RNA-seq soil from a military training ground
100 milligrams of each sample was transferred to a sterile 1.5 ml test tube. Metagenomic DNA was extracted using the DNeasy PowerSoil Pro Kits (Qiagen, Germany). DNA quality was analysed using the dsDNA BR fluorescent DNA concentration kit (Denovix, USA) and electrophoretically by agarose electrophoresis using a MIDI 1 device (Carl Roth, Germany). The V3-V4 variable region of the 16S rRNA gene was amplified using 341F (5- CCTAYGGGRBGCASCAG-3) and 806R (5-GGACTACNNGGGTATCTAAT-3) primers (Yu et al., 2005). Amplification was performed using Q5 DNA polymerase (NEB, USA) on a thermocycler Eppendorf MasterCycler gradient (Eppendorf, ). Reaction conditions: 98 C  2 min, with the following 20 cycles: 98 C  5 sec, 56 C  20 sec and 72 C  20  sec and the final step 72 C  5 min. Samples were purified using QIAquick Gel Extraction Kit (Qiagen, Germany). Sequencing adapters and individual barcodes were added by ligation. The amplicons were sequenced on an Illumina MiSeq instrument as a paired-end library (2 x 250 nucleotides) with a sequencing depth of 100000 reads from each end (Novogene Europe, Germany). The sequences of barcodes and adapters were removed using the program cutadapt version 3.3 (Martin, 2011) and united using the programme flash version 1.2.11 (Mago & Salzberg, 2011). Chimerical sequences were filtered using the software vsearch version 2.16.0 (Rognes et al., 2016).