Mapping

1
Mapping

• April 7, 2005
• Todd Scheetz

2
Introduction
What is mapping? determining the location of
elements within a genome, with respect to
identifiable landmarks. Resolution
(lowest)
(highest)
Present in the genome
Base-pair resolution
3
Introduction

• Earliest examples noticing that certain traits
  co-segregate across generations -- linked.
  (Mendels peas)
• Types of mapping
• genetic mapping
• physical mapping
• restriction mapping
• cytogenetic mapping
• somatic cell mapping
• radiation hybrid mapping
• comparative mapping

4
Introduction
Genetic mapping Utilize recombination events to
estimate distance between genetic markers. Look
at a population and estimate the recombination
fraction ? recombinants / total Best if a
measured between close markers. Unit of
distance in genetic maps centimorgans, cM 1 cM
1 chance of recombination between markers
5
Genetic mapping example I

• Genes on two different chromosomes
• Independent assortment during meiosis
• No linkage

F1
9

3

3

1
6
Genetic mapping example II

• Genes very close together on same chromosome
• Will usually end up together after meiosis
• Tightly linked

F1
1

2

1
7
Genetic mapping example III

• Genes on same chromosome, but not very close
  together
• Recombination will occur
• Frequency of recombination proportional to
  distance between genes
• Measured in centiMorgans

recombinants
8
Genetic Mapping

• Requires informative markers -- polymorphic
• and a population with known relationships
• RFLP random fragment length polymorphism
• STRP short tandem repeat polymorphism
• SNP single nucleotide polymorphism
• Greater polymorphism is desirable.

A2
A2
A2
A1
A2
A1
n
n
n
PIC 1 - ? pi2 - ? ? pi2 pj2
i1
i1
ji1
9
RFLP

• Restriction-fragment length polymorphism
• Cut genomic DNA from two individuals with
  restriction enzyme
• Run Southern blot
• Probe with different pieces of DNA
• Sequence difference creates different band pattern

1
2
200
400


GGATCC CCTAGG
GGATCC GCTAGG
GGATCC CCTAGG
1
600
400

200
400
GGATCC CCTAGG
GCATCC GGTAGG
GGATCC CCTAGG
200
2

10
SSLP

• Simple-sequence length polymorphism
• Most genomes contain repeats of three or four
  nucleotides
• Length of repeat varies
• Use PCR with primers external to the repeat
  region
• On gel, see difference in length of amplified
  fragment

1
2
ATCCTACGACGACGACGATTGATGCT
1
18
12
2
ATCCTACGACGACGACGACGACGATTGATGCT
11
SNP

• Single-nucleotide polymorphism
• One-nucleotide difference in sequence of two
  organisms
• Found by sequencing
• Example Between any two humans, on average one
  SNP every 1,000 base pairs

ATCGATTGCCATGAC ATCGATGGCCATGAC
1
2
SNP
12
Genetic Mapping
A2 B2
A2 B2
A2 B2
A1 B1
A1 B1
A1 B1
A1 B2
A2 B1
A2 B2
A1 B1
A1 B1
A1 B1
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 B2
A2 B1
NR
NR
NR
NR
NR
R
R
? recombinant / total 2/7 0.286
13
Genetic Mapping
Theta calculation with inbred populations/strains
bn
det
bn
det
x
det
bn
det
bn
bn
det
bn
det
x
det
bn
det
bn
bn
det
bn
det
bn
det
bn
det
det
bn
det
bn
det
bn
det
bn
? recombinant total
5/1000 0.005
banded
detached
banded, detached
wild-type
483
512
2
3
14
Genetic Mapping

• ? theoretical maximum of 50
• independent assortment
• Most accurate when measured between close
  markers.
• Unit of distance in genetic maps centimorgans,
  cM
• d - 0.5 ln(1 - 2?)
• d 0.25 ln(1 2?)/(1 - 2?)
• 1 cM 1 chance of recombination between markers

15
Genetic Mapping
16
Fundamental Genetics (Background for Linkage
Analysis)

• Rule of Segregation
• offspring receive ONE allele (genetic material)
  from the pair of alleles possessed by BOTH
  parents
• Rule of Independent Assortment
• alleles of one gene can segregate independently
  of alleles of other genes
• (Linkage Analysis relies on the violation of
  Independent Assortment Rule)

