The design and analysis of transposon insertion sequencing experiments

ABSTRACT Transposon insertion sequencing (TIS) is a powerful approach that can be extensively applied to the genome-wide definition of loci that are required for bacterial growth under

diverse conditions. However, experimental design choices and stochastic biological processes can heavily influence the results of TIS experiments and affect downstream statistical analysis.

In this Opinion article, we discuss TIS experimental parameters and how these factors relate to the benefits and limitations of the various statistical frameworks that can be applied to the

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Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A DECADE OF ADVANCES IN TRANSPOSON-INSERTION SEQUENCING Article 12 June 2020 TECHNICAL CONSIDERATIONS FOR

COST-EFFECTIVE TRANSPOSON DIRECTED INSERTION-SITE SEQUENCING (TRADIS) Article Open access 21 March 2024 MEASURING AND INTERPRETING TRANSPOSABLE ELEMENT EXPRESSION Article 23 June 2020

REFERENCES * Barquist, L., Boinett, C. J. & Cain, A. K. Approaches to querying bacterial genomes with transposon-insertion sequencing. _RNA Biol._ 10, 1161–1169 (2013). Article  CAS 

PubMed  PubMed Central  Google Scholar  * van Opijnen, T. & Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. _Nat. Rev. Microbiol._

11, 435–442 (2013). Article  CAS  PubMed  Google Scholar  * 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). Article  CAS  PubMed  PubMed Central  Google Scholar  * 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). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gawronski, J. D., Wong, S. M., Giannoukos, G.,

Ward, D. V. & Akerley, B. J. 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). Article  CAS  PubMed  PubMed Central  Google Scholar  * Langridge, G. C. et al. Simultaneous assay of every _Salmonella_ Typhi gene using one million transposon

mutants. _Genome Res._ 19, 2308–2316 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Chiang, S. L. & Rubin, E. J. Construction of a mariner-based transposon for

epitope-tagging and genomic targeting. _Gene_ 296, 179–185 (2002). Article  CAS  PubMed  Google Scholar  * Rubin, E. J. et al. _In vivo_ transposition of mariner-based elements in enteric

bacteria and mycobacteria. _Proc. Natl Acad. Sci. USA_ 96, 1645–1650 (1999). Article  CAS  PubMed  PubMed Central  Google Scholar  * Goryshin, I. Y., Miller, J. A., Kil, Y. V., Lanzov, V. A.

& Reznikoff, W. S. Tn5/IS50 target recognition. _Proc. Natl Acad. Sci. USA_ 95, 10716–10721 (1998). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, Y. J. et al. Global

assessment of genomic regions required for growth in _Mycobacterium tuberculosis_. _PLoS Pathog._ 8, e1002946 (2012). Article  PubMed  PubMed Central  Google Scholar  * Chao, M. C. et al.

High-resolution definition of the _Vibrio cholerae_ essential gene set with hidden Markov model-based analyses of transposon-insertion sequencing data. _Nucleic Acids Res._ 41, 9033–9048

(2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lodge, J. K., Weston-Hafer, K. & Berg, D. E. Transposon Tn5 target specificity: preference for insertion at G/C pairs.

_Genetics_ 120, 645–650 (1988). CAS  PubMed  PubMed Central  Google Scholar  * Green, B., Bouchier, C., Fairhead, C., Craig, N. L. & Cormack, B. P. Insertion site preference of Mu, Tn5,

and Tn7 transposons. _Mob. DNA_ 3, 3 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Christen, B. et al. The essential genome of a bacterium. _Mol. Syst. Biol._ 7, 528

(2011). Article  PubMed  PubMed Central  Google Scholar  * 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). Article  CAS  PubMed  PubMed Central  Google Scholar  * DeJesus, M. A. et al. Bayesian analysis of gene essentiality based on sequencing of

transposon insertion libraries. _Bioinformatics_ 29, 695–703 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lamichhane, G. et al. A postgenomic method for predicting

essential genes at subsaturation levels of mutagenesis: application to _Mycobacterium tuberculosis_. _Proc. Natl Acad. Sci. USA_ 100, 7213–7218 (2003). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Griffin, J. E. et al. High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. _PLoS Pathog._ 7, e1002251 (2011).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Zemansky, J. et al. Development of a mariner-based transposon and identification of _Listeria monocytogenes_ determinants, including

the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype. _J. Bacteriol._ 191, 3950–3964 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Johnson, C. M.

& Grossman, A. D. Identification of host genes that affect acquisition of an integrative and conjugative element in _Bacillus subtilis_. _Mol. Microbiol._ 93, 1284–1301 (2014). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Shevchenko, Y. et al. Systematic sequencing of cDNA clones using the transposon Tn5. _Nucleic Acids Res._ 30, 2469–2477 (2002). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Troy, E. B. et al. Understanding barriers to _Borrelia burgdorferi_ dissemination during infection using massively parallel sequencing. _Infect.

