High-Throughput Insertion Tracking by Deep Sequencing for the Analysis of Bacterial Pathogens
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.
Key wordsHITS Himar1 Mariner Transposon mutagenesis Bacteria Haemophilus influenzae Genetic footprinting Deep sequencing
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.
- 12.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995), John Wiley and Sons, Inc.Google Scholar
- 18.Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis, Mol Microbiol 22, 951– 965.PubMedCrossRefGoogle Scholar
- 20.Langridge, G. C., Phan, M. D., Turner, D. J., Perkins, T. T., Parts, L., Haase, J., Charles, I., Maskell, D. J., Peters, S. E., Dougan, G., Wain, J., Parkhill, J., and Turner, A. K. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants, Genome Res 19, 2308 –2316.PubMedCrossRefGoogle Scholar