Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
Open Access
- 28 September 2010
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 38 (21), e197
- https://doi.org/10.1093/nar/gkq807
Abstract
Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.Keywords
This publication has 54 references indexed in Scilit:
- Index-Free De Novo Assembly and Deconvolution of Mixed Mitochondrial GenomesGenome Biology and Evolution, 2010
- Tablet—next generation sequence assembly visualizationBioinformatics, 2009
- An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomesNucleic Acids Research, 2009
- PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular datingBioinformatics, 2009
- Multiple Alignment of DNA Sequences with MAFFTMethods in Molecular Biology, 2009
- A Comparative Analysis of Mitochondrial Genomes in Coleoptera (Arthropoda: Insecta) and Genome Descriptions of Six New BeetlesMolecular Biology and Evolution, 2008
- Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technologyNucleic Acids Research, 2008
- jModelTest: Phylogenetic Model AveragingMolecular Biology and Evolution, 2008
- A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexingNucleic Acids Research, 2007
- Microbial diversity in the deep sea and the underexplored “rare biosphere”Proceedings of the National Academy of Sciences of the United States of America, 2006