Environmental metagenomics with next generation sequencing
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Environmental metagenomics with next generation sequencing. 2009-11 SAGES and Soybean Rust Sampling Network. Lacombe. Agassiz. Saskatoon. Ottawa. Beloeil. Morden. St. Clotilde. Morrisburg. Forest. Harrington, PEI. Toronto. 350 air and rain samples processed

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Environmental metagenomics with next generation sequencing
Environmental metagenomics withnext generation sequencing


2009-11 SAGES and Soybean Rust Sampling Network

Lacombe

Agassiz

Saskatoon

Ottawa

Beloeil

Morden

St. Clotilde

Morrisburg

Forest

Harrington, PEI

Toronto

350 air and rain samples processed

60M DNA barcode sequences (bacteria & fungi)

Woodslee

Caledonia

St. Thomas

Harrow

Ridgetown


Types of samplers in the 2010 11 environmental sampling network
Types of Samplers in the 2010-11 Environmental Sampling Network

JB: rain is filtered directly in the field through a filter (0.45uM or 8uM)

BK: air samples collected directly into a sterile tube

YE: rainwater is collected in a bucket, vacuum-filtered in the labthrough a 0.45 µm filter


Matching barcode sequences from environment to references

Unresolved names

Gaps in collections and taxonomy

Biological Collections

DNA barcodes (>50K/ sample)

DNA

Environmental sample

NGS DNA sequencers


Samplers in different locations 2010 2011
Samplers in different locations (2010-2011*)

* Collector running for one week.

DNA sequenced every 4 week.

5 samples processed per summer.

2007-9 also available, but less sites.

http://www.encyclopedia.com/video/SS4ZnFAv7iY-classification-les-agaricales.aspx

http://www.encyclopedia.com/video/SS4ZnFAv7iY-classification-les-agaricales.aspx

JB BK YE_LD


Sampler efficiency in true f ungi total number of sequences on left and ratio between 3 sites
Sampler efficiency in trueFungitotal number of sequences on left and ratio between 3 sites

http://www.encyclopedia.com/video/SS4ZnFAv7iY-classification-les-agaricales.aspx

JB BK YE_LD


Sampler efficiency in trueFungi - total number of sequences on left and ratio between 3 sites

http://www.encyclopedia.com/video/SS4ZnFAv7iY-classification-les-agaricales.aspx

http://www.encyclopedia.com/video/SS4ZnFAv7iY-classification-les-agaricales.aspx

JB BK YE_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 national herbarium (DAOM) & in Quarantine and Invasive Species ProjectPATRICE BOUCHARD AND ANDRÉ LÉVESQUEAgriculture and Agri-Food CanadaNovember 21, 2012CFIA Plant Business Line Committee meeting


Scope
Scope national herbarium (DAOM) & in

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)

DNA extraction

PCR amplify with universal markers (e.g. 16S, ITS, cpn60, COI, matK)

Sequencing reactions

GRDI Barcode of Life Database (BOLD)

BOLD at Guelph, iBOL and GenBank

Sanger sequencing

@NRC

RNA/DNA whole genomes



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
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

Grain samples imported from Asia

PRELIMINARY RESULTS

Total of 32,307 OTU’s

Canadian grain samples

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 Canadian grains


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


Thank Canadian grains you

Native species Trichalophusgranicollis

Regulated pest Otiorhynchusligustici

Photos from Patrice Bouchard, AAFC Ottawa, Coordinator of GRDI project


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