454 GS FLX+ sequencing
Fungal intergenic transcribed spacer region of  rDNA gene, ITS275
triplicate PCR reaction (ITS1F/ITS4) final volume 50ul containing 1xGoTaqGreen Mastermix (Promega), 25pmol of each primer, 10ng DNA; initial activation 95C for 5 min, 10 cycles touchdown PCR 94C 30s+60C (-1C) 45s+72C 2min, 30 cycles  94C 30s+50C 45s+72C 2min, final extension 72C 10 min,QIAquick Gel Extraction Kit (Qiagen), Quant-iT PicoGreen dsDNA Assay Kit, equimolar pooling of samples, sequencing from gene end, trimming to 350bp containing ITS2 region, normalization, cd-hit clustering, GAST taxonomic assignment, uchime chimera check