IMIS

In the studied lake (280 m long and 100 m wide, maximum depth around 6 m) the brines were located only within a deep trough in correspondence of which a pingo like feature (PLF) occurs. PLF is a frost mound that intruded the lake ice surface reaching a maximum height of 45 cm and extending within an area of approximately of 500 m2. Below that mound, brines have been preliminarily identified using GPR data; they were then reached and sampled through a 51 mm diameter borehole that was drilled in the center of the frost mound using a semi-portable core auger. In particular, the first pocket of liquid brine (TF1) was found between 3.78 m, and 3.98 m. TF1 was separated by the second pocket of liquid brine (TF2) (0.84 m thick) by only a 12 cm layer of ice (containing some organic material inclusions). Below TF2 additional frozen sediments rich in organic content occurred between 4.94 m and the bottom of the borehole (5.68 m). Both brines were collected in sterile Pyrex bottles using a peristaltic pump and sterile tubing. After collections, the brines were stored at −20 °C in the dark at Mario Zucchelli Station (MZS) prior to their delivery to laboratories for chemical and microbiological analyses.
Study Extent: Three replicate samples were taken from two separate brine layers in a lake in Tarn Flat area (75°4′S 162°30′E), an ice-free area (ca. 100 km2) in the north to the McMurdo Dry Valley in Victoria Land.
Method step description:

1. Total DNA from brines samples was aseptically extracted using Power Water DNA Isolation Kit (Qiagen, Germany) following the operating instructions. Prior to DNA extraction, the brines were thawed at 4 °C and aseptically filtered in order to collect the biomass present in each brine on sterile cellulose acetate filter (cutoff = pore size 0.2 µm Sartorius Stedim, Biotech, Germany). The quality and quantity of DNA extracted was determined by using QuBit 2.0 Fluorometer Assay (Life Technologies Corporation) and by NanoDrop 2000 c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Three replicates for each brine were run on Illumina MiSeq.
2. Fungal internal transcribed spacer region 2 (ITS2) was amplified using IlluAdp_ITS31_NeXTf 5′-CATCGATGAAGAACGCAG-3′ and IlluAdp_ITS4_NeXTr5′-TCCTCCGCTTATTGATATGC-3′60. The PCR products were sequenced using the Illumina MiSeq platforms, following the standard protocols of the company STAB Vida Lda. (Caparica, Portugal).