Lecture-3 EXOME SEQUENCING Huseyin Tombuloglu, Phd GBE423 Genomics & Proteomics

Lecture-3EXOME SEQUENCING

Huseyin Tombuloglu, Phd

GBE423 Genomics & Proteomics

EXOME SEQUENCING

)͠ 1% of genome

Mutations or variations in this portion of genome have severe consequences than in the remaining 99%

Exome sequencing is especially effective in the study of rare Mendelian diseases

EXOME SEQUENCING

How it works?

How it works?

Target-enrichment strategies

1 PCR (Multiplex)2 Molecular inversion probes (MIP)3 Hybrid capture4 In-solution capture5 Sequencing

Target-enrichment strategies

1 PCR (Multiplex) 2 Molecular inversion probes (MIP)3 Hybrid capture4 In-solution capture5 Sequencing

3 Hybrid capture

Roche NimbleGen

~180,000 coding exons

Agilent Capture Array

Biotiniylated gene spesific probes

Array technology

4 In Solution CaptureProbes in solution can

capture all exons (exome) for high throughput sequencing

Biotin-streptovidin based

• 1-2% of whole genome seq• Easily multiplex 20 samples

in one lane

11

Comparative SequencingWGS & WES

Comparative Sequencing WGS & WES

• The CaseSomatic mutation detection between normal /

cancer pairs

WGS & WES ?

14Meyerson et al, Nat Rev Genet 2010

Figure 1 Steps in the generation of whole-genome or whole-exome sequencing data for analysis

Foo, J.-N. et al. (2012) Whole-genome and whole-exome sequencing in neurological diseasesNat. Rev. Neurol. doi:10.1038/nrneurol.2012.148

Clinical practices of Exome Sequencing

Rare diseases

• Some rare diseases affect as many as 200,000 people, but some are so rare that they affect one or two people in the world.

This was the first reported study that used exome sequencing as an approach to identify an unknown causal gene for a rare mendelian disorder (2009)

Strategy to mine the Causal Gene

Exome Sequencing ServicesThe pilot program announced by 23andMe costs $999 and requires no physician signature, but provides only raw data without analysis.

In November 2012, DNADTC, a division of Gene by Gene started offering exomes at 80X coverage and introductory price of $695. This price per DNADTC web site is currently $895.

In October 2013, Beijing Genomics Institute (BGI) announced a promotion for personal whole exome sequencing at 50X coverage for $499

1% of genome, limited sizeUsefull for large genomes, such as wheat, barley …etcLess complicated results, Cost effective

Advantages

Applications in other organisms