## 2 Genomics
### Mode of Action  
Advances in next generation sequencing technologies and the continued reduction in cost has placed whole genome sequencing (WGS) firmly into the drug discovery pipeline from mechanism of action identification through to monitoring drug resistance post approval [29]. Illumina sequencing (Illumina Inc., San Diego, CA, USA) generates short sequence reads (typically 150 bp long), and while the majority of sequencing data generated today is from Illumina platforms, alternative methodologies are also useful to antimicrobial drug discovery. Long read sequencing (-kilobases), available through platforms such as PacBio (Pacific Biosciences, Menlo Park, CA, USA) and Oxford Nanopore (Oxford Nanopore Technologies Ltd, Oxford, UK), are especially suited for sequencing repetitive regions and structural variations. WGS has been exploited to identify mutations that occur in spontaneous drug resistant colonies growing on solid agar containing a novel antimicrobial compound of interest, typically between 5-fold and 10-fold the minimum inhibitory concentration. Using drug exposure as the selective pressure, this methodology may reveal mutations that affect drug action, in the drug target itself or in gene functions that influence drug activation or efflux. [PERSON] et al. in the discovery of bedaquiline, employed WGS of _M. smegmatis_ and _M.tb_ bedaquiline-resistant mutants cultured in vitro to demonstrate that this diaryquinoline targets the product of _atpE_; a subunit of the mycobacterialATP synthase anchored in the mycobacterial membrane [30]. [PERSON] et al. further characterized this mode of action, showing that bedaquiline binds to the epsilon subunit [31]. Mutations can, however, be identified in other non-target genes that confer resistance, expanding understanding of drug action but complicating the interpretation of results. For example, in a murine model of TB infection, mutations in the putative Xaa-Pro aminopeptidase _pepQ_ were identified that conferred low level resistance to both bedaquiline and clofazimine, neither of which target PepQ directly [32]. While the function of PepQ remains to be determined in _M.tb_, [PERSON] et al. postulated that this mutation prevented protein degradation of the efflux pump MmpL5, enhancing drug efflux and inducing resistance to bedaquiline and clofazimine [32]. WGS has also been used to reveal the mechanism of action of repurposed licensed pharmaceuticals in _M.tb_. [PERSON] et al. identified lansoprazole from the Prestwick library of 1280 FDA approved drugs as protective to lung fibroblasts in an _M.tb_ intracellular model of infection [33]. Three lansoprazole resistant mutants were sequenced; each mutant showed the same SNP, a substitution of leucine for proline, in the \(\beta\)-subunit of the cytochrome bc1 complex gene _qcrB_, a key element of the mycobacterial respiratory chain. To demonstrate that exposure to this commonly prescribed proton pump inhibitor did not select for lansoprazole-resistant _M.tb_, [PERSON] et al. sequenced 13,559 _M.tb_ Complex clinical isolates revealing only one _M. bovis_ isolate with a mutation in _qcrB_[34]. Of course, this methodology to define the target of drugs with unknown mechanisms of action is only fruitful if drug-resistant colonies can be raised, which is not always the case. This was highlighted in the discovery of the novel antimicrobial drug, teixobactin from the bacterium _Eleftheria terrae_. Here, a novel iChip culture method was used to capture previously unculturable bacteria in their natural soil environment, which led to the identification of the cell wall-targeting agent teixobactin [35]. Neither _Staphylococcus aureus_ nor _M.tb_ drug resistant colonies could be generated even at sub-inhibitory concentrations. The authors hypothesized that this indicated general cell toxicity of the compound; however, no mammalian cell toxicity was observed. Subsequent biochemical approaches indicated that this compound exerted its antimicrobial effects through interactions with peptidoglycan pre-cursors--mostly lipid II.