Download Genetic Mapping of Mutations Genetic Maps Have Two Properties

Genetic Mapping of Mutations
Genetic Maps Have Two Properties:
1. Identifying candidate genes
2. Initiating positional cloning projects
• Distance between markers
• Marker order
Genetic distance is determined by recombination frequency
Resolution of map is determined by number of meioses scored
Haploid Mapping Cross
Classical Genetic Mapping
Mutants will tend to have the a allele, B and b will be 1:1
WT will tend to have the A allele, B and b will be 1:1
Markers used in initial mapping:
• SSLP Markers (Simple sequence length polymorphisms)
Also called:CA-repeats, SSRs (simple sequence repeats), microsatellites
Length of CA tract differs in different strains
Forward
200 bp
Reverse
200 bp
A
Reverse
Forward
Forward
CACACACACA
Single nucleotide polymorphisms
G
60 bp, 140bp
Reverse
• snip SNP’s (restriction enzyme polymorphisms)
Forward
CACACACACACACACA
206 bp
Reverse
• Co-dominant: both alleles can be detected in a heterozygote
• Co-dominant: both alleles can be detected in a heterozygote
• Abundant polymorphisms: ~1/100 bp
• Informative in most crosses: 50-90% polymorphic among strains
• Sequence information needed for assay design
• Robust markers: easily scored, reproducible banding patterns, easy
transfer information between crosses and labs
• With the advent of deep sequencing, millions of SNPs can be
scored in a single experiment
1
SSLP markers scored on
haploid individuals
8 different SSLP markers scored on
pools of haploid WT and mutants
0 recombinants among 20 meioses = 0 cM
Wild type
Mutant
3 recombinants among 20 meioses = 15 cM
Wild type
Mutant
Three classes of markers:
• non-polymorphic (uninformative)
• polymorphic unlinked
• polymorphic linked
For gel pictures from a previous year, see
http://people.fas.harvard.edu/~ianwoods/woods_hole_2010/
Testing pools with 48 primers
arranged in a 96-well format
WT
4
8 12 16 20 24 28 32 36 40 44 48
mutant
4
8 12 16 20 24 28 32 36 40 44 48
WT
3
7 11 15 19 23 27 31 35 39 43 47
mutant
3
7 11 15 19 23 27 31 35 39 43 47
WT
2
6 10 14 18 22 26 30 34 38 42 46
mutant
2
6 10 14 18 22 26 30 34 38 42 46
WT
1
5
9 13 17 21 25 29 33 37 41 45
mutant
1
5
9 13 17 21 25 29 33 37 41 45
Where did those primers come from?
Original link, now not maintained:http://zebrafish.mgh.harvard.edu/mapping/ssr_map_index.html
All data on SSLPs, available at zfin.org
~ 240 primer pairs = 5 96-well plates
Primers used in our lab
Original site:
MGH zebrafish server
http://people.fas.harvard.edu/~ianwoods/woods_hole_2010/
Woods Hole Zebrafish Course 2007
2
Diploid mapping cross
Mapping a mutation
1.
+/-
X
+/+
+/-
X
+/-
Establish a polymorphic mapping cross
2.
Prepare genomic DNA from wild-type and mutant
siblings
3.
Analyze SSLPs on WT and mutant pools
(3a optional: retest some markers on the pools)
4.
Analyze putatively linked SSLPs on moderate number of
individuals (50-100)
-/+/+
5.
Analyze more individuals for high-resolution mapping
+/+
-/-/-
-/-/-
-/-/-
-/-
-/-/-/-
+/+/+/-
+/-
+/+
+/-
+/+
+/+
+/-
+/+
+/+/-
+/-
+/-
+/+ +/+
-/-
+/-
-/-
+/+/-
+/-
+/-
+/-
+/+
Identify carriers and
intercross
+/+
+/-
+/+ +/-/-
-/+/+
+/-
-/-
-/-
-/-
+/-
+/-
-/-
+/-
+/-
-/+/+
+/-
+/+
+/+
+/-
-/-
+/-
Collect embryos
+/-
-/- +/-
+/-
Expected results from BSA on diploids
+/+/+
+/-
(Carefully) sort embryos
+/-
Marker Q:
Polymorphic
unlinked
m +
mut
WT
Prepare gDNA from embryos
+/-
+/-
-/- +/+
+/-
Bulk Segregant Analysis on diploids
+/-
-/-
Outcross ID’d carrier
to a divergent strain
m +
Marker X:
Not polymorphic
unlinked
m +
x
m +
x
Q q
Q q
mut
WT
x
x
x
Marker Y:
Polymorphic
linked
Marker Z:
Not polymorphic
linked
m + x m +
m + x m +
Y
z
y
Y
y
z
z
z
x
Pool DNA samples (20 each)
mut
WT
mut
WT
mut
WT
WT
mut
PCR with arrayed primers
WT
mut
WT
mut
WT
mut
Mapping a mutation
1.
