ITS2 amplicon sequencing of mock fungal community: pure cultivar fungus DNA
Modified CTAB DNA Extraction; ITS2 metabarcode sequencing using Illumina dual-indexed primers fITS7 and ITS4; Samples were amplified in triplicate 15 l reactions: Go-Taq DNA polymerase (Promega), 3.3 g bovine serum albumin (New England BioLabs), 0.1 pmol of non-indexed primers, 95 C for 2 minutes, then 5 cycles of 30 s at 95.0C, 60 s at 48.0C and 60 s at 72.0C, then 25 cycles of 30 s at 95.0C, 60 s at 55.0C and 60 s at 72.0C followed by final extension as 72.0C for 10 minutes; PCR products were normalized based on the concentration of DNA from 250-400 bp then pooled using the epMotion 3075 liquid handling robot, then cleaned using 0.8x Omega Bio-Tek Mag-Bind Beads according to the manufacturers protocol.