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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 total, each group will obtain five pots each: This specific protocol describes the molecular biology and a step-by-step guide for rapid barcoding of total native DNA extracted from the five treatment groups followed by Nanopore sequencing. We will perform sequencing of all five native DNA samples from three research groups to enable us to have three independent replicates per sample. Replications makes our observation and analyses more robust. Total native DNA contains all the DNA extracted from your leaf material plus any contamination that was part of the extraction process. We did not include a negative extraction control this time round. We will select the samples of three research groups based on their quality control values such as DNA concentration and DNA size measured by gel electrophoresis. Most of the class need not be present for these library preparations but will join in for loading the final library and starting sequencing runs. We will also explain more about the theory during the lab. This protocol is applicable for week 6. Conceptual overview: Bring all DNA samples to same concentration and same volume. Barcode individual samples with transposas loaded with specific DNA barcodes. Pool all samples and clean up with magnetic beads. Add sequencing adapters with click chemistry applied to the barcoded and pooled DNA samples. Prepare for loading onto the flowcell. Prime flowcell to make it ready for loading. Load library. Start of sequencing run. Basecall during or after the sequencing run. You can cite this protocol in the methods section of your report as for all other protocols. No need to write it all up again :).
Singh et al. (Sat,) studied this question.