The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

Lars Barquist,Matthew Mayho,Christine J Boinett,Michael A Quail,Gemma C Langridge,Julian Parkhill,Amy K Cain,Andrew J Page,Carla Cummins,Jacqueline A Keane

doi:10.1093/bioinformatics/btw022

Abstract

Summary: Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology.Availability and implementation: The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at https://github.com/sanger-pathogens/Bio-TradisContact: parkhill@sanger.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

Highlights

Steady improvements in high-throughput sequencing technologies have resulted in an increasing number of sequenced bacterial genomes, revealing extensive genetic diversity both within and between species
We previously developed a method for this purpose, called Transposon Directed Insertion Sequencing (TraDIS; Langridge et al, 2009)
TraDIS uses fragmentation of genomic DNA followed by specific PCR amplification of transposon-containing fragments to selectively enrich for transposon-flanking sequences, and can be adapted for any transposon of interest through a simple redesign of sequencing primers

Summary

Introduction

Steady improvements in high-throughput sequencing technologies have resulted in an increasing number of sequenced bacterial genomes, revealing extensive genetic diversity both within and between species. TraDIS has since been applied to a variety of target organisms and transposons in a wide variety of both in vivo and in vitro growth conditions. These include Tn5-based libraries in Salmonella (Barquist et al, 2013b; Chaudhuri et al, 2013; Langridge et al, 2009) and Escherichia (Dziva et al, 2013; Eckert et al, 2011) and Marinerbased libraries in Clostridia (Dembek et al, 2015) and Mycobacteria (Weerdenburg et al, 2015).

Library preparation and sequencing

The Bio-Tradis analysis pipeline

Summary