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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
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, No. 1, Article 68, Year 2014
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Description
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. © 2014 Clarke et al.; licensee BioMed Central Ltd.
Available Materials
https://efashare.b-cdn.net/share/pmc/articles/PMC3922791/bin/1471-2164-15-68-S1.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC3922791/bin/1471-2164-15-68-S2.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC3922791/bin/1471-2164-15-68-S3.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC3922791/bin/1471-2164-15-68-S4.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC3922791/bin/1471-2164-15-68-S5.zip
https://efashare.b-cdn.net/share/pmc/articles/PMC3922791/bin/1471-2164-15-68-S6.zip
Authors & Co-Authors
Clarke, Andrew C.
Unknown Affiliation
Prost, Stefan
Unknown Affiliation
Stanton, J. L.
Unknown Affiliation
White, W. Timothy J.
Unknown Affiliation
Kaplan, Matthew E.
Unknown Affiliation
Matisoo-Smith, Elizabeth A.
Unknown Affiliation
Adhikarla, Syama
Unknown Affiliation
Adler, Christina J.
Unknown Affiliation
Balanovska, Elena V.
Unknown Affiliation
Balanovsky, Oleg P.
Unknown Affiliation
Bertranpetit, Jaume
Unknown Affiliation
Comas, D.
Unknown Affiliation
Cooper, Alan C.
Unknown Affiliation
Der Sarkissian, Clio S.I.
Unknown Affiliation
Dulik, Matthew C.
Unknown Affiliation
Gaieski, Jill Bennett
Unknown Affiliation
GaneshPrasad, Arun Kumar
Unknown Affiliation
Haak, Wolfgang
Unknown Affiliation
Haber, Marc E.
Unknown Affiliation
Jin, Li
Unknown Affiliation
Li, Shilin
Unknown Affiliation
Martínez-Cruz, Begoña
Unknown Affiliation
Merchant, Nirav C.
Unknown Affiliation
Mitchell, Robert John
Unknown Affiliation
Owings, Amanda C.
Unknown Affiliation
Parida, Laxmi P.
Unknown Affiliation
Pitchappan, Ramasamy M.
Unknown Affiliation
Platt, Daniel E.
Unknown Affiliation
Quintana-Murci, Lluís
Unknown Affiliation
Renfrew, Colin
Unknown Affiliation
Lacerda, Daniela Rodrigues
Unknown Affiliation
Royyuru, Ajay K.
Unknown Affiliation
Santos, Fabrício Rodrigues Dos
Unknown Affiliation
Schurr, Theodore G.
Unknown Affiliation
Soodyall, Himla
Unknown Affiliation
Hernanz, Soria
Unknown Affiliation
Swamikrishnan, Pandikumar
Unknown Affiliation
Tyler-Smith, Chris
Unknown Affiliation
Santhakumari, Arun Varatharajan
Unknown Affiliation
Vieira, Pedro Paulo R.
Unknown Affiliation
Vilar, Miguel G.
Unknown Affiliation
Zalloua, Pierre A.
Unknown Affiliation
Ziegle, Janet S.
Unknown Affiliation
Wells, R. Spencer
Unknown Affiliation
Statistics
Citations: 44
Authors: 44
Affiliations: 24
Identifiers
Doi:
10.1186/1471-2164-15-68
e-ISSN:
14712164
Research Areas
Cancer
Genetics And Genomics
Health System And Policy