Single Nucleotide PolymorphismCopy Number Variations

and SNP Array

Xiaole Shirley Liu and

Jun Liu

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Outline• Definition and motivation• SNP distribution and characteristics

– Allele frequency, LD, population stratification• SNP discovery (unknown) and genotyping

(known)– CNV detection

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Polymorphism• Polymorphism: sites/genes with “common”

variation, less common allele frequency ≥1%, otherwise called rare variant and not polymorphic

• First discovered (early 1980): restriction fragment length polymorphism

• Some definitions: – Locus: position on chromosome where sequence

or gene is located– Allele: alternative form of DNA on a locus

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Polymorphism• Single Nucleotide Polymorphism

– Occasionally short (1-3 bp) indels are considered SNPs too

– Come from DNA-replication mistake individual germ line cell, then transmitted

– ~90% of human genetic variation• Copy number variations

– May or may not be genetic

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Why Should We Care• Disease gene discovery

– Association studies, certain SNPs are susceptible for diabetes

– Chromosome aberrations, duplication / deletion might cause cancer

• Personalized Medicine– Drug only effective if you have one allele

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SNP Distribution• Most common, 1 SNP / 100-300 bp

– Balance between mutation introduction rate and polymorphism lost rate

– Most mutations lost within a few generations• 2/3 are CT differences• In non-coding regions, often less SNPs at

more conserved regions• In coding regions, often more synonymous

than non-synonymous SNPs

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SNP Characteristics: Allele Frequency Distribution

• Most alleles are rare (minor allele frequency < 10%)

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Mode of inheritance

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SNP Characteristics:Hardy-Weinberg equilibrium (HWE)– In a population with genotypes BB, bb, and Bb, if p =

freq(B), q =freq(b), the frequencies of BB, bb and Bb will be p2, q2, and 2 pq respectively at equilibrium, and will not change.

– Assumptions for HWE: no mutation, no migration or emigration, infinite population size, no selective pressure, random mating. Could derivate from HWE if violated

– It provides a baseline against which to measure change, e.g., inbreeding index:

– More than 2 alleles:

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SNP Characteristics:Linkage Disequilibrium

• Equilibrium Disequilibrium

• LD: If Alleles occur together more often than can be accounted for by chance, then indicate two alleles are physically close on the DNA– In mammals, LD is often lost at ~100 KB– In fly, LD often decays within a few hundred

bases

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SNP Characteristics:Linkage Disequilibrium

• Statistical Significance of LD– Chi-square test with 1 df– eij = ni. n.j / nT

ji ij

ijij

een

,

22 )(

B1 B2 TotalA1 n11 n12 n1.A2 n21 n22 n2.Total n.1 n.2 nT

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SNP Characteristics:Linkage Disequilibrium

• Three ways to calculate LD

11 1 1

1 2 2 1max max

1 1 2 2

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1 2 1 2

max( , ) 0' / , where

max( , ) 0

D p p q

p q p q if DD D D D

p q p q if D

Drp p q q

ObservedExpected

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SNP Characteristics:Linkage Disequilibrium

• Haplotype block: a cluster of linked SNPs• Haplotype boundary: blocks of sequence

with strong LD within blocks and no LD between blocks, reflect recombination hotspots

• Haplotype size distribution

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SNP Characteristics:Linkage Disequilibrium

• Can see haplotype block: a cluster of linked SNPs

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SNP Characteristics:Linkage Disequilibrium

• [C/T] [A/G] T X C [A/C] [T/A]– Possible haplotype: 24

– In reality, a few common haplotypes explain 90% variations

• Tagging SNPs: – SNPs that capture

most variations in haplotypes

– removes redundancy

Redundant

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SNP Characteristics:Population Stratification

• Population stratification: individuals selected from two genetically different populations, stratification may be environmental, cultural, or genetic

• Could give spurious results in case control association studies – the example of “chopstick genes”

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Using genetic variation to study populations

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SNP Discovery Methods• Sequencing individuals for difference: too costly • First check whether big regions have SNPs

– Basic idea: denature and re-anneal two samples, detect heterduplex

– Can pool samples (e.g. 10 African with 10 Caucasians) to speed screening

• Resequence to verify• dbSNP: 12M RefSNP, 6M validated

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SNP Genotyping• For a known locus TT C/A AG, does this individual

have CC, AA or AC? Many methods• Hybridization-based methods

– Dynamic allele-specific hybridization– Molecular beacons– SNP-array chip (simultaneously genotype thousands of SNPs)

• Enzyme-based methods– RFLP– PCR-based methods– Flap endonuclease– Primer extension– Oligonucleotide ligase assay

• Other methods (based on physical properties of DNA)

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SNP Array• One SNP at a time or genome-wide (SNP array)

2.5kb5.8kb0.30

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40 Probes Used Per SNP• Allele call

– AA, BB, AB• Signal

– Theoretically 1A+1B, 2A, 2B– But couldhave 1A+3BAmplified!

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T

SNP Chip for LOH• Loss of Heterozygosity: tumor suppressor

gene inactivation by allelic loss in cancers

T T

Normal First genetic hit Cancer

XOR

T T X TX TXA B A A AA B

LOH

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SNP Array for CNV• Collect normal / diseased samples on SNP arrays• Probe normalization, background subtraction

• Use HMM to infer CNV

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Integrate CNV with Expression toIdentify oncogene MITF in melanoma

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Summary• SNP and CNV• SNP distribution and characteristics

– Allele frequency (minor allele > 1%)– LD: linkage ~ physical proximity– Population stratification

• SNP discovery: heteroduplex• SNP genotyping

– SNP array– CNV detection: HMM

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Acknowledgement• Stefano Monti• Tim Niu• Kenneth Kidd, Judith Kidd and Glenys

Thomson• Joel Hirschhorn• Greg Gibson & Spencer Muse