DECIPHER, a Search-Based Approach to Chimera Identification for 16S rRNA Sequences

Wright, Erik S.; Yılmaz, L. Şafak; Noguera, Daniel R.

doi:10.1128/aem.06516-11

Cited by 598 publications

(393 citation statements)

References 19 publications

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“…Our approach in combination with the initial analysis at the genomic centre provided us with high-quality reads ready for the downstream analyses. Screening for chimeras was initially carried out by using DECIPHER (Wright et al, 2012). However, this gave a number of apparent false positives.…”

Section: Resultsmentioning

confidence: 99%

Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition

et al. 2015

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We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago and Brachypodium) in compost and sand and three crops (Brassica, Pisum and Triticum) in compost alone. We used serial inoculation of 24 independent replicate microcosms over three plant generations for each plant/soil combination. Stochastic variation between replicates was surprisingly weak and by the third generation, replicate microcosms for each plant had communities that were very similar to each other but different to those of other plants or unplanted soil. Microbiota diversity remained high in compost, but declined drastically in sand, with bacterial opportunists and putative autotrophs becoming dominant. These dramatic differences indicate that many microbes cannot thrive on plant exudates alone and presumably also require carbon sources and/or nutrients from soil. Arabidopsis had the weakest influence on its microbiota and in compost replicate microcosms converged on three alternative community compositions rather than a single distinctive community. Organisms selected in rhizospheres can have positive or negative effects. Two abundant bacteria are shown to promote plant growth, but in Brassica the pathogen Olpidium brassicae came to dominate the fungal community. So plants exert strong selection on the rhizosphere microbiota but soil composition is critical to its stability. microbial succession/ plant-microbe interactions/rhizosphere microbiota/selection.

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Section: Resultsmentioning

confidence: 99%

Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition

et al. 2015

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“…SSU rRNA gene sequences were then reconstructed from the remainder Leptospirillum-subtracted reads using EMIRGE (Miller et al, 2011) with parameters -l 101 -i 300 -s 100 -phred33, and run until 40 iterations. Sequences classified as chimeras by uchime (Edgar et al, 2011) and DECIPHER (Wright et al, 2012) were excluded. Relative abundances and taxonomic classification are reported in Supplementary Table S3. SSU rRNA gene sequences assembled via Cufflinks and those reconstructed with EMIRGE were classified using the Ribosome Database Project web server with an 80% cutoff for the lowest classification level (Wang et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Community transcriptomics reveals unexpected high microbial diversity in acidophilic biofilm communities

et al. 2014

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A fundamental question in microbial ecology relates to community structure, and how this varies across environment types. It is widely believed that some environments, such as those at very low pH, host simple communities based on the low number of taxa, possibly due to the extreme environmental conditions. However, most analyses of species richness have relied on methods that provide relatively low ribosomal RNA (rRNA) sampling depth. Here we used community transcriptomics to analyze the microbial diversity of natural acid mine drainage biofilms from the Richmond Mine at Iron Mountain, California. Our analyses target deep pools of rRNA gene transcripts recovered from both natural and laboratory-grown biofilms across varying developmental stages. In all, 91.8% of the B254 million Illumina reads mapped to rRNA genes represented in the SILVA database. Up to 159 different taxa, including Bacteria, Archaea and Eukaryotes, were identified. Diversity measures, ordination and hierarchical clustering separate environmental from laboratory-grown biofilms. In part, this is due to the much larger number of rare members in the environmental biofilms. Although Leptospirillum bacteria generally dominate biofilms, we detect a wide variety of other Nitrospira organisms present at very low abundance. Bacteria from the Chloroflexi phylum were also detected. The results indicate that the primary characteristic that has enabled prior extensive cultivation-independent 'omic' analyses is not simplicity but rather the high dominance by a few taxa. We conclude that a much larger variety of organisms than previously thought have adapted to this extreme environment, although only few are selected for at any one time.

