design: The V4 hypervariable region of the 16S rRNA gene was amplified by PCR using specific barcoded primers (515F: 5-GTGCCAGCMGCCGCGGTAA-3; 806R: 5-GGACTACHVGGGTWTCTAAT-3). Each reaction contained 15 L Phusion High- Fidelity PCR Master Mix (New England Biolabs, EUA), 0.2 M of each primer, and 10 ng  of template DNA. The thermocycling protocol included an initial denaturation at 98C for 1 min, followed by 30 cycles of 98C for 10 s, 50C for 30 s, and 72C for 30 s, and a final extension at 72C for 5 min. PCR products were visualized on a 2% agarose gel containing SYBR Green I. The band corresponding to the amplified 16S rRNA gene (~300 bp) was excised and purified using the Qiagen Gel Extraction Kit. High-quality sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) with additional index codes. Library quality was assessed by both Qubit 2.0 Fluorometer (Thermo Scientific, EUA) and Agilent Bioanalyzer 2100. Subsequently, 250 bp paired-end reads were obtained via Illumina NovaSeq platform.