Computational protocol: How to Feed the Mammalian Gut Microbiota: Bacterial and Metabolic Modulation by Dietary Fibers

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Protocol publication

[…] The fastq files were processed using QIIME () as previously described (). Paired-end reads were merged and sequences that had passed quality control were kept with a length between 140 and 400 bp, mean sequence quality score >25, and with truncation of a sequence at the first base if a low quality rolling 10 bp window was found. Sequences with mismatched forward and/or reverse primers were removed.In order to calculate downstream diversity measures (alpha and beta diversity indices, UniFrac analysis), 16S rRNA Operational Taxonomic Units (OTUs) were defined at 97% sequence homology using uclust () and identified OTUs that were represented by less than 10 sequences were removed. All reads were classified to the lowest possible taxonomic rank using QIIME () and a reference dataset from the SILVA database version 123 (). Biodiversity of the samples (alpha-diversity) was calculated with Chao1 and Shannon indexes. For alpha-diversity analysis, samples with less than 20,000 reads were omitted (see Supplementary Table for details). Similarities between samples (beta-diversity) were calculated by weighted UniFrac (). Similarity scores were calculated as values between 0 and 1. Principal coordinate analysis (PCoA) representations of beta-diversity were performed using QIIME (). [...] Two-way ANOVA for repeated measures with “group” as between-subject factor (three levels: I, RS, and CP) was performed for: (i) BW changes, with “time” as within-subject factor (three levels: T1, T2, and T3); (ii) FI-to-BW ratio, with “time” as within-subject factor (four levels: week1, week2, week3, and week4). Follow up analysis was conducted using Student’s “t”-test, with a Bonferroni correction for multiple comparisons.For alpha- and beta-diversity data, PERMANOVA and ANOVA analyses were used, using Tuckey’s HSD post hoc test. Linear discriminant analysis (Lefse) was performed for microbial modulation by dietary fibers. Kendall’s tau and significance (two-tailed test) were calculated for co-occurrence analysis. All statistical analyses were performed with SPSS software. […]

Pipeline specifications

Software tools QIIME, UniFrac, UCLUST, LEfSe
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Homo sapiens, Rattus norvegicus
Chemicals Succinic Acid