title: Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing.
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.
authors.affiliation: 1] Unit for Medical Biotechnology, Department of Medical Biotechnology Center (MBC), VIB, Technologiepark 927, Ghent B-9052, Belgium [2] Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Ledeganckstraat 35, Ghent B-9000, Belgium.
authors.forename: Kristof
authors.initials: K
authors.lastname: Vandewalle
authors.affiliation: 1] Unit for Medical Biotechnology, Department of Medical Biotechnology Center (MBC), VIB, Technologiepark 927, Ghent B-9052, Belgium [2] Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Ledeganckstraat 35, Ghent B-9000, Belgium.
authors.forename: Nele
authors.initials: N
authors.lastname: Festjens
authors.affiliation: 1] Unit for Medical Biotechnology, Department of Medical Biotechnology Center (MBC), VIB, Technologiepark 927, Ghent B-9052, Belgium [2] Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Ledeganckstraat 35, Ghent B-9000, Belgium.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Plets
authors.affiliation: Gnomixx, Onafhankelijkheidslaan 38, Ghent B-9000, Belgium.
authors.forename: Marnik
authors.initials: M
authors.lastname: Vuylsteke
authors.affiliation: Unit for Immunoregulation and Mucosal Immunology, Inflammation Research Center (IRC), VIB, Technologiepark 927, Ghent B-9052, Belgium.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Saeys
authors.affiliation: 1] Unit for Medical Biotechnology, Department of Medical Biotechnology Center (MBC), VIB, Technologiepark 927, Ghent B-9052, Belgium [2] Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Ledeganckstraat 35, Ghent B-9000, Belgium.
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D015723:Gene Library; D055106:Genome-Wide Association Study; D016296:Mutagenesis; D009154:Mutation; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial