An optimized 16S rRNA sequencing protocol for vaginal microbiome to avoid biased abundance estimation

AbstractWe applied three 16S rRNA sequencing protocols on vaginal microbiome samples, to evaluate whether they produce unbiased estimation of vaginal microbiome composition. We modified the 27F primer (hereafter denoted as 27F’). Using vaginal samples from 28 healthy women and 10 women with bacterial vaginosis, we sequenced three 16S rRNA sequencing protocols, i.e., 27F-338R, 27F’-338R and 341F-806R protocols, naming after their PCR primer sets, to test whether the sequencing results are consistent with the clinical diagnostics, morphology and qPCR results. First, the 27F primer would not align with Gardnerlla vaginalis very well, leading to poor amplification of such species. By modifying the primer sequences, the modified 27F primer (27F’) was able to amplify Gardnerlla vaginalis very well. Second, the DNA sequence of characteristic species Lactobacillus crispatus is identical with Lactobacillus garrinarum, leading to biased estimation of abundance of Lactobacillus crispatus when using V3-V4 as PCR target region; in contrast, such bias did not occur when using V1-V2 as a target region. Third, optimized 27F’-338R avoided above-mentioned biases and restored the well-established community state types (CSTs) clustering.ImportanceVaginal microbiome has profound effects on the health of women and their newborns. Our study found that two well-established 16S rDNA sequencing protocols led to systementical biased estimation of characteristic species of vaginal microbiome. Subsequent analysis proved that the PCR primer fetching efficacy and target region identity were major contributor for such bias. With carefully selected target region and optimized PCR primer set, we were able to eliminate such biases and provide accurate estimation of vaginal microbiome, which showed high consistency with clinical diagnostics. We modified the 27F primer (27F’). Using the optimized PCR primer set of 27F’ and 338R to target the V1-V2 hyper-variable region, our 16S rRNA sequencing correctly evaluate the composition of vaginal microbiome.

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Haemophilus parainfluenzaeInfective Endocarditis Diagnosed by Direct 16S rRNA Sequencing of Vegetation

Laboratory Medicine Online ◽

10.3343/lmo.2012.2.2.111 ◽

2012 ◽

Vol 2 (2) ◽

pp. 111

Author(s):

Sung-Hee Oh ◽

Min-Chul Cho ◽

Jae-Wook Kim ◽

Dongheui An ◽

Mun-Hui Jeong ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Full-length versus short amplicon 16S rRNA sequencing: benefits, drawbacks and risks

10.26226/morressier.5f3288739a6b12016a74ff57 ◽

2020 ◽

Author(s):

Isabel Abellan-Schneyder ◽

Andrea Janina Bayer ◽

Sandra Reitmeier ◽

Klaus Neuhaus

Keyword(s):

16S Rrna ◽

Full Length ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Full-length versus short amplicon 16S rRNA sequencing: benefits, drawbacks and risks

10.26226/morressier.5f3392ca9d1718ca4c8b2f06 ◽

2020 ◽

Author(s):

Andrea Janina Bayer ◽

Sandra Reitmeier ◽

Klaus Neuhaus ◽

Isabel Abellan-Schneyder

Keyword(s):

16S Rrna ◽

Full Length ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Phenotypic and phylogenetic characterization of Lactobacillus species isolated from traditional Lighvan cheese

Food Production, Processing and Nutrition ◽

10.1186/s43014-021-00065-x ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Haleh Forouhandeh ◽

Sepideh Zununi Vahed ◽

Hossein Ahangari ◽

Vahideh Tarhriz ◽

Mohammad Saeid Hejazi

Keyword(s):

16S Rrna ◽

Random Amplified Polymorphic Dna ◽

Bacterial Species ◽

16S Rrna Sequencing ◽

Lactobacillus Species ◽

Culture Methods ◽

Phylogenetic Characterization ◽

Rrna Sequencing ◽

Specific Culture ◽

Lighvan Cheese

Abstract Lighvan cheese (Lighvan panir) is among the most famous traditional cheese in Iran for its desired aroma and flavor. Undoubtedly, the lactic acid bacteria especially the genus Lactobacillus are the critical factors in developing the aroma, flavor, and texture in Lighvan cheese. In this study, the Lactobacillus population of the main Lighvan cheese was investigated. The Lactobacillus of the main Lighvan cheese was isolated using specific culture methods according to previously published Guidelines. Then, the phylogenetic features were investigated and the phenotypic characteristics were examined using specific culture methods. Twenty-eight Gram-positive bacterial species were identified belonged to the genus Lactobacillus. According to the same sequences as each other, three groups (A, B, and C) of isolates were categorized with a high degree of similarity to L. fermentum (100%) and L. casei group (L. casei, L. paracasei, and L. rhamnosus) (99.0 to 100%). Random amplified polymorphic DNA (RAPD) fingerprint analysis manifested the presence of three clusters that were dominant in traditional Lighvan cheese. Cluster І was divided into 4 sub-clusters. By the result of carbohydrate fermentation pattern and 16S rRNA sequencing, isolates were identified as L. rhamnosus. The isolates in clusters II and III represented L. paracasei and L. fermentum, respectively as they were identified by 16S rRNA sequencing and fermented carbohydrate patterns. Our result indicated that the specific aroma and flavor of traditional Lighvan cheese can be related to its Lactobacillus population including L. fermentum, L. casei, L. paracasei, and L. rhamnosus. Graphical abstract

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Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed

Environmental Microbiome ◽

10.1186/s40793-021-00382-1 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Leena Malayil ◽

Suhana Chattopadhyay ◽

Emmanuel F. Mongodin ◽

Amy R. Sapkota

Keyword(s):

16S Rrna ◽

Irrigation Water ◽

Brackish Water ◽

Water Sources ◽

16S Rrna Sequencing ◽

Vbnc State ◽

Recycled Water ◽

Dna Labeling ◽

Rrna Sequencing ◽

Brdu Labeling

AbstractNontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including Vibrio spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2′-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active Vibrio spp. in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active Vibrio spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of V. cholerae, V. vulnificus, and V. parahaemolyticus using both methods, while V. aesturianus and V. shilonii were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including P. shigelloides, B. cereus and E. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in Vibrio spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active Vibrio spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.

