ITS amplicon metagenomics of corn, soy, switchgrass, and wheat
DNA was extracted from leaf fragments (up to 54 mg) using the Synergy 2.0 Plant DNA Kit (OPS Diagnostics LLC, Lebanon, NJ). Gene amplification of 20 ng DNA template was performed using duplicate reactions with the ITS1F (5 CTTGGTCATTTAGAGGAAGTAA 3) and ITS2 (5 GCTGCGTTCTTCATCGATGC 3) primers modified for Illumina. Technical replicates were pooled, cleaned with AmPure XP beads, and quantified. Equimolar samples were then pooled for 2x300 PE sequencing on Illumina MiSeq v3 at the NCSU Genome Sequencing Lab.