DNA SEQUENCING

-
o DNA Sequencing is Figuring out the order of DNA
nucleotides, or bases (A T G C ), in a genome that make
up an organism's DNA.
Strand to be sequenced
-- -"---- - Sangar
Sequencing
Preparelour reaction m xtures:
IncludeIn oacl\ a di11eren1
Prlmed DNA ropllcalion-stopplngr1uc1001ld9
+
7 T
A

Primer
opllcalion , Separate
ptodu<:IS 01 - prOdu<:LS by
·c·roaclion got electrophorQ$iS

F. Sangar
 History of DNA sequencing
- 1953
Discovery of the structure of the
1996
Pal Nyren and his student Mostafa
Ronaghi at the Royal Institute of
1998
Phil Green and Brent Ewing of
the University of Washington
Technology in Stockholm publish
DNA double helix publish "phred" for sequencer
their method of Pyrosequencing
data analysis.

1995
1972 Craig Venter Hamilton Smith 2001
and colleagues publish the I st
Development of Recombinant complete genome of bacterium
A draft sequence of the human
DNA technology,. H. irifluenzae (whole-genome genome is published.
shotgun sequencing.)

1977 1990
The first complete DNA genome to The U.S. National Institutes of 2004
be sequenced is that of Health (NIH) begins large-scale
Bacteriophage <pXI 74 & 454 Life Sciences markets a
sequencing trials on M
capricolum, E. coli parallelized version of
Frederick Sanger publishes "DNA
Caenorhabditis elegans and S. Pyrosequencing.
sequencing with chain­terminating
inhibitors" cerevisiae

1984
1987 2006
Medical Research Council
scientists decipher the complete Applied Biosystems markets first Era of Next Generation
DNA sequence of the Epstein­Barr automated sequencing machine, Sequencing- 454
virus, 170 kb. the model ABI 370. Sequencing, Illumina etc.
l )
 ERA OF SEQUENCING
-
1st
•
Generation sequencing
Sequence many identical molecules

• Sequencing in large gels or capillary tubing limits

scale
Sangar Chain Termination Maxam- Gilbert Sequencing
( 1977 ) (1977)

Strand to be sequenced
- - - ,.f'\.....
(B)
TheGrill< Dbeseq.J
DI 8l<1'd
O;w;:; lillallf'mp
Prepare tour reaction mixturos:
l!S)
lnciudo n t!-Ocil adltl'oront
ropllcation.-stopp ng nuclooUdo
tm
Primed DNA

l
+

J Id
!
DMS09
0'C
--
ii+.mr
Q!Mll 'filfl' lstlfal!

i fm
Me
 ERA OF SEQUENCING
ation Sequencing 2nd Generation Sequencing

• Sequence many identical • Sequence millions of clonally

molecules amplified molecules per run
• Sequencing in large gels or • Using a reversible, stepwise
capillary tubing limits scale sequencing chemistry I QIAGEN GeneReader
• Immobilized on a surface
I

Life Technologies/Applied Roche I 454

Biosystems; SOLID 5500 Pyrosequencer
 NEXT GENERATION SEQUENCING

- High throughput DNA Sequencing Technique.

"'-* Employs Micro and Nanotechnologies
Reduce sample size.
Low Reagent cost Less Time
_ Massive Parallel Sequencing
- Sequence thousands of sequences at
once.
Produce enormous amount of data .
 NGS WORI<FLOW
Sample Extraction , DNA fragmentation and invitro adapter ligation

========== ===

A B
Clonal Amplification by Clonal Amplification by
Emulsion PCR ., Bridge PCR

f.

DNA DNA polymerase

DNA
polymerase ligase
A C G T 3'
-
A.....111111111 Aee • 5'

11 35·'
Time
c e e ee
G ••
PP
ATP-su lphurylase
r ee••
i
I

J ATP
Luciferas
• • •
e Ligh G G + - T+ -A
t

Pyrosequencing Sequencing-by-ligation I Sequencing-by-synthesis

(454 Seguencing)_. L csoLiD Platform Solexa Technology)
 NGS WORI<FLOW
-
1. Create DNA fragments

2. Add platform-specific adapter sequences to every fragment.

'i!!!ldapter
igation
point

Adapter molecule bound

Adapter to DNA .
molecule

Adapter molecules : Bind library to a flowcell or bead; Add sequence primer

binding sites & Add barcodes for multiplexing.
Adapter Binding
300 bp sample DNA insert

