Dneasy powermax soil kit

DNA was extracted using the DNeasy PowerMax Soil Kit (QIAGEN, United States), following the manufacturer’s instructions. Prior to extraction, the membrane was cut into as small pieces as possible with a sterile scissor. For 16S rRNA gene amplification the primer set 515F (5′-GTG CCA GCM GCC GCG GTA A-3′)/907R (5′-CCG TCA ATT CMT TTR ADT TT-3′) was used for bacteria (Bates et al., 2011 (link); Xia et al., 2011 (link)) targeting V4-V5 region in order to minimize the overestimation due to intragenomic heterogeneity of 16S rRNA gene (Sun et al., 2013 (link)), and the primer set 519F (′-CAG CCG CGG TAA-3′)/915R (5′-GTG CTC CCC CGC CAA TTC CT-3′) was chosen for archaea due to its good specificity for the archaeal community (Yu et al., 2020 (link)). PCR amplification was performed with an initial denaturation step of 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s, after which there was a final extension step of 72°C for 10 min. Each sample was run in triplicate. Library construction was performed according to the NEBNext^® Ultra^™ II DNA Library Prep Kit for Illumina^® (New England Biolabs, United States). We selected Illumina HiSeq 2,500 and NovaSeq 6,000 platforms to perform the paired-end sequencing for bacterial and archaeal libraries, respectively.

From each terroir soil sample, total DNA extraction was performed using the DNeasy^® PowerMax^® Soil Kit (Qiagen, MD, USA), with slight changes to the manufacturer protocol. The protocol’s input material and lysis steps were adapted to fit different soil textures (for drier soils, 5 g of input soil were used instead of 10 g and an additional heating step was performed during lysis). DNA quality and concentration were assessed by NanoDrop^TM One and Qubit^TM 4 Flourometer analysis. Three biological replicates for each terroir were obtained and used as independent samples.

For metabarcoding, we extracted DNA from approximately 10 g of each soil sample using Qiagen DNeasy PowerMax Soil Kit. We used all samples collected or 21 per timepoint (63 total samples). Samples from each timepoint were extracted and sequenced separately (Table 1). We purified extracted genomic DNA using Qiagen DNeasy PowerClean Cleanup Kit. We used universal metazoan primers mlCOIint and HCO2198 from Leray et al. (2013 (link)) to amplify a fragment of the COI gene. Each reaction contained 10 µl of Phusion High‐Fidelity Master Mix (Thermo Fisher Scientific), 0.4 µl of each of the forward and reverse primers, 6.7 µl water, and 2.5 µl DNA. The reactions were run with the following protocol: [98°C × 3 min, 27× (98°C × 10 s, 46°C × 30 s, 72°C × 45 s), 72°C × 5 min]. Each sample was analyzed with three replicate PCRs. After verifying the PCRs’ success on 1.5% agarose gels, we pooled the three replicates together (21 total pools per timepoint) and quantified their DNA concentration using a Qubit. Sequencing was performed on a MiSeq v2 Nano flow cell with 2 × 250 bp paired‐end reads at the Genomics Core of the Research Technology Support Facility (RTSF) at Michigan State University. Raw reads were demultiplexed by the RTSF.

Sieved fresh soil (5.0 g) was used for DNA extraction with the DNeasy PowerMax Soil Kit (Qiagen, Hilden, Germany). The specific steps refer to the method of Duan et al. (2021) (link). The concentration of plasmid DNA was measured and converted to copy the concentration using the following equation as described by Whelan et al. (2003) (link): DNA (copy) = [6.02 × 10²³ (copies moL^–1) × DNA amount (g)]/[DNA length (bp) × 660 (g moL^–1 bp^–1)]. The primers and annealing temperatures are presented in Supplementary Table 3. Sterile water was used as a negative control to replace the template. All real-time PCR reactions were done in technical triplicates such that each treatment was analyzed nine times.

Total DNA was isolated from 1 mL of the examined cell suspension using the DNeasy PowerMax Soil Kit (Qiagen, Carlsbad, CA, USA). PCR fragments of the 16S rRNA gene were obtained with the universal primers 341F (5′-CCTAYGG-GDBGCWSCAG-3′) and 806R (5′-GGACTACNVG-GGTHTCTAAT-3′) [33 (link)]. The PCR fragments were barcoded with Nextera XT Index Kit v.2 (Illumina, San Diego, CA, USA) and purified using Agencourt AMPure beads (Beckman Coulter, Brea, CA, USA). The concentrations of the obtained PCR products were calculated using the Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). All PCR fragments were then mixed in equal amounts and sequenced on an Illumina MiSeq (2 × 300 nt from both ends). Pairwise reads were combined using FLASH v.1.2.11 [34 (link)]. The sequences were clustered into operational taxonomic units (OTUs) at 97% identity using Usearch [35 (link)]; low-quality reads, chimeras, and singletons were eliminated during clusterization using the Usearch algorithm. Taxonomic identification was carried out using the SILVA v.132 database and the VSEARCH algorithm [36 (link)].

Lesvos is located in the North Aegean Sea on the Aegean-Anatolian microplate between the converging African and Eurasian plates (Pe-Piper et al. 2019 (link)). Intense volcanic activity occurred between 18.5 and 17 Ma (Pe-Piper and Piper 1992 (link)), endowing a significant number of thermal springs. Wet soil samples from a hot spring in Polichnitos area (39°04′24.8″ N, 26°12′00.9″ E) were collected aseptically in sterile tubes in May 2019. The in situ temperature measurements reached 74 °C. The samples were stored at 4 °C upon return to the laboratory, and subsequent total DNA extraction was accomplished with a DNeasy^® PowerMax^® Soil kit (QIAGEN, Germany) using 10 g of each sample. Environmental DNA (eDNA) extraction was performed according to the manufacturer’s instructions. The extracted eDNA was concentrated by precipitation with NaCl/cold ethanol, and the resulting pellet was washed with ethanol solution (70% v/v) according to the manufacturer’s instructions. The quality of the purified eDNA was evaluated via agarose gel electrophoresis and quantity by measurements with a NanoDrop^® ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). The final yield was approximately 20 ± 7 ng/μL (average ± SD).

DNA was extracted from 10 g of soil using the DNeasy PowerMax Soil Kit^® (Qiagen) following the manufacturer’s instructions. DNA crude extract yields were calculated using Qubi^®t 2.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, United States) following the manufacturer’s instructions. DNA quality was evaluated using Nanodrop^TM (Invitrogen, Thermo Fisher Scientific, United States). After extraction, the total amount of eluted DNA (5 mL) was purified by using Amicon Ultra 5 mL Centrifugal Filters 30K NMWL (EMD Millipore Corporation, Billerica, MA, United States). The purified and concentrated DNA was diluted to 10 ng μL-1 and used in downstream steps.