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)

Abstract

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
• Status: Published
• Refereed: Yes
• 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: 2045-2322
• 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

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
170256		Understanding microbial community through in situ environmental 'omic data synthesis	Umer Zeeshan Ijaz	Natural Environment Research Council (NERC)	NE/L011956/1	ENG - Infrastructure & Environment