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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$32.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to the full article PDF.
USD 39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
van Opijnen, T., Bodi, K. L. & Camilli, A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat. Methods 6, 767–772 (2009).
Langridge, G. C. et al. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res. 19, 2308–2316 (2009).
Goodman, A. L. et al. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6, 279–289 (2009).
Gawronski, J. D. et al. Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc. Natl Acad. Sci. USA 106, 16422–16427 (2009).
van Opijnen, T. & Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat. Rev. Microbiol. 11, 435–442 (2013). A review on TIS that outlines the detail and difference of each variation of TIS.
Barquist, L., Boinett, C. J. & Cain, A. K. Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol. 10, 1161–1169 (2013). A review on TIS that outlines the use and all applications of TIS until 2013.
Chao, M. C., Abel, S., Davis, B. M. & Waldor, M. K. The design and analysis of transposon insertion sequencing experiments. Nat. Rev. Microbiol. 14, 119–128 (2016).
Kakkanat, A., Phan, M.-D., Lo, A. W., Beatson, S. A. & Schembri, M. A. Novel genes associated with enhanced motility of Escherichia coli ST131. PLoS One 12, e0176290 (2017).
Nolan, L. M. et al. A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa. Microb. Genomics https://doi.org/10.1099/mgen.0.000229 (2018). One of the first studies to apply physical separation methods to TIS, in this case to examine twitching motility-based biofilm formation.
Dorman, M. J., Feltwell, T., Goulding, D. A., Parkhill, J. & Short, F. L. The capsule regulatory network of Klebsiella pneumoniae defined by density-TraDISort. MBio 9, e01863-18 (2018).
Hassan, K. A. et al. Fluorescence-based flow sorting in parallel with transposon insertion site sequencing identifies multidrug efflux systems in Acinetobacter baumannii. MBio 7, e01200-16 (2016). One of the first methods to separate single mutant cells using FACS and sequence them using TIS, in this case to examine efflux of the biocide ethidium bromide from each cell.
Rego, E. H., Audette, R. E. & Rubin, E. J. Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity. Nature 546, 153–157 (2017).
Fowler, C. C. & Galán, J. E. Decoding a Salmonella typhi regulatory network that controls typhoid toxin expression within human cells. Cell Host Microbe 23, 65–76 (2018).
Duncan, M. C. et al. High-throughput analysis of gene function in the bacterial predator Bdellovibrio bacteriovorus. MBio 10, 1–12 (2019).
Thibault, D. et al. Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes. Nat. Commun. 10, 5729 (2019). The first method to separate and individually encapsulate single cells so as to assay each mutant’s effects independently from the population.
Judson, N. & Mekalanos, J. J. TnAraOut, a transposon-based approach to identify and characterize essential bacterial genes. Nat. Biotechnol. 18, 740–745 (2000).
Christen, B. et al. The essential genome of a bacterium. Mol. Syst. Biol. 7, 1–7 (2011).
Coe, K. A. et al. Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus. PLoS Pathog. 15, e1007862 (2019).
Santiago, M. et al. A new platform for ultra-high density Staphylococcus aureus transposon libraries. BMC Genomics 16, 1–18 (2015).
Yasir, M. et al. TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance. Genome Res. 30, 1–11 (2020).
Yasir, M. et al. A new massively-parallel transposon mutagenesis approach comparing multiple datasets identifies novel mechanisms of action and resistance to triclosan. bioRxiv https://doi.org/10.1101/596833 (2019).
Mutalik, V. K. et al. Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria. Nat. Commun. 10, 308 (2019).
Wetmore, K. M. et al. Sequencing randomly bar-coded transposons. MBio 6, 1–15 (2015).
Robinson, D. G., Chen, W., Storey, J. D. & Gresham, D. Design and analysis of Bar-seq experiments. G3 4, 11–18 (2014).
Price, M. N. et al. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 557, 503–509 (2018). A massively upscaled study that applies TIS to multiple bacterial species and more than 100 conditions so as to assign broad gene function en masse.
Liu, H. et al. Magic pools: parallel assessment of transposon delivery vectors in bacteria. mSystems 3, 1–17 (2018).
