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Rapid denoising of pyrosequencing amplicon data: exploiting the rank-abundance distribution. (2010)

by J Reeder, R Knight
Venue:Nat Methods
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by Jenny Ploeg, Lori Campbell, Margaret Denton, Anju Joshi, Sharon Davies, Jenny Ploeg, Lori Campbell, Margaret Denton, Gerontological Studies, Jenny Ploeg , 2003
"... SEDAP Research Paper No. 96For further information about SEDAP and other papers in this series, see our web site: ..."
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SEDAP Research Paper No. 96For further information about SEDAP and other papers in this series, see our web site:

Disturbance and temporal partitioning of the activated sludge metacommunity

by David C Vuono , Jan Benecke , Jochen Henkel , William C Navidi , Tzahi Y Cath , Junko Munakata-Marr , John R Spear , Jö Rg , E Drewes
"... The resilience of microbial communities to press disturbances and whether ecosystem function is governed by microbial composition or by the environment have not been empirically tested. To address these issues, a whole-ecosystem manipulation was performed in a full-scale activated sludge wastewater ..."
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The resilience of microbial communities to press disturbances and whether ecosystem function is governed by microbial composition or by the environment have not been empirically tested. To address these issues, a whole-ecosystem manipulation was performed in a full-scale activated sludge wastewater treatment plant. The parameter solids retention time (SRT) was used to manipulate microbial composition, which started at 30 days, then decreased to 12 and 3 days, before operation was restored to starting conditions (30-day SRT). Activated sludge samples were collected throughout the 313-day time series in parallel with bioreactor performance ('ecosystem function'). Bacterial small subunit (SSU) rRNA genes were surveyed from sludge samples resulting in a sequence library of 4417 000 SSU rRNA genes. A shift in community composition was observed for 12-and 3-day SRTs. The composition was altered such that r-strategists were enriched in the system during the 3-day SRT, whereas K-strategists were only present at SRTsX12 days. This shift corresponded to loss of ecosystem functions (nitrification, denitrification and biological phosphorus removal) for SRTsp12 days. Upon return to a 30-day SRT, complete recovery of the bioreactor performance was observed after 54 days despite an incomplete recovery of bacterial diversity. In addition, a different, yet phylogenetically related, community with fewer of its original rare members displaced the pre-disturbance community. Our results support the hypothesis that microbial ecosystems harbor functionally redundant phylotypes with regard to general ecosystem functions (carbon oxidation, nitrification, denitrification and phosphorus accumulation). However, the impacts of decreased rare phylotype membership on ecosystem stability and micropollutant removal remain unknown.
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... from pyrosequencing were binned by barcode and quality filtered using the ‘split_libraries.py’ script in the Quantitative Insights Into Microbial Ecology (QIIME v1.5-dev) software (Caporaso et al., 2010). Sequences with errors in the barcode or primer, Resilience of microbial communities DC Vuono et al 2 The ISME Journal shorter than 400 nt, longer than 460 nt, with a quality score o50, homopolymer run greater than 6 nt and sequences that contained ambiguous base calls were discarded from downstream analysis. Flowgrams for remaining sequences were denoised using DeNoiser version 1.3.0-dev by Reeder and Knight (2010). Chimeric sequences were identified using UCHIME under reference mode and de novo mode (Edgar et al., 2011) (for details, see Supplementary Information Materials and methods). The remaining 672 521 sequences were processed in Mothur as outlined by Schloss et al., (2009) Schloss SOP version data 15 February2013 (for details, see Supplementary Information Materials and methods). Singletons were discarded from downstream analysis prior to diversity calculations. A phylogenetic tree was constructed from the filtered alignment using FastTree. Unweighted and weighted UniFrac (WU) distance matrices ...

ecosystem development

by Kamlesh Jangid, William B. Whitman, Leo M. Condron, Benjamin L. Turner, Mark A. Williams
"... bacterial community succession during long-term ..."
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bacterial community succession during long-term

ORIGINAL ARTICLE Disturbance and temporal partitioning of the activated sludge metacommunity

by David C Vuono, Jan Benecke, Jochen Henkel, William C Navidi, Tzahi Y Cath, Junko Munakata-marr, John R Spear, Jörg E Drewes
"... The resilience of microbial communities to press disturbances and whether ecosystem function is governed by microbial composition or by the environment have not been empirically tested. To address these issues, a whole-ecosystem manipulation was performed in a full-scale activated sludge wastewater ..."
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The resilience of microbial communities to press disturbances and whether ecosystem function is governed by microbial composition or by the environment have not been empirically tested. To address these issues, a whole-ecosystem manipulation was performed in a full-scale activated sludge wastewater treatment plant. The parameter solids retention time (SRT) was used to manipulate microbial composition, which started at 30 days, then decreased to 12 and 3 days, before operation was restored to starting conditions (30-day SRT). Activated sludge samples were collected throughout the 313-day time series in parallel with bioreactor performance (‘ecosystem function’). Bacterial small subunit (SSU) rRNA genes were surveyed from sludge samples resulting in a sequence library of4417000 SSU rRNA genes. A shift in community composition was observed for 12- and 3-day SRTs. The composition was altered such that r-strategists were enriched in the system during the 3-day SRT, whereas K-strategists were only present at SRTsX12 days. This shift corresponded to loss of ecosystem functions (nitrification, denitrification and biological phos-phorus removal) for SRTsp12 days. Upon return to a 30-day SRT, complete recovery of the bioreactor performance was observed after 54 days despite an incomplete recovery of bacterial diversity. In addition, a different, yet phylogenetically related, community with fewer of its original

SPECIAL ISSUE: NATURE’S MICROBIOME

by Sarah L. Hicks, Emily C. Farrer, D. Lee Taylor, Andrea Porras-alfaro, Katherine N. Suding, Robert L. Sinsabaugh , 2013
"... Nitrogen deposition alters plant–fungal relationships: linking belowground dynamics to aboveground vegetation change ..."
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Nitrogen deposition alters plant–fungal relationships: linking belowground dynamics to aboveground vegetation change

Nutrient Addition Dramatically Accelerates Microbial Community Succession

by Joseph E. Knelman, Steven K. Schmidt, Ryan C. Lynch, John L. Darcy, Sarah C. Castle, Cory C. Clevel, Diana R. Nemergut
"... The ecological mechanisms driving community succession are widely debated, particularly for microorganisms. While successional soil microbial communities are known to undergo predictable changes in structure concomitant with shifts in a variety of edaphic properties, the causal mechanisms underlying ..."
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The ecological mechanisms driving community succession are widely debated, particularly for microorganisms. While successional soil microbial communities are known to undergo predictable changes in structure concomitant with shifts in a variety of edaphic properties, the causal mechanisms underlying these patterns are poorly understood. Thus, to specifically isolate how nutrients – important drivers of plant succession – affect soil microbial succession, we established a full factorial nitrogen (N) and phosphorus (P) fertilization plot experiment in recently deglaciated (,3 years since exposure), unvegetated soils of the Puca Glacier forefield in Southeastern Peru. We evaluated soil properties and examined bacterial community composition in plots before and one year after fertilization. Fertilized soils were then compared to samples from three reference successional transects representing advancing stages of soil development ranging from 5 years to 85 years since exposure. We found that a single application of +NP fertilizer caused the soil bacterial community structure of the three-year old soils to most resemble the 85-year old soils after one year. Despite differences in a variety of soil edaphic properties between fertilizer plots and late successional soils, bacterial community composition of +NP plots converged with late successional communities. Thus, our work suggests a mechanism for microbial succession whereby changes in resource availability drive shifts in community composition, supporting a role for nutrient colimitation in primary succession. These results suggest that nutrients alone, independent of other edaphic factors that change with succession, act as an
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...mopolymers, a minimum quality score of 25, and a maximum of ambiguous bases/primer mismatches of 0; reverse primers were removed, and all samples were then denoised using flowgram clustering in QIIME =-=[38]-=-. Chloroplast sequences were removed. OTUs were selected at a 97% identity level by clustering based on representative sequences via UCLUST [39]. The Ribosomal Database Classifier [40], a naı̈ve Bayes...

