16S amplicon metagenomics of Panicum virgatum leaf tissue
PCR reactions on negative controls used water instead of DNA template with the primers 515F and 806R (Caporaso et al. 2011). We also included universal peptide nucleic acid (PNA) blockers for host mitochondria and plastids (Lundberg et al. 2013). For root and leaf samples, the following PCR conditions were used to generate fungal amplicons: 1 KAPA Taq Ready Mix (Roche, Pleasanton, CA, USA), 0.2 M of each primer, 0.25 M of each PNA, 10 ng L-1 of DNA template, and water up to 25 L. For soils, PNA addition was replaced with an equal volume of water. Amplicons were 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) sequencing was performed on the Illumina MiSeq v2 platform in the Genomic Sciences Laboratory at North Carolina State University (Raleigh, NC, USA).