scholarly journals Investigation of MiSeq reproducibility on biomarker identification

2019 ◽  
Vol 62 (1) ◽  
Author(s):  
Hyejun Jo ◽  
Jiwan Hong ◽  
Tatsuya Unno
Keyword(s):  
16S Rrna ◽  
Beta Diversity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Human Feces ◽  
Differential Abundance ◽  
Biomarker Identification ◽  
Alpha And Beta Diversity ◽  
Operational Taxonomic Units ◽  
Bioinformatics Tools

Abstract MiSeq-derived artificial sequences appeared to be of good quality, thus bioinformatics tools failed to remove MiSeq artefacts. Even after removing singleton sequences or operational taxonomic units (OTUs), it is not clear how many sequence artefacts remained. Here, 16S rRNA genes were amplified from soil, human feces, pig feces, and groundwater. These were sequenced with five separate runs of MiSeq. Subsequently, each run of MiSeq was compared through alpha and beta-diversity analyses. We found more than half the OTUs were not in consensus through the multiple MiSeq runs, resulting in varying group-specific biomarker OTUs in each MiSeq run. Thus, differential abundance test should be interpreted with caution, and we suggest that results also should be verified further with other quantification methods such as qPCR.

Analysis of the Salivary Microbiome in the Periodontal Disease patients with hypertension and non-hypertension

2020 ◽  
Vol 15 ◽  
Author(s):  
Suhua Li ◽  
Rexidan Zaker ◽  
Xueqian Chu ◽  
Reyida Asihati ◽  
Chong Li ◽  
...  
Keyword(s):  
Periodontal Disease ◽  
Anaerobic Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Oral Microbiota ◽  
Periodontal Health ◽  
Differential Abundance ◽  
Operational Taxonomic Units ◽  
Hypervariable Regions ◽  
Microbiota Diversity

Background: An improved comprehension of the oral microbiota function in the pathogenesis of disease will contribute to diagnosis and treatment for both hypertension and periodontal disease. In our study, a comparison of the salivary microbiome between hypertension and Non-hypertension cohorts was designed to reveal microbial signatures. <P> Methods: Patients were divided into four sub-groups: Gingivitis, Periodontitis (stage 2, 3 and 4). Then the hypertension and Non-hypertension cohorts were split into periodontal health and periodontitis subgroups. The salivary samples were processed for DNA extraction (n=246). The V3-V4 hypervariable regions of microbiome 16S rRNA genes were amplified. Finally, sequencing libraries was constructed and subjected them to bioinformatics and statistical analyses. <P> Results: The oral microbial diversity decreased in both hypertension and periodontal disease groups compared to the healthy. At the genus level, the diversity showed 100 different operational taxonomic units (OTUs) for differential abundance testing. The first trend showed OTUs decreased in relative abundance with increasing periodontal disease, and as well as hypertension groups and non-hypertensive. For this trend OTUs comprised a mix of primarily anaerobic commensals and potential acute diarrhea pathogens. The second trend was that the diversity of genera was decreased in hypertension relative to non-hypertension, including other anaerobic bacteria related with periodontal disease. <P> Conclusions: Microbiota diversity decreased in both hypertension and different stages of periodontal disease groups, however, Neisseria and Solobacterium genera increased in co-existing hypertension and periodontal disease. Obviously, these findings indicate that the abundance of genera continues to change due to additional stresses caused by co-existing conditions

scholarly journals Bacteria Associated with Mucus and Tissues of the Coral Oculina patagonica in Summer and Winter

2006 ◽  
Vol 72 (8) ◽  
pp. 5254-5259 ◽  
Author(s):  
Omry Koren ◽  
Eugene Rosenberg
Keyword(s):  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Culturable Bacteria ◽  
Vibrio Splendidus ◽  
Marine Agar ◽  
Tissue Samples ◽  
Operational Taxonomic Units ◽  
Sybr Gold ◽  
16S Rrna Clone Library

ABSTRACT The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 × 107 and 8.3 × 108/cm2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different α-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (>99.5% identity). Of these, 295 were novel (<99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.

