Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

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Analysis of the Gut Microbiota by High-Throughput Sequencing of the V5–V6 Regions of the 16S rRNA Gene in Donkey

Current Microbiology ◽

10.1007/s00284-014-0528-5 ◽

2014 ◽

Vol 68 (5) ◽

pp. 657-662 ◽

Cited By ~ 24

Author(s):

Xinfeng Liu ◽

Hanlu Fan ◽

Xiangbin Ding ◽

Zhongshan Hong ◽

Yongwei Nei ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

High Throughput ◽

High Throughput Sequencing ◽

Rrna Gene ◽

The 16S Rrna Gene

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Monosodium Glutamate Induces Changes in Hepatic and Renal Metabolic Profiles and Gut Microbiome of Wistar Rats

Nutrients ◽

10.3390/nu13061865 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1865

Author(s):

Kanokwan Nahok ◽

Jutarop Phetcharaburanin ◽

Jia V. Li ◽

Atit Silsirivanit ◽

Raynoo Thanan ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Gut Microbiome ◽

Wistar Rats ◽

Monosodium Glutamate ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Male Wistar Rats ◽

The Impact

The short- and long-term consumption of monosodium glutamate (MSG) increases urinary pH but the effects on the metabolic pathways in the liver, kidney and the gut microbiota remain unknown. To address this issue, we investigated adult male Wistar rats allocated to receive drinking water with or without 1 g% MSG for 2 weeks (n = 10, each). We performed a Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolomic study of the jejunum, liver, and kidneys, while faecal samples were collected for bacterial DNA extraction to investigate the gut microbiota using 16S rRNA gene sequencing. We observed significant changes in the liver of MSG-treated rats compared to controls in the levels of glucose, pyridoxine, leucine, isoleucine, valine, alanine, kynurenate, and nicotinamide. Among kidney metabolites, the level of trimethylamine (TMA) was increased, and pyridoxine was decreased after MSG-treatment. Sequencing of the 16S rRNA gene revealed that MSG-treated rats had increased Firmicutes, the gut bacteria associated with TMA metabolism, along with decreased Bifidobacterium species. Our data support the impact of MSG consumption on liver and kidney metabolism. Based on the gut microbiome changes, we speculate that TMA and its metabolites such as trimethylamine-N-oxide (TMAO) may be mediators of the effects of MSG on the kidney health.

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Different analysis strategies of 16S rRNA gene data from rodent studies generate contrasting views of gut bacterial communities associated with diet, health and obesity

PeerJ ◽

10.7717/peerj.10372 ◽

2020 ◽

Vol 8 ◽

pp. e10372

Author(s):

Jose F. Garcia-Mazcorro ◽

Jorge R. Kawas ◽

Cuauhtemoc Licona Cassani ◽

Susanne Mertens-Talcott ◽

Giuliana Noratto

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Relative Abundance ◽

Bacterial Communities ◽

Future Research ◽

Rrna Gene ◽

Microbial Composition ◽

Analysis Strategies ◽

The Impact

Background One of the main functions of diet is to nurture the gut microbiota and this relationship affects the health of the host. However, different analysis strategies can generate different views on the relative abundance of each microbial taxon, which can affect our conclusions about the significance of diet to gut health in lean and obese subjects. Here we explored the impact of using different analysis strategies to study the gut microbiota in a context of diet, health and obesity. Methods Over 15 million 16S rRNA gene sequences from published studies involving dietary interventions in obese laboratory rodents were analyzed. Three strategies were used to assign the 16S sequences to Operational Taxonomic Units (OTUs) based on the GreenGenes reference OTU sequence files clustered at 97% and 99% similarity. Results Different strategies to select OTUs influenced the relative abundance of all bacterial taxa, but the magnitude of this phenomenon showed a strong study effect. Different taxa showed up to 20% difference in relative abundance within the same study, depending on the analysis strategy. Very few OTUs were shared among the samples. ANOSIM test on unweighted UniFrac distances showed that study, sequencing technique, animal model, and dietary treatment (in that order) were the most important factors explaining the differences in bacterial communities. Except for obesity status, the contribution of diet and other factors to explain the variability in bacterial communities was lower when using weighted UniFrac distances. Predicted functional profile and high-level phenotypes of the microbiota showed that each study was associated with unique features and patterns. Conclusions The results confirm previous findings showing a strong study effect on gut microbial composition and raise concerns about the impact of analysis strategies on the membership and composition of the gut microbiota. This study may be helpful to guide future research aiming to investigate the relationship between diet, health, and the gut microbiota.

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Differences in the bacterial profiles of infant gut by birth process, milk diet, and choice of 16S rRNA gene target region

Human Microbiome Journal ◽

10.1016/j.humic.2019.100062 ◽

2019 ◽

Vol 13 ◽

pp. 100062

Author(s):

Maze Ann Biol-Aquino ◽

Christine Jane Perdiz ◽

Melissa Borlagdan ◽

James David Alcantara ◽

Aida Mallillin

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Rrna Gene ◽

Milk Diet ◽

Birth Process ◽

Gene Target ◽

Target Region

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Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

Applied and Environmental Microbiology ◽

10.1128/aem.01177-06 ◽

2006 ◽

Vol 73 (1) ◽

pp. 278-288 ◽

Cited By ~ 343

Author(s):

Rebecca J. Case ◽

Yan Boucher ◽

Ingela Dahllöf ◽

Carola Holmström ◽

W. Ford Doolittle ◽

...

Keyword(s):

16S Rrna ◽

Molecular Marker ◽

16S Rrna Gene ◽

Microbial Ecology ◽

Gene Tree ◽

Single Copy ◽

Rrna Gene ◽

Phylogenetic Resolution ◽

The Impact ◽

The 16S Rrna Gene

ABSTRACT Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

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Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota

Scientific Reports ◽

10.1038/s41598-021-82726-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Francesco Durazzi ◽

Claudia Sala ◽

Gastone Castellani ◽

Gerardo Manfreda ◽

Daniel Remondini ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Shotgun Sequencing ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Experimental Conditions ◽

Rrna Gene Sequencing

AbstractIn this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.

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Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.052993-0 ◽

2014 ◽

Vol 64 (Pt_3) ◽

pp. 781-786 ◽

Cited By ~ 33

Author(s):

Maximo Sánchez ◽

Martha-Helena Ramírez-Bahena ◽

Alvaro Peix ◽

María J. Lorite ◽

Juan Sanjuán ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Similarity ◽

Carbon Sources ◽

Lotus Corniculatus ◽

Culture Conditions ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

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