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L’evoluzione del sequenziamento: Dal metodo Sanger alla Next Generation Roberto Fantozzi
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2. Principle of Sequencing Analysis
Standard PCR
Sequencing Reaction
2 Primers
1 Primer !
dNTPs
dNTPs
ddNTPs

3. Sanger Method:Chain Termination Sequencing
Template DNA
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
T - A - C - T - A- G - G - T - A -
Primer C
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
C - T
dATP
dTTP
dCTP
dGTP
ddATP
ddTTP
ddCTP
ddGTP
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
C - T - A
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
C - T - A - T

4. 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
A - T - G - A - T - C - C - A - T - G - A - T - A - G - C
x 25 cycles
60 °C
Elongation °C
Hybridization
T - A - C - T - A- G - G - T - A
Linear amplification

5. 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 - G
T - A - C - T - A- G - G - T - A -
C -
T -
A - 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

6. 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

7. Capillary Array:Detection Cell

8. Capillary Electrophoresis
Samples are ready for injection
Separation and detection of fluorescence-labeled DNA fragments

9. Sequencing Analysis Softwares
SeqScape 2.6

10. Principle of Sequencing Analysis
Workflow
PCR (and Product Purification)
Sequencing Reaction
Purification
Electrophoresis run

11. 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. 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. SOLiD™ System: Enabling New Applications by Redefining the Boundaries of Traditional Sequencing
Sequence
Analysis
Tag
Analysis
Whole Genome
Resequencing
Expression
Structural
Variation
The SOLiD system is much more than a sequencing instrument. The power of using millions of massively parallel reads allow the system to also be used for Tagging applications such as Gene Expression and Chip for example.
Methylation
Targeted
Resequencing
ChIP-Seq
de Novo
Sequencing
Copy Number

14. SOLiD™ Workflow Imaging and analysis Emulsion PCR & substrate
Application
specific
sample
preparation
SOLiD™ Workflow
Application
specific
Data analysis
Imaging and
analysis
Emulsion
PCR &
substrate
preparation
Sequencing
chemistry
This diagram highlights the split between the application specific requirements (the two boxes at the top) and the common components that are the same in any application (the bottom 3 boxes) . In order to add a new application to the system all that needs to be developed is a library generation protocol and a workflow for analyzing the data.
The SOliD system is truly an open platform for many applications. A workflow can be developed by clean sheet approach to preparing samples. The goal is to get samples and projects prepared to basic requirements of the fragment and mate-pair libraries for inclusion in the SOliD workflow.

15. 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.
Key Advantage is that SOLiD workflow eliminates need for Bacterial Cloning.
Still need to use PCR to amplify single clones, so some bias comes with that, but it is universal to all next gen sequencers that clonally amplifly.
Current bead density is modest, we can increase signficantly w/o any additional reagents.

16. Create fragment library Aplications: small genome resequencing-Tag counting
Fragmented
template
Ligate P1 and P2 primers to end
Complex sample
Application Specific Preperation.
The NGS system Requires the production of fragments of ~100 base pairs with two primers P1 and P2 ligated to each of these fragments . The 100 base pair is based on the requirements for emulsion PCR [longer fragments can be used but there is a dramatic drop off in PCR efficiency, leading to fewer successful beads ]
It should be noted that only fragments labeled P1 and P2 will successfully go through entire workflow. A sample preparation that enhances the probability of P1 and P2 will improve efficiency.
Complex samples (genomes, exons or amplicon pools) can be concatenated and sheared for directed resequencing of known populations of ‘genomes’
This type of library is sometimes referred to as a Fragment Library to distinguish it from a mate pair library
Fragmented template can be generated through random or targeted shearing e.g. sonication, mechanical, enzymatic digestion.

