Abstract
Transposon sequencing (TnSeq) is a next-generation deep sequencing-based method to quantitatively assess the composition of complex mutant transposon libraries after pressure from selection. Although this method can be used for any organism in which transposon mutagenesis is possible, this chapter describes its use in Mycobacterium tuberculosis. More specifically, the methods for generating complex libraries through transposon mutagenesis, design of selective pressure, extraction of genomic DNA, amplification and quantification of transposon insertions through next-generation deep sequencing are covered. Determining gene essentiality and statistical analysis on data collected are also discussed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ito M, Baba T, Mori H, Mori H (2005) Functional analysis of 1440 Escherichia coli genes using the combination of knock-out library and phenotype microarrays. Metab Eng 7:318–327
Sassetti CM, Boyd DH, Rubin EJ (2001) Comprehensive identification of conditionally essential genes in mycobacteria. Proc Natl Acad Sci U S A 98:12712–12717
van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767–772
Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84
Zhang YJ, Ioerger TR, Huttenhower C et al (2012) Global assessment of genomic regions required for growth in Mycobacterium tuberculosis. PLoS Pathog 8:e1002946
Akerley BJ, Rubin EJ, Camilli A et al (1998) Systematic identification of essential genes by in vitro mariner mutagenesis. Proc Natl Acad Sci U S A 95:8927–8932
van Opijnen T, Lazinski DW, Camilli A (2010) Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms. Curr Protoc Mol Biol Chapter 1:Unit1E.3–1E.3.16
Griffin JE, Gawronski JD, Dejesus MA et al (2011) High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog 7:e1002251
Gawronski JD, Wong SM, Giannoukos G et al (2009) Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc Natl Acad Sci U S A 106:16422–16427
Goodman AL, Wu M, Gordon JI (2011) Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat Protoc 6:1969–1980
McDonough E, Lazinski DW, Camilli A (2014) Identification of in vivo regulators of the Vibrio cholerae xds gene using a high-throughput genetic selection. Mol Microbiol 92:302–315
Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A 100:12989–12994
Joshi SM, Pandey AK, Capite N et al (2006) Characterization of mycobacterial virulence genes through genetic interaction mapping. Proc Natl Acad Sci U S A 103:11760–11765
Bardarov S, Kriakov J, Carriere C et al (1997) Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 94:10961–10966
DeJesus MA, Zhang YJ, Sassetti CM, Rubin EJ, Sacchettini JC, Ioerger TR (2013) Bayesian analysis of gene essentiality based on sequencing of transposon insertion libraries. Bioinformatics 29:695–703
DeJesus MA, Ioerger TR (2013) A Hidden Markov Model for identifying essential and growth-defect regions in bacterial genomes from transposon insertion sequencing data. BMC Bioinformatics 14:303
Zomer A, Burghout P, Bootsma HJ et al (2012) ESSENTIALS: software for rapid analysis of high throughput transposon insertion sequencing data. PLoS One 7:e43012
Acknowledgments
This work was supported by NIH awards F32A1093049 (J.E.L.), NIH AI064282 (C.M.S.), NIH AI095208 (T.I. and C.M.S.), NIH U19 AI107774 and HHMI.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Long, J.E., DeJesus, M., Ward, D., Baker, R.E., Ioerger, T., Sassetti, C.M. (2015). Identifying Essential Genes in Mycobacterium tuberculosis by Global Phenotypic Profiling. In: Lu, L. (eds) Gene Essentiality. Methods in Molecular Biology, vol 1279. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2398-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2398-4_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2397-7
Online ISBN: 978-1-4939-2398-4
eBook Packages: Springer Protocols