title: Fungal community analysis by high-throughput sequencing of amplified markers--a user's guide.
abstract: 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.
authors.affiliation: Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-750 07, Uppsala, Sweden.
authors.forename: Björn D
authors.initials: BD
authors.lastname: Lindahl
authors.affiliation: Department of Biological and Environmental Sciences, University of Gothenburg, Box 461, SE-405 30, Gothenburg, Sweden.
authors.forename: R Henrik
authors.initials: RH
authors.lastname: Nilsson
authors.affiliation: Institute of Ecology and Earth Sciences/Natural History Museum, University of Tartu, 46 Vanemuise St., 51014, Tartu, Estonia.
authors.forename: Leho
authors.initials: L
authors.lastname: Tedersoo
authors.affiliation: Institute of Ecology and Earth Sciences/Natural History Museum, University of Tartu, 46 Vanemuise St., 51014, Tartu, Estonia.
authors.forename: Kessy
authors.initials: K
authors.lastname: Abarenkov
authors.affiliation: Department of Biology, University of Oslo, PO Box 1066, Blindern, N-0316, Oslo, Norway.
authors.forename: Tor
authors.initials: T
authors.lastname: Carlsen
authors.affiliation: Department of Biology, University of Copenhagen, Øster Farimagsgade 2D, 1353, Copenhagen, Denmark.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Kjøller
authors.affiliation: Institute of Ecology and Earth Sciences/Natural History Museum, University of Tartu, 46 Vanemuise St., 51014, Tartu, Estonia.
authors.forename: Urmas
authors.initials: U
authors.lastname: Kõljalg
authors.affiliation: The Finnish Forest Research Institute, PL 18, FI-01301, Vantaa, Finland.
authors.forename: Taina
authors.initials: T
authors.lastname: Pennanen
authors.affiliation: Department of Biology, University of Copenhagen, Øster Farimagsgade 2D, 1353, Copenhagen, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Rosendahl
authors.affiliation: Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-750 07, Uppsala, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Stenlid
authors.affiliation: Department of Biology, University of Oslo, PO Box 1066, Blindern, N-0316, Oslo, Norway.
authors.forename: Håvard
authors.initials: H
authors.lastname: Kauserud
mesh_terms: D058448:Biota; D019295:Computational Biology / Q000379:methods*; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis; D021901:DNA, Intergenic; D005658:Fungi / Q000145:classification / Q000235:genetics*; D005819:Genetic Markers; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D038821:Mycorrhizae / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021901:DNA, Intergenic; D005819:Genetic Markers
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