Advertisement

Preparation of Next-Generation Sequencing Libraries Using Nextera™ Technology: Simultaneous DNA Fragmentation and Adaptor Tagging by In Vitro Transposition

  • Nicholas CaruccioEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 733)

Abstract

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

Key words

Next-generation sequencing DNA library preparation Roche/454 Illumina/Solexa Nextera 

Notes

Acknowledgments

The author would like to thank Jay Shendure and Hilary Morrison for their work characterizing the Illumina-compatible and Roche-compatible libraries and Haiying Grunenwald for technical advice and assistance during the development of this method.

Illumina and Solexa are registered trademarks of Illumina Inc., San Diego, CA. Tween is a registered trademark of ICI Americas Inc., Wilmington, DE. 454 and GS FLX are trademarks of Roche, Nutley, NJ. DNA Clean & Concentrator and Zymo-Spin are trademarks of Zymo Research., Orange, CA. Nextera and Tagmentation are trademarks of EPICENTRE, Madison, WI. Patent applications assigned to EPICENTRE and by US Patent Nos. 5,965,443, and 6,437,109; European Patent No. 0927258, and related patents and patent applications, exclusively licensed to EPICENTRE, cover Nextera™ Products.

References

  1. 1.
    Kahvehian, V., Quackenbush, J., and Thompson, J. F. (2008) What would you do if you could sequence everything? Nature Biotechnology. 26, 1125–1133.CrossRefGoogle Scholar
  2. 2.
    Metzker, M. L. (2010) Sequencing Technologies – the next generation. Nature Reviews – Genetics. 11, 31–  46.Google Scholar
  3. 3.
    Shendure, J. and Henlee, J. (2008) Next-generation DNA Sequencing. Nature Biotechnology. 26, 1135–1145.PubMedCrossRefGoogle Scholar
  4. 4.
    Mardis, E. (2008) Next-Generation DNA Sequencing Methods. (2008) Annual Review of Genomics and Human Genetics. 9, 387–  402.Google Scholar
  5. 5.
    Fuller, C. W., Meddendorf, L. R., Benner, S. A., Church, G. M., Harris, T., Huang, X., Jovanovich, S. B., Nelson, J. R., Schloss, J. A., Schwartz, D. C., and Vezenov, D. V. (2009) The challenges of sequencing by synthesis. Nature Biotechnology. 27, 1013  –1023.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Epicentre BiotechnologiesMadisonUSA

Personalised recommendations