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“…Sequences were then denoised and chimeras removed using the Chimera.uchime program in Mothur (http://www. mothur.org/wiki/Download_mothur) (Edgar et al, 2010;Wright et al, 2012). Operational taxonomic units were defined after removal of singleton sequences, clustering at 3% divergence (97% similarity) by using the pick_otus_through_otu_table.py script of QIIME.…”

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confidence: 99%

Bacterial Diversity and Composition in Oylat Cave (Turkey) with Combined Sanger/Pyrosequencing Approach

Gulecal-Pektas

2016

Polish Journal of Microbiology

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A b s t r a c tThe microbiology of caves is an important topic for better understanding subsurface biosphere diversity. The diversity and taxonomic composition of bacterial communities associated with cave walls of the Oylat Cave was studied first time by molecular cloning based on Sanger/pyrosequencing approach. Results showed an average of 1,822 operational taxonomic units per sample. Clones analyzed from Oylat Cave were found to belong to 10 common phyla within the domain Bacteria. Proteobacteria dominated the phyla, followed by Actino bacteria, Acidobacteria and Nitrospirae. Shannon diversity index was between to 3.76 and 5.35. The robust analysis conducted for this study demon strated high bacterial diversity on cave rock wall surfaces. This copy is for personal use only -distribution prohibited.Gulecal-Pektas Y. 70After collection, the samples were frozen on dry ice on site, and stored at -20°C upon return to the laboratory (Groth et al., 1999). Environmental DNA was extracted from samples using Fast DNA Spin kit for soil (MP biomedicals, Solon, OH USA).Clone library construction and Sanger sequence analysis. Total genomic DNA was used as a template for 16S rRNA PCR amplification using bacteria 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') universal pri mers (Weisburg et al., 1991). Each 20 µl reaction mixture contained: l µl environmental DNA template, 2.25 mM MgCl 2 , 2 µl GeneAmp 10X PCR Buffer II (Applied Biosystems, Foster City, CA, USA), 100 µM dNTPs (SigmaAldrich, Saint Louis, MO, USA), 0.2 µM each primer, 2.5 U AmpliTaq Gold DNA Polymerase (Applied Biosystems, Carlsbad, CA, USA). Thermal cycling was as follows: initial denaturation 5 min at 94°C, 25 cycles of 94°C for 1 min, hybridization at 50°C for 25 s and elongation at 72°C for 2 min followed by a final elongation at 72°C for 20 min. PCR products were purified using a QIAquick kit (QIAGEN, Valencia, CA, USA), and were cloned into Escherichia coli hosts using the TOPO TA Cloning kit with the pCR 2.1 Vector (Invitrogen Corporation, Carlsbad, CA, USA). Plasmid DNA was extracted and purified using the Ultra Clean Standard Mini Plasmid Prep Kit (MoBio Laboratories). Cloning products were sequenced by TUBITAK MAM DNA Services Facility at Gebze, Turkey, using standard M13 primers.Partial sequences were assembled with CodonCode Aligner v.1.2.4 (CodonCode, USA) and manually checked. Assembled sequences were checked for chimera by Bellerophon server (Huber et al., 2004) and Chimera_Check v 2.7 (Cole et al., 2005). Sample sequences were aligned by BioEdit (Ibis Biosciences, Carlsbad, CA, USA). Phylogenetic analysis was performed in PAUP (Sinauer Associates, Sunderland, MA) using parsimony, neighbor-joining, and maximum likelihood analyses. The 16S rRNA gene sequences were submitted to the NCBI Gen Bank database under accession numbers JQ065958-JQ065959 and JQ219081-JQ219137.454 pyrosequencing and sequence analysis. For the pyrosequencing, the V6 region of the 16S rRNA gene was amplified using PCR with a bacterial primer set 967f...

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DECIPHER, a Search-Based Approach to Chimera Identification for 16S rRNA Sequences

Cited by 598 publications

References 19 publications

Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition

Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition

Community transcriptomics reveals unexpected high microbial diversity in acidophilic biofilm communities

Bacterial Diversity and Composition in Oylat Cave (Turkey) with Combined Sanger/Pyrosequencing Approach

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