Developing a male-specific age predictive model based on Y-CpGs for forensic analysis

In forensic work, predicting the age of the criminal suspect or victim could provide beneficial clues for investigation. Epigenetic age estimation based on age-correlated DNA methylation has been one of the most widely studied methods of age estimation. However, almost all available epigenetic age prediction models are based on autosomal CpGs, which are only applicable to single-source DNA samples. In this study, we screened the available methylation data sets to identify loci with potential to meet the objectives of this study and then established a male-specific age prediction model based on 2 SNaPshot systems that contain 13 Y-CpGs and the mean absolute deviation (MAD) values were 4-6 years. The multiplex methylation SNaPshot systems and age-predictive model have been validated for sensitivity (the DNA input could be as low as 0.5 ng) and male specificity. They are supposed to have feasibility in forensic practice. In addition, it demonstrated that the method was also applicable to bloodstains, which were commonly found at crime scenes. The results showed good performance (the training set: R² = 0.9341, MAD = 4.65 years; the test set: R² = 0.8952, MAD = 5.73 years) in case investigation for predicting male age. For mixtures, when the male to female DNA ratio is 1:1, 1:10, the deviation between the actual age and the predicted age obtained by the model was less than 8 years, which offers great hope for future prediction of the age of males in mixtures and will be a powerful tool for special cases, such as sexual assault. Furthermore, the work provides a basis for the application of Y-CpGs in forensic science.