Fungal ITS2 metabarcoding of Potorous longipes (long-footed potoroo) scat specimens using Illumina NextSeq (control sample)
For negative extraction controls, DNA was extracted from empty PowerBead tubes using the DNeasy PowerLyzer PowerSoil kit (Qiagen) following the manufacturers instructions. One negative extraction control was used for each round of LFP scat sample DNA extractions. To maximise DNA extraction yield from tough fungal spores, we increased the lysis time with a heating step at 55C overnight, followed by tissue disruption using the TissueLyser II (Qiagen) at 30hz for 10 minutes. PCR amplification and sequencing were performed by the Australian Genome Research Facility (AGRF) in Melbourne, Australia. The rDNA ITS2 region was amplified with primers gITS7ngs/ITS4ngsUni (Tedersoo & Lindahl, 2016) using an Applied Biosystem 384 Veriti with Platinum SuperFi II mastermix (Life Technologies, Australia). Amplicon indexation was performed with Platinum SuperFi II mastermix (Life Technologies, Australia). PCR conditions were as follows: 30 cycles at 98C (30 sec), 98C (10 sec), 60C (10 sec), 72C (30 sec), and final extension at 72C (5 min). Samples were pooled at equimolar concentration and sequenced on an Illumina NextSeq 2000 (San Diego, CA, USA) with a P1 600 cycle kit (2 x 300 paired-end platform).