Browsing by Author "Breckell, Georgia"

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    Characterisation of epigenomic variation in natural isolates of E. coli : a thesis submitted in partial fulfilment of the requirements for the degree of Ph.D in Genetics, Massey University, College of Science, School of Natural Sciences, Auckland
    (Massey University, 2023) Breckell, Georgia
    DNA methylation is ubiquitous in bacteria and has a range of roles including self versus non-self recognition, DNA repair, and regulation of gene expression in response to internal and external cues. Regulation of gene expression by DNA methylation can lead to the establishment of phenotypic variation in otherwise isogenic populations. Until recently methods for the genome-wide study of DNA methylation in bacteria have been limited and therefore the full extent of DNA methylation's role in bacterial genomes is not well understood. In this thesis I use Oxford Nanopore Technologies sequencing to investigate the presence and activity of DNA methyltransferase in natural isolates of E. coli. The first aim of this thesis is to produce high quality genome assemblies that can be used to determine methylation patterns. To achieve this, in Chapter 2 I first use in silico methods to quantify the effects of different read length characteristics on assembly quality. I then optimise DNA isolation and library prep methods to obtain high quality DNA. In Chapter 3 I apply the results of Chapter 2 to sequence 49 natural isolates of E. coli from across the E. coli clade. I next benchmark five genome assembly methods for assembly accuracy. I base accuracy on five metrics designed to measure both the overall structural accuracy and the sequence accuracy of each assembly. The large number of isolates (49) used in this study, allows identification of the strengths associated with each assembly method. These results quantitatively describe best practices for bacterial genome assembly and highlight the current variability in genome assembly accuracy and therefore the importance of tailoring assembly methods to the study objectives. Finally, in chapter 4 I use the data produced in Chapter 3 to investigate DNA methylation in three E. coli natural isolates. After in silico identification of all the methyltransferases in each genome, I show that the activity of all predicted methyltransferases can be detected, as well as the activity of unexpected putative methyltransferases which are present in our isolates. Finally, I show that the genome wide DNA methylation patterns show consistent differences across growth conditions. These results suggest that E. coli exhibits transient DNA methylation patterns depending on growth environment and state. Overall this thesis establishes methods for assessing genome assemblies and broadens our understanding of genome wide DNA methylation patterns and the dynamics of these patterns in E. coli. Additionally this work provides insight into the possibility of transient epigenetic differentiation in E. coli which is reflected in the DNA methylation patterns across the genome.