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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


Principle of Sequencing Analysis

Standard PCR Sequencing Reaction

dNTPsdNTPs

2 Primers2 Primers 1 Primer !1 Primer !

dNTPsdNTPs ddNTPsddNTPs


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




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


Cycle Sequencing Reaction

x 25 cycles

Linear amplification

60 °C

Elongation



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


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


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)


Capillary Array:Detection Cell


Capillary Electrophoresis

• Samples are ready for injection• Separation and detection of fluorescence-labeled DNA fragments


Sequencing Analysis Softwares

Sequencing Analysis 5.3

SeqScape 2.6


PCR (and Product Purification)

Purification

Sequencing Reaction

Electrophoresis run

Principle of Sequencing Analysis

WorkflowWorkflow


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...”


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


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


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


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.


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.


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


Emulsion PCR (ii)

Mix PCR aqueous phase into a water-in-oil (w/o) emulsion and carry out emulsion PCR


Distribution of DNA and beads in emulsion droplets

Bead onlyBead + 2

DNADNA only

Removed by

Enrichment

Removed by

Enrichment

Removed

By

Analysis

Software


Enrichment

P1 P2

P2’

P2 P1Large (5µ)

Polystyrene

bead

Centrifuge in 60% glycerol

Supernatant Captured beads with templates

Pellet Beads with no template


Sequencing Array

3’-end modification

Beads covalently attached to glass surface in a random array

Template bead deposition


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


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


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


ligase

SOLiD 4-color ligationLigation reaction

ligase


P1 Primer

1µm

bead


1µm

bead

p5’

3’ 5’

3’ 5’

3’ 5’

5’

n n n G A z z z


n n n A C z z z

5’

n n n G A z z z


SOLiD 4-color ligation Visualization


P1 Primer

1µm

bead


1µm

bead

5’

n n n G A z z z

4-5


SOLiD 4-color ligation Cleavage


P1 Primer

1µm

bead


1µm

bead

p5’5’

n n n G A z z z

4-5


SOLiD 4-color ligationLigation (2nd cycle)

ligase


Adapter Oligo Sequence

1µm

bead


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 A C z z z

4-5


9-10

SOLiD 4-color ligation Visualization (2nd cycle)



1µm

bead

1µm

bead

4-5


n n n G A

5’

n n n A T z z z


SOLiD 4-color ligation Cleavage (2nd cycle)



1µm

bead


A T1µm

bead

p5’

9-104-5


SOLiD 4-color ligation interrogates every 5th base



1µm

bead


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 A C z z z

9-104-5


SOLiD 4-color ligation Reset



1µm

bead

1µm

bead


ligase

SOLiD 4-color ligation (1st cycle after reset)

ligase3’ p5’

universal seq primer n-1



1µm

bead


T1µm

bead

p5’

3’ 5’

3’ 5’

3’ 5’

5’

n n n G A z z z


n n n A C z z z


SOLiD 4-color ligation (1st cycle after reset)



1µm

bead


T1µm

bead

3-4


SOLiD 4-color ligation (2nd Round)



1µm

bead


1µm

bead

T C GTT

8-9 13-14 23-2418-193-4


Sequential rounds of sequencingMultiple cycles per round

3’

universal seq primer 4-5 9-10 14-15 21-20 24-25



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


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


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



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


No change

SNP

Single Mismatch


Why leave color space?Align color space reads against color space reference

Reference



ReferenceSNP 2 colors change



Reference

Incorrect call , single change in color space

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