2.2. Analysis of gut microbiota by 16S rRNA gene sequencing

Fecal DNA was extracted from frozen samples using the FastDNA® SPIN Kit for Feces (MP Biomedicals, Santa Ana, CA, USA). The V4 region of the 16S rRNA gene was amplified using dual-indexed primers. Amplicons were sequenced using an Illumina MiSeq with a MiSeq Reagent Kit V3 (Illumina, San Diego, CA, USA). Paired-end sequencing was performed using the Illumina MiSeq platform. Processing and quality filtering of reads was performed using Quantitative Insights into Microbial Ecology (QIIME) (v1.9.1) and the chimera-free sequences were aligned with the SILVA database (http://www.arb-silva.de) at Unclassified. Other data were used for further analysis at each level, which were then re-normalized. Principal component analysis (PCA) plots were generated using the function prcomp in the R package to identify clustering within each level. The raw data were deposited in the DNA Data Bank of Japan (DDBJ) under accession no. DRA011809. To detection of Muribaculum, Paramuribaculum, Duncaniella and Catenibacterium, the 16S rRNA gene copies of each sample were evaluated by real-time PCR using specific16S forward and reverse primers. The universal 16S rRNA gene was used as the internal control, and the genus was expressed as relative levels to 16S rRNA. Quantitative real-time-PCR analysis was performed using SYBR Premix Ex Taq II (TaKaRa, Shiga, Japan) and the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA). The bacterial primer sequences are listed in Supplementary Table S2.