DNA extraction and 16S rRNA gene amplicon sequencing

Fecal bacterial DNA was extracted using a QIAamp DNA Stool Mini kit (QIAGEN, Germany) according to the manufacturer's instructions. The DNA concentration was detected using a 7415 Nano spectrophotometer, and the 260/280 and 260/230 ratios were measured to assess and quantify the purity of the DNA samples. The DNA samples were stored at -20 °C before use. The amplicon library was constructed via the amplification of the V3-V4 hypervariable regions of the 16S rRNA gene sequences using the primers (319F/806R) ²⁶. The PCR amplification program was performed with DNA polymerase and a thermocycler using the following steps: initial denaturation at 98 °C for 2 min, 30 cycles of 98 °C for 15 s, 58 °C for 15 s, 72 °C for 15 s and a final extension at 72 °C for 3 min ²⁶. The qualities of the PCR products were assessed using an Agilent Bioanalyzer 2100 system (Agilent, CA, USA), and the amplicon pool of PCR products was prepared with AMPure XP beads. Subsequently, the pooled PCR products were sequenced by BGI Co., Ltd., using the Illumina MiSeq sequencing system ²⁷.