design: 16s sequencing for gut metagenome/Fecal samples from healthy blood donors, HBV carriers or OBI blood donors participating in follow-up were collected if the samples conformed to the requirements (smooth and soft stool that was sausage or snake shaped) and then immediately frozen at 80C. The collection and storage process was completed within 30 minutes. DNA was extracted using the TGuide S96 Magnetic Stool DNA Kit (Tiangen Biotech, Beijing, China) according to the instructions. DNA quantification and purity were assessed using NanoDrop 2000 (Thermo Scientific, MA, USA).The V3-V4 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene was amplified with the forward primers 338F (5-ACTCCTACGGGAGGCAGCA-3) and the reverse primers 806R (5-GGACTACHVGGGTWTCTAAT-3). The PCR conditions were as follows: initial denaturation at 95C for 5 min; 25 cycles of denaturation at 95C for 30 s, annealing at 50C for 30 s, and extension at 72C for 40 s; and a final extension at 72C for 7 min. The PCR products were extracted from 1.8% agarose gel and purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) according to the manufacturers protocol.Purified amplicons were pooled in equimolar amounts and were paired-end sequenced on an Illumina Novaseq 6000 platform (Illumina, San Diego, USA) according to the PE250 standard protocols by Biomarker Technology Co. Ltd. (Beijing, China). The raw gene sequencing reads were merged using FLASH version 1.2.11. The concatenated sequences were quality-filtered by Trimmomatic version 0.33, then chimeras were removed using UCHIME version 8.1. Operational taxonomic units (OTUs) with a 97% similarity cutoff were clustered using USEARCH version 10.0, and filtered with a threshold of 0.005%.