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Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics

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...

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Autores principales: Timmermans, M. J. T. N., Dodsworth, S., Culverwell, C. L., Bocak, L., Ahrens, D., Littlewood, D. T. J., Pons, J., Vogler, A. P.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995086/
https://www.ncbi.nlm.nih.gov/pubmed/20876691
http://dx.doi.org/10.1093/nar/gkq807
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author Timmermans, M. J. T. N.
Dodsworth, S.
Culverwell, C. L.
Bocak, L.
Ahrens, D.
Littlewood, D. T. J.
Pons, J.
Vogler, A. P.
author_facet Timmermans, M. J. T. N.
Dodsworth, S.
Culverwell, C. L.
Bocak, L.
Ahrens, D.
Littlewood, D. T. J.
Pons, J.
Vogler, A. P.
author_sort Timmermans, M. J. T. N.
collection PubMed
description 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.
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spelling pubmed-29950862010-12-01 Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics Timmermans, M. J. T. N. Dodsworth, S. Culverwell, C. L. Bocak, L. Ahrens, D. Littlewood, D. T. J. Pons, J. Vogler, A. P. Nucleic Acids Res Methods Online 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. Oxford University Press 2010-11 2010-09-28 /pmc/articles/PMC2995086/ /pubmed/20876691 http://dx.doi.org/10.1093/nar/gkq807 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Timmermans, M. J. T. N.
Dodsworth, S.
Culverwell, C. L.
Bocak, L.
Ahrens, D.
Littlewood, D. T. J.
Pons, J.
Vogler, A. P.
Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
title Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
title_full Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
title_fullStr Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
title_full_unstemmed Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
title_short Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
title_sort why barcode? high-throughput multiplex sequencing of mitochondrial genomes for molecular systematics
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995086/
https://www.ncbi.nlm.nih.gov/pubmed/20876691
http://dx.doi.org/10.1093/nar/gkq807
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