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Analysis of Gene Essentiality from TnSeq Data Using Transit

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Essential Genes and Genomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2377))

Abstract

TnSeq, or sequencing of transposon insertion libraries, has proven to be a valuable method for probing the functions of genes in a wide range of bacteria. TnSeq has found many applications for studying genes involved in core functions (such as cell division or metabolism), stress response, virulence, etc., as well as to identify potential drug targets. Two of the most commonly used transposons in practice are Himar1, which inserts randomly at TA dinucleotides, and Tn5, which can insert more broadly throughout the genome. These insertions cause putative gene function disruption, and clones with insertions in genes that cannot tolerate disruption (in a given condition) are eliminated from the population. Deep sequencing can be used to efficiently profile the surviving members, with insertions in genes that can be inferred to be non-essential. Data from TnSeq experiments (i.e. transposon insertion counts at specific genomic locations) is inherently noisy, making rigorous statistical analysis (e.g. quantifying significance) challenging. In this chapter, we describe Transit, a Python-based software package for analyzing TnSeq data that combines a variety of data processing tools, quality assessment methods, and analytical algorithms for identifying essential (or conditionally essential) genes.

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References

  1. van Opijnen T, Camilli A (2013) Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11(7):435–442

    Article  PubMed  CAS  Google Scholar 

  2. Barquist L, Boinett CJ, Cain AK (2013) Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol 10(7):1161–1169

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Chao M, Abel S, Davis B, Waldor M (2016) The design and analysis of transposon insertion sequencing experiments. Nat Rev Microbiol 14(2):119–128

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Langridge GC, Phan M, Turner D, Perkins T, Parts L, Haase J, Charles I, Maskell D, Peters S, Dougan G, et al. (2009) Simultaneous assay of every salmonella typhi gene using one million transposon mutants. Genome Res 19(12):2308–2316. http://www.ncbi.nlm.nih.gov/pubmed/19826075

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Gawronski JD, Wong SM, Giannoukos G, Ward DV, Akerley BJ (2009) 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(38):16422–16427

    Article  PubMed  PubMed Central  Google Scholar 

  6. Goodman AL, McNulty NP, Zhao Y, Leip D, Mitra RD, Lozupone CA, Knight R, Gordon JI (2009) Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6(3):279–289

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Wetmore K, Price M, Waters R, Lamson J, He J, Hoover C, Blow M, Bristow J, Butland G, Arkin A, Deutschbauer A (2015) Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 6(3):e00306-15

    Google Scholar 

  8. Jensen P, Zhu Z, van Opijnen T (2017) Antibiotics disrupt coordination between transcriptional and phenotypic stress responses in pathogenic bacteria. Cell Rep 20(7):1705–1716

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Zhang YJ, Reddy MC, Ioerger TR, Rothchild AC, Dartois V, Schuster BM, Trauner A, Wallis D, Galaviz S, Huttenhower C, Sacchettini JC, Behar SM, J RE (2013) Tryptophan biosynthesis protects mycobacteria from CD4 T-cell-mediated killing. Cell 155(6):1296–1308

    Google Scholar 

  10. Luo H, Lin Y, Gao F, Zhang CT, Zhang R (2014) DEG 10, an update of the Database of Essential Genes that includes both protein-coding genes and non-coding genomic elements. Nucleic Acids Research 42:D574–D580

    Article  PubMed  CAS  Google Scholar 

  11. Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, Mekalanos JJ (1999) In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. PNAS 96(4):1645–1650

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Sassetti CM, Boyd DH, Rubin EJ (2001) Comprehensive identification of conditionally essential genes in mycobacteria. PNAS 98(22):12712–12717. https://doi.org/10.1073/pnas.231275498. http://www.pnas.org/content/98/22/12712.abstract. http://www.pnas.org/content/98/22/12712.full.pdf+html

  13. Reznikoff WS (2003) Tn5 as a model for understanding DNA transposition. Mol Microbiol 43(5):1199–1206

    Article  Google Scholar 

  14. Lampe DJ, Churchill ME, Robertson HM (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. Eur Mol Biol Organ J 15(19):5470–5479

    Article  CAS  Google Scholar 

  15. Long J, DeJesus M, Ward D, Baker R, Ioerger T, Sassetti C (2015) Identifying essential genes in Mycobacterium tuberculosis by global phenotypic profiling. In: Lu LJ (ed) Methods in molecular biology: gene essentiality, vol 1279. Springer, Berlin, p 79–95

