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

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.