Optimized PCR Conditions for Fungal Diversity

Optimal PCR conditions were determined from DNA extracts of Agaricus bisporus and CIL Plus Mycoactive^™ Potting Soil (for LSU200-F/LSU481-R), and of a Harposporium sp. isolate for LSU200A-F /LSU476A-R. LSU PCR reactions were carried out in a total volume of 25 μL, with 0.5–4 μL template DNA, 12.5 μL of Toughmix (Quanta Biosciences), and 1.25 μL each of forward and reverse primers (5 μM). Optimal PCR conditions were 2 min at 94°C, followed by 29 cycles for 30 s at 94°C, 30 s at 62°C (55°C in first cycle followed by the remainder at 62°C for Ascomycota primers), with a final elongation temperature of 72°C for 18 s. ITS2 PCR reactions followed the protocol outlined by Toju et al. [41 (link)], scaled to a total volume of 25 μL, using 1 μL template DNA (20 ng/μL for peat and soil samples). Optimal PCR conditions were 3 min at 94°C, followed by 35 cycles for 20 s at 94°C, 30 s at 47°C, 20 s at 72°C, with a final elongation temperature of 72°C for 7 min [41 (link)]. PCR products were normalized with a Qubit fluorimeter with the dsDNA HS kit (Life Technologies) and submitted for paired-end MiSeq Illumina sequencing (2 x 300 bp with V3 chemistry) at the London Regional Genomics Centre (Robarts Research Institute, London, Canada).