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Transposon Insertion Site Sequencing for Synthetic Lethal Screening.

Yoshiharu Yamaichi1, Tobias Dörr2

  • 1Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Université Paris Sud, 1 Avenue de la Terrasse, 91198, Gif sur Yvette, France. yoshiharu.yamaichi@i2bc.paris-saclay.fr.

Methods in Molecular Biology (Clifton, N.J.)
|August 27, 2017
PubMed
Summary

Transposon insertion site sequencing (TIS) offers a powerful method for assessing bacterial gene function and genetic interactions. This study details a protocol for generating high-density TIS libraries for Vibrio cholerae, aiding genotype-phenotype relationship studies.

Keywords:
FitnessGenomicsHigh-throughput sequencingMariner transposonTISTnSeqTransposon mutagenesis

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Area of Science:

  • Microbiology
  • Genomics
  • Molecular Biology

Background:

  • Transposon insertion site sequencing (TIS) is a powerful technique for genome-wide fitness profiling in bacteria.
  • TIS aids in identifying essential genes and understanding complex genetic networks, including synthetic lethality and suppressor interactions.
  • Determining genotype-phenotype relationships is crucial for advancing bacterial research.

Purpose of the Study:

  • To describe a detailed protocol for generating high-density transposon insertion libraries.
  • To outline the subsequent DNA sample preparation for Illumina sequencing.
  • To demonstrate the application of this TIS protocol using the Gram-negative bacterium Vibrio cholerae.

Main Methods:

  • Generation of high-density transposon insertion libraries in Vibrio cholerae.
  • Preparation of genomic DNA from transposon mutant libraries.
  • Library preparation for high-throughput sequencing on Illumina platforms.

Main Results:

  • Successful generation of dense transposon mutant libraries in Vibrio cholerae.
  • Established a robust protocol for DNA preparation suitable for Illumina sequencing.
  • Demonstrated the feasibility of using TIS for high-throughput genotype-phenotype analysis in this bacterium.

Conclusions:

  • The described TIS protocol provides a valuable tool for high-throughput bacterial genetics.
  • This method facilitates the study of essential genes and genetic interactions in Vibrio cholerae.
  • The protocol is adaptable for similar analyses in other bacterial species.