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Next–generation DNA sequencing technologies – theory & practice

Next–generation DNA sequencing technologies – theory & practice. Outline. Next-Generation sequencing (NGS) technologies – overview NGS targeted re-sequencing – fishing out the regions of interest NGS workflow: data collection and processing – the exome sequencing pipeline.

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Next–generation DNA sequencing technologies – theory & practice

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  1. Next–generation DNA sequencing technologies – theory & practice

  2. Outline • Next-Generation sequencing (NGS) technologies – overview • NGS targeted re-sequencing – fishing out the regions of interest • NGS workflow: data collection and processing – the exome sequencing pipeline

  3. PART I: NGS technologies Next-Generation sequencing (NGS) technologies – overview

  4. DNA Sequencing – the next generation • The automated Sanger method is considered as a ‘first-generation’ technology, and newer methods are referred to as next-generation sequencing (NGS).

  5. Landmarks in DNA sequencing • 1953 Discovery of DNA double helix structure • 1977 • A Maxam and W Gilbert "DNA seq by chemical degradation" • F Sanger"DNA sequencing with chain-terminating inhibitors" • 1984 DNA sequence of the Epstein-Barr virus, 170 kb • 1987 Applied Biosystems - first automated sequencer • 1991 Sequencing of human genome in Venter's lab • 1996 P. Nyrén and M Ronaghi - pyrosequencing • 2001 A draft sequence of the human genome • 2003 human genome completed • 2004 454 Life Sciences markets first NGS machine

  6. DNA Sequencing – the next generation

  7. DNA Sequencing – the next generation • The newer technologies constitute various strategies that rely on a combination of • Library/template preparation • Sequencing and imaging

  8. DNA Sequencing – the next generation • Commercially available technologies • Roche – 454 • GSFLX titanium • Junior • Illumina • HiSeq2000 • MySeq • Life – SOLiD • 5500xl • Ion torrent • HelicosBioSciences– HeliScope • Pacific Biosciences – PacBio RS

  9. DNA Sequencing – the next generation

  10. Template preparation: STEP1 • Produce a non-biased source of nucleic acid material from the genome

  11. Template preparation: STEP1 • Produce a non-biased source of nucleic acid material from the genome

  12. Template preparation • Produce a non-biased source of nucleic acid material from the genome • Current methods: • randomly breaking genomic DNA into smaller sizes • Ligate adaptors • attach or immobilize the template to a solid surface or support • the spatially separated template sites allows thousands to billions of sequencing reactions to be performed simultaneously

  13. Template preparation • Clonal amplification • Roche – 454 • Illumina– HiSeq • Life – SOLiD • Single molecule sequencing • HelicosBioSciences– HeliScope • Pacific Biosciences – PacBio RS

  14. Template preparation: Clonal amplification • In solution – emulsion PCR (emPCR) • Roche – 454 • Life – SOLiD • Solid phase – Bridge PCR • Illumina – HiSeq

  15. Template preparation: Clonal amplification - emPCR

  16. Sequencing SOLiD 454

  17. Pyrosequencing Picotitre plate Pyrosequencing

  18. Pyrosequencing

  19. Sequencing by ligation

  20. Sequencing by ligation

  21. Sequencing by ligation

  22. Template preparation: Clonal amplification – Bridge PCR

  23. Template preparation: Single molecule templates Heliscope BioPac

  24. HiSeq Heliscope

  25. DNA Sequencing – the next generation • The major advance offered by NGS is the ability to cheaply produce an enormous volume of data • The arrival of NGS technologies in the marketplace has changed the way we think about scientific approaches in basic, applied and clinical research

  26. PART II: NGS targeted resequencing fishing out the regions of interest

  27. The beginning

  28. DNA Sequencing – the next generation • Library/template preparation • Library enrichment for target • Sequencing and imaging

  29. Target enrichment strategies

  30. Target enrichment strategies

  31. Target enrichment strategies

  32. Target enrichment strategies

  33. Target enrichment strategies: MIP

  34. Hybrid Capture

  35. Hybrid Capture

  36. Target enrichment strategies

  37. PCR based approaches

  38. PCR based approaches: Raindance

  39. PCR based approaches: Fluidigm • 48.48 Access Array

  40. PCR based approaches: Fluidigm • 48.48 Access Array

  41. PCR based approaches: Fluidigm • 48.48 Access Array

  42. Target enrichment strategies

  43. PART III: NGS workflow data collection and processing – the exome sequencing pipeline

  44. WholeExomeSequencing • The human genome • Genome = 3Gb • Exome = 30Mb • 180 000 exons • Protein coding genes • constitute only approximately 1% of the human genome • It is estimated that 85% of the mutations with large effects on disease-related traits can be found in exons or splice sites

  45. Exome sequencing

  46. The past, present & future

  47. Exomesequencingcapacity • HiSeq specifications: • 2 flow cells • 16 lanes (8 per flow cell) • 200-300 Gbases per flow cell • 10 days for a single run • Exome throughput • 96 @ 60x coverage per run • 3000 @ 60x coverage per year

  48. Data processing workflow

  49. Data processing

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