17. reverse primer, dNTPs, Taq
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

18. Emulsion PCR (ii)
Mix PCR aqueous phase into a water-in-oil (w/o) emulsion and carry out emulsion PCR
CLONAL Amplification
Water-in-oil emulsions (also referred to as “reverse emulsions”) consists of water droplets suspended in oil. In emulsion PCR each of these droplets is referred to as a microreactor an the concentrations of the reactants are set up so that the average microreactor contains less than 1 fragment of target DNA.
The presence of surfactants prevents the water droplets from coalescing.. The primers and Polymerase are in excess so every microreactor will contain adequate primer and enzyme, the concentration of target DNA is arranged so that each microreactor will contain <1 target, thus minimizing the number of times that multiple targets are placed on a bead.
Microreactor contents
Bead target Primers /polymerase result
1 1 in excess single product clonal bead
1 0 in excess No product
2 1 in excess multiple single product clonal beads (each bead lower signal)
1 2 in excess poly clonal bead
Note that there are a massive number of microreactors in 1ml of solution > 1010 thus as long as you can enrich for the successful reactions it is not an issue if many microreactors do not “work”

19. Distribution of DNA and beads in emulsion droplets
Removed by
Enrichment
Removed By
Analysis
Software
Bead + 2 DNA
DNA only
Bead only

20. Enrichment Centrifuge in 60% glycerol Supernatant
Large (5µ)
Polystyrene
bead P1 P2 P2 P1
Centrifuge in 60% glycerol
Supernatant
Captured beads with templates
Pellet
Beads with no template
Enrichment is an important step as the success of the enrichment step determines the ratio of good/bad beads in the sequencing reaction .
Enrichment is important as ~30% of beads contain sequence, after enrichment this is 70-90% (and improving )
Polystyrene beads are then removed from enriched beads by melting the P2 bonds and then using a magnetic field to capture the successful beads

21. Sequencing Array Template bead deposition 3’-end modification
An advantage of the bead-based system is the potential for high-density bead packing onto the surface of the slide.
Packing efficiency is one of the processes being optimized.
Current density range is 20,000 beads per panel; 1800 panels per full array.
Beads covalently attached to glass surface in a random array

22. 2 Base Pair Encoding Using 4 Dyes
Red-probe 5’
n n n A T z z z 3’ A C G T
2nd Base
1st Base
Blue-probe 5’
n n n T T z z z 3’
On our probes the 1st base encoded is position 4
the 2nd base encoded is position 5
What does two base encoding mean?
this chart shows the color seen when a specific pair of bases is interrogated
1st base refers to the first of the pair of bases (sometimes referred to as the leading base) and the 2nd base to the 5th base in the probe (sometimes referred to as the trailing base)
We are analyzing the 4th and 5th base the chart shows the color assigned if the 4th base is T and 5th base is T a blue signal is seen if the 4th base is A and 5th is a T then red will be seen
Note as we transition to AB dyes the color scheme will change (the colors are recorded as 0,1,2,3)

23. Properties of the Probes
Cleavage site is between 5th and 6th base 3’
3’ ligation site, cleavage site and dye are spatially separated
Fluorescent dye indicates base on 4th and 5th position
n n n A C z z z
green-probe
The last three bases are shown as z as they are universal bases and do not play a role in the specificity of the ligase. The ligase used (T4 DNA Ligase) needs a length of 8 nucleotides for ligation (oligos less than this length will not be ligated by T4 ligase). However, there is only fidelity in the first 5 bases (bases must be correct in these positions for the ligase to seal the junction), so the last 3 bases in the sequence need not be correct. Universal bases have no (significant) bias in hybridization potential, so these are used to reduce probe pool complexity, while also contributing to probe stacking/stability. Using this probe design reduces the probe pool complexity to 1 correct sequence per probes (256/dye) where full degeneracy (7 generate bases and 1 cleavable) would be 1 correct sequence per 16,384 probes).
Note in actuality we use a two base encoding system, this is complex and is being omitted from this slide deck as it has to many new concepts to introduce at once. The two base encoding system facilitates identification of sequencing errors as it enables discrimination between an incorrect base call and a SNP when using a reference sequence.
Why is the A probe called the A probe when it is actually measuring the presence of a T in the target? As we will end up constructing a 25bp complementary sequence the A probe is the so called as that will be the base present in the finished 25bp complementary sequence.
Probes are octamers
N=degenerate bases, Z=universal bases
1024 probes, 256 probes per color

