DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
27 06 2019
27 06 2019
Historique:
received:
04
11
2018
accepted:
28
05
2019
entrez:
29
6
2019
pubmed:
30
6
2019
medline:
27
10
2020
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 31249384
doi: 10.1038/s41598-019-44974-x
pii: 10.1038/s41598-019-44974-x
pmc: PMC6597572
doi:
Substances chimiques
DNA Primers
0
DNA, Fungal
0
RNA, Ribosomal, 18S
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
9328Références
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