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

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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?”

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)