Zomer, A., Burghout, P., Bootsma, H. J., Hermans, P. W. M. & van Hijum, S. A. F. T. Essentials: software for rapid analysis of high throughput transposon insertion sequencing data. PLoS One 7, 1–9 (2012).
McCoy, K. M., Antonio, M. L. & van Opijnen, T. MAGenTA: a galaxy implemented tool for complete Tn-Seq analysis and data visualization. Bioinformatics 33, 2781–2783 (2017).
DeJesus, M. A., Ambadipudi, C., Baker, R., Sassetti, C. & Ioerger, T. R. TRANSIT - a software tool for Himar1 TnSeq analysis. PLoS Comput. Biol. 11, 1–17 (2015).
Pritchard, J. R. et al. ARTIST: high-resolution genome-wide assessment of fitness using transposon-insertion sequencing. PLoS Genet. 10, e1004782 (2014).
Barquist, L. et al. The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 32, 1109–1111 (2016).
Anthony, J. S. & T. van O. Aerobio: an extensible full DAG streaming computation server with services and jobs for RNA-Seq, Tn-Seq, WG-Seq and Term-Seq. GitHub https://github.com/jsa-aerial/aerobio (2019).
van Opijnen, T. & Camilli, A. A fine scale phenotype-genotype virulence map of a bacterial pathogen. Genome Res. 22, 2541–2551 (2012).
Warr, A. R. et al. Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization. PLoS Pathogens 15, e1007652 (2019).
Subramaniyam, S. et al. Statistical analysis of variability in TnSeq data across conditions using zero-inflated negative binomial regression. BMC Bioinformatics 20, 603 (2019).
van den Berge, K. et al. Observation weights unlock bulk RNA-seq tools for zero inflation and single-cell applications. Genome Biol. 19, 24 (2018).
Hubbard, T. P., D’Gama, J. D., Billings, G., Davis, B. M. & Waldor, M. K. Unsupervised learning approach for comparing multiple transposon insertion sequencing studies. mSphere 4, e00031-19 (2019).
Page, A. J. et al. AlbaTraDIS: comparative analysis of large datasets from parallel transposon mutagenesis experiments. bioRxiv https://doi.org/10.1101/593624 (2019).
Canals, R. et al. The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid. PLoS Pathog. 15, e1007948 (2019).
Down, T. A., Piipari, M. & Hubbard, T. J. P. Dalliance: interactive genome viewing on the web. Bioinformatics 27, 889–890 (2011).
Surujon, D. & Van Opijnen, T. ShinyOmics: vollaborative exploration of omics-data. BMC Bioinformatics 21, 1–8 (2020).
Jana, B. et al. The secondary resistome of multidrug-resistant Klebsiella pneumoniae. Sci. Rep. 7, 42483 (2017).
Boinett, C. J. et al. Clinical and laboratory-induced colistin-resistance mechanisms in Acinetobacter baumannii. Microb. Genom. 5, e000246 (2019).
Coe, K. A. et al. Comparative Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus despite strain-dependent differences in essentiality of shared cell envelope genes. bioRxiv https://doi.org/10.1101/648246 (2019).
Geisinger, E., Mortman, N. J., Vargas-Cuebas, G., Tai, A. K. & Isberg, R. R. A global regulatory system links virulence and antibiotic resistance to envelope homeostasis in Acinetobacter baumannii. PLOS Pathog. 14, e1007030 (2018).
Xu, W. et al. Chemical genetic interaction profiling reveals determinants of intrinsic antibiotic resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 61, e01334-17 (2017).
Willcocks, S. et al. Genome-wide assessment of antimicrobial tolerance in Yersinia pseudotuberculosis under ciprofloxacin stress. Microb. Genomics https://doi.org/10.1099/mgen.0.000304 (2019).
Murray, J. L., Kwon, T., Marcotte, E. M. & Whiteley, M. Intrinsic antimicrobial resistance determinants in the superbug Pseudomonas aeruginosa. MBio 6, 1–10 (2015).
Rajagopal, M. et al. Multidrug intrinsic resistance factors in Staphylococcus aureus identified by profiling fitness within high-diversity transposon libraries. MBio 7, e00950-16 (2016).
