Testing new methods for degraded DNA recovery and next-generation sequencing

Human identification using DNA analysis is a central focus of forensic science. However, environmental, microbiological, and climatic factors often limit the amount of recoverable DNA (i.e. short DNA fragments and nucleotide damage) following death, especially in circumstances where only hard tissues, such as bones and teeth, are available for medico-legal assessment. Despite these limitations, the field of ancient DNA (aDNA) analysis has advanced DNA methodology to an unprecedented degree, allowing for the rapid quantification and genome-wide reconstruction of ancient samples (e.g. human, megafauna, and pathogen) dating as far back as the Middle Pleistocene (781,000 to 126,000 years ago). As a result, the applications of aDNA techniques to forensic cold cases have the potential to recover the complete genetic profiles of previously unidentified victims. These victims remained unidentified because traditional attempts failed to produce adequate DNA yields. We propose to apply aDNA techniques to forensic samples obtained from the American Southwest in Arizona, as this geographic region offers a unique environment from which to assess the breakdown of DNA obtained from unidentified cold case victims. In collaboration with Arizona’s Maricopa County Office of the Medical Examiner, we will obtain bone and teeth samples from 75 unidentified, open cases. To assess DNA degradation and identify the genetic profiles of these 75 individuals, our analysis will use state-of-the-art DNA extraction, library preparation, and targeted enrichment techniques (whole-genome SNP) coupled with Next-Generation Sequencing (NGS) technologies. In addition, we will use newly developed mini- and midi-STR multiplex amplification analysis to assess the DNA sequence damage and generate full STR profiles from these yet unknown individuals. We address these cases by asking the following questions: can we obtain mini- and midi-STR, mitochondrial genome, and whole-genome SNP data from cold cases using aDNA laboratory protocol? Can we ascertain the genetic profiles of these hitherto unknown cold case victims from Arizona? What is the success rate of the recovered DNA for standard CODIS and mini- and midi-STR analysis? Will single-stranded DNA (ssDNA) library construction produce higher quantities of host DNA than double-stranded DNA (dsDNA) techniques? What is the extent of fragmentation and damage of DNA obtained from cold case samples recovered from Arizona and, how do these arid and dry environments impact NGS sequencing and SNP data quality and quantity? Our optimized protocols will greatly benefit modern forensic laboratories across the country by providing detailed procedure to capture and sequence samples preserved in less than favorable desert conditions. 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