Cmmb Wk11 Mapping

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 Θ = # 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 -- polymorphic and 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 A2 B2 B2

A2 A1 B2 B1

A1 A1 B2 B1

A2 A1 B2 B1 A2 A1 B2 B1

NR

A1 A1 B1 B1

NR

A2 A1 B1 B1

A1 A1 B1 B1 A1 A1 B2 B1

A2 A1 B2 B1

R

NR

A1A1 B1 B1

NR

A2 A1 B1 B1

R

A2 A1 B2 B1

NR

Θ = # recombinant / # total = 2/7 = 0.286

Genetic Mapping Theta calculation with inbred population… bn

det+

bn

det+

bn

bn+ det

x

bn+ det

det+

x

bn+ det

bn

det

bn

det

bn

det+

bn+ det

bn

det

bn+ det+

bn

det

bn

bn

det

bn

banded

483

det

det

detached banded, wild-type detached

512

2

3

Θ = # recombinant # total = 5/1000 = 0.005

Genetic Mapping Θ : theoretical maximum of 50% Best if a measured between “close” markers. Unit of distance in genetic maps = centimorgans, cM d = - 0.5 ln(1 - 2Θ ) d = 0.25 ln[(1 + 2Θ )/(1 - 2Θ )] 1 cM = 1% chance of recombination between markers

Genetic Mapping

Restriction Mapping Background on restriction enzymes cut DNA at specific sites Ex. EcoRI cuts at GAATTC sites are often palindromic GAATTC CTTAAG may leave blunt ends GGCC CCGG

GG CC

CC GG

or overlaps GAATTC CTTAAG

G CTTAA

AATTC G

Restriction Mapping Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA. EcoRI HindIII PstII

BamHI

HindIII BamHI

PstII

Restriction Mapping BglII

BamHI

BglII +BamHI

PstI

BamHI +PstI

BglII +PstI

5.2 4.2 3.6

3.5

3.3 2.6

1.7

1.7 1.4 1.2 1.0

0.3

BamHI 0.7

PstI

BglII PstI

0.7

0.3 BglII

0.3

1.2 0.9 0.5 0.3

2.6

0.9

0.5

1.4 1.2 1.0

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 1 2 3 4 5 6 7 8

A! 1 2 6 24 120 720 5040 40,320

Restriction Mapping Multiple 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} 3

1

12

2

4

11 1

2

2

6

3

6

3

3

3 1

12

3 1 2

3

1

1

2 3

3

6

1

2

2

6

1

2

4 1

A

3

1

B A^B A’ B’ A^B’

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.

Cytogenetic Mapping 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 Probe1 Probe2 Probe3

1 0 0 1

Chromosome 2 3 4 1 0 0 0 1 1 1 1 1

5 0 0 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 genes

Somatic Hybrid Mapping EXP + +

WIL1 WIL6 WIL7 wil14 SIR3 … % discord

1 + +

2 + + +

3 + + +

4 + +

5 + + +

6 + + +

7 + + +

8 + + + + -

9 10 11 12 13 - - - - - + + - - + + - + - + - + + + + + +

14 15 16 + - + - + - + + - - +

17 18 19 20 21 22 + - - - + + - + + + + + - - + + - - - - + + + + + +

X +

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 cheap Con: 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 retention 0 or - for non-retention 2 or ? for ambiguous or unknown Ex. RN_ALB RN_HEM

0100110102010001100100100000102210010.. 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. Θ = A+B- + A-B+ TH(RA + RB - 2RARB) d = - ln (1 - Θ ) NOTE: Θ ∈ [0,1]

Summary of Mapping Strategies Mapping Strategy

Requires

Resolution How to increase resolution

Software

Genetic

Polymorphic Markers, and Pedigrees

GeneHunter

Restriction

Restriction Enzymes

Medium to Increase High number of markers or people High Increase number of enzymes.

Somatic Cell Hybrid

Somatic Hybrid Panel, and STSs

Low to Medium

Increase number of deletion hybrids

Radiation Hybrid

Radiation Hybrid Panel, and STSs

High

Use additional hybrids, or make a new panel.

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 Mapping Sources of Information sequence

BLAST

sequence

mapping

mapping potential orthologs

colocalization

Putative orthologs and syntenic segments

Comparative Mapping Sources 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 maps 13,973 placed ESTs 3057 significant mouse hits 9109 significant human EST hits 10,148 significant hits to GenBank’s nt database 2479 rat ESTs in preliminary human-rat comparative map 1671 rat ESTs in preliminary mouse-rat comparative map

Comparative Mapping Examples 0

RNO18

MMU18 1200

100 900 200 600 300 300 400

Comparative Mapping Examples

0

RNO4 } }

100 200 300

} HSA11 }

} } HSA7p } HSA4

HSA7 RNO12 { {

400

{ RNO12 { {

500

{ RNO5 {

600 700

Resources Genome browsers http://genome.ucsc.edu/goldenPath/hgTracks.html http://www.ensembl.org http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search GeneMap99 http://www.ncbi.nlm.nih.gov/genemap99