Whole Mitochondrial Genomes Assembled from Thermally Altered Forensic Bones and Teeth

The recovery and analysis of genetic material obtained from thermally altered human bones and teeth are increasingly important to forensic investigations, especially in cases where soft-tissue identification is no longer possible. Although little is known about how these fire-related processes affect DNA degradation over time, next-generation sequencing technology in combination with traditional osteobiographical applications may provide us clues to these questions. In this study, we compare whole-mitochondrial genome data generated using two different DNA extraction methods from 27 thermally altered samples from fire victims from Maricopa County, Arizona. DNA extracts were converted to double-stranded DNA libraries and enriched for whole-mitochondrial DNA using synthetic biotinylated RNA baits, then sequenced on an Illumina MiSeq. We processed the mitochondrial data using an in-house computational pipeline (MitoPipe1.0) composed of ancient DNA and modern genomics applications, then compared the resulting information across the two extraction types and five burn categories. Our analysis shows that DNA fragmentation increases with temperature, but that the acute insult from fire combined with the lack of water is insufficient to produce 5’ and 3’ terminal deamination characteristic of ancient DNA. Our data also suggest an acute and significant point of DNA degradation between 350˚C and 550˚C, and that the likelihood of generating high quality mtDNA haplogroup calls decreases significantly at temperatures >550˚C. This research is part of a concerted effort to understand how fire affects our ability to generate genetic profiles suitable for forensic identification purposes.