16S rDNA Amplicon Sequencing of Microbiome

16S rDNA amplicon sequencing of the V4 hypervariable region was performed as described previously⁵⁰ . Each sample was PCR amplified using 1 ng of template DNA with primers F515/R806, targeting the V4 hypervariable region of the 16 s ribosomal gene^{51 (link)}. In each batch of samples, a mixed pure culture isolates (mock), blanco extraction controls, pooled salivary extraction controls and pooled DNA amplification controls were included. The amount of DNA per sample was quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific). The amplicon libraries were pooled in equimolar amounts and purified using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare, Eindhoven, The Netherlands). Amplicon quality and size was analyzed on the Fragment Analyzer (Advanced Analytical). Paired-end sequencing of amplicons was conducted in five separate runs on the Illumina MiSeq platform (Illumina, Eindhoven, The Netherlands). Denoising of sequence data and identification of ASVs were performed using the DADA2 (1.12.1) pipeline^{52 (link)}.