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Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing

Author

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  • Kristof Vandewalle

    (Unit for Medical Biotechnology, VIB, Technologiepark 927
    Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ledeganckstraat 35)

  • Nele Festjens

    (Unit for Medical Biotechnology, VIB, Technologiepark 927
    Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ledeganckstraat 35)

  • Evelyn Plets

    (Unit for Medical Biotechnology, VIB, Technologiepark 927
    Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ledeganckstraat 35)

  • Marnik Vuylsteke

    (Gnomixx, Onafhankelijkheidslaan 38)

  • Yvan Saeys

    (Unit for Immunoregulation and Mucosal Immunology, Inflammation Research Center (IRC), VIB, Technologiepark 927)

  • Nico Callewaert

    (Unit for Medical Biotechnology, VIB, Technologiepark 927
    Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ledeganckstraat 35)

Abstract

Reverse genetics research approaches require the availability of methods to rapidly generate specific mutants. Alternatively, where these methods are lacking, the construction of pre-characterized libraries of mutants can be extremely valuable. However, this can be complex, expensive and time consuming. Here, we describe a robust, easy to implement parallel sequencing-based method (Cartesian Pooling-Coordinate Sequencing or CP-CSeq) that reports both on the identity as well as on the location of sequence-tagged biological entities in well-plate archived clone collections. We demonstrate this approach using a transposon insertion mutant library of the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. The method is applicable to any entity for which sequence-tagged identification is possible.

Suggested Citation

  • Kristof Vandewalle & Nele Festjens & Evelyn Plets & Marnik Vuylsteke & Yvan Saeys & Nico Callewaert, 2015. "Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing," Nature Communications, Nature, vol. 6(1), pages 1-7, November.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms8106
    DOI: 10.1038/ncomms8106
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