Abstract
Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1–V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacer and Enterococcus predominated in the community generated by V4–V6 primers, and the most numerous genera in the V7–V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4–V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7–V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1–V3 and V7–V9 primers providesd results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.
Highlights
Molecular approaches have revealed the presence of large numbers of as-yet-uncultivated organisms in the subgingival microbiome; creating a paradigm shift in our understanding of periodontal health and disease [1,2,3,4]
Subject selection Approval for this study was obtained from the Office of Responsible Research Practices at The Ohio State University. 10 current smokers with generalized moderate to severe chronic periodontitis were identified following clinical and radiographic examination and written informed consent was obtained
Bacteria were separated from the paper points by adding 200 ml of phosphate buffered saline (PBS) to the tubes and vortexing
Summary
Molecular approaches have revealed the presence of large numbers of as-yet-uncultivated organisms in the subgingival microbiome; creating a paradigm shift in our understanding of periodontal health and disease [1,2,3,4]. In recent years, sequencing of 16S rRNA genes by the Sanger method (16S cloning and sequencing) has been widely used to examine subgingival microbial profiles in periodontal health and disease, as well as to characterize compositional shifts in these communities [5,6,7,8,9]. In order to improve community coverage, various investigations have employed primers that target different regions of the gene [12,13,14] It has previously been shown, using Sanger sequencing, that the region of the 16S gene that is targeted for sequencing as well as the degeneracy of the sequencing primers introduce a level of bias into the community profile [2,15]. Since pyrosequencing provides an enormously increased depth-of-coverage, it is important to understand the extent and severity of bias introduced by primer selection on the profile of any given community
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