Computational protocol: Glial cell derived neuroregulators control type 3 innate lymphoid cells and gut defence

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Protocol publication

[…] Faeces were isolated from co-housed Retfl or RetΔ littermates. Sequencing of the 16S rRNA gene was performed as previously described. Briefly, barcoded primers were used to amplify the V4 region of the 16S rRNA gene, and the amplicons were sequenced on a MiSeq instrument (Illumina, San Diego, USA) using 150 bp, paired-end chemistry at the University of Pennsylvania Next Generation Sequencing Core. The paired ends were assembled and quality filtered, selecting for reads with a quality score ≥30. Reads with >10 bp homopolymers and reads shorter than 248 bp or longer than 255 bp were removed from the analysis. 16S rRNA sequence data were processed using mothur v 1.25.0 and QIIME v 1.8. Chimeric sequences were removed with ChimeraSlayer. Operational taxonomic units (OTUs) were defined with CD-HIT using 97% sequence similarity as a cut-off. Only OTUs containing ≥2 sequences were retained; OTUs assigned to Cyanobacteria or which were not assigned to any phylum were removed from the analysis. Taxonomy was assigned using the Ribosomal Database Project (RDP) classifier v 2.2, multiple sequence assignment was performed with PyNAST (v 1.2.2), and FastTree was used to build the phylogeny. Samples were rarified to 22,000 sequences per sample for alpha- and beta-diversity analyses. Taxonomic relative abundances are reported as the median with standard deviation. P values were calculated using the Wilcoxon rank-sum test. Statistical tests were conducted in R v. 3.2.0. To determine which factors were associated with microbial community composition, statistical tests were performed using the non-parametric analysis of similarities (ANOSIM) with weighted UniFrac distance metrics. […]

Pipeline specifications

Software tools mothur, QIIME, ChimeraSlayer, CD-HIT, RDP Classifier, PyNAST, FastTree, UniFrac
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Mus musculus