Abstract
Fungi constitute an important part of the human microbiota and they play a significant role for health and disease development. Advancements made in the culture-independent analysis of microbial communities have broadened our understanding of the mycobiota, however, microbiota analysis tools have been mainly developed for bacteria (e.g., targeting the 16S rRNA gene) and they often fall short if applied to fungal marker-gene based investigations (i.e., internal transcribed spacers, ITS). In the current paper we discuss all major steps of a fungal amplicon analysis starting with DNA extraction from specimens up to bioinformatics analyses of next-generation sequencing data. Specific points are discussed at each step and special emphasis is placed on the bioinformatics challenges emerging during operational taxonomic unit (OTU) picking, a critical step in mycobiota analysis. By using an in silico ITS1 mock community we demonstrate that standard analysis pipelines fall short if used with default settings showing erroneous fungal community representations. We highlight that switching OTU picking to a closed reference approach greatly enhances performance. Finally, recommendations are given on how to perform ITS based mycobiota analysis with the currently available measures.
Highlights
It is well-established that the microbiota contributes significantly to human health and disease
A recent comparison of the three different clustering strategies revealed the de novo approach based on a global distance matrix as the optimal method for clustering 16S rRNA gene sequences into operational taxonomic unit (OTU)
Fungal amplicon studies benefit greatly from the advancements made in the analysis of bacterial communities, many hurdles need still to be solved and standards are waiting to be defined
Summary
It is well-established that the microbiota contributes significantly to human health and disease. For amplification of fungal DNA various primers have been designed targeting different regions of the rRNA operon or other marker genes encoding translation elongation factor 1α, RNA polymerase II, β-tubulin, and the minichromosome maintenance complex component 7 (MCM7) protein (White et al, 1990; Tanabe et al, 2002; McLaughlin et al, 2009; O’Donnell et al, 2010; Schoch et al, 2012; Toju et al, 2012; Lindahl et al, 2013).
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