Single Nucleotide Polymorphism

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Single Nucleotide Polymorphism
Anshu Bhardwaj Research Fellow Centre for
Cellular Molecular Biology Hyderabad 8th
November, 2003
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Single Nucleotide Polymorphism
Single base-pair differences occurring in a
population with a frequency of gt1
...C C A T T G A C...
G G T A A C T G...
...C C G T T G A C...
G G C A A C T G...
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SNPs can be found in..

• NON-CODING REGION
• 5 and 3 UTRs
• Introns
• splice sites

• CODING REGION

Non-synonymous Amino acid substitution
Synonymous Silent
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Single base-pair differences occurring in a
population with a frequency of gt1
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GENOTYPIC FREQUENCY Relative distribution of
genotypes in a population for a particular locus
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ALLELIC FREQUENCY The relative abundance of an
allele of a particular gene with reference to its
other alleles
Let pf(M) and qf(N). Thus, pf(MM) ½ f(MN)
and qf(NN) ½ f(MN).
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ALLELIC FREQUENCY The relative abundance of an
allele of a particular gene with reference to its
other alleles
Percent
p
q
MM
MN
NN
Location
83.5
15.6
0.9
0.92
0.08
Greenland
Let pf(M) and qf(N). Thus, pf(MM) ½ f(MN)
and qf(NN) ½ f(MN).
GENOTYPIC FREQUENCY Relative distribution of
genotypes in a population for a particular locus
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WHY SNPs ? ?

• SNPs are distributed non-randomly throughout the
  genome

• On an average a significant SNP is found for
  every 1kb of
• the human genome, resulting in approximately
  3 million SNPs

• Large number
• Unambiguous assay techniques
• High levels of polymorphisms in population

• Most of the phenotypic differences arise from
  SNPs in
• genes, but these form only a small fraction of
  the total number

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dbSNP DENSITY DISTRIBUTION IN HUMAN

• Mean Density
• 0.001765 SNPs per base (17.652 SNPs per 10 kb)
• Mean Spacing
• 566.5118 bases per SNP

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SNP Discovery

• SNP Discovery refers to the initial
  identification of new
• SNPs
• The established method is electrophoresis(DNA
  sequencing)
• with subsequent data analysis. Some indirect
  Discovery
• techniques (e.g., dHPLC, SSCP) only indicate
  that a SNP
• (or other mutation) exists
• DNA sequencing of multiple individuals is used
  to determine
• the point and type of polymorphism

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SNP Validation

• SNP Validation refers to genetic validation, the
  
• process of ensuring that the SNP is not due
  to
• sequencing error
• Confirmation of SNPs found in discovery
• Larger numbers of individual samples to get
  statistical
• data on occurrence in the population

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• THE EXPERIMENTAL APPROACH
• RESTRICTION FRAGMENT LENGTH POLYMORPHISM
• SINGLE STRANDED CONFORMATIONAL POLYMORPHISM
• DENATURING HIGH PRESSURE LIQUID CHROMATOGRAPHY
• HYBRIDIZATION METHOD
• MALDI-TOF METHOD

SEQUENCING ALIGNMENT THEREAFTER
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• THE EXPERIMENTAL APPROACH
• RESTRICTION FRAGMENT LENGTH POLYMORPHISM
• SINGLE STRANDED CONFORMATIONAL POLYMORPHISM
• DENATURING HIGH PRESSURE LIQUID CHROMATOGRAPHY
• HYBRIDIZATION METHOD
• MALDI-TOF METHOD

SEQUENCING ALIGNMENT THEREAFTER
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IN SILICO SNP PREDICTION
POLYBAYES
SEAN SNP Prediction Program
SNP Finder
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IN SILICO SNP PREDICTION
POLYBAYES
SEAN SNP Prediction Program
SNP Finder
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Restriction Fragment Length Polymorphisms
Botstein et al (1980)
CHANGES IN MIGRATION PATTERNS THAT REPRESENT
ALLELIC VARIATION
A
3 Kb
Homolog 1
12 A
12 B
12 C
Homolog 2
1 Kb
2 Kb
PROBE
B
3 Kb
Homolog 1 2
C
Homolog 1 2
2 Kb
1 Kb
CAN BE USED TO DETECT SNPs DIFFERENTIALLY IN
HOMOZYGOUS HETEROZYGOUS INDIVIDUALS
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MALDI-TOF METHOD
Matrix-assisted laser desorption ionization-time
of flight
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SEQUENCING METHOD
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POLYBAYES BAYESIAN INFERENCE ENGINE TO CALCULATE
THE PROBABILITY THAT A GIVEN SITE IS POLYMORPHIC

• FRAGMENT CLUSTERING

• PARALOGUE IDENTIFICATION

• MULTIPLE ALIGNMENT

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SNP DETECTION IN REDUNDANT SEQUENCE DATA
SEQUENCE CLUSTERING CLUSTER REFINEMENT MULTIPL
E ALIGNMENT SNP DETECTION
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The PolyBayes Approach

• Use genomic sequence as reference
• cluster and align all available sequences
• remove repeats/paralogs
• Use Bayesian statistics to
• distinguish polymorphic sites from artifacts
• estimate likelihood
• Marth, GT, Korf, I, Yandell, MD, Yeh, RT, Gu, Z,
  Zakeri, H, Stitziel, NO, Hillier, L, Kwok, P-Y,
  Gish, WR A general approach to single-nucleotide
  polymorphism discovery. Nature Genet. 1999
  23452-456.

