454 seq of fungal LSU: soil
DNA was diluted by five fold in sterile water prior to PCR amplification. The fungal large subunit (LSU; 28S rRNA gene) region was amplified using the primers of LR0R (ACCCGCTGAACTTAAGC, Cubeta et al., 1991) and LR3 (CCGTGTTTCAAGACGGG, R. Vilgalys Website: http://sites.biology.duke.edu/fungi/mycolab/primers.htm) linked with adaptors A (5-CCATCTCATCCCTGCGTGTCTCCGAC-3) and B (5-CCTATCCCCTGTGTGCCTTGGCAGTC-3), respectively. A 10-base barcode (MID) sequence was inserted between adaptor A and the LR0R primer (Table S1). The 25 _L PCR cocktail consisted of 2.5 _L of 10_ buffer, 1.5 _L of Mg2+ (50 mM), 4.0 _L of dNTP mixture (each 2.5 mM), 1.0 _L of each primer (10 _M), 2.5 U BioTaq DNA polymerase (Bioline, London, UK), and 1.0 _L (c. 5 ng) of template DNA. Thermal cycling conditions were as follows: 95C for 6 min, 34 cycles of 95C for 40 s, 55C for 30 s and 72C for 1 min, followed by 72C for 10 min. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, San Luis Obispo, CA, USA), and quantified using the Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). A total of 50 ng of DNA from each sample was pooled and adjusted to 10 ng L-1 as required for pyrosequencing on a Roche Junior Genome Sequencer System (454 Life Sciences, Branford, CT, USA) using Titanium 454 chemistry by the Western Sydney University Next Generation Sequencing Facility.