Computational protocol: Dominant Effects of the Diet on the Microbiome and the Local and Systemic Immune Response in Mice

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Protocol publication

[…] Fecal DNA from 2 PD-, 2 TD-, 2 ABX PD-, and 2 ABX TD-fed mice were isolated and sequenced on a 454 Titanium sequencer at the Pennsylvania State University. The low numbers of samples sequenced was due in part to the high cost needed for sequencing. The goal of the metagenomic sequencing was to confirm the DGGE analysis that showed changes in the gut bacterial composition in mice treated with different diets or ABX and to give new information on the possible changes in phyla and families that occurred. The sequences obtained were analyzed using the MOTHUR software , and all analysis scripts and datasets are available on the NIH Sequence Read Archive (; accession number: SRX381841). Total sequencing reads were 278,327. Raw reads were filtered to have an average quality over 20 and with a length longer than 100 bp and to remove potential chimeric reads (ChimeraSlayer developed by Broad Institute). A preclustering step was applied that merged reads due to pyrosequencing errors. At the end of the quality filtering steps, 153,760 reads were retained with the reads for each group spanning from 28,578 to 45,368 reads. Taxonomical classification was performed with the rdp_multiclassfier using the RDP taxonomy. Statistical analysis was done using a statistical model based on Pearson Chi-Square Goodness of Fit test. Because of the small sample sizes, the cut-off value for statistical significance was more stringent and P<0.0001 was used. Thus, only large effects with small variance would be statistically significant in our model. […]

Pipeline specifications

Software tools mothur, ChimeraSlayer
Databases SRA
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Mus musculus, Citrobacter rodentium