title: 454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases.
abstract: • 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.
authors.affiliation: Department of Botany, Institute of Ecology and Earth Sciences, University of Tartu, 40 Lai St, 51005 Tartu, Estonia. leho.tedersoo@ut.ee
authors.forename: Leho
authors.initials: L
authors.lastname: Tedersoo
authors.forename: R Henrik
authors.initials: RH
authors.lastname: Nilsson
authors.forename: Kessy
authors.initials: K
authors.lastname: Abarenkov
authors.forename: Teele
authors.initials: T
authors.lastname: Jairus
authors.forename: Ave
authors.initials: A
authors.lastname: Sadam
authors.forename: Irja
authors.initials: I
authors.lastname: Saar
authors.forename: Mohammad
authors.initials: M
authors.lastname: Bahram
authors.forename: Eneke
authors.initials: E
authors.lastname: Bechem
authors.forename: George
authors.initials: G
authors.lastname: Chuyong
authors.forename: Urmas
authors.initials: U
authors.lastname: Kõljalg
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D015982:Bias; D058893:DNA Barcoding, Taxonomic; D021901:DNA, Intergenic / Q000235:genetics; D038821:Mycorrhizae / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature; D014329:Tropical Climate
chemical_list: D021901:DNA, Intergenic