2.3. Collection and Sequencing of Plaque Samples

The contents of the periodontal pocket in patients with chronic periodontitis (CP), chronic periodontitis associated with type 2 diabetes mellitus (CPT2DM) and the contents of the gingival sulcus in control subjects were the study material. The samples were collected from the patients in the morning on an empty stomach (between 9:00 and 11:00 a.m.) before they used a toothbrush and other hygiene products. The biological material was sampled from four spots of the periodontal pockets/sulcus at the level of the second molars using sterile paper endodontic posts, which were placed together into a test tube containing 0.2 mL of sterile physiological saline solution and shaken. Material was collected at six sites per tooth (mesio-, mid-, and disto-buccal area; mesio-, mid-, and disto-lingual area) for all teeth.

The samples were delivered to the laboratory and subsequently stored at −20 °C. Total DNA was extracted from the samples using a QIAamp DNA Investigator Kit (Qiagen, Düsseldorf, Germany) in accordance with the manufacturer’s protocol. Genomic DNA content was determined on a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. The enriched microbial DNA (50–100 ng) was fragmented using a Covaris S220 system (Covaris, Woburn, MA, USA). The final fragment size was determined using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa-Clara, CA, USA) in accordance with the manufacturer’s instructions. Briefly, the extracted DNA was amplified using standard 16S rRNA gene primers being complementary to the V3–V4 region and containing 5′-illumina adapter sequences. Sequencing was carried out on a HiSeq 2500 platform (Illumina) in accordance with the manufacturer’s instructions.

DNA samples for shotgun sequencing were pooled and prepared by ligating the genomic DNA of the samples within each study group taken at equimolar amounts. The amount of the mixed DNA pool was determined on a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. A NEBnext Microbiome DNA enrichment kit was used for enriching the microbial genomic DNA in the mixed pools in microbial genomic DNA in accordance with the manufacturer’s instructions. The libraries of paired terminal fragments were prepared in accordance with the manufacturer’s guidelines using a NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). The libraries were indexed using NEBNext multiplex Oligos for Illumina kits (96 Index Primers) (New England Biolabs, Ipswich, MA, USA). The distribution over library size and quality was assessed using a high-sensitivity DNA microarray (Agilent Technologies, Santa-Clara, CA, USA). Subsequent quantification of the libraries was performed using a high-sensitivity Quant-iT DNA Assay Kit (Thermo Scientific). Sequencing was conducted on a HiSeq 2500 platform (Illumina) in accordance with the manufacturer’s instructions using the following reagent kits: HiSeq Rapid PE Cluster Kit v2, HiSeq Rapid SBS Kit v2 (500 cycles), HiSeq Rapid PE FlowCell v2, and a 2% Phix spike in controls.