Click
to edit del
Master
title style
L’evoluzione
sequenziamento:
Dal metodo Sanger alla Next Generation
Click to edit Master subtitle style
Roberto Fantozzi
1
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Principle of Sequencing Analysis
Standard PCR
2 Primers
dNTPs
2
Ottobre 22, 2008
Sequencing Reaction
1 Primer !
dNTPs
© 2008 Applied Biosystems
ddNTPs
International School of Functional Genomics
Sanger Method:Chain Termination Sequencing
Template DNA
Primer
A-T-G-A-T-C-C-A-T-G-A-T-A-G-C
T - A - C - T - A- G - G - T - A - C
A-T-G-A-T-C-C-A-T-G-A-T-A-G-C
T - A - C - T - A- G - G - T - A - C - T
dATP
ddATP
dTTP
ddTTP
dCTP
ddCTP
dGTP ddGTP
A-T-G-A-T-C-C-A-T-G-A-T-A-G-C
T - A - C - T - A- G - G - T - A - C - T - A
A-T-G-A-T-C-C-A-T-G-A-T-A-G-C
T - A - C - T - A- G - G - T - A - C - T - A - T
3
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Cycle Sequencing Reaction
Denaturation 95 °C
T-A-C-T-A-G-G-T-A-C-T-A-T-C-G
A-T-G-A-T-C-C-A-T-G-A-T-A-G-C
x 25 cycles
Elongation
60 °C
A-T-G-A-T-C-C-A-T-G-A-T-A-G-C
T - A - C - T - A- G - G - T - A
Linear amplification
4
Ottobre 22, 2008
© 2008 Applied Biosystems
Hybridization
50 - 55 °C
International School of Functional Genomics
Cycle Sequencing Reaction: separation
Electrophoresis
●
separation matrix :
gel or polymer
●
Separation according to
the size of the DNA
fragment
●
1 bp resolution
T - A - C - T - A- G - G - T - A - C - T - A - T - C - G
T - A - C - T - A- G - G - T - A - C - T - A - T - C
T - A - C - T - A- G - G - T - A - C - T - A - T
T - A - C - T - A- G - G - T - A - C - T - A
T - A - C - T - A- G - G - T - A - C - T
T - A - C - T - A- G - G - T - A - C
5
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Electrokinetic Injection
Electrode (Cathode)
Capillary
● Capillary and electrode (cathode)
are placed into the sample
● Voltage is applied for a specified time
● Negatively-charged DNA enters the
capillary as it migrates toward the
postively-charged electrode (anode)
at the other end of the capillary
● Capillary is removed and placed into buffer
for electrophoresis
6
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Capillary Array:Detection Cell
7
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Capillary Electrophoresis
• Samples are ready for injection
• Separation and detection of fluorescence-labeled DNA fragments
8
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Sequencing Analysis Softwares
Sequencing Analysis 5.3
SeqScape 2.6
9
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Principle of Sequencing Analysis
Workflow
PCR (and Product Purification)
Sequencing Reaction
Purification
Electrophoresis run
10
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
E domani...?
“…Quando nel 2000 la Celera Genomics aveva terminato la
mappatura del DNA con una spesa di qualche centinaio di milione
di dollari…”
“...Oggi l’obiettivo è di avere l’intero genoma con 1000 dollari...”
11
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Next Generation System (NGS) - Overview
● The NGS is a genetic analysis platform that
enables massively parallel sequencing of
clonally amplified DNA fragments linked to
beads.
● Sequencing methodology is based on
sequential ligation with dye-labeled
oligonucleotide probes.
● The instrument Generates up to 20 GB of
mappable data/run
12
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD™ System: Enabling New Applications by Redefining the
Boundaries of Traditional Sequencing
Sequence
Analysis
13
Tag
Analysis
Whole Genome
Resequencing
Expression
Structural
Variation
Methylation
Targeted
Resequencing
ChIP-Seq
de Novo
Sequencing
Copy Number
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Application
specific
sample
preparation
Emulsion
PCR &
substrate
preparation
14
Ottobre 22, 2008
SOLiD™ Workflow
Sequencing
chemistry
© 2008 Applied Biosystems
Application
specific
Data analysis
Imaging and
analysis
International School of Functional Genomics
SOLiD - Workflow
1. Prepare a fragment or mate-paired library
from starting material.
