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Positional cloning: the rest of the story

Positional cloning: the rest of the story. a. a. a. a. a. a. a. a. X. http://faculty.ithaca.edu/iwoods/docs/wh. Today: So you have a map location … now what? Mapped Mutant Cloned Gene. Mapping: Ultimate Goal. = 0. Map Distance = # of recombinants

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Positional cloning: the rest of the story

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  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?”

  4. 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

  5. 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

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

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

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

  9. NCBI Nucleotide Query

  10. NCBI Nucleotide Query

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

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

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

  14. Find More Polymorphisms Find More Markers To Test . . .

  15. Simple Repeats: UCSC genome browser

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

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

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

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

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

  21. Defining the critical interval Z15270 Z11119

  22. 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

  23. 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

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

  25. Good candidate?

  26. calca at ZFIN

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

  28. calca expression: RNA-SEQ

  29. calca expression: RNA-SEQ

  30. calca expression: RNA-SEQ

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

  32. 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!

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

  34. 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

  35. 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?

  36. 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?

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

  38. 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?

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

  40. Obtaining gDNA from cDNA sequence: exporting from genome

  41. BLAT Result

  42. Good vs. Questionable Regions

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

  44. Obtaining gDNA from cDNA sequence: exporting from genome

  45. Obtaining gDNA from cDNA sequence: exporting from genome

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