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

Abstract Background: Waterlogging on the global environment has led to a significant decline in crop yields. However, the response of plant-associated microbes to waterlogging stress on different soils is not known. Moreover, there are few reports on whether this response is influenced by different sequencing methods. In this study, the effects of waterlogging on soybean rhizosphere microbial structure on two types of soil were examined, using a short reading 16S rRNA sequencing variable region V4 and two full-length 16S rRNA sequencing variable regions V1-V9.Results: The results revealed some similarities and differences in three sequencing methods for soybean rhizosphere microbial response to waterlogging stress. Based on CPCoA analysis, all the sequencing methods showed that waterlogging on both types of soil significantly affected the bacterial community structure of the soybean rhizosphere, and increased the relative abundance of Geobacter. However, the full-length sequencing methods had higher classification resolution than short-read sequencing (except phylum level of all sequencing methods and class level of LoopSeq sequencing). Further, analysis on OTU level and network showed that waterlogging increased the abundance of some microorganisms related to nitrogen cycle using V4 sequencing, and microorganisms related to phosphorus cycling when using two full-length sequencing methods. This is in line with the core microbial analysis. Environmental factors affecting the structure of microbial communities differed among sequencing methods.Conclusions: In summary, this piece of work detected the effects of waterlogging on soybean rhizosphere microbes using three sequencing methods. Some functional microbes were enriched in the rhizosphere, which may benefit soybean in resisting waterlogging stress. On the other hand, there were several differences in results among the three sequencing methods which might affect the response of rhizosphere microbial structure to stress. Our analysis of sequencing methods on various levels provides some useful information on environmental samples sequencing.

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Publisher Correction: The effect of taxonomic classification by full-length 16S rRNA sequencing with a synthetic long-read technology

Scientific Reports ◽

10.1038/s41598-021-90067-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jinuk Jeong ◽

Kyeongeui Yun ◽

Seyoung Mun ◽

Won‑Hyong Chung ◽

Song‑Yi Choi ◽

...

Keyword(s):

16S Rrna ◽

Full Length ◽

Taxonomic Classification ◽

16S Rrna Sequencing ◽

Rrna Sequencing ◽

Long Read

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Characterization of bacterial communities in wastewater with enhanced taxonomic resolution by full-length 16S rRNA sequencing

Scientific Reports ◽

10.1038/s41598-019-46015-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 19

Author(s):

Daniela Numberger ◽

Lars Ganzert ◽

Luca Zoccarato ◽

Kristin Mühldorfer ◽

Sascha Sauer ◽

...

Keyword(s):

16S Rrna ◽

Bacterial Communities ◽

Full Length ◽

16S Rrna Sequencing ◽

Taxonomic Resolution ◽

Rrna Sequencing

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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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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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