Validation of a Massively Parallel Sequencing Workflow for Mitochondrial DNA Analysis at UNTHSC Center for Human Identification for Missing Persons and Traditional Casework Analyses
Thursday September 27th, 2018 // 10:00 am - 10:20 am // North Ballroom
A number of studies have evaluated the applicability of massively parallel sequencing (MPS) technologies to analyze forensic biological evidence and the benefits afforded by these technologies. For mitochondrial DNA (mtDNA) sequencing, MPS technologies now make it feasible for forensic laboratories to analyze the entire mitochondrial genome (mtGenome) with increasingly streamlined and automated workflows, enhanced data analysis options, and the commercial availability of whole genome multiplex panels designed for challenged and degraded samples. Expanding analysis to the entire mtGenome offers the opportunity for an increase in discrimination power and phylogenetic resolution in mtDNA results, and the quantitative nature of MPS technologies brings new opportunities for mixture deconvolution in mtDNA analysis. As such, analysis of the mtGenome with MPS technologies can serve as the first step in transitioning from capillary electrophoresis-based to MPS-based technologies in forensic laboratories.
The Missing Persons and Forensic Units at UNTHSC’s Center for Human Identification (UNTCHI) have begun the implementation process for mtDNA testing of biological evidence using a MPS workflow. This workflow consists of the Precision ID mtDNA Whole Genome Panel, the Ion Chef, and the Ion S5 (Thermo Fisher Scientific). Forensic analysts’ experience from an extensive training program was used to develop SOPs and workflow and throughput considerations. Finally, validation studies were performed. Sensitivity and stochastic studies demonstrated the dynamic range and limit of detection with samples ranging from 300 pg to 2 pg of DNA. MPS-specific studies also addressed the limit of detection by evaluating the amount of library input and extent of sample multiplexing. Studies of reproducibility and repeatability were completed with multiple analysts and multiple instruments. The contamination assessment evaluated blanks and known haplotypes for evidence of exogenous DNA. Known and non-probative evidence samples, including family reference samples, human remains, and hairs, were sequenced and compared to previously generated Sanger sequencing results to determine performance with potentially challenging samples. Mixtures of ratios ranging from 1:2 to 1:20 were sequenced to evaluate the capability of the system to detect and resolve mixtures. Data from these studies support that this MPS workflow yields reliable results for the analysis of biological evidence.
Research Assistant Professor, University of North Texas Health Science Center
Dr. Jennifer Churchill is currently a Research Assistant Professor in GSBS at the UNT Health Science Center where she works in the research group at the Center for Human Identification. Her research interests lie in the development and application of human identification, population genetic analyses, and development of molecular biology technologies. Jennifer’s current research projects focus on defining the molecular biology of library preparation and the chemistry of massively parallel sequencing technologies.Submit Questions