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  1. Indications in green = Live content Indications in black = Edit in master Indications in white = Locked elements Template release: Date Click to edit Master title style Click to edit Master subtitle style L’evoluzione del sequenziamento:Dal metodo Sanger alla Next GenerationRoberto Fantozzi

  2. 2 Primers 1 Primer ! dNTPs dNTPs ddNTPs Principle of Sequencing Analysis Standard PCR Sequencing Reaction

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

  4. Denaturation 95 °C Elongation 60 °C A - T - G - A - T - C - C - A - T - G - A - T - A - G - C Hybridization 50 - 55 °C T - A - C - T - A- G - G - T - A Cycle Sequencing Reaction 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 Linear amplification

  5. T - A - C - T - A- G - G - T - A - C - 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 - 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 - G T - A - C - T - A- G - G - T - A - C - T - A Cycle Sequencing Reaction: separation Electrophoresis • separation matrix : gel orpolymer • Separation according tothe size of the DNA fragment • 1 bp resolution

  6. Electrokinetic Injection Electrode (Cathode) • 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

  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 Sequencing Analysis 5.3 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 ofmappable data/run

  13. SOLiD™ System: Enabling New Applications byRedefining the Boundaries of Traditional Sequencing Sequence Analysis Tag Analysis Whole Genome Resequencing Expression Structural Variation Methylation Targeted Resequencing ChIP-Seq Copy Number de Novo Sequencing

  14. SOLiD™ Workflow Application specific sample preparation Application specific Data analysis Imaging and analysis Emulsion PCR & substrate preparation Sequencing chemistry

  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.

  16. Create fragment libraryAplications: small genome resequencing-Tag counting Fragmented template Ligate P1 and P2 primers to end Complex sample Fragmented template can be generated through random or targeted shearing e.g. sonication, mechanical, enzymatic digestion.

  17. 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

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

  20. Enrichment P2’ Large (5µ) Polystyrene bead P1 P2 P2 P1 Centrifuge in 60% glycerol Supernatant Captured beads with templates Pellet Beads with no template

  21. Sequencing Array Template bead deposition 3’-end modification Beads covalently attached to glass surface in a random array

  22. Red-probe 2nd Base 5’ 3’ n n n A T z z z A C G T A 1st Base C Blue-probe 5’ 3’ G n n n TT z z z T On our probes the 1st base encoded is position 4 the 2nd base encoded is position 5 2 Base Pair Encoding Using 4 Dyes

  23. 3’ ligation site, cleavage site and dye are spatially separated Properties of the Probes Cleavage site is between 5th and 6th base 3’ Fluorescent dye indicates base on 4th and 5th position n n n AC z z z green-probe • Probes are octamers • N=degenerate bases, Z=universal bases • 1024 probes, 256 probes per color

  24. ligase n n n A T z z z n n n A C z z z n n n C C z z z n n n GA z z z 1µm bead 1µm bead 3’ 5’ universal seq primer P1 Primer Template Sequence 3’ p5’ SOLiD 4-color ligationLigation reaction 3’ 5’ 5’ 3’ 5’ 3’ 5’

  25. ligase ligase 5’ 5’ n n n A C z z z n n n A T z z z n n n C C z z z n n n GA z z z n n n GA z z z 1µm bead 1µm bead 3’ 5’ universal seq primer P1 Primer Template Sequence SOLiD 4-color ligationLigation reaction 3’ 5’ 3’ 5’ 3’ 5’ p5’

  26. 5’ n n n GA z z z 1µm bead 1µm bead 3’ 5’ universal seq primer P1 Primer Template Sequence 4-5 SOLiD 4-color ligation Visualization

  27. 5’ n n n GA z z z 1µm bead p5’ 1µm bead 3’ 5’ universal seq primer P1 Primer Template Sequence 4-5 SOLiD 4-color ligation Cleavage

  28. ligase 5’ n n n A T z z z n n n A C z z z n n n C C z z z n n n GA z z z 1µm bead A T 1µm bead 3’ 5’ universal seq primer Adapter Oligo Sequence Template Sequence 4-5 SOLiD 4-color ligationLigation (2nd cycle) 3’ 5’ 3’ 5’ 3’ 5’ p5’ n n n GA

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

  30. 1µm bead p5’ 1µm bead 3’ 5’ universal seq primer Adapter Oligo Sequence Template Sequence SOLiD 4-color ligation Cleavage (2nd cycle) A T 9-10 4-5

  31. T C 5’ n n n A T z z z n n n C C z z z n n n A C z z z n n n GA z z z 1µm bead A T G 1µm bead 3’ 5’ universal seq primer Adapter Oligo Sequence Template Sequence SOLiD 4-color ligation interrogates every 5th base 3’ 5’ 3’ 5’ 3’ 5’ 24-25 4-5 19-20 9-10 14-15

  32. 1µm bead 1µm bead 3’ 5’ Adapter Oligo Sequence Template Sequence SOLiD 4-color ligation Reset

  33. ligase ligase 5’ n n n C C z z z n n n GA z z z n n n A T z z z n n n A C z z z 1µm bead T 1µm bead 3’ 5’ universal seq primer n-1 universal seq primer n-1 Adapter Oligo Sequence Template Sequence 3’ p5’ SOLiD 4-color ligation (1st cycle after reset) 3’ 5’ 3’ 5’ 3’ 5’ p5’

  34. 1µm bead T 1µm bead 3’ 5’ universal seq primer n-1 Adapter Oligo Sequence Template Sequence 3-4 SOLiD 4-color ligation (1st cycle after reset)

  35. T C T T 1µm bead G 1µm bead 3’ 5’ universal seq primer n-1 Adapter Oligo Sequence Template Sequence 3-4 SOLiD 4-color ligation (2nd Round) 23-24 8-9 13-14 18-19

  36. universal seq primer n-1 3’ 1µm bead 1µm bead 3’ 5’ universal seq primer Adapter Oligo Sequence Template Sequence 3’ Sequential rounds of sequencingMultiple cycles per round 4-5 9-10 14-15 21-20 24-25 reset 3-4 8-9 13-14 18-19 23-24 reset universal seq primer n-2 2-3 7-8 12-13 17-18 22-23 3’ reset universal seq primer n-3 1-2 6-7 11-12 16-17 21-22 3’ reset universal seq primer n-4 0-1 5-6 10-11 15-16 20-21 3’

  37. Example of decoding (ii) 2nd Base A C G T A 1st Base C G T AACAAGCCTC 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

  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 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 T 1 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 Reference

  41. Why leave color space?Align color space reads against color space reference Reference SNP 2 colors change

  42. Why leave color space?Align color space reads against color space reference Reference Incorrect call , single change in color space