•-•dapter

-->
gation point

Bridg1e amplification to form

a cluster

IDNA >>
primer 1
Bind primer and sequence

I
 Cluster Amplification:
Bridge PCR
-

C A T
DNA fragments are flanked with adaptors (library)
G
A solid surface is coated with primers complementary to
the two adaptor sequences
Isothermal amplification, with one end of each "bridge"
tethered to the surface
Clusters of DNA molecules are generated on the chip.
Each cluster is originated from a single DNA fragment,
and is thus a clonal population. G A C A
TCGCGTA
Cydes - )
 Cluster Amplification :
Emulsion PCR
-
-

;- :-:;::(-
.

.:-
r-l .
Several thousand on average 1.6 million wells
copies of the same

*-.·-;
template sequence
oneach bead

Fragments with adaptors (the library) are PCR amplified within a water drop in oil.
One PCR primer is attached to the surface of a bead.
DNA molecules are synthesized on the beads in the water droplet. Each bead bears
clonal DNA originated from a single DNA fragment
Beads (with attached DNA) are then deposited into the wells of sequencing chips - one
well, one bead
 Sequencing & Imaging Technologies:
Chain Reversible Termination
-
Sequencing by Cyclic Reversible Termination (CRT): CRT uses
reversible terminators in a cyclic method that comprises nucleotide
incorporation, fluorescence imaging and cleavage. ioo-15obp reads are used.
•
a DNA polymerase, bound to the primed template, adds or incorporates just one
fluorescently modified nucleotide
Unincorporated nucleotides are washed away and a four­acquired
by total internal reflection fluorescence (TIFR) u
e ., a lllumina/Solexa- Reversible terminators
'.
..
(f)
A cleavage step (TCEP, a reducing agent) removes the t• restoring @a <D c ...T
the 3'-0H group and the fluorescent dye al>.
Incorporate all_.,.
four
" nucleotides,
each label with
a different dye

-
lncorpcnte
illlfour
nucleordes
exh bb"."'
Woth• dfft dye

Wash.four­
0
"'' I"0 00"1")
r 0 o",," colour imaging
\.--/
lfl

I I \ \ I

••
( _
Cleave dye
and term1nat1ng

•
groups. wash
I I I I I I I I

Repeat cycles'.... •
 Sequencing & Imaging Technologies:
Sequencing by Ligation
- Sequencing by Ligation (SBL) uses the enzyme DNA ligase to identify the nucleotide present

at a given position in a DNA sequence.

Cleavage agent *

D
Ligation cycle l 2
3 4 5
6 7... (n
cycles)
J'

Reset primer (n - 1),repeat ligation cycles

2nd base
Q
A C G U..._.,&Jseq pnm« C n I

V') A T 1· ''''''''
J
n:
:s c 'C
Jr
:
0• ie"l Pl''"" (n·l>
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Url -,..1.t!q prlmM-(n-l)--,,.......--.... .,--
....
V'l
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+-t-....,_,r+--f-+-'-
'''''''''''-r-"'-------,---t--t-.,._,-t-
,...
...0 -:-:-t-
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T E
4 Unl aq p11mtt ( n1J>
-r-t-t-t--t-+-
1....
l' iIIIIiiIIIIhIIii

!> Un1on;rlill t--t--r-r---r--:-

pNPirr I "°"") ---:--:-..,..--,
:J iiiiiiiiiiiiiiiii
 Sequencing & Imaging Technologies:
Pyrosequencing
- Pyrosequencing: non-electrophoretic, bioluminescence
release of inorganic pyrophosphate by proportionally converting it into visible light using a series of
enzymatic reaction

method that measures the

dNT dND dNMP + pho ph
P P t

Lag
luclfer m oxyluciferin
ht

APS+ AT AT
PPi P P

Nucleotide incorporation generates light seen as a peak in the

Pyrogram trace Tim
e
 Sequencing & Imaging Technologies:
Single Molecule-Real Time Sequencing
-
Single Molecule- Real Time (SMRT) is a parallelized single molecule DNA
sequencing method. A single DNA polymerase enzyme is affixed at the bottom of a ZMW
with a single molecule of DNA as a template

