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

BMC Microbiology

Research Article

Adam G. Clooney¹  Marcus J. Claesson¹  Fiona Fouhy²  Paul D. Cotter³  Catherine Stanton³ 

[1] School of Microbiology, University College Cork, Cork, Ireland;APC Microbiome Institute, University College Cork, Cork, Ireland;Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland;Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland;APC Microbiome Institute, University College Cork, Cork, Ireland;

关键词: Next-generation sequencing;    Mock communities;    16S rRNA;    MiSeq;    Ion PGM;    Gut microbiota;    Bias;    DNA extraction;   

DOI  :  10.1186/s12866-016-0738-z

 received in 2016-02-08, accepted in 2016-06-08,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundNext-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.ResultsThe 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.ConclusionsMicrobiota 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.

【 授权许可】

CC BY   
© The Author(s). 2016