Fecal DNA extraction and processing

Rat fecal DNA was extracted using the Qiagen DNA Stool Mini Kit following the protocol from the manufacturer (Qiagen, Valencia, CA). Total DNA concentration was determined by Qubit 2.0 Fluorometer (Life technologies, Norwalk, CT). The phylogenetically informative V3–V4 region of 16S rRNA gene was amplified using universal primer 347F/803R [54, 55]. We designed a dual-barcoding approach to label the 16S rRNA gene amplicons from each sample (illustrated in Additional file 1: Figure S1A). Briefly, the 6-mer barcodes were attached on the 5′ends of both forward and reverse PCR primers so that 16S rRNA gene PCR amplicons from each sample contained a unique dual barcode combination. PCR primers were designed against conserved sequences to amplify the flanking variable 16S rRNA gene regions. The primers were synthesized by IDT (Integrated DNA technology, Coralville, IA), and the sequences are shown in Additional file 1: Table S3.