1. A Tale of Two Worms: Comparing the Genomes of C. elegans & C. briggsae Lincoln Stein Cold Spring Harbor Laboratory

2. My Lab

3. International HapMap Project Find common regions of genetic variation in human population to reduce cost of genetic association studies. Reduce cost of genetic association studies. 600,000 SNPs x 270 individuals

4. Gramene Comparative genomics among monocots Rice as model system Rice genome, maps, proteins, mutants, QTLs, phenotypes Map alignments to maize, wheat, oats, barley &c.

5. Genome KnowledgeBase Biological pathways in human Curated by experts in the field Designed for Education Data mining & discovery Open data/Open software

6. WormBase Community database for C. elegans C. elegans genome C. briggsae genome Genetic maps Developmental anatomy RNAi screens Microarray screens Evolutionary biology

7. Generic Model Organism Db Reusable software for building model organism databases Used by WormBase, FlyBase, Gramene, RatDB, SGD, MGD… Genome browsers, genetic maps, curation tools…

10. male (lateral) male (ventral) hermaphrodite 200 µm 10 µm

11. Sequencing C. briggsae Isolate DNA, make libraries (2 mo) Map libraries (4 mo) Shotgun sequence genome (1 wk) Assemble genome (6 mo) Analyze genome (9 mo)

12. The Draft Contig TypeCountN 50 (kb)Length (% genome) Contigs534141105.6 Supercontigs899474107.5 Scaffolds1421450102.4 (98%) BAC Map Sequence Contigs Supercontigs Scaffolds Jim Mullikin, Sanger Center; LaDeana Hillier, WUSTL

13. Calling C. briggsae Genes

14. briggsae genes: “hybrid” strategy Elegans predictions Briggsae predictions Avril Coghlan, University of Dublin

15. How accurate is it? C. elegans gold standard 2,257 genes entirely confirmed by mRNA data Results on C. elegans set 92% of time, hybrid method picked the “gold standard gene” correctly 32 genes incorrectly split into 2 or more predictions (1.4%) 49 genes incorrectly merged into 1 prediction (1%)

16. Gene Sets Very Similar Table 1: Comparison of the C. briggsae and C. elegans Protein- Coding Gene Sets C. briggsaeC. elegans WS77* C. elegans Hybrid Number of genes 19,50718,80820,621 Median gene length 1.90 kbp1.91 kbp1.83 kbp Summed length of genes 55.7 Mbp52.5 Mbp55.6 Mbp Average gene density 5.4 kbp per gene 5.3 kbp per gene 4.9 kbp per gene

17. Identifying Orthologs Ce Cb ortholog pair ortholog vs paralog? best similarity match Use colinearity to resolve ambiguities (Todd Harris, CSHL)

18. Comparing Orthologs 12,155 orthologs 807 C. briggsae “orphans” 1,061 C. elegans “orphans” Divergence date: 80-110 Mya All genes under various degrees of purifying selection (Todd Harris, Jason Stajich)

19. Orthologs Similar but Differ in Detail Briggsae has 1 new intron every 5 th gene.

20. Comparing Gene Families: TRIBE-MCL

24. Cluster 1 Cluster 2 Cluster 3 Cluster 4Cluster 5

25. Cb/Ce protein clusters (Jason Stajich, rotation student) 2169 clusters of >= 2 members 24% of elegans single-copy genes 28% of briggsae single-copy genes

26. Protein Family Clusters ClusterElegansBriggsaeDescription 5112128Zn-finger 3128150Protein kinase 12151057TM receptor, subf 2 6122867TM receptor, subf 2 21691357TM receptor, subf 1 82204Unknown 1711622DUF38 >200 clusters unbalanced by more than 2-fold

27. A Rapidly Evolving Family: Olfactory Receptors PFAM Class C. elegansC. briggsae 7tm_4269222 7tm_5322163 sra3718 srb1612 sre5551 srg3230 Total718476

28. Putative ortholog pair elegans exclusive subtree Sra Olfactory Receptor Family

29. Synteny: Aligning C.b. to C.e. TypeIntergenUpstrDownstCDSIntron5' UTR3'UTRRepeatTotal Strong61,61527,51230,19249,358114,3232,7837,23928,313321,335 Coding41,81711,60015,571152,08649,1358551,55712,095284,716 Weak115,20053,18959,542188,601250,6035,88511,82349,624734,467 TOTAL218,63292,301105,305390,045414,0619,52320,61990,0321,340,518 (Todd Harris & Jason Stajich)

30. Synteny Reconstruction raw aligned segments (WABA) Merge overlaps Merge adjacent merged segments Reconstruct interrupted segments reconstructed segments (Yours truly)

31. Reconstructing briggsae 4,837 reconstructed segments ~85% of genome 0.5-0.7 bkpts/Mb/My)

32. Rearrangement is Local IIIIIIIVVX I3351722313216 II2562821309 III28942286 IV3143814 V27221 X170 Junctions of elegans chromosomes on briggsae contigs

