ITS amplicon metagenomics of Panicum virgatum leaf tissue
Fungal DNA was extracted from 40-50mg leaf and root material using the Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics, Lebanon, New Jersey, USA) and from 250mg soil material using the DNeasy PowerSoil Kit (Qiagen, Germantown, MD, USA). Amplicons to identify fungal taxa were generated using the primer set 5.8S_FUN (5'-AACTTYRRCAAYGGATCWCT-3') and ITS4_FUN (5'-AGCCTCCGCTTATTGATATGCTTAART-3') (Taylor et al 2016) with Illumina adapters. We also included a custom peptide nucleic acid (PNA) sequence (5'-CAGAATCCCGCGAACC-3') to block non-target amplification of host DNA (Supplementary Information: Custom PNA development, Figure S1). For root and leaf samples, the following PCR conditions were used to generate fungal amplicons: 12.5 uL of 2X KAPA Taq Ready Mix (Roche, Pleasanton, CA, USA), 0.625 uL of 20 mg/mL bovine serum albumin, 1 uL of 5mM forward primer, 1 uL of 5 mM reverse primer, 2.5 uL of 15 uM PNA, 25 ng of DNA template, and 2.375 uL water. For soils, PNA addition was replaced with an equal volume of water. Amplicons were then purified of fragments < 50 bp using the Agencourt AMpure XP system at sample to bead ratio of 1:1.8 (Beckman Coulter, Indianapolis, Indiana, USA) and genomic libraries were prepared using the Nextera XT Index Kit (Illumina, San Diego, CA, USA). Paired-end (2 x 250 bases) sequencing was performed on the Illumina MiSeq v2 platform in the Genomic Sciences Laboratory at North Carolina State University (Raleigh, NC, USA).