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Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

Harvey, N R ; Albury, C L ; Stuart, S ; Benton, M C ; Eccles, D A ; Connell, J R ; Sutherland, H G ; Allcock, R J N ; Lea, R A ; Haupt, L M ; Griffiths, L R Kalendar, Ruslan

PloS one, 2019-11, Vol.14 (11), p.e0224847-e0224847 [Periódico revisado por pares]

United States: Public Library of Science

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Título:
Ion torrent high throughput mitochondrial genome sequencing (HTMGS)
Autor: Harvey, N R ; Albury, C L ; Stuart, S ; Benton, M C ; Eccles, D A ; Connell, J R ; Sutherland, H G ; Allcock, R J N ; Lea, R A ; Haupt, L M ; Griffiths, L R
Kalendar, Ruslan
Assuntos: Analysis ; Bar codes ; Best practices ; Bioinformatics ; Biology and Life Sciences ; Computational biology ; Conductors ; Deoxyribonucleic acid ; DNA ; DNA sequencing ; DNA, Mitochondrial - genetics ; Elongation ; Gene sequencing ; Genetic Variation ; Genetics ; Genome, Mitochondrial ; Genomes ; Genomics ; Health ; High-Throughput Nucleotide Sequencing ; Humans ; Instrument industry ; Instrument industry (Equipment) ; International economic relations ; Methods ; Mitochondrial DNA ; Multiplexing ; Next-generation sequencing ; Nucleotide sequencing ; Optimization ; Parameter estimation ; Pedigree ; Quality Control ; Reagents ; Research and Analysis Methods
É parte de: PloS one, 2019-11, Vol.14 (11), p.e0224847-e0224847
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Current address: Human Genomics, Health and Environment Group, Institute of Environmental Science and Research, Wellington, New Zealand
Competing Interests: The authors have declared that no competing interests exist.
Current address: Peter MacCallum Cancer Centre, Parkville, Melbourne, Victoria, Australia
Descrição: 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.
Editor: United States: Public Library of Science
Idioma: Inglês

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Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

Harvey, N R ; Albury, C L ; Stuart, S ; Benton, M C ; Eccles, D A ; Connell, J R ; Sutherland, H G ; Allcock, R J N ; Lea, R A ; Haupt, L M ; Griffiths, L R Kalendar, Ruslan

United States: Public Library of Science

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