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

Filipe Cortes-Figueiredo,Friedemann Paul,Hansi Weissensteiner,Sebastian Schönherr,Filipa S Carvalho,Vanessa A Morais,José M Ferro,Ana Catarina Fonseca

doi:10.3390/ijms222112031

Abstract

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™ 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™ 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.

Highlights

Mitochondria are the primary source of cellular ATP, while prominently contributing to cell survival, differentiation, and apoptosis
We have developed a fully customizable pipeline—PCP—for the Precision ID mitochondrial DNA (mtDNA) Whole Genome Panel, which produced BAMs accurately mapped to the revised Cambridge Reference Sequence (rCRS) and that has revisited the heteroplasmy threshold of the current gold standard
Consistent with what we described previously, while mean coverage was significantly decreased in PCP (Figure 5B)—mean difference of 30.87%, 95% CI [29.17%–32.58%], an adjusted p-value of 3.01 × 10−34, we still observed a higher number of variants with our pipeline (Figure 5D)—mean difference of 15.11%, 95% [10.07–20.16], an adjusted p-value of

Summary

Introduction

Mitochondria are the primary source of cellular ATP, while prominently contributing to cell survival, differentiation, and apoptosis. They contain their own double-stranded circular DNA (mtDNA), with 16,569 base pairs (bps) in humans, responsible for encoding. Out of the existing bioinformatic tools for mtDNA, with quality control assessment, variant calling, and haplogroup assignment [6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21], very few incorporate user accessibility, integrative data analysis, and regular updates. A suitable option is mtDNA-Server [17], which allows for FASTQ and BAM files as input and, considering the revised Cambridge Reference Sequence (rCRS) [22] as a reference, identifies heteroplasmic and homoplasmic variants, assigns a haplogroup with HaploGrep

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