Whole saliva (5 ml) was collected into ice-chilled sterile test tubes while chewing on a 1-g piece of paraffin wax. Next, 100 μl of fresh saliva was transferred to a transport medium for cultivation, the remaining saliva centrifuged at 4 °C (13,000 rpm for 5 min), and the pellets stored at −80 °C until DNA extraction and amplicon sequencing by Illumina MiSeq (http://www.illumina.com ).
Supragingival tooth biofilm was collected using sterile wooden toothpicks, pooled by subject in 100 µl TE-buffer (10 mM Tris, 1 mM EDTA, pH 7.6), and stored at −80 °C until DNA extraction and 16S rDNA amplicon sequencing by Illumina MiSeq and PacBio RS II SMRT (Pacific Biosciences, Menlo Park, CA, USA) was conducted.
Supragingival tooth biofilm was collected using sterile wooden toothpicks, pooled by subject in 100 µl TE-buffer (10 mM Tris, 1 mM EDTA, pH 7.6), and stored at −80 °C until DNA extraction and 16S rDNA amplicon sequencing by Illumina MiSeq and PacBio RS II SMRT (Pacific Biosciences, Menlo Park, CA, USA) was conducted.