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