Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing

Sirichoat, Auttawit; Sankuntaw, Nipaporn; Engchanil, Chulapan; Buppasiri, Pranom; Faksri, Kiatichai; Namwat, Wises; Chantratita, Wasun; Lulitanond, Viraphong

doi:10.1007/s00203-020-02114-4

Cited by 31 publications

(31 citation statements)

References 30 publications

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“…With a similar way, we used in our study the Ion 16S™ Metagenomics Kit with both genomic DNA from ZymoBIOMICS mock standard sample and DNA extracted from soil samples originated from different habitat mainly the Moroccan phosphate mine and native plants in the ore. The obtained results in the mock samples presented in gure 1 were in accordance with the ndings of Barb et al [28] and Sirichoat et al [33], since the hypervariable regions V2, V3, V4 and V6-7 regions gave the best resolution up to both family and genus levels. V9 region gave less resolution and identi ed only Proteobacteria phylum.…”

Section: Discussionsupporting

confidence: 91%

“…Barb et al [28] used multiple hypervariable regions of the 16S rRNA gene to study the human microbiome and concluded that the use of multiple regions improve the results of identifying bacteria that compose complex communities such as those inhabiting human. The Ion 16S™ Metagenomics kit was also used to evaluate the performance of individual hypervariable regions of the 16S rRNA gene, for capturing human vaginal microbiota [33]. Authors of this study indicated that analyses using the V3 region generally indicated the highest bacterial diversity followed by the V6-V7 and V4 regions, while the V9 region gave the lowest bacterial resolution.…”

Section: Discussionmentioning

confidence: 98%

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16S Metagenomics investigation of Bacterial diversity and prediction of its functionalities in Moroccan phosphate mine ecosystem.

Azaroual

Kasmi

Aasfar

et al. 2021

Preprint

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Background Native plants in extreme environments may harbor some unique microbial communities with particular functions to sustain their growth and tolerance to harsh conditions. The aim of this study was to investigate the bacterial communities profiles in the Moroccan phosphate mine using 16S metagenomics sequencing. Taxonomic resolution of six hypervariable regions of the 16S rRNA was assessed in the collected samples and mock standard community. The bacterial functions enriched in the samples were also predicted to assess the contribution of the proposed low cost approach in the description of bacterial diversity and functionality in soil and plant. Methods The Ion 16S™ metagenomics kit was used to compare the discriminatory potential of the six targeted hypervariable regions of the 16S rRNA gene in the description of the bacterial communities. Alpha and beta diversity analysis were also performed accordingly in the studied samples. The later were collected from the bulk phosphate mine and the soils attached to roots of three different wild plants, that natively grow in this environment. Samples from the rhizosphere of wheat plants in cropping system were also studied. Actinobacteria related sequences were compared between the samples to investigate their possible origin between the bulk mine and the wild plants rhizosphere. The prediction of 82 bacterial functions related to nitrogen and phosphate metabolism along with stress was performed using Tax4fun bioinformatics tool. Results The rhizosphere of the three wild plants in the Moroccan phosphate mine is characterized by interesting bacterial diversity including Proteobacteria (62,24%, 71,15% and 65,61%), Actinobacteria (22,53%, 15,24%, 22,30%), Bacteroidetes (7,57% ; 4,23% ; 7,63%), and Firmicutes (5,82%; 1,17% ; 2,83%). Broad taxonomic diversity of minor phyla was also found in the native plants samples and included Acidobacteria, Armatimonadetes, Chloroflexi, Cyanobacteria, Gemmatimonadetes and Verrucomicrobia. The bulk phosphate mine samples were dominated by Actinobacteria with average relative abundance of 97,73% and Proteobacteria was Less abundant phylum in these samples. V3, V4 and V67 regions performed better in the taxonomic resolution at different levels e.g., phylum, family and genus. However, V8 and V9 regions gave the lesser taxonomic resolution and V9 region identified only Proteobacteria phylum in the mock standard sample. Alpha and beta diversity analysis indicated low abundance of observed OTUs and diversity in the bulk samples compared with the rhizosphere samples of the native plants in the phosphate mine. Moreover, significant (p = 0,021) differences in bacterial diversity were observed among at least three kind of samples : The bulk phosphate mine, the rhizosphere of the native wild plants of the phosphate mine and the rhizosphere of the wheat crop. The results indicated that both plant genotype and mainly soil conditions may be involved in the shaping of bacterial diversity. Such indication was also confirmed by the prediction of functional profiles that showed enrichment of many functions related to biological nitrogen fixation in the rhizosphere of native plants and the stress related functions in the bulk phosphate mine in comparison with the wheat rhizosphere samples Conclusion The proposed amplicon sequencing approach combined to bioinformatic prediction of bacterial functions could be considered as low-cost method to study the bacterial communities in soils and plants. The application of such approach to samples from the phosphate mines and wild plants revealed the need to consider at least V3, V4 and V6-7 hypervariable regions of the 16S rRNA gene in the taxonomic resolution of bacterial diversity in soil samples. This approach revealed also interesting bacterial diversity governed by the plant genotypes and the local conditions with interesting predicted functionalities involved in plant growth and development and stress tolerance in extreme environments. Further studies could target some interesting identified phyla and predicted bacterial functionalities, such studies could also deeply investigate the origin of the bacterial diversity in these native plants to develop new generation of bacterial inoculants for sustainable agriculture.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 98%

16S Metagenomics investigation of Bacterial diversity and prediction of its functionalities in Moroccan phosphate mine ecosystem.

