PCR-Free Nextera Di-Tagged DNA Library Preparation for NGS Applications

The Nextera™ technology for generating libraries of di-tagged DNA fragments is rapidly becoming the preferred method for massively parallel DNA sequencing. Despite rapid advances in sequencing instrument throughput, classic library preparation by step-wise ligation of adaptors is a time-intensive an...

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Bibliographic Details
Published in:Journal of biomolecular techniques 2011-10, Vol.22 (Suppl), p.S41-S41
Main Authors: Grunenwald, H., Baas, B., Goryshin, I., Caruccio, N., Maffitt, M., Kinross, C.
Format: Article
Language:English
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Poster Session Abstracts
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Summary:The Nextera™ technology for generating libraries of di-tagged DNA fragments is rapidly becoming the preferred method for massively parallel DNA sequencing. Despite rapid advances in sequencing instrument throughput, classic library preparation by step-wise ligation of adaptors is a time-intensive and throughput-limiting bottleneck. PCR amplification of libraries prior to cluster generation is also a major concern because of its possibility to reduce library complexity, particularly in regions of extreme G+C content (high or low), thereby producing uneven genome coverage and confounding mapping and assembly. In this study, we describe novel modifications of the Nextera™ library preparation system to address such library preparation bias by eliminating PCR amplification. Sequencer-ready libraries can be obtained from as little as 200 ng of genomic DNA in 3 hours with 90-minutes of hands-on time. Deep sequencing of genomic libraries indicates that this system reduces coverage bias and GC bias, as well as improves library diversity.
ISSN:1524-0215
1943-4731