scholarly journals Comparative Analysis of the Methanogen Diversity in Horse and Pony by UsingmcrAGene and Archaeal 16S rRNA Gene Clone Libraries

Archaea ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Khin-Ohnmar Lwin ◽  
Hiroki Matsui
Keyword(s):  
Comparative Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Clone Libraries ◽  
Operational Taxonomic Units ◽  
Gene 16S Rrna ◽  
Methanogen Population ◽  
Methyl Coenzyme M Reductase ◽  
16S Rrna Clone Libraries

Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing theα-subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. ThemcrAclone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (P<0.05). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both themcrAand the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds ofEquus caballus.

scholarly journals Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas C. A. Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Careful Attention ◽  
Operational Taxonomic Units ◽  
Basic Concepts ◽  
Gnotobiotic Mice ◽  
Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

scholarly journals Change in Bacterial Community Structure during In Situ Biostimulation of Subsurface Sediment Cocontaminated with Uranium and Nitrate

2004 ◽  
Vol 70 (8) ◽  
pp. 4911-4920 ◽  
Author(s):  
Nadia N. North ◽  
Sherry L. Dollhopf ◽  
Lainie Petrie ◽  
Jonathan D. Istok ◽  
David L. Balkwill ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Quantitative Pcr ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Clone Libraries ◽  
Subsurface Sediments

ABSTRACT Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the α, β, δ, and γ subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing δ-Proteobacteria increased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the δ-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.

scholarly journals Bacteroidales Diversity in Ring-Billed Gulls (Laurus delawarensis) Residing at Lake Michigan Beaches

2009 ◽  
Vol 75 (6) ◽  
pp. 1525-1533 ◽  
Author(s):  
Sonja N. Jeter ◽  
Colleen M. McDermott ◽  
Patricia A. Bower ◽  
Julie L. Kinzelman ◽  
Melinda J. Bootsma ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Lake Michigan ◽  
Rrna Gene ◽  
Fecal Indicator ◽  
Fecal Samples ◽  
Operational Taxonomic Units ◽  
The North ◽  
The Family ◽  
Sample Representation

ABSTRACT This study investigated the occurrence and diversity of Bacteroidales fecal bacteria in gulls residing in the Great Lakes region. Members of this bacterial order have been widely employed as human and bovine host-specific markers of fecal pollution; however, few studies have focused on gulls, which can be a major source of fecal indicator bacteria and pathogens at beaches. We found a low but consistent occurrence of Bacteroidales in gulls at five beaches in three different counties spanning the Wisconsin shoreline of Lake Michigan. The percentages of gulls positive for Bacteroidales were 4 to 8% at beaches in the southern part of the state and 8 to 50% at beaches in the north. Sequencing of 931 clones from seven gull Bacteroidales 16S rRNA gene libraries revealed a large amount of diversity in both individual and pooled gull fecal samples. Two libraries constructed from pooled gull fecal samples (n = 5 and n = 6) did not have a greater richness of sequences than individual samples, suggesting that even within a single gull diversity is high and an extensive sequencing effort is needed to characterize the populations. Estimates of the numbers of operational taxonomic units (OTUs) for the libraries obtained using different similarity levels revealed a large amount of microdiveristy with a limited number of OTUs at the 95% similarity level. Gull sequences were clustered by the beach from which they were collected, suggesting that there were geographic effects on the distribution of Bacteriodales. More than 53% of the 16S rRNA gene sequences from gulls at the southern beaches were associated with the family Porphyromonadaceae, primarily the genus Parabacteroides, whereas sequences from gulls at the northern beaches were comprised of Bacteroidaceae and Prevotellaceae sequences. Comparison of gull sequences with sequences from goose, canine, raccoon, and sewage sources revealed distinct clusters of closely related gull sequences; however, these sequences were widely dispersed across a dendrogram that included all other sources, including previously characterized gull Bacteroidales from other studies, suggesting that geographic influence or simply sample representation plays a greater role in the observed population structure than strictly the host gut environment.

scholarly journals Comparative Analysis of Bacterial Diversity in Freshwater Sediment of a Shallow Eutrophic Lake by Molecular and Improved Cultivation-Based Techniques

2005 ◽  
Vol 71 (4) ◽  
pp. 2162-2169 ◽  
Author(s):  
Hideyuki Tamaki ◽  
Yuji Sekiguchi ◽  
Satoshi Hanada ◽  
Kazunori Nakamura ◽  
Nakao Nomura ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Diversity ◽  
Clone Library ◽  
Eutrophic Lake ◽  
Freshwater Sediment ◽  
Gene Clone ◽  
Rrna Gene ◽  
Shallow Eutrophic Lake

ABSTRACT Comparative analysis of bacterial diversity in freshwater sediment collected from a shallow eutrophic lake was performed by using 16S rRNA gene clone library and improved cultivation-based techniques. Our study demonstrated that the use of gellan gum as a gelling reagent instead of agar was more effective at increasing culturability, cultivating a diverse array of novel microbes, and reducing the gaps of the results between molecular and cultivation-based analyses.

scholarly journals Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

2004 ◽  
Vol 70 (11) ◽  
pp. 6559-6568 ◽  
Author(s):  
Hector Castro ◽  
Andrew Ogram ◽  
K. R. Reddy
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Conservation ◽  
Phylogenetic Analyses ◽  
Florida Everglades ◽  
Rrna Gene ◽  
Conservation Area ◽  
Clone Libraries ◽  
Phylogenetic Characterization ◽  
Acetoclastic Methanogen

ABSTRACT Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.

scholarly journals Use of 16S rRNA Gene Based Clone Libraries to Assess Microbial Communities Potentially Involved in Anaerobic Methane Oxidation in a Mediterranean Cold Seep

2007 ◽  
Vol 53 (3) ◽  
pp. 384-398 ◽  
Author(s):  
Sander K. Heijs ◽  
Ralf R. Haese ◽  
Paul W. J. J. van der Wielen ◽  
Larry J. Forney ◽  
Jan Dirk van Elsas
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Methane Oxidation ◽  
Anaerobic Methane Oxidation ◽  
Cold Seep ◽  
Rrna Gene ◽  
Clone Libraries
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