amplicon16S Homo Sapiens black plaque
Samples were amplified using specific primers  for the V3-V4 regions of  the 16S rRNA gene . (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3) and  V3-V4-Reverse (5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3).  The PCR was performed in 10-L final volume with 0.2-M primer concentration. The PCR included: 3 min at 95 C (initial denaturation) followed by 25 cycles: 30 s at 95 C, 30 s at 55 C, and 30 s at 72 C, and a final elongation step of 5 min at 72 C. PCR products were purified using AMPure XP beads with a 0.9 ratio according to the manufacturers instructions. PCR products were eluted from the magnetic beads with 30 L of Milli-Q water. The above-described primers contain overhangs allowing the addition of full-length Nextera barcoded adapters for multiplex sequencing in a second PCR step, resulting in sequencing ready libraries with approximately 450 bp insert sizes. In brief, 5 L of the first PCR purified product were used as the template for a second PCR with Nextera XT v2 adaptor primers in a final volume of 30 L using the same PCR mix and thermal profile as for the first PCR but with only 8 cycles. 25 L of the second PCR product were purified with SequalPrep normalization kit, according to the manufacturers protocol. Libraries were eluted in 20 L final volume and pooled for sequencing.