SNPs and Variation

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SNPs and Variation
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• Most variation at the DNA level comes in the form
  of small insertions and deletions and single
  nucleotide polymorphisms (SNP).
• A substitutional variation must occur in at least
  1 of the population to be considered as a SNP.
• A haplotype is a cluster of SNPs.

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High-throughput Sequencing
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SNP
homozygous
A
C
A/C
A/C
A substitutional variation must occur in at least
1 of the population to be considered as a SNP.
heterozygous
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Haplotype a cluster of SNPs
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The Nature of Single Nucleotide Polymorphisms

• Classification of SNPs
• nucleotide change by way of transition (purine
  to purine, or pyrimidine to pyrimidine) and
  transversion (purine to pyrimidine, or pyrimidine
  to purine).
• 1. Noncoding SNP
• 2. Coding SNP

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Noncoding SNP
Flanking region
Flanking region
5'
3'

GT
AG
GT
AG
GC box
Initiation codon
Stop codon
Poly(A)-addition site
CAAT box
TSS
AATAA
GC box
TATA box
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Coding SNP
Flanking region
Flanking region
5'
3'

GT
AG
GT
AG
GC box
Initiation codon
Stop codon
Poly(A)-addition site
CAAT box
TSS
AATAA
GC box
TATA box
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Coding SNP
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3.1 (Part 1) Human promoter SNPs that affect
gene expression
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3.1 (Part 2) Human promoter SNPs that affect
gene expression
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SNP Database
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SNP database of disease related loci.
http//pga.mbt.washington.edu/
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Distribution of SNPs

• Population genetics study the distribution of
  genetic variants.
• Quantitative genetics study the relationship of
  variants and phenotypes.
• Quantitative trait loci (QTL) a region of DNA
  that is associated with a particular phenotypic
  trait - these QTLs are often found on different
  chromosomes. Knowing the number of QTLs that
  explains variation in the phenotypic trait tells
  us about the genetic architecture of a trait.

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• Neutral theory most SNPs are maintained in
  natural populations as a result of a balance
  between mutation and genetic drift.
• The dynamics of genotypic variation are described
  by the balance of mutation, drift, migration, and
  selection.
• Neutral theory supplies the null hypothesis when
  the pattern of variants are assessed.

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• Three key concepts for characterizing SNP
  variation
• 1. Allel frequency distribution
• 2. Linkage disequilibrium
• 3. Population stratification

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3.2 (Part 1) Nucleotide diversity in natural
populations
874 SNPs from 75 candidate human hypertension
loci.
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3.2 (Part 2) Nucleotide diversity in natural
populations
LD decays with time regarding recombination rate
r.
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Linkage disequilibrium and Haplotype Maps

• 1. Non-random assortment of alleles.
• 2. Typically occurs over kbs.
• 3. Measures based 2 SNPs system A/a B/b.

The probability of each haplotype (for two
allele)
chromosome recombination

• PAB ? PAPB
• PAb ? PAPb PA(1-PB)
• PaB ? PaPB (1-PA) PB
• Pab ? PaPb (1-PA) (1-PB)

A
b
There are 4 possible haplotypes for SNP sites A/a
and B/b.
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D Coefficient

• We can measure the non-randomness of two loci by
  means of a deviation, D, defined as follows
• D PAB PAPB or PABPab PAbPaB
• PAB PAPB D
• PAb PA(1-PB) - D
• PaB (1-PA) PB - D
• Pab (1-PA) (1-PB) D
• These two SNPs are linkage equilibrium iff D 0.

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Let D PAB PAPB , and D D/Dmax, where Dmax
stands for the absolute maximal possible value of
D.
D
D
0
-PAPB
PAPB

• D is independent of allele frequencies, 1 gt D
  gt -1.
• D allow us to compare LD at different
  combination of loci.

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• D is constrained between -1 and 1.
• D 1 (perfect positive LD between SNP alleles)
• D 0 (linkage equilibrium between SNP alleles)
• D -1 (perfect negative LD between SNP alleles)
• D 0.87 (strong positive LD between SNP
  alleles)
• D 0.12 (weak positive LD between SNP alleles)
• Other measures of D coefficient
• r2 or ?2
• Chi-square Test.
• Fisher exact test (for small samples).
• P value.

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• A set of closely linked SNPs located on one
  chromosome.

SNP 1
SNP 2
SNP 3
GATATTCGTACGGA-T GATGTTCGTACTGAAT GATATTCGTACGGA-T
GATATTCGTACGGAAT GATGTTCGTACTGAAT GATGTTCGTACTGAA
T
Haplotypes
AG- 2/6 GTA 3/6 AGA 1/6
DNASequences
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3.3 Distribution of linkage disequilibrium
across the human lipoprotein lipase (LPL) gene
The significance is inferred according to Fisher
exact test. Blue box significance,
p lt 0.001 Yellow box nonsignficance. White
unknown.
66 SNP sites in this genes
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Haplotype Blocks in Human Genome

• The human genome has been shown to contain
  regions of high LD interspersed by regions of low
  LD.
• The recombination occurs frequently in low LD
  regions.
• The high LD regions can form haplotype blocks.
• The International HapMap Project aims to build
  the haplotype map across human genome.

