title: Fluid ITS
design: 25 ng of DNA was used to amplify  ITS2 hypervariable regions using 100 nm final concentration of ITS3mix and ITS4ngs primers that have partial Illumina sequencing adapters as overhangs and KAPA HiFi HotStart Ready Mix. The PCR involved an initial denaturation of 95C for 5 min followed by 25 cycles of 95C for 30s, 55C for 45s and 72C for 30s and a final extension at 72C for 7 min. The amplicons were purified using Ampure beads to remove unused primers. Additional 8 cycles of PCR was performed using Illumina barcoded adapters to prepare the sequencing libraries