Total Microbial DNA Extraction and PCR Amplification Data

1. Total Microbial DNA Extraction and PCR Amplification: Microbial DNA was extracted from the fecal samples. The DNA concentration was quantified using Nanodrop, and the quality of the DNA extraction was assessed by 1.2% agarose gel electrophoresis. Primers (forward primer sequence: ACTCCTACGGGAGGCAGCA; reverse primer sequence: GGACTACHVGGGTWTCTAAT) were used to amplify the V3-V4 region of the 16S rRNA gene by PCR. After purification using magnetic beads, the amplification products were quantified using fluorescence.2. Library construction and high-throughput sequencing: The required libraries put into the machine were constructed using the standard Illumina TruSeq DNA library preparation protocol (Illumina TruSeq DNA Sample Preparation Guide). After quality control, the libraries were quantified using the Quant-iT PicoGreen dsDNA Assay Kit on the Promega QuantiFluor fluorescence quantification system, and qualified libraries were loaded onto the sequencing instrument for sequencing. 3. Data Analysis: The raw sequencing data retrieved from high-throughput sequencing were preliminarily screened based on sequence quality. The sequences were denoised or clustered into operational taxonomic units (OTUs) using either the QIIME2 dada2 analysis workflow or Vsearch software. Other software and corresponding statistical methods were employed to analyze alpha diversity, beta diversity, and differences in species abundance composition in the samples.