16S rRNA gene amplicon sequencing

The sample size for 16S rRNA gene amplicon sequencing is 20 per group. Genomic DNA was extracted from fecal samples by cetyltrimethylammonium bromide (CTAB) method. The purity and concentration of the extracted genomic DNA were detected by agarose gel electrophoresis. The V3 – V4 region of the gene sequence was amplified by specific primers 341F (5 ′ - CCTAYGGGRBGCASCAG-3 ′) and 806R (5 ′ - GGACTACNNGGGTATCTAAT-3 ′) and high fidelity enzyme to construct the library. After the library is qualified, it will be sequenced on the NovaSeq 6000 platform (PCR reaction will be completed by Beijing Novogene Technology Co., Ltd.). Using FLASH (v.1.2.11, http://ccb.jhu.edu/software/FLASH/ ）[20] The software concatenates the offline data of the sample to obtain raw tags. Subsequently, fastp (v.0.23.1) software was used to perform quality control on the raw tags, resulting in high-quality clean tags. Using Usearch software, clean tags were compared with the database to remove chimeras and obtain valid data [21]. Based on valid data, denoising was performed using DADA2 to filter out sequences with an abundance less than 5, resulting in the final amplification sequence variants (ASVs) and feature tables. Compare the obtained ASVs with the Silva138.1 database using the classify sklarn module in QIIME 2 software to obtain species information for each ASV. Calculate the alpha diversity index using QIIME 2 software and analyze the differences between groups; Calculate the weighted UniFrac distance and plot a principal coordinate analysis (PCoA) graph for beta diversity analysis. Use the Anosim function to analyze the significance of differences in community structure between groups; LEfSe was used to analyze significantly different species between groups, with an LDA score threshold of 4.