HW2: exome sequencing and complex disease

1. HW2: exomesequencing and complexdisease Jacquemin Jonathan de Bournonville Sébastien

2. Table of contents • Introduction • Study design: • Sampleselection • Sequencingstrategy • Variant calling • Association analysis • Approaches for follow-up of promisingsignals • The role of functionalassays in interpreting ESS • Conclusion Exome sequencing

3. Introduction • Exome = only 1% of the entiregenome Exome sequencing

4. Introduction Exomesequencing

5. Study design: Sampleselection • Objectives definition: • What kind of variants • What scale (survey range variations in individuals, find variants linked to specific trait) • Samples inventory with interesting traits • Focus on samples with extreme outcome • Quantitative traits → Extremes • Discrete traits → Unsuals • Sequencing related individuals? • Restricted geographic distribution • Founder populations Exome sequencing

6. Study design: Sequencingstrategy • High quality bases coverage • Reach >20x in ~90% of the proteincodingsequences • Enrichment→ Local variations → Aim for average depth of 60 to 80x • Light coverage of the rest of the genome Exome sequencing

7. Variant calling • Processrawsequencesinto high-qualitygenotypes: • Cleaning the DNA • Alignmentof the short sequencesreads to the referencegenome • Removal of the duplicate reads • Qualitymetrics of eachsample: Exome sequencing

8. Variant calling • Processrawsequencesinto high-qualitygenotypes: • New qualitymetrics • Identification of variant sites + inspection Exome sequencing

9. Variant calling Exome sequencing

10. Variant calling • Drawback: • Manychoices for thesesteps -> impacts the final result Exome sequencing

11. Association analysis • A same variant altersdifferenttranscripts→ focus on • Canonical transcript • Most deleterioustranscript, etc. • Shouldalwaysstartwithsingle variant association tests • In order to generate QQ plots and quality check • Population structure • Variants relevance • Groups variantsthat impact genefunction Exome sequencing

12. Association analysis • Consider two types of tests • Variable thresholds • Or variable cut-off frequencies • Analyses focused on homozygousindividuals • P-values ~ • Generatesummarizing QQ plots Exome sequencing

13. Approaches for follow-up of promisingsignals • Goal: identifyvery rare variants • Differentmethods: • Data fromothersamples • Statisticalimputation • Targetedsequencing Exome sequencing

14. Conclusion • Forwardgenetics Exome sequencing

15. Conclusion • Challenges for future: • Applyit to complex and commomdiseases • Methods to combine resultsfromdifferentstudies • New protocols or statisticalmethods Exome sequencing

16. Thankyou for listening Exome sequencing

17. References • http://www.geneticliteracyproject.org/2013/02/25/scientists-savage-each-other-over-junk-dna-study-while-journalists-mis-report-the-science/#.UqXNaeLZ2os • http://ueb.vhir.org/2012-06-18+Xavier+de+Pedro+Pipeline+for+Exome+Variant+Analysis • http://www.bioscience.org/2009/v14/af/3318/fulltext.php?bframe=figures.htm • https://www.my46.org/intro/whole-genome-and-exome-sequencing • http://en.wikipedia.org/wiki/Exome_sequencing Exome sequencing