Internal Validation of a Mitochondrial DNA Control Region Sequencing Workflow Using Precision ID mtDNA Whole Genome Panel, Ion Chef™ System and Ion S5™ XL System

<b>Background/Objectives</b>: Mitochondrial DNA (mtDNA) analysis is an essential tool for human identification in contexts such as disaster victim identification (DVI) and missing persons cases, where the remains may be highly degraded or even skeletonised. Traditionally, capillary electrophoresis (CE)-based Sanger sequencing has been the standard method for analysing the mtDNA control region. With the development of massively parallel sequencing (MPS) technologies, mtDNA sequencing using MPS offers advantages over traditional Sanger sequencing, such as increased sensitivity, higher throughput, and less sample consumption. The Ion Chef™ and Ion S5™ XL system from Thermo Fisher Scientific represents one such MPS system. <b>Methods</b>: We conducted an internal validation study evaluating key parameters including (a) concordance, repeatability and reproducibility; (b) potential cross-contamination; (c) sensitivity; (d) effects of library pooling on read depth; and (e) mixture sample analysis. Additionally, to mimic samples typically encountered during forensic investigations, case type samples were also used to evaluate the performance of this workflow. While the entire mitochondrial genome was sequenced in this validation study, considering that the international guidelines for full mtDNA genome analysis and interpretation have yet to be fully updated, our analysis, interpretation and subsequent implementation are limited to the control region only. <b>Results</b>: The results obtained demonstrated the reliability, sensitivity and reproducibility of this MPS workflow. <b>Conclusions</b>: This internal validation study supports the implementation of this workflow in our laboratory for the analysis of forensic casework samples.