Amplicon sequencing of soil: Virola nobilis
Locus-specific primers used for PCR1 included the Illumina sequencing primer sequence on the 5 ends. We amplified region 1 of the internal transcribed spacer (ITS) barcode using oomycete-specific primers ITS1oo and ITS2 as described previously (Riit et al. 2016). We used Platinum 2X Mastermix (Thermo) in PCR reactions with a final volume of 12.5l. The reaction was amplified as follows: initial denaturation at 95C for 3 min, 34 cycles of denaturation at 95C for 30s, annealing at 55C for 30s, extension at 72C for 1 min, followed by final extension at 72C for 10 min. PCR2 used 2ul of PCR1 as template and added on remaining Illumina adaptors and index sequences. PCR2 products were cleaned and normalized using PCR Normalization plates (CharmBiotech, USA) and pooled libraries concentrated using AMPure beads (Beckman Coulter, USA).