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From cheek swabs to consensus sequences : an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes
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The Genographic Consortium (Including: Clarke, Andrew C., Prost, Stefan, Stanton, Jo-Ann L., White, W Timothy J., Kaplan, Matthew E. and Matisoo-Smith, Lisa). (2014) From cheek swabs to consensus sequences : an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes. BMC Genomics, Volume 15 (Number 1). Article number 68. doi:10.1186/1471-2164-15-68
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WRAP_Clarke_1471-2164-15-68.pdf - Published Version Available under License Creative Commons Attribution 2.0.. Download (1083Kb) | Preview |
Official URL: http://dx.doi.org/10.1186/1471-2164-15-68
Abstract
Background: Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users.
Results: Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling).
Conclusions: All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources.
| Item Type: | Journal Article | ||||
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| Subjects: | Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH426 Genetics |
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| Divisions: | Faculty of Science > Life Sciences (2010- ) | ||||
| Library of Congress Subject Headings (LCSH): | DNA, Mitochondrial DNA, Genomes, Bioinformatics | ||||
| Journal or Publication Title: | BMC Genomics | ||||
| Publisher: | BioMed Central Ltd. | ||||
| ISSN: | 1471-2164 | ||||
| Official Date: | January 2014 | ||||
| Dates: |
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| Volume: | Volume 15 | ||||
| Number: | Number 1 | ||||
| Number of Pages: | 12 | ||||
| Article Number: | Article number 68 | ||||
| DOI: | 10.1186/1471-2164-15-68 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Open Access | ||||
| Funder: | International Business Machines Corporation (IBM), Waitt Family Foundation (U.S.), National Geographic Society (U.S.) |
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