Molecular community analysis of magnesium-rich bittern brine recovered from a Tunisian solar saltern

2011 ◽  
Vol 57 (12) ◽  
pp. 975-981 ◽  
Author(s):  
Houda Baati ◽  
Raja Jarboui ◽  
Néji Gharsallah ◽  
Abdelghani Sghir ◽  
Emna Ammar
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Archaeal Community ◽  
Community Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Solar Saltern ◽  
16S Rrna Gene Analysis ◽  
Operational Taxonomic Units

The microbial community of a magnesium-rich bittern brine saturated with NaCl (380–400 g/L) from a Tunisian solar saltern was investigated using a molecular approach based on 16S rRNA gene analysis and viability tests. The results revealed the existence of microbial flora. Viability test assessment showed that 46.4% of this flora was viable but not detectable by culturability tests. 16S rRNA genes from 49 bacterial clones and 38 archaeal clones were sequenced and phylogenetically analyzed. Eleven operational taxonomic units (OTUs) determined by the DOTUR program with 97% sequence similarity were generated for Bacteria. These OTUs were affiliated with Bacteroidetes and Gammaproteobacteria. The archaeal community composition exhibited more diversity with 38 clones, resulting in 13 OTUs affiliated with the Euryarchaeota phylum. Diversity measurement showed a more diverse archaeal than bacterial community at the saturated pond.

scholarly journals Genome-Derived Criteria for Assigning Environmental narG and nosZ Sequences to Operational Taxonomic Units of Nitrate Reducers

2009 ◽  
Vol 75 (15) ◽  
pp. 5170-5174 ◽  
Author(s):  
Katharina Palmer ◽  
Harold L. Drake ◽  
Marcus A. Horn
Keyword(s):  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
Nitrate Reducers ◽  
Multiple Copies ◽  
Tree Topologies ◽  
Gene Similarity ◽  
Very High

ABSTRACT Ninety percent of cultured bacterial nitrate reducers with a 16S rRNA gene similarity of ≥97% had a narG or nosZ similarity of ≥67% or ≥80%, respectively, suggesting that 67% and 80% could be used as standardized, conservative threshold similarity values for narG and nosZ, respectively (i.e., any two sequences that are less similar than the threshold similarity value have a very high probability of belonging to different species), for estimating species-level operational taxonomic units. Genus-level tree topologies of narG and nosZ were generally similar to those of the corresponding 16S rRNA genes. Although some genomes contained multiple copies of narG, recent horizontal gene transfer of narG was not apparent.

scholarly journals Phylogenetic Characterization of 16S rRNA Gene Clones from Deep-Groundwater Microorganisms That Pass through 0.2-Micrometer-Pore-Size Filters

2005 ◽  
Vol 71 (2) ◽  
pp. 1084-1088 ◽  
Author(s):  
Tatsuo Miyoshi ◽  
Teruki Iwatsuki ◽  
Takeshi Naganuma
Keyword(s):  
16S Rrna ◽  
Pore Size ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Deep Groundwater ◽  
Phylogenetic Characterization ◽  
Operational Taxonomic Units ◽  
Pass Through

ABSTRACT A total of 247 clones of 16S rRNA genes from microorganisms captured by 0.2- and 0.1-μm-pore-size filters from sedimentary and granite rock aquifers were amplified and yielded 37 operational taxonomic units (OTUs). Fifteen OTUs captured by 0.1-μm-pore-size filters were affiliated with the candidate divisions OD1 and OP11, representing novel lineages. On the other hand, OTUs captured by 0.2-μm-pore-size filters were largely affiliated with Betaproteobacteria.

Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study

2008 ◽  
Vol 54 (6) ◽  
pp. 479-482 ◽  
Author(s):  
Han-Bo Zhang ◽  
Chan-Wen Xu ◽  
Miao-Miao Wang ◽  
Tao Li ◽  
Zhi-Wei Zhao
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Taxonomic Rank ◽  
Operational Taxonomic Units

We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36–53 OTUs using the DOTUR program when sequence similarities of 95%–100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon–Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.

scholarly journals Impact of Helicobacter Pylori Infection on Duodenal Microbial Community Structure and Microbial Metabolic Pathways

2021 ◽  
Author(s):  
Tadashi Maeda ◽  
Hiroaki Zai ◽  
Yuto Fukui ◽  
Yoshifumi Kato ◽  
Eri Kumade ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Metabolic Pathways ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
H Pylori ◽  
The Impact

Abstract Background The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. Results Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection increased the relative mean abundance of Proteobacteria and Neisseria and decreased the abundance of the two other phyla (Actinobacteria and TM7) and nine genera (Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. Conclusions H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

scholarly journals 16S rRNA–Based Analysis Reveals Differences in the Bacterial Community Present in Tissues of Choromytilus chorus (Mytilidae, Bivalvia) Grown in an Estuary and a Bay in Southern Chile

Diversity ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 209
Author(s):  
Tamara Valenzuela ◽  
Joaquin I. Riling ◽  
Giovanni Larama ◽  
Jacquelinne J. Acuña ◽  
Marco Campos ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
Illumina Miseq ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Venn Diagram ◽  
Linear Discriminant ◽  
Operational Taxonomic Units ◽  
Marine Bivalves ◽  
Diagram Analysis

