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.

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…Terrestrial samples -mainly soil -were studied almost as often as marine samples, with the most common focus of these studies being the analysis of fungi (e.g. Teasdale et al 2013;Lazarus & James 2015;Song et al 2015) followed by prokaryotes (e.g. Lin et al 2010;Kanokratana et al 2011).…”

“…Nagahama et al 2011;Lazarus & James 2015), but the internal transcribed spacer (ITS) region 1 (ITS1, located between 18S and 5.8S rRNA genes) was the more commonly targeted (e.g. Bellemain et al 2010;Singh et al 2012;Bazzicalupo et al 2013;Song et al 2015), with more recent studies switching to the ITS2 region (between 5.8S and 28S rRNA genes; Bazzicalupo et al 2013). A majority of studies of plant DNA were based on the trnL UAA intron within the chloroplast transfer RNA, or tRNA gene (e.g.…”

“…The most commonly-adopted PCR purification approaches were the Agencourt AMPure XP system (e.g. Bazzicalupo et al 2013;Pochon et al 2015b;Song et al 2015), the Qiagen MinElute PCR purification kit (e.g. Stoeck et al 2010;Thomsen et al 2012a;Calvignac-Spencer et al 2013) and the Promega Wizard SV Gel and PCR Clean-Up system (e.g.…”

“…The 454 pyrosequencing platform was used with increasing frequency prior to 2013 but declined in popularity after this date (e.g. Stoeck et al 2010;Pawlowski et al 2011;Yu et al 2012;Parducci et al 2013;Pochon et al 2013;Yang et al 2014;Zimmermann et al 2015), coinciding with a rise in the use of Illumina sequencing platforms for DNA analysis; however, the maximum number of studies using the latter system in any year was just seven (Costa et al 2015;Deiner et al 2015;Dong et al 2015;Ficotela et al 2015;Pansu et al 2015a;Pochon et al 2015b;Song et al 2015). The Ion Torrent platform was used just three times (all since 2013) among the 167 studies reviewed (Deagle et al 2013;Young et al 2014;Zaiko et al 2015).…”

Ectomycorrhizal fungal diversity and saprotrophic fungal diversity are linked to different tree community attributes in a field‐based tree experiment

“…While the use of high-throughput sequencing has become a standard way to study soil fungal communities, there is growing recognition that multiple quality control steps are needed to ensure ecological accurate analyses (Lindahl et al 2013;Nguyen et al 2015;Song et al 2015). To address the issue of false positives in our data (i.e.…”