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Determination of Repeat Number and Expression States of Phase-Variable Loci Through Next Generation Sequencing and Bioinformatic Analysis

  • Luke R. Green
  • Richard D. Haigh
  • Christopher D. BaylissEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1969)

Abstract

Phase variation (PV) enables high frequency, reversible switches in expression of genetic loci across numerous species of bacteria. A major mechanism of PV in bacteria is the use of slipped strand mispairing across simple sequence repeats (SSRs). The generation and online availability of genomic datasets enables a comprehensive analysis of the distribution and composition of SSRs across multiple bacterial genomes of a species. PhasomeIt is a program that was developed to rapidly identify SSRs, to determine whether these SSRs mediate PV and to find homologous PV loci across multiple genomes. We describe use of this program for analysis of neisserial genomes. We further describe a method to reassemble specific PV loci to allow analysis of large repeat tracts which are often poorly assembled due to inherent drawbacks of the Illumina next generation sequencing (NGS) platform. These methodologies allow for rapid analysis of a major mechanism of PV across numerous species of Neisseria and other bacterial species.

Key words

Phase variation Next generation sequencing PhasomeIt Simple sequence repeats 

Notes

Acknowledgments

The authors would like to acknowledge Jack Aidley for the design and programming of PhasomeIt. We would further like to acknowledge Richard Haigh for his help and discussions regarding the rebuilding of PV loci. This publication made use of the Neisseria Multi Locus Sequence Typing website (https://pubmlst.org/neisseria/) developed by Keith Jolley and Martin Maiden and sited at the University of Oxford [6]. The development of this site has been funded by the Wellcome Trust and European Union. This research used the ALICE High Performance Computing Facility at the University of Leicester.

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Copyright information

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

Authors and Affiliations

  • Luke R. Green
    • 1
  • Richard D. Haigh
    • 1
  • Christopher D. Bayliss
    • 1
    Email author
  1. 1.Department of Genetics and Genome BiologyUniversity of LeicesterLeicesterUK

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