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

Lars Barquist, Matthew Mayho, Carla Cummins, Amy K. Cain, Christine J. Boinett, Andrew J. Page, Gemma C. Langridge, Michael A. Quail, Jacqueline A. Keane, Julian Parkhill
2016 Bioinformatics  
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.  ...  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  ...  Acknowledgements The authors would like to thank Mark Gibbs and Isabelle Rasolonjatovo (Illumina FAS) for expert assistance in developing the sequencing recipes described here, and Elizabeth Huckle and  ... 
doi:10.1093/bioinformatics/btw022 pmid:26794317 pmcid:PMC4896371 fatcat:wmejtnbxtjg7vgppqgkhmkubkq

Defining the ABC of gene essentiality in streptococci

Amelia R. L. Charbonneau, Oliver P. Forman, Amy K. Cain, Graham Newland, Carl Robinson, Mike Boursnell, Julian Parkhill, James A. Leigh, Duncan J. Maskell, Andrew S. Waller
2017 BMC Genomics  
Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses,  ...  Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality.  ...  Outputs files of tradis_essentiality and from orthologue and KEGG analysis are available in the Additional files.  ... 
doi:10.1186/s12864-017-3794-3 pmid:28569133 pmcid:PMC5452409 fatcat:sgaes5sygzc45g6fx45v6pjwya

Novel genes required for the fitness of Streptococcus pyogenes in human saliva [article]

Luchang Zhu, Amelia Charbonneau, Andrew Waller, Randall Olsen, Stephen Beres, James Musser
2017 bioRxiv   pre-print
Using transposon-directed insertion site sequencing (TraDIS), we identified 92 genes required for GAS fitness in saliva.  ...  The transposon mutant library was exposed to human saliva to screen for GAS genes required for wild-type fitness in this clinically relevant fluid.  ...  The collected mutant pools (4 replicates, 438 4 time points, n = 16) were stored at -80°C for the subsequent TraDIS analysis. 439 DNA preparation and massive parallel sequencing.  ... 
doi:10.1101/196931 fatcat:xzeun6frgzakbakq34gjhvqbhu

The Essential Genome of Escherichia coli K-12

Emily C. A. Goodall, Ashley Robinson, Iain G. Johnston, Sara Jabbari, Keith A. Turner, Adam F. Cunningham, Peter A. Lund, Jeffrey A. Cole, Ian R. Henderson, Kimberly A. Kline, Swaine L. Chen
2018 mBio  
We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. coli model  ...  To identify essential genes in Escherichia coli , we constructed a transposon mutant library of unprecedented density.  ...  ACKNOWLEDGMENTS We thank Discuva Ltd. for providing some of their large transposon mutant library. We thank N. Loman and J. Quick for help with optimization of our MiSeq protocol.  ... 
doi:10.1128/mbio.02096-17 pmid:29463657 pmcid:PMC5821084 fatcat:iyvfag5kffhi7fsrw5hp7dspa4

Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii

Karl A. Hassan, Amy K. Cain, TaoTao Huang, Qi Liu, Liam D. H. Elbourne, Christine J. Boinett, Anthony J. Brzoska, Liping Li, Martin Ostrowski, Nguyen Thi Khanh Nhu, Tran Do Hoang Nhu, Stephen Baker (+2 others)
2016 mBio  
A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside  ...  IMPORTANCE Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various  ...  Transposon mutant library generation and verification by TraDIS.  ... 
doi:10.1128/mbio.01200-16 pmid:27601573 pmcid:PMC5013296 fatcat:opqjdco3krayxj6tq2xrlikrje

Gene fitness landscape of group A streptococcus during necrotizing myositis [article]

Luchang Zhu, Randall Olsen, Stephen Beres, Jesus Eraso, Matthew Ojeda Saavedra, Samantha Kubiak, Concepcion Cantu, Leslie Jenkins, Amelia Charbonneau, Andrew Waller, James Musser
2018 bioRxiv   pre-print
Using transposon directed insertion-site sequencing (TraDIS) we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively.  ...  For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters.  ...  The 525 resulting sequencing reads were analyzed with the TraDIS toolkit (83). 600 qRT-PCR analysis of infected NHP and human muscle.  ... 
doi:10.1101/432526 fatcat:76t65tmedrgq7lreiscboz4nbq

The essential genome of Escherichia coli K-12 [article]

Emily Goodall, Ashley Robinson, Iain Johnston, Sara Jabbari, Keith Turner, Adam Cunningham, Peter Lund, Jeffrey Cole, Ian Henderson
2017 bioRxiv   pre-print
Currently, the TraDIS method has not been benchmarked against such libraries and therefore it remains unclear whether the two methodologies are comparable.  ...  Single gene deletion libraries are considered the gold standard for identifying essential genes.  ...  Tn-seq explorer: a tool for analysis of 661 high-throughput sequencing data of transposon mutant libraries. PLoS One 662 10:e0126070. 663 20. Liu G, Draper GC, Donachie WD. 1998.  ... 
doi:10.1101/237842 fatcat:abe5fuekwzgbzksyrg6yp45kty

Functional analysis of colonization factor antigen I positive enterotoxigenic Escherichia coli identifies genes implicated in survival in water and host colonization

Moataz Abd El Ghany, Lars Barquist, Simon Clare, Cordelia Brandt, Matthew Mayho, Enrique Joffre´, Åsa Sjöling, A. Keith Turner, John D. Klena, Robert A. Kingsley, Grant A. Hill-Cawthorne, Gordon Dougan (+1 others)
2021 Microbial Genomics  
Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian  ...  The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.  ...  The authors wish to thank the WTSI Sequencing team for their help with generating TraDIS sequencing data and the Pathogen Informatics Team for their assistance with bioinformatics analysis of TraDIS and  ... 
doi:10.1099/mgen.0.000554 pmid:34110281 pmcid:PMC8461466 fatcat:3xtfuxq7azcgramamst3t5yroq

