PCR reaction of marker regions (a.k.a metabarcodes) for bacteria and fungi v1
Authors: Graetz Abigail , Schwessinger Benjamin 
Published: February 2025

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 is a step by step guide to perform PCR reactions on the extracted DNA samples of the five TG. The PCR reaction target conserved marker regions (a.k.a. metabarcodes) for bacteria and fungi. These primers are designed to amplify "all" bacteria and fungi in the sample. The simplified idea of metabarcodes is that one can amplify a region that is conserved enough to generate primers that allow one to amplify this region from "all" members of the kindgom somewhat representatively. Within the amplicon though there are hypervariable DNA regions that are diagnostic to different between species or genera. Matching these amplicons against a databases let's one identify the organism of this specific kingdom present in the sample. At least this is the simple theory, however as you will see in the lectures there are many issues and biases to consider when applying these techniques in general and specifically to pathogen diagnostics for biosecurity purposes. The final goal is to achieve the following: To measure and adjust DNA concentration for each sample to allow for equal amount of DNA being added to each PCR reaction. Perform PCR reaction for the bacterial and fungal kingdom metabarcode using published 16S and ITS (Internal transcribed spacer) primers. Cross the fingers it all works well. This protocol is applicable for week 4. Protocols progress overview: Week 4 PCR reaction for 16S and ITS. You will measure double stranded DNA concentration with a dye based method. Here the dye binds to double stranded DNA only and this will change the fluorescent of the dye in a way directly proportional to the DNA concentration. You will use the pre-mixed broad range dye kit https://www.thermofisher.com/order/catalog/product/Q32853. The primers are as follows: ITS: For the ITS PCR reactions primers are called ITSFor and ITSRev. These are based on White et. al., 1989 and Ohta, Nishi, Hirota and Matsuo, 2023. Their sequences are as follows. ITSFor (a.k.a. ITS1-F_KYO2): 5'-TAGAGGAASTAAAAGTCGTAA-3' ITSRev (a.k.a.LR6): 5'-CGCCAGTTCTGCTTACC-3' The expected amplicon size is around 2 to 3 kb (kilobase pairs). 16S: For the 16S PCR reactions primers are called 16SFor and 16SRev. These are based on Wuyts et. al., 2002, Baker et. al., 2003, and Youssef et. al., 2009. 16SFor (a.k.a. 27F, E8F, or E8Fa): 5'-AGAGTTTGATCCTGGCTCAG-3' 16SRev (a.k.a. 1492R): 5'-ACCTTGTTACGACTT-3' The expected amplicon size is around 1.5 kb. Importantly, these 16S primers also partially bind and amplify 16S regions found in plant mitochondria and chloroplast. In research settings (not here in the teaching prac), the amplification of the plant 16S region can be suppressed with sequence specific probes called PNAs (Peptide mediated Nucleic Acids) https://www.pnabio.com/products/PCR_blocker.htm. We regularly would use PNAs for the 16S PCR reactions based on Lundberg et a. 2013. References: White, Bruns, Lee and Taylor, 1989, 'Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics', in PCR - Protocols and Applications - A Laboratory Manual, Academic Press Wuyts, Van de Peer, Winkelmans and De Wachter, 2002, 'The European database on small subunit ribosomal RNA', Nuc. Ac. Res., vol. 30 (1), pp. 183-185 Ohta, Nishi, Hirota and Matsuo, 2023, 'DNA metabarcoding workflow utilising nanopore long-read sequencing and consensus generation for rapid identification of fungal taxa with high phylogenetic resolution, BioRxiv, doi: https://doi.org/10.1101/2023.04.14.536971 Baker, Smith and Cowan, 2003, 'Review and re-analysis of domain-specific 16S primers', J. Microbiol. Meth., vol. 55 (3), pp. 541-555 Youssef, Sheik, Krumholz, Najar, Roe and Elshahed, 2009, 'Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys', App. Env. Microbiol., vol. 75 (16) Lundberg, Derek S., Scott Yourstone, Piotr Mieczkowski, Corbin D. Jones, and Jeffery L. Dangl. “Practical Innovations for High-Throughput Amplicon Sequencing.” Nature Methods 10, no. 10 (October 2013): 999–1002.