Targeted sequencing library preparation by genomic DNA circularization

BMC Biotechnology

Methodology Article

Samuel Myllykangas¹  Georges Natsoulis¹  Hanlee P Ji²  John M Bell³ 

[1] Division of Oncology, Department of Medicine, Stanford University School of Medicine, 94305, Stanford, CA, USA;Division of Oncology, Department of Medicine, Stanford University School of Medicine, 94305, Stanford, CA, USA;Stanford Genome Technology Center, Stanford University, 94304, Palo Alto, CA, USA;Stanford Genome Technology Center, Stanford University, 94304, Palo Alto, CA, USA;

关键词: Sequencing Library;    Hybridization Temperature;    Genomic Circularization;    Target Specific Site;    Molecular Inversion Probe;   

DOI  :  10.1186/1472-6750-11-122

 received in 2011-08-02, accepted in 2011-12-14,  发布年份 2011

来源: Springer

【 摘 要 】

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

【 授权许可】

Unknown   
© Myllykangas et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.