High-Throughput Next Generation Sequencing Methods and Applications
Due to their novel concepts and extraordinary high-throughput sequencing capacity, the “next generation sequencing” methods allow scientists to grasp system-wide landscapes of the complex molecular events taking place in various biological systems, includ
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Introduction Next-generation sequencing (variously known as deep sequencing, second-generation sequencing, or high-throughput sequencing) has led to genomic data acquisition on a scale never seen before, enabling “hypothesis-free” studies and changing the approaches used to answer the fundamental scientific questions ((1) and references therein). Currently available sequencing platforms, as well as platforms anticipated in the near future, utilize three basic chemistries to sequence DNA, including pyrosequencing, sequencing by synthesis (both cyclic and real time), and sequencing by ligation (recently reviewed in (2–4)).
Young Min Kwon and Steven C. Ricke (eds.), High-Throughput Next Generation Sequencing: Methods and Applications, Methods in Molecular Biology, vol. 733, DOI 10.1007/978-1-61779-089-8_17, © Springer Science+Business Media, LLC 2011
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Despite the diversity in sequencing chemistries used for sequencing polony arrays or single molecules, upstream sample preparation is generally very similar and follows a common threestep workflow. In the first step, large DNAs are sheared to appropriately sized fragments. Next, the DNA fragments are “repaired” to have blunt ends or A tails. In the final step, platform-specific adaptors are ligated onto the repaired fragments in order to attach the library to a solid surface in a spatially separated array via a complementary sequence (e.g., a tagged glass slide or bead). Each of these steps consists of multiple and distinct handling, incubation, and clean-up procedures, which result in a complex, multistep protocol. Thus, current sample preparation methods require microgram amounts of genomic DNA, significant hands-on time, and suffer from limited sample throughput (5). In vitro transposition using Nextera technology can be exploited to perform all of these steps in a single, 5-min reaction. In a classical transposition reaction, a hyperactive transposase enzyme is used under conditions that catalyze near-random insertion of excised transposons into DNA targets with high efficiency in vitro. When Transposome™ complexes are assembled with free transposon-end DNA instead, the target DNA is simultaneously fragmented and tagged with the transposon-end sequence, thereby generating a tagged DNA fragment library in a single reaction (Fig. 1). Sequencing libraries are prepared using a simple, two-step process. First, genomic DNA is fragmented and tagged using in vitro transposition. Second, platform-specific adaptors and a
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5’-Tagged DNA Fragments
Fig. 1. Simultaneous fragmenting and tagging of DNA by in vitro transposition. Transposomes assembled with free, double-stranded transposon end DNA (a) are sufficient to bind and integrate into target DNA (b). The resulting tagged fragments (c) serve as the foundation for subsequent adaptor addition.
NGS Library Preparation by In Vitro Transposition
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optional bar codes are added and the library is enriched using limited-cycle PCR. Methods are described to prepare DNA fragme
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