DNA samples limited in quantity or degraded are often encountered in forensic casework. The success of analyzing these samples has increased with the application of mini STR and bi-allelic SNPs. Although there is a clear advantage in targeting nuclear DNA (that is, its high discrimination power), degraded and quantity limited samples often cannot be effectively analyzed through such methods. Instead, quality limited and degraded samples can be more effectively analyzed through the use of mitochondrial DNA (mtDNA) because of its high copy number. Yet, mtDNA's small size and highly conserved genome lower its discrimination potential. Next generation sequencing technology allows for the analysis of the entire mitochondrial genome, compensating for limitations of current mtDNA analysis methods. In 2012, a colleague developed a probe capture 454 next generation sequencing assay for target enrichment and deep sequencing of the whole mitochondrial genome. She included the liquid phase probe capture step into the assay design to enrich for mtDNA and to eliminate the need for amplification primers that target the sample DNA. As a result, the designed assay has the potential to analyze highly degraded samples while bypassing amplification complications. To further improve the assay's capability of processing degraded samples, I incorporated a mechanical shearing fragmentation method independent of sample quality into the assay. The goal of my project was to optimize and validate this assay for use on limited, mixed, and degraded samples.
(Publisher abstract provided.)