title: Top-Down effect of salamanders on fungal communities
design: DNA extraction    Each sample containing both the leaf litter, wood, and the organic horizon of the soil was homogenized for 60 seconds using a Black & Decker HC306 1-1/2-Cup One-Touch Electric Chopper. The resulting particle size was approximately 1–3 mm. All components of the electric chopper were sterilized for 20 minutes in a 1:10 dilution of Clorox bleach and rinsed in molecular grade water between each sample. Total genomic DNA was isolated from 0.25 g wet weight of each sample (n = 16) using the MOBIO Power Soil DNA Extraction Kit (MOBIO Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions and stored at -20 C.    454 pyrosequencing methods    The ITS region of rDNA and partial ribosomal large subunit was amplified using the PCR primers ITS1F_KYO2 (Toju et al. 2012) and LR3 (http://biology.duke.edu/fungi/mycolab/primers.htm). Each PCR reaction consisted of 12.5 µL of HotStarTaq Master Mix (Qiagen), 1 µL (10uM) each primer, 9.5 µL sterile DIH2O, and 1 µL DNA quantified at 10-20 ng/µL using an Eppendorf Biospectrometer. Thermal cycler conditions were as follows: 95 C for 15 min, followed by 20 cycles at 94 C for 20 sec, 50 C for 30 sec, 72 C for 120 sec, and final extension at 72 C for 7 min. Each PCR product was subjected to a second round of PCR that targeted the ITS2 region using 454 fusion primers in the following configuration,  5’ - 454 Adaptor A + 8-bp multiplex tag + forward gene-specific primer - 3’ and 5’ - 454 Adaptor B + reverse gene-specific primer. Each DNA extraction/PCR reaction representative of a single organic matter sample was tagged with a unique multiplex tag (MID; n = 16). The forward gene-specific primer was ITS3_KYO2 (Toju et al. 2012) and reverse primer LR_KYO1b (Toju et al. 2013). Each PCR reaction consisted of 12.5 µL of HotStarTaq Master Mix (Qiagen), 1 µL (1µM) each primer, 9.5 µL sterile DIH2O, and 1 µL PCR product. The thermal cycler conditions were as follows: 95 C for 15 min, followed by 40 cycles at 94 C for 20 sec, 50 C for 30 sec, 72 C for 60 sec, and final extension at 72 C for 7 min. The PCR products were purified twice with AMPureXP beads, analyzed for short fragments with qPCR, and quantified with the Invitrogen Qubit Fluorometer (Invitrogen) at Oregon State University (OSU) Center for Genome Research and Biocomputing. 454 pyrosequencing was performed on a GS Junior sequencer (Roche, Basel, Switzerland) at OSU. Negative controls were run during both DNA extraction and PCR.     Sequence processing and analyses    Sequencing resulted in 141 376 ITS sequences (GenBank Short Reads Archive: SRA #). Reads were quality controlled using the PyroTrimmer software (Oh et al. 2012) under the following conditions: a minimum length of 150 bp, maximum length of 700 bp, average quality value cutoff of 20 for 3' end trimming, average quality value cutoff of 25 for full length sequences, 3 maximum mismatches for trimming primer sequences, 0 maximum ambiguous nucleotides, maximum homopolymer length of 8. Forward primers, MIDs, and reverse primers (if present) were removed before further analyses. All additional analyses were completed in CloVR-ITS (White et al. 2013), which is an automated pipeline for comparisons of ITS pyrosequencing data. Chimera detection and removal was completed in UCHIME (Edgar et al. 2011) according to White et al. (2013). All non-chimeric sequences were searched against the clovr-itsdb v1.0 curated database of ITS sequences (White et al. 2013) using BLASTN (Altschul et al. 1997) with an e-value cutoff of 1e-5. Each ITS sequence is assigned to a taxonomic group based on a minimum 90% query sequence length coverage and minimum identity of 75%, 70% and 60% for family, order, and class rank, respectively (see White et al. 2013 for justification). Five total analyses were run using the CloVR-ITS automated pipeline. The first analysis included the complete curated (PyroTrimmer) dataset consisting of 16 MIDs, which corresponds to sample from a single enclosure (n = 8) at each sampling time (Day 1, 21; n = 16). The remaining four analyses are outlined in Figure 2 and compare 4 vs. 4 enclosures at each of the two different sampling points.