Insights into the human gut microbial eukaryome

The microbial inhabitants of the human gut have long been the subject of considerable scrutiny and scientific research effort (Anderson 1917; Savage 1977) and as their importance continues to be realised, not just in terms of community structure and who is there, but also the associated function of these inhabitants (Lloyd-Price et al. 2017; Parfrey, Moreau and Russell 2018). Although, much of this research has focused on prokaryotic organisms in the gut, such as bacteria; a wealth of eukaryotic organisms have been described, including fungi, protists and helminths (Scanlan and Marchesi 2008; Bik et al. 2012; Andersen, Nielsen and Stensvold 2013; Hamad, Raoult and Bittar 2016). The methodologies to study these complex eukaryotic organisms vary and a consensus method was not initially described, thus a variety of approaches have been used both across different studies and within studies in order to capture the most diversity (Scanlan and Marchesi 2008; Chen et al. 2011; Gouba, Raoult and Drancourt 2013, 2014; Huseyin et al. 2017). The development of culture-independent approaches such as next-generation sequencing means that researchers no longer have to rely solely on culturing and microscopy to determine the presence of these organisms (Mardis 2013; Buermans and den Dunnen 2014). However, with the uptake of this new technology in the laboratory so too have researchers faced new challenges (Suhr and Hallen-Adams 2015; Clooney et al. 2016; Claesson, Clooney and O’Toole 2017; O’Toole and Flemer 2017). The reporting of factors that may introduce biases and the consequences of the lack of appropriately used controls are increasing in the literature with respect to NGS technology as it is realised that seemingly inconsequential methodological choices can have detrimental effects when it comes to the downstream analysis of the data generated (Yu and Morrison 2004; Parfrey et al. 2014; Tang et al. 2015; Hill et al. 2016; Goffau et al. 2018). Coupled with this, the use of multiple methods when surveying the human gut mycobiome, unless addressed whilst the field is still in its infancy will further affect the generalisation of results across multiple studies and hinder the translation of potentially clinically relevant findings. The initial aim for this thesis was to survey the scientific literature with respect to the human gut mycobiome, in order to determine the most important considerations facing researchers when undertaking new studies. Subsequently we sought to assess these methodological considerations by comparing the approaches that others had used in their published work in our laboratory to determine if a consensus method could be determined for use in future research. Upon determination of the most suitable approach to characterise the mycobiome of healthy individuals in Chapter 2, this methodological approach was applied to further cohorts of nondiseased individuals in Chapters 3 and 4 as well as further analysis of the microbial eukaryotic component in the gut by culture-independent methods to survey the presence and prevalence of a number of pathogenic protists and the protists of undetermined pathogenicity Blastocystis and Dientamoeba fragilis (Scanlan and Stensvold 2013; Scanlan et al. 2014, 2018).