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Mycobiome diversity: high-throughput sequencing and identification of fungi


Fungi are major ecological players in both terrestrial and aquatic environments by cycling organic matter and channelling nutrients across trophic levels. High-throughput sequencing (HTS) studies of fungal communities are redrawing the map of the fungal kingdom by hinting at its enormous — and largely uncharted — taxonomic and functional diversity. However, HTS approaches come with a range of pitfalls and potential biases, cautioning against unwary application and interpretation of HTS technologies and results. In this Review, we provide an overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis. We also discuss upcoming trends and techniques in the field and summarize recent and noteworthy results from HTS studies targeting fungal communities and guilds. Our Review highlights the need for reproducibility and public data availability in the study of fungal communities. If the associated challenges and conceptual barriers are overcome, HTS offers immense possibilities in mycology and elsewhere.

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Fig. 1: The main steps in a fungal metabarcoding project.
Fig. 2: Emerging sequencing techniques.
Fig. 3: Fungal diversity in different environments.


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M. Unterseher is acknowledged for valuable feedback on parts of the manuscript. R.H.N. and the UNITE community acknowledge support from the Alfred P. Sloan Foundation (grant no. G-2015-14062). M.B. received support from the Estonian Research Council (grant no. PUT1317).

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Nature Reviews Microbiology thanks G. Giardinali, P. Kennedy, H. Toju and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

BLAST (a BLAST search interface to the sequence data in INSDC):

Dryad Digital Repository (digital archive for scientific data):

Genomic Standards Consortium (set of metadata standards (notably MIxS and MIMARKS) for increased reproducibility in metabarcoding):

International Nucleotide Sequence Database Collaboration (primary database for molecular data in science):

RDP (reference database for prokaryotic and eukaryotic SSU and fungal LSU sequences; also provides RDP Classifier training sets (16S rRNA, fungal LSU, Warcup fungal ITS and UNITE fungal ITS):

SILVA (reference database for prokaryotic and eukaryotic SSU and LSU sequences):

UNITE (reference database for fungal ITS sequences and various HTS pipelines; provides DOI-tagged species hypotheses under the ‘Resources’ tab):

Supplementary Information



Organisms that cannot produce their own food (as most plants can through photosynthesis), relying instead on intake of nutrition and energy from external sources of organic carbon.


The process of obtaining nutrients and energy from decomposing non-living organic matter such as dead wood, detritus and fallen leaves.


A symbiotic and mutually beneficial interaction between organisms, such as mycorrhizal relationships between fungi and plants.


A relationship between two organisms in which one organism, the parasite, obtains an advantage at the expense of the other organism.


A rapid method of PCR-based biodiversity assessment powered by high-throughput DNA sequencing.


Organisms that live inside a plant for at least a part of their life cycle without specialized nutrient-exchange structures or symptoms of apparent harm or disease.

Arbuscular mycorrhizal fungi

Fungi of the phylum Glomeromycota (and the Endogonales of the Mucoromycota) that establish mutualistic symbioses with primarily herbaceous plants; plant cell walls are penetrated, and the fungi produce arbuscules and sometimes vesicles inside the plant cells.

Technical cross-contamination

Mutations that turn distinct indices into indices used for other samples will lead to errors in sample assignment.


Barcoded amplicon sequences are determined in different sequencing phases by adding spacers of different lengths to the primer sequences.

Species hypotheses

A species hypothesis is a group of similar sequences that is tentatively delimited at the species level.

Droplet digital PCR

A PCR approach in which the PCR solution is divided into smaller oil-covered droplets in which the PCRs are then carried out.

Ectomycorrhizal fungi

Fungi that form mutualistic symbioses between fungi and various species of primarily woody plants at the root tips of the plants, although the fungi do not penetrate the plant cell walls. The ability to form ectomycorrhiza is primarily found in the fungal phyla Ascomycota and Basidiomycota and has evolved and been lost multiple times independently.

Poly(A) tail

A stretch of mRNA that has only adenine (A) bases; it is important for the nuclear export, translation and stability of mRNA.


The branching filaments that collectively make up the mycelium of a fungus.


Asexual, non-motile fungal spores typically produced on specialized stalked cells (conidiophores) for survival and dispersal.


The fungal spore is the unit for sexual and asexual reproduction, as well as for dispersal and, at times, survival during unfavourable conditions through dormancy.

Saprotrophic fungi

Fungi deriving their energy and nutrients from decomposing non-living organic matter; these are found throughout the fungal tree of life and are often intermingled with species with other nutritional strategies in puzzling ways.


The process of soil formation as affected by the soil biota and the environment at large.

Extraradical phase

Scavenging fungal hyphae that emanate, for example, from ectomycorrhizal root tips.

Ericoid mycorrhizal symbionts

Organisms that participate in mutualistic symbiosis formed between members of the plant family Ericaceae and a number of fungal lineages mainly of the ascomycetes; plant cell walls are penetrated, and fungal coils are found within the plant cells.


Referring to fungi in their asexual state.

Marine snow

Organic matter falling from upper waters to the deep ocean; it is often the dominant external source of carbon in these nutrient-deprived systems.

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Nilsson, R.H., Anslan, S., Bahram, M. et al. Mycobiome diversity: high-throughput sequencing and identification of fungi. Nat Rev Microbiol 17, 95–109 (2019).

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