A simplified and efficient method for himar-1 transposon sequencing in bacteria, demonstrated by creation and analysis of a saturated transposon-mutant library in mycobacterium abscessus

Mark Foreman, Moran Gershoni, Daniel Barkan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

We present a technically simple, easy-to-perform method for generating the genomic libraries for Himar-1 transposon site sequencing (Tn-seq). In addition to being simpler than present methods in the technical aspect, it also allows more robust and straightforward identification of the insertion site, by generating a longer sequence surrounding the insertion TA in the genome. The method makes Tn-seq more user-friendly and accessible to laboratories with more-limited bioinformatic resources. Finally, we created a saturated transposon-mutant library in Mycobacterium abscessus and demonstrated the usefulness of the method in analysis of genes involved in colony morphology, as well as in analysis of the whole Tn-mutant library, with identification of over 8,000 unique mutants. IMPORTANCE Transposon insertion sequencing is a powerful tool, but many researchers are discouraged by the apparent technical complexity of preparing the genomic library for deep sequencing and by the complicated computational analysis needed for insertion site identification. Our proposed method makes the preparation of the library easy and straightforward, relying on well-known molecular biology techniques. In addition, the results obtained from the deep sequencing are easily analyzed in terms of transposon insertion site identification, placing library preparation and analysis within the reach of more researchers in the microbiology community, including those with less computational and bioinformatic resources and experience. This is demonstrated by analysis of the most saturated Tn-mutant library created to date in the emerging pathogen Mycobacterium abscessus.

Original languageAmerican English
Article numbere0097620
JournalmSystems
Volume5
Issue number5
DOIs
StatePublished - 20 Oct 2020

Bibliographical note

Publisher Copyright:
Copyright © 2020 Foreman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Keywords

  • Bacterial genetics
  • Bioinformatics
  • Genomics
  • Mycobacteria
  • Mycobacterium abscessus
  • Transposon
  • Transposon library

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