These authors contributed equally to this work.
454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases
Article first published online: 15 JUL 2010
© The Authors (2010). Journal compilation © New Phytologist Trust (2010)
Special Issue: Featured papers on ‘The Ectocarpus genome sequence’
Volume 188, Issue 1, pages 291–301, October 2010
How to Cite
Tedersoo, L., Nilsson, R. H., Abarenkov, K., Jairus, T., Sadam, A., Saar, I., Bahram, M., Bechem, E., Chuyong, G. and Kõljalg, U. (2010), 454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases. New Phytologist, 188: 291–301. doi: 10.1111/j.1469-8137.2010.03373.x
- Issue published online: 2 SEP 2010
- Article first published online: 15 JUL 2010
- Received: 14 April 2010, Accepted: 25 May 2010
- 454 massively parallel pyrosequencing;
- community structure;
- ectomycorrizal fungi;
- Korup National Park;
- PCR bias
- •Compared with Sanger sequencing-based methods, pyrosequencing provides orders of magnitude more data on the diversity of organisms in their natural habitat, but its technological biases and relative accuracy remain poorly understood.
- •This study compares the performance of pyrosequencing and traditional sequencing for species’ recovery of ectomycorrhizal fungi on root tips in a Cameroonian rain forest and addresses biases related to multi-template PCR and pyrosequencing analyses.
- •Pyrosequencing and the traditional method yielded qualitatively similar results, but there were slight, but significant, differences that affected the taxonomic view of the fungal community. We found that most pyrosequencing singletons were artifactual and contained a strongly elevated proportion of insertions compared with natural intra- and interspecific variation. The alternative primers, DNA extraction methods and PCR replicates strongly influenced the richness and community composition as recovered by pyrosequencing.
- •Pyrosequencing offers a powerful alternative for the identification of ectomycorrhizal fungi in pooled root samples, but requires careful selection of molecular tools. A well-populated backbone database facilitates the detection of biological and technical artifacts. The pyrosequencing pipeline is available at http://unite.ut.ee/454pipeline.tgz.