Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

Autoři: N. R. Harvey aff001;  C. L. Albury aff001;  S. Stuart aff001;  M. C. Benton aff001;  D. A. Eccles aff001;  J. R. Connell aff001;  H. G. Sutherland aff001;  R. J. N. Allcock aff003;  R. A. Lea aff001;  L. M. Haupt aff001;  L. R. Griffiths aff001
Působiště autorů: Genomics Research Centre, School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia aff001;  Health Sciences and Medicine faculty, Bond University, Robina, Queensland, Australia aff002;  School of Biomedical Sciences, University of Western Australia (M504), Crawley, Western Australia, Australia aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224847


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.

Klíčová slova:

DNA libraries – Genome sequencing – Genomic libraries – Mitochondria – Mitochondrial DNA – Next-generation sequencing – Polymerase chain reaction – High throughput sequencing


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Článek vyšel v časopise


2019 Číslo 11