16S rRNA amplicon sequencing of aquatic bacteria

Total genomic DNA was extracted using the DNeasy PowerSoil Pro Kit (Qiagen), and its quality and concentration were verified with a NanoDrop One spectrophotometer and agarose gel electrophoresis. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using barcoded specific primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). After library preparation, paired-end 250 bp sequencing was performed on an Illumina NovaSeq 6000 platform, with sequencing services provided by Shanghai Personal Biotechnology Co., Ltd. Raw reads were quality-filtered using Fastp (v0.23.2) and merged with FLASH (v1.2.11). Amplicon sequence variants (ASVs) were generated via the DADA2 pipeline in QIIME2 (v2022.11) and taxonomically annotated against the SILVA 138 database.