title: Genome-Wide Transposon Mutagenesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.
abstract: TnSeq, or transposon (Tn) insertion sequencing, is a powerful method for identifying the essential-as well as conditionally essential-regions in a genome, both coding and noncoding. The advent of accessible massively parallel DNA sequencing technologies in particular has resulted in the increased use of TnSeq-based approaches to elucidate various aspects of bacterial physiology and metabolism. Moreover, the availability of detailed protocols has enabled even nonspecialist laboratories to adapt and develop TnSeq approaches to address specific research questions. In this chapter, we describe a recently modified experimental protocol used in our laboratory for TnSeq in the major human pathogen, Mycobacterium tuberculosis, as well as the related non-pathogenic mycobacterium, M. smegmatis. The method, which was developed in close consultation with pioneers in the field of mycobacterial genetics, includes the steps involved in preparing a phage stock, generating a mutant library, selection of the library under a specific experimental condition, isolation of genomic DNA from the pooled population of mutants, amplification of the sites of Tn insertion and, finally, determining the essential genomic regions by next-generation sequencing.
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Majumdar
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors.forename: Rendani
authors.initials: R
authors.lastname: Mbau
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors.forename: Vinayak
authors.initials: V
authors.lastname: Singh
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. digby.warner@uct.ac.za.|Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa. digby.warner@uct.ac.za.
authors.forename: Digby F
authors.initials: DF
authors.lastname: Warner
authors.affiliation: Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Marte Singsås
authors.initials: MS
authors.lastname: Dragset
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. raju.mukherjee@uct.ac.za.|Division of Biology, Indian Institute of Science Education and Research, Tirupati, India. raju.mukherjee@uct.ac.za.
authors.forename: Raju
authors.initials: R
authors.lastname: Mukherjee
mesh_terms: D004251:DNA Transposable Elements / Q000235:genetics*; D015723:Gene Library; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D016296:Mutagenesis / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements
keywords: MycoMarT7; TnSeq analysis; Transposon mutagenesis