Determination of Repeat Number and Expression States of Phase-Variable Loci Through Next Generation Sequencing and Bioinformatic Analysis
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 wordsPhase variation Next generation sequencing PhasomeIt Simple sequence repeats
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 . 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.
- 2.Wanford JJ, Green LR, Aidley J, Bayliss CD (2018) Phasome analysis of pathogenic and commensal Neisseria species expands the known repertoire of phase variable genes, and highlights common adaptive strategies. PLoS One 13:e0196675Google Scholar
- 3.Jordan PW, Snyder LA, Saunders NJ (2005) Strain-specific differences in Neisseria gonorrhoeae associated with the phase variable gene repertoire. BMC Microbiol 5:21Google Scholar
- 4.De Bolle X, Bayliss CD, Field D, van de Ven T, Saunders NJ, Hood DW, Moxon ER (2000) The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases. Mol Microbiol 35:211–222Google Scholar
- 7.Aidley J, Wanford JJ, Green LR, Sheppard SK, Bayliss CD (2018) PhasomeIt: an 'omics' approach to cataloguing the potential breadth of phase variation in the genus Campylobacter. Microb Genom 4. https://doi.org/10.1099/mgen.0.000228
- 8.Green LR, Lucidarme J, Dave N, Chan H, Clark S, Borrow R, Bayliss CD (2018) Phase variation of NadA in invasive Neisseria meningitidis isolates impacts on coverage estimates for 4C-MenB, a MenB vaaccine. J Clin Microbiol 56:e00204-18Google Scholar