S rRNA gene level from high-throughput sequencing data, and explore whether there are specialized types for different ecological niches. Preliminary analysis of Megasphaera spp. has also revealed numerous oligotype distribution profiles among women with GS BV (results not shown), suggesting that applying the method described here to other species that are commonly found in the genitourinary microbiota could yield important new insights. Additionally, consideration should be given to oligotype analyses of other phylogenetically informative genes, such as recA , , in order to explore to which extent the oligotypes at the 16S rRNA gene level are able to predict genomic variation.
In summary, our study describes a novel method for revealing concealed diversity at a very low level of taxonomy by utilizing Shannon entropy to amplify weak signals of subtle but reproducible nucleotide variation within high-throughput sequencing reads. This oligotyping approach can be applied to existing sequence libraries to explore diversity at an ecologically meaningful level and investigate potential ecotypes and their diversity hidden within conventionally defined species.
Phylogenetic distribution of 46 oligotypes. Bars and numbers next to oligotypes indicate how many samples they were present at least once in all samples.
Hierarchical clustering results of samples from male patients. Penile skin (clustering significance: p = 0.011, UniFrac significance: p = 0.001) and urethra (clustering significance: p<0.001, UniFrac significance: p = 0.077) samples were clustered based on the UniFrac distance metric. Red, orange and green colors indicate samples that are sexual partners of GS-defined BV, GS-defined intermediate and GS-defined normal female patients, respectively.
Stacked bar representation of oligotype profiles among couples. While VS labeled bars represent female patients oligotype profile, for every couple MP (penile skin sample) or MU (urethra sample) bars represent male sexual partners oligotype profile. For the sake of compactness, only the more similar sample to vaginal sample from male partner were used when both MP and MU samples were available for a given couple.
Number of sequences in each sample. Number of sequences per sample in the original pyrosequencing library versus the number of sequences that were 99% or more similar to one of the full length G. vaginalis sequences in the local search database with minimum alignment length of 480bp.
Oligotype distribution among sample groups. Every column in this table shows the number of samples in a group in which the given oligotype was observed at least once. The total number of vaginal swab (VS), penile skin (PS), and urethral (U) samples are shown in parentheses in all three groups of Gram Stain (GS) BV, GS Intermediate and GS Normal.
We would like to thank our anonymous reviewers, as well as Susan M. Huse and David Mark-Welch from Marine Biological Laboratory for their valuable critiques and suggestions.
Conceived and designed the experiments: MJF AME DHM. Performed the experiments: MZ AME. Analyzed the data: AME MJF DHM MZ CMT. Contributed reagents/materials/analysis tools: AME SED MJF DHM. Wrote the paper: AME MJF MZ DHM SED CMT. Designed the software used in analysis: AME CMT.
- 1. Whitman WB, Coleman DC, Wiebe WJ (1998) Prokaryotes: the unseen majority. Proc Natl Acad Sci USA 95: 6578–6583.WB WhitmanDC ColemanWJ Wiebe1998Prokaryotes: the unseen majority.Proc Natl Acad Sci USA9565786583
- 2. Peterson J, Garges S, Giovanni M, McInnes P, et al. Group NIHHMPW (2009) The NIH Human Microbiome Project. Genome Res 19: 2317–2323.J. PetersonS. GargesM. GiovanniP. McInnesGroup NIHHMPW2009The NIH Human Microbiome Project.Genome Res1923172323
- 3. Frank DN, Pace NR (2008) Gastrointestinal microbiology enters the metagenomics era. Curr Opin Gastroenterol 24: 4–10.DN FrankNR Pace2008Gastrointestinal microbiology enters the metagenomics era.Curr Opin Gastroenterol24410
- 4. Savage DC (1977) Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 31: 107–133.DC Savage1977Microbia