Identification of novel skin/sweat-specific CpG sites and development of a skin/sweat identification multiplex system...

Identification of the biological origin of forensic stains provides critical contextual information for crime scene reconstruction and evidence interpretation. Among forensic biological materials, skin/sweat is particularly important for distinguishing intimate from social contact in sexual assault investigations. Although DNA methylation-based body fluid identification has advanced considerably, reliable CpG sites for skin remain scarce. Here, we systematically screened skin/sweat-specific CpG sites using Illumina Infinium MethylationEPIC (850 K) array data from five body fluids and developed a multiplex DNA methylation assay for forensic skin/sweat identification. Following site prioritization and assay optimization, a 10-CpG multiplex panel based on amplification refractory mutation system PCR (ARMS-PCR) was established. The assay was comprehensively validated using single-source samples, simulated degraded samples, and mixed body fluid samples representative of forensic casework. The limit of detection of the 10-CpG panel was 0.25 ng. Integration of a random forest classifier enabled robust interpretation of methylation profiles and achieved 100% accuracy in distinguishing skin/sweat-containing from non-skin/sweat samples, including degraded, mixed, and casework specimens. This study represents a promising addition to the forensic epigenetic toolbox for body fluid identification, particularly in cases involving trace or degraded skin/sweat evidence.