IMIS

Study Extent: Soils samples were taken from King George Island (Fildes Peninsula) and Nelson Island during the 61st Russian Antarctic expedition in 2016.

Method step description:

1. Following the manufacturer’s instructions, DNA was extracted from the 0.2 g soil samples using the PowerSoil DNA Isolation Kit (Mobio Laboratories, Solana Beach, CA, USA), which included a bead-beating step. The samples were homogenized twice using a Precellys 24 tissue homogenizer (Bertin Corp, USA) for 30 s at 6.5 m s-1. The purity and quantity of the DNA were tested by electrophoresis in 0.5 × TAE buffer on 1% agarose gels. DNA concentrations were measured at 260nm using a SPECTROStar Nano microplate reader (BMG Labtech, Ortenberg, Germany). The average DNA yield was 2–5 μg at concentrations of 10–50 ng μL-1. The purified DNA templates were amplified with the universal multiplex primers F515 5′-GTGCCAGCMGCCGCGGTAA-3′ and R806 5′-GGACTACVSGGGTATCTAAT-3′ (Bates et al. 2011), which target the variable V4 region of bacterial and archaeal 16S rRNA genes. Each multiplex primer contained the adapter, a 4 bp key (TCAG), a 10 bp barcode, and the primer sequences. The amplified product was expected to be 400 bp long. The purification, pooling and pyrosequencing of the amplicons were performed with specified reagents according to the manufacturer’s instructions (Roche, Branford, USA). Pyrosequencing was performed with a Roche 454 GS Junior system in the Core Centrum ‘Genomic Technologies, Proteomics and Cell Biology’ (All-Russia Research Institute for Agricultural Microbiology).