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

1. Nextera™ NGS Library Preparation From 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 Methods There are three types of sequencing chemistries in common use: Sequencing by synthesis Sequencing by ligation Real-time sequencing by synthesis

4. IlluminaSolexa®

5. Helicos

7. Real-Time Sequencing by Synthesis Pacific Biosciences Visigen

8. NGS Methods Have a Common Library Prep Workflow

9. Typical Workflow Suffers from Some Limitations Multiple steps, time-consuming Requires microgram amounts of DNA Not suitable for high-throughput processing

11. Sequencer-ready libraries in <2 hours from 20-50 ng of DNA

13. Roche 454™-Compatible Libraries Nextera™ EnzymeMix(free transposon ends) Add FLX or Ti sites by PCR (+/- bar codes) emPCR input

14. IlluminaSolexa®-Compatible Libraries Nextera™ Enzyme Mix (appended transposon ends) Add bPCR sites by PCR (+/- bar codes) bPCR input

15. Nextera™ DNA Sample Prep Kits Current kits available (February 2010) Roche/454-Compatible Libraries GS FLX™ Bar Coding Module (n = 12) GS FLX™ Titanium Bar Coding Module (n = 12) Illumina-Compatible Libraries GAII Bar Coding Module (n = 12) Nextera PCR Enzyme Required component

16. Library Preparation Comparison

17. Deep Sequencing of 454-Compatible Libraries Libraries prepared using Nextera™ Sample Prep E. coli, plasmid, or soybean genomic DNA Summary table of sequencing yield, coverage, and mapping data

18. Deep Sequencing of 454-Compatible Libraries Libraries prepared using Nextera™ Sample Prep E. coli, plasmid, or soybean genomic DNA Relative coverage of individual soybean chromosomes shows even coverage across all 20 chromosomes.

19. Deep Sequencing of Illumina-Compatible Libraries Library prepared using Nextera™ Sample Prep E. coli genomic DNA Coverage 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 Libraries Distribution of depth (nucleotides vs. depth) shows a near- Poisson distribution. GC bias of coverage is comparable to libraries generated using physical shearing

21. Additional Nextera™ Validation

23. Human, E. coli, phage

24. Illumina GAII and Roche FLX Titanium

25. Characterized:

26. Insertion bias

27. Fragment size

28. Coverage

29. Library complexity

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 Washington Emily Turner RuolanQiu Andrew Adey Jay Shendure MBL-Woods Hole Hilary Morrison Jessica Mark Welch Ekaterina Andreishcheva Bill Reznikoff EPICENTRE® Haiying Grunenwald Dilara Begum Igor Goryshin Les Hoffman Joanne Decker Mark Maffitt Jerry Jendrisak Acknowledgements © 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.