Computational protocol: Bifidobacteria Abundance-Featured Gut Microbiota Compositional Change in Patients with Behcet’s Disease

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Protocol publication

[…] We processed the files using QIIME software (version 1.9.1) with the default settings according to a tutorial for the microbiome study [].The sequencing data contained approximately 8.4 million reads in total and the average quality read per sample was 285,752. We filtered the reads and subjected to operational taxonomic units (OTU; species grouping according to the sequence) with the cut off similarity of 97%.We summarized the OTU data into several OTU tables using QIIME software for the evaluation of relative abundance of bacterial taxa [].We then estimated microbial alpha and beta diversity using QIIME software []. Alpha diversity is defined as the diversity within a community and is mainly measured by Chao 1 and Shannon diversity indexes [].Chao1 index estimates richness in species numbers and Shannon index estimates evenness (equitability) of species frequencies [, ].Beta diversity is defined as the distance between communities and we estimated the distance by principal coordinates analysis (PCoA) [–]. The analysis assigns each sample to a position in a three dimensional structure to reduce the multiple dimensions of otu_table.biom files using linear conversion formulas. We visualized each PCoA plot in the three dimensional structure and summed up the distances between the pair of plots. We compared the total distances within and between BD patients and normal individuals using a two-sided Student's two-sample t-test as an exploratory analysis of beta diversity [].We utilized a file (seqs.fna) filtered by QIIME software for a linear discriminant analysis (LDA) of LEfSe (LDA effect size, explained below). We created an OTU table by pick_closed_reference_otus.py with a Greengenes-formatted database (gg_13_5). We modified the file by dividing each OTU by known/predicted 16S rRNA gene copy number abundance using PICRUSt software (version 1.0.0, normalize_by_copy_number.py), and uploaded it to http://huttenhower.sph.harvard.edu/galaxy []. […]

Pipeline specifications

Software tools QIIME, LEfSe, PICRUSt
Databases Greengenes
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Homo sapiens, Bacteria, Firmicutes
Diseases Behcet Syndrome, Immune System Diseases