Immun._ 81, 2347–2357 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * 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). Article  CAS  PubMed  PubMed Central  Google Scholar  * Pritchard,

J. R. et al. ARTIST: high-resolution genome-wide assessment of fitness using transposon-insertion sequencing. _PLoS Genet._ 10, e1004782 (2014). Article  PubMed  PubMed Central  Google

Scholar  * van Opijnen, T. & Camilli, A. A fine scale phenotype-genotype virulence map of a bacterial pathogen. _Genome Res._ 22, 2541–2551 (2012). Article  CAS  PubMed  PubMed Central 

Google Scholar  * 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). Article  CAS  PubMed  PubMed Central  Google Scholar  * Abel, S. et al. Sequence tag-based analysis of microbial population dynamics. _Nat. Methods_

12, 223–226 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kaper, J. B., Morris, J. G. Jr & Levine, M. M. Cholera. _Clin. Microbiol. Rev._ 8, 48–86 (1995). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Ritchie, J. M., Rui, H., Bronson, R. T. & Waldor, M. K. Back to the future: studying cholera pathogenesis using infant rabbits. _mBio_ 1,

e00047-10 (2010). Article  PubMed  PubMed Central  Google Scholar  * Kamp, H. D., Patimalla-Dipali, B., Lazinski, D. W., Wallace-Gadsden, F. & Camilli, A. Gene fitness landscapes of

_Vibrio cholerae_ at important stages of its life cycle. _PLoS Pathog._ 9, e1003800 (2013). Article  PubMed  PubMed Central  Google Scholar  * Zomer, A., Burghout, P., Bootsma, H. J.,

Hermans, P. W. & van Hijum, S. A. ESSENTIALS: software for rapid analysis of high throughput transposon insertion sequencing data. _PLoS ONE_ 7, e43012 (2012). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Lazinski, D. W. & Camilli, A. Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library

construction. _Biotechniques_ 54, 25–34 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gallagher, L. A., Shendure, J. & Manoil, C. Genome-scale identification of

resistance functions in _Pseudomonas aeruginosa_ using Tn-seq. _mBio_ 2, e00315-10 (2011). Article  PubMed  PubMed Central  Google Scholar  * Yamaichi, Y. et al. High-resolution genetic

analysis of the requirements for horizontal transmission of the ESBL plasmid from _Escherichia coli_ O104:H4. _Nucleic Acids Res._ 43, 348–360 (2014). Article  PubMed  PubMed Central  Google

Scholar  * Fu, G. K. et al. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. _Proc. Natl Acad. Sci. USA_ 111,

1891–1896 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Shiroguchi, K., Jia, T. Z., Sims, P. A. & Xie, X. S. Digital RNA sequencing minimizes sequence-dependent bias

and amplification noise with optimized single-molecule barcodes. _Proc. Natl Acad. Sci. USA_ 109, 1347–1352 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, H. et al. The

sequence alignment/map format and SAMtools. _Bioinformatics_ 25, 2078–2079 (2009). Article  PubMed  PubMed Central  Google Scholar  * Baugh, L. et al. Combining functional and structural

genomics to sample the essential _Burkholderia_ structome. _PLoS ONE_ 8, e53851 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Valentino, M. D. et al. Genes contributing to

_Staphylococcus aureus_ fitness in abscess- and infection-related ecologies. _mBio_ 5, e01729-14 (2014). Article  PubMed  PubMed Central  Google Scholar  * Klein, B. A. et al. Identification

of essential genes of the periodontal pathogen _Porphyromonas gingivalis_. _BMC Genomics_ 13, 578 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Deng, J., Su, S., Lin, X.,

Hassett, D. J. & Lu, L. J. A statistical framework for improving genomic annotations of prokaryotic essential genes. _PLoS ONE_ 8, e58178 (2013). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Remmele, C. W. et al. Transcriptional landscape and essential genes of _Neisseria gonorrhoeae_. _Nucleic Acids Res._ 42, 10579–10595 (2014). Article  CAS  PubMed  PubMed

Central  Google Scholar  * DeJesus, M. A. & Ioerger, T. R. A. Hidden Markov Model for identifying essential and growth-defect regions in bacterial genomes from transposon insertion

sequencing data. _BMC Bioinformatics_ 14, 303 (2013). Article  PubMed  PubMed Central  Google Scholar  * Brutinel, E. D. & Gralnick, J. A. Anomalies of the anaerobic tricarboxylic acid

cycle in _Shewanella oneidensis_ revealed by Tn-seq. _Mol. Microbiol._ 86, 273–283 (2012). Article  CAS  PubMed  Google Scholar  * Zhang, Y. J. et al. Tryptophan biosynthesis protects

mycobacteria from CD4 T-cell-mediated killing. _Cell_ 155, 1296–1308 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mann, B. et al. Control of virulence by small RNAs in

