Improving the Recovery of Degraded DNA from Historical Skeletal Remains

DNA analysis has become an essential component for resolving unidentified human remains, disaster victim identification, and cold cases. Due to the high copy number of mitochondrial DNA (mtDNA) molecules in each cell compared to nuclear DNA, mtDNA is often used in these cases to aid with identification when nuclear DNA recovery fails. The Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL) routinely works to identify individuals from highly degraded skeletal remains. In order to increase success rates for these cases, next generation sequencing methods were validated and integrated into their casework pipeline in 2016. For this analysis, extracted DNA is converted to libraries which can be sequenced. However, the current methods used for DNA extraction and library preparation have been optimized for degraded samples with DNA averaging ~70 bp in size. When these techniques are applied to the most severely degraded samples that underwent postmortem chemical treatment, the shorter fragments are often lost, resulting in failed or incomplete profiles.

The ancient DNA field has optimized DNA extraction and library preparation methods for highly degraded DNA from both skeletal and sediment samples. The success of these methods has been demonstrated by the recovery of DNA sequences from individuals older than 300,000 years¹. In this study, we compare three common forensic extraction methods to one of the most widely used ancient DNA extraction methods² using 15 skeletal remains with DNA of varying quality. We found that the ancient DNA extraction method recovered between 2 and 37 times as many DNA fragments that mapped to the human genome than any of the other extraction protocols. In addition, we compared a double stranded DNA library preparation method that is typically used for mtDNA analysis at the AFMES-AFDIL to two single stranded library preparation methods used in the ancient DNA field. The results of this study demonstrate that the integration of ancient DNA techniques into forensic workflows will help increase success rates for generating DNA profiles from degraded remains.

1. Meyer, M. et al. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature 505, 403-406, doi:10.1038/nature12788 (2014).
2. Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of the National Academy of Sciences 110, 15758, doi:10.1073/pnas.1314445110 (2013).