DNA extraction and 16S rRNA sequencing
Total genomic DNA in students’ feces samples was extracted from the glass fiber filters using TIANamp Bacteria DNA Kit (QIAGEN) following the manufacturer’s instructions. The concentration and quality of the extracted genomic DNA were determined using spectrophotometer (NanoDrop, Thermo Fisher Scientific, Inc., USA). Extracted DNA was subjected to 16S sequencing at Shanghai Personal Biotechnology Cp. Ltd. The following universal 16S rRNA primers were used for the PCR reaction: 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) for the V4 region, and these PCR products were sequenced with an Illumina HiSeq 2000 platform. FLASH was applied to merge overlapped reads, and sequence analysis was performed using UPARSE software package. Reads with quality scores lower than 20, ambiguous bases, and improper primers were discarded prior to clustering. Simultaneously, chimaeras were checked and eliminated during clustering. The resulting high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% similarity. According to the annotation of taxa, the relative abundance of total bacteria in each sample was categorized into different classification levels (Phylum, Class, Order, Family, and Genera). The alpha diversity index (Shannon index and Chao1 index) was calculated, and the beta diversity index (principal component analysis, PCoA) was mapped in QIIME2 software. The raw reads were deposited in the Sequence Read Archive (SRA) database of NCBI (PRJNA883039).