Technical note: Methylation-based forensic age estimation in blood, buccal cells, saliva and semen: A comparison of two age estimation tools

Forensic age estimation of stain donors through DNA methylation has been intensively studied in recent years. To date, there are many published age estimation tools which are based on technologies including pyrosequencing, minisequencing, or MPS. With the implementation of such tools into routine forensic casework in many laboratories worldwide, there is a need for thorough evaluation and performance comparison. In this study, we tested published age estimation tools that are based on either minisequencing or MPS on four body fluids (blood, saliva, buccal cells and semen). All samples were analyzed with both technologies and the age estimates were compared. Biological replicates were taken from ten (blood, saliva, buccal cells) or 12 individuals (semen) to assess the reproducibility of each tool. Our study demonstrates high accuracy in estimating chronological age for various body fluids using both technologies, except for semen. The mean absolute errors (MAEs) ranged from three to five years for blood, saliva and buccal cells, while semen exhibited a higher MAE of seven to eight years. Despite the overall good performance for blood, saliva, and buccal cells, significant discrepancies were observed for some individuals both between the two technologies or when compared to their chronological age. Conclusively, we demonstrated that forensic age estimation tools based on two different technologies are similarly accurate for blood, saliva and buccal cells, while the semen tools need some adjustments before implementation into forensic casework. Our results could be helpful in the decision-making process for laboratories seeking to newly establish an age estimation workflow.