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1 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Click to edit Master title style Click to edit Master subtitle.

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Presentation on theme: "1 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Click to edit Master title style Click to edit Master subtitle."— Presentation transcript:

1 1 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Click to edit Master title style Click to edit Master subtitle style Indications in green = Live content Indications in black = Edit in master Indications in white = Locked elements Template release: Date Levoluzione del sequenziamento: Dal metodo Sanger alla Next Generation Roberto Fantozzi

2 2 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Principle of Sequencing Analysis Standard PCR Sequencing Reaction dNTPs 2 Primers 1 Primer ! dNTPs ddNTPs

3 3 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Sanger Method:Chain Termination Sequencing dATPdTTPdCTPdGTPddATPddTTPddCTPddGTP 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 - Primer CTAC - T - ACTAC - T - A C CTC - TCTC - T CTATC - T - A - TCTATC - T - A - T

4 4 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 50 - 55 °C Hybridization 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 5 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 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 -TT - A - C - T - A- G - G - T - A -C -T -A -T -CT - A - C - T - A- G - G - T - A -C -T -A -T -C -G Cycle Sequencing Reaction: separation

6 6 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 7 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Capillary Array:Detection Cell

8 8 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Capillary Electrophoresis Samples are ready for injection Separation and detection of fluorescence-labeled DNA fragments

9 9 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Sequencing Analysis Softwares Sequencing Analysis 5.3 SeqScape 2.6

10 10 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics PCR (and Product Purification) Purification Sequencing Reaction Electrophoresis run Principle of Sequencing Analysis Workflow

11 11 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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…DNA...Oggi lobiettivo è di avere lintero genoma con 1000 dollari...

12 12 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 13 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD System: Enabling New Applications by Redefining the Boundaries of Traditional Sequencing Sequence Analysis de Novo Sequencing Tag Analysis ChIP-Seq Copy Number Structural Variation Methylation Whole Genome Resequencing Targeted Resequencing Expression

14 14 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD Workflow Application specific sample preparation Application specific sample 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 15 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 16 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 17 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 18 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 19 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Distribution of DNA and beads in emulsion droplets Bead only Bead + 2 DNA DNA only Removed by Enrichment Removed by Enrichment Removed By Analysis Software

20 20 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Enrichment P1P2 P1 Large (5µ) Polystyrene bead Centrifuge in 60% glycerol Supernatant Captured beads with templates Pellet Beads with no template

21 21 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Sequencing Array 3-end modification Beads covalently attached to glass surface in a random array Template bead deposition

22 22 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics 2 Base Pair Encoding Using 4 Dyes ACGT A C G T 2 nd Base 1 st Base Red-probe 5 n n n A T z z z 3 Blue-probe 5 n n n T T z z z 3 On our probes the 1 st base encoded is position 4 the 2 nd base encoded is position 5

23 23 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 24 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD 4-color ligation Ligation reaction ligase 3 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 zn n n C C z z zn n n A T z z zn n n A C z z z 1µm bead

25 25 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics ligase SOLiD 4-color ligation Ligation 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 zn n n A T z z zn n n A C z z z 5 n n n G A z z z

26 26 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 27 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 28 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD 4-color ligation Ligation (2 nd cycle) ligase Template Sequence 5 3 Adapter Oligo Sequence 1µm bead universal seq primer AT 1µm 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 zn n n A T z z zn n n A C z z z 4-5

29 29 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics 9-10 SOLiD 4-color ligation Visualization (2 nd 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 30 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD 4-color ligation Cleavage (2 nd cycle) Template Sequence 5 3 Adapter Oligo Sequence 1µm bead universal seq primer AT 1µm bead p5 9-10 4-5

31 31 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD 4-color ligation interrogates every 5 th base Template Sequence 5 3 Adapter Oligo Sequence 1µm bead universal seq primer AT 19-20 C 14-15 T 1µm 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 zn n n A T z z zn n n A C z z z 9-10 4-5

32 32 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD 4-color ligation Reset Template Sequence 5 3 Adapter Oligo Sequence 1µm bead 1µm bead

33 33 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics ligase SOLiD 4-color ligation (1 st cycle after reset) ligase 3 p5 universal seq primer n-1 Template Sequence 5 3 Adapter Oligo Sequence 1µm bead universal seq primer n-1 T 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 zn n n A T z z zn n n A C z z z

34 34 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD 4-color ligation (1 st cycle after reset) Template Sequence 5 3 Adapter Oligo Sequence 1µm bead universal seq primer n-1 T 1µm bead 3-4

35 35 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics SOLiD 4-color ligation (2 nd Round) Template Sequence 5 3 Adapter Oligo Sequence 1µm bead universal seq primer n-1 1µm bead T C G TT 8-913-14 23-24 18-19 3-4

36 36 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Sequential rounds of sequencing Multiple cycles per round 3 universal seq primer 4-59-1014-1521-2024-25 Template Sequence 5 3 Adapter Oligo Sequence 1µm bead 1µm bead 3 universal seq primer n-1 reset 3-48-913-1418-1923-24 3 universal seq primer n-2 2-37-812-1317-1822-23 reset 3 universal seq primer n-3 1-26-711-1216-1721-22 reset 3 universal seq primer n-4 0-15-610-1115-1620-21 reset

37 37 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Example of decoding (ii) AA CC GG TT AC CA GT TG AC CA GT TG AA CC GG TT AA CC GG TT AG CT GA TC AG CT GA TC AG CT GA TC AT CG GC TA ACGT A C G T 2 nd Base 1 st Base AACAAGCCTC

38 38 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational 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 39 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics A C G G T C G T C G T G T G C G T 1 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 40 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Why leave color space? Align color space reads against color space reference Reference

41 41 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Why leave color space? Align color space reads against color space reference Reference SNP 2 colors change

42 42 Ottobre 22, 2008 © 2008 Applied BiosystemsInternational School of Functional Genomics Why leave color space? Align color space reads against color space reference Reference Incorrect call, single change in color space


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