Genome-wide Association Study
• Focus on association between SNPs and traits
genotyped for the majority of common known SNPs
Healthy control group
Case group
For each of SNPs, allele frequency alters?Odds ratio
• Tendency
Proportion of the same allele
Proportion of a specific allele
– Larger and larger sample size
– Use of more narrowly defined phenotypes(blood lipids, proinsulin
or similar biomarkers
• Limitations
– Sufficient sample size
– The massive number of statistical tests performed presents an
unprecedented potential for the positive results
– Search the entire genome-->not worth the expenditure
Advantage of Exome Sequecing
• Whole genome sequencing
– Redundant raw data(6 Gb in each human diploid
genome )
• Exome sequecing(targeted exome capture)
– Exons are short and 180,000 exons constitute 1%
of the human genome
– The goal is to identify the functional variation that
is responsible for both mendelian and common
diseases
Significance
• Exome sequencing can be used to identify causal
variants of rare disorders
• The first reported study that used exome sequencing as
an approach to identify an unknown causal gene for a
rare mendelian disorder
The Shendure Lab
• Next-generation human genetics
– A multiplex approach to genome sequencing
– Targeted sequence enrichment
• Protocols relying on molecular inversion probe
• Hybrid capture
– Novel analytical strategies to identify the genetic
basis of Mendelian disorders by exome sequecing
• Autosomal recessive disorders such as Miller syndrome
• Autosomal dominant disorders such as Kabuki syndrome
Hapmap project
• Focuse on common SNPs(at least 1% of the
population)
• Samples: 4 populations
– (30*3 YRI, 30*3 CEU, 45 JPT, 45 CHB)
• Data:
– SNP frequencies, genotypes
Work flow
DNA samples, targeted capture and
massively parallel sequencing
Read mapping and variant analysis
Direct identification of the causal gene for
FSS
Target enrichment
Methods
a. PCR-based approach
b. Molecular inversion probe(MIP)based approach
c. Hybrid capture-based approach
Mamanova et al.
Nat Method 7(2):112-118
Mamanova et al.
Nat Method 7(2):112-118
Figure.
①Probe list of array2
② Probe list of array1
③Exome on 1-22, X and Y
chromosomes
Work flow
DNA samples, targeted capture and
massively parallel sequencing
Read mapping and variant analysis
Direct identification of the causal gene for
FSS
Coming…
Comparison of sequence calls to
array genotypes, dbSNP and whole
genome sequencing
Direct identification of the causal
gene for FSS
Method
Genomic DNA samples
Oligonucleotides and adaptors
Shotgun library construction
Design of exon capture array
Targeted capture by hybridization of DNA microarrays
Sequencing
Read mapping
Target Masking
Variant calling
Comparison of sequence calls to array genotypes, dbSNP and whole genome sequencing
Variant annotation
Calculation of genome-wide estimates
Method
Method 2:MIP and resequencing
Method 3: Whole genome sequencing
Method 4:
Figure. Table of cSNPs of 8 HapMap individuals
Figure. Table of Splice Site Variants of 8 HapMap
individuals
Figure. Table of Coding Indels of 8 HapMap individuals
Figure. Table of coverage of
8 HapMap individuals and 4 FSS individual
Figure. Intervals that were exclued….
Figure.
①Probe list of array2
② Probe list of array1
③Exome on 1-22, X and Y
chromosomes
YRI: Nigeria - Yoruba people of Ibadan
CHB: China - Beijing
JPT: Japan - Tokyo
CEU: Centre d'Etude du Polymorphisme Humain (CEPH)
Eur: European–American ancestry
About mendelian disease
Traditional situation
Current situation
Considerations
•
•
•
•
Causal genes may be shared by case group.
Control group may not contain that mutation.
Common mutation may not be causal.
Causal mutation should cause animo acid
change.
Result
Further application
• Typical single gene disorder.
• Disorder caused by single but not uniform
gene.
• Multiple gene disorder.
• Complex disease.
• Cancer.