Environmental long read amplicons of soil fungi across Podzol soil profile

Study Extent

Soil samples were collected in mid-October 2013 from Ivantjärnsheden field station in Jädraås (60°49‘N, 16°30’E, altitude 185 m)

Sampling

We collected 5 soil cores (5 cm diameter and 15 cm deep) in each of 12 plots after peeling back the top shrub and moss layer (incl. most of the litter layer). Soil cores were separated into visually distinct podzol soil layers: organic soil (O), mineral elluvial soil (E) and mineral illuvial soil (B) before pooling the layers for each plot. This sampling rendered a total of 36 soil samples that were separately homogenized in ziplock bags before separating a 15 mL sample from each that was transported back to the laboratory on ice and stored at -20°C. Approximately 1500 bp of the rDNA from all soil DNA extracts was amplified using the primer set ITS1F (Gardes & Bruns, 1993) and LR5 (Hopple Jr & Vilgalys, 1994). PCR products from the separate soil horizons were pooled to generate three amplicon libraries (SwO, SwE and SwB) for sequencing at SciLifeLab/NGI (Uppsala, Sweden) on a PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA).

Method steps

1. Data analysis is described in more detail in Kalsoom-Khan et al. 2020 (DOI: 10.1186/s43008-020-00045-9). In short, raw sequence reads were filtered and trimmed using the tool cutadapt (Martin 2011; version 1.18) to de-multiplexed reads based on the forward and reverse barcodes, to keep only reads with both primers present, and to remove the actual primer sequences from the reads. Amplicons sequenced in reverse were reverse complemented before continuing the analyses. Amplified sequence variants (ASVs) were generated using DADA2 (Callahan et al. 2016; version 1.9.3). Default parameters were used for filtering the reads, but discarding sequences with more than 12 "expected errors" (maxEE=12). The tool ITSx (Bengtsson-Palme et al. 2013; version 1.1-beta) was used to extract the ITS2 region of ribosomal rDNA within each ASV, which was then used for taxonomy assignment.

Soil samples were collected from Ivantjärnsheden field station in Jädraås (60°49‘N, 16°30’E, altitude 185 m), in an area of around 150x150 m.