Blake, K. L. & O’Neill, A. J. Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents. J. Antimicrob. Chemother. 68, 12–16 (2012).
Gallagher, L. A., Shendure, J. & Manoil, C. Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. MBio 2, (2011).
Cain, A. K. et al. Transposon directed insertion-site sequencing (TraDIS) to elucidate the mode of action of the antimicrobial arenicin-3 (Arn-3). in 54th International Conference of Antimicrobial Agents and Chemotherapy (ICAAC) and Infectious Diseases Society of America (IDSA) (ASM, 2014).
Santiago, M. et al. Genome-wide mutant profiling predicts the mechanism of a lipid II binding antibiotic. Nat. Chem. Biol. 14, 601–608 (2018).
van Opijnen, T., Dedrick, S. & Bento, J. Strain dependent genetic networks for antibiotic-sensitivity in a bacterial pathogen with a large pan-genome. PLoS Pathog. 12, e1005869 (2016).
Fields, P. I., Swanson, R. V., Haidaris, C. G. & Heffron, F. Mutants of Salmonella Typhimurium that cannot survive within the macrophage are avirulent. Proc. Natl Acad. Sci. USA 83, 5189–5193 (1986).
Hensel, M. et al. Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403 (1995).
Goh, K. G. K. et al. Genome-wide discovery of genes required for capsule production by uropathogenic Escherichia coli. MBio 8, e01558-17 (2017).
McCarthy, A. J., Stabler, R. A. & Taylor, P. W. Genome-wide identification by transposon insertion sequencing of Escherichia coli K1 genes essential for in vitro growth, gastrointestinal colonizing capacity, and survival in serum. J. Bacteriol. 200, e00698-17 (2018).
Sharp, C. et al. O-antigen-dependent colicin insensitivity of uropathogenic Escherichia coli. J. Bacteriol. 201, e00545-18 (2019).
Dembek, M. et al. High-throughput analysis of gene essentiality and sporulation in Clostridium difficile. MBio. 6, 1–13 (2015).
Chaudhuri, R. R. et al. Comprehensive assignment of roles for Salmonella Typhimurium genes in intestinal colonization of food-producing animals. PLoS Genet. 9, e1003456 (2013).
Vohra, P. et al. Retrospective application of transposon-directed insertion-site sequencing to investigate niche-specific virulence of Salmonella Typhimurium in cattle. BMC Genomics 20, 20 (2019).
Shames, S. R. et al. Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries. Proc. Natl Acad. Sci. USA 114, E10446–E10454 (2017).
Zhu, L. et al. Novel genes required for the fitness of streptococcus pyogenes in human saliva. mSphere 2, e00460-17 (2017).
Subashchandrabose, S. et al. Acinetobacter baumannii genes required for bacterial survival during bloodstream infection. mSphere 1, e00013-15 (2016).
Charbonneau, A. R. L. et al. Identification of genes required for the fitness of Streptococcus equi subsp. equi in whole equine blood and hydrogen peroxide. Microb. Genomics https://doi.org/10.1099/mgen.0.000362 (2020).
Wang, H. et al. Hypermutation-induced in vivo oxidative stress resistance enhances Vibrio cholerae host adaptation. PLoS Pathog. 14, 1–22 (2018).
Wong, Y.-C. et al. Genetic determinants associated with in vivo survival of Burkholderia cenocepacia in the Caenorhabditis elegans model. Front. Microbiol. 9, 1118 (2018).
Shields, R. C., Zeng, L., Culp, D. J. & Burne, R. A. Genomewide identification of essential genes and fitness determinants of streptococcus mutans UA159. mSphere 3, e00031-18 (2018).
Crabill, E., Schofield, W. B., Newton, H. J., Goodman, A. L. & Roy, C. R. Dot/Icm-translocated proteins important for biogenesis of the Coxiella burnetii-containing vacuole identified by screening of an effector mutant sublibrary. Infect. Immun. 86, e00758-17 (2018).
Grant, A. J. et al. Modelling within-host spatiotemporal dynamics of invasive bacterial disease. PLoS Biol. 6, e74 (2008).
