Preparation of Transposon Library and Tn-Seq Amplicon Library for Salmonella Typhimurium

  • Sardar Karash
  • Tieshan Jiang
  • Deepti Samarth
  • Reena Chandrashekar
  • Young Min KwonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2016)


Transposon sequencing (Tn-seq) is a powerful tool for functional genomics of bacteria. Tn-seq combines transposon mutagenesis with next generation sequencing to assess genetic requirements at a genome-wide scale and identify essential and conditionally essential genes. An efficient application of this experimental approach relies on robust protocols for transposon mutagenesis system and Tn-seq amplicon library preparation method. However, the existing approaches for the Tn-seq amplicon library preparation have several shortcomings. Hence, we present a robust, fast, specific, and cost-effective approach for the transposon mutagenesis of Salmonella Typhimurium and Tn-seq amplicon library preparation for Illumina sequencing. Besides S. Typhimurium that was used here for illustration, this protocol can also be used for other bacteria. In particular, the procedure for Tn-seq amplicon library preparation can be broadly applicable to any transposon elements. We delineate comprehensive step-by-step protocols for transposon mutagenesis and Tn-seq amplicon library such that it can be reproduced effortlessly by other researchers.

Key words

Transposon mutagenesis Tn-seq amplicon library Illumina sequencing Functional genomics Salmonella Typhimurium 


  1. 1.
    van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767–772. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kwon YM, Ricke SC, Mandal RK (2016) Transposon sequencing: methods and expanding applications. Appl Microbiol Biotechnol 100(1):31–43. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Karash S, Liyanage R, Qassab A, Lay JO, Kwon YM (2017) A comprehensive assessment of the genetic determinants in Salmonella Typhimurium for resistance to hydrogen peroxide using proteogenomics. Sci Rep 7:17073. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Martínez-García E, Calles B, Arévalo-Rodríguez M, de Lorenzo V (2011) pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol 11(1):38. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chun JY, Kim KJ, Hwang IT, Kim YJ, Lee DH, Lee IK, Kim JK (2007) Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Res 35:e40. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Mandal RK, Kwon YM (2017) Global screening of Salmonella enterica serovar Typhimurium genes for desiccation survival. Front Microbiol 8:1723. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Karash S, Kwon YM (2018) Iron-dependent essential genes in Salmonella Typhimurium. bioRxiv:159921.
  8. 8.
    Mandal RK, Jiang T, Kwon YM (2017) Essential genome of Campylobacter jejuni. BMC Genomics 18:616. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Abel S, Abel zur Wiesch P, Davis BM, Waldor MK (2015) Analysis of bottlenecks in experimental models of infection. PLoS Pathog 11:e1004823. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Sardar Karash
    • 1
  • Tieshan Jiang
    • 2
  • Deepti Samarth
    • 2
  • Reena Chandrashekar
    • 2
  • Young Min Kwon
    • 1
    • 2
    Email author
  1. 1.Cell and Molecular Biology ProgramUniversity of ArkansasFayettevilleUSA
  2. 2.Department of Poultry ScienceUniversity of ArkansasFayettevilleUSA

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