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Research article - Peer-reviewed, 2013

Fungal community analysis by high-throughput sequencing of amplified markers - a user's guide

Lindahl BD, Nilsson RH, Tedersoo L, Abarenkov K, Carlsen T, Kjøller R, Kõljalg U, Pennanen T, Rosendahl S, Stenlid J, Kauserud H;


Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.


454-pyrosequencing; bioinformatics; barcoding; environmental sequencing; internal transcribed spacer (ITS) region; PCR

Published in

New Phytologist

2013, volume: 199, number: 1, pages: 288-299

Authors' information

Swedish University of Agricultural Sciences, Department of Forest Mycology and Pathology
Nilsson, Henrik
Tedersoo, Leho
Abarenkov, Kessy
University of Tartu
Carlsen, Tor
Kjøller, Rasmus
University of Copenhagen
Kõljalg, Urmas
University of Tartu
Pennanen, Taina
Rosendahl, Søren
University of Copenhagen
Swedish University of Agricultural Sciences, Department of Forest Mycology and Plant Pathology
Kauserud, Håvard

UKÄ Subject classification

Bioinformatics and Systems Biology
Biochemistry and Molecular Biology

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