Metagenomic Analysis of Subgingival Microbiome

The subgingival microbial DNA in a 500 µL transport medium was extracted by using the DNeasy PowerBiofilm kit (Qiagen, Hilden, Germany) with a preceding bead beating (45 s; speed: 3450 oscillations/min) and stored at −20 °C. Then, the DNA concentration was determined by a Colibri Microvolume spectrophotometer (Titertek Berthold, Pforzheim, Germany). The 16S rRNA gene of each sample was amplified using the primer pairs 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) targeting the V3–V4 region. Library preparation was achieved by the Illumina MiSeq platform generating 300 bp paired-end reads. The raw sequence data were imported into QIIME2 [51 (link)], in which paired-end reads were merged and denoised into amplicon sequence variants (ASVs) using the DADA2 plugin [52 (link)]. The reads were filtered based on exact matches to the barcode/primer and an average quality score of 30. Due to the issue of sample bleeding between the Illumina MiSeq runs [53 (link)], the low-abundance filters (a cutoff threshold of 0.1% of mean frequency 70,651) were applied to reduce the number of spurious ASVs in the data set.