Evaluation and Implementation of High Throughput Second Generation Sequencing for Mitochondrial DNA Testing in Missing Persons and Forensic Casework at the UNT Center for Human Identification.

As submitted by the applicant: UNT Center for Human Identification (UNTCHI) is one of the largest crime laboratories in the United States focused on aiding in the identification of Missing Persons through DNA testing of unidentified remains and family reference samples. The laboratory performs nuclear DNA analysis and mitochondrial DNA (mtDNA) sequence analysis and enters the DNA profiles into the Combined DNA Index System ensuring that profiles can be searched across the Nation. Since 2003, UNTCHI has received approximately 15,122 family reference samples, 7,109 unidentified decedent samples, 755 direct reference samples and 111 samples from unidentified living persons from US law enforcement agencies. Over 65% of the unidentified remains profiles and family reference pedigrees in the national databases were processed by UNTCHI. Between 2012-2014, a total of 1,462 law enforcement agencies have submitted samples to UNTCHI for missing persons cases. The ongoing successes in missing persons identifications have demonstrated that mtDNA analysis serves a pivotal role in most missing persons cases by providing a lineage-based genetic marker that solidifies pedigree relationships. Currently, we are aware of only seven public crime laboratories that perform mtDNA sequencing and regularly enter mtDNA results into CODIS. Between 2012-2015, submissions of unidentified remains and family reference samples to UNTCHI have increased by 55.3 and 74.5 percent, respectively. The problem that has developed is that although the current mtDNA workflow has been optimized using the traditional Sanger-based methods, the only mechanisms to respond to increased client demand are to increase personnel, laboratory space, and instrumentation to offset the highly manual analysis methods, or to upgrade to the current gold standard for DNA sequencing by implementing a high-throughput, automatable deep sequencing approach. The goal for this project is to begin the transition to fully sequence-based DNA typing at the UNTCHI by implementing high-throughput second-generation DNA sequencing in our mtDNA processing workflow. This project will address four major questions regarding mtDNA sequence analysis procedures: 1) How does the current Sanger-based workflow compare to high-throughput methods with regard to cost and efficiency; 2) Does sequence data produced on the second-generation platform acceptably compare to data previously generated from reference samples and unidentified remains with regard to sequence completeness, quality, and accuracy; 3) What is the learning curve for experienced mtDNA analysts in becoming proficient with the new workflow; and 4) What are the necessary Quality System considerations/changes that need to be adopted/implemented and how effective are they at insuring testing quality. Note: This project contains a research and/or development component, as defined in applicable law. ca/cf