DNA Contamination, Degradation, Damage and Associated Microbiomes: A Comparative Analysis through Massive Parallel Sequencing and Capillary Electrophoresis

Forensic Science has entered into an exciting new technological era, where Massive Parallel Sequencing (MPS)/Next Generation Sequencing (NGS) has the potential to retrieve increasingly more nuanced and detailed genetic profile data from co-mingled sources, poor quality samples, as well as associated microbiomes. However, adopting an MPS/NGS approach, especially to the study of low copy number (LCN), aged, degraded, and/or damaged DNA samples, does not negate the fundamental complications associated with their study, namely effective recovery and downstream authentication of results. The latter is of the utmost importance in the forensic community, as advancements in more sensitive DNA technology has led to a proportionate increase in the detection of non-target contamination. This proposal will utilize MPS/NGS "shotgun" sequencing technology to evaluate the degradation of DNA contamination, in the form of touch DNA placed on human and animal bone tissue, across the course of one year at ambient temperatures. The effect of additional decontamination treatments, such as sodium hypochlorite (bleach) and ultraviolet (UV) will also be analyzed. This will allow a determination of whether time and/or decontamination procedures can mimic the expected characteristics of low copy number (LCN), aged, damaged, and degraded DNA samples often encountered in forensic settings. Utilizing the same techniques, the endogenous DNA content of the bone samples will be evaluated, allowing a comparison of damage and degradation patterns between two different biological matrices common to crime scenes as well as forensic laboratories and storage facilities. The impact of damaged nucleotide bases, as well as decreased DNA strand length and copy number, on error rates and successful amplification of genomic profiles will also be examined across MPS and capillary electrophoresis platforms. This includes whole genome enrichment (WGE), lllumina's FGX Forenseq, and Life Technologies AmpFLSTR® ldentifiler kit. Using the data generated above, we will then assess our ability to deconvolute multiple DNA profiles represented by our damaged and LCN sample set. Finally, as the MPS "shotgun" reads simultaneously include microbial sequence data, this proposal can additionally address the stability, degradation, and alterations of skin (microbial touch DNA) and bone microbiomes due to time and decontamination treatments, further determining the forensic utility of such evidence. 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