454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases.
• 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.
Tedersoo
Leho
L
Department of Botany, Institute of Ecology and Earth Sciences, University of Tartu, 40 Lai St, 51005 Tartu, Estonia. leho.tedersoo@ut.ee
Nilsson
R Henrik
RH
Abarenkov
Kessy
K
Jairus
Teele
T
Sadam
Ave
A
Saar
Irja
I
Bahram
Mohammad
M
Bechem
Eneke
E
Chuyong
George
G
Kõljalg
Urmas
U
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
D021901:DNA, Intergenic