eDNA SCANZANO 1
Metabarcoding analysis was performed by IGA Technology Services s.r.l. The PCR amplification consisted of two steps, the first step using primers that amplify 16S rRNA region (Riaz et al. 2011).  The PCR mix (final volume of 25 l) contained 12.5 l 2 KAPA HiFi HotStart ReadyMix, 2.5 l of the forward primer 16S-515F 5- GTGYCAGCMGCCGCGGTAA -3 (with Illumina adaptor 2 M) and 2.5 l of the reverse primer 16S-805R 5- GGACTACNVGGGTWTCTAAT -3 16S (with Illumina adaptor 2 M). After adding 50 ng of the DNA extract, this mix was incubated with initial denaturation for 3 min at 95 C, 25 cycles of denaturation for 30 s at 95 C, annealing for 30 s at 55 C and extension for 30s at 72 C, and a final extension for 4 min at 72 C.  PCR products were purified using 1.6X Ampure XP beads and eluted in 35 l Tris-HCl pH 8.0 buffer. For the second step, the index PCR, 7.5 l of the purified PCR product was added to a PCR mix containing 12.5 l 2 KAPA HiFi HotStart ReadyMix and 2.5 l of each index primer (Nextera XT). PCR conditions were: initial denaturation for 3 min at 95 C, 9 cycles of denaturation for 30 s at 95 C, annealing for 30 s at 55 C and extension for 30 s at 72 C, and a final extension for 5 min at 72 C. After measuring with the Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA), the indexed PCR products were equimolarly pooled and sent for sequencing using the Illumina MiSeq 2*300 bp platform (Illumina, San Diego, CA). Base calling, demultiplexing and adapter masking were performed on-instrument with MiSeq Reporter.