Establish a polymorphic mapping cross
2.
Prepare genomic DNA from wild-type and mutant
siblings
3.
Analyze SSLPs on WT and mutant pools
(3a optional: retest some markers on the pools)
Mapping of mutant X
• Incompletely characterized somite phenotype, ventrally curved body axis
• Diploid mapping cross
• PCR: 48 markers on linkage groups 4-7
WT pool, mut pool
• Load and run gel (96 samples)
• Analyze BSA gels for linkage
4.
5.
Analyze putatively linked SSLPs on moderate number of
individuals (50-100)
• Analyze some markers on individuals
• Discuss follow-up experiments
Analyze more individuals for high-resolution mapping
3
Group 1
Group 2
Group 3
Group 4
Sample gel
What’s next?
Marker
Wild-type pool
Mutant pool
Test promising markers on panels of individual mutants
and wild-types
Not all markers in the following slides are “promising,” but
were chosen to show different kinds of possible results
First, the ones that were wrong . . .
4
Sample gel
Z22467
MUTANT
INDIVIDUALS
Marker
1
2
3 4 5
6
7
WILD-TYPE
INDIVIDUALS
8
Wild-type pool
Mutant pool
Not polymorphic
Uninformative marker
Test others in region, perhaps run gel longer
Sample gel
MUTANT
INDIVIDUALS
Marker
1
2
3 4 5
6
7
Z11988
WILD-TYPE
INDIVIDUALS
8
Wild-type pool
Mutant pool
Polymorphic, not linked
Likely not located near this marker
Sample gel
MUTANT
INDIVIDUALS
1
2
3 4 5
6
7
Z8693
WILD-TYPE
INDIVIDUALS
8
Marker
Wild-type pool
Mutant pool
Polymorphic & linked?
Marker segregating as an intercross
No recombinants in 16 meioses
Run more mutant individuals:
Confirm position and distance from mutation
5
Sample gel
MUTANT
INDIVIDUALS
1
2
3 4 5
6
7
Z11119
WILD-TYPE
INDIVIDUALS
8
Marker
Wild-type pool
Mutant pool
Polymorphic & linked?
Marker segregating as an intercross
No recombinants in 16 meioses
Run more mutant individuals:
Confirm position and distance from mutation
Sample gel
Z3057
MUTANT
INDIVIDUALS
Marker
1
2
3 4 5
6
7
WILD-TYPE
INDIVIDUALS
8
Wild-type pool
Mutant pool
Polymorphic & linked?
Marker segregating as an intercross
Potential recombinants in mutant #3 = 1 out of 16 meioses
Run more mutant individuals to confirm
Sample gel
MUTANT
INDIVIDUALS
Marker
1
2
3 4 5
6
7
Z4999
WILD-TYPE
INDIVIDUALS
8
Wild-type pool
Mutant pool
Polymorphic & linked?
Marker segregating as an intercross
Potential recombinants in mutant #3 = 1 out of 16 meioses
Run more mutant individuals to confirm
6
Sample gel
Z7109
MUTANT
INDIVIDUALS
Marker
1
2
3 4 5
6
7
WILD-TYPE
INDIVIDUALS
8
Wild-type pool
Mutant pool
Polymorphic & linked?
Marker segregating as an intercross
Potential recombinants in mutant #3 = 1 out of 16 meioses
Run more mutant individuals to confirm
Sample gel
MUTANT
INDIVIDUALS
Marker
1
2
3 4 5
6
Z13936
7
WILD-TYPE
INDIVIDUALS
8
Wild-type pool
Mutant pool
Polymorphic & linked?
Marker segregating as an intercross
Potential recombinants in mutant #4, 5, 7, 8 = 4 out of 16 meioses
Run more mutant individuals to confirm
Linked markers:
Marker
LG
Data
Z13936
Z8693
Z11119
Z3057
Z4999
Z7109
7
7
7
7
7
7
4 recs: #4, 5, 7, 8 (4/16)
0 recs (0/16)
0 recs (0/16)
1 rec: #3 (1/16)
1 rec: #3 (1/16)
1 rec: #3 (1/16)
Model of mutation location
LG 7
Z3057
Z4999
Z7109
Z11119
Z8693
Z13936
mutation
7
MUTANT
INDIVIDUALS
Mutant Individuals
Mutant locus
1 2
3 4 5 6 7 8
-
-
-
-
-
-
-
1
2
3 4 5
6
7
Z11119
WILD-TYPE
INDIVIDUALS
8
-
Polymorphic & linked?