17
Examples
18
Examples
19
Example
20
BBS4 Pedigree
21
Hardy-Weinberg Equilibrium

• Rule that relates allelic and genotypic
  frequencies in a population of diploid, sexually
  reproducing individuals if that population has
  random mating, large size, no mutation or
  migration, and no selection
• Assumptions
• allelic frequencies will not change in a
  population from one generation to the next
• genotypic frequencies are determined in a
  predictable way by allelic frequencies
• the equilibrium is neutral -- if perturbed, it
  will reestablish within one generation of random
  mating at the new allelic frequency

22
(No Transcript)
23
H-W

• f(AA) p2
• f(Aa) 2pq
• f(aa) q2
• (pq)2
• (p2 q2 r2 2pq 2pr 2qr) (pqr)2

24
Dominant and Recessive Penetrance
Modeledpenetrance P(pt gt)

• DD Dd dd
• 1 1 0
• DD Dd dd
• 0.9 0.9 0.0

• DD Dd dd
• 0 0 1
• DD Dd dd
• 0 0 0.8

25

• Given the following observations family
  structure, affection status, genotypes, and
  disease allele frequencies. Assuming a model for
  the disease, can we calculate the probability
  that these observations fit an assumed model???

26
Linkage Analysis

• Goal find a marker linked to a disease gene.
• LOD score log of likelihood ratio
• LR?data k Pdata ?
• theta estimate of genetic distance
  (recombination fraction) between marker and
  disease
• proportion of recombinant gametes/total gametes

27
Linkage Analysis

• Linkage analysis calculates the likelihood that
  the inheritance pattern of the phenotype
  (disease) is supported by the observed
  inheritance patterns (genotypes) in a pedigree.
• few monogenic models, easy to test
• more difficult to find models explaining
  inheritance in polygenic models
• parameter maximization

28
Linkage Analysis Programs

• FASTLINK - 2 point
• O(n2), where n number of markers
• GeneHunter - multipoint, 2 point
• O(n2), where n number of people

29
Allele Sharing

• tries to show that affected family members
  inherit the same chromosomal regions more often
  than expected by chance

30
Allele Sharing Example
Needs at least sibs.
31
Association Studies

• Allelic association studies provide the most
  powerful method for locating genes of small
  effect contributing to complex diseases and
  traits. Daniels, Am J Hum Genet 621189-1197,
  1998.
• Linkage analysis
• genome wide screen, 400 markers 10 cM (10 MB),
  association needs 4000 polymorphic markers
• generally need nuclear family or larger
• Association finds linkage disequilibruim

32
Association Studies

• Association is simply a statistical statement
  about the co-occurrence of alleles or phenotypes.
  Allele A is associated with disease D if people
  who have D also have A more (or maybe less) often
  than would be predicted from the individual
  frequencies of D and A in the population. Pg.
  286 Human Molecular Genetics 2, Tom Strachan

33
Examples

• HLA-DR4 (antigen marker)
• 36 in UK
• 78 with rheumatoid arthritis
• CF( RFLP markers XV2.c (X1,X2), KM19(K1,K2))
• Marker Alleles CF(case) Normal(control)
• X1, K1 3 49
• X1, K2 147 19
• X2, K1 8 70
• X2, K2 8 25
• CF associated with X1, K2 in 89 (Strachan)

34
Linkage Disequilibrium

• linkage equilibrium (aka Hardy-Weinberg) is true
  if
• P(gt1,gt1gt2,gt2) P(gt1,gt1)P(gt2,gt2)
  where P(haplotype)
• case vs controls
• TDT (heterozygous marker transmitted), HRR
  (untransmitted alleles as control)
• allelic associations (outbred populations)
  maintained at only lt 1cM

35
Homozygosity Mapping
36
BBS2 genetic mapping
C16
1 2 3 4 5 6 7 8 9 10 11 12
37
BBS2 genetic mapping
unaffected
affected
C16
1 2 3 4 5 6 7 8 9 10 11 12
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Introduction

• Physical mapping
• Relies upon observable experimental outcomes
• hybridization
• amplification
• May or may not have a distance measure.

