Positional cloning:the rest of the story
http://faculty.ithaca.edu/iwoods/docs/wh
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Today: So you have a map location … now what?
Mapped Mutant Cloned Gene
Mapping:Ultimate Goal
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Screen MANY markers on FEW meiosesLOW resolution = Potentially HIGH distanceGreat for “Which Marker is Linked?”
Map Distance = # of recombinants
# of meioses = 0
Screen NEARBY markers on MANY (1000’s) meiosesHIGH resolution = Potentially ZERO distance
Great for “Where is the Mutation?”
High-Resolution MappingBasic 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
mutantsFind the actual mutation? How to know . . . Generate more SNPs = more markers to map on more
mutants
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
From mutant map position to cloned gene
• Refining the map location with high-resolution mapping
• Trolling for candidate genes
• Testing candidates
From mutant map position to cloned gene
• Refining the map location with high-resolution mapping
• Trolling for candidate genes
• Testing candidates
What’s near Z15270?http://www.ncbi.nlm.nih.gov/nucleotide
Obtain sequence so we can localize it to Genome
NCBI Nucleotide Query
NCBI Nucleotide Query
Sequence Search at Ensembl Genome Browser
Start close and move out both ways
Sequence Search at Ensembl Genome Browser
Start close and move out both ways
Sequence Search at Ensembl Genome Browser
Find More Markers To Test . . .
Find More Polymorphisms
Find More Markers To Test . . .
Simple Repeats:UCSC genome browser
Designing PCR primers
http://frodo.wi.mit.edu/primer3/
Testing for informative
SSLPs
“Informative” = polymorphic
= PCR amplicons of different lengths from WT and mutants
Testing for informative
SSLPs
“Informative” = polymorphic
= PCR amplicons of different lengths from WT and mutants
Refining the map
More fish (i.e. embryos / larvae)
= more recombinants= higher resolving power
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Narrowing the critical interval
More fish = more better
5/1156 Z15270
7/1156 Z11119
Z11119
Z15270 Defining the critical interval
Now what?
• Identify more markers and do more high-res mappingKey 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
Now what?
• Identify more markers and do more high-res mappingKey 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
What’s nearby in the genome?http://www.ensembl.org/Danio_rerio/
Good candidate?
calca at ZFIN
calca expression
motor neuron expressionMutant = lack slow muscle fibers
what if . . . A secreted signal from motor neurons to developing muscle?!
calca expression: RNA-SEQ
calca expression: RNA-SEQ
calca expression: RNA-SEQ
What’s known about calca?
http://www.ncbi.nlm.nih.gov/gene
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!
How to test if this is the right
gene?
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
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?
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?
Single nucleotide polymorphisms
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200 bp
60 bp, 140bp
Forward
ForwardReverse
Reverse
SNPs = ~ 1 / 250 bp in genome
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 sequencingpooled WT and pooled mutant. which regions are differentially homozygous?
Obtaining gDNA from cDNA sequence: exporting from genome
http://genome.ucsc.edu/
Obtaining gDNA from cDNA sequence: exporting from genome
BLAT Result
Good vs. Questionable Regions
Good vs. Questionable Regions
Beware of shotgun (non-BAC, i.e. large clone) assembly
Here there be Monsters
Safe Sailing (mostly)
Obtaining gDNA from cDNA sequence: exporting from genome
Obtaining gDNA from cDNA sequence: exporting from genome
Designing PCR primers
http://frodo.wi.mit.edu/primer3/
PCR primers
Amplify from WT and mut, sequence . . .
Locating a SNP to map
. . . run on your mapping panel- still a candidate? (0 recombinants)- narrow the candidate interval?
Identifying a restriction enzyme to map your SNP
http://helix.wustl.edu/dcaps/dcaps.html
dCAPS results
Striking the right balancein positional cloning
Mapping:
lots of fish, lots of PCR, lots of gelsshould always give you an unambiguous answer
Functional:
Sequencing => often done concomitantly with mapping
mRNA rescue, CRISPR allele, Morpholinos => time, moneyAmbiguous, easy to make up lots of stories
Follow-up: Map? Or Biology?
Mapping:Ultimate Goal
X
Screen MANY markers on FEW meiosesLOW resolution = Potentially HIGH distanceGreat for “Which Marker is Linked?”
Map Distance = # of recombinants
# of meioses = 0
Screen NEARBY markers on MANY (1000’s) meiosesHIGH resolution = Potentially ZERO distance
Great for “Where is the Mutation?”
Mapping can do it all!
What if ZF genome turns outto be a dead end (RARE!)?
• Check other fish genomes
- more candidate genes?- fix a gap in the ZF data
• RNA-SEQ or HMFSeq?
• Start a chromosome walk
- iterative BAC screening
What if ZF genome turns outto be a dead end?
• Check other fish genomes
Pufferfish (Tetraodon, Fugu)
- smaller, more compact genome- good for getting enhancer regions
Tetraodon calca region
More Candidates to test: find and map zebrafish orthologs
Today: So you have a map location … now what?Mapped Mutant Cloned Gene
Tomorrow’s bioinformatics practical:
1) Virtual Positional Cloning
2) Navigate Genome browsers for information related to expression, Loss-of-function, Rescue
3) Zebrafish orthologs of your favorite human genesIdentification of enhancer elements Transgenic Lines
4) Doing cool things in big batches (batch BLAST, perl)