Transcript Présentation PowerPoint

Metabarcoding and HTS* for assessing river
ecological quality with diatom indices at the scale
of a regular monitoring network.
Agnès BOUCHEZ, Cécile CHARDON, François KECK,
Frédéric RIMET, Kalman TAPOLCZAI, Valentin VASSELON
INRA CARRTEL, Thonon, France
* High-Throughput Sequencing
SFS Sacramento 2016
Biomonitoring
Monitoring of water quality is
required (USA : Federal CWA 1972,
Europe : WFD – 2000)
Biological indicators
(as diatoms)
DNA approaches
Barcoding (Hebert et al 2003)
Metabarcoding (Taberlet et al 2012)
HTS
Metabarcoding & Biomonitoring
Proof of concept :
 macroinvertebrates (Hajibabaei et al 2011)
 diatoms (Kermarrec et al 2013 - rbcL)
More tests on diatom environmental communities :
 with rbcL barcode (Kermarrec et al 2014)
 with 18S barcode (Zimmermann et al 2015, Visco et al 2015)
What about environmental monitoring networks ?
European Water Framework Directive
WFD in France :
RCS : 2043 monitoring sites
RCO : 4618 monitoring sites
+ a new French oversea
territory being part of France
since 2011  WFD mandatory
Mayotte Island, Indian Ocean
 opportunity for a large scale test
RCS
REF
POL
(2008) 2011 ------------------15
2013-15
2014-15
144 mi2
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metabarcoding + HTS
on diatom communities
Taxonomic
inventory
Quality Indices
Molecular-indices vs Microscopy-indices ?
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Taxonomic
inventory
1. sampling
REF
RCS
POL
48 sites
from the 3 networks
in 2014 and 2015
 88 samples
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2. DNA barcodes
Taxonomic
inventory
DNA extraction : GenElute (Valentin’s talk)
DNA barcode : rbcL – 312 bp region (Diat_rbcL_708F / R3)
3. HTS
PGM in PGTB http://www.pgtb.u-bordeaux2.fr/
Ion Torrent PGM
318 chip
6.723.455 sequences for 88 samples
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4. Bioinformatics
Reference database
Taxonomic
inventory
Mothur software (Schloss et al. 2009)
Denoising : Precluster command
Chimera removal : Uchime (Edgar et al. 2011)
OTU clustering
REF database : linking DNA barcodes to diatom taxa
R-Syst::diatom
http://www.rsyst.inra.fr/en
(Rimet et al. 2016)
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R-Syst::diatom
 Linking up-to-date taxonomy / barcodes / phenotypes / ecology
 Open-access: www.rsyst.inra.fr/
Life-forms
Functionnal groups
Quality
indices
Sizes, biovolumes,
cellule shapes
DNA sequence
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Rimet et al 2016
R-Syst::diatom
 Integration of sequences in the database
 Curation procedure
Collection: TCC
Every 6 months
rbcL + 18s
NCBI
(Thonon Culture Collection)
Curation
procedure
R-Syst::diatom
database
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Rimet et al 2016
R-Syst::diatom
4670 barcodes (18s, rbcL)
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Rimet et al 2016
4. Bioinformatics
Reference database
Taxonomic
inventory
Mothur software (Schloss et al. 2009)
Denoising : Precluster command
Chimera removal : Uchime (Edgar et al. 2011)
OTU clustering
Linking DNA barcodes to diatom taxa
R-Syst::diatom database
http://www.rsyst.inra.fr/en
(Rimet et al. 2016)
 Taxonomic assignation of OTUs
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Taxonomic
inventory
5. Taxonomic
inventories
Monitoring
networks
POL
RCS
REF
 the dissimilarity matrix based on metabarcoding
is significantly correlated to
the dissimilarity matrix based on microscopy
Mantel statistic r : 0.4739
Significance : 0.001
NMDS on OTUs
(Braycurtis dissimilarity matrix)
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6. Index values
Taxonomic
inventory
 IPS index based on
taxonomic assignation
of reads
Ecological status
IPS
Bad
Poor
Medium
Good
Very good
Specific Pollution Sensitivity Index IPS
based on molecular inventories
IPS - Coste 1982
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Taxonomic
inventory
6. Index values
… which is hopefully very well correlated to
!
But for Mayotte:
• reference database
is uncomplete
• index to be adapted
to local ecological
gradients
R² = 0.722
p-value = 3.952e-14
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Taxonomic
inventory
6. Index values
Robustness of
20
20
5710 reads
: decreasing sequencing depth in silico
20
20
1000 reads
20
500 reads
100 reads
15
15
15
15
15
10
10
10
10
10
5
5
5
5
5
0
0
0
5
10
R2= 0.998
0
0
0 15
5 20
10
0
R2= 0.994
15
5
20
10
50 reads
0
0
R2= 0.991
15
5
20
10
0
R2= 0.953
15
5
20
10
15
20
R2= 0.941
 still good signal with 100-times less sequences
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PGM Technology
Multiplexing
optimization
Taxonomic
inventory
Sampling
CEN standard (TR)
rbcL
R-Syst::diatom
in open-access
CEN standard (TR)
Quality Indices
Correlation
Robustness
Taxa detection
abundances
Thank you for
your attention
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