Abstract
Amplification and sequencing of 16S amplicons are widely used for profiling the structure of oral microbiota. However, it remains not clear whether and to what degree DNA extraction and targeted 16S rRNA hypervariable regions influence the analysis. Based on a mock community consisting of five oral bacterial species in equal abundance, we compared the 16S amplicon sequencing results on the Illumina MiSeq platform from six frequently employed DNA extraction procedures and three pairs of widely used 16S rRNA hypervariable primers targeting different 16S rRNA regions. Technical reproducibility of selected 16S regions was also assessed. DNA extraction method exerted considerable influence on the observed bacterial diversity while hypervariable regions had a relatively minor effect. Protocols with beads added to the enzyme-mediated DNA extraction reaction produced more accurate bacterial community structure than those without either beads or enzymes. Hypervariable regions targeting V3-V4 and V4-V5 seemed to produce more reproducible results than V1-V3. Neither sequencing batch nor change of operator affected the reproducibility of bacterial diversity profiles. Therefore, DNA extraction strategy and 16S rDNA hypervariable regions both influenced the results of oral microbiota biodiversity profiling, thus should be carefully considered in study design and data interpretation.
Highlights
Oral microbiota are implicated in the aetiology of many oral and systemic diseases, such as dental caries, periodontal diseases, obesity and cancer[1,2,3,4]
In this study, based on a mock oral microbiota consisting of five oral bacterial species of equal abundance, we compared the 16S amplicon sequencing results from six frequently employed DNA extraction procedures and three pairs of widely used 16S rRNA hypervariable regions (Tables 1, 2; Fig. 1)
Pairwise comparisons showed that the traditional Phenol:chloroform-based method (M1) yielded the lowest amount of metagenomic DNA, which was followed by Kit DNA extraction method (M2) with a significant difference (p < 0.001; Fig. 2)
Summary
Oral microbiota are implicated in the aetiology of many oral and systemic diseases, such as dental caries, periodontal diseases, obesity and cancer[1,2,3,4]. For human intestinal microbiota[9,10,11,12,13] and environmental microbiota[14,15,16,17], various factors such as DNA extraction method, targeted 16S rRNA hypervariable regions and sample handling environment can all greatly influence the resulted biodiversity profiles. The choice of 16S rRNA hyper-variable regions targeted for sequencing, which have been the most widely used markers to assess the phylogenetic diversity of microbes, is an important decision to make. Critical assessment of the choice of hyper-variable regions will be important, so as to minimize distortion and conflicts in sequence-based analysis and comparison of oral microbiota
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