DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies

Frau, A. et al. (2019) DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies. Scientific Reports, 9, 9328. (doi: 10.1038/s41598-019-44974-x) (PMID:31249384) (PMCID:PMC6597572)

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Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Ijaz, Dr Umer
Authors: Frau, A., Kenny, J. G., Lenzi, L., Campbell, B. J., Ijaz, U. Z., Duckworth, C. A., Burkitt, M. D., Hall, N., Anson, J., Darby, A. C., and Probert, C. S.J.
College/School:College of Science and Engineering > School of Engineering > Infrastructure and Environment
Journal Name:Scientific Reports
Publisher:Nature Research
ISSN (Online):2045-2322
Copyright Holders:Copyright © 2019 The Authors
First Published:First published in Scientific Reports 9: 9238
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
170256Understanding microbial community through in situ environmental 'omic data synthesisUmer Zeeshan IjazNatural Environment Research Council (NERC)NE/L011956/1ENG - Infrastructure & Environment