    Google Scholar 

  16. DeJesus MA, Ioerger TR (2016) Normalization of transposon-mutant library sequencing datasets to improve identification of conditionally essential genes. J Bioinform Comput Biol 14(3):1642004

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Zomer A, Burghout P, Bootsma HJ, Hermans PW, van Hijum SA (2012) ESSENTIALS: software for rapid analysis of high throughput transposon insertion sequencing data. PLoS ONE 7(8):e43012

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Solaimanpour S, Sarmiento F, Mrazek J (2015) Tn-seq explorer: a tool for analysis of high-throughput sequencing data of transposon mutant libraries. PLoS ONE 10(5):e0126070

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Barquist L, Mayho M, Cummins C, Cain AK, Boinett CJ, Page AJ, Langridge GC, Quail MA, Keane JA, Parkhill J (2016) The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 32(7):1109

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Zhao L, Anderson MT, Wu W, T Mobley HL, Bachman MA (2017) TnseqDiff: identification of conditionally essential genes in transposon sequencing studies. BMC Bioinformatics 18(1):326

    Google Scholar 

  21. van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6(10):767–772

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Pritchard JR, Chao MC, Abel S, Davis BM, Baranowski C, Zhang YJ, Rubin EJ, Waldor MK (2014) ARTIST: high-resolution genome-wide assessment of fitness using transposon-insertion sequencing. PLoS Genet 10(11):e1004782

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. DeJesus MA, Ambadipudi C, Baker R, Sassetti C, Ioerger TR (2015) TRANSIT–a software tool for Himar1 TnSeq analysis. PLoS Comput Biol 11(10):e1004401

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Griffin JE, Gawronski JD, DeJesus MA, Ioerger TR, Akerley BJ, Sassetti CM (2011) High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog 7(9):e1002251. https://doi.org/10.1371/journal.ppat.1002251

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25(14):1754–1760

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. DeJesus MA, Zhang YJ, Sassetti CM, Rubin EJ, Sacchettini JC, Ioerger TR (2013) Bayesian analysis of gene essentiality based on sequencing of transposon insertion libraries. Bioinformatics 29(6):695–703

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. DeJesus MA, Ioerger TR (2013) A Hidden Markov Model for identifying essential and growth-defect regions in bacterial genomes from transposon insertion sequencing data. BMC Bioinformatics 14:303

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Cole ST, Brosch R, Parkhill J (1998) Deciphering the biology of mycobacterium tuberculosis from the complete genome sequence. Nature 393(6685):537–544. http://dx.doi.org/10.1038/31159

    Article  PubMed  CAS  Google Scholar 

  29. Galperin MY, Makarova KS, Wolf YI, Koonin EV (2015) Expanded microbial genome coverage and improved protein family annotation in the COG database. Nucl Acids Res 43:D261–D269

    Article  PubMed  CAS  Google Scholar 

  30. Irizarry RA, Wang C, Zhou Y, Speed TP (2009) Gene set enrichment analysis made simple. Stat Methods Med Res 18(6):565–575

    Article  PubMed  PubMed Central  Google Scholar 

  31. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. PNAS 102(43):15545–15550

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Grossmann S, Bauer S, Robinson PN, Vingron M (2007) Improved detection of overrepresentation of Gene-Ontology annotations with parent-child analysis. Bioinformatics 23(22):3024–3031

    Article  PubMed  CAS  Google Scholar 

  33. Zhang L, Hendrickson RC, Meikle V, Lefkowitz EJ, Ioerger TR, Niederweis M (2020) Comprehensize analysis of iron utilization by Mycobacterium tuberculosis. PLoS Pathog 16(3):e1008337

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. DeJesus MA, Nambi S, Smith CM, Baker RE, Sassetti CM, Ioerger TR (2017) Statistical analysis of genetic interactions in Tn-Seq data. Nucl Acids Res 45(11):e93

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Tukey J (1949) Comparing individual means in the analysis of variance. Biometrics 5(2):99–114

    Article  PubMed  CAS  Google Scholar 

  36. Subramaniyam S, DeJesus MA, Zaveri A, Smith CM, Baker RE, Ehrt S, Schnappinger D, Sassetti CM, Ioerger TR (2019) Statistical analysis of variability in tnseq data across conditions using zero-inflated negative binomial regression. BMC Bioinf 20(1):603

