Computational protocol: Rapid change of fecal microbiome and disappearance of Clostridium difficile in a colonized infant after transition from breast milk to cow milk

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Protocol publication

[…] The sequencing results were processed using Quantitative Insights Into Microbial Ecology (QIIME) []. The bi-directional reads were joined using QIIME’s default join_paired_ends script. The merged reads were filtered based on Phred quality score ≥20, which corresponds to sequencing error rate ≤0.01. Chimeras were identified using USEARCH61 [] and removed from downstream analysis. After the removal of chimeras, the remaining sequences were mapped into operational taxonomic units (OTUs) against the Greengenes reference sequences (version 2013.5) with the program UCLUST []. Microbial taxonomy was assigned using a naïve Bayesian classifier trained with the Greengenes 2013.5 reference sequences [, ]. The level of species was classified by running BLAST [] against the Greengenes data set. OTUs which showed significant change in relative abundance was identified using QIIME’s “group_significance.py” script with Kruskal-Wallis test. Principle coordinates were calculated using unweighted UniFrac metrics []. QIIME commands used to generate the principal coordinate plots were included in Additional file . […]

Pipeline specifications

Software tools QIIME, USEARCH, UCLUST, UniFrac
Databases Greengenes
Application 16S rRNA-seq analysis
Organisms Escherichia coli, Clostridioides difficile, Homo sapiens, Bos taurus
Diseases Enterocolitis, Pseudomembranous, Escherichia coli Infections, Infection