Development of a DNA Methylation Multiplex Assay for Body Fluid Identification and Age Determination

For forensic laboratories, the determination of body fluid origin of samples collected at a crime scene are typically presumptive and often destructive. However, given that in certain cases the presence of DNA is not in dispute and rather where the DNA came from is of primary concern, new methodologies are needed. Epigenetic modifications, such as DNA methylation, affect gene expression in every cell of every mammal. These DNA methylation patterns typically are observed as the addition of a methyl group on the 5’ carbon of a cytosine followed by guanine (CpG). Methylation patterns have been observed to change in response to the needs of the cell as well as to external stimulus. The investigation of DNA methylation patterns for forensic applications is a relatively new field, with the first publication in 2010. Since then, enormous growth in knowledge and technology has allowed for new and sensitive applications. Two of the primary branches of DNA methylation analysis for forensic applications are body fluid identification and age determination. In this study, we have designed, optimized and validated a body fluid identification multiplex capable of identifying saliva, blood, vaginal epithelia, and semen samples via pyrosequencing. This multiplex assay gives results consistent with the literature and the interpretation of the results can be automated by classification modeling which reduces human error. The results of the multiplex represent the first multiplex assay via pyrosequencing for body fluid identification. Lastly, the construction of a Targeted Methyl Sequencing assay for body fluid identification and age determination using next generation sequencing was explored in order to push this branch of research into the future of forensic DNA methylation analysis. As the cost of next generation sequencing begins to come down, it is important that work begins now to ensure that the tools for tomorrow’s forensic DNA analyst exist. It is our hope that the results of the targeted methyl sequencing assay serve as a starting point for an exciting future for forensic laboratories across the world