Environmental metagenomics with next generation sequencing
Jan 07, 2016
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