Ion torrent high throughput mitochondrial genome sequencing (HTMGS).
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 fu...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2019-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0224847 |
| _version_ | 1818833675990597632 |
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| author | N R Harvey C L Albury S Stuart M C Benton D A Eccles J R Connell H G Sutherland R J N Allcock R A Lea L M Haupt L R Griffiths |
| author_facet | N R Harvey C L Albury S Stuart M C Benton D A Eccles J R Connell H G Sutherland R J N Allcock R A Lea L M Haupt L R Griffiths |
| author_sort | N R Harvey |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-19T02:22:42Z |
| format | Article |
| id | doaj.art-dfe5f28153ed49adafb408dcd6145d52 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-19T02:22:42Z |
| publishDate | 2019-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-dfe5f28153ed49adafb408dcd6145d522022-12-21T20:40:06ZengPublic Library of Science (PLoS)PLoS ONE1932-62032019-01-011411e022484710.1371/journal.pone.0224847Ion torrent high throughput mitochondrial genome sequencing (HTMGS).N R HarveyC L AlburyS StuartM C BentonD A EcclesJ R ConnellH G SutherlandR J N AllcockR A LeaL M HauptL R GriffithsThe 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.https://doi.org/10.1371/journal.pone.0224847 |
| spellingShingle | N R Harvey C L Albury S Stuart M C Benton D A Eccles J R Connell H G Sutherland R J N Allcock R A Lea L M Haupt L R Griffiths Ion torrent high throughput mitochondrial genome sequencing (HTMGS). PLoS ONE |
| title | Ion torrent high throughput mitochondrial genome sequencing (HTMGS). |
| title_full | Ion torrent high throughput mitochondrial genome sequencing (HTMGS). |
| title_fullStr | Ion torrent high throughput mitochondrial genome sequencing (HTMGS). |
| title_full_unstemmed | Ion torrent high throughput mitochondrial genome sequencing (HTMGS). |
| title_short | Ion torrent high throughput mitochondrial genome sequencing (HTMGS). |
| title_sort | ion torrent high throughput mitochondrial genome sequencing htmgs |
| url | https://doi.org/10.1371/journal.pone.0224847 |
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