16S rRNA Metagenome analysis
Raw paired-end Illumina reads were trimmed for 16S rRNA gene primer
sequences using Cutadapt (https://cutadapt.readthedocs.org/en/stable/).
Pre-processed sequence data were filtered using the Quantitative
Insights Into Microbial Ecology (QIIME), software suite. Sequences were
clustered into OTUs on the basis of similarity to known bacterial
sequences available in the Greengenes database (v13.8;
http://greengenes.secondgenome.com/;) (at 97% sequence similarity
cut-off) using UCLUST clustering software. Sequences that could not be
matched to references in the Greengenes databases were clustered de novo
based on pair-wise sequence identity. Total sum scaling (TSS)
normalization was applied followed by CSS to correct biases introduced
by TSS, and log2 transformation to account for the non-normal
distribution of taxonomic counts. Statistical analyses were executed
using the Calypso software V8.7241(cgenome.net/calypso/).