Male-specific age prediction based on Y-chromosome DNA methylation with blood using pyrosequencing

Age prediction is an important aspect of forensic science that offers valuable insight into identification. In recent years, extensive studies have been conducted on age prediction based on DNA methylation, and numerous studies have demonstrated that DNA methylation is a reliable biomarker for age prediction. However, almost all studies on age prediction based on DNA methylation have focused on age-related CpG sites in autosomes, which are concentrated on single-source DNA samples. Mixed samples, especially male-female mixed samples, are common in forensic casework. The application of Y-STRs and Y-SNPs can provide clues for the genetic typing of male individuals in male-female mixtures, but they cannot provide the age information of male individuals. Studies on Y-chromosome DNA methylation can address this issue. In this study, we identified five age-related CpG sites on the Y chromosome (Y-CpGs) and developed a male-specific age prediction model using pyrosequencing combined with a support vector machine algorithm. The mean absolute deviation of the model was 5.50 years in the training set and 6.74 years in the testing set. When we used a male blood sample to predict age, the deviation between the predicted and chronological age was 1.18 years. Then, we mixed the genomic DNA of the male and a female at ratios of 1:1, 1:5, 1:10, and 1:50, the range of deviation between the predicted and chronological age of the male in the mixture was 1.16-1.74 years. In addition, there was no significant difference between the methylation values of bloodstains and blood in the same sample, which indicates that our model is also suitable for bloodstain samples. Overall, our results show that age prediction using DNA methylation of the Y chromosome has potential applications in forensic science and can be of great help in predicting the age of males in male-female mixtures. Furthermore, this work lays the foundation for future research on age-related applications of Y-CpGs.