Abel, S., Abel zur Wiesch, P., Davis, B. M. & Waldor, M. K. Analysis of bottlenecks in experimental models of infection. PLoS Pathog. 11, e1004823 (2015).
Carter, P. B. & Collins, F. M. The route of enteric infection in normal mice. J. Exp. Med. 139, 1189–1203 (1974).
Maier, L. et al. Granulocytes impose a tight bottleneck upon the gut luminal pathogen population during salmonella typhimurium colitis. PLoS Pathog. 10, e1004557 (2014).
Lawley, T. D. et al. Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse. PLoS Pathog. 2, e11 (2006).
Rowe, H. M. et al. Bacterial factors required for transmission of streptococcus pneumoniae in mammalian hosts. Cell Host Microbe 25, 884–891 (2019). The first TIS study to identify bacterial factors for transmission between hosts and exploit these factors for a transmission-blocking vaccine.
Carter, R. et al. Genomic analyses of pneumococci from children with sickle cell disease expose host-specific bacterial adaptations and deficits in current interventions. Cell Host Microbe 15, 587–599 (2014).
Hancock, S. J. et al. Identification of IncA/c plasmid replication and maintenance genes and development of a plasmid multilocus sequence typing scheme. Antimicrob. Agents Chemother. 61, e01740-16 (2017).
Liu, Y. et al. Transposon insertion sequencing reveals T4SS as the major genetic trait for conjugation transfer of multi-drug resistance pEIB202 from Edwardsiella. BMC Microbiol. 17, 1–15 (2017).
Cowley, L. A. et al. Transposon insertion sequencing elucidates novel gene involvement in susceptibility and resistance to phages T4 and T7 in Escherichia coli O157. MBio 9, e00705-18 (2018).
Pickard, D. et al. A genomewide mutagenesis screen identifies multiple genes contributing to Vi capsular expression in Salmonella enterica serovar typhi. J. Bacteriol. 195, 1320–1326 (2013).
Klein, B. A., Duncan, M. J. & Hu, L. T. Defining essential genes and identifying virulence factors of Porphyromonas gingivalis by massively parallel sequencing of transposon libraries (Tn-seq). Methods Mol. Biol. 1279, 25–43 (2015).
Wong, Y.-C. et al. Candidate essential genes in Burkholderia cenocepacia J2315 identified by genome-wide TraDIS. Front. Microbiol. 7, 1288 (2016).
Willcocks, S. J., Stabler, R. A., Atkins, H. S., Oyston, P. F. & Wren, B. W. High-throughput analysis of Yersinia pseudotuberculosis gene essentiality in optimised in vitro conditions, and implications for the speciation of Yersinia pestis. BMC Microbiol. 18, 46 (2018).
Charbonneau, A. R. L. L. et al. Defining the ABC of gene essentiality in streptococci. BMC Genomics 18, 426 (2017).
Mesarich, C. H. et al. Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae. PLoS One 12, e0172790 (2017).
Goodall, E. C. A. et al. The essential genome of Escherichia coli K-12. MBio 9, e02096-17 (2018). Using TIS, the essential genome of the most well-studied bacterial model strain, E. coli K12, is defined in detail.
Ruiz, L. et al. The essential genomic landscape of the commensal Bifidobacterium breve UCC2003. Sci. Rep. 7, 5648 (2017).
Barquist, L. et al. A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium. Nucleic Acids Res. 41, 4549–4564 (2013).
Poulsen, B. E. et al. Defining the core essential genome of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 116, 10072–10080 (2019).
Geisinger, E. et al. The landscape of phenotypic and transcriptional responses to ciprofloxacin in Acinetobacter baumannii: acquired resistance alleles modulate drug-induced SOS response and prophage replication. MBio 10, 1–19 (2019).
Carey, A. F. et al. TnSeq of mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities. PLoS Pathog. 14, e1006939–e1006939 (2018).
Deutschbauer, A. et al. Towards an informative mutant phenotype for every bacterial gene. J. Bacteriol. 196, 3643–3655 (2014).
Basta, D. W., Bergkessel, M. & Newman, D. K. Identification of fitness determinants during energy-limited growth arrest in Pseudomonas aeruginosa. MBio 8, e01170-17 (2017).
