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From forensics to clinical research: expanding the variant calling pipeline for the precision ID mtDNA whole genome panel

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Item Type:Article
Title:From forensics to clinical research: expanding the variant calling pipeline for the precision ID mtDNA whole genome panel
Creators Name:Cortes-Figueiredo, F. and Carvalho, F.S. and Fonseca, A.C. and Paul, F. and Ferro, J.M. and Schönherr, S. and Weissensteiner, H. and Morais, V.A.
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.
Keywords:Mitochondrial DNA, Next-Generation Sequencing, Massively Parallel Sequencing, Whole Genome Sequencing, Precision ID, Thermo Fisher Scientific, Variant Calling, Mixture, Performance Metrics
Source:International Journal of Molecular Sciences
ISSN:1422-0067
Publisher:MDPI
Volume:22
Number:21
Page Range:12031
Date:6 November 2021
Official Publication:http://doi.org/10.3390/ijms222112031
PubMed:View item in PubMed

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