Microbial activity and survivability in heated and irradiated bentonite under deep geological repository relevant conditions
All the bentonite samples, together with all the positive culture samples, were sequenced. For samples with a low DNA yield (< 0.5 ng/L and Cq values > 18), two PCR reactions were performed with standard and barcoded fusion primers, while only one PCR reaction containing barcode fusion primers was performed in the case of samples with higher DNA concentrations ( 0.5 ng/L and Cq values < 18). PCR conditions were as follows: an initial cycle at 95C for 3 min, followed by 10 (first PCR)/35 (second PCR) cycles at 98C for 20 s, 50C for 15 s, and 72C for 45 s, with a final extension at 72C for 1min. The thermocycling conditions were the same for both the first and second PCR reactions, except for a difference in the number of cycles. For both PCR runs, EliZyme HS HIFI MIX polymerase (Elizabeth Pharmacon, Czech Republic) and the universal primers 515F (Dowd et al., 2008) and 802R (Claesson et al., 2010) were used for amplification of the hypervariable V4 region of the 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). The amplified PCR product was then purified using the Agencourt Ampure XP system at a 50:50 ratio of PCR product: Ampure XP paramagnetic beads (Beckman Coulter, USA), following the manufacturers protocol. The concentration of purified PCR product was measured using a Qubit 2.0 fluorimeter (Life Technologies, USA). Finally, barcode-tagged amplicons from different samples were mixed at equimolar concentrations (25nM solution in 20 L), and sequencing was performed on an Ion Torrent Genexus system (Thermo Fisher Scientific, USA) using the Genexus sequencing kit combo with the Ion GX5 chip (Thermo Fisher Scientific, USA), following the manufacturers instructions.