ITS2 amplicon sequencing of wooly fungal pathogen positive control
Modified CTAB DNA Extraction; ITS2 metabarcode sequencing using Illumina dual-indexed primers fITS7 and ITS4; Amplified in triplicate 15 l reactions: 0.4 mM MgSO4, 0.16 g/L BSA (New England BioLabs Inc.), 50 M each dNTP, 0.2 M of nonbarcoded primers, and 1.25 units of AccuPrime polymerase; PCR conditions: 2 min at 95 C, 30 cycles of 95 C for 15 s, 55 C for 1 min, and 68 C for 1 min, followed by final extension at 68 C for 5 min; PCR products (250-400bp) were concentration-normalized then pooled using the epMotion 3075 liquid handling robot; pools were cleaned using 0.8x Mag-Bind RXNPure plus beads (OMEGA) according to the manufacturers protocol.