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1. Known repeat sequences are masked using
RepeatMasker
2. FRAGMENT CLUSTERING (a) WU-BLAST used to
search against dbEST (b) Sequence traces
processed with PHRED base-calling values
(c) Distinct group of matching ESTs registered
as clusters
3. Each cluster member pair-wise aligned to the
genomic anchor sequence with CROSS_MATCH
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PARALOGUE IDENTIFICATION
1. May give rise to false SNP predictions
points to difficulties during marker development
2. Calculate probability PNAT that a cluster
member is derived from genomic region.
3. Distinguish between less accurate sequences
that nevertheless originate from the same
underlying genomic location More accurate
sequences with high-quality discrepancies that
are likely to be paralogous
4. Using a threshold value PNAT,MIN paralogous
cluster members are removed
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DNAT L PPOLY.2 E (PPOLY.2 0.001) DPAR
L PPAR E (PPAR 0.02)
d discrepancies
P(MODELNATD)
PNAT,MIN 0.75
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MULTIPLE ALIGNMENT

1. Depth of coverage
2. The base-quality values of the sequences
3. The a priori expected rate of polymorphic sites
   in the region

• PSNP ? PROBABILITY THAT THE SITE IS POLYMORPHIC
  

• DISTRIBUTION OF PROBABILITY SCORES EXHIBITS A
• HIGH LEVEL OF SPECIFICITY

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THRESHOLD VALUE PSNP 0.4
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THE POLYBAYES SOFTWARE
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OTHER SNP PREDICTION SNP FINDING SOFTWARE

• SEAN Search for localized SNPs
• and predict SNPs
• (http//zebrafish.doc
  .ic.ac.uk/Sean/)
• SNP Finder For analyzing user-submitted
• trace data
• (http//gai.nci.nih.g
  ov/)

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SIGNIFICANCE OF SNPs

• IN DISEASE DIAGNOSIS
• IN FINDING PREDISPOSITION TO DISEASES
• IN DRUG DISCOVERY DEVELOPMENT
• IN DRUG RESPONSES
• INVESTIGATION OF MIGRATION PATTERNS
• ALL THESE ASPECT WILL HELP TO LOOK FOR MEDICATION
  DIAGNOSIS AT INDIVIDUAL LEVEL

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SNP Screening

• Two different screening strategies
• - Many SNPs in a few individuals
• - A few SNPs in many individuals
• Different strategies will require different
  tools
• Important in determining markers for complex
  genetic
• states

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SNP genotyping methods for detecting genes
contributing to susceptibility or resistance to
multifactorial diseases, adverse drug reactions
gt case-control association analysis
.GCCGTTGAC. .GCCATTGAC. .GCCATTGAC. .GCCAT
TGAC.
case
control
allele frequency genotype frequency
haplotype frequency A , G AA ,
AG , GG SNP1, SNP2, SNP3
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HAPLOTYPE
A set of closely linked genetic markers present
on one chromosome which tend to be inherited
together (not easily separable by recombination)
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SNP-Haplotype
SNP
SNP
BLACK EYE BROWN EYE BLACK EYE BLUE EYE BROWN
EYE BROWN EYE
GATATTCGTACGGA-T GATGTTCGTACTGAAT GATATTCGTACGGA-T
GATATTCGTACGGAAT GATGTTCGTACTGAAT GATGTTCGTACTGAA
T
Haplotypes
AG 2/6(BLACK EYE) GTA 3/6(BROWN EYE) AGA 1/6
(BLUE EYE)
1 2 3 4 5 6
DNA Sequence
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HAPLOTYPE CORRELATION WITH PHENOTYPE

• The Haplotype centric approach combines the
  information of adjacent SNPs into composite
  multilocus haplotypes.

• Haplotypes are not only more informative but
  also capture the regional LD information, which
  is assumed to be robust and powerful

• Association of haplotype frequencies with the
  presence of desired phenotypic frequencies in the
  population will help in utilizing the maximum
  potential of SNP as a marker.

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ADVANTAGES

1. SNPs ARE THE MOST FREQUENT FORM OF DNA VARIATIONS
2. THEY ARE THE DISEASE CAUSING MUTATIONS IN MANY
   GENES
3. THEY ARE ABUNDANT HAVE SLOW MUTATION RATES
4. EASY TO SCORE
5. MAY WORK AS THE NEXT GENERATION OF GENETIC
   MARKERS

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LIMITATIONS
1. EXPERIMENTAL DETECTION OF SNPs REQUIRES
IMPLEMENTATION OF EXPENSIVE TECHNOLOGIES
2. NEED FOR LARGE POPULATION DATASETS FOR
ASSOCIATION STUDIES
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Some important SNP database Resources
1. dbSNP (http//www.ncbi.nlm.nih.gov/SNP/)
LocusLink (http//www.ncbi.nlm.nih.gov/LocusLink/l
ist.cgi) 2. TSC (http//snp.cshl.org/) 3. SNPper
(http//snpper.chip.org/bio/) 4. JSNP
(http//snp.ims.u-tokyo.ac.jp/search.html) 5.
GeneSNPs (http//www.genome.utah.edu/genesnps/) 6.
HGVbase (http//hgvbase.cgb.ki.se/) 7. PolyPhen
(http//dove.embl-heidelberg.de/PolyPhen/)
OMIM (http//www.ncbi.nlm.nih.gov/entrez/query.fcg
i?dbOMIM)
8. Human SNP database (http//www-genome.wi.
mit.edu/snp/human/)
Feb. 25. 2003 SI Hung