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Open Access Highly Accessed Methodology article

Targeted sequencing library preparation by genomic DNA circularization

Samuel Myllykangas1, Georges Natsoulis1, John M Bell2 and Hanlee P Ji12*

Author affiliations

1 Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA

2 Stanford Genome Technology Center, Stanford University, Palo Alto, CA, 94304, USA

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Citation and License

BMC Biotechnology 2011, 11:122  doi:10.1186/1472-6750-11-122

Published: 14 December 2011

Abstract

Background

For next generation DNA sequencing, we have developed a rapid and simple approach for preparing DNA libraries of targeted DNA content. Current protocols for preparing DNA for next-generation targeted sequencing are labor-intensive, require large amounts of starting material, and are prone to artifacts that result from necessary PCR amplification of sequencing libraries. Typically, sample preparation for targeted NGS is a two-step process where (1) the desired regions are selectively captured and (2) the ends of the DNA molecules are modified to render them compatible with any given NGS sequencing platform.

Results

In this proof-of-concept study, we present an integrated approach that combines these two separate steps into one. Our method involves circularization of a specific genomic DNA molecule that directly incorporates the necessary components for conducting sequencing in a single assay and requires only one PCR amplification step. We also show that specific regions of the genome can be targeted and sequenced without any PCR amplification.

Conclusion

We anticipate that these rapid targeted libraries will be useful for validation of variants and may have diagnostic application.