NGx Sequencing 101-platforms

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The field of next-generation sequencing (NGS) has been experiencing explosive growth over the past several years and shows little sign of slowing down. The increasing capabilities and dramatically lowered costs have expanded NGS's reach beyond that of the human genome into nearly every corner of biological research. An overview of the platforms on the market today, including an assessment of their relative strengths and weaknesses, will be presented. The presentation will conclude with a peek into where the technology is going and what will be available in the future.

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NGx Sequencing 101-platforms

  1. 1. Sequencing 101 – NGS Platforms Shawn C. Baker, Ph.D.
  2. 2. Overview Review Major Applications Review Major Platforms Future Trends Asking the Right Questions
  3. 3. Applications
  4. 4. Whole Genome Sequencing  30X coverage  90 Gb  Long reads  Paired-end  Mate pairCredits: Darryl Leja (NHGRI), Ian Dunham (EBI)
  5. 5. Exome Sequencing  100X+ coverage  5 Gb  Long readsCredits: Darryl Leja (NHGRI), Ian Dunham (EBI)
  6. 6. Small Genome Sequencing  1+ Gb  Long reads  Paired-end  multiplexingCredit: Rocky Mountain Laboratories, NIAID, NIH Credit: Graham Colm (Wikipedia)
  7. 7. Targeted DNA Sequencing  10+ Mb  multiplexingCredits: Darryl Leja (NHGRI), Ian Dunham (EBI)
  8. 8. Transcriptome Sequencing  Lots of reads  Long readsCredits: Darryl Leja (NHGRI), Ian Dunham (EBI)
  9. 9. RNA Profile Sequencing  Lots of reads  multiplexingCredits: Darryl Leja (NHGRI), Ian Dunham (EBI)
  10. 10. ChIP Sequencing  Lots of reads  multiplexingCredits: Darryl Leja (NHGRI), Ian Dunham (EBI)
  11. 11. Metagenome Sequencing  Lots of reads  Long reads  Paired-end
  12. 12. Applications
  13. 13. Platforms
  14. 14. Platforms
  15. 15. Platforms
  16. 16. Illumina – Chemistry Library Prep Amplification Sequencing
  17. 17. Illumina – Performance Specs HiSeq HiSeq GAIIx HiScanSQ MiSeq 2000/2500 1000/1500Gb/run 600/120 300/60 95 150 7Run time 11d/27hrs 8.5d/27hrs 14d 8.5d 35hrsGb/day 55/120 35/60 ~7 18 ~7Read length 2x100/150 2x100/150 2x150 2x100 2x250# of single 3B/600M 1.5B/300M 320M 750M 15Mreads/runInstrument $690k/$74 $590k/$640 ~$300k ~$400k $125kcost 0k kRun cost ~$23k ~$11k ~$17k ~$11k ~$1k
  18. 18. Illumina – Applications HiSeq HiSeq GAIIx HiScanSQ MiSeq 2000/2500 1000/1500Whole GenomeExomeSmall GenomeTargetedTranscriptomeRNA ProfilingChIP-SeqMetagenomics
  19. 19. Illumina – Future Developments HiSeq HiSeq GAIIx HiScanSQ MiSeq 2000/2500 1000/1500  Focus on desktop system  Longer reads  Push into diagnostics  Chemistry A = fast reads?  Chemistry B = long reads?
  20. 20. Platforms
  21. 21. Life: SOLiD – Chemistry Library Prep Amplification Sequencing
  22. 22. Life: SOLiD – Performance Specs SOLiD SOLiD SOLiD SOLiD 5500 5500xl 5500xl W 5500 W Gb/run 95 240 48 120 Run time 6 days 10 days 6 days 10 days Gb/day ~16 24 ~8 12 Read length 2X60 2X50 2X60 2X50 # of single ~800M 2.4B ~400M 1.2B reads/run Instrument $595k $70k $349k $70k cost upgrade upgrade Run cost ~$10k ~$5k ~$5k ~$2.5k
  23. 23. Life: SOLiD – Applications SOLiD SOLiD 5500xl(W) 5500(W) Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
  24. 24. Life: SOLiD – Future Developments SOLiD SOLiD 5500xl(W) 5500(W)  Probably very little  Main focus is on Ion Torrent technology
  25. 25. Platforms
  26. 26. Life: Ion Torrent – Chemistry Library Prep Amplification Sequencing
