Transposon sequencing (Tn-seq) methodology. A Tn library is prepared by infecting M. tuberculosis with the temperature-sensitive MycoMarT7 bacteriophage, which results in transposon (Tn) insertion at genomic loci that contain TA sites. The Tn, denoted as an inverted red arrowhead, contains a kanamycin resistance gene (kan) that is utilized to select cells that contain a Tn insertion, the E. coli oriR6K origin of replication, two outward facing T7 promoters (red arrows in (4)), and 29-bp inverted repeats. Tn insertions that disrupt four genes, A to D, are represented in the library. The library is then subjected to selection under any condition of interest. Tn mutants carrying an insertion in a gene that is essential under that condition will not survive, as illustrated in this schematic by gene C. After selection, genomic DNA is extracted from surviving organisms, sheared, and T-tailed adapters (denoted by green lines) are then ligated to the DNA ends. Adapter-specific and Tn-specific primers with extensions homologous to Illumina sequencing primers (orange lines) are then used for direct sequencing on Illumina platforms. Sequence reads are trimmed at the Tn region, and mapped to the parental strain genome. Genes that have no or few insertions are likely to be important for survival under the selective condition. The schematic is adapted from Zhang et al. .