1. High-throughput bisulfite sequencing reveals relationships between gene expression and DNA methylation in the bivalve, Crassostrea gigas Mackenzie Gavery & Steven Roberts University of Washington School of Aquatic and Fishery Sciences Seattle, WA USA

2. Open Science Slides, links and related materials can be found at: oystergen.es/norway

3. Background DNA methylation in invertebrates Results Characterization of DNA methylation in Pacific oysters Discussion & Future Directions Outline

4. GENES (DNA) TRAITS color growth disease resistance ENVIRONMENT nutrition pathogens temperature Background

5. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT nutrition pathogens Background temperature

6. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT nutrition pathogens Background temperature

7. Me C G C G DNA Methylation

8. Me C G C G Gene A TF X DNA Methylation

9. Me C G C G Gene A TF X DNA Methylation VERTEBRATE

10. Gene A TF X VERTEBRATE DNA Methylation

11. Gene A TF X VERTEBRATE Gene A INVERTEBRATE DNA Methylation

12. Gene A TF X VERTEBRATE Gene A INVERTEBRATE DNA Methylation

13. Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation

14. Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation

15. Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation Ancestral pattern

16. Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation Ancestral pattern Important regulator of phenotype

17. DNA Methylation Objectives: Characterize DNA methylation in C. gigas Gain an understanding of the functional role

18. Part 1

19. Approach In silico analysis Experimental analysis: MBD-Seq

20. CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

21. CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

22. CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

23. CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

24. Part 2

25. genomic DNA Approach High-throughput bisulfite sequencing: Gill tissue Additional resources: RNA-seq data: gill tissue (Zhang et al, 2012)

26. genomic DNA Part 2 Approach High-throughput bisulfite sequencing: Gill tissue Additional resources: RNA-seq data: gill tissue (Zhang et al, 2012)

27. > 2.5 million CG dinucleotides Part 2: Results

28. scaffold 86 (Galaxy Trackster) Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0%

29. Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster)

30. Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster)

31. Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster)

32. Distribution in genomic elements Part 2: Results

33. Distribution in genomic elements Part 2: Results

34. Relationship with expression

35. RNA-Seq data (Zhang et al., 2012) Part 2: Results Relationship with expression Gene expression (Deciles) DNA methylation/gene

36. Part 3

37. Approach: High-throughput bisulfite sequencing: Gill tissue Part 3

38. Approach: High-throughput bisulfite sequencing: Gill tissue Male gamete (sperm) tissue Part 3

39. Part 3: Results genes %methylation: gill CG %methylation: sperm 0bp 6,000bp

40. Part 3: Results genes %methylation: gill CG %methylation: sperm 0bp 6,000bp

41. Part 3: Results genes %methylation: gill CG %methylation: sperm 0bp 6,000bp

42. Part 3: Results Identify differential methylation (DM)

43. Part 3: Results Identify differential methylation (DM)

44. Part 3: Results Identify differential methylation 1/3 of all genes had DM

45. Summary

46. unmethylatedmethylated

47. Gene function: Summary unmethylatedmethylated

48. Gene function: Summary unmethylated inducible housekeeping methylated

49. Gene function: Expression: Summary unmethylated inducible housekeeping methylated

50. Gene function: Expression: Summary unmethylated inducible low housekeeping high methylated

51. Gene function: Expression: Summary unmethylated inducible low housekeeping high methylated Tissue specific methylation:

52. Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated patterns change at fine-scale

53. Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated patterns change at fine-scale Role of methylation in introns:

54. Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated unknown Role of methylation in introns: patterns change at fine-scale

55. Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated Role of methylation in inter-genic regions: unknown Role of methylation in introns: patterns change at fine-scale

56. Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated Role of methylation in inter-genic regions: unknown Role of methylation in introns: unknown patterns change at fine-scale

57. Next Steps GENES (DNA) EPIGENOME (DNA methylation)

58. Explore relationships between DNA methylation and alternative splicing Determine if DNA methylation mediates response to environmental stress in shellfish Tool development: DNA tiling array (MeDIP-Chip) Next Steps GENES (DNA) EPIGENOME (DNA methylation)

59. Acknowledgements Roberts Lab: Steven Roberts Samuel White Brent Vadopalas Emma Timmins-Schiffman Claire Ellis Lisa Crosson Taylor Shellfish: Jonathan Davis Molly Jackson website: oystergen.es/norway