title: High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors.
abstract: Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.
authors.affiliation: Division of Molecular Biology and Cancer Systems Biology Center, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
authors.forename: Marco J
authors.initials: MJ
authors.lastname: Koudijs
authors.forename: Christiaan
authors.initials: C
authors.lastname: Klijn
authors.forename: Louise
authors.initials: L
authors.lastname: van der Weyden
authors.forename: Jaap
authors.initials: J
authors.lastname: Kool
authors.forename: Jelle
authors.initials: J
authors.lastname: ten Hoeve
authors.forename: Daoud
authors.initials: D
authors.lastname: Sie
authors.forename: Pramudita R
authors.initials: PR
authors.lastname: Prasetyanti
authors.forename: Eva
authors.initials: E
authors.lastname: Schut
authors.forename: Sjors
authors.initials: S
authors.lastname: Kas
authors.forename: Theodore
authors.initials: T
authors.lastname: Whipp
authors.forename: Edwin
authors.initials: E
authors.lastname: Cuppen
authors.forename: Lodewyk
authors.initials: L
authors.lastname: Wessels
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
authors.forename: Jos
authors.initials: J
authors.lastname: Jonkers
mesh_terms: D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics*; D030541:Databases, Genetic; D008324:Mammary Tumor Virus, Mouse; D051379:Mice; D016254:Mutagenesis, Insertional; D012192:Retroviridae Infections / Q000235:genetics*; D014412:Tumor Virus Infections / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm