Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0738-z) contains supplementary material, which is available to authorized users.
Highlights
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
The percentage of retained reads was similar across platforms and primer sets, with the notable exception of the V4-V5 primers on the Ion PGM, where 80–90 % were retained following chimera removal, compared to an average of 99 % retained for the other primers on both platforms
The misidentified species were, in the majority of cases, closely related to species known to be present in the mock samples
Summary
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. Roche 454 platforms were employed in numerous important and enlightening human microbiome studies [1,2,3,4], the Illumina MiSeq [5] and Life Technologies Ion PGM [6] platforms are most commonly used for 16S rRNA gene-based investigations of microbiota composition [7,8,9,10,11]. Numerous studies have investigated the effects of different factors on 16S rRNA gene microbiota data including, in the case of gut microbiota studies, sample type [12] (e.g. faecal vs cecal), sample storage prior to DNA extraction [13], DNA extraction procedure [14, 15], primers (sequences and 16S rRNA gene regions) [16,17,18] and the sequencing platform used [19]. Recent studies have shown the region of the 16S rRNA gene that is sequenced will impact on the results achieved [22]
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