Identifying Essential Genes in Mycobacterium tuberculosis by Global Phenotypic Profiling
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 wordsMycobacterium tuberculosis Himar1 mutagenesis Transposon sequencing (TnSeq) Illumina next-generation sequencing Essentiality
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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