ABSTRACT 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.
Zhang et al. (Thu,) studied this question.