Background: Genetic testing at crime scenes is an instrumental molecular technique to identify or eliminate suspects, as well as to overturn wrongful convictions. Yet, genotyping alone cannot reveal the age of a sample, which could help advance the utility of crime scene samples for suspect identification. The distribution of cytosine methylation within a DNA sample can be leveraged to determine the epigenetic age of someone's blood. Methodology: We sought to demonstrate the ability of DNA methylation markers to accurately discern the age of blood spots from an actual crime scene, a "mock" crime scene, and also from a tube of blood stored in ethylenediaminetetraacetic acid for >20 years. This was achieved by quantifying methylation within known age-associated genetic loci across each DNA sample. We observed a strong linear coefficient (0.91) and high overall correlation (R 2 = 0.963) between the known age of a sample and the predicted age. Conclusion: We show that novel methods for targeted methylation and low-input whole-genome bisulfite sequencing can enable a novel and improved forensic profile of a crime scene that discerns not only who was present at the crime, but also their age. Finally, we use this model to discern the age and provenance of a blood sample that was used in a criminal investigation.
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