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NGx Sequencing 101-platforms
The document provides an overview of next-generation sequencing (NGS) platforms, detailing applications, performance specifications, and future trends. Key highlights include various sequencing types like whole genome, exome, small genome, and targeted sequencing, along with comparisons of different platform technologies such as Illumina, Life Technologies, and Ion Torrent. Major trends indicate a shift towards desktop machines, faster runs, and increased usage in diagnostic applications.
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NGx Sequencing 101-platforms
- 1. Sequencing 101 –NGS Platforms Shawn C. Baker, Ph.D.
- 2. Overview Review MajorApplications Review Major Platforms Future Trends Asking the Right Questions
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- 4. Whole Genome Sequencing 30X coverage 90 Gb Long reads Paired-end Mate pair Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
- 5. Exome Sequencing 100X+ coverage 5 Gb Long reads Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
- 6. Small Genome Sequencing 1+ Gb Long reads Paired-end multiplexing Credit: Rocky Mountain Laboratories, NIAID, NIH Credit: Graham Colm (Wikipedia)
- 7. Targeted DNA Sequencing 10+ Mb multiplexing Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
- 8. Transcriptome Sequencing Lots of reads Long reads Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
- 9. RNA Profile Sequencing Lots of reads multiplexing Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
- 10. ChIP Sequencing Lots of reads multiplexing Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
- 11. Metagenome Sequencing Lots of reads Long reads Paired-end
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- 16. Illumina – Chemistry Library Prep Amplification Sequencing
- 17. Illumina – PerformanceSpecs HiSeq HiSeq GAIIx HiScanSQ MiSeq 2000/2500 1000/1500 Gb/run 600/120 300/60 95 150 7 Run time 11d/27hrs 8.5d/27hrs 14d 8.5d 35hrs Gb/day 55/120 35/60 ~7 18 ~7 Read length 2x100/150 2x100/150 2x150 2x100 2x250 # of single 3B/600M 1.5B/300M 320M 750M 15M reads/run Instrument $690k/$74 $590k/$640 ~$300k ~$400k $125k cost 0k k Run cost ~$23k ~$11k ~$17k ~$11k ~$1k
- 18. Illumina – Applications HiSeq HiSeq GAIIx HiScanSQ MiSeq 2000/2500 1000/1500 Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
- 19. Illumina – FutureDevelopments 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?
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- 21. Life: SOLiD –Chemistry Library Prep Amplification Sequencing
- 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. Life: SOLiD –Applications SOLiD SOLiD 5500xl(W) 5500(W) Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
- 24. Life: SOLiD –Future Developments SOLiD SOLiD 5500xl(W) 5500(W) Probably very little Main focus is on Ion Torrent technology
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- 26. Life: Ion Torrent– Chemistry Library Prep Amplification Sequencing
- 27. Life: Ion Torrent– Performance Specs PGM 314 PGM 316 PGM 318 Proton 1 Proton 2 Gb/run 10-40 Mb 100-400 Mb 1 Gb ~10 Gb ~100 Gb Run time 2 hours 2 hours 2 hours ~4 hours ~4 hours Gb/day ~120 Mb ~1.2 Gb ~3 Gb ~30 Gb ~ 300 Gb Read length 200b 200b 200b 200b >200b # of single ~0.6M ~3M ~5.5M ~82M ~330M reads/run Instrument $50k $50k $50k $149k $149k cost Run cost $349 $549 $749 ~$1k ~$1k
- 28. Life: Ion Torrent– Applications PGM 314 PGM 316 PGM 318 Proton 1 Proton 2 Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
- 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
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- 31. 454/Roche – Chemistry Library Prep Amplification Sequencing
- 32. 454/Roche – PerformanceSpecs 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. 454/Roche – Applications GS FLX+ GS Jr. Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
- 34. 454/Roche – FutureDevelopments GS FLX+ GS Jr. Probably very little Roche has signaled the desire for new technology
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- 36. Pacific Biosciences –Chemistry Library Prep Amplification Sequencing
- 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. Pacific Biosciences –Applications PacBio RS Whole Genome Exome Small Genome Targeted Transcriptome RNA Profiling ChIP-Seq Metagenomics
- 39. Pacific Biosciences –Future Developments PacBio RS Longer reads Reduced error rate Increased chip density Non-fluorescent detection?
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- 41. More Information… www.blueseq.com
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- 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. 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. 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. 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. 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. 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. 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. Metagenome Sequencing ILMN HiSeq ✔ ILMN MiSeq SOLiD ✔ Ion PGM Ion Proton ✔ 454 GS FLX+ 454 GS Jr. PacBio RS
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- 52. Major Trends Desktopmachines Ease of use Faster runs Diagnostic/clinical use Longer reads Nanopore technologies
- 53. Asking the RightQuestions
- 54. Asking the RightQuestions 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?