design: DNA was extracted from each sample using the NucleoSpin DNA Stool Kit, and 5 ng of DNA was used for the first PCR targeting the V3-V4 region of the 16S rRNA gene with Universal primers. The PCR products were purified using AMPure XP beads, and subjected to index PCR using the Nextera XT index kit. The DNA was denatured by adjusting the final denatured DNA concentration to 6 pM using 25% PhiX spike-in. Sequencing was performed using a MiSeq Reagent v2 kit with a 250-bp paired-end strategy.