Using Next Generation Sequencing to uncover real fungal diversity at the contaminated site of abandoned antimony mine in Dubrava, Slovakia.
The presented dataset includes fungal ITS sequences retrieved by metabarcoding of the soil samples at the Dúbrava locality in Central Slovakia. The locality has been contaminated by antimony mining in the past and the recent research has focused on the impact of such contamination on the fungal biodiversity. Sampling methods – sampling description: For each site, 10 soil samples approximately 5 cm diam. and 15 cm depth were collected in a randomly selected grid with samples at least 5m apart using a sterile gardening tool/spade. Samples from the same site were mixed into one large bulk soil sample and frozen immediately after collection. Several subsamples stored at -45 °C were used for DNA extraction; i.e. after thawing, each sample was well vortexed, centrifuged at 14 000 rpm for 5 min at the room temperature, and excess water was removed. Next the samples were dryied at 37 °C for 20 min in a Concentrator plus/Vacufuge® plus (Eppendorf). DNA was extracted from the dryied samples with DNeasy PowerSoil Kit from Qiagen according to the manufacturer's protocol. The final DNA concentration and purification were determined using spectrophotometry (Biodrop and Qubit) and the DNA quality was checked using agarose gel electrophoresis. The fungal ITS rRNA gene region was amplified using ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3') (White et al., 1990; Motooka et al., 2017. The PCR was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 °C for 3 minutes, followed by 30 cycles of 94 °C for 30 seconds, 53 °C for 40 seconds and 72 °C for 1 minute, after which a final elongation step at 72 °C for 5 minutes was performed. The samples were purified using calibrated Agencourt AMPure XP beads and the purified PCR product was used to prepare Illumina MiSeq DNA library according to the manufacturer's guidelines. Sequencing and library preparation was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer's guidelines.