16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform - PubMed (original) (raw)

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

Fiona Fouhy et al. BMC Microbiol. 2016.

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.

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

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Figures

Fig. 1

Fig. 1

Rarefaction curves based on sample ID and number of observed species for mock cells (a) and mock DNA samples (b). Curves are approaching or are horizontal with the x axis indicating that additional sequencing would not yield additional novel data

Fig. 2

Fig. 2

Percentage relative abundance of expected species (n = 20) detected in mock community DNA based on sequencing platform and primer set used

Fig. 3

Fig. 3

Heat map of species abundance. Only the 20 expected taxa from mock DNA (HM-782D) were included. Hierarchical clustering was performed using hclusing default parameters (complete linkage). The blue colours represent samples sequenced on MiSeq platform while green represent the Ion PGM

Fig. 4

Fig. 4

Percentage relative abundance of expected species based on extraction procedure

Fig. 5

Fig. 5

Heat map of species abundance by sequencer and extraction method for mock community cells. Only expected taxa were included and hierarchical clustering was performed using hclust default parameters (complete linkage). The top colour legend depicts the sequencing technology and primer set. The blue colours represent samples sequenced on MiSeq platform while green represent the Ion PGM. The bottom legend represents the samples and extraction method

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