sequences of bacteria
Fecal samples (0.20.5 g) were homogenized using a TissueLyser-48 (Shanghai Jingxin) at 60 Hz. Total genomic DNA was extracted using the OMEGA Soil DNA Kit (M5635-02, Omega Bio-Tek) following the manufacturer's protocol. The bacterial 16S rRNA gene V3V4 hypervariable region was amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'), with a 710 bp barcode incorporated into the forward primer for sample multiplexing. PCR was performed using ABclonal DNA polymerase with the following program: initial denaturation at 98C for 5 min; 25 cycles of 98C for 30 s, 52C for 30 s, and 72C for 45 s; final extension at 72C for 5 min. PCR products were purified using magnetic beads. Sequencing libraries were constructed using the TruSeq Nano DNA LT Library Prep Kit (Illumina) according to the manufacturer's instructions, and quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Paired-end sequencing (2  250 bp) was performed on the Illumina NovaSeq 6000 platform using the NovaSeq 6000 SP Reagent Kit (500 cycles). Raw reads were demultiplexed, primer-trimmed, quality-filtered, denoised, merged, and chimera-removed using the DADA2 plugin in QIIME2 (2019.10). Amplicon sequence variants (ASVs) were generated and taxonomically classified using the Greengenes database.