, Volume 22, Issue 4, pp 309–315 | Cite as

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

  • Håvard Kauserud
  • Surendra Kumar
  • Anne K. Brysting
  • Jenni Nordén
  • Tor Carlsen
Original Paper


In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.


Ectomycorrhiza Community ecology 454 pyrosequencing Sanger sequencing Bistorta vivipara 



The University of Oslo is acknowledged for financial support and three anonymous reviewers for helpful comments. We thank biology students at the University of Oslo who did the field work and initiated this work as a part of a course project.

Supplementary material

572_2011_403_MOESM1_ESM.doc (188 kb)
Table S1 Number of reads obtained from all OTUs, distributed on plant root systems and replicates. Top blast hits are shown along with identity, query coverage, and GC content of the representative sequence. (DOC 188 kb)


  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Amend A, Samson R, Seifert K, Bruns T (2010a) Deep sequencing reveals diverse and geographically structured assemblages of fungi in indoor dust. PNAS 107:13748–13753PubMedCrossRefGoogle Scholar
  3. Amend AS, Seifert KA, Bruns TD (2010b) Quantifying microbial communities with 454 pyrosequencing: does read abundance count? Mol Ecol 19:5555–5565PubMedCrossRefGoogle Scholar
  4. Avis PG, Branco S, Tang Y, Mueller GM (2010) Pooled samples bias fungal community descriptions. Mol Ecol Res 10:135–141CrossRefGoogle Scholar
  5. Buée M, Reich M, Murat C, Nilsson RH, Uroz S, Martin F (2009) 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytol 184:449–456PubMedCrossRefGoogle Scholar
  6. Chao A, Chazdon RL, Colwell RK, Shen T (2005) A new statistical approach for assessing similarity of species composition with incidence and abundance data. Ecol Lett 8:148–159CrossRefGoogle Scholar
  7. Chase M, Fay M (2009) Barcoding of plants and fungi. Science 325:682–683PubMedCrossRefGoogle Scholar
  8. Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F, Ochman H, Hugenholtz P (2010) Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J 4:642–647PubMedCrossRefGoogle Scholar
  9. Eriksen M, Bjureke KE, Dhillion SS (2002) Mycorrhizal plants of traditionally managed boreal grasslands in Norway. Mycorrhiza 12:117–123PubMedCrossRefGoogle Scholar
  10. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118PubMedCrossRefGoogle Scholar
  11. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  12. Hesselmann H (1900) Om mycorrhizabildningar hos artiska växter. Bihang Till K Svenska Vet Akad Handlingar 26:1–46Google Scholar
  13. Horton T, Bruns T (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black box. Mol Ecol 10:1855–1871PubMedCrossRefGoogle Scholar
  14. Jumpponen A, Jones KL (2009) Massively parallel 454 sequencing indicates hyperdiverse fungal communities in temperate Querqus macrocarpa phylloshere. New Phytol 184:438–448PubMedCrossRefGoogle Scholar
  15. Kumar S, Carlsen T, Mevik B, Enger P, Blaalid R, Shalchian-Tabrizi K, Kauserud H (2011) CLOTU: an online pipeline for processing and clustering of 454 amplicon reads into OTUs followed by taxonomic annotation. BMC Bioinformatics 12:82CrossRefGoogle Scholar
  16. Massicotte HB, Melville LH, Peterson RL, Luoma DL (1998) Anatomical aspects of field ectomycorrhizas on Polygonum viviparum (Polygonaceae) and Kobresia bellardii (Cyperaceae). Mycorrhiza 7:287–292CrossRefGoogle Scholar
  17. Mühlmann O, Bacher M, Peintner U (2008) Polygonum viviparum mycobionts on an alpine primary successional glacier forefront. Mycorrhiza 18:87–95PubMedCrossRefGoogle Scholar
  18. Murray MG, Thompson WF (1980) Rapid isolation of high weight plant DNA. Nucl Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  19. Oksanen J, Kindt R, Legendre P, O’Hara B, Simpson GL, Stevens MHH (2008) vegan: Community Ecology Package. R packageGoogle Scholar
  20. Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740PubMedCrossRefGoogle Scholar
  21. Pukkila PJ, Skrzynia C (1993) Frequent changes in the number of reiterated ribosomal-rna genes throughout the life-cycle of the Basidiomycete Coprinus cinereus. Genetics 133:203–211PubMedGoogle Scholar
  22. Tedersoo L, Nilsson RH, Abarenkov K, Jairus T, Sadam A, Saar I, Bahram M, Bechem E, Chuyong G, Kóljag U (2010) 454 Pyrosequencing and Sanger sequencing of tropical mycorrhiza fungi provide similar results but reveal substantial methodological biases. New Phytol 188:291–301PubMedCrossRefGoogle Scholar
  23. Venables WN, Ripley BD (2002) Modern applied statistics with S. Springer, New YorkGoogle Scholar
  24. Wallander H, Johansson U, Sterkenburg E, Durling MB, Lindahl B (2010) Production of ectomycorrhizal mycelium peaks during canopy closure in Norway spruce. New Phytol 187:1124–1134PubMedCrossRefGoogle Scholar
  25. White TJ, Bruns T, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninski JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, San Diego, pp 315–322Google Scholar
  26. Zhou J, Wu L, Deng Y, Zhi X, Jiang YH, Tu Q, Xie J, Van Nostrand JD, He Z, Yang Y (2011) Reproducibility and quantitation of amplicon sequencing-based detection. ISME J 1:11Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Håvard Kauserud
    • 1
  • Surendra Kumar
    • 1
  • Anne K. Brysting
    • 2
  • Jenni Nordén
    • 1
  • Tor Carlsen
    • 1
  1. 1.Department of Biology, Microbial Evolution Research Group (MERG)University of OsloOsloNorway
  2. 2.Department of BiologyCentre for Ecological and Evolutionary Synthesis (CEES)OsloNorway

Personalised recommendations