RT Journal
A1 Koudijs, Marco J.
A1 Klijn, Christiaan
A1 van der Weyden, Louise
A1 Kool, Jaap
A1 ten Hoeve, Jelle
A1 Sie, Daoud
A1 Prasetyanti, Pramudita R.
A1 Schut, Eva
A1 Kas, Sjors
A1 Whipp, Theodore
A1 Cuppen, Edwin
A1 Wessels, Lodewyk
A1 Adams, David J.
A1 Jonkers, Jos
T1 High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors
JF Genome Research 
JO Genome Research 
YR 2011 
FD December 01 
VO 21 
IS 12 
SP 2181 
OP 2189 
DO 10.1101/gr.112763.110 
UL http://genome.cshlp.org/content/21/12/2181.abstract 
AB 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.