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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 ISSN 1471-2164.

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Official URL: http://dx.doi.org/10.1186/1471-2164-15-68

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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
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Divisions: Faculty of Science, Engineering and Medicine > 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:
DateEvent
January 2014Published
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 (Creative Commons)
Date of first compliant deposit: 26 December 2015
Date of first compliant Open Access: 26 December 2015
Funder: International Business Machines Corporation (IBM), Waitt Family Foundation (U.S.), National Geographic Society (U.S.)

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