DNA-Seq of glacier, 3119 m
Identification of microorganisms in samples was carried out based on analysis of the nucleotide sequence of the 16S rRNA gene. DNA from the samples was isolated according to the method described by I.A. Vladimirov. (Vladimirov, 2014). Primers used: 16S Amplicon PCR Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, 16S Amplicon PCR Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.Amplicons were produced in a volume of 20 l using a Tertsik amplifier. The reaction mixture contained 1 M forward and reverse primers, 2.5 units of a. "Taq" polymerase ("Beagle", Russia), buffer offered by the enzyme manufacturer (Mg++ concentration in 1x buffer 2.5 mM), additional 2% BSA and 5 mM of each dNTP and an agarose fragment containing total DNA of the sample under study (in in the case of primary amplification) or 1 l of the primary amplification in the case of the second stage of amplification. Amplicons were purified (removal of unproduced primers) using the size selection procedure on magnetic particles. AMPure XP (Beckman) was used according to the manufacturer's protocol.Diversity analysis was performed using the Qiime software package (version 1.9.0) according to a standard protocol. The classification was carried out based on comparison with the Greengenes database.