Faculty Opinions recommendation of PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample.

Author(s):  
Daniel Vaulot
Archaea ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Khin-Ohnmar Lwin ◽  
Hiroki Matsui

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.


2004 ◽  
Vol 70 (8) ◽  
pp. 4911-4920 ◽  
Author(s):  
Nadia N. North ◽  
Sherry L. Dollhopf ◽  
Lainie Petrie ◽  
Jonathan D. Istok ◽  
David L. Balkwill ◽  
...  

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.


2008 ◽  
Vol 74 (21) ◽  
pp. 6663-6671 ◽  
Author(s):  
Lisa M. Steinberg ◽  
John M. Regan

ABSTRACT Methanogens play a critical role in the decomposition of organics under anaerobic conditions. The methanogenic consortia in saturated wetland soils are often subjected to large temperature fluctuations and acidic conditions, imposing a selective pressure for psychro- and acidotolerant community members; however, methanogenic communities in engineered digesters are frequently maintained within a narrow range of mesophilic and circumneutral conditions to retain system stability. To investigate the hypothesis that these two disparate environments have distinct methanogenic communities, the methanogens in an oligotrophic acidic fen and a mesophilic anaerobic digester treating municipal wastewater sludge were characterized by creating clone libraries for the 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes. A quantitative framework was developed to assess the differences between these two communities by calculating the average sequence similarity for 16S rRNA genes and mcrA within a genus and family using sequences of isolated and characterized methanogens within the approved methanogen taxonomy. The average sequence similarities for 16S rRNA genes within a genus and family were 96.0 and 93.5%, respectively, and the average sequence similarities for mcrA within a genus and family were 88.9 and 79%, respectively. The clone libraries of the bog and digester environments showed no overlap at the species level and almost no overlap at the family level. Both libraries were dominated by clones related to uncultured methanogen groups within the Methanomicrobiales, although members of the Methanosarcinales and Methanobacteriales were also found in both libraries. Diversity indices for the 16S rRNA gene library of the bog and both mcrA libraries were similar, but these indices indicated much lower diversity in the 16S digester library than in the other three libraries.


2004 ◽  
Vol 70 (11) ◽  
pp. 6559-6568 ◽  
Author(s):  
Hector Castro ◽  
Andrew Ogram ◽  
K. R. Reddy

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.


2010 ◽  
Vol 1 (2) ◽  
pp. 197-207 ◽  
Author(s):  
W. Fernando ◽  
J. Hill ◽  
G. Zello ◽  
R. Tyler ◽  
W. Dahl ◽  
...  

The effects of diets supplemented with either chickpea or its main oligosaccharide raffinose on the composition of the faecal microbial community were examined in 12 healthy adults (18-65 years) in a randomised crossover intervention study. Subjects consumed their usual diet supplemented with soups and desserts that were unfortified, or fortified with either 200 g/d of canned chickpeas or 5 g/d of raffinose for 3 week periods. Changes in faecal bacterial populations of subjects were examined using 16S rRNA-based terminal restriction fragment length polymorphisms (T-RFLP) and clone libraries generated from the diet pools. Classification of the clone libraries and T-RFLP analysis revealed that Faecalibacterium prausnitzii, reported to be an efficient butyrate producer and a highly metabolically active bacterium in the human intestinal microbiota, was more abundant in the raffinose diet and the chickpea diet compared to the control diet. However, no significant difference was observed in the faecal total short chain fatty acid concentration or in the levels of the components (butyrate, acetate and propionate) with the chickpea diet or the raffinose diet compared to the control diet. Bifidobacterium species were detected by T-RFLP in all three diet groups and quantitative real-time PCR (qPCR) analysis showed a marginal increase in 16S rRNA gene copies of Bifidobacterium with the raffinose diet compared to control (P>0.05). The number of individuals showing TRFs for the Clostridium histolyticum - Clostridum lituseburense groups, which include pathogenic bacteria species and putrefactive bacteria, were lower in the chickpea diet compared to the other two treatments. Diet appeared to affect colonisation by a high ammonia-producing bacterial isolate which was detected in 83%, 92% and 42% of individuals in the control, raffinose and chickpea groups, respectively. Our results indicate that chickpea and raffinose have the potential to modulate the intestinal microbial composition to promote intestinal health in humans.


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

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