Sequencing bacterial and fungal metabarcode with PCR primer based barcoding and Nanopore v1
Authors: Graetz Abigail , Jones Ashley , 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 describes the molecular biology and a step-by-step guide for PCR barcoding of the 16S and ITS regions using primers that contain barcodes, followed by ligation based amplicon sequencing with Nanopore. The initial steps of repeated PCR with high-fidelity polymerase and purification of the PCR amplicons has been performed by your demonstrator due to time limitations. We selected DNA samples of three research groups in week 5 so that we have three replicates per treatment group. This gives us 2x (16S and ITS) 15 PCR reactions plus 1 negative control. We skip the extraction control for this practical mini-research project. This is step 1 of the protocol. The three groups selected in week 5 will collaborate to perform the library preparation on the two PCR barcoded and equimolar pooled amplicons (16S and ITS). Most of the class need not be present for this library preparation [Step 2] 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: Repeat PCR reactions on the samples of three research groups using primers containing barcodes in them (see below). Clean and pool amplicons at equimolar ratios. End-prep the pooled amplicons to make them ready to ligate sequencing adapters. Ligate sequencing adaptars with DNA ligase. Make amplicon library ready for loading. 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 :). Primers used: We used ITS and 16S primers that directly include DNA barcodes as follows, where N15-16 represents a unique barcode sequence ITSFor (a.k.a. ITS1-F_KYO2_BARCODE): 5'-N15-16-TAGAGGAASTAAAAGTCGTAA-3' ITSRev (a.k.a.LR6_BARCODE): 5'-N15-16-CGCCAGTTCTGCTTACC-3' 16SFor (a.k.a. 27F_BARCODE): 5'-N15-16-AGAGTTTGATCCTGGCTCAG-3' 16SRev (a.k.a. 1492R_BARCODE): 5'-N15-16-ACCTTGTTACGACTT-3' References: 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 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) Hebert, P.D.N., Floyd, R., Jafarpour, S., Prosser, S.W.J., 2023. Barcode 100K Specimens: In aSingle Nanopore Run, BioRxiv, doi: https://doi.org/10.1101/2023.11.29.569282