Optimizing the Analysis of DNA From Burned Bone Using Ancient DNA Techniques

Thermal alteration of the human skeleton reduces the amount of DNA available for STR (short tandem repeat) genotyping and downstream NGS (next-generation sequencing) analyses. As a consequence, the identity of many who died as the result of fire, such as some recent victims recovered from the California wildfires in 2018, remain unknown. Our preliminary applications of ancient DNA extraction methods to burned victims (subjected to temperatures >600°C) has shown that it is possible to reconstruct partial STR profiles using ultrashort DNA molecules. Despite these promising results, little is known about how these severely degraded DNA extracts will perform when analyzed with new STR amplification kits, such as Promega’s PowerPlex® Fusion Systems, or how ancient single- and double stranded DNA library build coupled with targeted enrichment will impact genome-wide mitochondrial DNA and single nucleotide polymorphism (SNP) reconstruction using NGS. Our prospective analysis proposes to test the limits of DNA recovery from fire-death victims using a suite of these cutting-edge genomic technologies. Our project joins researchers from Arizona State University with the Maricopa County Office of the Medical Examiner (MCOME) and the Forensic Anthropology Center (FAC) at the University of Tennessee, Knoxville. Degraded DNA from burned skeletal remains collected from the MCOME (n=15-40), in addition to experimentally burned cadavers from FAC (n=5), will be extracted using an ancient and forensic DNA purification protocol. DNA extracts will be genotyped using the newly designed PowerPlex® Fusion Systems STR kit. The extracted DNA will then be converted into double and single-stranded DNA libraries according to established ancient DNA methods. All DNA libraries will be enriched using two custom synthetic RNA bait-sets targeting the whole mitochondrial genome and 4,700 SNPs across the nuclear genome. Downstream analysis of the sequenced libraries will undergo a series of computational algorithms designed to assemble our genomic targets and detect degradation patterns in NGS data. This research probes the margins of obtaining DNA from burned human remains using novel ancient genomic technologies optimized for maximum ultra-short molecular recovery. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF