Amplification targeted the internal transcribed spacer (ITS) region 1 of the nuclear ribosomal coding cistron, part of the region formally proposed as a universal DNA barcode for fungi (Schoch et al., 2012 (link)). Forward primers comprised the 454 Fusion Primer A-adaptor, a specific 8-bp multiplex identifier (MID) barcode, and the ITS1F primer (5′-CTTGGTCATTTAGAGGAAGTAA-3′ Gardes and Bruns, 1993 (link)), while the reverse primer was composed of the B-adapter and ITS2 primer (5′-GCTGCGTTCTTCATCGATGC-3′ White et al., 1990 ).
Pyrosequencing PCR mixtures contained 0.25 μl of HotStarTaq polymerase (Qiagen, Valencia, CA, USA), 2.5 μl of 10 × PCR buffer supplied by manufacturer, 2.5 μl 10 × each dNTPs (200 μℳ), 0.2 μl of 50 μℳ reverse primer, 1 μl of 10 μℳ forward primer, 0.25 μl of 100 mg ml−1 BSA, 5 μl DNA template (some samples diluted 1:10 to overcome inhibitors) and water up to 25 μl. Following an initial denaturation at 95 °C for 15 min to activate the polymerase, samples were amplified by 35 cycles of 94 °C for 1 min, 51 °C for 1 min and 72 °C for 1 min, and subjected to a final extension at 72 °C for 10 min. Samples were PCR-amplified in triplicate and pooled before cleaning using AMPure magnetic beads (Beckman Coulter Genomics, Danvers, MA, USA). Amplicon samples were then individually quantified using the Qubit flourometer (Invitrogen, Carlsbad, CA, USA) and pooled to an equimolar concentration. Sequences were run on 1/8th of a 454 FLX Titanium pico-titer plate at the Duke Institute for Genomic Sciences and Policy (Durham, NC, USA) and submitted to the National Center for Biotechnology Information Sequence Read Archive under accession number SRA059097.
Pyrosequencing PCR mixtures contained 0.25 μl of HotStarTaq polymerase (Qiagen, Valencia, CA, USA), 2.5 μl of 10 × PCR buffer supplied by manufacturer, 2.5 μl 10 × each dNTPs (200 μℳ), 0.2 μl of 50 μℳ reverse primer, 1 μl of 10 μℳ forward primer, 0.25 μl of 100 mg ml−1 BSA, 5 μl DNA template (some samples diluted 1:10 to overcome inhibitors) and water up to 25 μl. Following an initial denaturation at 95 °C for 15 min to activate the polymerase, samples were amplified by 35 cycles of 94 °C for 1 min, 51 °C for 1 min and 72 °C for 1 min, and subjected to a final extension at 72 °C for 10 min. Samples were PCR-amplified in triplicate and pooled before cleaning using AMPure magnetic beads (Beckman Coulter Genomics, Danvers, MA, USA). Amplicon samples were then individually quantified using the Qubit flourometer (Invitrogen, Carlsbad, CA, USA) and pooled to an equimolar concentration. Sequences were run on 1/8th of a 454 FLX Titanium pico-titer plate at the Duke Institute for Genomic Sciences and Policy (Durham, NC, USA) and submitted to the National Center for Biotechnology Information Sequence Read Archive under accession number SRA059097.
