Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.

The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques...

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Autores principales:	Bjorn Tytgat, Elie Verleyen, Dagmar Obbels, Karolien Peeters, Aaike De Wever, Sofie D'hondt, Tim De Meyer, Wim Van Criekinge, Wim Vyverman, Anne Willems
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ff535c37accc48a2a8b57174bbcfe4e4
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spelling	oai:doaj.org-article:ff535c37accc48a2a8b57174bbcfe4e42021-11-18T08:17:35ZBacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.1932-620310.1371/journal.pone.0097564https://doaj.org/article/ff535c37accc48a2a8b57174bbcfe4e42014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24887330/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques. By contrast, cultivation-based approaches allow the retrieval of both common and uncommon bacteria that can grow in the conditions used and provide access to strains for biotechnological applications. We performed bidirectional pyrosequencing of the bacterial 16S rRNA gene diversity in two terrestrial and seven aquatic Antarctic microbial mat samples previously studied by heterotrophic cultivation. While, not unexpectedly, 77.5% of genera recovered by pyrosequencing were not among the isolates, 25.6% of the genera picked up by cultivation were not detected by pyrosequencing. To allow comparison between both techniques, we focused on the five phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Deinococcus-Thermus) recovered by heterotrophic cultivation. Four of these phyla were among the most abundantly recovered by pyrosequencing. Strikingly, there was relatively little overlap between cultivation and the forward and reverse pyrosequencing-based datasets at the genus (17.1-22.2%) and OTU (3.5-3.6%) level (defined on a 97% similarity cut-off level). Comparison of the V1-V2 and V3-V2 datasets of the 16S rRNA gene revealed remarkable differences in number of OTUs and genera recovered. The forward dataset missed 33% of the genera from the reverse dataset despite comprising 50% more OTUs, while the reverse dataset did not contain 40% of the genera of the forward dataset. Similar observations were evident when comparing the forward and reverse cultivation datasets. Our results indicate that the region under consideration can have a large impact on perceived diversity, and should be considered when comparing different datasets. Finally, a high number of OTUs could not be classified using the RDP reference database, suggesting the presence of a large amount of novel diversity.Bjorn TytgatElie VerleyenDagmar ObbelsKarolien PeetersAaike De WeverSofie D'hondtTim De MeyerWim Van CriekingeWim VyvermanAnne WillemsPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 6, p e97564 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Bjorn Tytgat Elie Verleyen Dagmar Obbels Karolien Peeters Aaike De Wever Sofie D'hondt Tim De Meyer Wim Van Criekinge Wim Vyverman Anne Willems Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.
description	The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques. By contrast, cultivation-based approaches allow the retrieval of both common and uncommon bacteria that can grow in the conditions used and provide access to strains for biotechnological applications. We performed bidirectional pyrosequencing of the bacterial 16S rRNA gene diversity in two terrestrial and seven aquatic Antarctic microbial mat samples previously studied by heterotrophic cultivation. While, not unexpectedly, 77.5% of genera recovered by pyrosequencing were not among the isolates, 25.6% of the genera picked up by cultivation were not detected by pyrosequencing. To allow comparison between both techniques, we focused on the five phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Deinococcus-Thermus) recovered by heterotrophic cultivation. Four of these phyla were among the most abundantly recovered by pyrosequencing. Strikingly, there was relatively little overlap between cultivation and the forward and reverse pyrosequencing-based datasets at the genus (17.1-22.2%) and OTU (3.5-3.6%) level (defined on a 97% similarity cut-off level). Comparison of the V1-V2 and V3-V2 datasets of the 16S rRNA gene revealed remarkable differences in number of OTUs and genera recovered. The forward dataset missed 33% of the genera from the reverse dataset despite comprising 50% more OTUs, while the reverse dataset did not contain 40% of the genera of the forward dataset. Similar observations were evident when comparing the forward and reverse cultivation datasets. Our results indicate that the region under consideration can have a large impact on perceived diversity, and should be considered when comparing different datasets. Finally, a high number of OTUs could not be classified using the RDP reference database, suggesting the presence of a large amount of novel diversity.
format	article
author	Bjorn Tytgat Elie Verleyen Dagmar Obbels Karolien Peeters Aaike De Wever Sofie D'hondt Tim De Meyer Wim Van Criekinge Wim Vyverman Anne Willems
author_facet	Bjorn Tytgat Elie Verleyen Dagmar Obbels Karolien Peeters Aaike De Wever Sofie D'hondt Tim De Meyer Wim Van Criekinge Wim Vyverman Anne Willems
author_sort	Bjorn Tytgat
title	Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.
title_short	Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.
title_full	Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.
title_fullStr	Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.
title_full_unstemmed	Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.
title_sort	bacterial diversity assessment in antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/ff535c37accc48a2a8b57174bbcfe4e4
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Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation.

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