TraDIS-validate: a method for curating ordered gene-replacement libraries [article]

Emily C A Goodall, Faye C Morris, Samantha A McKeand, Rudi Sullivan, Isabel A Warner, Emma Sheehan, Gabriela Boelter, Christopher Icke, Adam F. Cunningham, Jeffrey A Cole, Manuel Banzhaf, Jack Alfred Bryant (+1 others)
2022 bioRxiv   pre-print
This dataset provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of an antibiotic resistance cassette in any ordered library.  ...  We applied our method (TraDIS-Validate) to the well-known Keio library of Escherichia coli gene-deletion mutants.  ...  Acknowledgements We thank Professor Mori for his helpful tables and insight into the Keio library.  ... 
doi:10.1101/2022.02.16.479736 fatcat:ypzxibuwlnb6vnoeqrfpfkhy2i

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis

Colette E. O'Neill, Rachel J. Skilton, Jade Forster, David W. Cleary, Sarah A. Pearson, David J. Lampe, Nicholas R. Thomson, Ian N. Clarke
2021 Wellcome Open Research  
This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis.  ...  Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and  ...  We would also like to thank Prof Beatrix Suess for useful discussions around the use of riboswitches and for sending materials for use in early developmental experiments.  ... 
doi:10.12688/wellcomeopenres.16068.1 pmid:35087955 pmcid:PMC8767425 fatcat:hd5jcsmrkfevnnryfde7nmjpbu

The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid [article]

Rocío Canals, Roy R. Chaudhuri, Rebecca E. Steiner, Siân V. Owen, Natalia Quinones-Olvera, Melita A. Gordon, Michael Ibba, Jay C. D. Hinton
2019 bioRxiv   pre-print
We used a genome-wide 26 transposon library with insertions every 10 nucleotides to identify genes required for survival and growth 27 in vitro and during infection of murine macrophages.  ...  By infecting murine macrophages 33 with the transposon library, we identified 68 genes that were required for intra-macrophage replication 34 but did not impact fitness in vitro.  ...  index was calculated for each gene as explained in [19] , using the Bio::Tradis toolkit 605 [78] .  ... 
doi:10.1101/689075 fatcat:avfge6nldzcexi4qnu5hftrakq

Tools and Approaches for Dissecting Protein Bacteriocin Import in Gram-Negative Bacteria

Iva Atanaskovic, Colin Kleanthous
2019 Frontiers in Microbiology  
Here, we review the various approaches that now exist for investigating how protein bacteriocins translocate into Gram-negative bacteria and highlight areas of research that will need methodological innovations  ...  In order to translocate across the cell envelope and cause cell death, protein bacteriocins hijack nutrient uptake pathways.  ...  ACKNOWLEDGMENTS The authors would like to thank Ms. Hannah Behrens, Dr. Joanna Szczepaniak, Dr. Nathalie Reichmann, and Dr. Nick Housden for useful discussions and feedback on this manuscript.  ... 
doi:10.3389/fmicb.2019.00646 pmid:31001227 pmcid:PMC6455109 fatcat:tsmvcecf2vdbdnv4ttoyemcvqe

Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs

Susan L. Brockmeier, Crystal L. Loving, Tracy L. Nicholson, Jinhong Wang, Sarah E. Peters, Lucy Weinert, Roy Chaudhuri, David J. Seilly, Paul R. Langford, Andrew Rycroft, Brendan W. Wren, Duncan J. Maskell (+2 others)
2017 Infection and Immunity  
The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated  ...  Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations.  ...  The TraDIS toolkit: sequencing and analysis for dense 573 transposon mutant libraries. Bioinformatics 32:1109-1111. 574 17.  ... 
doi:10.1128/iai.00559-17 pmid:29203546 pmcid:PMC5820948 fatcat:cob4ng2ivrdf3n74l5awhd73ki

High-throughput experimental and computational studies of bacterial evolution

Lars Barquist, Apollo-University Of Cambridge Repository, Apollo-University Of Cambridge Repository
In the first section, consisting of three chapters, I describe a recently developed high-throughput technology for probing gene function, transposon-insertion sequencing, and its application to the study  ...  In a second study I more directly probe the genomic features responsible for differences in serovar pathogenicity by analyzing transposon-insertion sequencing data produced following a two hour infection  ...  Acknowledgements I have been tremendously fortunate to spend the past four years on the Wellcome Trust Genome Campus at the Sanger Institute and the European Bioinformatics Institute.  ... 
doi:10.17863/cam.16351 fatcat:zi7lq5a57fdjznteahj3clqb7y

Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks

FungMin Liew, Michael E. Martin, Ryan C. Tappel, Björn D. Heijstra, Christophe Mihalcea, Michael Köpke
2016 Frontiers in Microbiology  
Both of these strategies, demonstrated at bench-scale, have abundant potential to rapidly expand the commercial product spectrum of gas fermentation and further improve efficiencies and yields.  ...  As such, the technology can utilize a wide range of feedstocks including gasified organic matter of any sort (e.g., municipal solid waste, industrial waste, biomass, and agricultural waste residues) or  ...  When coupled with next generation sequencing, transposon-directed insertion site sequencing (TraDIS) was performed on C. difficile and identified 404 essential genes for growth and 798 genes that are likely  ... 
doi:10.3389/fmicb.2016.00694 pmid:27242719 pmcid:PMC4862988 fatcat:ch2m6xrimngtfbadgrpiuimfhe
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