_Streptococcus pneumoniae_. _PLoS Pathog._ 8, e1002788 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * McDonough, E., Lazinski, D. W. & Camilli, A. Identification of _in

vivo_ regulators of the _Vibrio cholerae_ xds gene using a high-throughput genetic selection. _Mol. Microbiol._ 92, 302–315 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Moll, A. et al. Cell separation in _Vibrio cholerae_ is mediated by a single amidase whose action is modulated by two nonredundant activators. _J. Bacteriol._ 196, 3937–3948 (2014). Article

  PubMed  PubMed Central  Google Scholar  * Dorr, T. et al. A novel peptidoglycan binding protein crucial for PBP1A-mediated cell wall biogenesis in _Vibrio cholerae_. _PLoS Genet._ 10,

e1004433 (2014). Article  PubMed  PubMed Central  Google Scholar  * Skurnik, D. et al. A comprehensive analysis of _in vitro_ and _in vivo_ genetic fitness of _Pseudomonas aeruginosa_ using

high-throughput sequencing of transposon libraries. _PLoS Pathog._ 9, e1003582 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * 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). Article  PubMed  PubMed

Central  Google Scholar  * Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. _Nat. Rev. Genet._ 10, 57–63 (2009). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. _Nat. Biotechnol._ 31, 46–53 (2013). Article  CAS  PubMed 

Google Scholar  * Anders, S. & Huber, W. Differential expression analysis for sequence count data. _Genome Biol._ 11, R106 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Khatiwara, A. et al. Genome scanning for conditionally essential genes in _Salmonella enterica_ serotype Typhimurium. _Appl. Environ. Microbiol._ 78, 3098–3107 (2012). Article  CAS  PubMed

  PubMed Central  Google Scholar  * Santa Maria, J. P. Jr et al. Compound-gene interaction mapping reveals distinct roles for _Staphylococcus aureus_ teichoic acids. _Proc. Natl Acad. Sci.

USA_ 111, 12510–12515 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hsiao, A. et al. Members of the human gut microbiota involved in recovery from _Vibrio cholerae_

infection. _Nature_ 515, 423–426 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Moxon, E. R. & Murphy, P. A. _Haemophilus influenzae_ bacteremia and meningitis resulting

from survival of a single organism. _Proc. Natl Acad. Sci. USA_ 75, 1534–1536 (1978). Article  CAS  PubMed  PubMed Central  Google Scholar  * Barnes, P. D., Bergman, M. A., Mecsas, J. &

Isberg, R. R. _Yersinia pseudotuberculosis_ disseminates directly from a replicating bacterial pool in the intestine. _J. Exp. Med._ 203, 1591–1601 (2006). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Kaiser, P., Slack, E., Grant, A. J., Hardt, W. D. & Regoes, R. R. Lymph node colonization dynamics after oral _Salmonella typhimurium_ infection in mice. _PLoS

Pathog._ 9, e1003532 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Grant, A. J. et al. Modelling within-host spatiotemporal dynamics of invasive bacterial disease. _PLoS

Biol._ 6, e74 (2008). Article  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the Howard Hughes Medical Institute, the US National

Institutes of Health (AI R37-042347 to M.K.W.; 5F32 GM108355-02 to M.C.C.), and the Swiss Foundation for Grants in Biology and Medicine (PASMP3_142724/1 to S.A.). AUTHOR INFORMATION AUTHORS

AND AFFILIATIONS * Department of Microbiology and Immunobiology, USA; the Division of Infectious Disease, Harvard Medical School, Boston, Massachusetts 02115, Brigham and Women's

Hospital, Boston, Massachusetts 02115, USA; and the Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA., Michael C. Chao, Brigid M. Davis & Matthew K. Waldor * the

Division of Infectious Disease, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA, Michael C. Chao, Brigid M. Davis & Matthew K. Waldor * Department of Pharmacy,

University of Tromsø, The Arctic University of Norway, 9019, Tromsø, Norway Sören Abel Authors * Michael C. Chao View author publications You can also search for this author inPubMed Google

Scholar * Sören Abel View author publications You can also search for this author inPubMed Google Scholar * Brigid M. Davis View author publications You can also search for this author

inPubMed Google Scholar * Matthew K. Waldor View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Matthew K. Waldor.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. RELATED LINKS DATABASES Sequencing Read Archive (SRA) FURTHER INFORMATION Picard POWERPOINT

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design and analysis of transposon insertion sequencing experiments. _Nat Rev Microbiol_ 14, 119–128 (2016). https://doi.org/10.1038/nrmicro.2015.7 Download citation * Published: 19 January

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