COSA: A consolidated methylation-based prediction panel for body fluid/tissue of origin, smoking status, and...

DNA methylation at CpG sites has emerged as a powerful epigenetic biomarker for predicting forensically relevant traits, including chronological age, the biological origin of forensic samples encompassing body fluid and tissue sources, and lifestyle-associated factors such as smoking. Existing models for age estimation, body fluid and tissue of origin identification, and smoking inference have demonstrated robust performance, but their reliance on separate assays limits practical application. To address this gap, we developed COSA (a Consolidated prediction panel for Origin, Smoking, and Age), an integrated methylation-based assay implemented through amplicon-based massively parallel sequencing (MPS). COSA consolidates 126 previously reported CpG markers from multiple validated models into 67 amplicons, thereby enabling the simultaneous prediction of body fluid and tissue of origin, smoking status, and chronological age from a single analysis. By unifying these established markers, COSA provides a scalable and streamlined solution for comprehensive forensic epigenetic profiling. The panel comprises three functional modules. First, body fluid identification incorporates 9 CpG markers specific to blood, semen, saliva, menstrual blood, and vaginal fluid, supporting accurate determination of sample origin in forensic framework. Additionally, body fluid and tissue of origin inference extends to internal organs through 18 CpGs targeting blood, liver, skeletal muscle, heart, brain, epidermis, dermis, kidney, and lung. Second, lifestyle inference is supported by 13 CpGs, including the well-characterized cg05575921 locus in the AHRR gene for smoking prediction. Third, age estimation is incorporated through three fluid-specific models optimized for blood, saliva, and semen, which are the fluids most frequently encountered in forensic investigations. Methodological refinements were essential to achieve balanced multiplex amplification. Multiplex PCR for bisulfite-converted DNA is challenged by issues related to primer compatibility and GC-content variation. To overcome this, we implemented a touchdown PCR strategy that improved amplification balance and coverage uniformity across multiple loci. Several primer sets were newly designed or modified to optimize amplicon length and annealing temperature, ensuring robust co-amplification within the 67-amplicon panel. Importantly, using as little as 20 ng of bisulfite-converted DNA, the COSA panel supported inference of biological origin, smoking status, and chronological age. Overall, COSA integrates three major forensic prediction modules, including origin classifiers for body fluids and organ tissues, fluid-specific age estimators, and a smoking-status predictor within a single DNA workflow. This panel represents a practical and scalable tool for forensic laboratories seeking to maximize information yield from limited DNA, advancing the application of epigenetics in human identification and investigative intelligence.