From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

Persistent Link:
http://hdl.handle.net/10150/610024
Title:
From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes
Author:
Clarke, Andrew; Prost, Stefan; Stanton, Jo-Ann; White, W. T.; Kaplan, Matthew; Matisoo-Smith, Elizabeth; The, Genographic Consortium
Affiliation:
Department 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
Issue Date:
2014
Publisher:
BioMed Central
Citation:
Clarke et al. BMC Genomics 2014, 15:68 http://www.biomedcentral.com/1471-2164/15/68
Journal:
BMC Genomics
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)
Collection Information:
This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.
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.
EISSN:
1471-2164
DOI:
10.1186/1471-2164-15-68
Keywords:
Human; Mitochondrial DNA; Next-generation sequencing; 454 sequencing; Long-range PCR; Bioinformatics
Version:
Final published version
Additional Links:
http://www.biomedcentral.com/1471-2164/15/68

Full metadata record

DC FieldValue Language
dc.contributor.authorClarke, Andrewen
dc.contributor.authorProst, Stefanen
dc.contributor.authorStanton, Jo-Annen
dc.contributor.authorWhite, W. T.en
dc.contributor.authorKaplan, Matthewen
dc.contributor.authorMatisoo-Smith, Elizabethen
dc.contributor.authorThe, Genographic Consortiumen
dc.date.accessioned2016-05-20T08:56:45Z-
dc.date.available2016-05-20T08:56:45Z-
dc.date.issued2014en
dc.identifier.citationClarke et al. BMC Genomics 2014, 15:68 http://www.biomedcentral.com/1471-2164/15/68en
dc.identifier.doi10.1186/1471-2164-15-68en
dc.identifier.urihttp://hdl.handle.net/10150/610024-
dc.description.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.en
dc.language.isoenen
dc.publisherBioMed Centralen
dc.relation.urlhttp://www.biomedcentral.com/1471-2164/15/68en
dc.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)en
dc.subjectHumanen
dc.subjectMitochondrial DNAen
dc.subjectNext-generation sequencingen
dc.subject454 sequencingen
dc.subjectLong-range PCRen
dc.subjectBioinformaticsen
dc.titleFrom cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomesen
dc.typeArticleen
dc.identifier.eissn1471-2164en
dc.contributor.departmentDepartment of Anatomy, University of Otago, Dunedin, New Zealanden
dc.contributor.departmentAllan Wilson Centre for Molecular Ecology and Evolution, Dunedin, New Zealanden
dc.contributor.departmentCurrent address: School of Life Sciences, University of Warwick, Coventry, United Kingdomen
dc.contributor.departmentDepartment of Integrative Biology, University of California, Berkeley, California, USAen
dc.contributor.departmentDepartment of Mathematics and Statistics, University of Otago, Dunedin, New Zealanden
dc.contributor.departmentHuman Origins Genotyping Laboratory, Arizona Research Laboratories, Division of Biotechnology, University of Arizona, Arizona, USAen
dc.identifier.journalBMC Genomicsen
dc.description.collectioninformationThis item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
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