  27. 27. Life: Ion Torrent – Performance Specs PGM 314 PGM 316 PGM 318 Proton 1 Proton 2Gb/run 10-40 Mb 100-400 Mb 1 Gb ~10 Gb ~100 GbRun time 2 hours 2 hours 2 hours ~4 hours ~4 hoursGb/day ~120 Mb ~1.2 Gb ~3 Gb ~30 Gb ~ 300 GbRead length 200b 200b 200b 200b >200b# of single ~0.6M ~3M ~5.5M ~82M ~330Mreads/runInstrument $50k $50k $50k $149k $149kcostRun cost $349 $549 $749 ~$1k ~$1k
  28. 28. Life: Ion Torrent – Applications PGM 314 PGM 316 PGM 318 Proton 1 Proton 2Whole GenomeExomeSmall GenomeTargetedTranscriptomeRNA ProfilingChIP-SeqMetagenomics
  29. 29. Life: Ion Torrent – Future Developments PGM 314 PGM 316 PGM 318 Proton 1 Proton 2  Focus on desktop systems  Longer reads  Increased chip densities  New machine?  Push into diagnostics
  30. 30. Platforms
  31. 31. 454/Roche – Chemistry Library Prep Amplification Sequencing
  32. 32. 454/Roche – Performance Specs GS FLX+ GS Jr. Mb/run 700 35 Run time 23 hours 10 hours Mb/day 700 35 Read length Up to 1kb ~400b # of single 1M 0.1M reads/run Instrument ~$500k $125k cost Run cost ~$6k ~$1k
  33. 33. 454/Roche – Applications GS FLX+ GS Jr. Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
  34. 34. 454/Roche – Future Developments GS FLX+ GS Jr.  Probably very little  Roche has signaled the desire for new technology
  35. 35. Platforms
  36. 36. Pacific Biosciences – Chemistry Library Prep Amplification Sequencing
  37. 37. Pacific Biosciences – Performance Specs PacBio RS ‘C2’ Mb/run 120 Run time 40 min Gb/day ~1 Gb Read length 3kb (avg) # of single ~50k reads/run Instrument ~$700k cost Run cost $100
  38. 38. Pacific Biosciences – Applications PacBio RS Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
  39. 39. Pacific Biosciences – Future Developments PacBio RS  Longer reads  Reduced error rate  Increased chip density  Non-fluorescent detection?
  40. 40. Other Platforms…
  41. 41. More Information… www.blueseq.com
  42. 42. Matching Applicationswith Platforms
  43. 43. Whole Genome Sequencing ILMN HiSeq ✔ ILMN MiSeq SOLiD ✔ Ion PGM Ion Proton ✔ 454 GS FLX+Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr. ✔ PacBio RS
  44. 44. Exome Sequencing ILMN HiSeq ✔ ILMN MiSeq ✔ SOLiD ✔ Ion PGM Ion Proton ✔ 454 GS FLX+Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr. PacBio RS
  45. 45. Small Genome Sequencing ILMN HiSeq ILMN MiSeq ✔ SOLiD Ion PGM ✔ Ion Proton ✔Credit: Rocky Mountain Laboratories, NIAID, NIH Credit: Graham Colm (Wikipedia) 454 GS FLX+ ✔ 454 GS Jr. ✔ PacBio RS ✔
  46. 46. Targeted DNA Sequencing ILMN HiSeq ILMN MiSeq ✔ SOLiD Ion PGM ✔ Ion Proton ✔ 454 GS FLX+ ✔Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr. ✔ PacBio RS ✔
  47. 47. Transcriptome Sequencing ILMN HiSeq ✔ ILMN MiSeq SOLiD ✔ Ion PGM Ion Proton ✔ 454 GS FLX+Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr. PacBio RS
  48. 48. RNA Profile Sequencing ILMN HiSeq ✔ ILMN MiSeq SOLiD ✔ Ion PGM Ion Proton ✔ 454 GS FLX+Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr. PacBio RS
  49. 49. ChIP Sequencing ILMN HiSeq ILMN MiSeq ✔ SOLiD Ion PGM Ion Proton ✔ 454 GS FLX+Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr. PacBio RS
  50. 50. Metagenome Sequencing ILMN HiSeq ✔ ILMN MiSeq SOLiD ✔ Ion PGM Ion Proton ✔ 454 GS FLX+ 454 GS Jr. PacBio RS
  51. 51. Major Trends
  52. 52. Major Trends Desktop machines Ease of use Faster runs Diagnostic/clinical use Longer reads Nanopore technologies
  53. 53. Asking the Right Questions
  54. 54. Asking the Right Questions Before you buy a platform  Do I need to buy a platform?  What are my major applications?  What are my colleagues using? Before you start sequencing  What am I trying to answer?  How will I analyze the data?  Where is my variation coming from?  How many samples do I need?

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