Metabarcoding of mock community amplified 18 PCR cyles and 2  g input
Genomic DNA was extracted from fungal isolates using three different methods (Qiagen Dneasy Plant Mini Kit, peqGOLD Tissue DNA Mini Kit, MasterPure Yeast DNA Purification kit). The resulting DNA was pooled to form the mock community. PCRs were conducted in 25- l reactions using 0.5  l Herculase II Fusion enzyme (Agilent Technologies), 5  l of 5x PCR buffer, 0.62  l each primer (10 uM), 0.25  l dNTPs (250 mM each), 0.3  l BSA (20mg/ml BSA) on a SensoQuest labcycler with 2 min denaturation at 95  C, 13-30 cycles of 94  C for 30 sec, 55  C for 30 sec and 70  C for 4 min, and a final elongation at 70  C for 10 min. Multiple PCR reactions (up to 50) were required for each environmental sample to ensure sufficient product for library preparation (1 g purified PCR product).