Marker segregating as an intercross
No recombinants in 16 meioses
Run more mutant individuals:
Confirm position and distance from mutation
Z3057
Mutant Individuals
1 2
Mutant locus
Z11119
3 4 5 6 7 8
MUTANT
INDIVIDUALS
1
2
3 4 5
6
7
WILD-TYPE
INDIVIDUALS
8
- - - - - - - m m m m m m m m
Polymorphic & linked?
Marker segregating as an intercross
Potential recombinants in mutant #3 = 1 out of 16 meioses
Run more mutant individuals to confirm
Mutant Individuals
1 2
3 4 5 6 7 8
Z11119
- - - - - - - m m m m m m m m
Z3057
m m M m m m m m
Mutant locus
MUTANT
INDIVIDUALS
1
2
3 4 5
6
7
Z13936
WILD-TYPE
INDIVIDUALS
8
Polymorphic & linked?
Marker segregating as an intercross
Potential recombinants in mutant #4, 5, 7, 8 = 4 out of 16 meioses
Run more mutant individuals to confirm
8
Mutant Individuals
1 2
3 4 5 6 7 8
Z11119
m m m M M m M M
- - - - - - - m m m m m m m m
Z3057
m m M m m m m m
Z13936
Mutant locus
Mutant Individuals
1 2
Z13936
Mutant locus
Z11119
Z3057
3 4 5
6 7 8
m m m M M m M M
X
X
X
X
- - - - - - - m m m
m m m m m
X
m m M m m m m m
Overview of zebrafish genome and genomic resources
Mutant Individuals
Genome size: 1.4 x 109 bp (for comparison, C.elegans 108, Drosophila 1.7 x 108, pufferfish 0.4 x 109, mammals 3.3 x 109)
25 chromosomes
Maps and other genomic resources
1 2
Mutant locus
Z11119
3 4 5 6 7 8
- - - - - - - m m m
m m m m m
X
Z3057
m m M m m m m m
Z13936
m m m M M m M M
X
X
X
X
1994
First genetic map (Postlethwait et al.), ~400 RAPDs, several genes and mutations
First mutation cloned by the candidate gene approach (Schulte-Merker et al.)
1996
Centromeres mapped, more RAPDs added, remaining gaps closed (Johnson et al.)
First SSLP map (Knapik et al.), ~100 markers
Large-insert genomic libraries become available (Amemiya, Zon, Fishman et al.)
Insertional mutagenesis used to clone mutated genes (Hopkins and colleagues)
1998
~140 genes mapped (Postlethwait et al.)
SSLP map expanded to ~700 markers (Knapik et al.)
Large-scale generation of ESTs begins (Washington Univ. Sequencing Group)
First gene identified by positional cloning (Zhang et al.)
1999
SSLP map expanded to 2000 markers (Shimoda et al.)
~250 genes and ESTs genetically mapped (Gates et al.)
Radiation hybrid maps become available (Geisler et al., Hukreide et al.)
~200 additional genes mapped in RH panels (Geisler et al., Hukreide et al.)
X
Genome Resource Overview, continued
2000
pre-1994 No two genes or markers were known to be linked
Deep sequencing will change everything!
More than 50,000 ESTs generated (WashU)
~2000 genes and ESTs genetically mapped (Woods et al.; Stanford, Oregon)
~4000 genes and ESTs mapped in RH panel (WashU, Children’s, NIH, Tübingen)
~50 mutations identified by candidate approach (Many groups)
5-10 mutations identified by positional cloning (Many groups)
Dozens of mutations cloned by insertional mutagenesis (Hopkins et al.)
2005-07 >1,400,000 ESTs defining ~20,000 genes
~3400 genes and ESTs genetically mapped (Stanford, Oregon)
>5000 genes mapped in RH panels (Hukreide et al. 2001, Children’s, NIH, Tübingen)
Hundreds of mutated genes cloned by insertional mutagenesis (Hopkins/MIT)
>200 mutated genes cloned by candidate approach and positional cloning
7,823 BAC clones sequenced, totaling 1.02 Gb of sequence (June 2007)
2010
(Almost) entire 1.4 Gb genome in high-quality assembly
>11,500 genes sequenced as full-length cDNAs (NIH/ZGC)
~150,000 SNPs mapped at high-resolution, used for assembly framework (Sanger)
9
Strategy: sequence wildtype and mutant pools of genomic DNA
Wild type pool
Mutant pool
A
+
A
+
G
mut
A
+
ATCGGCATGCA TCGTAGCACGA
ATCGGCATGCA TCGTAGCACGA
ATCGGCATGCA TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCA TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
G
mut
G
mut
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
ATCGGCATGCG TCGTAGCACGA
10
Positional cloning:
the rest of the story
X
Today: So you have a map location … now what?
Mapped Mutant
Cloned Gene
http://faculty.ithaca.edu/iwoods/docs/wh!
