Miseq 2x300bp paired end sequencing

PCR amplicons for individual samples were generated by nested PCR using primers listed in Supplementary Table 3 and starting from >50-100 ng of purified gDNA. The first PCR step was performed with GoTaq G2 DNA Polymerase (Promega) according to manufacturer instruction using the following amplification protocol: 95°C x 5’, (95°C x 0.5’, 60°C x 0.5’, 72°C x 0.25’) x 20 cycles, 72°C x 5’. The second PCR step was performed with GoTaq G2 DNA Polymerase (Promega) according to manufacturer instruction using 5 µl of the first-step PCR product and the following amplification protocol: 95°C x 5’, (95°C x 0.5’, 60°C x 0.5’, 72°C x 0.3’) x 20 cycles, 72°C x 5’. Second-step PCR primers were endowed with tails containing P5/P7 sequences, i5/i7 Illumina tags to allow multiplexed sequencing and R1/R2 primer binding sites (Supplementary Table 3). PCR amplicons were separately purified performing double-side selection with AmpPure XP beads (Beckman Coulter). Library quality was assessed by LabChip® GX Touch HT (Perkin Elmer). Amplicons were multiplexed and sequenced by GeneWiz on MiSeq 2x300bp paired end sequencing (Illumina).