Genetic Variations

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Genetic Variations

• Lakshmi K Matukumalli

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Human Mouse Comparison
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Structural Variations
Ploidy (Downs Syndrome)
Inversions Translocations Segmental duplications
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Molecular Variations
Single nucleotide polymorphisms Short
Indels Simple sequence repeats Copy number
variants Loss of heterozygosity
Copy number variants
Microsatellite (2-9 bp core repeat)
Minisatellite (10-60 bp core repeat)
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Type of polymorphisms
Insertion/deletion polymorphism (indel)
Nonsynonymous polymorphism
Synonymous polymorphism
Single-nucleotide Polymorphism (SNP)
TAACGGTA GG
GAG Asp GUG Val
GAU Asp GAC Asp
TC
5 Untranslated region
3 Untranslated region
ATG
End
5 Flanking region
3 Flanking region
Promoter
Coding
Intron
Coding
Transcript
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Choosing the Technology
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Extent of Variation (Human Genome)

• gt 5 million SNPs (dbSNP)
• Recent genome analysis of diploid individual
  showed 4.1 million DNA variants, encompassing
  12.3 Mb.
• - 3,213,401 single nucleotide polymorphisms
  (SNPs),
• - 53,823 block substitutions (2206 bp),
• - 292,102 heterozygous insertion/deletion events
  (indels)(1571 bp),
• - 559,473 homozygous indels (182,711 bp),
• - 90 inversions,
• - Plus segmental duplications and copy number
  variations.
• Non-SNP DNA variation accounts for 22 of all
  events, however they involve 74 of all variant
  bases. This suggests an important role for
  non-SNP genetic alterations in defining the
  diploid genome structure.
• Moreover, 44 of genes were heterozygous for one
  or more variants.

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Importance of SNPs and other variants

• Study Genetic variation in diverse populations in
  any species to
• understand evolutionary origins and history,
• estimate population size,
• breeding structure, or life-history characters
• Migration within and between sub-populations
• Understand evolutionary basis for maintenance of
  genetic variation and speciation.
• Applications
• Genetic association of traits
• Effects on gene expression (e.g., synonymous vs
  nonsynonymous / TF binding sites)
• DNA finger printing or sample tracking

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Fine Mapping with SNP Markers

• Advantages of SNPs as genetic markers
• as compared to microsatellites.
• High abundance
• Distribution throughout the genome
• Ease of genotyping
• Improved accuracy
• Availability of high throughput
• multiplex genotyping platforms

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SNP Discovery - Sanger sequencing (EST)
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SNP Discovery - Diploids (heterozygous loci)
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SNP-PHAGE (Software package)
SNP Pipeline for Haplotype Analysis and GEnbank
(dbSNP) submissions.

• Important steps are
• Primer development
• Primer testing
• Sequencing
• Base calling,
• Sequence assembly
• Polymorphisms analysis
• Haplotype analysis
• GenBank submission of confirmed polymorphisms

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Application of Machine Learning in SNP Discovery
Objective Reduce human intervention by using
expert annotated dataset for training a Machine
learning (ML) program and use it to differentiate
good/bad polymorphisms

• Steps
• Parameter Selection
• Parameter Optimization
• Testing
• Implementation.
• Results
• Achieved substantial improvement in the
  accuracies as compared to using only polybayes or
  polyphred.

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SNP Discovery using next generation sequencers

• Short sequences 23-35 bp long at a fraction of
  cost.
• Reduced Representation Sequencing
• Digest genomic DNA with restriction enzyme
• Screen based on in silico digestion
• Size select based on
• Repetitive DNA
• Number of fragments
• Sequencing platform
• Allows targeted deep sequencing of pools of DNA
• Randomly distributed

Cost / Mb ABI 880 454 160 Solexa 5
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SNP Discovery - Bioinformatics

• Strategies to maximize performance
• High quality score stringencies
• For each read
• At base for putative SNP
• Require single map location of a 23-bp tag (and
  4-bp restriction site)
• Allow only one single base pair difference match
  for a putative SNP
• Reduces repeat content
• Reduces gene family/paralog false positives
• Require 2 copies of each allele assembly can
  count as 1

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Predicted Observed Minor Allele Frequency
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Population Genetics

• Population genetics is the study of the
  allele frequency distribution and change under
  the influence of the four evolutionary forces
  natural selection, genetic drift, mutation and
  gene flow. It attempts to explain phenomena as
  adaptation and speciation.
• (www.wikipedia.org)

Variation
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Population Genetics
Neutral theory Rate at which new genetic
variants are formed is equal to the loss of
genetic diversity due to drift.
Genotypes CT, CC, TT Alleles C and T
C/T C/C T/T
Genotyping of a population of 1000 individuals
for a SNP resulted in 100, 500 and 400 genotypes
for CC, CT and TT respectively Genotype
Frequencies CC (0.1), CT (0.5) and
TT(0.4) Allele Frequencies C (p)
(200500)/2000 0.35 (minor allele -- MAF)
T (q) (500800)/2000 0.65 (major
allele) Hardy-Weinberg Equilibrium Expected
genotype frequencies are p2, 2pq and q2 (122,
422 and 455) HWE Deviations Drift, Selection,
Admixture etc.,
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Fst
Useful to partition genetic variation into
components within populations between
populations among populations Sewall Wrights
Fixation index (Fst is a useful index of genetic
differentiation and comparison of overall effect
of population substructure. Measures reduction
in heterozygosity (H) expected with non-random
mating at any one level of population hierarchy
relative to another more inclusive hierarchical
level. Fst (HTotal - Hsubpop)/HTotal Fst
ranges between minimum of 0 and maximum of 1
0 ? no genetic differentiation ltlt 0.5 ?
little genetic differentiation gtgt 0.5 ?
moderate to great genetic differentiation 1.0
? populations fixed for different alleles
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Genotype Phenotype Association (Significance of
Haplotypes)
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Haplotype inference

• The solution to the haplotype phasing problem is
  not straightforward due to resolution ambiguity

• Computational and statistical algorithms for
  addressing ambiguity in Haplotype Phasing
• 1) parsimony
• 2) phylogeny
• 3) maximum-likelihood
• 4) Bayesian inference

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Linkage disequilibrium (LD)

• Non-random association of alleles at two or more
  loci, not necessary in the same chromosome.
• LD is generally caused by interactions between
  genes genetic linkage and the rate of
  recombination random drift or non-random mating
  and population structure.

Let A and B be two loci segregating two alleles
each a1 and a2 with frequencies p1 and p2 in
A, and b1 and b2 with frequencies q1 and q2 in B.
B1 B2 Total A1 p11 p1 q1 D p12 p1
q2 - D p1 A2 p21 p2 q1 - D p22 p2 q2
D p2 Total q1 q2 1
A
B
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Linkage disequilibrium (cont)

• D p11 - p1q1
• D depends on the allele frequencies at A and B.
• D a scaled version of D

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Linkage disequilibrium (cont)

• Squared correlation coefficient

D2
r2
p1p2q1q2
The measure preferred by population
geneticists Is independent of of allele
frequencies Ranges between 0 and 1 r2 1
implies the markers provide exactly the same
information r2 0 when they are in perfect
equilibrium
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2.4 Linkage disequilibrium (cont)

• Visualizing LD

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2.4 Linkage disequilibrium (cont)

• Visualizing LD

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