16s data and Bile acid metabolomics

Bile samples were obtained from 42 patients, comprising three groups: 10 normal controls (undergoing right hemihepatectomy), 9 patients with cholesterol polyps (post-cholecystectomy with pathological confirmation), and 23 patients with gallstones. Patients with acute cholecystitis were excluded. Additionally, 18 gallbladder tissue specimens were paraffin-embedded for spatial transcriptomic analysis, of which 13 passed quality control and were included in subsequent experiments.Total microbial DNA was extracted from bile samples using the Bacterial Genome Extraction Kit (TIANGEN DP302-02) according to the manufacturer’s instructions. DNA concentration was quantified using a Qubit fluorometer (Invitrogen, USA). The 16S rRNA gene V3-V4 region was amplified with primers 341F/805R. PCR products were purified, quantified, and sequenced on an Illumina NovaSeq 6000 platform (PE250). Raw reads were processed using cutadapt, FLASH, and fqtrim for quality control, followed by DADA2 denoising within QIIME2 to generate amplicon sequence variants (ASVs). Taxonomic assignment was performed against the SILVA and NT-16S databases. Alpha diversity metrics (Chao1, Shannon) were compared using Wilcoxon or Kruskal-Wallis tests. Beta diversity was assessed via principal coordinate analysis (PCoA) based on UniFrac distances, with significance evaluated by ANOSIM and PERMANOVA. Differentially abundant genera were identified using Wilcoxon or Kruskal-Wallis tests with FDR correction, as well as LEfSe (LDA > 3.0, p < 0.05). Microbial functional potential was predicted using PICRUSt2 and BugBase.