A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites (original) (raw)

Nature Protocols volume 4, pages 789–798 (2009)Cite this article

Abstract

Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds of thousands of insertion sites from mouse tumors and, unlike other methods, has been specifically optimized for the murine leukemia virus (MuLV), and can easily be performed in a 96-well plate format for the efficient multiplex isolation of insertion sites.

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References

  1. Greenman, C. et al. Patterns of somatic mutation in human cancer genomes. Nature 446, 153 (2007).
    Article CAS PubMed PubMed Central Google Scholar
  2. Mikkers, H. & Berns, A. Retroviral insertional mutagenesis: tagging cancer pathways. Adv. Cancer Res. 88, 53 (2003).
    CAS PubMed Google Scholar
  3. Dupuy, A.J. et al. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436, 221 (2005).
    Article CAS PubMed Google Scholar
  4. Keng, V.W. et al. Forward genetic screen for hepatocellular carcinoma associated genes. Proceedings of the 99th Annual Meeting of the American Association for Cancer Research (San Diego, 2008).
    Google Scholar
  5. Wu, X. et al. Murine metastatic medulloblastoma driven by Sleeping Beauty transposition identifies genes and pathways important for medulloblastoma initiation and progression. Proceedings of the 99th Annual Meeting of the American Association for Cancer Research (San Diego, 2008).
    Google Scholar
  6. Bender, A.M. et al. Sleeping Beauty mediated gliomagenesis. Proceedings of the 99th Annual Meeting of the American Association for Cancer Research (San Diego, 2008).
    Google Scholar
  7. Collier, L.S. et al. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272 (2005).
    Article CAS PubMed Google Scholar
  8. Cuypers, H.T. et al. Murine leukemia virus-induced T-cell lymphomagenesis: integration of proviruses in a distinct chromosomal region. Cell 37, 141 (1984).
    Article CAS PubMed Google Scholar
  9. Ochman, H., Gerber, A.S. & Hartl, D.L. Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621 (1988).
    CAS PubMed PubMed Central Google Scholar
  10. McAleer, M.A., Coffey, A.J. & Dunham, I. DNA rescue by the vectorette method. Methods Mol. Biol. 226, 393 (2003).
    CAS PubMed Google Scholar
  11. Devon, R.S., Porteous, D.J. & Brookes, A.J. Splinkerettes-improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res. 23, 1644 (1995).
    Article CAS PubMed PubMed Central Google Scholar
  12. Hui, E.K., Wang, P.C. & Lo, S.J. Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants. Cell Mol. Life Sci. 54, 1403 (1998).
    Article CAS PubMed Google Scholar
  13. Hengen, P.N. Vectorette, splinkerette and boomerang DNA amplification. Trends Biochem. Sci. 20, 372 (1995).
    Article CAS PubMed Google Scholar
  14. Uren, A.G. et al. Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks. Cell 133, 727 (2008).
    Article CAS PubMed PubMed Central Google Scholar
  15. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376 (2005).
    Article CAS PubMed PubMed Central Google Scholar
  16. Wang, G.P. et al. HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res. 17, 1186 (2007).
    Article CAS PubMed PubMed Central Google Scholar
  17. Mikkers, H. et al. High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat. Genet. 32, 153 (2002).
    Article CAS PubMed Google Scholar
  18. Marra, M.A., Kucaba, T.A., Hillier, L.W. & Waterston, R.H. High-throughput plasmid DNA purification for 3 cents per sample. Nucleic Acids Res. 27, e37 (1999).
    Article CAS PubMed PubMed Central Google Scholar
  19. Mullikin, J.C. & McMurragy, A.A. Techview: DNA sequencing. Sequencing the genome, fast. Science 283, 1867 (1999).
    Article CAS PubMed Google Scholar
  20. Parameswaran, P. et al. A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res. 35, e130 (2007).
    Article PubMed PubMed Central Google Scholar
  21. Giordano, F.A. et al. New bioinformatic strategies to rapidly characterize retroviral integration sites of gene therapy vectors. Methods Inf. Med. 46, 542 (2007).
    Article CAS PubMed Google Scholar
  22. Kong, J., Zhu, F., Stalker, J. & Adams, D.J. iMapper: a web application for the automated analysis and mapping of insertional mutagenesis sequence data against Ensembl genomes. Bioinformatics 24, 2923 (2008).
    Article CAS PubMed PubMed Central Google Scholar
  23. Kustikova, O.S., Modlich, U. & Fehse, B. Retroviral insertion site analysis in dominant haematopoietic clones. Methods Mol. Biol. 506, 373 (2009).
    Article CAS PubMed Google Scholar
  24. Ambrosi, A., Cattoglio, C. & Di Serio, C. Retroviral integration process in the human genome: is it really non-random? A new statistical approach. PLoS Comput. Biol. 4, e1000144 (2008).
    Article PubMed PubMed Central Google Scholar

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Acknowledgements

A.G.U., J.K., M.v.L. and A.B. are funded by the NWO Genomics program and the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO). D.J.A. and his laboratory are funded by Cancer Research-UK and the Wellcome Trust. L.v.d.W. is funded by the Kay Kendall Leukemia Fund. We thank L. Collier for critically reading this manuscript.

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Author notes

  1. Anthony G Uren and Harald Mikkers: These authors contributed equally.

Authors and Affiliations

  1. Division of Molecular Genetics, Cancer Genomics Centre, Netherlands Cancer Institute, Plesmanlaan, Amsterdam, The Netherlands
    Anthony G Uren, Jaap Kool, Maarten van Lohuizen & Anton Berns
  2. Department of Molecular Cell Biology and Regenerative Medicine Program, Leiden University Medical Centre, Leiden, The Netherlands
    Harald Mikkers
  3. Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
    Louise van der Weyden, Catherine H Wilson, Richard Rance & David J Adams
  4. BRIC—Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, Denmark
    Anders H Lund
  5. Division of Molecular Biology, Cancer Genomics Centre, Netherlands Cancer Institute, Amsterdam, Plesmanlaan, The Netherlands
    Jos Jonkers

Authors

  1. Anthony G Uren
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  2. Harald Mikkers
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  3. Jaap Kool
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  4. Louise van der Weyden
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  5. Anders H Lund
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  6. Catherine H Wilson
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  7. Richard Rance
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  8. Jos Jonkers
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  9. Maarten van Lohuizen
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  10. Anton Berns
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  11. David J Adams
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Corresponding authors

Correspondence toAnton Berns or David J Adams.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

10 bp sequences suitable for 'barcoding' splinkerette products for 454 sequencing. These sequences facilitate the computational 'demultiplexing' of pooled splinkerette-PCR products following 454 sequencing. (PDF 18 kb)

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Uren, A., Mikkers, H., Kool, J. et al. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.Nat Protoc 4, 789–798 (2009). https://doi.org/10.1038/nprot.2009.64

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