16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform
Fouhy F, Clooney AG, Stanton C, Claesson MJ, Cotter PD
BMC Microbiol. 2016. doi: 10.1186/s12866-016-0738-z
COMMENT: Next generation sequencing technologies are being widely used in the study of microbial communities. The accuracy of the results achieved by the use of these techniques could be affected by numerous factors such as sample type, sample storage, DNA extraction procedure, primers and the sequencing platform used.
By the use of mock communities and mock community DNA, the authors analyze the effects of the combination of 3 factors on sequencing results:
1. Three 16S rRNA gene primer sets (V4-V5, V1-V2 and V1-V2 degenerate primers).
2. Two commonly used DNA extraction procedures.
3. The use of Illumina MiSeq and Life Technologies Ion PGM sequencers.
Data here obtained demonstrated that the choice of PCR primers and sequencing platform had more impact on the results than either of the DNA extraction method, probably due to the ease of extraction of DNA from the mock community cells.
This study reveals the differences obtained in the microbiota composition by the use of different sequencing approaches. Considering these results, authors recommend the use of a standardized approach for sequencing when a comparison across multiple sequencing runs is required.