DNA-metabarcoding of soil fungi
For amplification of the ITS2 region, the ITS3 (forward): GCATCGATGAAGAACGCAGC and ITS4 (reverse): TCCTCCGCTTATTGATATGC primers were used. The KAPA HiFi HotStart polymerase (Roche Molecular Systems) was used, applying the following temperature protocol: initial denaturation at 92C for 2 minutes, 30 cycles of denaturation at 98C for 20 seconds, hybridization at 55C for 30 seconds and extension at 72C for 20 seconds, and final extension at 72C for 5 minutes. The PCR products were cleaned with 1x AMPure XP Beads (Beckman Coulter) and eluted in 30 l of nuclease-free water, followed by a second PCR to ligate Illumina UD indexes. The same polymerase was used, and the temperature protocol was as follows: initial denaturation at 92C for 2 minutes, 6 cycles of denaturation at 98C for 20 seconds, hybridization at 60C for 30 seconds and extension at 72C for 30 seconds, and final extension at 72C for 2 minutes.