title: Illumina MiSeq of rhizosphere fungi
design: A two-step PCR approach using frameshifting nucleotide primers was used for sequencing with barcode tagged reverse primers. Primers for bacterial PCRs included 8 forward and 6 reverse 515F/806R primers for the V4 region and 11 forward and 6 reverse primers for fungal ITS2 at equal molar concentrations (0.5 mM). Thermal cycler conditions for primary PCRs consisted of 5 cycles at 95C for 1 min, 50C for 2 min, and 72C for 1 min. Secondary PCRs consisted of denaturation at 95C for 45 s followed by 32 cycles of 95C for 15 s, 60C for 30 s, 72C for 30 s, and final extension at 72C for 30 s. Experimental units were pooled based on gel band intensity and then purified using Agencourt AMPure XP beads system (0.7:1 ratio; Beckman Coulter Inc., Pasadena, CA).