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Updated: Jun 6, 2026
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Nanopore DNA Sequencing for Metagenomic Soil Analysis
Published on: December 14, 2017
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Transposon-directed insertion-site sequencing (TraDIS) analysis of Enterococcus faecium using nanopore sequencing and
Alexandra L Krause1, Wytamma Wirth1,2, Adrianna M Turner1
1Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
Microbiology Spectrum
|June 10, 2025
Summary
We developed a new genetic tool, transposon-directed insertion-site sequencing (TraDIS), for studying vancomycin-resistant Enterococcus faecium (VREfm). This platform helps uncover new genes involved in VREfm resistance and persistence.
Area of Science:
- Microbiology
- Genomics
- Infectious Diseases
Background:
- Vancomycin-resistant Enterococcus faecium (VREfm) are significant healthcare-associated pathogens.
- Understanding VREfm's molecular mechanisms for persistence and antimicrobial resistance requires advanced genetic tools.
Purpose of the Study:
- To develop and optimize a transposon-directed insertion-site sequencing (TraDIS) platform for Enterococcus faecium.
- To enable genome-scale forward genetic screens for identifying genes involved in VREfm phenotypes.
Main Methods:
- Engineered a novel transposon delivery plasmid (pIMTA(tetM)) for high-density mutant library generation.
- Utilized Oxford Nanopore Technology amplicon sequencing for precise insertion site mapping.
- Developed a bioinformatic analysis suite with a visualization tool (Diana) for data exploration.
Main Results:
- Successfully generated and analyzed a library of 48,458 unique transposon mutants.
- Confirmed the role of the vanB operon in vancomycin resistance.
- Identified essential roles for vanWB and vanYB in VREfm vancomycin resistance, challenging previous designations.
Conclusions:
- The developed TraDIS platform provides an accessible, end-to-end solution for functional genomic analysis in VREfm.
- This platform will accelerate the investigation of VREfm pathobiology, persistence, and resistance mechanisms.
- The methodology is potentially applicable to other Gram-positive bacteria.
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