Exome seuencing (steps, method, and applications)

Presented By: Hamza Khan

Registration # FA16-R02-019

1

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

2

Methodology

2 steps:

1. Select only the subset of DNA that encodes proteins

(exons)

2. Sequencing of the exonic DNA using DNA sequencing

technology

3

1. Selection of Exons (Target Enrichment)

2 strategies

Array-based capture

In-solution capture

4

Array-Based Capture

5

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

6

7

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)

8

Sanger Sequencing

Chain termination

method

Use of labeled ddNTPs

Lack 3’-OH group required for

phosphodiesterbond formation

9

10

Next Generation Sequencing

11

DNAfragmentation and in vitro adaptor ligation

11 Library preparation

12

DNAfragmentation and in vitro adaptor ligation

emulsion PCR

1

2

1

2

Library preparation

Clonal amplification

13

DNAfragmentation and in vitro adaptor ligation

emulsion PCR bridge PCR

1

2

1

2

Library preparation

Clonal amplification

14

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

DNAfragmentation and in vitro adaptor ligation

emulsion PCR bridge PCR

Pyrosequencing

1

2

3

1

2

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

1

emulsion PCR

2

3

1

2

3

454 sequencing SOLiD platform

Cyclic array sequencing

Library preparation

Clonal amplification

16

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

DNAfragmentation and in vitro adaptor ligation

bridge PCR

Pyrosequencing Sequencing-by-ligation Sequencing-by-synthesis

1

emulsion PCR

2

3

1

2

3

454 sequencing SOLiD platform Solexa technology

Cyclic array sequencing

Library preparation

Clonal amplification

17

Roche 454 pyrosequencing

18

Illumina Sequencing

19

SOLiD Sequencing

20

Applications Rare variant mapping in complex disorders

Discovery of Mendelian disorders

Clinical Diagnostics

21

22