DNA metabarcoding of fungi: sample 4
Fungal DNA was amplified using the forward primer fITS7 (5-GTGARTCATCGAATCTTTG-3) (Ihrmark et al. 2012) and the revers primer ITS4 (5-TCCTCSSCTTATTGATATGC-3), targeting the ITS2 region of ribosomal DNA (White et al. 1990). The forward primers were designed to include fITS7, an adapter and a barcode. PCR reactions and thermocycler program was the same as for bacteria, except that the annealing temperature was increased from 50C to 56C. Amplicons were pooled and DNA yield was quantified fluorometrically with a Qubit 3.0 fluorometer (Invitrogen, USA) and purified using AMPure XP (Beckman Coulter, USA). For preparation of the libraries (end-repair, adaptor ligation and nick repair) Ion Plus Fragment Library Kit was used (Thermo Fisher Scientific, USA) according to the manufacturers instructions. The Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific, USA) was used to quantify the libraries before pooling. The final library got further preparation by Ion Chef System before sequencing on Ion GeneStudio S5 (Thermo Fisher Scientific, USA).