High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors

Division of Molecular Biology and Cancer Systems Biology Center, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Genome Research (Impact Factor: 14.63). 08/2011; 21(12):2181-9. DOI: 10.1101/gr.112763.110
Source: PubMed


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.

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Available from: Jos Jonkers,
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    • "59 out of 92 (64%) integrations supported by chimeric fusions were confirmed by DNA analyses (Figure 1D and E), while the majority of these (~90%) showed an expected distribution close to enhancer midpoints (Figure 6). While the number of integrations in only four tumors may seem high comparably high numbers of integrations in transposon and retroviral tumor models have been reported previously [45,66]: e.g. deep sequencing analysis of mouse mammary tumor virus (MMTV)-induced tumors, revealed an average of 27 integrations per tumor, a number that was significantly higher when including integrations supported by only a single chimeric sequencing read [45]. "
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    ABSTRACT: Background Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined. Results We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues. Conclusions We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.
    Retrovirology 05/2014; 11(1):36. DOI:10.1186/1742-4690-11-36 · 4.19 Impact Factor
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    • "In order to obtain an improved quantitative representation of insertion sites by their sequence read numbers, we combined two technical advancements of recent reports – Illumina sequencing to obtain >106 reads per sample [7], and acoustic shearing to reduce PCR biases [7,8] - and developed a protocol for efficient high-throughput sequencing of transposon insertions. We applied our protocol for the analysis of eleven various solid tumors that we had generated by mutagenesis with the PiggyBac transposon array ATP1-S2 [5]. "
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    ABSTRACT: Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10(6) reads corresponding to >10(4) insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.
    PLoS ONE 08/2013; 8(8):e72338. DOI:10.1371/journal.pone.0072338 · 3.23 Impact Factor
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    • "We employed a splinkerette PCR-based method to identify retroviral insertion sites, broadly as described by Koudijs and colleagues [9]. Briefly, genomic DNA was extracted from 1 million KBM7 cells (Gentra Puregene kit) and sheared by sonication (Bioruptor®, Diagenode) to an average fragment size of ∼400 bp. "
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    ABSTRACT: The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.
    PLoS ONE 06/2012; 7(6):e39651. DOI:10.1371/journal.pone.0039651 · 3.23 Impact Factor
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