Genome-wide Association S tudy

Genome-wide Association Study• Focus on association between SNPs and traits

• Tendency– 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

For each of SNPs, allele frequency alters?Odds ratio

Proportion of the same allele Proportion of a specific allele

genotyped for the majority of common known SNPs

Healthy control group Case group

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

Direct identification of the causal gene for FSS

Read mapping and variant analysis

DNA samples, targeted capture and massively parallel sequencing

a. PCR-based approachb. Molecular inversion probe(MIP)-

based approachc. Hybrid capture-based approach

Mamanova et al. Nat Method 7(2):112-118

Target enrichment Methods

Mamanova et al. Nat Method 7(2):112-118

Figure. ①Probe list of array2

② Probe list of array1③Exome on 1-22, X and Y chromosomes

Work flow

Direct identification of the causal gene for FSS

Read mapping and variant analysis

DNA samples, targeted capture and massively parallel sequencing

Coming…

Direct identification of the causal gene for FSS

Comparison of sequence calls to array genotypes, dbSNP and whole

genome sequencing

Method

Calculation of genome-wide estimates

Variant annotation

Comparison of sequence calls to array genotypes, dbSNP and whole genome sequencing

Variant calling

Target Masking

Read mapping

Sequencing

Targeted capture by hybridization of DNA microarrays

Design of exon capture array

Shotgun library construction

Oligonucleotides and adaptors

Genomic DNA samples

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 array1③Exome on 1-22, X and Y chromosomes

YRI: Nigeria - Yoruba people of IbadanCHB: China - BeijingJPT: Japan - TokyoCEU: 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.