Abstract
BackgroundNext-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.ResultsHere 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).ConclusionsAll 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.
Highlights
Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology
In addition to evolutionary applications, complete mitochondrial DNA (mtDNA) genomes are being sequenced to identify markers associated with mitochondrial disease, and the advent of NGS has seen the significant expansion of research in this area [6]
The bioinformatics steps offer their own challenges because many of the individual components/programs are available it is often difficult to get the outputs from one component into a format where they can be used as inputs for other components
Summary
Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. 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. In addition to evolutionary applications, complete mtDNA genomes are being sequenced to identify markers associated with mitochondrial disease, and the advent of NGS has seen the significant expansion of research in this area [6]. Despite the increasing use of NGS technologies and a better appreciation of their importance, there remain significant obstacles to the successful implementation of NGS-based projects. These challenges often relate to assembling the constituent components of a NGS sequencing protocol into a single workflow to suit a given study. The challenges in assembling a complete NGS protocol represent a major source of inertia for researchers wanting to undertake NGS studies for the first time
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