metagenome from the oil reservoir
Total genomic DNA of the samples was extracted as described previously. The samples were stored at 20 C or analyzed immediately. Genomic DNA was extracted from both precipitates and membrane filters by using the method described previously. DNA concentration was determined by using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and confirmed by electrophoresis on a 1.5 % (w/v) agarose gel. Preparation of the libraries was performed in accordance with the 16S Metagenomic Sequencing Library Preparation Protocol recommended for Illumina MiSeq. The first round of amplification of the V3-V4 region of the 16S rRNA gene was performed using the DNA Engine Tetrad 2 cycler (Bio-Rad, Munich, Germany) with specific primers  (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and B (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). PCR amplification was carried out according to the following protocol: initial denaturation for 3 min at 95C, 27 cycles of 30 s at 95C, 30 s at 55C, and 30 s at 72C; and a final extension for 5 min at 72C. The purification of amplicons was performed using the Agencourt AMPure XP purification kit (Beckman Coulter, USA). The second round of amplification was performed for double indexing of samples using the same cycle parameters. The library obtained was validated using the Bioanalyzer 2100 system and the Agilent DNA 1000 Kit (Agilent, USA) and was quantified using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and Quant-iT dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Purified amplicons were pooled at equal concentration. Further preparation of samples and sequencing were performed using MiSeq Reagent Kit v2 (300 cycles) and MiSeq-device (Illumina, USA) according to the manufacturers protocol.