Metabarcoding of fungal ITS2: Willow_Girdled5
DNA was extracted from a 50 mg sub-sample using the NucleoSpin Soil Kit (Macherey-Nagel, Dren, Germany). ITS2 markers were amplified by PCR using the fungal gITS7 forward (Ihrmark et al., 2012) and reverse primer mix of 3/4 of ITS4 (for general eukaryotes) and 1/4 of ITS4arc (Aadapted for Archaeorhizomycetes (Sterkenburg et al. 2018)) with minimal cycle numbers (23-25) in order to minimise biases in the community data. PCR reactions were run in duplicates per sample. Pooled duplicate The duplicate PCR products from each sample were pooled and purified using AMPure magnetic beads (Beckman Coulter, Carlsbad, CA, USA), concentrations measured with Qubit (Invitrogen, Carlsbad, CA, USA) and equal DNA amounts mixed into a single pool. The pool was further cleaned using the E.Z.N.A CyclePure kit (Omega, Nocross, GA, USA) and sequenced with the Pacific Biosciences SMRT Sequel  technology after adaptor ligation by SciLifeLab NGI (Uppsala, Sweden).
W5G2018