DNA metabarcoding of nematodes: sample 17
Nematode DNA was amplified using a nested PCR protocol. The primers NemF (5-GGGGAAGTATGGTTGCAAA-3) and 18Sr2b (5-TACAAAGGGCAGGGACGTAAT-3) were used in the first PCR. For the second PCR NF1 (5-GGGGAAGTATGGTTGCAAA-3) and 18Sr2b were used. The NF1 forward primers was designed like the bacteria and fungi forward primers with an adapter and a unique barcode. The PCR reactions were as described by Sapkota & Nicolaisen (2015).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).