16S Subgingival plaque
DNA was extracted and the V4 hypervariable region of the 16S rRNA gene was amplified with primers 515F/806R for multiplexed amplicon sequencing. PCRs (triplicates per sample to reduce bias) used KAPA2G Robust HotStart ReadyMix (12.5 L) with 1.5 L each of 10 M forward/reverse primers, 7.5 L nuclease-free water, and 2 L template DNA. Cycling: 95 C 3 min; 22 cycles of 95 C 15 s, 50 C 15 s, 72 C 15 s; final 72 C 5 min. Triplicates were pooled, verified on 1% TBE agarose, quantified by PicoGreen, normalized by equal concentration, purified with AMPure XP, and sequenced on an Illumina MiSeq (Centre for the Analysis of Genome Evolution and Function, University of Toronto) using the V2 chemistry kit (2150 bp). Run controls included a single-species control (Pseudomonas aeruginosa DNA), a mock community (ZymoBIOMICS Microbial Community DNA Standard), and a template-free negative control.
SUB15674644
SUB15617535