Environmental metagenomics with next generation sequencing

Agassiz Lacombe Saskatoon Morden Harrow Woodslee Forest Ridgetown St. Thomas Caledonia Toronto Morrisburg Ottawa St. Clotilde Beloeil Harrington, PEI SAGES and Soybean Rust Sampling Network 350 air and rain samples processed 60M DNA barcode sequences (bacteria & fungi)

Types of Samplers in the Environmental Sampling Network JB: rain is filtered directly in the field through a filter (0.45uM or 8uM) YE: rainwater is collected in a bucket, vacuum-filtered in the lab through a 0.45 µm filter BK: air samples collected directly into a sterile tube

Matching barcode sequences from environment to references Environmental sample NGS DNA sequencers Biological Collections 4 DNA Gaps in collections and taxonomy

Samplers in different locations ( *) ProvinceCity Sampler Air Passive Rain Active Rain PEIHarrington_PEIBKJBYE QCSainteClotilde_QCBKJBYE ON Woodslee_ONJB Ottawa_ONBK YE/LD Ridgetown_ONBK YE/LD MBMorden_MBBK YE SKSaskatoon_SKJB ABLacombe_ABJB BCAgassiz_BCBKJBYE -classification-les-agaricales.aspx JBBKYE_LD * Collector running for one week. DNA sequenced every 4 week. 5 samples processed per summer also available, but less sites.

-classification-les-agaricales.aspx JBBKYE_LD Sampler efficiency in true Fungi total number of sequences on left and ratio between 3 sites

-classification-les-agaricales.aspx Sampler efficiency in true Fungi - total number of sequences on left and ratio between 3 sites -classification-les-agaricales.aspx JBBKYE_LD CitySampler Total number of sequence AgassizBK AgassizJB AgassizYE_LD HarringtonBK HarringtonJB HarringtonYE_LD SainteClotildeBK SainteClotildeJB SainteClotildeYE_LD

Numbers of unique fungal species represented in AAFC national herbarium (DAOM) & in GenBank (NCBI) with at least one ITS sequence Downy Mildews Rusts Powdery Mildews

GENOMICS R&D INITIATIVE Quarantine and Invasive Species Project PATRICE BOUCHARD AND ANDRÉ LÉVESQUE Agriculture and Agri-Food Canada November 21, 2012 CFIA Plant Business Line Committee meeting

Scope Building targeted reference libraries of genetic data (Barcodes) to aquatic and terrestrial organisms of greatest concern to Canada Libraries will ultimately be made available to the national and international scientific community Develop focused metagenomic approaches for the direct detection of invasive species from complex environmental samples Finfish (2.1) Tunicates (2.2) Parasites (2.3) Arthropods (3.1) Nematodes (3.2) Fungi (3.3) Plants (3.5) Viruses (3.4) Sequencing reactions DNA extraction RNA/DNA whole genomes PCR amplify with universal markers (e.g. 16S, ITS, cpn60, COI, matK) Sanger sequencing GRDI Barcode of Life Database (BOLD) BOLD at Guelph, iBOL and

Distribution of species of associated with agriculture primarily found in each country

Number of species shared with Eurasia is lower than among our three countries and impact of introduction is higher between continents

Average count of each OTU (Fungi) comparing imported and Canadian grains Each dot represents a known or putative new species Sample numbers: Imported from Asia = 8; Canadian samples = 130 Canadian grain samples Grain samples imported from Asia Total of 32,307 OTU’s PRELIMINARY RESULTS OTU’s absent in Canada: Potential alien risk for NA crops if becomes established OTU’s present in Canada but absent in imports tested so far: Potential risk for NA trade Present both in Canada and in imports – low risk

Global Initiative

Unknown sweet potato viruses in “clean” plants – The first reality check about potential trade impact

Thank you Native species Trichalophus granicollis Regulated pest Otiorhynchus ligustici Photos from Patrice Bouchard, AAFC Ottawa, Coordinator of GRDI project