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1 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
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L’evoluzione del sequenziamento:Dal metodo Sanger alla Next Generation
Roberto Fantozzi
2 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Principle of Sequencing Analysis
Standard PCR Sequencing Reaction
dNTPsdNTPs
2 Primers2 Primers 1 Primer !1 Primer !
dNTPsdNTPs ddNTPsddNTPs
3 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Sanger Method:Chain Termination Sequencing
dATPdATP
dTTPdTTP
dCTPdCTP
dGTPdGTP
ddATPddATP
ddTTPddTTP
ddCTPddCTP
ddGTPddGTP
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
Template DNA
T - A - C - T - A- G - G - T - A -
T - A - C - T - A- G - G - T - A -
T - A - C - T - A- G - G - T - A -
T - A - C - T - A- G - G - T - A -
Primer
CC - TT - AA
CC
CC - TT
CC - TT - AA - TT
4 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Cycle Sequencing Reaction
x 25 cycles
Linear amplification
60 °C
Elongation
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
50 - 55 °CHybridization
T - A - C - T - A- G - G - T - A
T - A - C - T - A - G - G - T - A - C - T - A - T - C - G
Denaturation 95 °C
5 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
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 - C - T - A- G - G - T - A -C - T
T - A - C - T - A- G - G - T - A -C - T - A
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 - C
T - A - C - T - A- G - G - T - A -C - T - A - T - C - G
Cycle Sequencing Reaction: separation
6 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Electrokinetic Injection
● 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
Capillary
Electrode (Cathode)
7 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Capillary Array:Detection Cell
8 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
9 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Sequencing Analysis Softwares
Sequencing Analysis 5.3
SeqScape 2.6
10 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
PCR (and Product Purification)
Purification
Sequencing Reaction
Electrophoresis run
Principle of Sequencing Analysis
WorkflowWorkflow
11 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...”
12 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
13 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD™ System: Enabling New Applications by Redefining the Boundaries of Traditional Sequencing
SequenceAnalysis
de Novo Sequencing
TagAnalysis
ChIP-Seq
Copy Number
Structural Variation
Methylation
Whole GenomeResequencing
TargetedResequencing
Expression
14 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD™ WorkflowApplication
specificsample
preparation
Application specificsample
preparation
Emulsion PCR &
substrate preparation
Emulsion PCR &
substrate preparation
Imaging and analysis
Imaging and analysis
Application specific
Data analysis
Application specific
Data analysis
Sequencing chemistry
Sequencing chemistry
15 Ottobre 22, 2008 © 2008 Applied Biosystems 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.
16 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Create fragment library Aplications: small genome resequencing-Tag counting
Fragmented template
Complex sample
Ligate P1 and P2 primers to end
Fragmented template can be generated through random or targeted shearing e.g. sonication, mechanical, enzymatic digestion.
17 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
PCR set up
Super paramagnetic polystirene beads
Covered with biotinilated primers-1
Dna fragments with adaptors
PCR mix with
reverse primer, dNTPs, Taq
Oil
Polymerase
18 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
19 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Distribution of DNA and beads in emulsion droplets
Bead onlyBead + 2
DNADNA only
Removed by
Enrichment
Removed by
Enrichment
Removed
By
Analysis
Software
20 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Enrichment
P1 P2
P2’
P2 P1Large (5µ)
Polystyrene
bead
Centrifuge in 60% glycerol
Supernatant Captured beads with templates
Pellet Beads with no template
21 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Sequencing Array
3’-end modification
Beads covalently attached to glass surface in a random array
Template bead deposition
22 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
2 Base Pair Encoding Using 4 Dyes
A C G T
A
C
G
T
2nd Base
1st
Bas
e
Red-probe
5’
n n n A T z z z3’
Blue-probe
5’
n n n T T z z z3’
On our probes the 1st base encoded is position 4the 2nd base encoded is position 5