33. Rearrangement is Local left armcenterright arm left arm494174123 center592163 right arm445 Junctions of elegans chromosome arms on briggsae contigs

34. big map Syntenic blocks Genes & meiotic map Orthologs Orphans Essential genes Repetitive elements K A /K S KSKS

35. Improving elegans: new gene?

36. Improving elegans: bad exon?

37. Corrections to C. elegans Table 11. Updating the C. elegans Gene Set Using C. briggsae Similarity Gene SetWS77WS103 New genes 1,275985 New exons in existing genes 1,7631,243 Exon extensions in existing genes 1,115845 Exon deletions in existing genes 2,0931,600 Exon truncations in existing genes 1,6751,114

38. Recent Work: Chemosensory Receptors PFAM Class C. elegansC. briggsae 7tm_4269222 7tm_5322163 sra3718 srb1612 sre5551 srg3230 Third largest C. elegans protein family. Subclass of GPCR 7TM receptors.

39. Questions Are these differences real? Mechanism of the differences? Amplification vs gene loss Why are some subfamilies unbalanced and not others? Phenotypic consequences of the differences?

40. Putative ortholog pair elegans exclusive subtree Sra Olfactory Receptor Family

41. Are the Differences Real? Intensive search for missing sra family members. C. elegansC. briggsae sra elegans genome briggsae genome (Jack Chen, Postdoc; Shraddha Pai, URP) similarity searching new

42. Results Family size differences real (still roughly twice as manyelegans sra as briggsae sra) Differences due to species-specific tandem duplications, not due to conversion into pseudogenes. But…

43. Hitting non-sra elegans genes? 18 non-sra C. elegans genes 17 non-sra C. briggsae genes

44. A New Nematode Chemosensory (Sub)family? sra family sra-like genes

45. 7TM Domain Structure C36C5.7 Most candidates showed 7 transmembrane domain signatures characteristic of GPCR membrane receptors.

46. …Usually

47. Repairing Incomplete Genes missed exon

48. Before & After After repairing: 7 TMsBefore repairing: 6 TMs

49. After Repair

50. Expression Patterns Co-Cluster with sra Family Genes sra genes sra-like genes Kim et al Science, 293: 2087-2092. 2001Science, 293: 2087-2092. 2001

51. Anatomic Expression Pattern

52. Promoter/GFP Fusion Analysis (Collaboration w/ David Baillie) GeneHeadTail T20D4.1- T21H8.4- C33G8.5 C36C5.6 C47A10.6 C05F5.4- T20D4.18--

53. Phasmid Neuron Expression anus PHA/PHB T21H8.4-GFP

54. Amphid Neuron Expression axon dendrite ASx Cell body C05F5.4

55. Conclusion Likely new olfactory receptor subfamily Closely related to sra subfamily ~50% more members in elegans than briggsae Specific expression in both amphid & phasmid sensory neurons

56. Next Steps Continue refining families in the 2 species More novel candidate (sub)families, one with ~100 members. Characterize expression patterns

57. Longer Term Deconvolute odorant combinatorial code. chemotaxisaversion odorant chemosensory receptors sensory neurons interneurons

58. Resources to Apply Neuronal wiring chart. Receptor promoter::GFP fusions. Calcium-flux sensitive GFP constructs (“Cameleons”) Phenotypic assays (elegans & briggsae) Transgenesis/rescue

59. And Coming Soon

60. Who Dunnit Zhirong BaoAlan CoulsonShraddha Pai Thomas BlumenthalRichard DurbinBob Plumb Michael Brent Sam Griffith-JonesJane Rogers Jack Chen Todd HarrisMark Sohrmann Laura Clarke LaDeana HillierJason Stajich Chris Clee Patricia KuwabaraRobert Waterston Avril CoghlanJames Mullikin David Willey CSHL, WUGSC, Trinity College Dublin, Wellcome Trust/Sanger Institute, NIH, Duke University, UW Funding from: NIH & Wellcome Trust

61. Who Dunnit Zhirong BaoAlan CoulsonShraddha Pai Thomas BlumenthalRichard DurbinBob Plumb Michael Brent Sam Griffith-JonesJane Rogers Jack Chen Todd HarrisMark Sohrmann Laura Clarke LaDeana HillierJason Stajich Chris Clee Patricia KuwabaraRobert Waterston Avril CoghlanJames Mullikin David Willey CSHL, WUGSC, Trinity College Dublin, Wellcome Trust/Sanger Institute, NIH, Duke University, UW Funding from: NIH & Wellcome Trust

62. Who Dunnit Zhirong BaoAlan CoulsonShraddha Pai Thomas BlumenthalRichard DurbinBob Plumb Michael Brent Sam Griffith-JonesJane Rogers Jack Chen Todd HarrisMark Sohrmann Laura Clarke LaDeana HillierJason Stajich Chris Clee Patricia KuwabaraRobert Waterston Avril CoghlanJames Mullikin David Willey David Baillie, University of British Columbia