BioSpace Collaborative - Generation of Long Insert Pairs Using a Cre-LoxP Inverse PCR Approach

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Generation of Long Insert Pairs Using a Cre-LoxP Inverse PCR Approach
Published: Monday, January 09, 2012
Author: Ze Peng et al.

by Ze Peng, Zhiying Zhao, Nandita Nath, Jeff L. Froula, Alicia Clum, Tao Zhang, Jan-fang Cheng, Alex C. Copeland, Len A. Pennacchio, Feng Chen

Large insert mate pair reads have a major impact on the overall success of de novo assembly and the discovery of inherited and acquired structural variants. The positional information of mate pair reads generally improves genome assembly by resolving repeat elements and/or ordering contigs. Currently available methods for building such libraries have one or more of limitations, such as relatively small insert size; unable to distinguish the junction of two ends; and/or low throughput. We developed a new approach, Cre-LoxP Inverse PCR Paired-End (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) the use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends and to improve the self-ligation efficiency. We have successfully created CLIP-PE libraries up to 22 kb that are rich in informative read pairs and low in small fragment background. These libraries have demonstrated the ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms. More...