title: 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 anin silicoITS1 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.
authors.affiliation: Institute of Pathology, Medical University of GrazGraz, Austria; Theodor Escherich Laboratory for Medical Microbiome Research, Medical University of GrazGraz, Austria; BioTechMed-Graz, Interuniversity CooperationGraz, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Halwachs
authors.affiliation: Institute of Pathology, Medical University of GrazGraz, Austria; Theodor Escherich Laboratory for Medical Microbiome Research, Medical University of GrazGraz, Austria.
authors.forename: Nandhitha
authors.initials: N
authors.lastname: Madhusudhan
authors.affiliation: Section of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Medical University of Graz Graz, Austria.
authors.forename: Robert
authors.initials: R
authors.lastname: Krause
authors.affiliation: Department of Biological and Environmental Sciences, University of Gothenburg Gothenburg, Sweden.
authors.forename: R Henrik
authors.initials: RH
authors.lastname: Nilsson
authors.affiliation: BioTechMed-Graz, Interuniversity CooperationGraz, Austria; Section of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Medical University of GrazGraz, Austria.
authors.forename: Christine
authors.initials: C
authors.lastname: Moissl-Eichinger
authors.affiliation: Theodor Escherich Laboratory for Medical Microbiome Research, Medical University of GrazGraz, Austria; BioTechMed-Graz, Interuniversity CooperationGraz, Austria; Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of GrazGraz, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Högenauer
authors.affiliation: BioTechMed-Graz, Interuniversity CooperationGraz, Austria; Institute of Molecular Biotechnology, Graz University of TechnologyGraz, Austria.
authors.forename: Gerhard G
authors.initials: GG
authors.lastname: Thallinger
authors.affiliation: Institute of Pathology, Medical University of GrazGraz, Austria; Theodor Escherich Laboratory for Medical Microbiome Research, Medical University of GrazGraz, Austria; BioTechMed-Graz, Interuniversity CooperationGraz, Austria.
authors.forename: Gregor
authors.initials: G
authors.lastname: Gorkiewicz
keywords: 16S rRNA gene; DNA isolation; OTU picking; formalin-fixed paraffin-embedded tissue (FFPE); internal transcribed spacer (ITS); microbiota; multiple sequence alignment (MSA); mycobiota