Intestinal microbiota analysis by 16S sequencing

Intestinal microbiota composition in fecal samples was assessed using 16S rRNA sequencing as previously described²⁴. DNA was isolated using the Qiagen DNeasy PowerSoil kit (Qiagen). Individual amplicons were pooled in equal concentrations and cleaned using Ampure XP beads (Beckman Coulter). The band of interest was subjected further to isolation via gel electrophoresis on a 1.5% Blue Pippin HT gel (Sage Science). The library was quantified using qPCR followed by 300-bp paired-end sequencing using Illumina MiSeq in the Genome Center DNA Technologies Core, University of California, Davis. Demultiplexing, removal of chimeras, rarification, and quality filter of low-abundance sequencing reads were performed by the UC Davis Host Microbe Systems Biology Core Facility. Analysis of alpha and beta-diversity were performed on R software²⁵. Differential abundances were analyzed using DESeq2 with phlyoseq and omu on R^26–28.