Homo sapiens saliva in-vitro biofilm
Samples were amplified using  16S rRNA V3-V4 regions specific primers  (V3-V4-Forward    5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3,V3-V4- Reverse 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3) The PCR was performed in 10 l final volume with 0.2-M primer concentration.  The PCR cycle included:  3 min at 95 C (initial denaturation) followed by 25 cycles: 30 s at 95 C 30s at 55 C, and 30s at 72 C,  and a final elongation step of 5 min at 72 C. PCR  products were purified using AMPure XP beads (Beckman Coulter, Nyon, Switzerland) with a 0.9x ratio according to manufacturers instructions.  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 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 (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer's protocol. Libraries were eluted in 20 l final volume and pooled for sequencing. The final pool was quantified by qPCR using Kapa library quantification kit for Illumina Platforms (Kapa Biosystems, SigmaAldrich, Saint Louis, MO, USA) on an ABI 7900HT real-time cycler (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA). Sequencing was performed using Illumina MiSeq (2300 bp) and v3 chemistry with a loading concentration of 10 pM. In all cases, 15% of PhIX control libraries   were used  to increase the diversity of the sequenced sample. Negative controls included sample collection buffer, DNA extraction, and PCR amplification steps, PRC products after both PCR steps were visualized by electrophoresis  gel (1.5% agarose) with SYBR Safe (ThermoFisher Scientific, Waltham, MA, USA).  No visible bands were observed.