A Cost-Effective Approach to Sequence Hundreds of Complete Mitochondrial Genomes

PLoS One. 2016 Aug 9;11(8):e0160958. doi: 10.1371/journal.pone.0160958. eCollection 2016.

Abstract

We present a cost-effective approach to sequence whole mitochondrial genomes for hundreds of individuals. Our approach uses small reaction volumes and unmodified (non-phosphorylated) barcoded adaptors to minimize reagent costs. We demonstrate our approach by sequencing 383 Fundulus sp. mitochondrial genomes (192 F. heteroclitus and 191 F. majalis). Prior to sequencing, we amplified the mitochondrial genomes using 4-5 custom-made, overlapping primer pairs, and sequencing was performed on an Illumina HiSeq 2500 platform. After removing low quality and short sequences, 2.9 million and 2.8 million reads were generated for F. heteroclitus and F. majalis respectively. Individual genomes were assembled for each species by mapping barcoded reads to a reference genome. For F. majalis, the reference genome was built de novo. On average, individual consensus sequences had high coverage: 61-fold for F. heteroclitus and 57-fold for F. majalis. The approach discussed in this paper is optimized for sequencing mitochondrial genomes on an Illumina platform. However, with the proper modifications, this approach could be easily applied to other small genomes and sequencing platforms.

MeSH terms

  • Animals
  • Chromosome Mapping
  • Cost-Benefit Analysis*
  • Fundulidae / genetics
  • Genome, Mitochondrial / genetics*
  • Reference Standards
  • Sequence Alignment
  • Sequence Analysis, DNA / economics*
  • Sequence Analysis, DNA / standards

Grants and funding

This work was funded by the National Science Foundation (NSF) grants MCB-1158241 and IOS-1147042 to MFO and the Rosenstiel School of Marine and Atmospheric Science (RSMAS) grants SURGE 2014 and SURGE 2015 to JCBN. Data analysis was supported by the NSF (collaborative research grants DEB-1120512, DEB-1265282, DEB-1120013, DEB-1120263, DEB-1120333, DEB-1120398). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.