Comparative Analysis of Eukaryotic Marine Microbial Assemblages from 18S rRNA Gene and Gene Transcript Clone Libraries by Using Different Methods of Extraction

ABSTRACTEukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.

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Comparison of Eukaryotic Phytobenthic Community Composition in a Polluted River by Partial 18S rRNA Gene Cloning and Sequencing

Microbial Ecology ◽

10.1007/s00248-002-2024-x ◽

2002 ◽

Vol 44 (4) ◽

pp. 372-380 ◽

Cited By ~ 30

Author(s):

U. Dorigo ◽

A. Bérard ◽

J.F. Humbert

Keyword(s):

Gene Cloning ◽

Community Composition ◽

18S Rrna ◽

18S Rrna Gene ◽

Rrna Gene ◽

Cloning And Sequencing ◽

Polluted River ◽

Partial 18S

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Molecular Characterization of Subject-Specific Oral Microflora during Initial Colonization of Enamel

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2837-2848.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2837-2848 ◽

Cited By ~ 240

Author(s):

Patricia I. Diaz ◽

Natalia I. Chalmers ◽

Alexander H. Rickard ◽

Colin Kong ◽

Craig L. Milburn ◽

...

Keyword(s):

Community Composition ◽

Bacterial Species ◽

Microbial Community Composition ◽

Microbial Colonization ◽

Rrna Gene ◽

Dependent Manner ◽

Clone Libraries ◽

Tooth Surfaces ◽

Biofilm Development

ABSTRACT The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.

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Bacterial, archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

Polar Biology ◽

10.1007/s00300-008-0509-x ◽

2008 ◽

Vol 32 (1) ◽

pp. 93-103 ◽

Cited By ~ 47

Author(s):

Fei Tian ◽

Yong Yu ◽

Bo Chen ◽

Huirong Li ◽

Yu-Feng Yao ◽

...

Keyword(s):

16S Rrna ◽

18S Rrna ◽

18S Rrna Gene ◽

Gene Clone ◽

Rrna Gene ◽

Clone Libraries ◽

Eukaryotic Diversity ◽

Arctic Sediment

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Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp.)

Applied and Environmental Microbiology ◽

10.1128/aem.69.10.6007-6017.2003 ◽

2003 ◽

Vol 69 (10) ◽

pp. 6007-6017 ◽

Cited By ~ 101

Author(s):

Dirk Schmitt-Wagner ◽

Michael W. Friedrich ◽

Bianca Wagner ◽

Andreas Brune

Keyword(s):

Intestinal Tract ◽

Companion Paper ◽

Rrna Gene ◽

Molecular Fingerprinting ◽

Axial Distribution ◽

Phylogenetic Groups ◽

Clone Libraries ◽

Gram Positive Bacteria ◽

Good Agreement

ABSTRACT The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (Termitidae: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.

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Bacterial Community Structure of Acid-Impacted Lakes: What Controls Diversity?

Applied and Environmental Microbiology ◽

10.1128/aem.01719-07 ◽

2008 ◽

Vol 74 (6) ◽

pp. 1856-1868 ◽

Cited By ~ 72

Author(s):

Sascha F. Percent ◽

Marc E. Frischer ◽

Paul A. Vescio ◽

Ellen B. Duffy ◽

Vincenzo Milano ◽

...

Keyword(s):

Species Richness ◽

Community Structure ◽

Community Composition ◽

The United States ◽

Trophic Levels ◽

Community Diversity ◽

Rrna Gene ◽

Clone Libraries ◽

Bacterioplankton Community ◽

Bacterioplankton Communities

ABSTRACT Although it is recognized that acidification of freshwater systems results in decreased overall species richness of plants and animals, little is known about the response of aquatic microbial communities to acidification. In this study we examined bacterioplankton community diversity and structure in 18 lakes located in the Adirondack Park (in the state of New York in the United States) that were affected to various degrees by acidic deposition and assessed correlations with 31 physical and chemical parameters. The pH of these lakes ranged from 4.9 to 7.8. These studies were conducted as a component of the Adirondack Effects Assessment Program supported by the U.S. Environmental Protection Agency. Thirty-one independent 16S rRNA gene libraries consisting of 2,135 clones were constructed from epilimnion and hypolimnion water samples. Bacterioplankton community composition was determined by sequencing and amplified ribosomal DNA restriction analysis of the clone libraries. Nineteen bacterial classes representing 95 subclasses were observed, but clone libraries were dominated by representatives of the Actinobacteria and Betaproteobacteria classes. Although the diversity and richness of bacterioplankton communities were positively correlated with pH, the overall community composition assessed by principal component analysis was not. The strongest correlations were observed between bacterioplankton communities and lake depth, hydraulic retention time, dissolved inorganic carbon, and nonlabile monomeric aluminum concentrations. While there was not an overall correlation between bacterioplankton community structure and pH, several bacterial classes, including the Alphaproteobacteria, were directly correlated with acidity. These results indicate that unlike more identifiable correlations between acidity and species richness for higher trophic levels, controls on bacterioplankton community structure are likely more complex, involving both direct and indirect processes.

