Using Genome Scale Mutant Libraries to Identify Essential Genes

Methods Mol Biol. 2022;2377:215-236. doi: 10.1007/978-1-0716-1720-5_12.


Identification of essential genes is key to understanding the required processes and gene products of organisms under one or more conditions. Transposon sequencing (Tn-seq) has been used to predict essential genes or ones that conditionally impact fitness in a wide variety of organisms. Here, we describe the generation of genome-scale mutant libraries and the analysis of Tn-seq data to identify essential genes from cultures grown in a single condition as well as those that are conditionally important by analyzing the behavior of these mutant libraries in different growth environments. While we illustrate the approach using data derived from Tn-seq analysis of the α-proteobacteria Rhodobacter sphaeroides and Zymomonas mobilis, the protocols and systems we describe should be generally applicable to a variety of organisms.

Keywords: Bacteria; Computational biology; Conditional fitness; Essential genes; Gene disruption; Genomics; Microbial lifestyles; Sequencing; Transposon.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.