ITS2 Sequencing Protocol for Fungal Identification

The ITS2 gene was sequenced using the fITS7b (5′-GTGARTCATCGAATCTTTG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers. Before sequencing, PCR was conducted utilizing the Phusion Flash High-Fidelity PCR master mix from Thermo Fisher Scientific. The following PCR protocol was followed: 2 min of initialisation, 35 repetitions of denaturation at 98 °C for 10 s, annealing at 54 °C for 20 s, and elongation at 72 °C for 30 s, with the final elongation lasting for 7 min. Amplicon sequencing was executed using an IonTorrent PGM platform. Sequencing was performed in four separate runs. Details about DNA extraction, PCR, and sequencing have been previously published [31 (link)].