16S rRNA sequencing of grape marc at zero days of incubation (T0)
Grape marcs preparation.  Prosecco grapes were mechanically harvested and crushed in a commercial winery. The must was separated from the juice by pressing, and the marc was transported to a nearby distillery. The grape marc was stored into four 30 kg-capacity plastic bags, sealed hermetically, and placed at room temperature (10–18 °C). Grape marc samples (250 g) were collected the day of processing (T0).  Extraction of DNA for metagenomic analysis.  The genomic DNA was extracted starting from 20 g of grape marcs. 25 ml of physiological saline buffer solution were added to the marcs and the mixture was stirred with a vortex mixer for approximately ten minutes. The mixture obtained was filtered through a 50 um filter to remove plant debris. The preparation obtained was centrifuged for 15 min at RT at maximum speed and, genomic DNA was extracted from the pellet obtained (~0.04 g each) using the kit Mobio (power soil) using the standard protocol.  16s rRNA gene amplification.  Two regions of the bacterial 16s rRNA gene were amplified using primers "F357" and "926R" for the hypervariable regions V3 and V4 and using primers “B1046R” and “UF89F” for the V5 and V6 regions. The amplification was performed independently for the two parts of the 16S gene (V3-V4 and V5-V6) using the two couples of primers and using the following reaction mixture: 2 ul of genomic DNA (the template concentration was not determined due to the low concentration), 0.4 ul of 10 mM each dNTPs, 0.2 ul of Phusion DNA polymerase (Finzimes), 0.6 ul of primer forward (10 uM), 0.6 ul of primer reverse (10 uM), 4 ul of 5x Phusion HF buffer, final volume 20 ul. Three separate reactions were set up for each of the amplicons and amplified using three different melting temperatures (58, 59, 60° C) to allow the degenerate primers to operate more efficiently. The final number of PCR cycles used used to generate the amplicon for FLX library production was 26. The PCR cycle used in an Eppendorf Mastercycler thermocycler EP was: 95°C 2'; (95°C 20''; 58-60°C, 45'', 72°C 20'') x 26 cycles, 72°C 120''. The amplified product obtained was purified with the Agencourt AMPure kit with a final DNA elution performed with 40 ul of water. Each sample has been "tagged" during PCR amplification using "MID2" for the “V5-V6” sample at T0 and with “MID80” for the “V3-V4” sample at T0. Identical amounth of each amplicon was pooled and sent to the Ramaciotti Centre (UNSW, Sydney) for GS FLX amplicon sequencing using Titanium chemistry.