Price, M. N. et al. Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics. PLoS Genet. 14, 1–23 (2018).
Byrne, R. T., Chen, S. H., Wood, E. A., Cabot, E. L. & Cox, M. M. Escherichia coli genes and pathways involved in surviving extreme exposure to ionizing radiation. J. Bacteriol. 196, 3534–3545 (2014).
Kingsley, R. A. et al. Functional analysis of Salmonella Typhi adaptation to survival in water. Environ. Microbiol. 20, 4079–4090 (2018).
Mandal, R. K. & Kwon, Y. M. Global screening of Salmonella enterica serovar Typhimurium genes for desiccation survival. Front. Microbiol. 8, 1723 (2017).
Fabian, B. K., Tetu, S. G. & Paulsen, I. T. Application of transposon insertion sequencing to agricultural science. Front. Plant Sci. 11, 291 (2020).
Royet, K., Parisot, N., Rodrigue, A., Gueguen, E. & Condemine, G. Identification by Tn-seq of Dickeya dadantii genes required for survival in chicory plants. Mol. Plant Pathol. 20, 287–306 (2019).
Duong, D. A., Jensen, R. V. & Stevens, A. M. Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn-Seq analysis. Mol. Plant Pathol. https://doi.org/10.1111/mpp.12669 (2018).
Liu, H. et al. Large-scale chemical-genetics of the human gut bacterium Bacteroides thetaiotaomicron. bioRxiv https://doi.org/10.1101/573055 (2019).
Goodman, A. L., Wu, M. & Gordon, J. I. Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat. Protoc. 6, 1969–1980 (2011).
Zimmermann, M., Zimmermann-Kogadeeva, M., Wegmann, R. & Goodman, A. L. Separating host and microbiome contributions to drug pharmacokinetics and toxicity. Science 363, eaat9931 (2019). An applied study that uses a TIS arrayed approach to dissect complex host–microbiome interactions during the response to drug treatment.
Stacy, A., Fleming, D., Lamont, R. J., Rumbaugh, K. P. & Whiteley, M. A commensal bacterium promotes virulence of an opportunistic pathogen via cross-respiration. MBio 7, e00782-16 (2016).
Ibbersona, C. B. et al. Co-infecting microbes dramatically alter pathogen gene essentiality during polymicrobial infection. Nat. Microbiol. 2, 17079 (2017). One of the first studies to use TIS in a co-infection model, in this case two key pathogens in a wound infection.
Duncan, M. C. et al. Vibrio cholerae motility exerts drag force to impede attack by the bacterial predator Bdellovibrio bacteriovorus. Nat. Commun. 9, 4757 (2018).
Chatterjee, A. et al. Parallel genomics uncover novel enterococcal-bacteriophage interactions. MBio 11, e03120-19 (2020).
Fu, Y., Waldor, M. K. & Mekalanos, J. J. Tn-seq analysis of vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe 14, 652–663 (2013).
Nolan, L. M. et al. Discovery of a pseudomonas aeruginosa type VI secretion system toxin targeting bacterial protein synthesis using a global genomics approach. bioRxiv https://doi.org/10.1101/733030 (2019).
Mann, B. et al. Control of virulence by small RNAs in Streptococcus pneumoniae. PLoS Pathog. 8, 34 (2012). One of the first studies to combine RNA-seq and TIS to investigate small RNAs in S. pneumoniae.
Warrier, I. et al. The Transcriptional landscape of Streptococcus pneumoniae TIGR4 reveals a complex operon architecture and abundant riboregulation critical for growth and virulence. PLoS Pathog. 14, 1–25 (2018).
Capel, E. et al. Comprehensive identification of meningococcal genes and small noncoding RNAs required for host cell colonization. MBio 7, e01173-16 (2016).
Costanzo, M. et al. Global genetic networks and the genotype-to-phenotype relationship. Cell 177, 85–100 (2019).
Fenton, A. K., El Mortaji, L., Lau, D. T. C., Rudner, D. Z. & Bernhardt, T. G. CozE is a member of the MreCD complex that directs cell elongation in Streptococcus pneumoniae. Nat. Microbiol 2, 16237 (2016).
