The gut microbiome plays a vital role in host homeostasis and an improved understanding of its biology is essential for a better comprehension of disorders such as foetal ethanol spectrum disorder (FASD). While next-generation sequencing has been used to assess the role of the gut microbiome, differences in experimental approaches may induce undesired biases. In this dissertation, the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of fungal and bacterial communities were assessed. A fungal mock community comprised of Candida glabrata, Candida parapsilosis, Clavispora lusitaniae, Meyerozyma guilliermondii, and Pichia kudriavezevii, was used to optimize targeted nanopore sequencing approaches based on the internal transcribed spacer (ITS) genomic region. Additionally, using a FASD mouse model, one untargeted (shotgun/metagenomic) and two targeted (based on the full length 16S rRNA gene and the 16S-ITS-23S region from the ribosomal RNA operon) nanopore sequencing approaches were performed to sequence PCR products amplified from DNA extracts of gut content of 12 infant mice exposed to ethanol in utero. Faecalibaculum rodentium and Duncaniella sp. were the two most prevalent taxa detected using targeted sequencing approaches, while bacterial taxa were more evenly represented when using the metagenomic approach. The targeting of the full-length 16S rRNA gene provided the most comprehensive results. The nanopore sequencing optimization results suggest that experimental and/or bioinformatics steps, can introduce biases, misrepresenting certain taxa depending on the genomic region used for sequencing. Afterwards, the optimized 16S rRNA gene-targeted nanopore sequencing was performed to sequence DNA extracts from the gut content of nine infant mice exposed to ethanol in utero and eight non-exposed infant mice and compared with Illumina sequencing to determine the differences in the composition of gut microbiota between ethanol treated and untreated mice pups. No fungi were detected in the infant mice gut which implicate that fungi failed to reach the PCR detection threshold. Nanopore and Illumina sequencing retrieved different microbial profiles, probably explained by the different experimental and bioinformatical setups employed. Specifically, Prevotella and Bidifobacterium were not detected by nanopore sequencing and Akkermansia muciniphila was not detected by Illumina sequencing. Only Parabacteroides distasonis, Muribaculum intestinale, and Duncaniella sp. were simultaneously detected by both sequencing platforms. Neither platform detected any relevant effect of ethanol exposure. Concluding, this dissertation highlightsthe experimental handicaps of nanopore sequencing while detailing sequencing biases that could be observed towards certain taxa, therefore nanopore targeted approaches should be used with caution to avoid inaccurate microbial profiles.
|Date of Award||Jun 2022|
|Supervisor||Fergus Guppy (Supervisor), Joao Inacio Silva (Supervisor) & Lucas Bowler (Supervisor)|