16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform
Keyword
Next-generation sequencingMock communities
16S rRNA
MiSeq
Ion PGM
Gut microbiota
Bias
DNA extraction
Date
24/06/2016
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Fouhy et al. 16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform. BMC Microbiology. 2016 Jun 24;16(1):123Abstract
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.Funder
European Union; Science Foundation IrelandGrant Number
603038; SFI/12/RC/2273; SFI/11/PI/1137ae974a485f413a2113503eed53cd6c53
http://dx.doi.org/10.1186/s12866-016-0738-z