24. SOLiD 4-color ligation Ligation reaction
3’ p5’
universal seq primer
ligase 3’ 5’ 5’
n n n A C z z z
n n n G A z z z 3’ 5’ 3’ 5’
n n n A T z z z
n n n C C z z z
Note: Here is the first slide where we point out the fact that ligation is working in the opposite orientation that many people are used to. Standard sequencing is done using polymerases to extend strands in the 5’3’ direction. In the SOLiD system ligated products are built in the 3’ to 5’ direction.
[An aside about this technology: The ligation can be done in either direction as the ligase needs only a phosphorylated junction (or ‘nick’). Therefore, ligation-based sequencing can be done using a 5’ phosphorylated sequencing primer (as shown) but it can also be done in the other direction using 5’-phosphorylated probes (and nonphsphorylated sequencing primer).]
For clarity only one target is shown, each bead will have many targets (10,000s) per bead which leads to very bright fluorescent signal.
1µm
bead
Template Sequence 5’ 3’
P1 Primer
1µm
bead

25. SOLiD 4-color ligation Ligation reaction
ligase 5’
n n n G A z z z 3’ 5’
n n n A C z z z 3’ 5’ 3’ 5’
n n n A T z z z
n n n C C z z z
ligase 5’
n n n G A z z z
universal seq primer
After ligation, there is a capping step (with phosphatase) which will remove the 5’ phosphate from any un-extended primer. Removing the 5’phosphate from strands not extended in the previous sequencing round will render them inactive in the next ligation reaction. This capping step therefore reduces dephasing of template strands.
1µm
bead
p5’
Template Sequence 5’ 3’
P1 Primer
1µm
bead

26. SOLiD 4-color ligation Visualization5’
n n n G A z z z
universal seq primer
Wash out unligated probes, and image array using a powerful xenon light source (no lasers). Each bead will light up in 1 of 4 colors (color corresponds to either A, C, G or T). Bead images are recorded with the color of each defined at each cycle.
1µm
bead
Template Sequence 5’ 3’
P1 Primer
1µm
bead
4-5

27. SOLiD 4-color ligation Cleavage
p5’ 5’
n n n G A z z z
universal seq primer
Probes are cleaved in an efficient chemical reaction and occur between the 5th and 6th base. This generates a 5’ phosphate that is essential for the next cycle of ligation to occur. Any primer (or DNA end) that did not ligate had its 5’ phosphate removed earlier during the phosphatase step. Only strands with a 5’ phosphate can participate in the next round of ligation.
1µm
bead
Template Sequence 5’ 3’
P1 Primer
1µm
bead
4-5

28. SOLiD 4-color ligation Ligation (2nd cycle)
ligase 5’
n n n G A z z z 3’ 5’
n n n A C z z z 3’ 5’ 3’ 5’
n n n A T z z z
n n n C C z z z
universal seq primer
Note that as first ligation probe has been cleaved it is now 5 base pairs long so second base queried will be at the 10th position
1µm
bead
p5’
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
n n n G A A T
4-5

29. SOLiD 4-color ligation Visualization (2nd cycle)
universal seq primer
Read second position (10th base) 5’
1µm
bead
n n n A T z z z
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
n n n G A
4-5
9-10

30. SOLiD 4-color ligation Cleavage (2nd cycle)
p5’
Cleavage occurs between 5th and 6th base. It generates a 5’ phosphate (any primer that did not ligate had its 5’ phosphate removed earlier , the net effect is that it is blocked from future ligation, thus preventing dephasing)
universal seq primer
1µm
bead
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead A T
4-5
9-10

31. SOLiD 4-color ligation interrogates every 5th base5’
n n n G A z z z 3’ 5’
n n n A C z z z 3’ 5’ 3’ 5’
n n n A T z z z
n n n C C z z z G
Note when giving this presentation after the 5th probe (25) shows up you want to start discussing the reset reaction as the next click will reveal the cleaned up target
Note the final probe does not need to be cleaved as it will be removed at reset.
universal seq primer
1µm
bead
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead A T T C
4-5
9-10
14-15
19-20
24-25

32. SOLiD 4-color ligation Reset
This is the result of a reset: the extended template strand is melted off and the sequencing template re-exposed as a fresh, clean template and devoid of any noise generated by previous sequencing cycles. In this method, noise is generated by the attrition of strands at each cycle (uncompleted extensions) that serve to reduce template number (and therefore, fluorescence intensity) on each bead, and increases the noise-to-signal ratio of each bead.
The ability to ‘reset’ is one of the major benefits of this chemistry. A major problem that limits read length of all NGS systems is that as the read gets longer, the signal falls and noise rises, until you can no longer accurately call a base. By resetting the system every 5 cycles we remove all the accumulated noise at each sequencing round.
1µm
bead
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead

