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Title: Effort versus reward: preparing samples for fungal community characterization in high-throughput sequencing surveys of soils

Author
item SONG, ZEWEI - University Of Minnesota
item SCHLATTER, DANIEL - University Of Minnesota
item KENNEDY, PETER - University Of Minnesota
item KINKEL, LINDA - University Of Minnesota
item Kistler, Harold
item NGUYEN, NHU - University Of Minnesota
item BATES, SCOTT - University Of Minnesota

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2015
Publication Date: 5/14/2015
Citation: Song, Z., Schlatter, D., Kennedy, P., Kinkel, L.L., Kistler, H.C., Nguyen, N., Bates, S.T. 2015. Effort versus reward: preparing samples for fungal community characterization in high-throughput sequencing surveys of soils. PLoS One. 9(11):1-13.

Interpretive Summary: While historically, plant pathologists have focused on understanding fungi that cause disease in plants, many fungi are known to be helpful to plant health and vigor. However, little is known about the overall composition of fungi inhabiting the soil or growing endophytically within healthy plants. Many fungal species grow slowly, lack readily distinguishable traits and/or are recalcitrant to isolation in pure culture and so only a fraction of fungal species have been described even from well-studied agricultural plants and soils. This study tests high thoroughput DNA sequencing methodologies used to characterize the fungi associated with soil at the roots of plants. The goal of this work was to established generally accepted protocols for measuring fungal communities in the plant root environment. These studies likely will influence thinking and planning of cropping systems arising from agricultural monoculture and intercropping and their impact on fungal soil communities and plant health. The primary users of the research in this publication will be other scientists engaged in research to improve disease management on small grain crops.

Technical Abstract: Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in PCR, and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal taxon richness or community structure at either site. Physical pooling captured more taxon richness compared to individual samples, but total taxon richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence taxon richness estimates, it is important to carefully consider the study aims when planning sampling procedures.