Identification and quantification of abundant species from pyrosequences of 16S rRNA by consensus alignment

ABSTRACTThe animal gastrointestinal tract houses a large microbial community, the gut microbiota, that confers many benefits to its host, such as protection from pathogens and provision of essential metabolites. Metagenomic approaches have defined the chicken fecal microbiota in other studies, but here, we wished to assess the correlation between the metagenome and the bacterial proteome in order to better understand the healthy chicken gut microbiota. Here, we performed high-throughput sequencing of 16S rRNA gene amplicons and metaproteomics analysis of fecal samples to determine microbial gut composition and protein expression. 16 rRNA gene sequencing analysis identifiedClostridiales,Bacteroidaceae, andLactobacillaceaespecies as the most abundant species in the gut. For metaproteomics analysis, peptides were generated by using the Fasp method and subsequently fractionated by strong anion exchanges. Metaproteomics analysis identified 3,673 proteins. Among the most frequently identified proteins, 380 proteins belonged toLactobacillusspp., 155 belonged toClostridiumspp., and 66 belonged toStreptococcusspp. The most frequently identified proteins were heat shock chaperones, including 349 GroEL proteins, from many bacterial species, whereas the most abundant enzymes were pyruvate kinases, as judged by the number of peptides identified per protein (spectral counting). Gene ontology and KEGG pathway analyses revealed the functions and locations of the identified proteins. The findings of both metaproteomics and 16S rRNA sequencing analyses are discussed.

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Identification and Quantification of Methanogenic Archaea in Adult Chicken Ceca

Applied and Environmental Microbiology ◽

10.1128/aem.01931-06 ◽

2006 ◽

Vol 73 (1) ◽

pp. 353-356 ◽

Cited By ~ 59

Author(s):

Suwat Saengkerdsub ◽

Robin C. Anderson ◽

Heather H. Wilkinson ◽

Woo-Kyun Kim ◽

David J. Nisbet ◽

...

Keyword(s):

16S Rrna ◽

Copy Number ◽

Methanogenic Archaea ◽

Molecular Methods ◽

16S Rrna Genes ◽

Rrna Genes ◽

Adult Chicken ◽

Wet Weight ◽

Identification And Quantification

ABSTRACT By using molecular methods for the identification and quantification of methanogenic archaea in adult chicken ceca, 16S rRNA genes of 11 different phylotypes, 10 of which were 99% similar to Methanobrevibacter woesei, were found. Methanogen populations, as assessed by cultivation, and the 16S rRNA copy number were between 6.38 and 8.23 cells/g (wet weight) and 5.50 and 7.19 log10/g (wet weight), respectively.

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Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

Applied and Environmental Microbiology ◽

10.1128/aem.63.4.1268-1273.1997 ◽

1997 ◽

Vol 63 (4) ◽

pp. 1268-1273 ◽

Cited By ~ 163

Author(s):

P Kaufmann ◽

A Pfefferkorn ◽

M Teuber ◽

L Meile

Keyword(s):

16S Rrna ◽

Colony Hybridization ◽

Bifidobacterium Species ◽

Identification And Quantification

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Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton

Applied and Environmental Microbiology ◽

10.1128/aem.00214-07 ◽

2007 ◽

Vol 73 (12) ◽

pp. 4055-4065 ◽

Cited By ~ 56

Author(s):

Natalia V. Ivanikova ◽

Linda C. Popels ◽

R. Michael L. McKay ◽

George S. Bullerjahn

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Lake Superior ◽

Abundant Species ◽

Rrna Gene ◽

Neighbor Joining ◽

16S Rrna Gene Sequences ◽

The World ◽

Gene Data ◽

The 16S Rrna Gene

ABSTRACT Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 105 cells ml−1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.

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A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION platform

F1000Research ◽

10.12688/f1000research.28216.3 ◽

2021 ◽

Vol 10 ◽

pp. 79

Author(s):

Boy M. Bachtiar ◽

Citra F. Theodorea ◽

Dicky L. Tahapary ◽

Cindy Astrella ◽

Natalina ◽

...

Keyword(s):

Pilot Study ◽

16S Rrna ◽

Host Response ◽

Active Role ◽

Amplicon Sequencing ◽

Abundant Species ◽

Periodontal Pocket ◽

Periodontal Tissue ◽

16S Rrna Amplicon Sequencing ◽

Systemic Condition

Background: Subgingival niche is one biofilm habitat containing rich microbiota, which plays an active role in maintaining the health of periodontal tissue and determining host response. As such, a study of changing subgingival biofilms is important for understanding the effect of a systemic condition. In this study, we compared the occurrence of six bacteria cohabiting in the subgingival area of periodontitis subjects, with (DP, n = 8) and without (NDP, n = 4) diabetes. Methods: The six genus and species of targeted bacteria were confirmed by 16S rRNA amplicon sequencing on MinION nanopore platform. Descriptive statistic was used to describe the obtained data. Results: We found that the six genus and species of targeted bacteria were detected but in different quantities in either group's periodontal pocket. Our data showed that Tannerella forsythia was the most abundant species in subgingival biofilms of the DP group of the red complex bacteria. In contrast, Aggregatibacter sp., which belongs to the phylum of proteobacteria, was present at a relatively lower level. In contrast, Fusobacterium sp., which belongs to orange complex bacteria, showed relative similarities in subgingival biofilms of both groups tested, while Veillonella sp., were abundant in the DP groups. Conclusions: Our data show that the diversity of classic periodontopathogens increased in the subgingival niche of periodontitis subjects with diabetes. It is the first study in Indonesia to apply MinION-based, full-length 16S rRNA amplicon sequencing in periodontitis patients with and without diabetes.

