Applications of Next Generation

DNA Sequencing Technology in

Medicine
Central dogma of molecular biology

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Polymeric organization in bio-macromolecules

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Answers of many questions
asked in biology are
in the “sequence”.

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Mutations: Changes in DNA sequences
Important event during life processes
major cause of diversity; key reason for many diseases

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 Adapted
Adapted from
from Eric
Eric Green,
Green, NIH;
NIH; Adapted
Adapted from
from Messing
Messing &
& Llaca,
Llaca, PNAS
PNAS (1998)
(1998)
History
History of
of DNA
DNA Sequencing
Sequencing
1870 Miescher: Discovers DNA

1940 Avery: Proposes DNA as ‘Genetic Material’

Efficiency
1953 Watson & Crick: Double Helix Structure of DNA
(bp/person/year)

1 1965 Holley: Sequences Yeast tRNAAla

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1970 Wu: Sequences  Cohesive End DNA
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Sanger: Dideoxy Chain Termination

1,500 1977
Gilbert: Chemical Degradation

1980 Messing: M13 Cloning

15,000

25,000
1986 Hood et al.: Partial Automation

50,000 1990
• Cycle Sequencing
200,000
• Improved Sequencing Enzymes
• Improved Fluorescent Detection Schemes

50,000,000 2002
• Next Generation Sequencing
• Improved enzymes and chemistry
100,000,000,000 2008
• Improved image processing
 Sanger method based DNA sequencing technologies
ABI Genetic Analyzers

ABI 310 ABI Prism 3100 ABI 3130 ABI3130xl

Beckman Genetic Analyzer

CEQ 8800
CEQ 8000
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 Advantages and short comings of Sanger based DNA
sequencing technologies
 Deciphering the complete genes and later entire
genome sequencing.
 Involved in completion of human genome project
(HGP, 2003).
 Considered to be gold standard from last 30 years
because of accurate base calling and read length.
 Prohibitively, expensive and time consuming method
for routine sequencing of human genomes in case of
disease genomics.

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Next Generation DNA Sequencing:
Concepts and Applications
A paradigm shift in sequencing
 The Next Generation DNA sequencing
 Demand for faster, affordable DNA sequencing has led to the
development of so-called “next generation” sequencing technologies.

 These technologies are delivering DNA sequencing at unprecedented

speed, thereby enabling impressive scientific achievements and novel
biological applications.

 To date, these technologies have been applied in a variety of contexts,

 Whole-genome sequencing
 Targeted resequencing
 Transcriptome analysis
 Discovery of transcription factor binding sites
&
 Discoveries of small Non-coding RNAs.

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Work flow of conventional versus
second-generation sequencing
Current Commercially available Next Generation
DNA sequencing platform

The Illumina/Solexa genome analyzer

Roche 454 technology

ABI SoliD Helicos tSMS Technology

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The Illumina Next Generation DNA
Sequencing Technology
Introduced in 2006
Sequencing by synthesis chemistry

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 Illumina sequencing concept: An overview
1. Prepare genomic DNA
Library

2. Attach DNA to surface

2. Bridge amplification

3. Fragments become
double stranded

4. Denature the double-

stranded molecules

5. Complete amplification
Randomly fragment genomic DNA ligated to adapters at both ends

Source: http://www.illumina.com/downloads/SS_DNAsequencing.pdf 14
 1. Prepare genomic DNA

2. Attach DNA to surface

3. Bridge amplification

4. Fragments become
double stranded

5. Denature the double-

stranded molecules

6. Complete amplification

Bind single-stranded fragments randomly to the inside surface

of the flow cell channels 15
 1. Prepare genomic DNA

2. Attach DNA to surface

3. Bridge amplification

4. Fragments become
double stranded

5. Denature the double-

stranded molecules

6. Complete amplification

Add unlabeled nucleotides and enzyme to initiate solid-phase

bridge amplification
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 1. Prepare genomic DNA

2. Attach DNA to surface

3. Bridge amplification

4. Fragments become
double stranded

5. Denature the double-

stranded molecules

6. Complete amplification

The enzyme incorporates nucleotides to build

double-stranded bridges on the solid-phase substrate
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 1. Prepare genomic DNA

2. Attach DNA to surface

3. Bridge amplification

4. Fragments become
double stranded

5. Denature the double-

stranded molecules

6. Complete amplification

Denaturation leaves single-stranded

templates anchored to the substrate
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 1. Prepare genomic DNA

The 35 cycles of 2. Attach DNA to surface

bridge
amplification 3. Bridge amplification
process result in
cluster formation
of ~2000
4. Fragments become
molecules. double stranded

