Functional Genomic Approaches to
Better Understand
Shellfish-Pathogen-Environment
Interactions
Steven Roberts
University 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
Examine by looking at
differential gene and Pathogen physiology
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 seqs
big enough?

Case Studies
qPCR Proteomics NGS
Naturally
Disease Resistant Clam: gene discovery
Vibrio
Oysters Vibrio: gene discovery
tubiashii
Oyster: expression analysis
Salmon: expression analysis

Oysters
Offspring
Offspring of Naive
of Survivors Oysters
of Heavy
Disease Pressure

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
Naturally
Disease Resistant Clam: gene discovery
Vibrio
Oysters Vibrio: gene discovery
tubiashii
Oyster: expression analysis
Salmon: expression analysis

Approach - Protein Expression
Protein Extraction
2D gels
ID differences

Approach - Protein Expression
Experiment
Response to Host
Protein Extraction
2D gels
ID differences

Response to Host
oysters
control

pI MW
3.7 29k
4.5 49k
4.8 26k

pI MW
3.7 29k
4.5 49k
4.8 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
Naturally
Disease Resistant Clam: gene discovery
Vibrio
Oysters Vibrio: gene discovery
tubiashii
Oyster: expression analysis
Salmon: expression analysis

Hard Clams
photo: Dale Leavitt (RWU)

Hard Clams
photo: Dale Leavitt (RWU)

Vibrio tubiashii
Gene Discovery
226 million bp

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
Naturally
Disease Resistant Clam: gene discovery
Vibrio
Oysters Vibrio: gene discovery
tubiashii
Oyster: expression analysis
Salmon: expression analysis

Oyster Larvae challenged
with Oyster Herpes Virus
Larvae mRNA Library
Construction
Compare Reads
Larvae mRNA ~10 million each
Library
challenged Construction
Colleen Burge
Carolyn Friedman

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
Funding
USDA-NRAC
California SeaGrant
School of Aquatic and Fishery Sciences
NOAA- S/K
Technical
Sam White
UW HTSU - Michael Dorschner