Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms

Abstract

Our knowledge of gene function has increasingly lagged behind gene discovery, hindering our understanding of the genetic basis of microbial phenotypes. Recently, however, massively parallel sequencing has been combined with traditional transposon mutagenesis in techniques referred to as transposon sequencing (Tn-seq), high-throughput insertion tracking by deep sequencing (HITS), insertion sequencing (INSeq) and transposon-directed insertion site sequencing (TraDIS), making it possible to identify putative gene functions in a high-throughput manner. Here, we describe the similarities and differences of these related techniques and discuss their application to the probing of gene function and higher-order genome organization.

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

“…However, genome wide screening and identification of all genes involved in tolerance has been a limiting factor, which in the last years have been overcome through the use of next generation sequencing (NGS) techniques. Transposon insertion sequencing (Tn‐seq) (Figure 1a), enables high‐throughput identification of genomic regions involved in the survival of cells when exposed to various conditions (Barquist, Boinett, & Cain, 2013; van Opijnen & Camilli, 2013). A number of organisms have been investigated using this technique, thereby successfully identifying genes important for growth under different stress conditions, essentially by simultaneously investigating the fitness of all single mutants (Gawronski, Wong, Giannoukos, Ward, & Akerley, 2009; Langridge et al, 2009; Lennen & Herrgård, 2014; Santiago et al, 2015).…”

Compound-gene interaction mapping reveals distinct roles forStaphylococcus aureusteichoic acids

“…Transposondirected insertion site sequencing (TraDIS, similar to Tn-seq), a PCR-based next generation sequencing technique that determines the location and abundance of transposon insertions in a population, showed that the library contained 60,000 independent insertion sites ( Fig. 2A), which represents approximately 20-fold coverage of the S. aureus genome (12,13). The high saturation of the transposon library enabled us to screen for genes that interact with WTAs by using tunicamycin to turn off WTA synthesis.…”

“…We reasoned that insight into distinct functions of TAs could be gleaned by identifying their genetic interactions. The recent merging of classical transposon mutagenesis with high-throughput next-generation sequencing (e.g., TraDIS, Tn-seq) has enabled parallel genotype-phenotype profiling and functional mapping for large collections of mutants (12,13). These methods have been used to identify genetic factors that influence susceptibility to lethal antibiotics (14), but can also be applied to map cell envelope networks.…”

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum

“…High-throughput genomic approaches such as transposon sequencing (Tn-seq) have been used to identify genetic elements required for in vitro and in vivo fitness (8). Tn-seq allows for simultaneous assessment of the abundance of tens or hundreds of thousands of individual transposon mutants after growth in a selective condition (e.g., in vivo infection model) (9)(10)(11).…”