Santa Maria, J. P. J. et al. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids. Proc. Natl Acad. Sci. USA 111, 12510–12515 (2014).
Lorenz, A. et al. Importance of flagella in acute and chronic Pseudomonas aeruginosa infections. Environ. Microbiol. 21, 883–897 (2019).
DeJesus, M. A. et al. Statistical analysis of genetic interactions in Tn-Seq data. Nucleic Acids Res. 45, e93 (2017).
Jensen, P. A., Zhu, Z. & van Opijnen, T. Antibiotics disrupt coordination between transcriptional and phenotypic stress responses in pathogenic bacteria. Cell Rep. 20, 1705–1716 (2017).
Turner, K. H., Everett, J., Trivedi, U., Rumbaugh, K. P. & Whiteley, M. Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection. PLoS Genet. 10, e1004518 (2014).
Deutschbauer, A. et al. Evidence-based annotation of gene function in Shewanella oneidensis MR-1 using genome-wide fitness profiling across 121 conditions. PLoS Genet. 7, e1002385 (2011).
Smith, J. J. et al. Expression and functional profiling reveal distinct gene classes involved in fatty acid metabolism. Mol. Syst. Biol. 2, 2006.0009 (2006).
Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).
Keren, L. et al. Massively parallel interrogation of the effects of gene expression levels on fitness. Cell 166, 1282–1294.e18 (2016).
Price, M. N. et al. Indirect and suboptimal control of gene expression is widespread in bacteria. Mol. Syst. Biol. 9, 660 (2013).
Z. Zhu et al. Entropy of a bacterial stress response is a generalizable predictor for fitness and antibiotic sensitivity. bioRxiv https://doi.org/10.1101/813709 (2019).
Paulsen, I. T., Cain, A. K. & Hassan, K. A. Physical enrichment of transposon mutants from saturation mutant libraries using the TraDISort approach. Mob. Genet. Elements 7, 1–7 (2017).
O’Connor, T. J., Boyd, D., Dorer, M. S. & Isberg, R. R. Aggravating genetic interactions allow a solution to redundancy in a bacterial pathogen. Science 338, 1440–1444 (2012).
Lee, S. Y. et al. Dense transposon integration reveals essential cleavage and polyadenylation factors promote heterochromatin formation. Cell Rep. 30, 2686–2698 (2020).
Michel, A. H. et al. Functional mapping of yeast genomes by saturated transposition. Elife 6, e23570 (2017).
Guo, Y. et al. Integration profiling of gene function with dense maps of transposon integration. Genetics 195, 599–609 (2013).
Zhang, C., Phillips, A. P. R., Wipfler, R. L., Olsen, G. J. & Whitaker, R. J. The essential genome of the crenarchaeal model Sulfolobus islandicus. Nat. Commun. 9, 1–11 (2018).
Sarmiento, F., Mrázek, J. & Whitman, W. B. Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis. Proc. Natl Acad. Sci. USA 110, 4726–4731 (2013).
Carette, J. E. et al. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing. Nat. Biotechnol. 29, 542–546 (2011).
Carette, J. E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009).
Friedrich, M. J. et al. Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice. Nat. Protoc. 12, 289–309 (2017).
Rad, R. et al. A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer. Nat. Genet. 47, 47–56 (2015).
Zhang, M. et al. Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis. Science 360, eaap7847 (2018).
Smith, A. M. et al. Quantitative phenotyping via deep barcode sequencing. Genome Res. 19, 1836–1842 (2009).
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Liu, X. et al. High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Mol. Syst. Biol. 13, 931 (2017).
Peters, J. M. et al. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165, 1493–1506 (2016).
Lee, H. H. et al. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. Nat. Microbiol. 4, 1105–1113 (2019).
Rousset, F. et al. Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors. PLoS Genet. 14, e1007749 (2018).
Wang, T. et al. Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance. Nat. Commun. 9, 2475 (2018).
Steen, A. D. et al. High proportions of bacteria and archaea across most biomes remain uncultured. ISME J. 13, 3126–3130 (2019).
