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

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.


Background
Standard AMPure XP is used to remove DNA fragments smaller than 100 bp. This cut-off can be increased or decreased (e.g., up to 300 bp, down to 50 bp) by isolating the beads from AMPure XP and resuspending them in PEG-Bead Solution containing polyethylene glycol (PEG) 8000. It is the concentration of PEG in the solution that defines the size cut-off, with increasing PEG concentration meaning a lower cut-off. Fragments up to 50 bp can be removed using a PEG concentration of approx. 30% (w/v) and up to 300 bp with a PEG concentration of approx. 10% (w/v). The relationship between fragment cut-off and PEG concentration is not linear however, with concentrations close to 10% having much larger effects than those close to 30%.
The optimum PEG concentration can be determined by trialling concentrations at 5% increments, and then narrowed down to 0.5-1.0% increments. The template that will ultimately be sequences can be used for testing, or alternatively a DNA ladder can be used as this makes it much easier to visualise the effects of varying PEG concentration. If using a ladder, do not use NEB ladders because the fragments are modified in a way that causes fragments of all sizes to be retained (Sebastian Lippold, pers. comm.) Both Fermantas and Invitrogen ladders are OK to use. Results can be visualised on an agarose gel (ladders only) or using the Bioanalyzer 2100.

Materials
Where a supplier is mentioned, this represents the supplier we have used; other suppliers' products may produce comparable results. http://www.biomedcentral.com/1471-2164/15/68 Reagents All reagents should be an appropriate molecular biology grade.
3. Carefully remove the buffer, taking care not to disturb the beads.
4. While still in the SPRIStand, wash the beads by adding 500 μL TET buffer. Incubate for 2 min and then completely remove the TET without disturbing the beads. Repeat this step.

Bead resuspension in PEG buffer
5. Remove the tube(s) from the SPRIStand and resuspend the beads in the desired PEG-Bead Solution. Use same volume of PEG-Bead Solution as the amount of AMPure XP originally used (e.g., if beads were isolated from 1.5 mL AMPure XP then resuspend the beads in 1.5 mL PEG-Bead Solution). Vortex the solution to ensure the beads are completely resuspended. The PEG-Bead Solution is stable at 4°C for at least two months or the expiry date of the AMPure XP, whichever is earlier.  [2]. Elute the fragments in 20 μL 10 mM Tris.
Bioanalzyer 2100 analysis of fragmented products 7. To visualise the results from a ladder trial, the eluate can be run on an agarose gel. Trials containing more complex mixtures of fragments (e.g., products from a Fragmentase reaction) should be run on the Bioanalyzer 2100 using a DNA 7500 chip according to the manufacturer's instructions.