Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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. https://doi.org/10.1038/nmeth.1377
Kwon YM, Ricke SC, Mandal RK (2016) Transposon sequencing: methods and expanding applications. Appl Microbiol Biotechnol 100(1):31–43. https://doi.org/10.1007/s00253-015-7037-8
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. https://doi.org/10.1038/s41598-017-17149-9
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. https://doi.org/10.1186/1471-2180-11-38
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. https://doi.org/10.1093/nar/gkm051
Mandal RK, Kwon YM (2017) Global screening of Salmonella enterica serovar Typhimurium genes for desiccation survival. Front Microbiol 8:1723. https://doi.org/10.3389/fmicb.2017.01723
Karash S, Kwon YM (2018) Iron-dependent essential genes in Salmonella Typhimurium. bioRxiv:159921. https://doi.org/10.1101/159921
Mandal RK, Jiang T, Kwon YM (2017) Essential genome of Campylobacter jejuni. BMC Genomics 18:616. https://doi.org/10.1186/s12864-017-4032-8
Abel S, Abel zur Wiesch P, Davis BM, Waldor MK (2015) Analysis of bottlenecks in experimental models of infection. PLoS Pathog 11:e1004823. https://doi.org/10.1371/journal.ppat.1004823
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Karash, S., Jiang, T., Samarth, D., Chandrashekar, R., Kwon, Y.M. (2019). Preparation of Transposon Library and Tn-Seq Amplicon Library for Salmonella Typhimurium. In: Ricke, S., Park, S., Davis, M. (eds) Microbial Transposon Mutagenesis. Methods in Molecular Biology, vol 2016. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9570-7_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9570-7_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9569-1
Online ISBN: 978-1-4939-9570-7
eBook Packages: Springer Protocols