2. Amplify library onto beads using emulsion
PCR
3. Deposit bead clones onto slide surface.
4. Sequence clones by ligation-based
sequencing.
15
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Create fragment library
Aplications: small genome resequencing-Tag counting
Complex
sample
Fragmented
template
Ligate P1 and P2
primers to end
Fragmented template can be generated through random or
targeted shearing e.g. sonication, mechanical, enzymatic
digestion.
16
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
PCR set up
Dna fragments with adaptors
Super paramagnetic polystirene beads
Covered with biotinilated primers-1 
Polymerase
PCR mix with
reverse primer, dNTPs, Taq
Oil
17
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Emulsion PCR (ii)
Mix PCR aqueous phase into a water-in-oil (w/o)
emulsion and carry out emulsion PCR
18
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Distribution of DNA and beads in emulsion droplets
Removed
by
Enrichment
Removed
By
Removed
by
Enrichment
Analysis
Software
Bead only
19
Ottobre 22, 2008
Bead + 2
DNA
© 2008 Applied Biosystems
DNA only
International School of Functional Genomics
Enrichment
P2’
P1
P2 Large (5µ)
P2
Polystyrene
P1
bead
Centrifuge in
60% glycerol
Supernatant
Captured beads with templates
Pellet
Beads with no template
20
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Sequencing Array
Template bead
deposition
3’-end
modification
21
Ottobre 22, 2008
Beads covalently attached to
glass surface inInternational
a random
array
© 2008 Applied Biosystems
School
of Functional Genomics
2 Base Pair Encoding
Using 4 Dyes
Red-probe
2nd Base
A
C
G
5’
3’
n n n A T z z z
T
1st Base
A
Blue-probe
C
5’
3’
n n n T T z z z
G
T
On our probes the 1st base encoded is
position 4
the 2nd base encoded is position 5
22
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Properties of the Probes
Cleavage site is between
5th and 6th base
3’ ligation site,
cleavage site and
dye are spatially
separated
3’
n n n A C z z z
Fluorescent dye
indicates base on 4th
and 5th position
green-probe
Probes are octamers
N=degenerate bases, Z=universal bases
1024 probes, 256 probes per color
23
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD 4-color ligation
Ligation reaction
universal seq primer
3’
p5’
ligase
3’
5’
5’
nnnACzzz
nnnGAzzz
3’
5’
nnnCCzzz
1µm 1µm
beadbead
24
5’
Ottobre 22, 2008
P1 Primer
3’
nnnATzzz
Template Sequence
© 2008 Applied Biosystems
5’
3’
International School of Functional Genomics
SOLiD 4-color ligation
Ligation reaction
ligase
3’
5’
5’
nnnACzzz
nnnGAzzz
3’
5’
nnnCCzzz
3’
5’
nnnATzzz
ligase
universal seq primer
p5’
1µm 1µm
beadbead
25
5’
nnnGAzzz
5’
Ottobre 22, 2008
P1 Primer
Template Sequence
© 2008 Applied Biosystems
3’
International School of Functional Genomics
SOLiD 4-color ligation
Visualization
universal seq primer
1µm 1µm
beadbead
5’
nnnGAzzz
5’
P1 Primer
Template Sequence
3’
4-5
26
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD 4-color ligation
Cleavage
universal seq primer
p5’
1µm 1µm
beadbead
5’
nnnGAzzz
5’
P1 Primer
Template Sequence
3’
4-5
27
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD 4-color ligation
Ligation (2nd cycle)
ligase
3’
5’
5’
nnnACzzz
nnnGAzzz
3’
5’
nnnCCzzz
3’
5’
nnnATzzz
universal seq primer
p5’
1µm 1µm
beadbead
nnnG
5’
A
A
T
Adapter Oligo Sequence
Template Sequence
3’
4-5
28
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD 4-color ligation
