Positional cloning the rest of the story

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

From mutant map position to cloned gene

• Refining the map location with high-resolution mapping • Trolling for candidate genes • Testing candidates

Refining the map position Two basic strategies: • Establish boundaries: Test other markers

- SSLPs – likely polymorphic, no sequence needed - SNPs – require sequence data

• Improve resolution: Test more meioses

- generate more mutants One advanced strategy: • NextGen sequencing of WT and mutants =>

RNA SEQ => focus on exons or gDNA, esp. combined with hybrid capture . . . SNPs = more markers to map actual mutation? How to know . . .

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

What SSLPs are in the region? http://www.zfin.org!

Ciick on ‘Genetic maps’

ZFIN map query

MGH = microsatellite / SSLP map

ZFIN map view

Start close and move out both ways

ZFIN marker view

GenBank Marker View

Obtaining FASTA sequence

Designing PCR primers

http://frodo.wi.mit.edu/primer3/!

Testing for informative

SSLPs

Informa(ve = polymorphic Different lengths on WT and mut chromosome

Identifying polymorphic

markers

Informa(ve = polymorphic … some will have same SSLP allele,

not good for mapping

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 7/1156

Z11119

Z15270

Defining the critical interval

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 [pretty good] MODEL of reality

No luck in genome sequence? (rare these days) 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 mapping Key point = continually refine boundaries by recombination • Look in genome for potential candidates

What’s nearby in genome? (as a MODEL of reality)

No luck in genome sequence? (rare these days) misassembly 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 expression muscle phenotype

what if . . . signal from motor neurons to developing muscle?!

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 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? - 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? - if not, can it narrow your interval?

How to turn it into a map marker? What’s a good candidate?

Single nucleotide polymorphisms

A

G

200 bp

60 bp, 140bp

Forward

Forward Reverse

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

Obtaining gDNA from cDNA sequence: exporting from genome

http://genome.ucsc.edu/!







Beware of shotgun (non-BAC, i.e. large clone) assembly

Here there be Monsters

Safe Sailing (mostly)



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 balance in positional cloning

Mapping:

lots of fish, lots of PCR, lots of gels should always give you an unambiguous answer

Functional:

Sequencing => often done concomitantly with mapping mRNA cloning/rescue Morpholinos => time, money Ambiguous, easy to make up lots of stories

Follow-up: Map? Or Biology?

What if ZF genome turns out to be a dead end?

• Check other fish genomes

- more candidate genes? - fix a gap in the ZF data

• RNA-SEQ? • Check genetic/RH maps on ZFIN • Start a chromosome walk

- iterative BAC screening

What if ZF genome turns out to 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) Positional cloning in 2 (mostly) easy steps 2) Design morpholinos (ATG, Splice) and rescue

for LOF, probably better to do TALEN/CRISPR . . . 3) Zebrafish orthologs of your favorite human genes Identification of enhancer elements Transgenic Lines 4) Doing cool things in big batches (batch BLAST, perl)

Documents

Positional cloning the rest of the story