A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.

<h4>Background</h4>The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. W...

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Autores principales:	Michiel Vos, Christopher Quince, Agata S Pijl, Mattias de Hollander, George A Kowalchuk
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fb6bcccc0315456ab2fbf2533c563b40
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spelling	oai:doaj.org-article:fb6bcccc0315456ab2fbf2533c563b402021-11-18T07:28:15ZA comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.1932-620310.1371/journal.pone.0030600https://doaj.org/article/fb6bcccc0315456ab2fbf2533c563b402012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22355318/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria.<h4>Methodology/principal findings</h4>Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster.<h4>Conclusions/significance</h4>The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.Michiel VosChristopher QuinceAgata S PijlMattias de HollanderGeorge A KowalchukPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 2, p e30600 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Michiel Vos Christopher Quince Agata S Pijl Mattias de Hollander George A Kowalchuk A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.
description	<h4>Background</h4>The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria.<h4>Methodology/principal findings</h4>Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster.<h4>Conclusions/significance</h4>The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.
format	article
author	Michiel Vos Christopher Quince Agata S Pijl Mattias de Hollander George A Kowalchuk
author_facet	Michiel Vos Christopher Quince Agata S Pijl Mattias de Hollander George A Kowalchuk
author_sort	Michiel Vos
title	A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.
title_short	A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.
title_full	A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.
title_fullStr	A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.
title_full_unstemmed	A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.
title_sort	comparison of rpob and 16s rrna as markers in pyrosequencing studies of bacterial diversity.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/fb6bcccc0315456ab2fbf2533c563b40
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A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.

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