Correlagen next gen presentation 042711

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Correlagen next gen presentation 042711

  1. 1. Sequencing technologies past, current and next generation•Introduction to Helicos, Illumina and Solid sequencing technology •Applications Robert Pinard Some slides were adapted from Karen Staehling-Hampton The Stowers Genome Center
  2. 2. Introduction• Genomics research has entered a new age, in which deciphering the genome’s effect on biology and medicine requires not only the detection of mutations and sequence variation, but also understanding the dynamic nature of genome biology.• The “Next Generation Sequencing” should be called the Current Generation Sequencing
  3. 3. Overview of three major next Generation Sequencing Technologies• Illumina / GAII• Helicos / Heliscope• Life Tech / Solid• (briefly Ion Torrent, PacBio & Complete Genomics)
  4. 4. • The principle at the heart of all these technologies is similar• (sequence by synthesis) Sanger Sequencing
  5. 5. (Except Solid)Detect stopped fluorescent fragments (using ddNTP spikes)
  6. 6. • It is similar with the next generation sequencers where the different platforms either detect the incorporation of a fluorescent nucleotide or a bi-product of the reaction like the PPi or the release of a proton by the DNA polymerase
  7. 7. Common Steps (to all platforms)• Library Preparation• Amplification Steps• Attachment to a matrix (FlowCell)• Sequencing & Detection• Interpretation
  8. 8. Common Steps• Library Preparation• Amplification Steps• Attachment to a matrix (FlowCell)• Sequencing & Detection• Interpretation
  9. 9. Overall Steps Library Preparation Modify ends/Adaptors Selection Shear AttachAmplification Sequence & Detection
  10. 10. Library Preparation Workflow (Illumina & Solid)
  11. 11. Helicos Library Preparation
  12. 12. Common Steps• Library Preparation• Selection/Amplification Steps• Attachment to a matrix (FlowCell)• Sequencing & Detection• Interpretation
  13. 13. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation AttachAmplification Sequence & Detection
  14. 14. Selection Step (Principles)POND BAITs ENRICHED POND
  15. 15. Step ARegion Selection: using Sure Select, capture region that wereally want to look at (complete exomes), subset of genes (Familial Cardiac Genes).
  16. 16. Step B some PCR involved• Pre-Hybrid Selection and post-Hybrid Selection amplification PCR. A- Pre-(to increase the pond of fragment DNA) B- Post- (to increase the DNA that was captured)
  17. 17. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation Amplify and or AttachAmplification Sequence & Detection
  18. 18. STEP C: Clonal amplification to increase signal detection• EMULSION PCR or CLUSTER AMPLIFICATION Illumina 454/Roche Solid/Life Helicos
  19. 19. Amplification: where the technologies differ?
  20. 20. Emulsion PCRAmplified Materials deposited in picotiter plate oron slide via 3’ modification of the 3’end SOLID; 454; ION TORRENT
  21. 21. ILLUMINAAmplification on Slide and Cluster Generation
  22. 22. ILLUMINA
  23. 23. Illumina
  24. 24. Common Steps• Library Preparation• Amplification Steps• Attachment to a matrix (FlowCell)• Sequencing & Detection• Interpretation
  25. 25. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation Amplify and or AttachAmplification Sequence & Detection
  26. 26. ILLUMINA
  27. 27. Helicos
  28. 28. 454 & Solid Solid454/Roche Amplified Materials deposited in picotiter plate or on slide via 3’ modification of the 3’end
  29. 29. Common Steps• Library Preparation• Amplification Steps• Attachment to a matrix (FlowCell)• Sequencing & Detection• Interpretation
  30. 30. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation Amplify and or AttachAmplification Sequence & Detection
  31. 31. Illumina
  32. 32. Illumina
  33. 33. Helicos
  34. 34. Helicos
  35. 35. The addition of one of the four 454deoxynucleotide triphosphates (dNTPs)(in thecase of dATP we add dATPαS which is not asubstrate for a luciferase) initiates the secondstep. DNA polymerase incorporates thecorrect, complementary dNTPs onto thetemplate. This incorporation releasespyrophosphate (PPi) stoichiometrically.ATP sulfurylase quantitatively converts PPi toATP in the presence of adenosine 5´phosphosulfate. This ATP acts as fuel to theluciferase-mediated conversion of luciferin tooxyluciferin that generates visible light inamounts that are proportional to the amountof ATP. The light produced in the luciferase-catalyzed reaction is detected by a camera andanalyzed in a program.Unincorporated nucleotides and ATP aredegraded by the apyrase, and the reaction canrestart with another nucleotide.
  36. 36. Solid
  37. 37. Common Steps• Library Preparation• Amplification Steps• Attachment to a matrix (FlowCell)• Sequencing & Detection• Interpretation
  38. 38. Alignment et Sequence ReconstructionAll fragments put together andalign to region of interest
  39. 39. Utilities• •Whole Genome re-sequencing• –Bacterial genomes to identify SNPs that confer drug resistance• •Targeted Re-sequencing (Cardio Gen Scan)• –Sure Select• –Regular PCR & Long Range PCR (small panels/ Patch Assay)• •Coding exons (Whole Exome and Clinical Exomes)• •Detect Rare variants• –Can detect a 1/20 event (1 het among 10 samples)• –Somatic mutations in cancer samples
  40. 40. Other emergent platforms• Ion Torrent• PacBio• Complete Genomics
  41. 41. Ion Torrent
  42. 42. Pacific BioSciences Sequence multiple time same fragment
  43. 43. NanoBall (Complete Genomics)
  44. 44. Conclusions• Several new platforms are emerging (variation on a same theme) that will increase throughput and reduce cost.• The Next Gen Sequencing approaches are really the Now Gen Sequencing approaches and they are making a real impact in life sciences and soon in clinical diagnostics (starting with our own CGS test).

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