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Correlagen next gen presentation 042711
- 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. Introduction • Genomics researchhas 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. Overview of threemajor next Generation Sequencing Technologies • Illumina / GAII • Helicos / Heliscope • Life Tech / Solid • (briefly Ion Torrent, PacBio & Complete Genomics)
- 6. • The principleat the heart of all these technologies is similar • (sequence by synthesis) Sanger Sequencing
- 7. (Except Solid) Detect stoppedfluorescent fragments (using ddNTP spikes)
- 9. • It issimilar 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
- 10. Common Steps (toall platforms) • Library Preparation • Amplification Steps • Attachment to a matrix (FlowCell) • Sequencing & Detection • Interpretation
- 11. Common Steps • Library Preparation • Amplification Steps • Attachment to a matrix (FlowCell) • Sequencing & Detection • Interpretation
- 12. Overall Steps Library Preparation Modify ends/Adaptors Selection Shear Attach Amplification Sequence & Detection
- 13. Library Preparation Workflow (Illumina & Solid)
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- 15. Common Steps • Library Preparation • Selection/Amplification Steps • Attachment to a matrix (FlowCell) • Sequencing & Detection • Interpretation
- 16. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation Attach Amplification Sequence & Detection
- 17. Selection Step (Principles) POND BAITs ENRICHED POND
- 18. Step A Region Selection:using Sure Select, capture region that we really want to look at (complete exomes), subset of genes (Familial Cardiac Genes).
- 19. Step B somePCR 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)
- 20. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation Amplify and or Attach Amplification Sequence & Detection
- 21. STEP C: Clonalamplification to increase signal detection • EMULSION PCR or CLUSTER AMPLIFICATION Illumina 454/Roche Solid/Life Helicos
- 22. Amplification: where thetechnologies differ?
- 23. Emulsion PCR Amplified Materialsdeposited in picotiter plate or on slide via 3’ modification of the 3’end SOLID; 454; ION TORRENT
- 24. ILLUMINA Amplification on Slideand Cluster Generation
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- 27. Common Steps • Library Preparation • Amplification Steps • Attachment to a matrix (FlowCell) • Sequencing & Detection • Interpretation
- 28. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation Amplify and or Attach Amplification Sequence & Detection
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- 32. 454 & Solid Solid 454/Roche Amplified Materials deposited in picotiter plate or on slide via 3’ modification of the 3’end
- 33. Common Steps • Library Preparation • Amplification Steps • Attachment to a matrix (FlowCell) • Sequencing & Detection • Interpretation
- 34. Overall Steps Modify ends/Adaptors Selection Shear Library Preparation Amplify and or Attach Amplification Sequence & Detection
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- 40. The addition ofone of the four 454 deoxynucleotide triphosphates (dNTPs)(in the case of dATP we add dATPαS which is not a substrate for a luciferase) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase- catalyzed reaction is detected by a camera and analyzed in a program. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.
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- 42. Common Steps • Library Preparation • Amplification Steps • Attachment to a matrix (FlowCell) • Sequencing & Detection • Interpretation
- 43. Alignment et SequenceReconstruction All fragments put together and align to region of interest
- 47. 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
- 49. Other emergent platforms •Ion Torrent • PacBio • Complete Genomics
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- 51. Pacific BioSciences Sequence multiple time same fragment
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- 53. Conclusions • Several newplatforms 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).