title: ITS amplicon: apple twigs
design: DNA was extracted with the MagAttract PowerSoil DNA extraction kit (Qiagen) according to the manufacturers protocol, DNA solution was diluted 1:1 and the fungal ITS2 region was amplified using primers ITS2KYO2F (5-GATGAAGAACGYAGYRAA-3) and ITS4R (5-TCCTCCGCTTATTGATATGC-3) (White et al. 1990) tagged with Illumina adapters in PCR reactions using a Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). The 25 amplification cycles were as follows: A denaturation step of 30 sec at 98C followed by 25 amplification cycles of 30 sec 98C, 30 sec 50C, 30 sec 72C and a final elongation step of 2 min at 72C.