5. Denature the double-

stranded molecules

6. Complete amplification

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 1. Prepare genomic DNA

2. Attach DNA to surface

3. Bridge amplification

4. Fragments become
double stranded

5. Denature the double-

stranded molecules

6. Complete amplification

7. Cleavage of reverse
strand

Several million dense clusters of double-stranded DNA

are generated in each channel of the flow cell
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 8. Determine first base

 The labeled 9. Image first base

reversible
terminator s 10. Determine second base
contain a blocking
groups which
11. Image second
prevent the
chemistry cycle
incorporation of
next base during
each cycle. 12. Sequencing over
multiple chemistry cycles

13. Align data

The first sequencing cycle begins by adding four labeled

reversible terminators (corresponding to four bases),
primers, and DNA polymerase. 21
The emitted 7. Determine first base
fluorescence via
Laser excitation 8. Image first base
from each cluster
is captured and 9. Determine second base
the first base is
identified.
10. Image second
chemistry cycle

11. Sequencing over

multiple chemistry cycles

12. Align data

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Determination of 7. Determine first base
next base become
possible by 8. Image first base
removing the
florescent tag of
previous cycle and 9. Determine second base
hence unblock the
terminator. 10. Image second
chemistry cycle

11. Sequencing over

multiple chemistry cycles

12. Align data

The next cycle repeats the incorporation of four labeled

reversible terminators, primers, and DNA polymerase
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 7. Determine first base

8. Image first base

9. Determine second base

10. Image second

chemistry cycle

11. Sequencing over

multiple chemistry cycles

12. Align data

After laser excitation the image is captured as before,

and the identity of the second base is recorded.

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 7. Determine first base
Imaging data of
multiple cycles
from each
8. Image first base
cluster is stored
and converted 9. Determine second base
in to linear
sequence. 10. Image second
chemistry cycle

11. Sequencing over

multiple chemistry cycles

12. Align data

The sequencing cycles are repeated to determine the

sequence of bases in a fragment, one base at a time.

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 7. Determine first base
Reference
sequence
8. Image first base

9. Determine second base

10. Image second

chemistry cycle
Unknown variant Known
identified and called SNP 11. Sequencing over
called
multiple chemistry cycles

12. Align data

The data are aligned and compared to a reference, and

sequencing differences are identified. 26
 Three routes of Illumina sequencing technology

Single read sequencing Paired end sequencing Mate pair sequencing

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 Advantages and disadvantages of Illumina technology

 The Illumina genome Analyzer generates Giga bases of sequencing data with
read length of up to 100 bases.

 Impressive throughput with almost 430X coverage of an E. coli genome.

 As a result of reversible terminators approach homopolymer sequences

can precisely determined.

 Library preparation is less laborious.

 Per-base cost is cheaper compare to Roche technology.

 Small fragments size (75-100 bp) from Illumina create difficulty in case of
denovo genome assembling & need sophisticated algorithm /super computers.

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Applications of NGS
in medicine

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The most widely used applications are
 Whole Genome sequencing (de novo sequencing and
resequencing of human, microbial and plant
genomes)
 Tartgeted resequencing (Exome sequencing)
 Transcriptome sequencing (RNA-Seq)
 Gene Expression Analysis
 Small RNA sequencing
 Chip sequencing
 Epigenomics
 more

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Chromatin Immunoprecipitation Sequencing (ChlP-Seq)

 A technique to determine the locations where proteins interact

with DNA.

 The transcription factors interact with DNA to regulate gene

expression.

 Determining such location of DNA is essential for

understanding many biological processes and disease states.

 Mostly the Illumina/ABI Solid technologies are being used for

Chromatin immunoprecipitation sequencing (ChIP-Seq).

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Chromatin Immunoprecipitation sequencing

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 Transcriptome Sequencing
 During transcriptome analysis cDNA is sequenced rather then genomic DNA.
 Transcriptome sequencing have wide applications in
Gene expression profiling
Genome annotations
&
Non coding RNA discoveries (tRNA, rRNA, snRNA, microRNA).

 Due to short read length illumina and ABI solid technology are ideal for the
analysis of snRNA, micro RNA and small interfering RNA with deeper
coverage.

 NGS transcriptome analysis is more versatile, more throughput and cost

effective then Sanger based analysis.

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 Exome Sequencing
A Targeted Next Generation Resequencing
technique widely used in medicine.
The term “Exome” refers to all the exons in
the human genome.
The “Exome” are functionally relevant ~1% of
the genome where the majority of diease
causing muations reside.

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Major challenges in genomics research are

development of fast computational tools and

algorithm for assembling of huge NGS data and

comprehensive data analysis. .

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Thanks……..

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