Roberts AFS 09

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Overview of application of NGS in shellfish research at UW

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Roberts AFS 09

  1. 1. Functional Genomic Approaches to Better Understand Shellfish-Pathogen-Environment Interactions Steven Roberts University of Washington
  2. 2. Where are the limits to the application of functional genomic approaches for non-model species?
  3. 3. Scientific Focus Shellfish immune response Pathogen physiology
  4. 4. Scientific Focus Shellfish immune response Examine by looking at differential gene and Pathogen physiology protein expression
  5. 5. Bigger Issues • Improve shellfish broodstock selection • Predict disease outbreaks • Understand consequences of environmental change
  6. 6. Outline qPCR Proteomics NGS
  7. 7. Challenges $ $$$ qPCR Proteomics NGS what genes? what’s the peptide? are the seqs big enough?
  8. 8. Case Studies qPCR Proteomics NGS Naturally Disease Resistant Clam: gene discovery Vibrio Oysters Vibrio: gene discovery tubiashii Oyster: expression analysis Salmon: expression analysis
  9. 9. Oysters Offspring Offspring of Naive of Survivors Oysters of Heavy Disease Pressure
  10. 10. 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
  11. 11. qPCR Proteomics* NGS Naturally Disease Resistant Clam: gene discovery Vibrio Oysters Vibrio: gene discovery tubiashii Oyster: expression analysis Salmon: expression analysis
  12. 12. Approach - Protein Expression Protein Extraction 2D gels ID differences
  13. 13. Approach - Protein Expression Experiment Response to Host Protein Extraction 2D gels ID differences
  14. 14. Response to Host oysters control
  15. 15. pI MW 3.7 29k 4.5 49k 4.8 26k
  16. 16. pI MW 3.7 29k 4.5 49k 4.8 26k
  17. 17. 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
  18. 18. qPCR Proteomics* NGS Naturally Disease Resistant Clam: gene discovery Vibrio Oysters Vibrio: gene discovery tubiashii Oyster: expression analysis Salmon: expression analysis
  19. 19. Hard Clams photo: Dale Leavitt (RWU)
  20. 20. Hard Clams photo: Dale Leavitt (RWU)
  21. 21. Vibrio tubiashii Gene Discovery 226 million bp
  22. 22. Genes
  23. 23. Vibrio tubiashii Reference Assembly
  24. 24. Vibrio tubiashii Reference Assembly
  25. 25. 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
  26. 26. qPCR Proteomics* NGS Naturally Disease Resistant Clam: gene discovery Vibrio Oysters Vibrio: gene discovery tubiashii Oyster: expression analysis Salmon: expression analysis
  27. 27. 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
  28. 28. RNA-Seq Analysis
  29. 29. 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
  30. 30. Acknowledgements Funding USDA-NRAC California SeaGrant School of Aquatic and Fishery Sciences NOAA- S/K Technical Sam White UW HTSU - Michael Dorschner

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