week 11: mapping november 8, 2001 todd scheetz. introduction what is mapping? determining the...
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Week 11: Mapping
November 8, 2001
Todd Scheetz
Introduction
What is mapping?determining the location of elements within a genome, with respect to identifiable landmarks.
Types of mapping…• genetic mapping• physical mapping
• restriction mapping• cytogenetic mapping• somatic cell mapping• radiation hybrid mapping
• comparative mapping
Introduction
Genetic mapping
Utilize recombination events to estimate distance between genetic markers.
• RFLP• STRP• SNP
Look at a population and estimate the recombination fraction = # recombinants / # total
Introduction
Physical mapping
Relies upon observable experimental outcomes• hybridization• amplification
May or may not have a distance measure.
Genetic Mapping
Requires informative markers -- polymorphicand a population with known relationships
Best if a measured between “close” markers.
Unit of distance in genetic maps = centimorgans, cM
1 cM = 1% chance of recombination between markers
Genetic Mapping
A2
B2
A2
B2
A2
B2
A2
B2
A2
B2
A2
B2
A2
B2
A1
B1
A1
B1
A1
B1
A1
B1
A1
B1
A1
B1
A1
B1
A1
B1
A1
B1
A1
B1
A1
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A1
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A1
B1
A1
B1
A1
B1
A1
B2
A2
B1
A1
B2
A2
B1
NR NRNRNRNR RR
= # recombinant / # total = 2/7 = 0.286
Genetic Mapping
Theta calculation with inbred population…
bn det+
det+bn
bn+ det
detbn+
bn det+
detbn+
bn det
detbn
bn+ det
detbn
bn det+
detbn
bn det
detbn
bn+ det+
detbn
banded detached banded,detached
wild-type
483 512 2 3
x
x
= # recombinant # total = 5/1000 = 0.005
Genetic Mapping
: theoretical maximum of 50%
Best if a measured between “close” markers.
Unit of distance in genetic maps = centimorgans, cMd = - 0.5 ln(1 - 2)d = 0.25 ln[(1 + 2)/(1 - 2)]
1 cM = 1% chance of recombination between markers
Genetic Mapping
Restriction Mapping
Background on restriction enzymes
cut DNA at specific sitesEx. EcoRI cuts at GAATTC
sites are often palindromicGAATTCCTTAAG
may leave blunt ends or overlaps
GGCC GG CCCCGG CC GG
GAATTC G AATTCCTTAAG CTTAA G
Restriction Mapping
Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA.
HindIII BamHI PstIIPstII BamHI
HindIII
EcoRI
Restriction Mapping
BglII BamHI PstIBglII+BamHI
BglII+PstI
BamHI+PstI
4.2
5.2
3.6 3.53.3
2.6
1.7 1.71.4 1.41.2 1.2
1.0 1.01.2
0.7 0.9
0.5
0.3 0.30.3
BglII BamHI PstI BglII PstI
0.3 0.7 2.6 0.9 0.5 1.2
Restriction Mapping
Creating a restriction map from a double digest experiment is NP-complete.
No polynomial-time solution.
As the number of fragments increase, the complexity increases as A!B!.
if the two single-enzyme reactions generate 6 and 8 times respectively,
29,030,400 potential permutations to evaluate
A A!1 12 23 64 245 1206 7207 50408 40,320
Restriction MappingMultiple valid solutions possible.• Reflections• Equivalence
A = {1,3,3,12} B = {1,2,3,3,4,6}A^B = {1,1,1,1,2,2,2,3,6}
A
B
A^B
A’
B’
A^B’
1
1 1 1
1
1
1
1
1 1 1 1
3 3
3 3
3
3 3
3 3
3
2
2 2
2
2 2
2
2
12
12
4
4
6
6
6
6
4320 map configurations, but only 208 distinct solutions.
Cytogenetic Mapping
Cytogenetic mapping refers to observing a map location in reference to a chromosomal banding pattern.
CytogeneticMapping
These methods allow a rough determination of location, but to not yield a direct measure of distance.
Cytogenetic Mapping
Somatic Hybrid Mapping
Somatic cell mapping can be used to map an element to a portion of a genome.
typically with chromosome resolution
Exploits the ability of rodent (hamster) cells to stably integrate genetic material from other species.
Cells from the target genome are fused with hamster cells. The resulting cells are then screened for cells (hybrids) that have retained one or more of the chromosomes from the target genome.
Ideally, a complete set of hybrids can be constructed such that each has retained a single chromosome from the target genome.