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A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION platform

F1000Research ◽

10.12688/f1000research.28216.4 ◽

2021 ◽

Vol 10 ◽

pp. 79

Author(s):

Boy M. Bachtiar ◽

Citra F. Theodorea ◽

Dicky L. Tahapary ◽

Cindy Astrella ◽

Natalina ◽

...

Keyword(s):

Pilot Study ◽

16S Rrna ◽

Host Response ◽

Active Role ◽

Amplicon Sequencing ◽

Abundant Species ◽

Periodontal Pocket ◽

Periodontal Tissue ◽

16S Rrna Amplicon Sequencing ◽

Systemic Condition

Background: Subgingival niche is one biofilm habitat containing rich microbiota, which plays an active role in maintaining the health of periodontal tissue and determining host response. As such, a study of changing subgingival biofilms is important for understanding the effect of a systemic condition. In this study, we compared the occurrence of six bacteria cohabiting in the subgingival area of periodontitis subjects, with (DP, n = 8) and without (NDP, n = 4) diabetes. Methods: The six genus and species of targeted bacteria were confirmed by 16S rRNA amplicon sequencing on MinION nanopore platform. Descriptive statistic was used to describe the obtained data. Results: We found that the six genus and species of targeted bacteria were detected but in different quantities in either group's periodontal pocket. Our data showed that Tannerella forsythia was the most abundant species in subgingival biofilms of the DP group of the red complex bacteria. In contrast, Aggregatibacter sp., which belongs to the phylum of proteobacteria, was present at a relatively lower level. In contrast, Fusobacterium sp., which belongs to orange complex bacteria, showed relative similarities in subgingival biofilms of both groups tested, while Veillonella sp., were abundant in the DP groups. Conclusions: Our data show that the diversity of classic periodontopathogens increased in the subgingival niche of periodontitis subjects with diabetes. It is the first study in Indonesia to apply MinION-based, full-length 16S rRNA amplicon sequencing in periodontitis patients with and without diabetes.

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A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION platform

F1000Research ◽

10.12688/f1000research.28216.1 ◽

2021 ◽

Vol 10 ◽

pp. 79

Author(s):

Boy M. Bachtiar ◽

Citra F. Theodorea ◽

Dicky L. Tahapary ◽

Cindy Astrella ◽

Natalina ◽

...

Keyword(s):

Pilot Study ◽

16S Rrna ◽

Host Response ◽

Active Role ◽

Amplicon Sequencing ◽

Abundant Species ◽

Periodontal Pocket ◽

Periodontal Tissue ◽

16S Rrna Amplicon Sequencing ◽

Systemic Condition

Background: Subgingival niche is one biofilm habitat containing rich microbiota, which plays an active role in maintaining the health of periodontal tissue and determining host response. As such, a study of changing subgingival biofilms is important for understanding the effect of a systemic condition. In this study, we compared the occurrence of six bacteria cohabiting in the subgingival area of periodontitis subjects, with (DP, n = 8) and without (NDP, n = 4) diabetes. Methods: The six genus and species of targeted bacteria were confirmed by 16S rRNA amplicon sequencing on MinION nanopore platform. Descriptive statistic was used to describe the obtained data. Results: We found that the six genus and species of targeted bacteria were detected but in different quantities in either group's periodontal pocket. Our data showed that Tannerella forsythia was the most abundant species in subgingival biofilms of the DP group of the red complex bacteria. In contrast, Aggregatibacter sp., which belongs to the phylum of proteobacteria, was present at a relatively lower level. In contrast, Fusobacterium sp., which belongs to orange complex bacteria, showed relative similarities in subgingival biofilms of both groups tested, while Veillonella sp., were abundant in the DP groups. Conclusions: Our data show that the diversity of classic periodontopathogens increased in the subgingival niche of periodontitis subjects with diabetes. It is the first study in Indonesia to apply MinION-based, full-length 16S rRNA amplicon sequencing in periodontitis patients with and without diabetes.

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A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION platform

F1000Research ◽

10.12688/f1000research.28216.2 ◽

2021 ◽

Vol 10 ◽

pp. 79

Author(s):

Boy M. Bachtiar ◽

Citra F. Theodorea ◽

Dicky L. Tahapary ◽

Cindy Astrella ◽

Natalina ◽

...

