA was possible for 18 of 22 STRs. We identified D1S1677, FGA, D5S2800, SE33, D8S1179 and D19S433 flanking sites with varying DNA methylation between body fluids, but not sufficient discrimination power for use as exclusive body fluid identification markers. A first exemplary experiment showed STR potential for assignment of the corresponding contributor in mixtures, most promising for specific mixture cases including semen and second pre-determined body fluid. We confirmed known tDMPs and linked iSNPs as valuable targets for contributor assignment in mixtures. FDSTools was used with a newly generated library file for the naming of STRs in bisulfite converted DNA and allele-linked DNAm analysis usable and editable even for other research questions and applications.