Computational protocol: Intestinal microbiota sustains inflammation and autoimmunity induced by hypomorphic RAG defects

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Protocol publication

[…] For the evaluation of intestinal microbiota, weaned Rag2R229Q and Rag2+/+ littermates were cohoused in the same cages until analysis. The bacterial microbiota of 20 fecal samples from 10 Rag2+/+ and 10 Rag2R229Q mice was investigated by sequencing the V5-V6 hypervariable regions of 16S rDNA gene by using the Illumina MiSeq platform as described previously (). The obtained 250 × 2 paired-end reads were taxonomically profiled by applying the BioMaS pipeline (). Paired-end reads of each sample were merged into consensus sequences using Flash (Adobe; ) and then dereplicated by Usearch (drive5; ). The unmerged reads were trimmed of low-quality regions (Phred score cutoff of 25), and paired ends containing reads shorter than 50 nt were removed. Both the merged sequences and the unmerged reads were mapped against the RDP II database (release 10.32; ) by using Bowtie 2 (). The mapping data were filtered according to query coverage (≥70%) and similarity percentage (≥97%) and were stored in a file format suitable for the taxonomic assignment steps. Finally, the Tango tool processed the similarity analysis data to assign the sequences to a taxonomic clade in the NCBI taxonomy (; ). Statistically significant differences of the bacterial population in Rag+/+ and Rag2R229Q mice were determined at family, genus, and species levels by using the R/Bioconductor DESeq package (). […]

Pipeline specifications

Software tools BioMaS, USEARCH, Bowtie, TANGO, DESeq
Applications Genome annotation, 16S rRNA-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Congenital Abnormalities, Autoimmune Diseases, Immunologic Deficiency Syndromes, Inflammatory Bowel Diseases, Severe Combined Immunodeficiency