Keyword(s):

Pilot Study ◽

16S Rrna ◽

Host Response ◽

Active Role ◽

Amplicon Sequencing ◽

Abundant Species ◽

Periodontal Pocket ◽

Periodontal Tissue ◽

16S Rrna Amplicon Sequencing ◽

Systemic Condition

Background: Subgingival niche is one biofilm habitat containing rich microbiota, which plays an active role in maintaining the health of periodontal tissue and determining host response. As such, a study of changing subgingival biofilms is important for understanding the effect of a systemic condition. In this study, we compared the occurrence of six bacteria cohabiting in the subgingival area of periodontitis subjects, with (DP, n = 8) and without (NDP, n = 4) diabetes. Methods: The six genus and species of targeted bacteria were confirmed by 16S rRNA amplicon sequencing on MinION nanopore platform. Descriptive statistic was used to describe the obtained data. Results: We found that the six genus and species of targeted bacteria were detected but in different quantities in either group's periodontal pocket. Our data showed that Tannerella forsythia was the most abundant species in subgingival biofilms of the DP group of the red complex bacteria. In contrast, Aggregatibacter sp., which belongs to the phylum of proteobacteria, was present at a relatively lower level. In contrast, Fusobacterium sp., which belongs to orange complex bacteria, showed relative similarities in subgingival biofilms of both groups tested, while Veillonella sp., were abundant in the DP groups. Conclusions: Our data show that the diversity of classic periodontopathogens increased in the subgingival niche of periodontitis subjects with diabetes. It is the first study in Indonesia to apply MinION-based, full-length 16S rRNA amplicon sequencing in periodontitis patients with and without diabetes.

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The Limits and Avoidance of Biases in Metagenomic Analyses of Human Fecal Microbiota

Microorganisms ◽

10.3390/microorganisms8121954 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1954

Author(s):

Emma Bergsten ◽

Denis Mestivier ◽

Iradj Sobhani

Keyword(s):

16S Rrna ◽

Dna Extraction ◽

Fecal Microbiota ◽

16S Rrna Sequencing ◽

Marker Genes ◽

Bacteria Identification ◽

16S Rna ◽

Rrna Sequencing ◽

The Impact ◽

Identification And Quantification

An increasing body of evidence highlights the role of fecal microbiota in various human diseases. However, more than two-thirds of fecal bacteria cannot be cultivated by routine laboratory techniques. Thus, physicians and scientists use DNA sequencing and statistical tools to identify associations between bacterial subgroup abundances and disease. However, discrepancies between studies weaken these results. In the present study, we focus on biases that might account for these discrepancies. First, three different DNA extraction methods (G’NOME, QIAGEN, and PROMEGA) were compared with regard to their efficiency, i.e., the quality and quantity of DNA recovered from feces of 10 healthy volunteers. Then, the impact of the DNA extraction method on the bacteria identification and quantification was evaluated using our published cohort of sample subjected to both 16S rRNA sequencing and whole metagenome sequencing (WMS). WMS taxonomical assignation employed the universal marker genes profiler mOTU-v2, which is considered the gold standard. The three standard pipelines for 16S RNA analysis (MALT and MEGAN6, QIIME1, and DADA2) were applied for comparison. Taken together, our results indicate that the G’NOME-based method was optimal in terms of quantity and quality of DNA extracts. 16S rRNA sequence-based identification of abundant bacteria genera showed acceptable congruence with WMS sequencing, with the DADA2 pipeline yielding the highest congruent levels. However, for low abundance genera (<0.5% of the total abundance) two pipelines and/or validation by quantitative polymerase chain reaction (qPCR) or WMS are required. Hence, 16S rRNA sequencing for bacteria identification and quantification in clinical and translational studies should be limited to diagnostic purposes in well-characterized and abundant genera. Additional techniques are warranted for low abundant genera, such as WMS, qPCR, or the use of two bio-informatics pipelines.

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Stromatoporoids from the Famennian (Devonian) Wabamun Formation, Normandville oilfield, north–central Alberta, Canada

Journal of Paleontology ◽

10.1017/s0022336000018321 ◽

1988 ◽

Vol 62 (03) ◽

pp. 411-419 ◽

Cited By ~ 5

Author(s):

Colin W. Stearn

Keyword(s):

Patch Reef ◽

Abundant Species ◽

The Other ◽

North Central ◽

Biotic Crisis

Stromatoporoids are the principal framebuilding organisms in the patch reef that is part of the reservoir of the Normandville field. The reef is 10 m thick and 1.5 km2in area and demonstrates that stromatoporoids retained their ability to build reefal edifices into Famennian time despite the biotic crisis at the close of Frasnian time. The fauna is dominated by labechiids but includes three non-labechiid species. The most abundant species isStylostroma sinense(Dong) butLabechia palliseriStearn is also common. Both these species are highly variable and are described in terms of multiple phases that occur in a single skeleton. The other species described areClathrostromacf.C. jukkenseYavorsky,Gerronostromasp. (a columnar species), andStromatoporasp. The fauna belongs in Famennian/Strunian assemblage 2 as defined by Stearn et al. (1988).

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