A hexaplex droplet digital PCR assay for DNA methylation-based age prediction from blood samples targeting ELOVL2,...

DNA methylation analysis has emerged as a promising approach, offering valuable information in forensic investigations. This study presents the development of a hexaplex droplet digital PCR (ddPCR) assay for forensic age prediction based on three well-established age-associated markers: ELOVL2, FHL2, and TRIM59. The assay reliably distinguished methylation levels as low as 1 %, with no cross-reactivity or signal interference in the multiplex format. To evaluate the assay's predictive performance, two statistical methods were used: hold-out cross-validation and 5-fold cross-validation. In both methods, all three markers showed strong correlations with chronological age. In the hold-out method, the model achieved a mean absolute error (MAE) of 2.80 years for the test set. Comparable performance was observed in 5-fold cross-validation, with an average MAE of 3.13 years. Prediction accuracy was higher in younger individuals and decreased with older age. The hexaplex assay allows all markers to be analyzed under identical experimental conditions, enhancing consistency, minimizing technical variability, and reducing reagent costs. Future work will include validation studies in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and application to real forensic casework to further confirm the assay's practical utility.