Dataset Information

Influence of 16S rRNA Hypervariable Region on Estimates of Bacterial Diversity and Community Composition in Seawater and Marine Sediment.

ABSTRACT: To assess the influence of 16S ribosomal RNA (rRNA) tag choice on estimates of microbial diversity and/or community composition in seawater and marine sediment, we examined bacterial diversity and community composition from a site in the Central North Atlantic and a site in the Equatorial Pacific. For each site, we analyzed samples from four zones in the water column, a seafloor sediment sample, and two subseafloor sediment horizons (with stratigraphic ages of 1.5 and 5.5 million years old). We amplified both the V4 and V6 hypervariable regions of the 16S rRNA gene and clustered the sequences into operational taxonomic units (OTUs) of 97% similarity to analyze for diversity and community composition. OTU richness is much higher with the V6 tag than with the V4 tag, and subsequently OTU-level community composition is quite different between the two tags. Vertical patterns of relative diversity are broadly the same for both tags, with maximum taxonomic richness in seafloor sediment and lowest richness in subseafloor sediment at both geographic locations. Genetic dissimilarity between sample locations is also broadly the same for both tags. Community composition is very similar for both tags at the class level, but very different at the level of 97% similar OTUs. Class-level diversity and community composition of water-column samples are very similar at each water depth between the Atlantic and Pacific. However, sediment communities differ greatly from the Atlantic site to the Pacific site. Finally, for relative patterns of diversity and class-level community composition, deep sequencing and shallow sequencing provide similar results.

SUBMITTER: Kerrigan Z

PROVIDER: S-EPMC6646839 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Influence of 16S rRNA Hypervariable Region on Estimates of Bacterial Diversity and Community Composition in Seawater and Marine Sediment.

Kerrigan Zak Z Kirkpatrick John B JB D'Hondt Steven S

Frontiers in microbiology 20190716

To assess the influence of 16S ribosomal RNA (rRNA) tag choice on estimates of microbial diversity and/or community composition in seawater and marine sediment, we examined bacterial diversity and community composition from a site in the Central North Atlantic and a site in the Equatorial Pacific. For each site, we analyzed samples from four zones in the water column, a seafloor sediment sample, and two subseafloor sediment horizons (with stratigraphic ages of 1.5 and 5.5 million years old). We ...[more]

PMID: 31379788

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Project description:16S rRNA gene sequences are commonly analyzed for taxonomic and phylogenetic studies because they contain variable regions that can help distinguish different genera. However, intra-genus distinction using variable region homology is often impossible due to the high overall sequence identities among closely related species, even though some residues may be conserved within respective species. Using a computational method that included the allelic diversity within individual genomes, we discovered that certain Escherichia and Shigella species can be distinguished by a multi-allelic 16S rRNA variable region single nucleotide polymorphism (SNP). To evaluate the performance of 16S rRNAs with altered variable regions, we developed an in vivo system that measures the acceptance and distribution of variant 16S rRNAs into a large pool of natural versions supporting normal translation and growth. We found that 16S rRNAs containing evolutionarily disparate variable regions were underpopulated both in ribosomes and in active translation pools, even for an SNP. Overall, this study revealed that variable region sequences can substantially influence the performance of 16S rRNAs and that this biological constraint can be leveraged to justify refining taxonomic assignments of variable region sequence data. IMPORTANCE This study reevaluates the notion that 16S rRNA gene variable region sequences are uninformative for intra-genus classification and that single nucleotide variations within them have no consequence to strains that bear them. We demonstrated that the performance of 16S rRNAs in Escherichia coli can be negatively impacted by sequence changes in variable regions, even for single nucleotide changes that are native to closely related Escherichia and Shigella species; thus, biological performance is likely constraining the evolution of variable regions in bacteria. Further, the native nucleotide variations we tested occur in all strains of their respective species and across their multiple 16S rRNA gene copies, suggesting that these species evolved beyond what would be discerned from a consensus sequence comparison. Therefore, this work also reveals that the multiple 16S rRNA gene alleles found in most bacteria can provide more informative phylogenetic and taxonomic detail than a single reference allele.

Project description:Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to -80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.

Dataset Information

Influence of 16S rRNA Hypervariable Region on Estimates of Bacterial Diversity and Community Composition in Seawater and Marine Sediment.

Publications

Influence of 16S rRNA Hypervariable Region on Estimates of Bacterial Diversity and Community Composition in Seawater and Marine Sediment.

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