23 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Properties of the Probes
green-probe
3’
n n n A C z z z
Probes are octamers
N=degenerate bases, Z=universal bases
1024 probes, 256 probes per color
Fluorescent dye indicates base on 4th and 5th position
Cleavage site is between 5th and 6th base
3’ ligation site, cleavage site and dye are spatially separated
24 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligationLigation reaction
ligase3’ p5’
universal seq primer
Template Sequence 5’ 3’
P1 Primer
1µm
bead
5’
3’ 5’
3’ 5’
3’ 5’
n n n G A z z z
n n n C C z z z n n n A T z z z
n n n A C z z z
1µm
bead
25 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
ligase
SOLiD 4-color ligationLigation reaction
ligase
Template Sequence 5’ 3’
P1 Primer
1µm
bead
universal seq primer
1µm
bead
p5’
3’ 5’
3’ 5’
3’ 5’
5’
n n n G A z z z
n n n C C z z z n n n A T z z z
n n n A C z z z
5’
n n n G A z z z
26 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligation Visualization
Template Sequence 5’ 3’
P1 Primer
1µm
bead
universal seq primer
1µm
bead
5’
n n n G A z z z
4-5
27 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligation Cleavage
Template Sequence 5’ 3’
P1 Primer
1µm
bead
universal seq primer
1µm
bead
p5’5’
n n n G A z z z
4-5
28 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligationLigation (2nd cycle)
ligase
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
universal seq primer
A T1µm
bead
p5’
n n n G A
3’ 5’
3’ 5’
3’ 5’
5’
n n n G A z z z
n n n C C z z z n n n A T z z z
n n n A C z z z
4-5
29 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
9-10
SOLiD 4-color ligation Visualization (2nd cycle)
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
1µm
bead
4-5
universal seq primer
n n n G A
5’
n n n A T z z z
30 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligation Cleavage (2nd cycle)
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
universal seq primer
A T1µm
bead
p5’
9-104-5
31 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligation interrogates every 5th base
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
universal seq primer
A T
19-20
C
14-15
T1µm
bead
24-25
G
3’ 5’
3’ 5’
3’ 5’
5’
n n n G A z z z
n n n C C z z z n n n A T z z z
n n n A C z z z
9-104-5
32 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligation Reset
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
1µm
bead
33 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
ligase
SOLiD 4-color ligation (1st cycle after reset)
ligase3’ p5’
universal seq primer n-1
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
universal seq primer n-1
T1µm
bead
p5’
3’ 5’
3’ 5’
3’ 5’
5’
n n n G A z z z
n n n C C z z z n n n A T z z z
n n n A C z z z
34 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligation (1st cycle after reset)
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
universal seq primer n-1
T1µm
bead
3-4
35 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
SOLiD 4-color ligation (2nd Round)
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
universal seq primer n-1
1µm
bead
T C GTT
8-9 13-14 23-2418-193-4
36 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Sequential rounds of sequencingMultiple cycles per round
3’
universal seq primer 4-5 9-10 14-15 21-20 24-25
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
1µm
bead
3’
universal seq primer n-1 reset
3-4 8-9 13-14 18-19 23-24
3’
universal seq primer n-2 2-3 7-8 12-13 17-18 22-23
reset
3’
universal seq primer n-3 1-2 6-7 11-12 16-17 21-22
reset
3’
universal seq primer n-40-1 5-6 10-11 15-16 20-21
reset
37 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Example of decoding (ii)
AACCGGTT
ACCAGTTG
ACCAGTTG
AACCGGTT
AACCGGTT
AGCTGATC
AGCTGATC
AGCTGATC
ATCGGCTA
A C G T
A
C
G
T
2nd Base
1st
Bas
e
AACAAGCCTC
38 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
A C G G T C G T C G T G T G C G T
Advantages of 2 base pair encoding Real SNP
reference
expected
observed
A SNP to be real must be encoded by two color changes
A C G G T C G C C G T G T G C G T
39 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
A C G G T C G T C G T G T G C G T
A C G G T C G T C G T G T G C G T1
2
3
A C G G T C G C C G T G T G C G T
A C G G T C G T C G T G T G C G T
No change
SNP
Single Mismatch
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
41 Ottobre 22, 2008 © 2008 Applied Biosystems International School of Functional Genomics
Why leave color space?Align color space reads against color space reference
ReferenceSNP 2 colors change
42 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