Critical Issues in Mycobiota Analysis

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.

Analysis of the otic mycobiota in dogs with otitis externa compared to healthy individuals

“…A limitation of the present study was the difficulty of obtaining adequate DNA and sequence yields from samples. This could be because of low fungal DNA burdens or the presence of inhibitors in samples . One study reported that bead‐beating leads to significant decrease in fungal DNA yield .…”

Commensal Fungi in Health and Disease

“…This effort is frustrated by a lack of quality-controlled reference databases, evolving recognition and definition of new fungal species/complexes, and confusion as to the taxonomic lineage of many species as well as common use of very different names for sexual and asexual forms of many frequently-encountered fungi. (Halwachs et al, 2017) Because GenBank acts primarily as an archive, many sequences submitted have been annotated with incorrect or poorly-defined species names. It is estimated that more than 10% of the publicly-available fungal ITS sequences are annotated incorrectly at the species level (Nilsson et al, 2006).…”

The mammalian mycobiome: A complex system in a dynamic relationship with the host

“…A recent study evaluating DNA extraction methods from saliva samples demonstrated that diversity of fungi, unlike that of their bacterial counterparts, is heavily influenced by the method employed (Vesty, Biswas, Taylor, Gear, & Douglas, ). Based on our experiences and other comparative studies of DNA extraction methods (Halwachs et al, ; Huseyin, Rubio, O'Sullivan, Cotter, & Scanlan, ), at least an extra bead‐beating step is required to ensure mechanical lysis of fungal cell walls for effective genomic content recovery. The best practices for culture‐dependent and ‐independent methods are covered in greater depth elsewhere (Huseyin et al, ).…”