    Article  Google Scholar 

  37. Xu W, DeJesus MA, Rücker N, Engelhart CA, Wright MG, Healy C, Lin K, Wang R, Park SW, Ioerger TR, Schnappinger D, Ehrt S (2017) Chemical genetic interaction profiling reveals determinants of intrinsic antibiotic resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 61(22):e01334–17

    PubMed  PubMed Central  Google Scholar 

  38. Matern WM, Rifat D, Bader JS, Karakousis PC (2018) Gene enrichment analysis reveals major regulators of Mycobacterium tuberculosis gene expression in two models of antibiotic tolerance. Front Microbiol 9:610

    Article  PubMed  PubMed Central  Google Scholar 

  39. Kieser KJ, Boutte CC, Kester JC, Baer CE, Barczak AK, Meniche X, Chao MC, Rego EH, Sassetti CM, Fortune SM, Rubin EJ (2015) Phosphorylation of the peptidoglycan synthase PonA1 governs the rate of polar elongation in mycobacteria. PLoS Pathog 11(6):e1005010

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Nambi S, Long JE, Mishra BB, Baker R, Murphy KC, Olive AJ, Nguyen HP, Shaffer SA, Sassetti CM (2015) The oxidative stress network of mycobacterium tuberculosis reveals coordination between radical detoxification systems. Cell Host Microbe 17(6):829–837

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Baranowski C, Welsh MA, Sham LT, Eskandarian HA, Lim HC, Kieser KJ, Wagner JC, McKinney JD, Fantner GE, Ioerger TR, Walker S, Bernhardt TG, Rubin EJ, Rego EH (2018) Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape. Elife 7:e37516

    Article  PubMed  PubMed Central  Google Scholar 

  42. Fu Y, Waldor M, Mekalanos J (2013) Tn-seq analysis of vibrio cholerae intestinal colonization reveals a role for t6ss-mediated antibacterial activity in the host. Cell Host Microbe 14(6):652–663

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Dragset MS, Ioerger TR, Loevenich M, Haug M, Sivakumar N, Marstad A, Cardona PJ, Klinkenberg G, Rubin EJ, Steigedal M, Flo TH (2019) Global assessment of Mycobacterium avium subsp. hominissuis genetic requirement for growth and virulence. mSystems 4:e00402-19

    Google Scholar 

  44. DeJesus MA, Gerrick ER, Xu W, Park SW, Long JE, Boutte CC, Rubin EJ, Schnappinger D, Ehrt S, Fortune SM, Sassetti CM, Ioerger TR (2017) Comprehensive essentiality analysis of the mycobacterium tuberculosis genome via saturating transposon mutagenesis. MBio 8(1):e02133-16

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Lampe DJ, Grant TE, M RH (1998) Factors affecting transposition of the Himar1 mariner transposon in vitro. Genetics 149(1):179–187

    Google Scholar 

  46. Ason B, Reznikoff WS (2004) DNA sequence bias during Tn5 transposition. J Mol Biol 335:1213–1225

    Article  PubMed  CAS  Google Scholar 

  47. Chao MC, Pritchard JR, Zhang YJ, Rubin EJ, Livny J, Davis BM, Waldor MK (2013) 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(19):9033–9048

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Pickands J (1975) Statistical inference using extreme order statistics. Ann Stat 3:119–131

    Google Scholar 

  49. Warr AR, Hubbard TP, Munera D, Blondel CJ, zur Wiesch PA, Abel S, Wang X, Davis BM, Waldor MK (2019) Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization. PLoS Pathog 15(8):e1007652

    Google Scholar 

  50. Benjamini Y, Yekutieli D (2005) False discovery rate controlling confidence intervals for selected parameters. J Am Stat Assoc 100(469):71–81

    Article  CAS  Google Scholar 

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Acknowledgements

This work has been supported by NIH grants U19 AI107774 and P01 AI143575.

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Authors and Affiliations

  1. Department of Computer Science, Texas A&M University, College Station, TX, USA

    Thomas R. Ioerger

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  1. Thomas R. Ioerger
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Correspondence to Thomas R. Ioerger .

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  1. Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA

    Ren Zhang

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Ioerger, T.R. (2022). Analysis of Gene Essentiality from TnSeq Data Using Transit. In: Zhang, R. (eds) Essential Genes and Genomes. Methods in Molecular Biology, vol 2377. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1720-5_22

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  • DOI: https://doi.org/10.1007/978-1-0716-1720-5_22

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1719-9

  • Online ISBN: 978-1-0716-1720-5

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