Fungal ITS2 amplicon sequencing of soil metagenome: resin-contaminated soil from inactive resin processing site
DNA was extracted from 0.5 g composite soil samples (n = 3) using an optimized phenol/chloroform/CTAB methodology [34]. The ITS2 fungal rDNA region was amplified in two steps: initial PCR with standard primers, followed by a second PCR using the same primers carrying a barcode: gITS7 (5'-GTGARTCATCGARTCTTTG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). In the second amplification conditions were modified: denaturation at 95C, 25 cycles, annealing at 55C. PCR products were purified (Zymo DNA Clean & Concentrator-5) and quality-checked by gel electrophoresis and NanoDrop spectrometry (One Microvolume UV-Vis Spectrophotometer, Thermo Fisher Scientific Inc., USA).