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Next Generation Sequencing, Direct Detection and Genotyping of Fungi, Bacteria and Nematodes in the Agri-Food System

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Next Generation Sequencing, Direct Detection and Genotyping of Fungi, Bacteria and Nematodes in the Agri-Food System Next generation sequencing, direct detection and genotyping of fungi, bacteria and nematodes in the agri-food system Prepared by: André Lévesque, et al. Agriculture and Agri-Food Canada K.W. Neatby Bldg Ottawa, ON K1Y 4X2 Contract Project Number: CRTI 09-462RD CSA: Nezih Mrad, Portfolio Manager Chem/Bio, DKTS, Centre for Security Science The scientific or technical validity of this Contract Report is entirely the responsibility of the Contractor and the contents do not necessarily have the approval or endorsement of the Department of National Defence of Canada. Contract Report DRDC-RDDC-2015-C148 April 2015 IMPORTANT INFORMATIVE STATEMENTS Next generation sequencing, direct detection and genotyping of fungi, bacteria and nematodes in the agri- food system (CRTI 09-462RD) was supported by the Canadian Safety and Security Program which is led by Defence Research and Development Canada’s Centre for Security Science, in partnership with Public Safety Canada. The project was led by Agriculture & AgriFood Canada in partnership with CFIA Charlottetown, CFIA Ottawa, Canadian Grain Commission, AAFC Lethbridge, AAFC Saskatoon, AAFC London. Canadian Safety and Security Program is a federally-funded program to strengthen Canada’s ability to anticipate, prevent/mitigate, prepare for, respond to, and recover from natural disasters, serious accidents, crime and terrorism through the convergence of science and technology with policy, operations and intelligence. © Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2015 © Sa Majesté la Reine (en droit du Canada), telle que représentée par le ministre de la Défense nationale, 2015 CRTI 09-462RD / CSSP 30vv01 Agri-food pathogen detection and next-gen sequencing CRTI 09-462RD CSSP 30vv01 Next generation sequencing, direct detection and genotyping of fungi, bacteria and nematodes in the agri-food system Final - 30 April 2015 Led by Defence Research and Development Canada - Centre for Security Science page 1 CRTI 09-462RD / CSSP 30vv01 Agri-food pathogen detection and next-gen sequencing Co-authors André Lévesque, Project Manager, Research Scientist, AAFC Ottawa Wen Chen, Deputy Project Manager, Research Scientist, AAFC Ottawa Sarah Hambleton, Research Scientist (Mycology: cereal rusts, smuts and bunts), AAFC Ottawa Keith Seifert, Research Scientist (Mycology: Penicillium), AAFC Ottawa Zaky Adam, post doctoral fellow (Bacteriology: plant pathogens), AAFC Ottawa Guillaume Bilodeau, Research Scientist, Ottawa Plant Laboratory (Fallowfield), CFIA, Ottawa Jeff Cullis, Bioinformatics Programmer, AAFC Ottawa Tigst Demeke, Research Scientist- Molecular Biology, Grain Research Laboratory, CGC Winnipeg Tim Dumonceaux, Research Scientist (Bacteriology: Endophytes of plants), AAFC Saskatoon Marie-Claude Gagnon, post doctoral fellow, Ottawa Plant Laboratory (Fallowfield), CFIA, Ottawa Tom Graefenhan, Research Scientist-Mycologist, Grain Research Laboratory, CGC Winnipeg Larry Kawchuk, Research Scientist (Mycology: potato pathogens), AAFC Lethbridge Izhar Khan, Research Scientist (Bacteriology: human/animal pathogens), AAFC, Ottawa, , Christopher Lewis, Chief Bioinformatician for Biodiversity, AAFC Ottawa Xiang Li, Research Scientist, Charlottetown Laboratory - Plant Health, CFIA, Charlottetown Matthew Links, Biologist (Molecular Bioinformatics), AAFC Saskatoon Benjamin Mimee, Research Scientist (nematology), AAFC Saint Jean sur Richelieu Mike Rott, Research Scientist, Sidney Laboratory - Plant Health, CFIA, Saanich James Tambong, Research Scientist (Bacteriology: plant pathogens), AAFC Ottawa Ed Topp, Research Scientist (Bacteriology: human/animal pathogens), AAFC London Qin Yu, Research Scientist (Nematology: plant pathogens), AAFC Ottawa Kat Yuan, post doctoral fellow, Charlottetown Laboratory - Plant Health, CFIA, Charlottetown © Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, © Sa Majesté la Reine (en droit du Canada), telle que représentée par le ministre de la Défense nationale, page 2 CRTI 09-462RD / CSSP 30vv01 Agri-food pathogen detection and next-gen sequencing Abstract …….. This project engaged Agriculture and Agri-Food Canada (AAFC) and first responders, the Canadian Food Inspection Agency (CFIA) and the Canadian Grain Commission (CGC) to deliver on a collection of sub- projects unified by the technology of Next Generation Sequencing (NGS). The objectives focused on developing innovative methods to counter threats from fungal, bacterial and nematode pathogens. The outputs covered the full spectrum of Technology Readiness Levels (TRL). For example, de novo sequencing was accomplished for some high