Oral combinatorial antibiotic treatment on the constituency of gut commensal bacteria
16S rRNA metagenomic analysis was performed at Repertoire Genesis (Osaka, Japan). In brief, microbial DNA was extracted from frozen murine fecal samples of SI using Isospin fecal DNA (NIPPON GENE Co., Ltd., Tokyo, Japan). For bioinformatics analysis of 16SrRNA, the V3V4 region of 16S rRNA genes was amplified and sequenced by MiSeq Deep sequencer and MiSeq Reagent Kit v3 (Illumina) following the manufacturer's instructions. The sequence data were preprocessed and analyzed using the Flora Genesis software (Repertoire Genesis Inc.). In brief, the R1 and R2 read pairs were joined and chimera sequences were removed. Operational taxonomic unit (OTU) picking was performed by the open-reference method using the 97% ID prefiltered Greengenes database and uclust. Representative sequences of each OTU were chosen and taxonomy assignment was performed by the ribosomal database project (RDP) classifier using a threshold score of 0.5 or more. The OTUs were grouped if their annotation was the same regardless of the RDP score.