Nextera Overview Feb 2010
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The Nextera technology streamlines NGS library preparation, enabling the creation of sequencer-ready libraries from 20-50 ng of DNA in under two hours, making it suitable for high-throughput processing. The method integrates fragmentation, end-repair, and ligation into a single workflow and has been validated against conventional preparation methods, showing comparable results in coverage and sequencing yield. Feedback from early adopters highlights the protocol's simplicity and robustness in producing barcoded libraries from limited DNA amounts.
Nextera Overview Feb 2010
- 1. Nextera™ NGS Library PreparationFrom Nanograms of DNA to Sequencer-Ready Libraries in under Two Hours© 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.
- 2. Deep Sequencing MethodsThere are three types of sequencing chemistries in common use:Sequencing by synthesisSequencing by ligationReal-time sequencing by synthesis
- 3. Sequencing by SynthesisRoche 454™
- 5. Helicos
- 6. Intelligent Bio SystemsSequencing by LigationAB SOLiD™PolonatorComplete Genomics
- 7. Real-Time Sequencing by SynthesisPacific BiosciencesVisigen
- 8. NGS Methods Have a Common Library Prep Workflow
- 9. Typical Workflow Suffers from Some LimitationsMultiple steps, time-consumingRequires microgram amounts of DNANot suitable for high-throughput processing
- 10. Nextera™ Technology Streamlines Library PreparationNextera™ TechnologyCombines fragmentation, end-repair, and ligation steps
- 11. Sequencer-ready libraries in <2 hours from 20-50 ng of DNA
- 12. Compatible with 96-well plate processing“Tagmentation”: Fragmentation/Tagging by In Vitro TranspositionTransposomecomplexes with free transposon ends5’-Tagged DNA fragments
- 13. Roche 454™-Compatible LibrariesNextera™ EnzymeMix(free transposon ends)Add FLX or Ti sites by PCR (+/- bar codes)emPCR input
- 14. IlluminaSolexa®-Compatible LibrariesNextera™ Enzyme Mix (appended transposon ends)Add bPCR sites by PCR (+/- bar codes)bPCR input
- 15. Nextera™ DNA Sample Prep KitsCurrent kits available (February 2010)Roche/454-Compatible LibrariesGS FLX™Bar Coding Module (n = 12)GS FLX™ TitaniumBar Coding Module (n = 12)Illumina-Compatible LibrariesGAIIBar Coding Module (n = 12)Nextera PCR EnzymeRequired component
- 17. Deep Sequencing of 454-Compatible LibrariesLibraries prepared using Nextera™ Sample PrepE. coli, plasmid, or soybean genomic DNASummary table of sequencing yield, coverage, and mapping data
- 18. Deep Sequencing of 454-Compatible LibrariesLibraries prepared using Nextera™ Sample PrepE. coli, plasmid, or soybean genomic DNARelative coverage of individual soybean chromosomes shows even coverage across all 20 chromosomes.
- 19. Deep Sequencing of Illumina-Compatible LibrariesLibrary prepared using Nextera™ Sample PrepE. coli genomic DNACoverage plot indicating sequencing depth vs. position on reference genome.(Stray reads from unrelated libraries in same channel mapped to LacZ and nohB promoters.)
- 20. Deep Sequencing of Illumina-Compatible LibrariesDistribution of depth (nucleotides vs. depth) shows a near- Poisson distribution. GC bias of coverage is comparable to libraries generated using physical shearing
- 22. NGS Library Analysis Conducted by Early AdoptersCompared: Nebulization, NEB Fragmentase, Nextera™
- 23. Human, E. coli, phage
- 24. Illumina GAII and Roche FLX Titanium
- 25. Characterized:
- 26. Insertion bias
- 27. Fragment size
- 28. Coverage
- 30. Manuscript in preparation for peer review and publication.“…the insertion sites are essentially random for all three methods of library preparation.”
- 31. Early Adopter Feedback“… 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“We're very excited by what we've seen from it so far.” – Jay Shendure, Univ. Washington“It's incredibly easy as compared to conventional library construction methods.”
- 32. Univ. of WashingtonEmily TurnerRuolanQiuAndrew AdeyJay ShendureMBL-Woods HoleHilary MorrisonJessica Mark WelchEkaterina AndreishchevaBill ReznikoffEPICENTRE®Haiying GrunenwaldDilara BegumIgor GoryshinLes HoffmanJoanne DeckerMark MaffittJerry JendrisakAcknowledgements© 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.