33. SOLiD 4-color ligation (1st cycle after reset)
3’ p5’
universal seq primer n-1
ligase 5’
n n n G A z z z 3’ 5’
n n n A C z z z 3’ 5’ 3’ 5’
n n n A T z z z
n n n C C z z z
ligase T
The cycles are now exactly as before but the primer is set one base back (n-1) note the primer is not shorter it is offset by one base. The length of the P1 oligo is 41-bp an therefore allows 19-bp sequencing primers to nest back for several rounds of sequencing.
universal seq primer n-1
1µm
bead
p5’
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead

34. SOLiD 4-color ligation (1st cycle after reset)
The cycles are now exactly as before but the primer is set one base back (n-1) note the primer is not shorter it is offset by one base. The length of the P1 oligo is 41-bp an therefore allows 19-bp sequencing primers to nest back for several rounds of sequencing.
universal seq primer n-1
1µm
bead
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead
3-4

35. SOLiD 4-color ligation (2nd Round)
The cycles are now exactly as before but the primer is set one base back (n-1) note the primer is not shorter it is offset by one base. The length of the P1 oligo is 41-bp an therefore allows 19-bp sequencing primers to nest back for several rounds of sequencing.
universal seq primer n-1
1µm
bead
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead T
3-4
8-9
13-14
18-19
23-24

36. Sequential rounds of sequencing Multiple cycles per round
bead
Template Sequence 5’ 3’
Adapter Oligo Sequence
1µm
bead 3’
universal seq primer
4-5
9-10
14-15
21-20
24-25
reset 3’
universal seq primer n-1
3-4
8-9
13-14
18-19
23-24
reset
universal seq primer n-2 3’
2-3
7-8
12-13
17-18
22-23
Again the reset is one of the unique attributes of Ligation based sequencing, A first round and four resets give a total of 5 rounds of sequencing that records the color at every 5th position to generate a 25 base read length. Feasibility out to 50 bases has been demonstrated (5 rounds of 10 cycles).
reset
universal seq primer n-3 3’
1-2
6-7
11-12
16-17
21-22
reset
universal seq primer n-4 3’
0-1
5-6
10-11
15-16
20-21

37. Example of decoding (ii)T
2nd Base
1st Base
Example of decoding (ii) 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
This slide explains how to decode a sequence. All you need to decode a base sequence is to know
IN this instance we will say we know the first base is an A [important it does not need to be the first base as long as you are certain of one of the bases then the decoding is automatic]
Remember that the 2nd base in each decoded pair is the first base of the next pair
In this example we know the first base is an A using the lookup table 1st base A and blue color tells us second base is an A
Now moving to the second observed color we know the first base this pair must be an A so if we see 1st base A and green signal 2nd base must be a C and so on ………………………………………….
AACAAGCCTC

38. Advantages of 2 base pair encoding Real SNP
A C G G T C G T C G T G T G C G T
reference
expected
observed
A C G G T C G C C G T G T G C G T
In the case of a real SNP there will always be two color changes
A SNP to be real must be encoded by two color changes

39. 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
No change
A C G G T C G C C G T G T G C G T 2
SNP
A C G G T C G T C G T G T G C G T
Single Mismatch 3

40. Why leave color space? Align color space reads against color space reference
There is no need to ever leave color space as you can convert your reference sequence to its color space and then align the two sequences. This is in fact desirable given the error properties of color space, our analysis software will use color space. note the misalignment in here are explained in next two slides)
Reference

41. Why leave color space? Align color space reads against color space reference
This is a SNP because you are sequencing amplified single molecules you see heterozygotes as differences between individual reads in this case 4/3 so its approx 50;50. As the sequencing depth will be much greater you would be able to easily detect variations form the 50:50 ratio e.g. cancer resequencing can see an allele present at 1% (or even less if sequence to great depth)
Reference
SNP 2 colors change

42. Why leave color space? Align color space reads against color space reference
In this case there is a single error, this shows up as a single changed color call in color space with rest of the calls still aligning. {note in base space the single error in color space causes all the sequence to change)
Reference
Incorrect call , single change in color space