Azaroual

Kasmi

Aasfar

et al. 2021

Preprint

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“…Human milk is a low microbial load sample, so precautions to avoid contamination and primer selection are crucial factors in this culture-independent technique. The choice of the 16S rRNA region can significantly affect the estimates of taxonomic diversity [ 38 , 39 ]. For instance, V2–V3 or V3–V4 regions compute similar numbers or reads per phyla but at lower taxonomic ranks the differences become larger [ 38 ].…”

Section: Resultsmentioning

confidence: 99%

Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum

et al. 2021

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The objective of this work was to characterize the microbiota of breast milk in healthy Spanish mothers and to investigate the effects of lactation time on its diversity. A total of ninety-nine human milk samples were collected from healthy Spanish women and were assessed by means of next-generation sequencing of 16S rRNA amplicons and by qPCR. Firmicutes was the most abundant phylum, followed by Bacteroidetes, Actinobacteria, and Proteobacteria. Accordingly, Streptococcus was the most abundant genus. Lactation time showed a strong influence in milk microbiota, positively correlating with Actinobacteria and Bacteroidetes, while Firmicutes was relatively constant over lactation. 16S rRNA amplicon sequencing showed that the highest alpha-diversity was found in samples of prolonged lactation, along with wider differences between individuals. As for milk nutrients, calcium, magnesium, and selenium levels were potentially associated with Streptococcus and Staphylococcus abundance. Additionally, Proteobacteria was positively correlated with docosahexaenoic acid (DHA) levels in breast milk, and Staphylococcus with conjugated linoleic acid. Conversely, Streptococcus and trans-palmitoleic acid showed a negative association. Other factors such as maternal body mass index or diet also showed an influence on the structure of these microbial communities. Overall, human milk in Spanish mothers appeared to be a complex niche shaped by host factors and by its own nutrients, increasing in diversity over time.

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“…We used the universal primer pair 337F/518R (see Section 2.3. Amplification of the 16S rRNA gene) to amplify a fragment of the 16S rRNA gene that includes the V3 region, which is considered one of the most effective hypervariable regions for phylogenetic analysis and taxonomic classification of bacterial species [65,66].…”

Section: Discussionmentioning

confidence: 99%

Changes in the Microbiome Profile in Different Parts of the Intestine in Piglets with Diarrhea

Gryaznova

Smirnova

Syromyatnikov

et al. 2022

Animals

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Determining the taxonomic composition of microbial consortia of the piglet intestine is of great importance for pig production. However, knowledge on the variety of the intestinal microbiome in newborn piglets is limited. Piglet diarrhea is a serious gastrointestinal disease with a high morbidity and mortality that causes great economic damage to the pig industry. In this study, we investigated the microbiome of various sections of the piglet intestine and compared the microbiome composition of healthy and diarrheal piglets using high-throughput sequencing of the 16S rRNA gene. The results showed that bacteria of the Lactobacillus genus were the most common in the ileum, while Fusobacterium and Bacteroides dominated in the rectum. Comparing the microbiome composition of healthy and diarrheal piglets revealed a reduced number of Lactobacillus bacteria as a hallmark of diarrhea, as did an increased content of representatives of the Escherichia-Shigella genus and a reduced number of Bacteroides, which indicates the contribution of these bacteria to the development of diarrhea in piglets. The relative abundance of Enterococcus bacteria was higher in the diarrhea group. Although some bacteria of this genus are commensals, a small number of species may be associated with the development of diarrhea in piglets. Therefore, our results indicate that the gut microbiome may be an important factor in the development of diarrhea in piglets.

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Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing

Cited by 31 publications

References 30 publications

16S Metagenomics investigation of Bacterial diversity and prediction of its functionalities in Moroccan phosphate mine ecosystem.

16S Metagenomics investigation of Bacterial diversity and prediction of its functionalities in Moroccan phosphate mine ecosystem.

Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum

Changes in the Microbiome Profile in Different Parts of the Intestine in Piglets with Diarrhea

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Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing

Cited by 31 publications

References 30 publications

16S Metagenomics investigation of Bacterial diversity and ﻿prediction of its functionalities in Moroccan phosphate mine ecosystem.

16S Metagenomics investigation of Bacterial diversity and ﻿prediction of its functionalities in Moroccan phosphate mine ecosystem.

Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum

Changes in the Microbiome Profile in Different Parts of the Intestine in Piglets with Diarrhea

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Product

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16S Metagenomics investigation of Bacterial diversity and prediction of its functionalities in Moroccan phosphate mine ecosystem.

16S Metagenomics investigation of Bacterial diversity and prediction of its functionalities in Moroccan phosphate mine ecosystem.