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Identifying Essential Genes in Mycobacterium tuberculosis by Global Phenotypic Profiling

  • Jarukit E. Long
  • Michael DeJesus
  • Doyle Ward
  • Richard E. Baker
  • Thomas Ioerger
  • Christopher M. Sassetti
Part of the Methods in Molecular Biology book series (MIMB, volume 1279)

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.

Key words

Mycobacterium tuberculosis Himar1 mutagenesis Transposon sequencing (TnSeq) Illumina next-generation sequencing Essentiality 

Notes

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.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Jarukit E. Long
    • 1
  • Michael DeJesus
    • 2
  • Doyle Ward
    • 3
  • Richard E. Baker
    • 1
  • Thomas Ioerger
    • 2
  • Christopher M. Sassetti
    • 1
    • 4
  1. 1.Department of Microbiology and Physiological SystemsUniversity of Massachusetts Medical SchoolWorcesterUSA
  2. 2.Department of Computer ScienceTexas A&M UniversityCollege StationUSA
  3. 3.Broad InstituteCambridgeUSA
  4. 4.Howard Hughes Medical InstituteChevy ChaseUSA

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