Fungal ITS2 metabarcoding of Potorous longipes (long-footed potoroo) scat specimens using Illumina NextSeq (control sample)
Empty wells were sequenced to provide negative sequencing controls. 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).