Exome seuencing (steps, method, and applications)

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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emulsion PCR

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2

1

2

Library preparation

Clonal amplification

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emulsion PCR bridge PCR

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2

1

2

Library preparation


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Introduction to NGS http://ueb.ir.vhebron.net/NGS


emulsion PCR bridge PCR

Pyrosequencing

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2

3

1

2

3 Cyclic array sequencing

Library preparation


454 sequencing 15

http://ueb.ir.vhebron.net/NGS



bridge PCR

Pyrosequencing Sequencing-by-ligation

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emulsion PCR

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3

1

2

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454 sequencing SOLiD platform

Cyclic array sequencing

Library preparation


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bridge PCR

Pyrosequencing Sequencing-by-ligation Sequencing-by-synthesis

1

emulsion PCR

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3

1

2

3

454 sequencing SOLiD platform Solexa technology

Cyclic array sequencing

Library preparation


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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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