Computational protocol: The Enterocyte-Associated Intestinal Microbiota of Breast-Fed Infants and Adults Responds Differently to a TNF-α-Mediated Pro-Inflammatory Stimulus

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Protocol publication

[…] Sequencing reads were analyzed using the QIIME pipeline [], as described in Claesson et al. []. Briefly, V4 sequences were filtered according to the following criteria: (i) exact matches to primer and barcode sequences; (ii) read length not shorter than 150 bp and not longer than 350 bp; (iii) no ambiguous bases (Ns); (iv) a minimum average quality score over a 50-bp rolling window of 25. For bacterial taxonomy assignment we utilized RDP-classifier (version 2.2) with 50% as confidence value threshold. Trimmed reads were clustered into OTUs at 97% identity level and further filtered for chimeric sequences using ChimeraSlayer (http://microbiomeutil.sourceforge.net/#A_CS). Alpha-diversity and rarefaction plots were computed using four different metrics: Shannon, PD whole tree, chao1 and observed species. Weighted and unweighted UniFrac distances were used to perform Principal Coordinates Analysis (PCoA) and Procrustes superimposition in order to evaluate β-diversity and the correlation between pyrosequencing and phylogenetic microarray data. PCoA, Procrustes, heatmap and bar plots were built using the packages Made4 [] and Vegan [] in R 3.0.0. The R packages Stats and Vegan were used to perform statistical analysis. In particular, Wilcoxon signed-rank test was used to compare infant and adult gut microbiota for α- and β-diversity; data separation in the PCoA was tested using a permutation test with pseudo F-ratios (function adonis in the Vegan package); Fisher’s exact test was used to assess the significance of differences between clusters from the hierarchical clustering analysis; Student’s t-test or Mann-Whitney U test were used to carry out significant differences at phylum, group or genus level. When appropriate, P values were adjusted for multiple comparison using the Benjamini-Hochberg correction. False discovery rate (FDR) < 0.05 was considered as statistically significant. The function protest was utilized to validate the Procrustes analysis. A P value < 0.05 was considered as statistically significant. […]

Pipeline specifications

Software tools QIIME, RDP Classifier, ChimeraSlayer
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Neoplasms