Recruitment was conducted at the Second Affiliated Hospital of Dalian Medical University and the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. Patients with autoimmune diseases were enrolled based on confirmed diagnoses by licensed physicians, adhering to the 2019 European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria for ankylosing spondylitis (AS), systemic lupus erythematosus (SLE), and primary Sjögren’s syndrome (pSS). Exclusion criteria included diabetes, severe hypertension, severe obesity or metabolic syndrome, inflammatory bowel disease (IBD), cancers, abnormal liver or kidney function, and recent use of antibiotics or probiotic products within the preceding four weeks. Based on these criteria, the study included 130 AS patients, 73 SLE patients, 66 pSS patients, and 118 healthy controls for further analysis. Fecal samples were collected from participants, temporarily stored on dry ice, and transported to the laboratory within 24 hours, where they were stored at -80°C until further processing. DNA extraction was performed using the TIANamp Stool DNA Kit (TIANGEN, China), and DNA quality was evaluated using the Qubit 2.0 Fluorometer. Metagenomic sequencing libraries were prepared using the NEB Next Ultra DNA Library Prep Kit (NEB, USA) following the manufacturer’s protocol, with unique index codes assigned to each sample. Library quality was verified using an Agilent 2100 Bioanalyzer. Indexed samples were clustered on a cBot Cluster Generation System using an Illumina PE Cluster Kit (Illumina, USA) according to the manufacturer’s instructions. After cluster generation, DNA libraries were sequenced on the Illumina NovaSeq platform, producing 150 bp paired-end reads.