Sequencing of mitochondrial genomes using the Precision ID mtDNA Whole Genome Panel

Abstract

Massively parallel sequencing offers a fast and cost-effective method for sequencing of the whole mtDNA genome. The Precision ID mtDNA Whole Genome Panel amplifies the entire mtDNA genome in two multiplex PCRs with 81 primer sets using the Ion AmpliSeq™ technology. In this study, the performance of the panel was evaluated by testing different amplification methods (two-in-one or conservative), the number of PCR cycles (21, 23, and 25), and different reaction volumes (recommended volume or half-volume). Furthermore, a dilution series, controlled mtDNA mixtures, and casework samples were also sequenced. The normalised read depths of the individual fragments were highly consistent irrespectively of the amplification methods or reaction volumes used. The sequencing output matched the mixture ratios of the controlled mtDNA mixtures indicating that the sequencing results were a loyal representation of the input DNA. Complete mtDNA profiles were generated from <10 pg genomic DNA. Seven fragments with relatively low read depths and large variations in read depth were identified. One of these fragments covered part of the control region and the hypervariable region II.

Developments in forensic DNA analysis

“…The Verogen ForenSeq™ DNA Signature Prep Kit runs on the MiSeq FGx™ Forensic Genomics System, which uses Illumina technology to sequence a combination of autosomal STRs, Y-/X-STRs, and identity SNPs, and can be expanded to include phenotype-and ancestry-informative SNPs [15,16]. Kits have also been developed to target part or all of the mitochondrial genome [17][18][19]. All three of these alleles would be classified as 14 alleles on the basis of their length but determining their nucleotide sequence allows them to be discriminated.…”

Methods for the analysis of mitochondrial DNA

“…Multiple evaluations of this panel have been carried out (Gouveia et al, 2017; Pereira, Longobardi, & Borsting, 2018; Strobl et al, 2018, 2019; Woerner et al, 2018). These studies demonstrate that the panel is able to generate consistent, concordant, and reliable mitochondrial genome haplotypes.…”

“…These studies demonstrate that the panel is able to generate consistent, concordant, and reliable mitochondrial genome haplotypes. These evaluations included compromised sample types (Gouveia et al, 2017; Pereira et al, 2018; Strobl et al, 2019).…”

Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy

“…The NGS workflows were selected for this study as they are commonly used in forensics for mitogenome sequencing (e.g. [42][43][44][45][46][47][48][49][50]). These well-established NGS methods offer a quantitative analysis of mitogenome sequence data, which allowed for an investigation of the effects of the sequencing platform as well as enrichment strategy on LHP.…”