title: Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils.
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 OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU 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 OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.
authors.affiliation: Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108, United States of America.
authors.forename: Zewei
authors.initials: Z
authors.lastname: Song
authors.affiliation: Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108, United States of America.
authors.forename: Dan
authors.initials: D
authors.lastname: Schlatter
authors.affiliation: Department of Plant Biology, University of Minnesota, Saint Paul, MN 55108, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: Kennedy
authors.affiliation: Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108, United States of America.
authors.forename: Linda L
authors.initials: LL
authors.lastname: Kinkel
authors.affiliation: Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108, United States of America; USDA ARS Cereal Disease Laboratory, Saint Paul, MN 55108, United States of America.
authors.forename: H Corby
authors.initials: HC
authors.lastname: Kistler
authors.affiliation: Department of Plant Biology, University of Minnesota, Saint Paul, MN 55108, United States of America.
authors.forename: Nhu
authors.initials: N
authors.lastname: Nguyen
authors.affiliation: Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108, United States of America.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Bates
mesh_terms: D044822:Biodiversity; D004271:DNA, Fungal / Q000032:analysis*; D021903:DNA, Ribosomal Spacer; D005658:Fungi / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D012987:Soil; D012988:Soil Microbiology
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D012987:Soil