Exome Sequence

Analysis
Introduction
Exome sequencing, also known as whole exome
Identifying all the available genes in the genome
sequencing

Sequencing all the expressed genes in a genome

Humans have about 180,000 exons, 30 million base

pairs

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Key terms & terminologies
 Copy number variants (CNVs) are genetic variations that occur when
sections of the genome are repeated. The number of repeats varies
between individuals of the same species.

 It can be categorized into two main groups: short repeats and long
repeats. However, there are no clear boundaries between the two groups
and the classification depends on the nature of the loci of interest.

 A DNA sequence variation (SNVs) that occurs when a single nucleotide

(adenine, thymine, cytosine, or guanine) in the genome sequence is
altered. Single nucleotide variants may be rare or common in a
population.
Methodolo
gy
2
steps:

1. Select only 2. Sequencing of

the subset of the exonic DNA
DNA that using DNA
encodes sequencing
proteins technology
(exons) 4
1. Selection of Exons (Target
Enrichment)
Array-
based
2 strategies capture
In-
solution
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Array Based Capture
 Array-based sequence capture is a technology that allows for
the sequencing of large DNA regions in a more efficient and
less time-consuming way.

 It is a sequence capture technology that utilizes high-density

oligonucleotide to capture a subset of the genome, such as
the exome, a particular chromosome, a set of genes or a
region of interest.
Array-Based Capture

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In- Solution Capture
 In-solution capture sequencing is a sensitive method for detecting single
nucleotide variants, insertions and deletions, and copy number
variations.

 It can also be used to identify sequence variants in coding and noncoding

regions of very large genomes.

 It uses biotinylated DNA or RNA molecules (baits) to capture target

regions. The resulting heteroduplexes are captured on streptavidin-
coated magnetic beads.

 In-solution capture-enrichment can be used to retrieve sequences of

interest and reduce the fraction of microbial DNA. This is advantageous
for ancient biological samples, which often have low levels of
endogenous DNA.
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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Steps in Sanger sequencing
 DNA Sequence For Chain Termination PCR. The DNA
sequence of interest is used as a template for a
special type of PCR called chain-termination PCR.

 Size Separation by Gel Electrophoresis.

 Gel Analysis & Determination of DNA Sequence.

Chain termination
 This is a technique for DNA sequencing based upon the selective
incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA
polymerase during in vitro DNA replication.

 The sequencing method is based on the use of chain terminators, the

dideoxynucleotides (ddNTPs). The dideoxynucleotides, or ddNTPSs, differ
from the deoxynucleotides by the lack of a free 3′ OH group on the five-
carbon sugar.

 ddNTPs are randomly incorporated in the DNA strands during elongation,

thereby terminating strand elongation at each location along the sequence.
Subsequent capillary electrophoresis separates the DNA strands with
respect to the size, and the terminating nucleotides are identified using
each fluorescent dye.
Sanger Sequencing

Lack 3’-OH
Chain
Use of group
terminati
labeled required for
on
ddNTPs phosphodieste
method
r bond
formation

1
2
1
3
Next Generation Sequencing

1
4
1 Library 1 DNA
preparation fragmentation
and in
vitro adaptor
ligation

1
5
1 Library 1 DNA
preparation
2 fragmentation
Clonal and in
amplification vitro adaptor
ligation
emulsion
PCR
2

1
6
1 Library 1 DNA
preparation
2 fragmentation
Clonal and in
amplification vitro adaptor
ligation
emulsion bridge
PCR PCR
2

1
7
1 Library preparation 1 DNA
2 Clonal amplification fragmentation
and in
3 Cyclic array vitro adaptor
sequencing ligation
emulsion bridge
PCR PCR
2

3 Pyrosequenci
ng

454 1
8
1 Library preparation 1 DNA
2 Clonal amplification fragmentation
and in
3 Cyclic array vitro adaptor
sequencing ligation
emulsion bridge
PCR PCR
2

3 Pyrosequenci Sequencing-by-
ng ligation

454 IntroductionSOLiD platf

to NGS http://ueb.ir.vhebron.net/ 1
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1 Library preparation 1 DNA
2 Clonal amplification fragmentation
and in
3 Cyclic array vitro adaptor
sequencing ligation
emulsion bridge
PCR PCR
2

3 Pyrosequenci Sequencing-by- Sequencing-by-

ng ligation synthesis

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0
 Emulsion PCR

 Emulsion PCR allows simultaneous amplification of each

sequence without risk of contamination.

 Here, each bead acts as a microreactor for PCR, each

containing one strand of DNA.

 It reduces formation of unwanted, chimeric products caused

by templates annealing to each other
Bridge PCR
 Bridge PCR, also known as jumping PCR or bridge
amplification, is a technique that allows for different single
stranded DNA templates to be sequenced and amplified in
tandem.

 The fragments are about 200-600 nucleotides long and contain

two different adapters at both ends.

 Bridge PCR is a method used to amplify sequences prior to

NGS.
Pyrosequencing
Pyrosequencing is a method of DNA sequencing that differs from Sanger
sequencing, in that it relies on the detection of pyrophosphate release and
the generation of light on nucleotide incorporation, rather than chain
termination with dideoxynucleotides.

Pyrosequencing can be used to:

 Reveal the genetic code of a section of DNA

 Detect single nucleotide polymorphisms

 Detect insertion-deletions or other sequence variations

 Quantify DNA methylation and allele frequency

 Sequencing by ligation (SBL)

 It is a DNA sequencing method that uses the enzyme DNA

ligase to identify the nucleotide present at a given position in
a DNA sequence. Unlike most other DNA sequencing
methods, SBL does not use a DNA polymerase to create a
second strand.

 Sequencing by ligation is a DNA sequencing method that

harnesses the mismatch sensitivity of DNA ligase to determine
the underlying sequence of nucleotides in each DNA sequence
(Ho et al., 2011).
 Sequencing by synthesis (SBS)

 Sequencing by synthesis (SBS) is a technique that uses DNA

polymerase to extend a primer that is hybridized to a
template by a single nucleotide. It then determines the
identity of the nucleotide and incorporates the next
nucleotide. This process is repeated until the entire DNA
sequence is read out serially.

 During analysis, the computer will group all reads with the
same index together. Illumina uses a "sequence by synthesis"
approach. This process takes place inside of an acrylamide-
coated glass flow cell.
Applications
⚫ Rare variant mapping in complex
disorders

⚫ Discovery of Mendelian disorders

⚫ Clinical Diagnostics

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