Research project Exploring practical applications of metabarcoding with MinION to support the surveillance of plant pathogens

In progress METAMINSURV 01/09/2022 → 28/02/2025

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Annelies Haegeman

ILVO researcher

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General introduction

METAMINSURV evaluates the feasibility of MinION metabarcoding as a fast and accurate diagnostic tool for pathogen detection and identification. For several years, the world of metagenomic DNA and RNA sequencing has been accelerating and becoming more accessible. For example, very fast so-called nanopore sequencing relies on analyzing rather long strands of DNA through a very small hole called a nanopore. With that technique, UK researchers have developed a portable DNA sequencing device (MinION) that can already provide reliable real-time diagnostic analysis for certain (human) diseases. The aim of this project is to test the potential of MinION using two cases: 1) bacteria located in the tissue type of the bark of a tree, specifically Candidatus Phytoplasma and Candidatus Liberibacter species and 2) pathogenic fungi in forests in complex communities of different origins (seeds of conifers and spore traps). Numerous issues surrounding the metabarcoding process are now being clarified and finalized.

Research approach

Within both case studies, ILVO answers the following specific research questions: Which barcode(s), primer(s) and reference database(s) combinations are most appropriate? What is the sensitivity and specificity of MinION metabarcoding as a function of detection and identification of pathogens of interest while varying 4 groups of parameters? Those parameters are: 1) the type of samples (mock communities, spiked samples, real samples); 2) different library preparation protocols and sequence depths; 3) different (combinations of multiplexed) barcodes and/or primers and 4) different bioinformatics pipelines and/or reference databases. What are the advantages and limitations of using MinION metabarcoding for the detection and identification of plant pathogens (i.e., what is the sensitivity, specificity, speed, cost)? Finally, how useful are the developed protocols for other barcode/organism combinations?

Relevance/Valorization

The main outcomes of METAMINSURV will be protocols and accompanying bioinformatics workflows to detect and identify plant pathogens (phloem bacteria / fungi) using MinION metabarcoding. Since the project will use mock communities, spike-ins and real samples on different case studies, and will benchmark with alternative methodologies, it will reveal the potential of MinION metabarcoding for routine plant pathogen diagnostics and surveillance. Research results will be disseminated through scientific publications in peer-reviewed journals, and protocols and bioinformatics pipelines will be shared with the authorities (NPPO (FPS Public Health, FAFSC) and Flemish Government) and the scientific community. By developing general guidelines for the optimization of MinION metabarcoding protocols and analysis pipelines, we expect a spill-over effect to other plant pathogens, in particular for the detection of multiple (closely related) pathogens at once.