Monday, August 22, 2011

High-throughput semi-quantitative analysis of insertional mutations in heterogeneous tumors [METHODS]

High-throughput semi-quantitative analysis of insertional mutations in heterogeneous tumors [METHODS]:

Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice, and for the identification of drug and pathogen resistance genes in cultured cells. 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 splinkerette adapter 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 semi-quantitative 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 semi-quantitative measure of the relative clonality of individual insertions within heterogeneous samples and permits discrimination between (near) clonal insertions and those insertions that occur in single, or just a few cells, within the tumor. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV) induced tumors showed that shear-splink enables the semi-quantitative 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.

(Via Genome research (advanced).)