ITS-MiSeq of agricultural soil metagenome
Total soil DNA was extracted from 0.25 g lyophilized soil using the Qiagen DNeasy PowerSoil kit and checked for quality (260:280 > 1.75; 260:230  > 1.70) on a spectrophotometer (DS-11 FX+, DeNovix, Wilmington, DE, USA). Quantity of DNA per sample was determined using Invitrogen PicoGreen Quanti-IT dsDNA assay kits (ThermoFisher Scientific, Waltham, MA, USA). Equimolar equivalents of DNA were used as templates for ITS region amplicon libraries. Fungal ITS regions were targeted using primers ITS1f-ITS2 (Bokulich and Mills, 2013). Reactions utilized Platinum Hot Start high fidelity PCR master mix (ThermoFisher Scientific, Waltham, MA, USA), and were pooled in equimolar concentrations prior to running 2 x 250 bp paired-end sequencing on the Illumina MiSeq platform. ITS sequencing was performed at the University of California, Davis DNA Technologies Core laboratory.