SLIDE 1
Coral holobiont analysis with MinION sequencer onboard Tara
Quentin Carradec Julie Poulain Laboratoire d’analyses génomiques des eucaryotes CEA/Genoscope
Workshop MinIon 13 december 2017
SLIDE 2 2016-2018 2009-2012 2014 2006-2008
Tara Expeditions
2013
- 15 years of scientific expeditions
- Study and understand the impact
- f climate change on the world's
- ceans
- Multi-disciplinary
- Oceanography
- Chemistry
- Biology (genomics, imaging)
- Communication
- Inform populations about consequences of global warming
- Education programs for children (500 French teacher involved)
SLIDE 3 Tara Pacific
- 100,000 km
- More than 100 researchers
- 23 research institutes (France, Monaco, USA, Germany, Switzerland, South Arabia, Japan)
- 15,000 samples of corals and other species
« Genetic complexity of coral holobiont across the Pacific »
SLIDE 4 collecting, freezing and shipping Coral sampling Water sampling
freezing shipping
SLIDE 5 Molecular analysis of corals on board ?
- Species identification via marker gene sequencing
- Direct RNA sequencing to evaluate coral health and response to stress.
SLIDE 6
Triangle of coral diversity
50% of total coral diversity
SLIDE 7
Corals
Corals diversity
798 species identified Coral diversity
SLIDE 8
Pocillopora
Symbiodinium
Corals
Corals diversity
Zooxanthella (Symbiodinium)
>150 species
SLIDE 9
Corals
Corals diversity
Molecular identification of coral holobiont
ribosomal 18S ITS2 Ribosomal 16S Symbiodinium Bacterial community
genus or species level species level species level
Coral
SLIDE 10
DNA extraction Corals sampling Grinding (1minute) DNA extraction (2h30 for 12 samples)
TerraLyzer Emilie Boissin
96 samples in 12 days
SLIDE 11
PCR and library preparation 3 PCR
18S 16S ITS2
Sample 1 3 PCR
18S 16S ITS2
Sample 2
Barcode 1 Barcode 2
3 PCR
18S 16S ITS2
Sample 3
Barcode 3
Sample 12 3 PCR
18S 16S ITS2 Barcode 12 Pool of all amplicons PCR – adapter ligation 24-48h Run Julie Poulain
SLIDE 12 Bio-informatic analysis
Barcoded reads Demultiplexing (Porechop)
Sample 1 Sample 2 Sample 3
Mapping (BWA-MEM)
ITS2 18S 16S
Consensus
18S Porites lobata
Identical to the reference New species ?
1 RUN = 12 samples and 3 Marker genes / sample
Symbiodinium Coral bacteria
SLIDE 13
Kimbe island reef
SLIDE 14
Morphological identification
Diploastrea Physogyra Galaxea Favites Porites Fungia Symphilyllia Pocillopora ??? Seriatopora Fugiidae 1 2 3 4 5 6 7 8 10 11 9 ??? 12 Emilie Boissin
SLIDE 15 Sequencing results
- 862 639 reads
- 127 974 (15%) with
detected barcodes on each side ITS2 size (300 bp) Non-specific reads 16S size (1300 bp) 18S size (1800 bp)
SLIDE 16 Mapping results
Number of reads Barcode
BWA :
- 80% of read length aligned
- >70% of identity
coral symbiodinium bacteria
SLIDE 17 Fungia 6 Morphology 18S sequencing
- Fungia paumotensis
- Fungia horrida
Symbiodinium -Clade C161 (+3 SNPs) ITS2 sequencing
Bacteria community : not yet analyzed…. 16S sequencing
Results for sample 6
SLIDE 18 Conclusion
Prospects:
- Daily use of MinION for species identification.
- Less samples to conserved onboard
- Less samples to send.
- Immediate answer.
Conclusion:
- 96 DNA extractions
- 5 MinION runs
- More than 30 corals identified + symbiodinium + bacterial community.
limitations:
- Flow cells quality (over than 2 month at 4°C)
- Reactive conservation at low temperature.
- Parallelization of library preparation (VolTrax ?).
SLIDE 19
Pocillopora
Environmental diagnostic with RNA sequencing ?
SLIDE 20
4 2 1 3
Environmental diagnostic with RNA sequencing ?
SLIDE 21 Direct RNA sequencing
RNA extraction
PolyA RNA selection
Library preparation for RNA sequencing
run 1 run 2
11 325 reads aligned 34 452 reads 27 712 reads mapping on Pocillopora reference transcriptome
RNA extraction
PolyA RNA selection
RNA extraction
PolyA RNA selection
RNA extraction
PolyA RNA selection
Library preparation for RNA sequencing
18 247 reads aligned
SLIDE 22
Genoscope, CEA Patrick Wincker
Corinne Cruaud (LBioMeg) Valerie Barbe (LBioMeg) Arnaud Lemainque (LBioMeg) Stefan Engelen (R&D bioseq team) Denis Debaussart (informatic team) Tara Pacific Consortium Serge Planes (CNRS) Denis Allemand (Centre scientifique de Monaco) web site: http://oceans.taraexpeditions.org/ CRIOBE, Perpignan Emilie Boissin
Thank you for your attention
