Development and preliminary validation of a 12-CpG methylation SNaPshot multiplex assay for forensic age estimation from semen

Accurate age estimation from semen is crucial for forensic investigations of sexual assault cases. This study aimed to develop and validate a single-reaction capillary electrophoresis (CE)-based methylation SNaPshot multiplex assay for age estimation from semen in a Han Chinese population. Twelve age-related CpG (AR-CpG) markers identified from our prior genome-wide screening studies were incorporated into a 13-plex SNaPshot assay (12 AR-CpGs plus one bisulfite conversion control). Semen samples from 260 Han Chinese males aged 22.00-67.19 years were analyzed. Three regression models-multiple linear regression (MLR), support vector regression (SVR), and multiple quantile regression (MQR)-were developed and evaluated using a 70/30 train-test split and 100-repeat nested 10-fold cross-validation. Sensitivity (n = 5 donors, triplicate conversions, 20-1 ng inputs) and 118-day ambient stain stability (n = 5 pairs) were initially characterized. Cross-platform concordance was assessed by comparing SNaPshot and bisulfite amplicon sequencing (BSAS) data from 247 shared samples. In the independent testing set (n = 76), SVR achieved the lowest mean absolute error (MAE) of 2.82 years (root mean squared error = 3.74; r = 0.927), confirmed by cross-validated MAE of 2.86 ± 0.44 years. Sensitivity data indicated adequate reproducibility at ≥ 10 ng inputs (inter-replicate SD ≤ 1.55 years), and 118-day stains showed ~1-year mean deviation from fresh semen. Cross-platform analysis confirmed that age estimation models are platform-specific and must be applied within their validated framework. The 12-CpG methylation SNaPshot multiplex assay provides an accurate, cost-effective, and practically implementable tool for semen donor age estimation that is directly compatible with existing forensic laboratory CE infrastructure.