Critical Issues in mycobiota analysis

• 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.

Subject headings

NATURVETENSKAP  -- Biologi -- Biologisk systematik (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biological Systematics (hsv//eng)

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Mikrobiologi inom det medicinska området (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Microbiology in the medical area (hsv//eng)

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

NATURVETENSKAP  -- Biologi -- Botanik (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Botany (hsv//eng)

NATURVETENSKAP  -- Biologi -- Genetik (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Genetics (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Klinisk medicin -- Infektionsmedicin (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Clinical Medicine -- Infectious Medicine (hsv//eng)

NATURVETENSKAP  -- Biologi -- Ekologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Ecology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Bioinformatik och systembiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)

Keyword

microbiota

mycobiota

internal transcribed spacer (ITS)

16S rRNA gene

multiple sequence alignment (MSA)

OTU picking

formalin-fixed paraffin-embedded tissue (FFPE)

DNA isolation

Publication and Content Type

ref (subject category)

art (subject category)