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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
Notes
- 1.
The sequences of Primer 1 and Primer 2 and portions of Adaptor 1 and Adaptor 2 correspond to Illumina bPCR sequences and are copyrighted by Illumina, Inc. Oligonucleotide sequences© 2006–2010 Illumina, Inc. All rights reserved.
References
Kahvehian, V., Quackenbush, J., and Thompson, J. F. (2008) What would you do if you could sequence everything? Nature Biotechnology. 26, 1125–1133.
Metzker, M. L. (2010) Sequencing Technologies – the next generation. Nature Reviews – Genetics. 11, 31– 46.
Shendure, J. and Henlee, J. (2008) Next-generation DNA Sequencing. Nature Biotechnology. 26, 1135–1145.
Mardis, E. (2008) Next-Generation DNA Sequencing Methods. (2008) Annual Review of Genomics and Human Genetics. 9, 387– 402.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Caruccio, N. (2011). Preparation of Next-Generation Sequencing Libraries Using Nextera™ Technology: Simultaneous DNA Fragmentation and Adaptor Tagging by In Vitro Transposition. In: Kwon, Y., Ricke, S. (eds) High-Throughput Next Generation Sequencing. Methods in Molecular Biology, vol 733. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-089-8_17
Download citation
DOI: https://doi.org/10.1007/978-1-61779-089-8_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-088-1
Online ISBN: 978-1-61779-089-8
eBook Packages: Springer Protocols