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Selection of validated hypervariable regions is crucial in 16S-based microbiota studies of the female genital tract.

ABSTRACT: Next-generation sequencing-based methods are extensively applied in studies of the human microbiota using partial 16?S rRNA gene amplicons. However, they carry drawbacks that are critical to consider when interpreting results, including differences in outcome based on the hypervariable region(s) used. Here, we show that primers spanning the V3/V4 region identify a greater number of taxa in the vaginal microbiota than those spanning the V1/V2 region. In particular, taxa such as Gardnerella vaginalis, Bifidobacterium bifidum and Chlamydia trachomatis, all species that influence vaginal health and disease, are not represented in V1/V2-based community profiles. Accordingly, missing or underestimating the frequency of these species overestimates the abundance of other taxa and fails to correctly assess the bacterial diversity in the urogenital tract. We elaborate that covering these taxa using the V3/V4 region leads to profound changes in the assignment of community state types. Altogether, we show that the choice of primers used for studying the vaginal microbiota has deep implications on the biological evaluation of the results.

SUBMITTER: Graspeuntner S

PROVIDER: S-EPMC6018735 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Selection of validated hypervariable regions is crucial in 16S-based microbiota studies of the female genital tract.

Graspeuntner Simon S Loeper Nathalie N Künzel Sven S Baines John F JF Rupp Jan J

Scientific reports 20180626 1

Next-generation sequencing-based methods are extensively applied in studies of the human microbiota using partial 16 S rRNA gene amplicons. However, they carry drawbacks that are critical to consider when interpreting results, including differences in outcome based on the hypervariable region(s) used. Here, we show that primers spanning the V3/V4 region identify a greater number of taxa in the vaginal microbiota than those spanning the V1/V2 region. In particular, taxa such as Gardnerella vagina ...[more]

PMID: 29946153

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Project description:The shrimp has become the most valuable traded marine product in the world, and its microbiota plays an essential role in its development and overall health status. Massive high-throughput sequencing techniques using several hypervariable regions of the 16S rRNA gene are broadly applied in shrimp microbiota studies. However, it is essential to consider that the use of different hypervariable regions can influence the obtained data and the interpretation of the results. The present study compares the shrimp microbiota structure and composition obtained by three types of amplicons: one spanning both the V3 and V4 hypervariable regions (V3V4), one for the V3 region only (V3), and one for the V4 region only (V4) using the same experimental and bioinformatics protocols. Twenty-four samples from hepatopancreas and intestine were sequenced and evaluated using the GreenGenes and silva reference databases for clustering and taxonomic classification. In general, the V3V4 regions resulted in higher richness and diversity, followed by V3 and V4. All three regions establish an apparent clustering effect that discriminates between the two analyzed organs and describe a higher richness for the intestine and a higher diversity for the hepatopancreas samples. Proteobacteria was the most abundant phyla overall, and Cyanobacteria was more common in the intestine, whereas Firmicutes and Actinobacteria were more prevalent in hepatopancreas samples. Also, the genus Vibrio was significantly abundant in the intestine, as well as Acinetobacter and Pseudomonas in the hepatopancreas suggesting these taxa as markers for their respective organs independently of the sequenced region. The use of a single hypervariable region such as V3 may be a low-cost alternative that enables an adequate description of the shrimp microbiota, allowing for the development of strategies to continually monitor the microbial communities and detect changes that could indicate susceptibility to pathogens under real aquaculture conditions while the use of the full V3V4 regions can contribute to a more in-depth characterization of the microbial composition.

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.

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Selection of validated hypervariable regions is crucial in 16S-based microbiota studies of the female genital tract.

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Selection of validated hypervariable regions is crucial in 16S-based microbiota studies of the female genital tract.

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