A Five-Color Multiplex Droplet Digital PCR Assay With Support Vector Regression for Forensic Age Prediction

Current forensic methylation analysis faces challenges: Traditional SNaPshot assays often suffer from limited inhibitor tolerance and high reagent costs, whereas massively parallel sequencing (MPS) involves a labor-intensive workflow and long turnaround time. Furthermore, conventional droplet digital PCR (ddPCR) assays typically lack high-order multiplexing capability, and most existing models are restricted to single tissue types. To address this, we validated 98 age-associated CpGs in 1398 Chinese blood methylomes and established a novel 17-plex assay utilizing an advanced 5-color fluorescence ddPCR platform. This assay was applied to 325 saliva, 107 blood, and 91 buccal swab samples. Results showed strong age correlations across tissues (Spearman's |r|: 0.554-0.956). Comparative modeling using support vector regression (SVR) versus multiple linear regression (MLR) demonstrated that SVR significantly outperformed MLR by effectively capturing non-linear methylation changes. The SVR model explained >98% of age variation with mean absolute deviations (MADs) of 2.396 years (saliva), 2.006 years (blood), and 2.044 years (buccal swabs), maintaining robust performance across broad age groups. This five-color multiplex strategy provides a rapid, cross-tissue, and cost-effective analytical method, offering a robust alternative to SNaPshot and MPS workflows for both forensic investigation and potential health-related age estimation.