G T A G C
Cl>
DNA 0
c C
Q)

Polymerase 0
II)

ZMW
0
(Zero Mode ::
u:
>
Waveguide DNA)
 NGS Technologies Overview
- NGS differs in template preparation, sequencing and imaging, and data analysis
Commercially available technologies:
Illumina /Solexa Roche/454
Helicos BioSciences
Life/APG - SOLiD system Pacific Biosciences
Ion Torrent technology
 ILLUMINA/SOLEXA SEQUENCING
d-phase amplification can produce 100-200 million spatially separated clusters, providing free
ends to which a universal sequencing primer can be hybridized to initiate the NGS reaction

lllumtna/otoxa

' '
ohd phas• llmpliflcatlon
0Me ONA rriut.,,cu l!'" p"'r clu )l"'t

S;:i. p e prep;inrt1on
DNA (S·g)
Templllte (
dl'-ITPt.
.:ond
polyn1C:<"o,.,,

'
T D Run time: 1-10 days
Bridge PCR Clustal
D Produces: 2-1000 Gb of sequence
(i

... -+ •••• --+

Amplification c
... D Read length: 2 x 50 hp - 2 x 250 hp
'T
. ,. (paired-end)
.
D Cost: $0.05-$0.40/Mb
c
a lllumina/Solexa -Reversible terminators
® o Applications
@ C ee'l'
D DNA sequencing
Incorporate all
four
nucleotides.
,..A c Gene Regulation Analysis
IS:>G
each label
wrth a
different dye c Sequencing-based Transcriptome Analysis
c SNPs and SVs discovery
c Cytogenetic Analysis
c ChIP-sequencing
Wash.four­
colour imaging D Small RNA discovery analysis

T T .-\ :\ I"
Cleave dye
and terminating
groups,wash

Repeat cycles '.... •

II 1111
1111111 111
T T 'I T T T I'
Over 1800
publications . • A whole human genome sequence was
determined in 8weeks to an ·1average depth
of - 40X, discovering - 4 new million SNPs
and -400000 SVs (with an accuracy <1%
for both over-calls and under-calls)
 ROCHE/454 SEQUENCING
o
-
Sequence much longer reads by sequencing multiple reads at once by reading optical signals as bases
are added.

oThe DNA or RNA is fragmented into shorter reads up to 1kb. o

Uses Emulsion PCR for Clustal Amplificication .
oPYROSEQUENCING as sequencing approach.
A
G

i
T
T

-
c
G
A
G
T
cUNA'
pol T
A T
A T
T

on average 1.6 mill on wells

Several thousand copies
of the same template
sequence on each bead
 .... ' ' Polymerase

T A G c ONA capture beoo

conla.inlng mllions cl
copies of a aingle clonally
amphfoed
lro9.-n1

All of the sequence reads we get from 454 will be different Pyrosequencing reaction

lengths, because different numbers of these bases will be added

with each cycle.
Tomplettt
Over 1300
publications...
1, Polym8T&•e 2.sunurylaao

Apyro&o
enzym•

Nucleotide incorporation generates light seen as a peak in the

Pyrogram trace .

Ct' • Applications
lc:
>

-
·u;
:
oWhole genome sequencing o Targeted
Q)
u
<n resequencing
Q
..
c::
0
..::J).
u: oSequencing-based Transcriptome Analysis o
: Metagenomics

nucleotides
 ION TORRENT SEQUENCING
Ion torrent and ion proton sequencing do not make use of optical signal exploit
the fact that addition of a dNTP to a DNA polymer releases an H+
Run time: 3 h; no termination or deprotection steps Clustal Amplification-
Emulsion PCR
T T
Read length: ioo-300 bp

Throughput determined by chip size :ioMb - 5 Gb Cost: $1-$20/Mb

dNTP

• • •
1
1
T A
c G
The pHchange, if any, is used to determine how many bases
(if any) were added with each cycle.
 LIFE/APG/ABI- SOLiD SEQUENCING
-
D AB SOLID™ 3 System generates over 20 gigabases & 400 M tags per run .
Library Preparation
Emulsion PCR/ Bead Enrichment Bead deposition
Sequencing by Ligation

2. Chemical crosslinking to
an amino-coated glass
surface
 SANGERS Vs. NGS
- Features
Sequencing
Sanger
Clones, PCR
NGS
DNA Libraries
Samples
Preparation Steps Few, Sequencing reactions clean up Many, Complex
procedures
Data Collection Samples in plates : Samples on slides
96, 384 1-16+
Data 1Read/ Sample Thousands & Millions of
Reads/ Samples.