Zhu, Z., Surujon, D., Pavao, A., Bento, J. & van Opijnen, T. Forecasting bacterial survival-success and adaptive evolution through multi-omics stress-response mapping, network analyses and machine learning. bioRxiv https://doi.org/10.1101/387910 (2018).
Acknowledgements
The authors acknowledge funding from various sources that supported the composition of this article: Australian Research Council Discovery Early Career Research Award fellowship DE180100929 and Australian National Health and Medical Research Council grant APP1159752 to A.K.C.; US National Institutes of Health grants U01AI124302, R01AI110724 and R21AI117247 to T.v.O.; a bayresq.net Bavarian research network grant to L.B.; UK Medical Research Council grant G1100100/1 to J.P.; Australian National Health and Medical Research Council grants GNT1060895 and GNT1120298 to I.T.P.; and US National Institutes of Health grant R35GM118159 to A.L.G.
Author information
Authors and Affiliations
Contributions
A.K.C., L.B. and T.v.O. substantially contributed to the discussion of the content. All authors researched content for the article, contributed to writing and reviewed/edited the manuscript before submission.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information
Nature Reviews Genetics thanks B. Akerley, A. Deutschbauer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
SalmonellaTyphimurium strain D23580: https://hactar.shef.ac.uk/D23580_2/
TraDIS-vault: https://tradis-vault.qfab.org/apollo/jbrowse/
Glossary
- Transposon
-
A mobile genetic element that inserts itself into a genome and disrupts genes or genetic features at that site.
- Next-generation sequencing
-
DNA sequencing using a massively parallel platform that separates DNA templates on a flow cell and clonally amplifies clusters for sequencing.
- Genomic features
-
Every component of the genome that can be annotated as being a feature, whether it be a gene, coding RNA, non-coding RNA or promoter region.
- Fragmentation
-
Breaking up DNA into smaller pieces in order to be sequenced. This can be done by physical shearing methods, such as sonication, or enzymatic digestion.
- Fluorescence-activated cell sorting
-
(FACS). A specialized type of flow cytometry that separates cells, one cell at time, by their fluorescent characteristics on the basis of light scattering.
- Sliding window
-
A window of arbitrary length is set and events within that window are assayed. This window is then moved around the genome. This approach provides a more objective method of assessing the genome that is independent of annotation.
- Bottlenecks
-
When a population size is drastically reduced through stochastic processes and the surviving cells will make up the new population but will have reduced genetic diversity.
- Overdispersion
-
When data exhibit greater variability than would be expected under a statistical model. In the context of sequencing data, overdispersion is often used in reference to the negative binomial distribution, which can be understood as a generalization of the Poisson distribution that allows a larger variance relative to the mean.
- Reverse genetics
-
Determining the phenotypic effects of a genetic feature by altering the genetic feature and observing changes in the organism compared with a wild type. ‘Reverse’ refers to the genotype-to-phenotype mode of investigation, being opposite the classical phenotype-to-genotype genetic investigations (‘forward genetics’).
- Organoid
-
A three-dimensional, simplified replica of an organ derived from stem cells to realistically model the organ in vitro.
- Pan-genome
-
The complete set of genes in all strains within a species, in contrast to the core genome, which is the set of genes shared by all strains within a species.
- Essential genome
-
The complete set of genes and genetic features in a genome that are essential for a cell to survive and grow, the examplar of which are ‘housekeeping’ genes for core processes such as replication and division.
- Gnotobiotic
-
An environment for culturing microorganisms, such as an animal model, where all microorganisms are either defined or removed.
- Synthetic lethal
-
Where individual mutants have no or little fitness effect, but when two or more of these mutations are combined, this leads to arrest in cell growth or to cell death.
- Antagonistic antibiotic combinations
-
When the activity of an antibiotic combination is lower than would be predicted from the effects of the individual antibiotics.
Rights and permissions
About this article
Cite this article
Cain, A.K., Barquist, L., Goodman, A.L. et al. A decade of advances in transposon-insertion sequencing. Nat Rev Genet 21, 526–540 (2020). https://doi.org/10.1038/s41576-020-0244-x
Accepted:
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s41576-020-0244-x