Recombination hot spots(Low LD regions, D lt 0)
Haplotype blocks(High LD regions, D gt 0)
Chromosome
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3.4 (Part 1) Distribution of LD in the human
genome
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3.4 (Part 2) Distribution of LD in the human
genome
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3.5 Tagging SNPs and the haplotype clusters
Tagging SNPs capture most of the nucleotide
variation included in haplotypes.
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3.6 Haplotype structure in the human lipoprotein
lipase (LPL) gene
(82 unrelated humans)
Only 8 of all haplotypes are unique regarding
racial groups
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3.7 Human diversity and population structure
Genotyping of 377 microsatellites in 1056
individuals.
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Applications of SNP Technology

• Population Genetics
• Recombination Mapping
• QTL Mapping
• Linkage Disequilibrium Mapping

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SNP Discovery

• Resequencing
• SNP identification
• SNP verification
• Sequence-free Polymorphism Detection

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Resequencing

• SNP are discovered by comparing sequences derived
  from different chromosomes.
• The probability of detecting a SNP in the
  condition of two alleles is given by
• P1-(1-p)2n
• where p is the frequency of rare allele and n
  is the comparison number of individuals.

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SNP identification

• Two major methods can be used to identify SNPs.
• 1. Comparison of whole genome sequence with
  cDNA (EST) sequences which from different
  strands.
• 2. Comparison of genomic sequences derived
  from different individuals.
• There is no way to determine whether a difference
  is the result of a sequencing error in original
  sequences.

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SNP verification

• PCR amplification of the specific fragment
  including SNP in a sample of at least 10
  individuals and resequencing.
• Sequencing by hybridization use sequencing chip.

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3.15 Sequencing by hybridization
One chip per individual
25-mer probe, N represent the variation site.
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Sequence-free Polymorphism Detection

• Denaturing High Performance Liquid Chromatography
  (DHPLC)
• Denaturing Gradient Gel Electrophoresis (DGGE)
  and Single-Strand Conformation Polymorphism
  (SSCP)
• Targeting Induced Local Lesion IN Genomes
  (TILLING)

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DHPLC
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DGGE and SSCP analysis
acrylamide gel
W and C are complementary strands
genotypes
Single-strand secondary structures
urea gradient
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GC-clamp
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AT, CG, AG, CT Four types of CG clamps
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Tilling
EthylMethaneSulfonate
Pools of rows and columns
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3.17 (Part 2) Tilling
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SNP Genotyping

• Low-Technology Methods
• Minisequencing Methods
• Homogeneous Fluorogenic Dye-Based Methods
• Haplotype Phasing Methods

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Low-Technology Methods

• All current SNP genotyping methods except Invader
  assay depend on specific amplification of the DNA
  sequence surrounding the site to be genotyped,
  which is generally achieved using the PCR.
• 1. PCR-RFLP method
• 2. dCAPS method
• 3. ASO method

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3.18 (Part 1) Bulked segregant mapping of
mutations using Snip-SNPs
PCR-RFLP method
1/5 1/2 of all SNPs contained in palindromic
segments
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3.18 (Part 2) Bulked segregant mapping of
mutations using Snip-SNPs
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3.19 dCAPS
dCAPS method
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ASO method
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Minisequencing Methods

• A philosophically different approach to
  genotyping is to actually resequence each allele
  only one or a few bases in a sample of
  individuals.
• 1. Single-base extension
• 2. Pyrosequencing

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3.20 (Part 1) Single-base extension methods
Single-base extension
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3.20 (Part 2) Single-base extension methods
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3.21 Pyrosequencing
Pyrosequencing
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Homogeneous Fluorogenic Dye-Based Methods

• Homogeneous assays are those that are carried out
  in a single reaction in solution.
• 1. TaqMan
• 2. Molecular beacons
• 3. Dye-labeled oligonucleotide ligation
• 4. The Invader assay

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3.22 (Part 1) Fluorogenic dye-based genotyping
methods
TaqMan
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3.22 (Part 2) Fluorogenic dye-based genotyping
methods
Molecular beacons
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3.22 (Part 3) Fluorogenic dye-based genotyping
methods
Dye-labeled oligonucleotide ligation
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3.23 (Part 1) The Invader assay
The Invader assay
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3.23 (Part 2) The Invader assay
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3.23 (Part 3) The Invader assay
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Haplotype Phasing Methods

• Consider two adjacent SNPs A/T and A/C, the
  haplotypes could be AA and TC or TA and AC.
• SNPs --------A/T--------- ----------A/C--------
  --
• --------A-------------------------A---------
  ----
• --------T-------------------------C---------
  ----
• or
• --------T-------------------------A---------
  ----
• --------A-------------------------C---------
  ----