Genome-Wide Transposon Mutagenesis in Mycobacterium tuberculosis and Mycobacterium smegmatis

: 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.