Positional cloning: the rest of the story

1. Positional cloning:the rest of the story a a a a a a a a X http://faculty.ithaca.edu/iwoods/docs/wh

2. Today: So you have a map location … now what? Mapped Mutant Cloned Gene

3. Mapping:Ultimate Goal = 0 Map Distance = # of recombinants # of meioses X Screen NEARBY markers on MANY (1000’s) meioses HIGH resolution = Potentially ZERO distance Great for “Where is the Mutation?” Screen MANY markers on FEW meioses LOW resolution = Potentially HIGH distance Great for “Which Marker is Linked?”

5. High-Resolution Mapping Basic strategies: • more markers: Refine boundaries - SSLPs – likely polymorphic, no sequence needed - SNPs – require sequence data • more mutants: Increase resolution One fancy strategy: • NextGen sequencing of pooled WT and pooled mutants => RNA SEQ => focus on exons “Homozygosity Mapping”: Define region homozygous in mutants Find the actual mutation? How to know . . . Generate more SNPs = more markers to map on more mutants

7. Data so far: Mutant with defects in slow muscle specification Initial Mapping: Out of 16 meioses: 1 recombinants: Z3057, Z4999, Z7109 0 recombinants: Z8693, Z11119 4 recombinants: Z13936

8. From mutant map position to cloned gene • Refining the map location with high-resolution mapping • Trolling for candidate genes • Testing candidates

9. From mutant map position to cloned gene • Refining the map location with high-resolution mapping • Trolling for candidate genes • Testing candidates

10. What’s near Z15270? http://www.ncbi.nlm.nih.gov/nucleotide Obtain sequence so we can localize it to Genome

11. NCBI Nucleotide Query

12. NCBI Nucleotide Query

13. Sequence Search at Ensembl Genome Browser Start close and move out both ways

14. Sequence Search at Ensembl Genome Browser Start close and move out both ways

15. Sequence Search at Ensembl Genome Browser Find More Markers To Test . . .

16. Find More Polymorphisms Find More Markers To Test . . .

19. Simple Repeats: UCSC genome browser

20. Designing PCR primers http://frodo.wi.mit.edu/primer3/

21. Testing for informative SSLPs “Informative” = polymorphic = PCR amplicons of different lengths from WT and mutants

22. Testing for informative SSLPs “Informative” = polymorphic = PCR amplicons of different lengths from WT and mutants

23. Refining the map a a a a a a a a More fish (i.e. embryos / larvae) = more recombinants = higher resolving power

24. Narrowing the critical interval 5/1156 Z15270 7/1156 Z11119 More fish = more better

25. Defining the critical interval Z15270 Z11119

26. Now what? • Identify more markers and do more high-res mapping Key point = continually refine boundaries by recombination • Look in genome for potential candidates What’s nearby in genome? . . . a [good] MODEL of reality No luck in genome sequence? (very rare) misassembly or gaps • conserved synteny with other fish • Physical map: BAC clones • genetic or RH maps

27. Now what? • Identify more markers and do more high-res mapping Key point = continually refine boundaries by recombination • Look in genome for potential candidates What’s nearby in genome? . . . a [good] MODEL of reality No luck in genome sequence? (very rare) misassembly or gaps • conserved synteny with other fish • Physical map: BAC clones • genetic or RH maps

28. What’s nearby in the genome? http://www.ensembl.org/Danio_rerio/

29. Good candidate?

30. calca at ZFIN

31. calca expression motor neuron expression Mutant = lack slow muscle fibers what if . . . A secreted signal from motor neurons to developing muscle?!

32. calca expression: RNA-SEQ

33. calca expression: RNA-SEQ

34. calca expression: RNA-SEQ

35. What’s known about calca? http://www.ncbi.nlm.nih.gov/gene

36. What’s known about calca? Cool new biology: it’s a secreted peptide with a novel role in directing slow muscle specification! Alert Cell, Science, and Nature!

37. How to test if this is the right gene?

38. Is calca the right gene? High resolution mapping - no recombinants between mutation and gene in lots of meioses Phenocopy with new mutant (or MO injection) or noncomplementation with another allele Rescue with mRNA injection Find mutation in coding sequence Picking the right strategy often is determined by balance of . . . - Available Resources - Number of Candidates These are often determined by size of candidate interval

39. Now what? Test potential candidates: • Turn the candidate into a new map marker - could it be the right gene? - even if not, can it narrow your interval? How to turn it into a map marker? What’s a good candidate?

40. Now what? Test potential candidates: • Turn the candidate into a new map marker - could it be the right gene? - even if not, can it narrow your interval? How to turn it into a map marker? What’s a good candidate?

41. Single nucleotide polymorphisms Forward 200 bp A Reverse Forward G 60 bp, 140bp Reverse SNPs = ~ 1 / 250 bp in genome

42. Generating map markers from ESTs/Genes/other sequences • Find or design primers for PCR (from gDNA) • Sequence PCR product on WT and mut • Find RE polymorphism • or use your huge list of markers from nextGen sequencing pooled WT and pooled mutant. which regions are differentially homozygous?

43. Obtaining gDNA from cDNA sequence: exporting from genome http://genome.ucsc.edu/

44. Obtaining gDNA from cDNA sequence: exporting from genome

46. BLAT Result

47. Good vs. Questionable Regions

48. Good vs. Questionable Regions Beware of shotgun (non-BAC, i.e. large clone) assembly Safe Sailing (mostly) Here there be Monsters

49. Obtaining gDNA from cDNA sequence: exporting from genome

50. Obtaining gDNA from cDNA sequence: exporting from genome