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Functional Genomic Approaches to Better Understand Shellfish-Pathogen-Environment Interactions Steven Roberts University of Washington
Functional Genomic Approaches to Better Understand Shellfish-Pathogen-EnvironmentInteractions
Steven RobertsUniversity of Washington
Where are the limits to the application of functional genomic approaches for non-model species?
Scientific Focus
Shellfish immune response
Pathogen physiology
Scientific Focus
Shellfish immune response
Pathogen physiology
Examine by looking at differential gene and protein expression
Bigger Issues
•Improve shellfish broodstock selection
•Predict disease outbreaks
•Understand consequences of environmental change
Outline
qPCR Proteomics NGS
Challenges
$ $$$
qPCR Proteomics NGS
what genes?what’s the peptide?
are the seqsbig enough?
Case Studies
qPCR Proteomics NGS
NaturallyDisease Resistant
OystersVibrio
tubiashii
Clam: gene discovery
Vibrio: gene discovery
Oyster: expression analysis
Salmon: expression analysis
Oysters
Offspringof Survivors
of HeavyDisease Pressure
Offspringof NaiveOysters
Disease Resistant Oysters
• Constitutive differences in gene expressions patterns exist across strains regardless of exposure
• Over-time, differences in gene expression patterns decrease
• Apoptosis and Protease pathways involved in host response
qPCR Proteomics* NGS
NaturallyDisease Resistant
OystersVibrio
tubiashii
Clam: gene discovery
Vibrio: gene discovery
Oyster: expression analysis
Salmon: expression analysis
Approach - Protein Expression
Protein Extraction
2D gels
ID differences
Approach - Protein Expression
Protein Extraction
2D gels
ID differences
ExperimentResponse to Host
Response to Host
oysters
control
pI MW3.7 29k4.5 49k4.8 26k
pI MW3.7 29k4.5 49k4.8 26k
Vibrio tubiashii proteomics
• Comparative proteomic approaches are viable for less studied taxa
• Several virulence associated pathways identified via protein expression
• polar flagellin, lateral flagellin (motility),
• OMP (survival & host invasion)
• In general, significant barriers still exist
qPCR Proteomics* NGS
NaturallyDisease Resistant
OystersVibrio
tubiashii
Clam: gene discovery
Vibrio: gene discovery
Oyster: expression analysis
Salmon: expression analysis
Hard Clams
photo: Dale Leavitt (RWU)
Hard Clams
photo: Dale Leavitt (RWU)
Vibrio tubiashii
226 million bp
Gene Discovery
Genes
Vibrio tubiashii
Reference Assembly
Vibrio tubiashii
Reference Assembly
NGS for Discovery
• Viable option, particularly for species with related taxa with sequence information
• Percentage of usable information could be considered low, however possibly still cost effective
qPCR Proteomics* NGS
NaturallyDisease Resistant
OystersVibrio
tubiashii
Clam: gene discovery
Vibrio: gene discovery
Oyster: expression analysis
Salmon: expression analysis
Oyster Larvae challenged with Oyster Herpes Virus
Colleen BurgeCarolyn Friedman
Larvae mRNA
Larvae mRNAchallenged
Library Construction
Library Construction
Compare Reads~10 million each
RNA-Seq Analysis
NGS for Expression Analysis
• Effective when have ability to use with annotated reference
• Offers simultaneous gene discovery and expression
• Compared to traditional approaches, has advantages
Acknowledgements
FundingUSDA-NRACCalifornia SeaGrantSchool of Aquatic and Fishery SciencesNOAA- S/K
TechnicalSam WhiteUW HTSU - Michael Dorschner
