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Protocol publication

[…] Image processing was performed with Imaris Software 8.3.1 (Bitplane, Zurich, Switzerland) after deconvolution using Huygens Remote Manager (Scientific Volume Imaging B.V. 2.1.2). Image data sets were quantified using the JImage Analyzer extension of ImageJ ( In each z-stack, the volumes stained with the labeled lectin and with SYTO® 60 were calculated. For subsequent analysis, biovolume ratios of lectin-specific glycoconjugates/bacteria were taken. [...] Forward and reverse reads were assembled using PEAR 0.9.6 []. Unassembled reads and chimeras were discarded. The assembled data set was quality screened using fastQC software v0.9.6 [], and a sequence length covering most of the data set was selected. Reads were trimmed to 400 bp and then filtered and clustered with usearch v8.1.1861 and the UPARSE pipeline []. Operational taxonomic units (OTUs) were generated on the basis of a high-quality subset of the data (maxee = 1.0), and the remaining reads were mapped onto these OTUs with a 97% similarity cutoff. OTUs were classified to genus level using mothur v1.36.1 [], with the Silva SSU Ref NR release 123 database serving as a reference []. Further data analysis was done in R []. Data from the same patient were pooled (two samples each for subjects 1–8, one sample for subject 9). The data were normalized by rarefaction to a sampling depth of 18,000 OTUs per patient. […]

Pipeline specifications

Software tools PEAR, FastQC, USEARCH, UPARSE, mothur
Organisms Solanum lycopersicum
Diseases Wiskott-Aldrich Syndrome
Chemicals Sucrose