Transcript positionalCloning15

Positional cloning:
the rest of the story
X
a
a
a
a
a
a
a
a
http://faculty.ithaca.edu/iwoods/docs/wh
Today: So you have a map location … now what?
Mapped Mutant
Cloned Gene
Mapping:
Ultimate Goal
Map Distance = # of recombinants
# of meioses
X
Screen MANY markers on FEW meioses
LOW resolution = Potentially HIGH distance
Great for “Which Marker is Linked?”
=0
Screen NEARBY markers on MANY (1000’s) meioses
HIGH resolution = Potentially ZERO distance
Great for “Where is the Mutation?”
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 homozygous region in mutants
Find the actual mutation? How to be sure . . . ?
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
2013
What’s near Z15270?
http://www.ncbi.nlm.nih.gov/nucleotide
Goal: 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 . . .
Additional validated
Polymorphisms
Simple Repeats:
UCSC genome browser
Designing PCR primers
http://bioinfo.ut.ee/primer3-0.4.0/primer3/
Where do we
go from here?
Can get sequences and
test each of these
Not all will be useful
“Informative” =
polymorphic
= PCR amplicons of
different lengths from
WT and mutants
Markers you’ve seen already
Testing for
informative
SSLPs
“Informative” =
polymorphic
= PCR amplicons of
different lengths from
WT and mutants
More fish =
refine the map
More fish (i.e. embryos / larvae)
= more recombinants
= higher resolving power
a
a
a
a
a
a
a
a
Narrowing the
critical interval
5/1156 Z15270
More fish = more better
7/1156 Z11119
Z15270
Z11119
Defining the BOUNDARIES in the genome
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 [very good] MODEL of reality
No luck in genome sequence? (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 mapping
Key point = continually refine boundaries by recombination
• Look in genome for potential candidates
What’s nearby in genome?
. . . a [very good] MODEL of reality
No luck in genome sequence? (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 expression
Mutant = lack slow muscle fibers
what if . . . A secreted signal from motor neurons to developing muscle?!
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
Forward
Forward
200 bp
A
Reverse
G
60 bp, 140bp
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 sequencing
pooled 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
Safe Sailing (mostly)
Here there be Monsters
Obtaining gDNA from cDNA sequence:
exporting from genome
Obtaining gDNA from cDNA sequence:
exporting from genome
Designing PCR primers
http://bioinfo.ut.ee/primer3-0.4.0/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
Follow-up: Map? Or Biology?
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 rescue, CRISPR allele, Morpholinos => time, money
Ambiguous, easy to make up lots of stories
Mapping:
Ultimate Goal
Map Distance = # of recombinants
# of meioses
X
Screen MANY markers on FEW meioses
LOW resolution = Potentially HIGH distance
Great for “Which Marker is Linked?”
=0
Screen NEARBY markers on MANY (1000’s) meioses
HIGH resolution = Potentially ZERO distance
Great for “Where is the Mutation?”
Mapping can do it all!
What if ZF genome turns out
to 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 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:
0) Virtual Positional Cloning
1) Navigate Genome browsers for information related to
expression, Loss-of-function, Rescue
2) Zebrafish orthologs of your favorite human genes
Identification of enhancer elements
Transgenic Lines
3) BLAST on your own computer, and blast parsing via
Python script
4) From transcriptome profiling, identify genes, download
upstream sequences, visualize overrepresented motifs
Today: So you have a map location … now what?
Mapped Mutant
Cloned Gene
Tomorrow’s bioinformatics practical:
0) Virtual Positional Cloning
A review of what we did today, with some extra stuff
Today: So you have a map location … now what?
Mapped Mutant
Cloned Gene
Tomorrow’s bioinformatics practical:
1) Navigate Genome browsers for information related to
expression, Loss-of-function, Rescue
Your favorite Zebrafish Gene =>
sequence / exon-intron boundaries, conservation
expression
morpholino design
obtaining mRNA clones for rescue
Today: So you have a map location … now what?
Mapped Mutant
Cloned Gene
Tomorrow’s bioinformatics practical:
2) Zebrafish orthologs of your favorite human genes
Identification of enhancer elements
Transgenic Lines
Human gene
ZF ortholog
location in genome
putative promoter / enhancer =>
conservation of noncoding DNA from other fish
Today: So you have a map location … now what?
Mapped Mutant
Cloned Gene
Tomorrow’s bioinformatics practical:
3) BLAST on your own computer, and blast parsing via
Python script
All human proteins associated with HH signaling
Identification of ALL putative ZF orthologs of these
proteins via local BLAST
Parse BLAST to get top result and genome location for
each ZF protein
Determine if genome location matches map position of
mutant
Today: So you have a map location … now what?
Mapped Mutant
Cloned Gene
Tomorrow’s bioinformatics practical:
4) From transcriptome profiling, identify genes, download
upstream sequences, visualize overrepresented motifs
List of short unidentified sequence
Assign to Ensembl ID via BLAST and parsing
Download 5’UTR and 2k upstream sequences for batch
of Ensembl ID’s
Search through these for enriched motifs
Visualize locations of enriched motifs
Tomorrow’s Informatics Practical
http://faculty.ithaca.edu/iwoods/docs/wh/