From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes
White, W. T.
The, Genographic Consortium
AffiliationDepartment of Anatomy, University of Otago, Dunedin, New Zealand
Allan Wilson Centre for Molecular Ecology and Evolution, Dunedin, New Zealand
Current address: School of Life Sciences, University of Warwick, Coventry, United Kingdom
Department of Integrative Biology, University of California, Berkeley, California, USA
Department of Mathematics and Statistics, University of Otago, Dunedin, New Zealand
Human Origins Genotyping Laboratory, Arizona Research Laboratories, Division of Biotechnology, University of Arizona, Arizona, USA
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CitationClarke et al. BMC Genomics 2014, 15:68 http://www.biomedcentral.com/1471-2164/15/68
Rights© 2014 Clarke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)
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AbstractBACKGROUND: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.
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