PacBio RS II amplicon sequencing of soil samples
PCR was performed using three forward primers ITS1ngs, ITS1Fngs (Oja et al. 2015), ITS1Oo (Riit et al. 2016) and a reverse primer ITS4ngs (Tedersoo et al. 2014). Both forward and reverse primers were tagged with one of the 93 identifiers (MIDs, 10-12 bases). All samples were amplified in duplicate using a reaction mixture consisting of 1 l DNA, 0.5 l each of the primers (20 pmol), 5 l 5xHOT FIREPol Blend Mastermix (Solis Biodyne, Tartu, Estonia) and 18 l double-distilled water in thermal cyclers. The cycling conditions for PCR amplification were as follows: initial 15 min at 95 C; 28 cycles of 30 s at 95 C, 30 s at 55 C, 1 min at 72 C; and a final cycle of 10 min at 72 C.
Eerika_soil_II