RESEARCH ARTICLE Metabarcoding Analysis of Fungal Diversity in the Phyllosphere and Carposphere of Olive

by Ahmed Abdelfattah, Maria Giulia, Li Destri Nicosia, Santa Olga Cacciola, Samir Droby, Leonardo Schena
"... The fungal diversity associated with leaves, flowers and fruits of olive (Olea europaea) was investigated in different phenological stages (May, June, October and December) using an implemented metabarcoding approach. It consisted of the 454 pyrosequencing of the fungal ITS2 region and the subsequen ..."
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The fungal diversity associated with leaves, flowers and fruits of olive (Olea europaea) was investigated in different phenological stages (May, June, October and December) using an implemented metabarcoding approach. It consisted of the 454 pyrosequencing of the fungal ITS2 region and the subsequent phylogenetic analysis of relevant genera along with vali-dated reference sequences. Most sequences were identified up to the species level or were associated with a restricted number of related taxa enabling supported speculations regard-ing their biological role. Analyses revealed a rich fungal community with 195 different OTUs. Ascomycota was the dominating phyla representing 93.6 % of the total number of detected sequences followed by unidentified fungi (3.6%) and Basidiomycota (2.8%). A higher level of diversity was revealed for leaves compared to flowers and fruits. Among plant pathogens the genus Colletotrichum represented by three species (C. godetiae syn. C. clavatum, C. acutatum s.s and C. karstii) was the most abundant on ripe fruits but it was also detected in other organs. Pseudocercospora cladosporioides was detected with a high frequency in all leaf samples and to a less extent in ripe fruits. A much lower relative frequency was revealed for Spilocaea oleagina and for other putative pathogens including Fusarium spp., Neofusicoccum spp., and Alternariaspp. Among non-pathogen taxa, Aureobasidium pullu-lans, the species complex of Cladosporium cladosporioides and Devriesia spp. were the most represented. This study highlights the existence of a complex fungal consortium including both phytopathogenic and potentially antagonistic microorganisms that can have a significant impact on olive productions.
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...ly, the sliding window test of quality scores (-w) was enabled with a value of 50 to discard sequences with bad windows according to the "-g" command. Sequences were denoised using the denois wrapper =-=[25]-=- and the ITS2 region was extracted using ITSx software [26]. Chimeric sequences were identified and filtered using USEARCh 6.1 [27]. The most abundant sequences were picked as representative sequences...

RESEARCH ARTICLE Open Access Changes in microbiota during experimental human

by Rhinovirus Infection, J. J. Hofstra, S. Matamoros, M. A. Van De Pol, B. De Wever, M. W. Tanck, H. Wendt-knol, M. Deijs, L. Van Der Hoek, K. C. Wolthers, R. Molenkamp, C. E. Visser, P. J. Sterk, R. Lutter, M. D. De Jong
"... Background: Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbio ..."
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Background: Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbiota. Methods: Six healthy volunteers were infected with HRV16. We performed 16S ribosomal RNA-targeted pyrosequencing on throat swabs taken prior, during and after infection. We compared overall community diversity, phylogenetic structure of the ecosystem and relative abundances of the different bacteria between time points. Results: During acute infection strong trends towards increases in the relative abundances of Haemophilus parainfluenzae and Neisseria subflava were observed, as well as a weaker trend towards increases of Staphylococcus aureus. No major differences were observed between day-1 and day 60, whereas differences between subjects were very high. Conclusions: HRV16 infection is associated with the increase of three genera known to be associated with secondary infections following HRV infections. The observed changes of upper respiratory tract microbiota could help explain why HRV infection predisposes to bacterial otitis media, sinusitis and pneumonia. Background
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...titative Insights into Microbial Ecology” (QIIME 1.8.0) [22]. After removal of low quality reads (quality score <25) and chimera (ChimeraSlayer) our data was denoised using the QIIME denoiser program =-=[23]-=-. Then we clustered the sequences into Operational Taxonomical Units (OTU’s) based on 97 % sequence similarity (Uclust) [24]. The resulting OTU table was then condensed by removing all OTUs representi...

