title: Fungal metagenomic DNA sequencing of first round PCR amplicons produced from the internal transcribed spacer 2 (ITS2) region of the ribosomal RNA (rRNA) gene in fungi:  Template DNA derived from root of a seedling whose seed was collected under mother tree FM7 and grown in soil containing an inoculum of forest soil from under mother tree FM7
design: Immediately after soil removal by washing with water, seedling roots were placed in sterile plastic tubes and flash frozen in liquid nitrogen.  They were then stored at -80 degrees C until shipped in a liquid nitrogen dry shipping container to the Marden Lab Penn State where they were transferred without thawing to a -80 freezer for storage.  In the Marden Lab, roots were ground under liquid nitrogen to a powder in a porcelain mortar and pestle.  The powder was transferred under liquid nitrogen to a sterile plastic tube and then without thawing to a -80 freezer for storage.  An aliquote of about 50 mg of sample powder was transferred without thawing to a sterile nuclease free 1.7 ml microcentrifuge tubes.  DNA in the sample aliquote was isolated using the Machery-Nagel NucleoSpin Plant II Mini Kit and subsequently cleaned using the Machery-Nagel NucleoSpin gDNA Clean-up Kit.  Isolated DNA was stored at -20 degrees C.  This DNA was used to produce amplicons from the fungal ITS2 region in a simplex first round of PCR using Phusion Hot Start II High Fidelity Polymerase and primer annealing at 50 degrees C.  Primers were ITS9 (5-GAACGCAGCRAAIIGYGA-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) that had a portion of the respective Illumina universal adapter followed by a heterogeneity spacer attached to the 5end. Amplicons were cleaned using Agencourt AMPure XP beads cleaned.  First round amplicons were supplied to the The Ohio State University, Molecular and Cellular Imaging Center (MCIC) at Wooster, OH who performed second round PCR (Phusion Hot Start II High Fidelity Polymerase, 8 cycles, primer annealing at 50 degrees C) to add the Illumina sequencing adapters and tag each sample with a unique combination of Nextera duel indices.  Agencourt AMPure XP bead cleaned products were quantified using the Qubit dsDNA Broad Range Assay Kit according to the manufacturers protocol and pooled at equimolar ratios.  These pools were further purified using the Sage Science Pippin Prep Size selection system to remove primer dimers, requantified with the Qubit dsDNA Broad Range Assay Kit, and sequenced.  Sequencing at the MCIC was performed on an Illumina MiSeq running the MiSeq Reagent Kit v3, 600 cycles and a final library concentration of 14.8 pM spiked with PhiX at 5%.