B6
Total genomic DNA was extracted using DNA Extraction Kit following the manufacturers instructions. Quality and quantity of DNA was verified with NanoDrop and agarose gel. Extracted DNA was diluted to a concentration of 1 ng/l and stored at-20C until further processing. The diluted DNA was used as template for PCR amplification of bacterial 16S rRNA genes with the barcoded primers and Takara Ex Taq (Takara). For bacterial diversity analysis, V3-V4 variable regions of 16S rRNA genes was amplified with universal primers 343F and 798R .Amplicon quality was visualized using gel electrophoresis, purified with AMPure XP beads (Agencourt), and amplified for another round of PCR. After purified with the AMPure XPbeads again, the final amplicon was quantified using Qubit dsDNA assay kit. Equal amounts of purified amplicon were pooled for subsequent sequencing.Raw sequencing data were in FASTQ format. Paired-end reads were then preprocessed using cutadapt software to detect and cut off the adapter. After trimming, paired-end reads were filtering low quality sequences, denoised , merged and detect and cut off the chimera reads using DADA2 with the default parameters of QIIME2 (2020.11). At last, the software output the representative reads and the ASV abundance table. The representative read of each ASV was selected using QIIME2 package. All representative reads were annotated and blasted against Silva database Version 138 using q2-feature-classifier with the default parameters. The microbial diversity in bile and pancreatic juice samples was estimated using the alpha diversity that include Chao1 index and Shannon index. The Unifrac distance matrix performed by QIIME software was used for weighted Unifrac Principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS). The 36S rRNA gene amplicon sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).