ORIGINAL ARTICLE Contemporary environmental variation determines microbial diversity patterns in acid mine drainage

by Jia-liang Kuang, Li-nan Huang, Lin-xing Chen, Zheng-shuang Hua, Sheng-jin Li, Min Hu, Jin-tian Li, Wen-sheng Shu
"... A wide array of microorganisms survive and thrive in extreme environments. However, we know little about the patterns of, and controls over, their large-scale ecological distribution. To this end, we have applied a bar-coded 16S rRNA pyrosequencing technology to explore the phylogenetic differentiat ..."
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A wide array of microorganisms survive and thrive in extreme environments. However, we know little about the patterns of, and controls over, their large-scale ecological distribution. To this end, we have applied a bar-coded 16S rRNA pyrosequencing technology to explore the phylogenetic differentiation among 59 microbial communities from physically and geochemically diverse acid mine drainage (AMD) sites across Southeast China, revealing for the first time environmental variation as the major factor explaining community differences in these harsh environments. Our data showed that overall microbial diversity estimates, including phylogenetic diversity, phylotype richness and pairwise UniFrac distance, were largely correlated with pH conditions. Furthermore, multivariate regression tree analysis also identified solution pH as a strong predictor of relative lineage abundance. Betaproteobacteria, mostly affiliated with the ‘Ferrovum ’ genus, were explicitly predominant in assemblages under moderate pH conditions, whereas Alphaproteobac-teria, Euryarchaeota, Gammaproteobacteria and Nitrospira exhibited a strong adaptation to more acidic environments. Strikingly, such pH-dependent patterns could also be observed in a subsequent comprehensive analysis of the environmental distribution of acidophilic microorgan-isms based on 16S rRNA gene sequences previously retrieved from globally distributed AMD and associated environments, regardless of the long-distance isolation and the distinct substrate types. Collectively, our results suggest that microbial diversity patterns are better predicted by contemporary environmental variation rather than geographical distance in extreme AMD systems.
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...as not been captured. It is not likely that this is due to inflation of biodiversity estimate by sequencing errors generated by noise introduced during pyrosequencing and the PCR amplification stage (=-=Reeder and Knight, 2010-=-), as such bias should have been limited after our stringent denoizing of data. Similar results have been reported in other extreme habitats such as hydrothermal chimneys (Brazelton et al., 2010) and ...

RESEARCH ARTICLE Spatial Homogeneity of Bacterial Communities Associated with the Surface Mucus Layer of the Reef-Building Coral

by Acropora Palmata, Dustin W. Kemp, Adam R. Rivers, Keri M. Kemp, Jamesw Porter, P. Wares , 1371
"... Coral surface mucus layer (SML) microbiota are critical components of the coral holobiont and play important roles in nutrient cycling and defense against pathogens. We sequenced 16S rRNA amplicons to examine the structure of the SML microbiome within and between colonies of the threatened Caribbean ..."
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Coral surface mucus layer (SML) microbiota are critical components of the coral holobiont and play important roles in nutrient cycling and defense against pathogens. We sequenced 16S rRNA amplicons to examine the structure of the SML microbiome within and between colonies of the threatened Caribbean reef-building coral Acropora palmata in the Florida Keys. Samples were taken from three spatially distinct colony regions—uppermost (high irradiance), underside (low irradiance), and the colony base—representing microhabitats that vary in irradiance and water flow. Phylogenetic diversity (PD) values of coral SML bac-teria communities were greater than surrounding seawater and lower than adjacent sedi-ment. Bacterial diversity and community composition was consistent among the three microhabitats. Cyanobacteria, Bacteroidetes, Alphaproteobacteria, and Proteobacteria, respectively were the most abundant phyla represented in the samples. This is the first time spatial variability of the surface mucus layer of A. palmata has been studied. Homogeneity in the microbiome of A. palmata contrasts with SML heterogeneity found in other Caribbean corals. These findings suggest that, during non-stressful conditions, host regulation of SML microbiota may override diverse physiochemical influences induced by the topographical complexity of A. palmata. Documenting the spatial distribution of SML microbes is essential to understanding the functional roles these microorganisms play in coral health and adapt-ability to environmental perturbations.
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...ecticut, USA). Sequences from the V1-V2 16S rRNA region were analyzed using a suite of tools available in QIIME version 1.8 [19]. Sequences libraries were demultiplexed and processed through Denoiser =-=[20]-=- to remove homopolymer errors characteristic of pyrosequencing. The initial assignment of operational taxonomic units at the 97% similarity level (OTUs0.03) was made using the denovo OTU picker script...

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