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 – easy, do first
- SNPs – require sequence data
• Improve resolution: Test more meioses
- generate more mutants
One advanced strategy:
• Deep sequencing of WT and mutants =>
SNPs = more markers to map
actual mutation?
esp. combined with hybrid capture . . .
1!
Data so far:
What SSLPs are in the region?
http://www.zfin.org!
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
Ciick on ‘Genetic maps’
ZFIN map query
MGH = microsatellite / SSLP map
ZFIN map view
Start close and move out both ways
2!
ZFIN marker view
GenBank Marker View
Obtaining FASTA sequence
Designing PCR primers
http://frodo.wi.mit.edu/primer3/!
3!
Testing for
informative
SSLPs!
Informa(ve+=+polymorphic+
Different+lengths+on+WT+and+mut+chromosome+
Refining the
map
Identifying
polymorphic
markers
Informa(ve+=+polymorphic+
…+some+will+have+same+SSLP+allele,+
++not+good+for+mapping+
Narrowing the
critical interval
5/1156
7/1156
More fish (i.e. embryos / larvae)
= more recombinants
= higher resolving power
More fish = more better
4!
Now what?
Now what?
• Identify more markers and do more high-res mapping
Key point = continually refine boundaries by recombination
• Identify more markers and do more high-res mapping
Key point = continually refine boundaries by recombination
• Look in genome for potential candidates
• Look in genome for potential candidates
What’s nearby in genome? (as a MODEL of reality)
What’s nearby in genome? (as a MODEL of reality)
No luck in genome sequence? (rare these days)
misassembly
gaps
No luck in genome sequence? (rare these days)
misassembly
gaps
• conserved synteny with other fish
• Physical map: BAC clones
• genetic or RH maps
• conserved synteny with other fish
• Physical map: BAC clones
• genetic or RH maps
What’s nearby in the genome?
Ensembl marker search
http://www.ensembl.org/Danio_rerio/!
No luck! Let’s try a sequence-based search: BLAST/BLAT!
5!
Ensembl BLAT search
Scroll down
And hit “Run”!
Genome assembly
6!
Good candidate?
calca at ZFIN
External references
calca expression
motor neuron expression
muscle phenotype
what if . . . signal from motor neurons to developing muscle?!
7!
What’s known about calca?
http://www.ncbi.nlm.nih.gov/gene!
How to test if
this is the right 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!!
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
8!
Now what?
Now what?
Test potential candidates
Test potential candidates
• Turn the candidate into a new map marker
- could it be the right gene?
- if not, can it narrow your interval?
• 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?
How to turn it into a map marker?
What’s a good candidate?
What’s a good candidate?
Single nucleotide
polymorphisms
Forward
Forward
Generating map markers
from ESTs/Genes/other sequences
200 bp
A
• Find or design primers for PCR (from gDNA)
Reverse
G
60 bp, 140bp
• Sequence PCR product on WT and mut
Reverse
• Find RE polymorphism
SNPs+=+~+1+/+250+bp+in+genome+
9!
Obtaining gDNA from cDNA sequence:
exporting from genome!
Obtaining gDNA from cDNA sequence:
exporting from genome!
http://genome.ucsc.edu/!
Obtaining gDNA from cDNA sequence:
exporting from genome!
Obtaining gDNA from cDNA sequence:
exporting from genome!
10!
Obtaining gDNA from cDNA sequence:
exporting from genome!
Obtaining gDNA from cDNA sequence:
exporting from genome!
Obtaining gDNA from cDNA sequence:
exporting from genome!
Obtaining gDNA from cDNA sequence:
exporting from genome!
11!
Obtaining gDNA from cDNA sequence:
exporting from genome!
Designing PCR primers
Beware of shotgun (non-BAC, i.e. large clone) assembly
Safe Sailing (mostly)
Here there be Monsters
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?
12!
Identifying a restriction enzyme to
map your SNP
dCAPS results!
http://helix.wustl.edu/dcaps/dcaps.html
Striking the right balance
in positional cloning
Mapping vs. Functional follow-up!
Mapping:
lots of fish, lots of PCR, lots of gels
should always give you an unambiguous answer
Functional:
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
• Check genetic/RH maps on ZFIN
• Start a chromosome walk
Sequencing => often done concomitantly with mapping
mRNA cloning/rescue Morpholinos => time, money
Ambiguous, easy to make up lots of stories
- iterative BAC screening
13!
What if ZF genome turns out
to be a dead end?
Tetraodon calca region
• Check other fish genomes
Pufferfish (Tetraodon, Fugu)
- smaller, more compact genome
- good for getting enhancer regions
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) Morpholinos (ATG, Splice) and rescue
3) Zebrafish orthologs of your favorite human genes
Identification of enhancer elements
Transgenic Lines
4) Doing cool things in big batches
14!