Fusarium EF1alpha mock community: experiment 1 10 species single replicate 1: Sample Fusarium10_single
"Amplicons of the Fusarium transcription elongation factor (EF-1?) were generated from DNA mock communities of either 10% of ten Fusarium species, 90% F. oxysporum and 10% F. graminearum or 90% F. graminearum and 10% F. oxysporum. Mock communities were amplified either with a nested PCR consisting of primer pairs EF1 and EF2 (O’Donnell et al. 1998, DOI: 10.2307/3761407) and Fa and Ra (Edel-Hermann et al 2015, DOI: 10.1016/j.mimet.2015.01.026) or a single PCR (ten-species community) with only Fa/Ra (ten- and two-species communities). Mock communities were run in three individually tagged PCR replicates. PCR with EF1/EF2 was run in 25 µl reactions with 0.8 ng/µl template, 150 µM of each nucleotide, 1.5 mM MgCl2, 400 nM of each primer and 0.12 U/µl polymerase (HotStarTaq Plus, QIAGEN, Germany) in PCR buffer. PCR conditions were 5 min at 95°C, 30 cycles of [1 min at 95°C, 75 s at 57°C, 1 min at 72°C] and 10 min at 72°C. PCR with Fa and Ra primers was run in 25 µl  reactions with 1 µl PCR product or 0.8 ng/µl template, 150 µM of each nucleotide, 1.5 mM MgCl2, 200 nM of each primer and 0.12 U/µl polymerase in PCR buffer. PCR conditions were 5 min at 95°C, 40 cycles of [1 min at 94°C, 1 min at 67°C, 1 min at 72°C] and 10 min at 72°C. The Fa primer was tagged with a 9 bp sample identifying tag. PCR products were cleaned using AMPure (Beckman Coulter, CA, USA). DNA concentration was measured on a NanoDrop 1000 spectrophotometer (Thermo Scientific, MA, USA). The sample pool was sent to LGC Genomics (Germany) for adaptor ligation and sequencing on 1/16th of a plate on a GS FLX Titanium sequencer (Roche, Switzerland). "