Computational protocol: Wheat bran promotes enrichment within the human colonic microbiota of butyrate‐producing bacteria that release ferulic acid

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Protocol publication

[…] Analysis of the sequence data was carried out using the mothur software package (Schloss et al., ), as described previously (Cooper et al., ), except that the minimum sequence length requirement was 200 bases. Following quality control steps, a total of 45 917 sequences remained (median of 1119.5 sequences per sample, range of 54–2808), which formed 406 OTUs at a 97% similarity threshold (Table S1). Data from the solid and liquid phase samples from the 2 h, 4 h, 24 h and 48 h time points from each of the four faecal donors were initially compared using metastats (White et al., ) analyses in mothur. There were 32 samples included in our comparison between the S and L fractions, and the median Good's coverage for the 16S rRNA gene sequence data from the individual S and L samples was 97.9% (mean 95.8%). metastats‐based comparison of all 406 OTUs detected in the sequence dataset revealed that there were no significant differences between samples recovered from the solid and liquid phase at each time point. As a result, pooled liquid/solid phase data for each donor from each time point were then compared using the LEfSe (Segata et al., ) software package, as implemented within mothur. Diversity metrics, such as observed OTU diversity, Chao estimates of total diversity, and Shannon and Simpson diversity indices, were calculated in mothur after first subsampling the pooled 4, 24 and 48 h datasets to 1502 reads to ensure equal sampling depth for these comparisons. The Good's coverage estimate at this sequencing depth was greater than 96% for all samples (median: 98.1%). The Kruskal–Wallis test as implemented in minitab (v16) was used to assess whether or not there were differences in diversity between incubation time points and between individual faecal donors. Diversity data were plotted using boxplotr (Spitzer et al., ). Community structures were compared between faecal donors and between incubation time points using the Bray–Curtis calculator and the dendrogram clustering (tree.shared), PCoA, and AMOVA commands as implemented in mothur. […]

Pipeline specifications

Software tools mothur, Metastats, LEfSe, BoxPlotR
Applications Miscellaneous, Metagenomic sequencing analysis
Organisms Triticum aestivum, Homo sapiens, Escherichia coli, Bacteria, Lachnospiraceae, Firmicutes
Chemicals Butyrates