1. 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 alleleProportion of a specific allele genotyped for the majority of common known SNPs Healthy control groupCase group

2. 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

3. 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

4. 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

5. 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

7. Work flow Direct identification of the causal gene for FSS Read mapping and variant analysis DNA samples, targeted capture and massively parallel sequencing

8. 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 Target enrichment Methods

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

10. Figure. ① Probe list of array2 ② Probe list of array1 ③ Exome on 1-22, X and Y chromosomes

11. Work flow Direct identification of the causal gene for FSS Read mapping and variant analysis DNA samples, targeted capture and massively parallel sequencing

12. Coming… Direct identification of the causal gene for FSS Comparison of sequence calls to array genotypes, dbSNP and whole genome sequencing

13. 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

14. Method

15. Method 2:MIP and resequencing

16. Method 3: Whole genome sequencing

17. Method 4:

18. Figure. Table of cSNPs of 8 HapMap individuals

19. Figure. Table of Splice Site Variants of 8 HapMap individuals

20. Figure. Table of Coding Indels of 8 HapMap individuals

21. Figure. Table of coverage of 8 HapMap individuals and 4 FSS individual

22. Figure. Intervals that were exclued….

23. Figure. ① Probe list of array2 ② Probe list of array1 ③ Exome on 1-22, X and Y chromosomes

24. YRI: Nigeria - Yoruba people of Ibadan CHB: China - Beijing JPT: Japan - Tokyo CEU: Centre d'Etude du Polymorphisme Humain (CEPH) Eur: European–American ancestry

30. About mendelian disease

31. Traditional situation

32. Current situation

33. 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.

34. Result

35. Further application Typical single gene disorder. Disorder caused by single but not uniform gene. Multiple gene disorder. Complex disease. Cancer.