16S rRNA gene sequencing of mock microbial populations-impact of DNA extraction method, primer choice and sequencing platform

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Date
2016-06-24
Authors
Fouhy, Fiona
Clooney, Adam G.
Stanton, Catherine
Claesson, Marcus J.
Cotter, Paul D.
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Publisher
BioMed Central
Published Version
10.1186/s12866-016-0738-z
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Abstract
Background: Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. Results: The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. Conclusions: Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.
Description
Keywords
Next-generation sequencing , Mock communities , 16S rRNA , MiSeq , Ion PGM , Gut microbiota , Bias , DNA extraction
Citation
Fouhy, F., Clooney, A. G., Stanton, C., Claesson, M. J. and Cotter, P. D. (2016) '16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform', BMC Microbiology, 16, 123 (13pp). doi: 10.1186/s12866-016-0738-z
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https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-016-0738-z
URI
https://hdl.handle.net/10468/4126
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Microbiology - Journal Articles
APC Microbiome Ireland - Journal Articles
Copyright
© 2016, the Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.