risk microbes for which there were no genomics data available (TRL1) and some tests developed from the genomics data were used by Canadian first responders to resolve trade disputes, and were either used or are currently being evaluated by first responders in other countries (>TRL7). The genomes and transcriptomes of high risk plant and animal / human pathogens found on crop commodities and in agro-ecosystems were sequenced. DNA and RNA from multiple strains of high profile pathogenic species were processed for NGS from the following genera: Arcobacter, Ditylenchus, Globodera, Pantoea, Pectobacterium, Penicillium, Phytophthora, Puccinia, Ralstonia, Streptomyces, Synchytrium, and Tilletia. Reductions in sequencing costs over the time span of the project and an in-kind contribution for genomics and for data analysis infrastructure installed mid-project, allowed us to increase the number of strains processed for full genome sequencing to more than four times our original commitment. We also annotated the genomes of additional species that were not originally planned. Quantitative PCR, single nucleotide polymorphism (SNP) detection and microsatellite-based assays were developed for these targets. The threat from a few targeted species, namely, Ralstonia solanacearum race 3 biovar 2 (brown rot of potato), Synchytrium endobioticum (potato wart), Globodera pallida (the pale cyst nematode of potato), G. rostochiensis (the golden nematode of potato), and Ditylenchus destructor (the potato rot nematode), increased nationally during the project. Therefore, some diagnostic technologies were transferred to end users and made operational much earlier than originally planned. The project also generated universal 'DNA barcode' sequences from environmental samples using NGS. Automated bioinformatics pipelines were developed and improved for the processing of next generation genomic and metagenomic data, an added deliverable leading to higher assembly efficiency than originally expected. A total of 322 environmental samples, 30% more than planned, from cereal grains, soils from potato fields and agricultural watersheds were processed to validate the NGS approach. This project generated comprehensive sequence databases, providing an up- to-date inventory for high risk plant pathogens. It also delivered state-of-the-art diagnostic technologies and routine monitoring strategies to improve the safety and security of the Canadian food and agriculture system, to counter challenges to international trade regulations, and to benefit the agriculture industry and communities in Canada. page 3 CRTI 09-462RD / CSSP 30vv01 Agri-food pathogen detection and next-gen sequencing Résumé …..... Ce projet a amené Agriculture et Agroalimentaire Canada (AAC) et les premiers intervenants, soit l’Agence canadienne d’inspection des aliments (ACIA) et la Commission canadienne des grains (CCG), à réaliser une série de sous-projets reposant sur la technologie du séquençage de nouvelle génération (SNG). Les objectifs étaient axés sur la mise au point de méthodes novatrices visant à contrer les menaces posées par des champignons, des bactéries et des nématodes pathogènes. Les résultats obtenus couvrent tous les niveaux de maturité technologique (NMT). Ainsi, un séquençage de novo a été réalisé pour des microorganismes à risque élevé pour lesquels on ne disposait d’aucune donnée génomique (NMT 1). Par ailleurs, des essais mis au point à partir des données génomiques obtenues ont été utilisés par des premiers intervenants canadiens afin de résoudre des différends commerciaux, et ont été utilisés ou sont en cours d’évaluation par les premiers intervenants d’autres pays (> NMT 7). On a également séquencé les génomes et les transcriptomes d’agents pathogènes à risque élevé pour les végétaux, les animaux et les humains, trouvés dans des produits végétaux et dans des agroécosystèmes. L’ADN et l’ARN de nombreuses souches d’espèces pathogènes importantes ont été traités par SNG. Les espèces visées appartenaient aux genres suivants : Arcobacter, Ditylenchus, Globodera, Pantoea, Pectobacterium, Penicillium, Phytophthora, Puccinia, Ralstonia, Streptomyces, Synchytrium et Tilletia. Grâce à la réduction des coûts de séquençage pendant la durée du projet et à la contribution en nature liée à la génomique et au cadre d’analyse de données mis à place à mi-parcours, nous avons pu multiplier par plus de quatre le nombre de souches traitées aux fins du séquençage du génome complet par rapport à notre engagement initial. Nous avons également annoté les génomes d’espèces additionnelles qui n’étaient pas prévues au départ. Nous avons mis au point des épreuves de PCR quantitatives, de détection de SNP et de génotypage par analyse de microsatellites pour les espèces ciblées. La menace posée par certaines d’entre elles, en l’occurrence le Ralstonia solanacearum race 3 biovar 2 (pourriture
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