• ••c •
•cc•e -0

c •
·
• •
••c-•
c

,
 Comparison Of NGS Platforms
Sina i -rno lec.u l Sequ nctn · by S quenc. by
Ion Py rose u nclng t rmlnatlon (S
M thod re Itlm yn the !ls bg tJon (SOLtO
s m 1Gond uc:tor (4S4 ) r-.g r
uenc1nc (lllumin ) s;equ ncin&)
s Q nc.I )

I
S0.-95 or 50-i-50
Readle nsth
2900 b p r_a_e-1f2-_oo
87% (rcd I ngthbp
t__oo
b_P I
·SO to_2_so b_P bp 400 to 900 bp

Accuracy mode). 98% 99.9% 98%

lcaccu racy mock!>
Rads p r run 5-75 thoL&Sand up to S m11t1on 1mlIlion 1.2 to 1..4 b1ll1on N/A
up to 3
l to 10 days.
billion
dcpendl upon
30 minutes to 2 20 minutes to 3
Time p.urW\ 2 hours 24 nours sequcnc r nd l to 2 w
hours hours
specified re d ks
length
cos.. pti' l SO.OS to $0.15 $0.13
m Oion ba es
$2 $1 $10 $2400

Potent..ia l for
Longest read Long 1nd1Yldual
nigh S<!q uencc
le.ngt h. Fast. Le:s.s pcnsf\f c Long read s1m yield, depending Low cost per reads.Usefulror ma
Advantaae-s
Dctect.s 4 mC, qulpment. Fast. Fast. base. ny
upon sc quc neer
5mC. 6mA. ppIleatlons.
mode.I

Low yJcld at h\gh Runs am ecpc More cxpcnstv«?

Homopolym r Equlpmcnt can Slow.er than and lmpractlc.a l for
DTud"" nU•• accuracy.
Equipment
b
... -rv can c rrors.
rt.Slve.
Homopolym r
be very
xpens ve .
ot rm
largcr
sc.qlJ(!ncing
thods.
1
JcpensN. .. errors_. _._ ...P__ro _
c_. c_c_ts .
 ADVANTAGES OF
NGS
a
-
--+-
- b

-
--+-
DNA fragmentation

-
== -- -
= =
--
In vivo cloning and amplification
= --
In vitro adaptor ligation
=
- -
DNA fragmentation

Cycle sequencing
--- Generation of polony array
- Low Reagent Cost
3' ... GACTAGATACGAGCGTGA ...-5' (template)
5'-... CTGAT
p r

L
...CTGATC ,P_
...CTGATCT

---
...CTGATCTA ..P_
(primer)
- Reduced Sample Size
...CTGATCTAT
...CTGATCTATG n
...CTGATCTATGC ,,,..._,

c(
Polymerase dNTPs ...CTGATCTATGCT ...f-'I
r·a
Labeled ddNTPs ...CTGATCTATGCTC
-
l
...CTGATCTATGCTCG ,,-
ss Time
Eleclrophorsesis (1 Cycl c array sequencing
read/capillary) (>106 reads/array)
Cycle 1 Cycle 2 Cycle 3 Le
c
T

What is base 1? What is base 2? What is base 3?

 APPLICATIONS OF NGS
Mutation discovery
oTranscriptome Analysis - RNA-Seq
oSequencing clinical isolates in strain-to-reference mechanisms. o
Enabling Metagenomics
oDefining DNA-Protein interactions - ChIP-Seq o Discovering
non-coding RNAs
oMolecular diagnostics for Oncology & Inherited Disease study. o

Gene Regulation Analysis

oWhole Genome Sequencing
oExploring Chromatin Packaging
 FUTURE APPLICATIONS OF NGS

Tumo
DNA
"h ro1u 1cs PCR

-- PCRi--...i
- Sornatic mutation detection

Ta1geted re sequenc11tg•Exomo
DNA SeqCap
Mendahan d&SordQrs/HlJ\ typ og

rary creahon

of. ng

PCA
--- .

"""'