This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics".
You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments.
In brief, we mapped the long-ITS reads against two databases using minimap2 with a winner takes all approach only reporting the best hit in the databases for each read. Not all reads might have a hit in the database as we learned that the primers also amplify plant and human DNA.
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