Computational protocol: No difference in small bowel microbiota between patients with irritable bowel syndrome and healthy controls

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Protocol publication

[…] Pyrosequenced amplicon reads were de-multiplexed by stripping barcode and primer from each read. Reads which had one or more mismatches in barcode or primer were discarded. The remaining reads were quality filtered using the fastq_filter command of USEARCH v7.0.1001 with parameters set to truncate reads to 200 basepairs whilst discarding shorter reads, and with maximum expected errors set to 0.5. The script from QIIME v1.7.0 was used to pick operational taxonomic units (OTUs). For this step the Greengenes May 2013 reference OTUs database at 97% identity was employed. Chimera filtering was performed on the representative OTU reads using the uchime_ref command of USEARCH against the ChimeraSlayer “Gold” database from the Broad Microbiome Utilities r20110519. The resulting data was analysed using R [] and the packages phyloseq 1.4.5, Metastats, cluster 1.14.4, fpc 2.1-7, ggplot2, plyr 1.8.1, grid 3.0.1 and VGAM 0.9-3. Analyses at phylum and genus level were performed by: (1) aggregating all OTUs which had identical classification at the given taxonomic level (suggested classifications were heeded, e.g. [Prevotella] was considered identical with Prevotella), (2) normalizing samples by their total abundance, (3) discarding unclassified taxa and taxa with mean relative abundance lower than 0.01%. Step (3) was also performed on OTUs before analysis. Low abundance taxa were discarded to avoid having sampling depth effects influencing the analysis. Also, since the sample size was fairly small the analysis focused on taxa that were expressed in most samples in order to get meaningful results. […]

Pipeline specifications

Software tools USEARCH, QIIME, UCHIME, ChimeraSlayer, phyloseq, Metastats, Ggplot2
Databases Greengenes
Applications Miscellaneous, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Escherichia coli Infections, Haemophilus Infections, Irritable Bowel Syndrome