A strategy for body fluid mixture analysis using semen- and epidermis-specific tDMRs and adjacent...

Forensic evidence derived from semen or epidermis is often found mixed with other body fluids, and analysis becomes particularly challenging when DNA from multiple contributors is present in similar proportions. In addition, even when tissue or body fluid are successfully identified from mixtures, determining the donor of each tissue remains difficult. To overcome these limitations, we developed and applied a combined genetic marker containing semen- or epidermis-specific methylated CpGs and nearby microhaplotypes (MHs) or single nucleotide polymorphisms (SNPs). DNA methylation array data from 29 samples-including semen, epidermis, blood, saliva, vaginal fluid, and menstrual blood-were used to identify tissue-specific CpGs. Public SNP database and blood DNA from 300 Korean individuals were further used to identify nearby MHs. In total, 21 CpG-MH loci including 16 semen-specific methylated CpGs and five epidermis-specific methylated CpGs were selected. Enzymatic conversion followed by massively parallel sequencing (MPS) was performed on 49 samples, including 16 single-source samples and 33 semen- or epidermis-containing mixtures with a 1:1 ratio, to simultaneously analyze the target CpGs and MHs. We performed tissue-specific DNA profiling using methylation status at the target CpGs. The assay demonstrated that, in mixed profiles with a 1:1 ratio-previously difficult to interpret using conventional methods-non-target tissue alleles could be effectively excluded, leaving predominantly target tissue alleles. Although tissue-specific DNA profiling requires more sophisticated modeling that accounts for allele zygosity, allele dropout, and a larger number of markers to ensure sufficient individual discrimination power, the CpG-MH assay is capable of extracting valuable information even from mixtures with similar proportions of two contributors, including semen or epidermis.