16S rRNA gene amplicon sequencing of bioaerosol from WWTP1 CS site
Total bioaerosol samples were collected using a high-volume liquid impinger sampler (P-1000, Dinglan Technology, China) operated at 1,000 L/min for 30 min, utilizing sterile mineral oil-coated dishes as the collection medium. Particulate matter was recovered by centrifugation and resuspended in 0.01 M phosphate-buffered saline (PBS, pH 7.2). Total genomic DNA was extracted using the OMEGA Soil DNA Kit (Omega Bio-Tek, USA) according to the manufacturer's protocol. DNA integrity and concentration were assessed via 1% agarose gel electrophoresis and NanoDrop NC2000 spectrophotometry (Thermo Fisher Scientific, USA). The hypervariable V3-V4 regions of the bacterial 16S rRNA gene were amplified using barcoded universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Purified PCR amplicons were quantified using PicoGreen, pooled equimolarly, and constructed into sequencing libraries using the TruSeq Nano DNA LT Library Prep Kit (Illumina, USA). Libraries were sequenced on an Illumina NovaSeq 6000 platform using a 250 bp paired-end (PE250) strategy. Raw fastq files were processed through the DADA2 pipeline in QIIME2 (v2022.11) for quality filtering, denoising, dereplication, and chimera removal to generate amplicon sequence variants (ASVs).