DNA extraction and amplicon production strategies deeply inf\u200aluence the outcome of gut mycobiome studies

Alessandra Frau,Carrie A Duckworth,Luca Lenzi,Michael D Burkitt,Alistair C Darby,John G Kenny,Christopher S J Probert,Barry J Campbell,Jim Anson,Neil Hall,Umer Z Ijaz

doi:10.1038/s41598-019-44974-x

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.

Highlights

The microbiota inhabiting the gut is mainly made of prokaryotes, but micro-eukaryotes are present, including fungi
It has been shown that ITS1F40 and ITS241 primers, commonly used to characterise the gut mycobiome[3,28,29,30], have mismatches to species relevant to the human mycobiome, including yeasts such as Saccharomycetes (ITS1F) and Basidiomycetes (ITS2) (i.e. Malassezia and Cryptococcus)[5]
The first improved the extraction of C. albicans, while the second greatly improved the extraction of A. fumigatus

Summary

Introduction

The microbiota inhabiting the gut is mainly made of prokaryotes, but micro-eukaryotes are present, including fungi. In early studies describing the gut mycobiome, three main approaches have been applied to extract DNA from faecal samples: (i) published protocols, which have often included bead beating[4,16,17], (ii) commercial kits used as per manufacturer defined protocols[18,19,20,21,22,23] and (iii) commercial kit protocols which have been modified with the addition of extra initial steps, such as bead beating and/or enzymatic degradation, to improve efficiency of cell lysis[24,25,26,27,28,29,30]. In order to achieve classification at species level and to enable phylogenetic analysis, the use of two markers, one from ITS and another from a region of the large or small rRNA subunit, could be considered; this would allow us to understand amplification biases between these approaches[39].

Objectives

Methods

Results

Conclusion