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HW2: exome sequencing and complex disease
Jacquemin Jonathan de Bournonville Sébastien
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Exome sequencing 2
Table of contents• Introduction• Study design:
o Sample selectiono Sequencing strategy
• Variant calling• Association analysis• Approaches for follow-up of promising signals• The role of functional assays in interpreting ESS• Conclusion
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Exome sequencing 3
Introduction• Exome = only 1% of the entire genome
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4
Introduction
Exome sequencing
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Exome sequencing 5
Study design: Sample selection
• Objectives definition:o What kind of variantso What scale (survey range variations in individuals, find variants linked
to specific trait)
• Samples inventory with interesting traits• Focus on samples with extreme outcome
o Quantitative traits → Extremeso Discrete traits → Unsuals
• Sequencing related individuals?• Restricted geographic distribution• Founder populations
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Exome sequencing 6
Study design: Sequencing strategy
• High quality bases coverage
• Reach >20x in ~90% of the protein coding sequences
• Enrichment → Local variations → Aim for average depth of 60 to 80x
• Light coverage of the rest of the genome
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Exome sequencing 7
Variant calling• Process raw sequences into high-quality
genotypes:
o Cleaning the DNA
o Alignment of the short sequences reads to the reference genome
o Removal of the duplicate reads
o Quality metrics of each sample:
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Exome sequencing 8
Variant calling
• Process raw sequences into high-quality genotypes:
o New quality metrics
o Identification of variant sites + inspection
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Exome sequencing 9
Variant calling
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Exome sequencing 10
Variant calling
• Drawback:
o Many choices for these steps -> impacts the final result
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Exome sequencing 11
Association analysis• A same variant alters different transcripts → focus
ono Canonical transcripto Most deleterious transcript, etc.
• Should always start with single variant association tests
• In order to generate QQ plots and quality checko Population structureo Variants relevance
• Groups variants that impact gene function
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Exome sequencing 12
Association analysis• Consider two types of tests• Variable thresholds• Or variable cut-off frequencies• Analyses focused on homozygous individuals• P-values ~• Generate summarizing QQ plots
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Exome sequencing 13
Approaches for follow-up of promising
signals• Goal: identify very rare variants
• Different methods:
o Data from other samples
o Statistical imputation
o Targeted sequencing
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Exome sequencing 14
Conclusion• Forward genetics
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Exome sequencing 15
Conclusion
• Challenges for future:
o Apply it to complex and commom diseases
o Methods to combine results from different studies
o New protocols or statistical methods
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Exome sequencing 16
Thank you for listening
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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