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 polymerase chain reaction (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 operational taxonomic unit (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.

This data set consists of four data files. The FASTA file, Representative OTU sequences.fa, contains representative sequences from the operational taxonomic units (OTUs) shown in the OTU table. FASTA files can be opened in simple text editors, and sequences can be aligned using the BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or open source software, like AliView (http://www.ormbunkar.se/aliview/). There are two Excel data files: OTU table and heatmaps.xlsx and Diversity Indexes.xlsx. The former contains the raw abundance data for the observed OTUs from the different experimental sites. The latter is a breakdown of various diversity indices that are grouped based on experimental characteristics, such as extraction volume, extraction method, etc. Excel_Archive.zip is a compressed version of the two Excel data files that have been converted to more archival-friendly formats using Excel Archival Tool.