From Forensics to Clinical Research: Expanding the Variant Calling Pipeline for the Precision ID mtDNA Whole Genome Panel

Despite a multitude of methods for the sample preparation, sequencing, and data analysis of mitochondrial DNA (mtDNA), the demand for innovation remains, particularly in comparison with nuclear DNA (nDNA) research. The Applied Biosystems (TM) Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, USA) is an innovative library preparation kit suitable for degraded samples and low DNA input. However, its bioinformatic processing occurs in the enterprise Ion Torrent Suite (TM) Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), with a heteroplasmy threshold level of 10%. Here, we present an alternative customizable pipeline, the PrecisionCallerPipeline (PCP), for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, we achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. We further validated our findings with 50 samples from an ongoing independent cohort of stroke patients, with PCP finding 98.31% of TSS's variants (TSS found 57.92% of PCP's variants), with a significant correlation between the variant levels of variants found with both pipelines.

Automated summary

Despite the various methods available for mtDNA sample preparation, sequencing, and data analysis, there is still a need for innovation compared to nDNA research. The Precision ID mtDNA Whole Genome Panel by Applied Biosystems is a novel library preparation kit suitable for degraded samples and low DNA input, however, its bioinformatic processing occurs in Ion Torrent Suite Software, producing BAM files aligned to an unorthodox version of rCRS with a heteroplasmy threshold of 10%. A customizable pipeline called PrecisionCallerPipeline (PCP) was presented in this study for processing samples correctly after Ion Torrent sequencing with the Precision ID library kit, achieving improved performance metrics compared to the proprietary software, with optimal performance at a 2.5% heteroplasmy threshold.