Nextera™ NGS Library Preparation<br />From Nanograms of DNA to Sequencer-Ready Libraries in under Two Hours<br />© 2010, E...
Deep Sequencing Methods<br />There are three types of sequencing chemistries in common use:<br />Sequencing by synthesis<b...
Sequencing by Synthesis<br /><ul><li>Roche 454™
IlluminaSolexa®
Helicos
Intelligent Bio Systems</li></li></ul><li>Sequencing by Ligation<br />AB SOLiD™<br />Polonator<br />Complete Genomics<br />
Real-Time Sequencing by Synthesis<br />Pacific Biosciences<br />Visigen<br />
NGS Methods Have a Common Library Prep Workflow<br />
Typical Workflow Suffers from Some Limitations<br />Multiple steps, time-consuming<br />Requires microgram amounts of DNA<...
Nextera™ Technology Streamlines  Library Preparation<br />Nextera™ Technology<br /><ul><li>Combines fragmentation, end-rep...
Sequencer-ready libraries in <2 hours from 20-50 ng of DNA
Compatible with 96-well plate processing</li></li></ul><li>“Tagmentation”: Fragmentation/Tagging by In Vitro Transposition...
Roche 454™-Compatible Libraries<br />Nextera™ EnzymeMix(free transposon ends)<br />Add FLX or Ti sites by PCR (+/- bar cod...
IlluminaSolexa®-Compatible Libraries<br />Nextera™ Enzyme Mix (appended transposon ends)<br />Add bPCR sites by PCR (+/- b...
Nextera™ DNA Sample Prep Kits<br />Current kits available (February 2010)<br />Roche/454-Compatible Libraries<br />GS FLX™...
Library Preparation Comparison<br />
Deep Sequencing of 454-Compatible Libraries<br />Libraries prepared using Nextera™ Sample Prep<br />E. coli, plasmid, or s...
Deep Sequencing of 454-Compatible Libraries<br />Libraries prepared using Nextera™ Sample Prep<br />E. coli, plasmid, or s...
Deep Sequencing of Illumina-Compatible Libraries<br />Library prepared using Nextera™ Sample Prep<br />E. coli genomic DNA...
Deep Sequencing of Illumina-Compatible Libraries<br />Distribution of depth (nucleotides vs. depth) shows a near- Poisson ...
Additional Nextera™ Validation<br />
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Nextera Overview Feb 2010

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An introduction to Nextera technology: preparing DNA samples for sequencing on next-generation platforms.

