ITS2 metabarcoding of airborne fungi: neg. sampling control
The fungal ITS2 region was amplified using the forward primer ITS3_KYO2 (GATGAAGAACGYAGYRAA) supplemented with additional variants matching more taxonomic groups (GATGAAGRACGCMGCGAA, GATGACGAACGCAGCGAA, GATGAAGAACACAGYGAA, GATGAAGAGCGYAGCRAA, GATGAAGAACGCGGCGAA, TCGATGAAGAMCGTWGC). The reverse primer was ITS4 (TCCTCCGCTTATTGATATGC), supplemented with following variants: TCCTCCGCTTATTAATATGC, TCCTCCGCTTAVTGATATGC, TCCTCCACTTATTGATATGC, TCCTCTGCTTATTGATATGC, TCCTGCGCTTATTGATATGC, TCCTCCGCTGAWTAATATGC, TCCTCGCCTTATTGATATGC. A first amplification step using primers with Nextera XT adapters was done using the KAPA Hifi HotStart ReadyMix in four replicate reactions (4 x 20 l). 27 PCR cycles were done. The indexed amplicon was normalized to equal concentrations, pooled and sequenced on the MiSeq (Illumina) using the reagent kit v3 (600 cycles) to generate paired-end reads of 2 x 300 bp.