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Methods

Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples

Heath E. O'Brien, Jeri Lynn Parrent, Jason A. Jackson, Jean-Marc Moncalvo, Rytas Vilgalys
Heath E. O'Brien
1Department of Biology, Duke University, Durham, North Carolina 27708
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  • For correspondence: heo3@duke.edu
Jeri Lynn Parrent
1Department of Biology, Duke University, Durham, North Carolina 27708
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Jason A. Jackson
1Department of Biology, Duke University, Durham, North Carolina 27708
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Jean-Marc Moncalvo
2Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, and Department of Botany, University of Toronto, Toronto, Canada
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Rytas Vilgalys
1Department of Biology, Duke University, Durham, North Carolina 27708
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DOI: 10.1128/AEM.71.9.5544-5550.2005
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  • FIG. 1.
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    FIG. 1.

    Proportions of SSU sequences belonging to different fungal phyla and other eukaryotic kingdoms. The width of triangles corresponds to the number of sequences in each, and the height corresponds to the maximum amount of sequence divergence within clades (see scale bars).

  • FIG. 2.
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    FIG. 2.

    Proportional distribution of different taxonomic groups in ITS clone libraries. The x axis indicates the proportion of sequences assigned to each taxonomic group, and bar width corresponds to the proportion of all sequences belonging to each library. A. Proportion of all ITS sequences belonging to each fungal kingdom. B. Proportion of Ascomycota ITS sequences belonging to each subclass. C. Proportion of Basidiomycota ITS sequences belonging to each order.

  • FIG. 3.
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    FIG. 3.

    Species-effort curves of richness for fungal OTUs. A. Mixed hardwood plot. B. Pine plot. C. Pooled data from both plots.

  • FIG. 4.
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    FIG. 4.

    Species-effort curves for observed and estimated fungal OTU richness in each plot.

Tables

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  • TABLE 1.

    Number of sequences and OTUs recovered from each library

    LibraryAmt of template DNA (μg)No. of sequencesNo. of OTUsHaNo. of chimeras
    MH-L-13043183.982
    MH-L-2b240101675.600
    MH-O-1325234.450
    MH-O-2350395.150
    MH-O-3318164.041
    MH-O-415072535.573
    MH-O-5c329254.560
    MH-O-6c323163.580
    MH-A-1b240d101393.6213
    MH-A-2b240d87625.600
    MH-B-1b240d70334.332
    P-L-1b240d68485.349
    P-O-1b240d62435.144
    P-A-130103625.613
    P-B-130123615.443
    • a Shannon-Weiner diversity index.

    • ↵ b Library generated from two pooled PCR products amplified using the modified “soil PCR” thermal cycler profile (see Materials and Methods).

    • ↵ c PCR primers ITS1F (MH-O-5 and -6) and ITS4B (MH-O-6 only) were used in place of ITS1 and ITS4.

    • ↵ d Template consisted of three pooled Mo Bio DNA preps.

  • TABLE 2.

    Number of sequences recovered for the most common genera encountereda

    Genus (order/subclass)No. of sequences in library:
    MH-LP-LMH-OP-OMH-AP-AMH-BP-BTotal
    Russula (Russulales)004221221335
    Mycena (Agaricales)11200000316
    Athelia (Polyporales)124100019
    Phialophora (Leotiomycetidae)302000038
    Tomentella (Thelephorales)003021017
    Hormonema (Dothidiomycetidae)330000006
    Alternaria (Dothidiomycetidae)410000005
    Cryptococcus (Tremellales)021000115
    Hygrocybe (Agaricales)000032005
    Tylospora (Polyporales)004000015
    • ↵ a Excluding MH-O-5, MH-O-6, and MH-A-1.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - Identities of ITS OTUs (greater than or equal to 97% similar) and distributions across libraries (Table S1).
      PDF document, 313K.
    • Supplemental file 2 - Identities of ITS OTUs (100% similarity) and distributions across libraries (Table S2).
      PDF document, 64K.
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Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples
Heath E. O'Brien, Jeri Lynn Parrent, Jason A. Jackson, Jean-Marc Moncalvo, Rytas Vilgalys
Applied and Environmental Microbiology Sep 2005, 71 (9) 5544-5550; DOI: 10.1128/AEM.71.9.5544-5550.2005

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Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples
Heath E. O'Brien, Jeri Lynn Parrent, Jason A. Jackson, Jean-Marc Moncalvo, Rytas Vilgalys
Applied and Environmental Microbiology Sep 2005, 71 (9) 5544-5550; DOI: 10.1128/AEM.71.9.5544-5550.2005
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    • ABSTRACT
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KEYWORDS

DNA, Ribosomal Spacer
Ecosystem
fungi
Sequence Analysis, DNA
soil microbiology

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