High-throughput sequencing of fungal community DNA from soils of beetle-killed forests: EF-GP2016-Site 21, sample 7
Soil cores (20 cm deep, 2.5 cm diameter) were randomly collected from ten locations within 30 30 m x 30 m sites, dominated by lodgepole pine (Pinus contorta) suffering high mortality from mountain pine beetle (Dendroctonus ponderosae). Soil cores were homogenized (organic, A, and B horizons combined), and approximately 4 g of soil per core were freeze dried and twice-ground. Genomic DNA was isolated from 250 mg of ground soil using QIAGEN DNeasy PowerSoil Kits. 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 AMPure XP 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.