Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

Abstract

The implementation and popularity of next generation sequencing (NGS) has led to the development of various rapid whole mitochondrial genome sequencing techniques. We summarise an efficient and cost-effective NGS approach for mitochondrial genomic DNA in humans using the Ion Torrent platform, and further discuss our bioinformatics pipeline for streamlined variant calling. Ion 316 chips were utilised with the Ion Torrent semi-conductor platform Personal Genome Machine (PGM) to perform tandem sequencing of mitochondrial genomes from the core pedigree (n = 315) of the Norfolk Island Health Study. Key improvements from commercial methods focus on the initial PCR step, which currently requires extensive optimisation to ensure the accurate and reproducible elongation of each section of the complete mitochondrial genome. Dual-platform barcodes were incorporated into our protocol thereby extending its potential application onto Illumina-based systems. Our bioinformatics pipeline consists of a modified version of GATK best practices tailored for mitochondrial genomic data. When compared with current commercial methods, our method, termed high throughput mitochondrial genome sequencing (HTMGS), allows high multiplexing of samples and the use of alternate library preparation reagents at a lower cost per sample (~1.7 times) when compared to current commercial methodologies. Our HTMGS methodology also provides robust mitochondrial sequencing data (>450X average coverage) that can be applied and modified to suit various study designs. On average, we were able to identify ~30 variants per sample with 572 variants observed across 315 samples. We have developed a high throughput sequencing and analysis method targeting complete mitochondrial genomes; with the potential to be platform agnostic with analysis options that adhere to current best practices.

Investigating the influence of mtDNA and nuclear encoded mitochondrial variants on high intensity interval training outcomes

“…Briefly, the concentration and purity of genomic DNA (gDNA) from all samples was assessed via Nanodrop spectrophotometry and Qubit fluorometry. We used an in-house sequencing method recently developed by our group at the Genomics Research Centre, Queensland University of Technology, Australia to sequence the whole mitochondrial genome of each participant 42 . Illumina Infinium Microarray was used on HumanCoreExome-24v1.1 bead chip to genotype all samples for ~ 550,000 loci.…”

“…Mutations within tRNA genes have previously been associated with reduction in organelle quantity and downregulation of protein synthesis 38 . Secondly, both synonymous variants in the ND4 (g.A11467G) and ND5 (g.G12372A) genes result in a codon that is used far less frequently (CUA [276] > CUG [42] ) in mitochondrial translation processes 39 . As the biosynthesis of tRNAs is costly with respect to intracellular energy levels, it is possible that the combination of the dysregulation of the tRNA leu and the codon usage frequency change in two subunits of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I) may result in premature intracellular energy (ATP) deficiency and contribute to the poor response to exercise training associated with these traits.…”

“…A bioinformatics pipeline (SAMtools, BCFtools) was utilised to generate variant call files (VCF) for all samples as described previously 42 . VCF files were then aligned to the revised Cambridge Reference Sequence (rCRS) and all sequences were stringently left aligned back to this reference genome to account for the single end (SE) reads generated from Ion Torrent sequence information.…”

Identification of novel mitochondrial and mitochondrial related genetic loci associated with exercise response in the Gene SMART study

“…Briefly, the concentration and purity of genomic DNA (gDNA) from all samples was assessed via Nanodrop spectrophotometry and Qubit fluorometry. We used an in-house sequencing method recently developed by our group at the Genomics Research Centre, Queensland University of Technology, Australia to sequence the whole mitochondrial genome of each participant (38). Illumina Infinium Microarray was used on HumanCoreExome-24v1.1 bead chip to genotype all samples for ∼550,000 loci.…”

“…A bioinformatics pipeline ( SAMtools, BCFtools ) was utilised to generate variant call files (VCF) for all samples as described previously (38). VCF files were then aligned to the revised Cambridge Reference Sequence (rCRS) and all sequences were stringently left aligned back to this reference genome to account for the single end (SE) reads generated from Ion Torrent sequence information.…”