Somatic Hybrid Mapping
Probe1Probe2Probe3
1 2 3 4 5Chromosome
0 11 1
1 1 1 1 1
000000
Probe1 -- maps to chromosome 2Probe2 -- maps to chromosomes 3 and 4 -- possible paralogs,
pseudogene, or low-copy repeatProbe3 -- maps to all chromosomes -- possible high-copy repeat
or ribosomal genes
Somatic Hybrid Mapping
EXP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 XWIL1 - - - - - - - - + - - - - - + - - + - - - + - +WIL6 - - + - + + + + + - + + - - + - - + - + + + -WIL7 - - + + - + + - + - + + - + + - - + + - - + - +wil14 + + - + - - - + + - + - + - + + - + - - - - - +SIR3 + + + + + + + + - + + + + + - - + + + + + + + +…% discord 0 32 17 24 31 21 21 31 21 24 30 21 21 28 14 24 21 28 17 34 41 21 27
A subset of the data used to map the Blood Coagulating Factor III to human chromosome 1.
Somatic Hybrid Mapping
Finer mapping (higher resolution) can be obtained if hybrids are present in the panel that contain partial chromosomes.(E.g., translocations)
Such a strategy is expensive, because numerous hybrids have to be screened to identify hybrids containing the partially retained chromosomes.
A more cost-effective and high-resolution alternative is Radiation Hybrid Mapping.
Radiation Hybrid Mapping
Radiation hybrid mapping is a method for high-resolution mapping.
Exploits the ability of rodent cells (hamster cells) to stably incorporate genetic material from fused cells.
Pro: Resolution is “tunable”, relatively cheapCon: Difficult to compare results from different groups
Radiation Hybrid Mapping
Radiation Hybrid Mapping
The data obtained from a radiation hybrid experiment is similar to that from a somatic cell hybrid. It is the retention data for the given locus for each hybrid.
This data is generally displayed as a vector of numbers or letters…
1 or + for retention0 or - for non-retention2 or ? for ambiguous or unknown
Ex.RN_ALB 0100110102010001100100100000102210010..RN_HEM 0101110102000100101100200010100110010..
Radiation Hybrid Mapping
Analytical methods --Many ranging from minimizing the number of obligate breaks to sophisticated methods relying on maximum likelihood or maximum posterior probability methods.
= A+B- + A-B+
TH(RA + RB - 2RARB)
d = - ln (1 - )
NOTE: [0,1]
Summary of Mapping StrategiesMappingStrategy
Requires Resolution How toincreaseresolution
Software
Genetic PolymorphicMarkers, andPedigrees
Medium toHigh
Increasenumber ofmarkers orpeople
GeneHunter
Restriction RestrictionEnzymes
High Increasenumber ofenzymes.
SomaticCell Hybrid
SomaticHybrid Panel,and STSs
Low toMedium
Increasenumber ofdeletion hybrids
RadiationHybrid
RadiationHybrid Panel,and STSs
High Use additionalhybrids, ormake a newpanel.
RHMap,MultiMap,RHMAPPER
Comparative Mapping
Can be very useful in utilizing animal models of human disease, and also in exploring the causes of complex diseases.
Comparing gene content, localization and ordering among multiple species.
Comparative MappingSources of Information
sequence
mapping
BLAST
potentialorthologs
colocalization
sequence
mapping
Putative orthologs andsyntenic segments
Comparative MappingSources of Information
GeneMap 99 (human)• 42,000 ESTS• 12,500 genes
Mouse RH consortium (mouse)• 14,000 ESTs
UIowa EST placements (rat)• 13,793 ESTs
Current Status
Initial comparative map (Welcome Trust and Otsuka Lab)about 500 previously identified orthologs
human-mouse-rat
University of Iowa comparative maps13,973 placed ESTs3057 significant mouse hits9109 significant human EST hits10,148 significant hits to GenBank’s nt database
2479 rat ESTs in preliminary human-rat comparative map1671 rat ESTs in preliminary mouse-rat comparative map
Comparative MappingExamples
RNO18 MMU18
300
600
900
1200
0
100
200
300
400
Comparative MappingExamples
} HSA11
400
500
600
700
}
{
100
200
300
RNO4 HSA7
}
{
{}
}
{
} HSA7p
0
RNO5 {
RNO12 {
RNO12 {
} HSA4
Resources
Genome browsershttp://genome.ucsc.edu/goldenPath/hgTracks.htmlhttp://www.ensembl.orghttp://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search
GeneMap99http://www.ncbi.nlm.nih.gov/genemap99