Searching the undetected mtDNA variants in forensic MPS data

Area of Science:

• Forensic Genetics
• Bioinformatics
• Mitochondrial DNA Analysis

Background:

• Massively parallel sequencing (MPS) is efficient for forensic mtDNA analysis, but data interpretation remains challenging.
• Existing bioinformatics tools often have high costs or specific system requirements, with few free, user-friendly options.
• Accurate variant calling is crucial for reliable forensic genetic analysis.

Purpose of the Study:

• To compare the performance of two bioinformatics tools, Ion Torrent Variant Caller (TVC) and mtDNA-Server (mDS), for analyzing forensic mtDNA data obtained via MPS.
• To assess the accuracy of these tools against manual inspection and Sanger sequencing confirmation.
• To evaluate the effectiveness of these tools in identifying polymorphisms and heteroplasmic variants.

Main Methods:

• Sequencing of 43 full mitochondrial genomes (mtGenomes) using the Ion Personal Genome Machine™ (PGM™) System.
• Analysis of MPS data using the TVC plug-in and the web-based mDS tool.
• Comparison of tool outputs with manual inspection using Integrative Genomics Viewer (IGV) and confirmation via Sanger sequencing.

Main Results:

• Thirty-nine discordant variant calls were identified between the tools and manual inspection, with Sanger sequencing confirming all but 7 deletions.
• Combined use of IGV, Sanger sequencing, TVC, and mDS improved variant assignment accuracy, detecting 32 additional true polymorphisms.
• Regarding heteroplasmy, TVC detected 28 out of 30 variants, while mDS detected 22.

Conclusions:

• Neither TVC nor mDS alone provided a perfect solution for forensic mtGenome MPS data evaluation.
• Manual inspection (IGV) and Sanger sequencing confirmation are essential for accurate variant assignment.
• Expert review remains critical for comprehensive analysis of forensic mtGenome MPS data.