16S amplicon of biofilm treated with phosphate-buffered-saline
Libraries were sequenced (v3 chemistry: 2 × 300 bp paired-end) on the Illumina MiSeq (Illumina, USA). The unprocessed sequences were demultiplexed using unique barcodes and converted into fastq format via the bcl2fastq software. The reverse reads were determined to have low quality (<Q25), preventing the achievement of the recommended 50 bp overlap ideal for merging both reads. Therefore, only forward reads with quality scores >Q30 were retained for downstream analyses to ensure the reliability of the data. Forward reads were processed and quality-filtered using the DADA2 software package version 1.28.0 in R version 4.3.1. Reads with more than two expected errors were removed, and sequences were trimmed at 28 bp and truncated at 200 bp to retain high-quality reads for denoising. Error rates of the filtered dereplicated reads were estimated using 1,000,000 sequences. Amplicon sequence variants (ASVs) were inferred exactly, resolving variants differing by as little as one nucleotide. Chimeric ASVs were discarded. Taxonomic assignment was performed using the naïve Bayesian classifier method with the Human Oral Microbiome Database (eHOMD) as a reference. Mitochondria and chloroplast taxa were removed. Additionally, ASVs with read counts totaling less than 0.001% of the total dataset read count were discarded to minimize noise. Sample read numbers were rarefied to the minimum sequencing depth obtained for this study (64,798 reads).