title: Fungal ITS + LSU PacBio amplicons from soil, litter, root, and wood samples
design: Total genomic DNA was extracted using different methods across the four substrates: the litter and soil samples with PowerSoil kits (MoBio, Carlsbad, CA, USA), the wood samples with the PowerPlant Pro kit (MO-BIO laboratories, Carlsbad, CA, USA, and the root samples with a standard CTAB chloroform extraction as outlined in Kennedy et al. (2003). For the three kit-based extractions, manufacturer's instructions were followed with no modifications. The starting material for each sample was as follows: 250 mg litter, soil, and wood and 20 mg of fine root material. All samples were pre-homogenized via grinding and/or bead-beating. Negative controls for each sample type were also extracted. For the PacBio sequencing, the full-length ITS gene region plus a significant portion of the 28S LSU gene (~750 bp) was amplified using the primers ITS1F and TW13 (Taylor and Bruns 1999) and the protocols provided by Pacific Biosciences (http://www.pacb.com/wp-content/uploads/Unsupported-Full-Length-16S-Amplification-SMRTbell-LibraryPreparation-and-Sequencing.pdf). Based on preliminary tests, the forward ITS1F primer was modified by adding a thiol bond between the third and fourth base pairs on the 3' end to prevent any primer editing activity of the KAPA polymerase (KAPA Biosystems, Sigma-Aldrich, St. Louis, MO, USA). PacBio PCR reagents and conditions were as follows: 2 μl template DNA, 15 μl nuclease-free water, 5 μl 5x KAPA HiFi buffer (Kapa Biosystems, Woburn, MA), 0.75 μl 10 mM dNTPs, 0.75 μl KAPA HiFi Polymerase, 0.75 μl forward primer (10 μM), 0.75 μl reverse primer (10 μM). PCR cycling conditions included 95°C for 30 seconds, followed by 27 cycles of 95°C for 20 seconds, 55°C for 30 seconds, and 72°C for 1 minute. After confirming amplification via gel electrophoresis, samples were cleaned using the Agencourt AMPure XMP kit (Beckman Coulter Life Sciences, Indianapolis, IN, USA) for PacBio sequencing. Following cleaning, samples were added in equal concentration to respective libraries for sequencing at the University of Minnesota Genomic Center. The PacBio sequencing was done using Magbead loading on the Sequel platform.