Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing

Kathleen Sim, Michael J Cox, Harm Wopereis, Rocio Martin, Jan Knol, Ming-Shi Li, William O C M Cookson, Miriam F Moffatt, J Simon Kroll, Kathleen Sim, Michael J Cox, Harm Wopereis, Rocio Martin, Jan Knol, Ming-Shi Li, William O C M Cookson, Miriam F Moffatt, J Simon Kroll

Abstract

The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, 'bifidobacteria-optimised' universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.

Conflict of interest statement

Competing Interests: HW, RM and JK are employed by Danone Research. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Proportions of 454 sequencing reads…
Figure 1. Proportions of 454 sequencing reads obtained using both primer sets.
Increased detection rate of Bifidobacterium dentium demonstrated using the ‘bifidobacteria-optimised’ universal primers (b) compared to regular universal primers (u).
Figure 2. Heatmap displaying the relative abundance…
Figure 2. Heatmap displaying the relative abundance of OTUs per sample.
Samples are grouped by hierarchical cluster analysis on the x-axis and by neighbour-joining phylogenetic tree with nearest neighbour interchange on the y-axis. Samples amplified with ‘bifidobacteria-optimised’ primers are in red and with the standard primers in blue. Bifidobacterial OTUs are highlighted in the red box.
Figure 3. Principal Coordinate Analysis using the…
Figure 3. Principal Coordinate Analysis using the weighted UniFrac metric.
(A) Sample pairs P9, P10 and in particular P1 and P5 cluster tightly together. These samples contain small or moderate numbers of bifidobacteria reads. (B) After removing bifidobacteria sequences from the analysis, all sample pairs cluster tightly showing that the main differences between the sets are due to the bifidobacteria sequences. U = regular universal primers (926R), B – ‘bifidobacteria-optimised’ universal primers (926Rb).

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