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A novel metabarcoding strategy for studying nematode communities

10.1101/2020.01.27.921304 ◽

2020 ◽

Author(s):

Md. Maniruzzaman Sikder ◽

Mette Vestergård ◽

Rumakanta Sapkota ◽

Tina Kyndt ◽

Mogens Nicolaisen

Keyword(s):

18S Rrna ◽

High Throughput Sequencing ◽

Soil Samples ◽

Coi Gene ◽

Rrna Gene ◽

Nematode Communities ◽

Sequencing Platform ◽

Spiked Soil ◽

Primer Sets ◽

Mock Communities

AbstractNematodes are widely abundant soil metazoa and often referred to as indicators of soil health. While recent advances in next-generation sequencing technologies have accelerated research in microbial ecology, the ecology of nematodes remains poorly elucidated, partly due to the lack of reliable and validated sequencing strategies. Objectives of the present study were (i) to compare commonly used primer sets and to identify the most suitable primer set for metabarcoding of nematodes; (ii) to establish and validate a high-throughput sequencing strategy for nematodes using Illumina paired-end sequencing. In this study, we tested four primer sets for amplicon sequencing: JB3/JB5 (mitochondrial, I3-M11 partition); SSU_04F/SSU_22R (18S rRNA, V1-V2 region); Nemf/18Sr2b (18S rRNA, V6-V8 region) from earlier studies; and MMSF/MMSR (18S rRNA, V4-V5 region), a newly developed primer set from this study. In order to test the primer sets, we used 22 samples of individual nematode species, 20 mock communities, 20 soil samples, 20 spiked soil samples (mock communities in soil), and 4 root/rhizosphere soil samples. We successfully amplified the target regions (I3-M11 partition of the COI gene; V1-V2, V4-V8 region of 18S rRNA gene) from these 86 DNA samples with the four different primer combinations and sequenced the amplicons on an Illumina MiSeq sequencing platform. We found that the MMSF/MMSR and Nemf/18Sr2b were efficient in detecting nematode compared to JB and SSU primer sets based on annotation of sequence reads at genus and in some cases at species level. Therefore, these primer sets are suggested for studies of nematode communities in agricultural environments.

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A quest for the biological sources of the ubiquitous long chain alkyl diols in the marine realm

10.5194/bg-2018-97 ◽

2018 ◽

Author(s):

Sergio Balzano ◽

Julie Lattaud ◽

Laura Villanueva ◽

Sebastiaan Rampen ◽

Corina P. D. Brussaard ◽

...

Keyword(s):

Water Column ◽

18S Rrna ◽

Amplicon Sequencing ◽

18S Rrna Gene ◽

Rrna Gene ◽

Phylogenetic Groups ◽

Suspended Particulate ◽

Marine Realm ◽

Biological Sources ◽

Long Chain

Abstract. Long chain alkyl diols (LCDs) are widespread in the marine water column and sediments but their biological sources are mostly unknown. Here we combine lipid analyses with 18S rRNA gene amplicon sequencing on suspended particulate matter (SPM) collected in the photic zone of the tropical North Atlantic at 24 stations to infer relationships between LCDs and potential LCD-producers. The C30 1,15-diol was detected in all SPM samples and accounted for > 95 % of the total LCDs, while minor proportions of C28 and C30 1,13-diols, C28 and C30 1,14-diols as well as C32 1,15-diol were found. The concentration of the C30 and C32 diols was higher in the mixed layer of the water column compared to the deep chlorophyll maximum (DCM), whereas concentrations of C28 diols were comparable. Sequencing analyses revealed extremely low contributions (≈ 0.1 % of the 18S rRNA gene reads) of known LCD-producers but the contributions from two taxonomic classes to which known producers are affiliated, i.e. Dictyochophyceae and Chrysophyceae, followed a trend similar to that of the concentrations of C30 and C32 diols. Statistical analyses indicated that the abundance of 4 operational taxonomic units (OTUs) of the Chrysophyceae and Dictyochophyceae, along with 23 OTUs falling in other phylogenetic groups, were significantly correlated with C30 diol concentrations. However, it is not clear whether some of these OTUs might indeed correspond to LCD-producers or whether these correlations are just indirect. Furthermore, based on the average LCD-content measured in cultivated LCD-producing algae, the detected concentrations of LCDs in SPM are too high to be explained by the abundances of the suspected LCD-producing OTUs. This is likely explained by the slower degradation of LCDs compared to DNA in the oxic water column and suggests that some of the LCDs found here were likely to be associated to suspended debris, while the DNA from the related LCD-producers had been already fully degraded. This suggests that care should be taken in constraining biological sources of relatively stable biomarker lipids by quantitative comparisons of DNA and lipid abundances.

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