Soil fungal ITS sequences Little Desert NP, Australia
DNA was extracted from 200 mg of soil samples using a DNeasy Powersoil Pro kit (Qiagen, GmBH, Hilden, Germany) according to the manufacturers instructions. Sequencing was processed through the Western Sydney Universitys Next-Generation Sequencing Facilitys (Richmond, NSW, Australia) liquid handling pipeline using in-house optimised protocols. The genomic DNA was purified using the Agencourt AMPure XP Beads (Beckman Coulter), followed by a quality assessment using the Qunat-iT  PicoGreen fluorescence-based analysis (ThermoFisher Scientific, North Ryde, NSW, Australia). The purified DNA then underwent amplification using polymerase chain reaction (PCR) targeting the internal transcribed spacer (ITS) region 2 (Blaalid et al., 2013) using the primer set FITS7/ITS4 (Ihrmark et al., 2012). Briefly, PCR reactions consisted of 5l Q5 High-Fidelity DNA Polymerase 2 x master mix (New England Biolabs, Notting Hill, Victoria, Australia), 0.2l of 10 M forward and reverse primer with 2l of DNA, total volume of the reaction was 10l. PCR reactions were as described in Caporaso et al. (2018). The amplified PCR product then underwent a short second PCR to attach the Illumina Nextera XT v2 index set (Illumina Australia, Melbourne, Australia), as per manufacturers instructions (https://support.illumina.com).  Each reaction consisted of 3.8l of Q5 High-Fidelity DNA Polymerase 2x master mix (NEB), 1.5l of each index (Illumina) and 2.3l of PCR product, total reaction volume was 7.6l. The amplicons were then diluted and again assessed using the Qunat-iT  PicoGreen (ThermoFisher Scientific) assay and normalised library pools were constructed using the Eppendorf epMotion.  The libraries were cleaned-up and prepared for sequencing following the Illumina MiSeq protocol. Sequencing was performed on the Illumina MiSeq platform using the Illumina MiSeq reagent kit v3 2x 300bps paired-end chemistry as per manufacturers instructions.