Fungal metabarcoding of disturbed pine forests and paired controls
Soil cores (25 cm deep, 2.5 cm diameter) were collected from 28 locations within 30 m x 30 m sites, dominated by lodgepole pine (Pinus contorta) disturbed within 6 years of collection by  mountain pine beetle (Dendroctonus ponderosae) outbreak, wildfire, clear-cur logging, or slavage-logging. Soil cores were homogenized (organic, A, and B horizons combined), and approximately 4 g of soil per core were freeze dried and ground. Genomic DNA was isolated from 250 mg of ground soil using E.Z.N.A. Soil DNA Kit. A two-step polymerase chain reaction (PCR) amplified the ITS1 (Internal Transcribed Spacer) region of nuclear rDNA extracted from soil cores. The first amplification of the ITS1 region was performed using primers ITS1F (Gardes and Bruns, 1993) for forward and ITS2 (White et al., 1990) for reverse directions. For the second PCR, Illumina Nextera Index barcode primers were used, N7xx for forward and N5xx for reverse directions. Amplicons generated during the first and second PCR were purified using Mag-Bind TotalPure NGS beads. Amplicon library sequencing was performed by the University of Alberta Molecular Biological Sciences Unit, using 2 x 300 bp paired-end reads on an Illumina MiSeq sequencing platform.