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Nextera Overview Feb 2010

  1. 1. Nextera™ NGS Library Preparation<br />From Nanograms of DNA to Sequencer-Ready Libraries in under Two Hours<br />© 2010, EPICENTRE Biotechnologies. This presentation may be linked or embedded as long as no modifications are made to the content, and credit is given with a link back to the source. <br />
  2. 2. Deep Sequencing Methods<br />There are three types of sequencing chemistries in common use:<br />Sequencing by synthesis<br />Sequencing by ligation<br />Real-time sequencing by synthesis<br />
  3. 3. Sequencing by Synthesis<br /><ul><li>Roche 454™
  4. 4. IlluminaSolexa®
  5. 5. Helicos
  6. 6. Intelligent Bio Systems</li></li></ul><li>Sequencing by Ligation<br />AB SOLiD™<br />Polonator<br />Complete Genomics<br />
  7. 7. Real-Time Sequencing by Synthesis<br />Pacific Biosciences<br />Visigen<br />
  8. 8. NGS Methods Have a Common Library Prep Workflow<br />
  9. 9. Typical Workflow Suffers from Some Limitations<br />Multiple steps, time-consuming<br />Requires microgram amounts of DNA<br />Not suitable for high-throughput processing<br />
  10. 10. Nextera™ Technology Streamlines Library Preparation<br />Nextera™ Technology<br /><ul><li>Combines fragmentation, end-repair, and ligation steps
  11. 11. Sequencer-ready libraries in <2 hours from 20-50 ng of DNA
  12. 12. Compatible with 96-well plate processing</li></li></ul><li>“Tagmentation”: Fragmentation/Tagging by In Vitro Transposition<br />Transposomecomplexes with free transposon ends<br />5’-Tagged DNA fragments<br />
  13. 13. Roche 454™-Compatible Libraries<br />Nextera™ EnzymeMix(free transposon ends)<br />Add FLX or Ti sites by PCR (+/- bar codes)<br />emPCR input<br />
  14. 14. IlluminaSolexa®-Compatible Libraries<br />Nextera™ Enzyme Mix (appended transposon ends)<br />Add bPCR sites by PCR (+/- bar codes)<br />bPCR input<br />
  15. 15. Nextera™ DNA Sample Prep Kits<br />Current kits available (February 2010)<br />Roche/454-Compatible Libraries<br />GS FLX™<br />Bar Coding Module (n = 12)<br />GS FLX™ Titanium<br />Bar Coding Module (n = 12)<br />Illumina-Compatible Libraries<br />GAII<br />Bar Coding Module (n = 12)<br />Nextera PCR Enzyme<br />Required component<br />
  16. 16. Library Preparation Comparison<br />
  17. 17. Deep Sequencing of 454-Compatible Libraries<br />Libraries prepared using Nextera™ Sample Prep<br />E. coli, plasmid, or soybean genomic DNA<br />Summary table of sequencing yield, coverage, and mapping data <br />
  18. 18. Deep Sequencing of 454-Compatible Libraries<br />Libraries prepared using Nextera™ Sample Prep<br />E. coli, plasmid, or soybean genomic DNA<br />Relative coverage of individual soybean chromosomes shows even coverage across all 20 chromosomes.<br />
  19. 19. Deep Sequencing of Illumina-Compatible Libraries<br />Library prepared using Nextera™ Sample Prep<br />E. coli genomic DNA<br />Coverage plot indicating sequencing depth vs. position on reference genome.<br />(Stray reads from unrelated libraries in same channel mapped to LacZ and nohB promoters.)<br />
  20. 20. Deep Sequencing of Illumina-Compatible Libraries<br />Distribution of depth (nucleotides vs. depth) shows a near- Poisson distribution. <br />GC bias of coverage is comparable to libraries generated using physical shearing<br />
  21. 21. Additional Nextera™ Validation<br />
  22. 22. NGS Library Analysis Conducted by Early Adopters<br /><ul><li>Compared: Nebulization, NEB Fragmentase, Nextera™
  23. 23. Human, E. coli, phage
  24. 24. Illumina GAII and Roche FLX Titanium
  25. 25. Characterized:
  26. 26. Insertion bias
  27. 27. Fragment size
  28. 28. Coverage
  29. 29. Library complexity
  30. 30. Manuscript in preparation for peer review and publication.</li></ul>“…the insertion sites are essentially random for all three methods of library preparation.”<br />
  31. 31. Early Adopter Feedback<br />“… The Nextera protocol meets [our] requirements [for making bar coded libraries from limiting amounts of DNA] and is so simple and robust that we would use it in preference to the standard method, even if we had DNA in excess.” – Hilary Morrison, MBL<br />“We're very excited by what we've seen from it so far.” – Jay Shendure, Univ. Washington<br />“It's incredibly easy as compared to conventional library construction methods.”<br />
  32. 32. Univ. of Washington<br />Emily Turner<br />RuolanQiu<br />Andrew Adey<br />Jay Shendure<br />MBL-Woods Hole<br />Hilary Morrison<br />Jessica Mark Welch<br />Ekaterina Andreishcheva<br />Bill Reznikoff<br />EPICENTRE®<br />Haiying Grunenwald<br />Dilara Begum<br />Igor Goryshin<br />Les Hoffman<br />Joanne Decker<br />Mark Maffitt<br />Jerry Jendrisak<br />Acknowledgements<br />© 2010, EPICENTRE Biotechnologies. This presentation may be linked or embedded as long as no modifications are made to the content, and credit is given with a link back to the source. <br />

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