TY - JOUR
T1 - From cheek swabs to consensus sequences
T2 - An A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes
AU - Clarke, Andrew C.
AU - Prost, Stefan
AU - Stanton, Jo Ann L.
AU - White, W. Timothy J.
AU - Kaplan, Matthew E.
AU - Matisoo-Smith, Elizabeth A.
AU - Adhikarla, Syama
AU - Adler, Christina J.
AU - Balanovska, Elena
AU - Balanovsky, Oleg
AU - Bertranpetit, Jaume
AU - Comas, David
AU - Cooper, Alan
AU - Der Sarkissian, Clio S.I.
AU - Dulik, Matthew C.
AU - Gaieski, Jill B.
AU - GaneshPrasad, Arun Kumar
AU - Haak, Wolfgang
AU - Haber, Marc
AU - Jin, Li
AU - Li, Shilin
AU - Martínez-Cruz, Begoña
AU - Merchant, Nirav C.
AU - Mitchell, R. John
AU - Owings, Amanda C.
AU - Parida, Laxmi
AU - Pitchappan, Ramasamy
AU - Platt, Daniel E.
AU - Quintana-Murci, Lluis
AU - Renfrew, Colin
AU - Lacerda, Daniela R.
AU - Royyuru, Ajay K.
AU - Santos, Fabrício R.
AU - Schurr, Theodore G.
AU - Soodyall, Himla
AU - Hernanz, Soria
AU - Swamikrishnan, Pandikumar
AU - Tyler-Smith, Chris
AU - Santhakumari, Arun Varatharajan
AU - Vieira, Pedro Paulo
AU - Vilar, Miguel G.
AU - Zalloua, Pierre A.
AU - Ziegle, Janet S.
AU - Wells, R. Spencer
PY - 2014/1/25
Y1 - 2014/1/25
N2 - 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.
AB - 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.
KW - 454 sequencing
KW - Bioinformatics
KW - Human
KW - Long-range PCR
KW - Mitochondrial DNA
KW - Next-generation sequencing
UR - http://www.scopus.com/inward/record.url?scp=84896721788&partnerID=8YFLogxK
U2 - 10.1186/1471-2164-15-68
DO - 10.1186/1471-2164-15-68
M3 - Article
C2 - 24460871
AN - SCOPUS:84896721788
SN - 1471-2164
VL - 15
JO - BMC Genomics
JF - BMC Genomics
IS - 1
M1 - 68
ER -