A decade of advances in transposon-insertion sequencing.

Amy K Cain,Tim Van Opijnen,Ian T Paulsen,Lars Barquist,Andrew L Goodman,Julian Parkhill

doi:10.1038/s41576-020-0244-x

Abstract

It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.

Highlights

Abstract | It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome
Four variations on the TIS method were published in 2009: transposon sequencing (Tn-Seq)[1], transposon-directed insertion site sequencing (TraDIS)[2], insertion sequencing (INSeq)[3] and high-throughput insertion tracking by deep sequencing (HITS)[4]
The way DNA undergoes fragmentation for library preparation differs: Tn-Seq and INSeq use the type II restriction enzyme MmeI to yield uniform-length shorter reads, which can remove PCR amplification bias, whereas TraDIS and HITS use random-sized shearing via sonication, which can have the advantage of improved transposon mapping owing to longer reads

Summary

B Challenge with condition

Growth/ sorting which inserts itself into thymine–adenine dinucleotide (TA) sites but otherwise does not have a sequence preference, and the others have the flexibility that they can use any transposon, but they commonly use Tn5 as it is commercially available and does not have a insertion site bias. TIS methods have been developed to incorporate other technologies and techniques to answer complex biological questions in creative ways These include physical separation and sorting of individual mutant cells, using inducible promoters to study essential genes and scaling of current techniques to simultaneously screen multiple environments and different species, facilitating pan-organism analysis (Fig. 2). A high-throughput method for gain-of-function assays was recently developed, dual-barcoded shotgun expression library sequencing (Dub-Seq), where barcoded overexpression libraries of E. coli were mapped, barcoded and used to assign gene function in 52 experimental conditions on the basis of mutant fitness changes due to increased gene dosage[22]. All of these tools implement variations on both gene essentiality and conditional fitness analyses, they differ in the details of preprocessing and

B Gain-of-function screens Ba Tn insertions show essential genes

Conclusions and future perspectives

Findings

A Predicting phenotypes