title: Generation of long insert pairs using a Cre-LoxP Inverse PCR approach.
abstract: 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.
authors.affiliation: Joint Genome Institute, United States Department of Energy, Walnut Creek, California, United States of America.
authors.forename: Ze
authors.initials: Z
authors.lastname: Peng
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhao
authors.forename: Nandita
authors.initials: N
authors.lastname: Nath
authors.forename: Jeff L
authors.initials: JL
authors.lastname: Froula
authors.forename: Alicia
authors.initials: A
authors.lastname: Clum
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.forename: Jan-Fang
authors.initials: JF
authors.lastname: Cheng
authors.forename: Alex C
authors.initials: AC
authors.lastname: Copeland
authors.forename: Len A
authors.initials: LA
authors.lastname: Pennacchio
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
mesh_terms: D001287:Attachment Sites, Microbiological / Q000235:genetics*; D019605:Euryarchaeota / Q000235:genetics; D015723:Gene Library; D019426:Integrases / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: C045073:Cre recombinase; D019426:Integrases