Computational protocol: Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation

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Protocol publication

[…] Mice were given 1 g/l ampicillin in their drinking water during 7 d, followed by a recovery period with normal drinking water for 14 d. The dominant Lactobacillus strain from the small intestine of B6 mice (The Jackson Laboratory) was isolated by plating contents under anaerobic conditions on plates with Lactobacilli MRS agar (BD). 16S rRNA was sequenced and classified using the ribosomal RDP classifier, and confirmed using MOTHUR to be identical to the operational taxonomic unit (OTU) most predominant in B6 mice (The Jackson Laboratory). 1 d after stopping ampicillin treatment, 108 CFUs of the isolated Lactobacillus strain were given to mice by oral gavage every other day during the 14-d recovery period. [...] Sequence data were compiled and processed using MOTHUR (). Sequences were aligned to the 16S rRNA gene, using as a template the SILVA reference alignment and the Needleman-Wunsch algorithm with the default scoring options. Potentially chimeric sequences were removed using the ChimeraSlayer program. To minimize the effect of pyrosequencing errors in overestimating microbial diversity, rare abundance sequences that differ in 1 or 2 nt from a high abundant sequence were merged to the high abundant sequence using the pre.cluster option in MOTHUR. Sequences were grouped into OTUs using the average neighbor algorithm. Sequences with distance-based similarity of 97% or greater were assigned to the same OTU. Mouse samples were processed and sequenced as individual experiments, and resulting sequences were analyzed together with all other samples within each figure panel. Human samples were processed and sequenced in batches, and resulting sequences were analyzed together. Sequences from all experiments have been deposited in the Sequence Read Archive of National Center for Biotechnology Information, submission number SRA049925. [...] OTU-based microbial diversity was estimated by calculating the Shannon diversity index () using MOTHUR. Phylogenetic classification was performed for each sequence, using the Bayesian classifier algorithm described by with the bootstrap cutoff at 60%. A phylogenetic tree was inferred using clearcut on the 16S sequence alignment generated by MOTHUR. Microbial chaos was quantified by mean Bray-Curtis time index, calculated as follows: Bray-Curtis dissimilarity index () between temporally adjacent samples was quantified using MOTHUR and divided by the length of the time interval (in days) between samples, starting with the last sample obtained before the transplant and all samples obtained until day 13. Unweighted UniFrac was run using the resulting tree (). PCoA was performed on the resulting matrix of distances between each pair of samples. […]

Pipeline specifications

Software tools RDP Classifier, mothur, ChimeraSlayer, Clearcut, UniFrac
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Graft vs Host Disease