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Antibiotic-Induced Dysbiosis of Microbiota Promotes Chicken Lipogenesis by Altering Metabolomics in the Cecum

Metabolites ◽

10.3390/metabo11080487 ◽

2021 ◽

Vol 11 (8) ◽

pp. 487

Author(s):

Tao Zhang ◽

Hao Ding ◽

Lan Chen ◽

Yueyue Lin ◽

Yongshuang Gong ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

High Performance ◽

Fat Deposition ◽

16S Rrna Sequencing ◽

Oral Antibiotics ◽

Ms Analysis ◽

Rrna Sequencing ◽

Cecal Microbiota ◽

Metabolite Network

Elucidation of the mechanism of lipogenesis and fat deposition is essential for controlling excessive fat deposition in chicken. Studies have shown that gut microbiota plays an important role in regulating host lipogenesis and lipid metabolism. However, the function of gut microbiota in the lipogenesis of chicken and their relevant mechanisms are poorly understood. In the present study, the gut microbiota of chicken was depleted by oral antibiotics. Changes in cecal microbiota and metabolomics were detected by 16S rRNA sequencing and ultra-high performance liquid chromatography coupled with MS/MS (UHPLC–MS/MS) analysis. The correlation between antibiotic-induced dysbiosis of gut microbiota and metabolites and lipogenesis were analysed. We found that oral antibiotics significantly promoted the lipogenesis of chicken. 16S rRNA sequencing indicated that oral antibiotics significantly reduced the diversity and richness and caused dysbiosis of gut microbiota. Specifically, the abundance of Proteobacteria was increased considerably while the abundances of Bacteroidetes and Firmicutes were significantly decreased. At the genus level, the abundances of genera Escherichia-Shigella and Klebsiella were significantly increased while the abundances of 12 genera were significantly decreased, including Bacteroides. UHPLC-MS/MS analysis showed that antibiotic-induced dysbiosis of gut microbiota significantly altered cecal metabolomics and caused declines in abundance of 799 metabolites and increases in abundance of 945 metabolites. Microbiota-metabolite network revealed significant correlations between 4 differential phyla and 244 differential metabolites as well as 15 differential genera and 304 differential metabolites. Three metabolites of l-glutamic acid, pantothenate acid and N-acetyl-l-aspartic acid were identified as potential metabolites that link gut microbiota and lipogenesis in chicken. In conclusion, our results showed that antibiotic-induced dysbiosis of gut microbiota promotes lipogenesis of chicken by altering relevant metabolomics. The efforts in this study laid a basis for further study of the mechanisms that gut microbiota regulates lipogenesis and fat deposition of chicken.

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Probiotic supplements alleviate gestational diabetes mellitus by restoring the diversity of gut microbiota: a study based on 16S rRNA sequencing

The Journal of Microbiology ◽

10.1007/s12275-021-1094-8 ◽

2021 ◽

Author(s):

Qing-Xiang Zheng ◽

Xiu-Min Jiang ◽

Hai-Wei Wang ◽

Li Ge ◽

Yu-Ting Lai ◽

...

Keyword(s):

Diabetes Mellitus ◽

16S Rrna ◽

Gestational Diabetes ◽

Gut Microbiota ◽

Gestational Diabetes Mellitus ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Alterations in intestinal microbiota diversity, composition, and function in patients with sarcopenia

Scientific Reports ◽

10.1038/s41598-021-84031-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lin Kang ◽

Pengtao Li ◽

Danyang Wang ◽

Taihao Wang ◽

Dong Hao ◽

...

Keyword(s):

Skeletal Muscle ◽

16S Rrna ◽

Intestinal Microbiota ◽

Muscle Growth ◽

Growth Function ◽

16S Rrna Sequencing ◽

Fecal Samples ◽

Lipopolysaccharide Biosynthesis ◽

Rrna Sequencing ◽

And Function

Abstract16S rRNA sequencing of human fecal samples has been tremendously successful in identifying microbiome changes associated with both aging and disease. A number of studies have described microbial alterations corresponding to physical frailty and nursing home residence among aging individuals. A gut-muscle axis through which the microbiome influences skeletal muscle growth/function has been hypothesized. However, the microbiome has yet to be examined in sarcopenia. Here, we collected fecal samples of 60 healthy controls (CON) and 27 sarcopenic (Case)/possibly sarcopenic (preCase) individuals and analyzed the intestinal microbiota using 16S rRNA sequencing. We observed an overall reduction in microbial diversity in Case and preCase samples. The genera Lachnospira, Fusicantenibacter, Roseburia, Eubacterium, and Lachnoclostridium—known butyrate producers—were significantly less abundant in Case and preCase subjects while Lactobacillus was more abundant. Functional pathways underrepresented in Case subjects included numerous transporters and phenylalanine, tyrosine, and tryptophan biosynthesis suggesting that protein processing and nutrient transport may be impaired. In contrast, lipopolysaccharide biosynthesis was overrepresented in Case and PreCase subjects suggesting that sarcopenia is associated with a pro-inflammatory metagenome. These analyses demonstrate structural and functional alterations in the intestinal microbiota that may contribute to loss of skeletal muscle mass and function in sarcopenia.

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