Abstract
Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis. Application of this approach to a model pathogen, Haemophilus influenzae, has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis.
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Acknowledgments
This work was supported in part by grant from the NIH (AI49437) to B.J.A. Initial development of the method was conducted in collaboration with Georgia Giannoukos and Doyle Ward of the Broad Institute.
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Wong, S.M.S., Gawronski, J.D., Lapointe, D., Akerley, B.J. (2011). High-Throughput Insertion Tracking by Deep Sequencing for the Analysis of Bacterial Pathogens. In: Kwon, Y., Ricke, S. (eds) High-Throughput Next Generation Sequencing. Methods in Molecular Biology, vol 733. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-089-8_15
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