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Application of Whole-Genome Sequencing to Transposon Mutants of Salmonella Heidelberg

  • Bryna Rackerby
  • Sang In Lee
  • Ian Moppert
  • Steven C. Ricke
  • KwangCheol C. Jeong
  • Si Hong ParkEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2016)

Abstract

Transposons are elements widely dispersed among organisms which are able to move and replicate fragments of genomes. The extensive variability in transposons present in most organisms requires extensive identification and interpretation of the resulting transposon mutants after transposon mutagenesis. However, much of this is reliant on utilizing randomness characteristics of transposon to identify essential genes for the organism of interest. Integration of the transposon mutant approach with commercialized next-generation sequencing (NGS) technology has helped to advance transposon identification by sequencing millions of reads generated from a single run on an NGS platform. Transposon sequencing is defined as a gene sequencing methodology that allows for the identification of nonessential genes and the determination of gene function using a random transposon insertional mutagenesis followed by massively parallel sequencing. The detailed protocol will be outlined in this chapter. The genomic DNA integrated with the transposons is sequenced using an NGS platform in order to determine the location of each mutation.

Key words

Salmonella Heidelberg Transposon mutant Next-generation sequencing (NGS) Whole-genome sequencing (WGS) 

References

  1. 1.
    Pray LA (2008) Transposons: the jumping genes. Nature Education 1(1):204Google Scholar
  2. 2.
    Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O (2007) A unified classification system for eukaryotic transposable elements. Nat Rev Genet 8(12):973CrossRefGoogle Scholar
  3. 3.
    Finnegan DJ (1989) Eukaryotic transposable elements and genome evolution. Trends Genet 5:103–107CrossRefGoogle Scholar
  4. 4.
  5. 5.
    Nguyen DH (2018) Four ways to insert foreign DNA into cells. Hearst Seattle Media, LLC, Seattle, WA. https://education.seattlepi.com/four-ways-insert-foreign-dna-cells-4064.html. Accessed 6 Sep 2018
  6. 6.
    Rivera AL, Gómez-Lim M, Fernández F, Loske A (2014) Genetic transformation of cells using physical methods. J Genet Syndr Gene Ther 5(4):237–242Google Scholar
  7. 7.
    Freed NE (2017) Creation of a dense transposon insertion library using bacterial conjugation in enterobacterial strains such as Escherichia coli or Shigella flexneri. J Vis Exp (127)Google Scholar
  8. 8.
    Voelkerding KV, Dames SA, Durtschi JD (2009) Next-generation sequencing: from basic research to diagnostics. Clin Chem 55(4):641–658CrossRefGoogle Scholar
  9. 9.
    Van Opijnen T, Camilli A (2013) Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11(7):435CrossRefGoogle Scholar
  10. 10.
    Barquist L, Boinett CJ, Cain AK (2013) Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol 10(7):1161–1169CrossRefGoogle Scholar
  11. 11.
    Kwon YM, Ricke SC, Mandal RK (2016) Transposon sequencing: methods and expanding applications. Appl Microbiol Biotechnol 100(1):31–43CrossRefGoogle Scholar
  12. 12.
    Khatiwara A, Jiang T, Sung S-S, Dawoud T, Kim JN, Bhattacharya D, Kim H-B, Ricke SC, Kwon YM (2012) Genome scanning for conditionally essential genes in Salmonella. Appl Environ Microbiol 78:3098–3107.13CrossRefGoogle Scholar
  13. 13.
    Rothrock MJ, Fan P, Jeong KC, Kim SA, Ricke SC, Park SH (2018) Complete genome sequence of Listeria monocytogenes strain MR310, isolated from a pastured-flock poultry farm system. Genome Announc 6(10):e00171–e00118CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Bryna Rackerby
    • 1
  • Sang In Lee
    • 2
  • Ian Moppert
    • 2
  • Steven C. Ricke
    • 3
    • 4
  • KwangCheol C. Jeong
    • 5
  • Si Hong Park
    • 2
    Email author
  1. 1.Department of MicrobiologyOregon State UniversityCorvallisUSA
  2. 2.Department of Food Science and TechnologyOregon State UniversityCorvallisUSA
  3. 3.Department of Food Science, Center for Food SafetyUniversity of ArkansasFayettevilleUSA
  4. 4.Cell and Molecular Biology ProgramUniversity of ArkansasFayettevilleUSA
  5. 5.Department of Animal Science, Institute of Food and Agricultural SciencesUniversity of FloridaGainesvilleUSA

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