Kingdom specific mucosal microbiota analysis

Del Castillo Izquierdo, Angela (2021) Kingdom specific mucosal microbiota analysis. Masters thesis, University of East Anglia.

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Across the field of microbiome research, the ways in which bacteria influence
the host in health and disease are well known. However, knowledge of the
involvement of other kingdoms is still relatively poor and methods to isolate,
identify and quantify them efficiently are needed. Nevertheless, the low abundance
in which these microorganisms are estimated makes their study even

Sample handling and DNA extraction methods are common factors that introduce
variability to sample processing workflows. In this thesis these factors
were examined using mucus isolated from mice colonic tissue. We tested three
DNA extraction methods and compared their performance in terms of DNA
quantity and quality, number of bacterial copies and microbial composition.
We found significant differences between the kits tested which were attributed
to different lysis and purification methods. Our results highlighted the importance
of introducing controls to account for contaminants. We quantitatively
compared two sample storage solutions: PBS and RNAlater. Samples stored
in RNAlater yielded significantly more DNA and bacterial copies than those
stored in PBS, making RNAlater a good alternative to conserve samples.

These findings were corroborated during the study of bacterial and fungal
communities in colorectal cancer. We analysed the differences in microbial
communities between carcinoma and adjacent tissue using 16S rRNA gene and
ITS1 region sequencing along with qPCR. We identified a large proportion of
off-targets and contaminants, which hampered the detection and identification
of fungi. Additionally, the observed bacterial and fungal communities were
driven by the different DNA extraction methods used. We were unable to
detect differences between carcinoma and adjacent tissue. However, trends in
our bacterial communities were observed, such as the increase of Fusobacteria
in tumour samples, as previously reported.

The results of this thesis highlighted the importance of establishing standard
methods for characterising microbial kingdoms in samples susceptible to
host contamination.

Item Type: Thesis (Masters)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Jackie Webb
Date Deposited: 29 Apr 2022 10:55
Last Modified: 29 Apr 2022 10:55

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