39
Restriction Mapping
Background on restriction enzymes cut DNA at
specific sites Ex. EcoRI cuts at GAATTC sites
are often palindromic 5- GAATTC -3 3- CTTAAG
-5 may leave blunt ends
or overlaps
GGCC GG CC CCGG CC GG
GAATTC G AATTC CTTAAG CTTAA G
40
Restriction Mapping
Restriction maps show the relative location of a
selection of restriction sites along linear or
circular DNA.
EcoRI
HindIII
BamHI
PstII
HindIII
BamHI
PstII
41
Restriction Mapping
BamHI PstI
BglII BamHI
BglII PstI
BglII
BamHI
PstI
5.2
4.2
3.6
3.5
3.3
2.6
1.7
1.7
1.4
1.4
1.2
1.2
1.2
1.0
1.0
0.9
0.7
0.5
0.3
0.3
0.3
BglII
BamHI
PstI
BglII
PstI
0.3
0.7
2.6
0.9
0.5
1.2
42
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
fragments respectively, 29,030,400 potential
permutations to evaluate
A A! 1 1 2 2 3 6 4 24 5 120 6 720 7 5040 8 40,320
43
Restriction Mapping

• Multiple valid solutions possible.
• Reflections
• Equivalence
• A 1,3,3,12 B 1,2,3,3,4,6
• AB 1,1,1,1,2,2,2,3,6

3
3
12
1
4320 map configurations, but only 208 distinct
solutions.
A
1
3
3
6
2
4
B
1
1
3
2
1
1
2
2
6
AB
1
3
3
12
A
1
3
3
2
4
6
B
1
1
1
3
1
2
2
2
6
AB
44
Cytogenetic Mapping
Cytogenetic mapping refers to observing a map
location in reference to a chromosomal banding
pattern.
45
CytogeneticMapping
These methods allow a rough determination of
location, but to not yield a direct measure of
distance.
46
Cytogenetic Mapping
47
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.
48
Somatic Hybrid Mapping
Chromosome
1
2
3
4
5
Probe1
0
1
0
0
0
Probe2
1
1
0
0
0
Probe3
1
1
1
1
1
Probe1 -- maps to chromosome 2 Probe2 -- maps to
chromosomes 3 and 4 -- possible paralogs,
pseudogene, or low-copy repeat Probe3 -- maps
to all chromosomes -- possible high-copy
repeat or ribosomal gene
49
Somatic Hybrid Mapping
A subset of the data used to map the Blood
Coagulating Factor III to human chromosome 1.
50
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.
51
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
cheap Con Difficult to compare results from
different groups
52
Radiation Hybrid Mapping
53
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 retention 0 or - for non-retention 2 or ?
for ambiguous or unknown Ex. RN_ALB 0100110102010
001100100100000102210010.. RN_HEM 0101110102000100
101100200010100110010..
54
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. ? AB- A-B TH(RA RB -
2RARB) d - ln (1 - ?) NOTE ? ? 0,1
RA retention of A RB retention of B TH
total number of hybrids
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Summary of Mapping Strategies
56
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.
57
Comparative MappingSources of Information
sequence
sequence
BLAST
mapping
mapping
potential orthologs
colocalization
Putative orthologs and syntenic segments
58
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

59
Current Status
Initial comparative map (Welcome Trust and Otsuka
Lab) about 500 previously identified
orthologs human-mouse-rat University of Iowa
comparative maps 13,973 placed ESTs 3057
significant mouse hits 9109 significant human EST
hits 10,148 significant hits to GenBanks nt
database 2479 rat ESTs in preliminary human-rat
comparative map 1671 rat ESTs in preliminary
mouse-rat comparative map
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Comparative MappingExamples
RNO18
MMU18
0
1200
100
900
200
600
300
300
400
61
Comparative MappingExamples
HSA7
RNO4
400
0
RNO12




100
500
RNO12
HSA11


600
200

RNO5

700
300
HSA7p
HSA4
62
Resources
Genome browsers http//genome.ucsc.edu http//ww
w.ensembl.org http//www.ncbi.nih.gov/