Microbiota associated with bivalves have drawn considerable attention because studies have suggested their relevance to the fitness and growth of marine bivalves. Although the mussel Choromytilus chorus is a valuable resource for Chilean aquaculture and fisheries, its microbiota is still unknown. In this study, the composition and predicted functions of the bacterial community in tissues of C. chorus specimens grown in an estuary (Nehuentue) and a bay (Hueihue) were investigated. Using 16S rRNA genes as targets, the bacterial abundance in tissues was estimated by quantitative PCR and sequenced via Illumina MiSeq. The abundances of bacteria ranged from 103 to 105 copies of 16S rRNA genes g−1 tissue. In the Nehuentue estuary, the bacterial communities in the tissues were dominated by the Tenericutes phylum, whereas the Tenericutes and Proteobacteria phyla dominated in mussels from Hueihue Bay. Higher numbers of operational taxonomic units (OTUs) were observed in tissues from the Nehuentue Estuary than in those from Hueihue Bay. Differences in bacterial community compositions in tissues between both locations were confirmed by nonmetric multidimensional scaling (nMDS) and Venn diagram analysis. In addition, linear discriminant analysis effect size (LEfSe) revealed that the Mollicutes class and Actynomycetales order were key phylotypes in tissues from the Nehuentue Estuary and Hueihue Bay, respectively. Our analysis also predicted a high abundance of sequences assigned to heterotrophy; however, relatively high functional diversity was also found in tissues from Hueihue Bay. This work represents our first attempt to elucidate the C. chorus microbiota in contrasting Chilean aquatic environments.

scholarly journals Composition and variation of the skin microbiota in sympatric species of European newts (Salamandridae)

2015 ◽  
Vol 36 (1) ◽  
pp. 5-12 ◽  
Author(s):  
Miguel Vences ◽  
Miguel Vences ◽  
Anja B. Dohrmann ◽  
Miguel Vences ◽  
Anja B. Dohrmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
Sympatric Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Triturus Cristatus ◽  
Operational Taxonomic Units ◽  
Lissotriton Vulgaris ◽  
Rdna Sequences ◽  
High Throughput Dna Sequencing

The mucous skin of amphibians provides a habitat for microorganisms which may interact with their hosts and thereby affect their condition and health. Cultivation-independent analyses of the bacterial communities based on the detection of PCR-amplified bacterial 16S rRNA genes provides a direct approach to characterize their diversity. In the present pilot study we utilized this approach in combination with a high-throughput DNA sequencing technology (454 pyrosequencing), to characterize the bacterial community structure of the skin of three newt species (Lissotriton vulgaris, Ichthyosaura alpestris, Triturus cristatus), collected near Braunschweig, Germany. 16S rDNA sequences were obtained from 19 unique samples. On average, 6113 amplicon sequences were obtained per sample and these could phylogenetically be assigned to a total of 1615 different operational taxonomic units (OTUs). Altogether, most samples were rather similar in their dominant bacterial taxa. Most represented were Betaproteobacteria (46%; mostly Janthinobacterium), Gammaproteobacteria (28%; mostly Pseudomonas), Flavobacteria (phylum Bacteroidetes: 19%, mostly Flavobacterium), and Sphingobacteria (Bacteroidetes: 5%, mostly Pedobacter). We found no significant differences between the three newt species, or between hemi-nested vs. non-nested PCR, but a strong difference among sampling dates (15 and 17 April 2013) which might be explained by the ongoing transition of the newts from their terrestrial to aquatic phase which coincided with this period, or by differences between sexes as these were unevenly sampled on the two dates. 16S rRNA gene sequences retrieved in this study in several cases were identical or very similar to those previously found on the skin of North American salamanders.

scholarly journals Clustering 16S rRNA for OTU prediction: A similarity based method

2019 ◽  
Vol 1 (2) ◽  
pp. 78-83
Author(s):  
Mehmet Can ◽  
Osman Gürsoy
Keyword(s):  
16S Rrna ◽  
Distance Measure ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Complex Environments ◽  
Operational Taxonomic Units ◽  
Heuristic Clustering ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

To study the phylogeny and taxonomy of samples from complex environments Next-generation sequencing (NGS)-based 16S rRNA sequencing , which has been successfully used  jointly with the PCR amplification and NGS technology. First step for many downstream analyses is clustering 16S rRNA sequences into operational taxonomic units (OTUs). Heuristic clustering is one of the most widely employed approaches for generating OTUs in which one or more seed sequences to represent each cluster are selected. In this work we chose five random seeds for each cluster from a genes library, and  we present a novel distance measure to cluster bacteria in the sample. Artificially created sets of 16S rRNA genes selected from databases are successfully clustered with more than %98 accuracy, sensitivity, and specificity.

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