Computational protocol: Culture-Dependent and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils

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Protocol publication

[…] Quality processing (read quality trimming, chimera check) of partial 16S rDNA and ITS1 sequences was performed in Mothur v.1.33.2 following the protocol used in Schloss et al. [] and accessed online (http://www.mothur.org/wiki/454_SOP) in September 2014. Sequences obtained from Sanger sequencing were edited, cleaned, and assembled in Geneious Pro v.6.1.5 (Biomatters). For both 454 sequences and Sanger sequences, clustering analyses were performed in Geneious, and OTUs were defined at 97% similarity. For 454-pyrosequencing datasets, singletons were not considered, and doubletons with a nucleotide similarity <100% were excluded. OTU rarefaction curves, Venn diagrams, taxonomic identifications, relative abundance analyses, and richness and diversity indices were performed in Mothur. Taxonomic identifications for bacterial and fungal sequences were performed in Mothur using the SILVA [] and UNITE [] databases, respectively. Prior to performing community comparisons based on the 454-pyrosequencing datasets, each library was randomly subsampled to the minimum number of sequences observed per composite soil sample. Heatmaps and Venn diagrams were created using the R statistical language v.3.0.0 [] with the packages gplots [] and VennDiagram []. Krona charts were calculated using the KronaTools available from http://krona.sourceforge.net []. To partition the datasets between abundant and rare OTUs, the number of reads observed for each OTU was divided by the number of reads counted for the most abundant OTU. Rare OTUs were defined as those with a read proportion of < 5% (). […]

Pipeline specifications

Software tools mothur, Geneious, gplots, VennDiagram, Krona
Application Phylogenetics
Diseases Mycoses