Environmental metagenomics with next generation sequencing.

Environmental metagenomics withnext generation sequencing

Agassiz

Lacombe

Saskatoon

Morden

Harrow

Woodslee

Forest

RidgetownSt. Thomas

CaledoniaToronto

Morrisburg

Ottawa

St. Clotilde

Beloeil

Harrington, PEI

2009-11 SAGES and Soybean Rust Sampling Network

350 air and rain samples processed60M DNA barcode sequences

(bacteria & fungi)

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)

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

Unresolved

names

Environmental sample NGS DNA sequencers

DNA barcodes (>50K/ sample)Biol

ogica

l Col

lect

ions

4

DNA

Gaps in collections and taxonomy

Samplers in different locations (2010-2011*)

Province City 

Sampler

AirPassive Rain

Active Rain

PEI Harrington_PEI BK JB YEQC SainteClotilde_QC BK JB YEON

Woodslee_ON JB Ottawa_ON BK YE/LD

Ridgetown_ON BK YE/LDMB Morden_MB BK YESK Saskatoon_SK JB AB Lacombe_AB JB BC Agassiz_BC BK JB YE

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

JB BK YE_LD

* 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

JB BK YE_LD

Sampler efficiency in true Fungi

total number of sequences on left and ratio between 3 sites

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

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

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

JB BK YE_LD

City Sampler Total number of sequence

Agassiz BK 912 349Agassiz JB 637 894Agassiz YE_LD 314 073Harrington BK 743 859Harrington JB 568 708Harrington YE_LD 597 009SainteClotilde BK 1 098 310SainteClotilde JB 518 834SainteClotilde YE_LD 826 872

Numbers of unique fungal species represented in AAFC national herbarium (DAOM) & in GenBank (NCBI) with at

least one ITS sequence

DAOM 206

NCBI 221

41

Peronosporaceae

DAOM 206

NCBI 221

41

Peronosporaceae

Erysiphales

DAOM 209

NCBI 16680

Erysiphales

DAOM 209

NCBI 16680

DAOM 2,896

NCBI 240

Pucciniales

166DAOM 2,896

NCBI 240

Pucciniales

166

Downy Mildews

Rusts

Powdery Mildews

GENOMICS R&D INITIATIVE

Quarantine and Invasive Species Project

PATRICE BOUCHARD AND ANDRÉ LÉVESQUEAgriculture and Agri-Food Canada

November 21, 2012CFIA 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

DN

A extraction

RNA/DNA whole genomes

PCR amplify with

universal markers

(e.g. 16S, ITS, cpn60, COI, matK)

Sanger

sequencing

GR

DI B

arcode of Life D

atabase (BO

LD)

BO

LD at G

uelph, iB

OL and G

enBank

@NRC

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 speciesSample numbers: Imported from Asia = 8; Canadian samples = 130

Canadian grain samples

Grain sam

ples im

ported

 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

Globa

l Initia

tive

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