Effect of temperature and pressure on proliferation of microorganisms in bentonite
For the library preparation, protocol with either one or two subsequent PCR reactions was used. For samples with low DNA yield (i.e., DNA concentration below 0.5 ng/l and Cq values above 18 approximately), two PCR reactions with standard and barcoded fusion primers were performed. In the case of samples with higher DNA concentration (i.e. DNA concentration  0.5 ng/l and Cq values approximately below 18), only one PCR reaction containing barcode fusion primers was performed. The PCR conditions were as follows: one cycle at 95 C for 3 min followed by 10/35 cycles at 98 C for 20 s, 50 C for 15 s, and 72 C for 45 s; finished by a final extension step at 72 C for 1min. The thermocycling conditions were the same for both 1st and 2nd PCR reactions except for the difference in the number of cycles, where the 1st reaction had 10 cycles and 2nd reaction had 35 cycles, respectively. In both the PCR's, universal primers 515F (Dowd et al., 2008) and 802R (Claesson et al., 2010) were used for the amplification of the hypervariable V4 region of 16S rDNA gene. The size of the amplicon was kept below 400 bp to cover as much microbial diversity as possible (Nmeek et al., 2017). EliZyme HS HIFI MIX polymerase (Elizabeth Pharmacon, Czech Republic) was used for library amplification. The amplified PCR product was purified using Agencourt Ampure XP system in 50: 50 ratio (PCR product: Ampure XP paramagnetic beads) (Beckman Coulter, Brea, CA, USA) following the manufacturer's protocol, and the concentration of the purified PCR products was measured with a Qubit 2.0 fluorimeter (Life Technologies, MA, USA). Gel-electrophoresis on 1% agarose gel was used to check the quality of the final library product. Finally, for sequencing, barcode-tagged amplicons from different samples were mixed in equimolar concentrations (25nM solution in 20 l). Sequencing of the amplicons was performed on an Ion Torrent Personal Genome platform (Thermo Fisher Scientific, USA) using Ion PGM Hi-Q Sequencing Kit with the Ion 314 Chip v2 (Thermo Fisher Scientific, USA) following the manufacturer's instructions.