Fungal community analysis by high-throughput sequencing of amplified markers – a user's guide
Article first published online: 28 MAR 2013
© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust
Volume 199, Issue 1, pages 288–299, July 2013
How to Cite
Lindahl, B. D., Nilsson, R. H., Tedersoo, L., Abarenkov, K., Carlsen, T., Kjøller, R., Kõljalg, U., Pennanen, T., Rosendahl, S., Stenlid, J. and Kauserud, H. (2013), Fungal community analysis by high-throughput sequencing of amplified markers – a user's guide. New Phytologist, 199: 288–299. doi: 10.1111/nph.12243
- Issue published online: 28 MAY 2013
- Article first published online: 28 MAR 2013
- Manuscript Accepted: 18 FEB 2013
- Manuscript Received: 3 JAN 2013
- European Forest Institute
- Swedish Research Council FORMAS
- environmental sequencing;
- internal transcribed spacer (ITS) region;
- 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.