Visualization (2nd cycle)
universal seq primer
1µm 1µm
beadbead
5’
n n n G An n n A T z z z
5’
Adapter Oligo Sequence
Template Sequence
4-5
29
Ottobre 22, 2008
3’
9-10
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD 4-color ligation
Cleavage (2nd cycle)
universal seq primer
1µm 1µm
beadbead
A
5’
Ottobre 22, 2008
p5’
Adapter Oligo Sequence
Template Sequence
4-5
30
T
3’
9-10
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD 4-color ligation
interrogates every 5th base
3’
5’
5’
nnnACzzz
nnnGAzzz
3’
3’
5’
5’
nnnATzzz
nnnCCzzz
universal seq primer
1µm 1µm
beadbead
A
5’
Ottobre 22, 2008
T
Adapter Oligo Sequence
C
G
Template Sequence
4-5
31
T
9-10
14-15 19-20
© 2008 Applied Biosystems
3’
24-25
International School of Functional Genomics
SOLiD 4-color ligation
Reset
1µm 1µm
beadbead
32
5’
Ottobre 22, 2008
Adapter Oligo Sequence
© 2008 Applied Biosystems
Template Sequence
3’
International School of Functional Genomics
SOLiD 4-color ligation
(1st cycle after reset)
universal seq primer n-1
3’
ligase
p5’
3’
5’
5’
nnnACzzz
nnnGAzzz
3’
5’
nnnCCzzz
3’
5’
nnnATzzz
ligase
universal seq primer n-1
p5’
1µm 1µm
beadbead
33
5’
Ottobre 22, 2008
T
Adapter Oligo Sequence
© 2008 Applied Biosystems
Template Sequence
3’
International School of Functional Genomics
SOLiD 4-color ligation
(1st cycle after reset)
universal seq primer n-1
1µm 1µm
beadbead
T
5’
Adapter Oligo Sequence
Template Sequence
3’
3-4
34
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
SOLiD 4-color ligation
(2nd Round)
universal seq primer n-1
1µm 1µm
beadbead
T
5’
Ottobre 22, 2008
T
Adapter Oligo Sequence
C
G
Template Sequence
3-4
35
T
8-9
13-14
© 2008 Applied Biosystems
18-19
3’
23-24
International School of Functional Genomics
Sequential rounds of sequencing
Multiple cycles per round
1µm 1µm
beadbead
5’
Adapter Oligo Sequence
Template Sequence
3’
universal seq primer
4-5
3’
reset
14-15
21-20
24-25
universal seq primer n-1
3-4
3’
reset
8-9
13-14
18-19
23-24
universal seq primer n-2
2-3
3’
reset
7-8
12-13
17-18
22-23
universal seq primer n-3
3’
reset
1-2
6-7
11-12
16-17
21-22
universal seq primer n-4
3’
36
9-10
Ottobre 22, 2008
0-1
5-6
10-11
© 2008 Applied Biosystems
15-16
20-21
International School of Functional Genomics
2nd Base
Example of decoding (ii)
A
C
G
T
1st Base
A
C
G
T
AA
CC
GG
TT
AC
CA
GT
TG
AC
CA
GT
TG
AA
CC
GG
TT
AG
CT
GA
TC
AT
CG
GC
TA
AA
CC
GG
TT
AG
CT
GA
TC
AG
CT
GA
TC
AACAAGCCTC
37
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Advantages of 2 base pair encoding
Real SNP
ACGGTCGTCGTGTGCGT
reference
expected
observed
ACGGTCGCCGTGTGCGT
A SNP to be real must be encoded by
two color changes
38
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
ACGGTCGTCGTGTGCGT
ACGGTCGTCGTGTGCGT
1
No change
ACGGTCGCCGTGTGCGT
2
SNP
ACGGTCGTCGTGTGCGT
Single
Mismatch
3
39
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Why leave color space?
Align color space reads against color space reference
Reference
40
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Why leave color space?
Align color space reads against color space reference
Reference
SNP 2 colors change
41
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics
Why leave color space?
Align color space reads against color space reference
Reference
Incorrect call , single change in color space
42
Ottobre 22, 2008
© 2008 Applied Biosystems
International School of Functional Genomics