Post on 23-Jan-2018
Presented By: Hamza Khan
Registration # FA16-R02-019
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IntroductionExome sequencing, also known as whole exomesequencing
Sequencing all of the expressed genes in a genome
Humans have about 180,000 exons, 30 million base pairs
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Methodology
2 steps:
1. Select only the subset of DNA that encodes proteins
(exons)
2. Sequencing of the exonic DNA using DNA sequencing
technology
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1. Selection of Exons (Target Enrichment)
2 strategies
Array-based capture
In-solution capture
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Array-Based Capture
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In-Solution Capture Use pool of custom oligonucleotides (probes)
Synthesized and hybridized in solution
Probes are labeled with beads
After hybridization beads can be pulled down and washed
Fragments can be sequenced after beads removal
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2. Sequencing Several sequencing platforms are available:
1. Sanger Sequencing
2. Next Generation Sequencing: 454 Pyrosequencing (454 Life Sciences) Illumina (Solexa) sequencing SOLiD Sequencing (Applied Bio-systems)
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Sanger Sequencing
Chain termination
method
Use of labeled ddNTPs
Lack 3’-OH group required for
phosphodiesterbond formation
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Next Generation Sequencing
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DNAfragmentation and in vitro adaptor ligation
11 Library preparation
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DNAfragmentation and in vitro adaptor ligation
emulsion PCR
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2
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Library preparation
Clonal amplification
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DNAfragmentation and in vitro adaptor ligation
emulsion PCR bridge PCR
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Library preparation
Clonal amplification
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Introduction to NGS http://ueb.ir.vhebron.net/NGS
DNAfragmentation and in vitro adaptor ligation
emulsion PCR bridge PCR
Pyrosequencing
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3 Cyclic array sequencing
Library preparation
Clonal amplification
454 sequencing 15
Introduction to NGS http://ueb.ir.vhebron.net/NGS
DNAfragmentation and in vitro adaptor ligation
bridge PCR
Pyrosequencing Sequencing-by-ligation
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emulsion PCR
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454 sequencing SOLiD platform
Cyclic array sequencing
Library preparation
Clonal amplification
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Introduction to NGS http://ueb.ir.vhebron.net/NGS
DNAfragmentation and in vitro adaptor ligation
bridge PCR
Pyrosequencing Sequencing-by-ligation Sequencing-by-synthesis
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emulsion PCR
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3
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2
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454 sequencing SOLiD platform Solexa technology
Cyclic array sequencing
Library preparation
Clonal amplification
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Roche 454 pyrosequencing
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Illumina Sequencing
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SOLiD Sequencing
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Applications Rare variant mapping in complex disorders
Discovery of Mendelian disorders
Clinical Diagnostics
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