IMIS

Access through the ice lid was accomplished using a 50-mm diameter Kovacs ice drill, which was cleaned between each hole with antibacterial wipes. Water samples were collected directly into three 60-mL centrifuge tubes for determination of major ions and isotopes, using a manual vacuum apparatus. A sample (1 L) was collected by vacuum into an acid-washed PVC bottle. A subsample (100 mL) filtered (Whatman GF/F) into an acid-washed polyethylene bottle and frozen for later nutrient analysis.

Study Extent: Cryoconite holes were sampled in the austral summer (December–January), during Antarctic field seasons between2010 and 2013 at the Koettlitz Glacier and Diamond Glacier (Southern Victoria Land, Antarctica). Only cryoconite holes with a diameter of greater than 30 cm (which were typically also greater than 35 cm deep) routinely contained liquid water, and were targeted for sampling. Selection was based on replicate N+1 being the next encountered appropriately sized closed cryoconite hole encountered in a random direction from replicate N.

Method step description:

1. DNA was extracted from sediment using the MoBio Power Soil kit (USA). Biosystems, USA). A region of the 16S rRNA gene covering the V3 and 4 sections was ampli ed by PCR (iCycler; Biorad) using bacterial-speci c primers 515F and 806R (Caporaso et al. 2011). PCR reactions were performed in 50 μL volumes with the reaction mixture containing; 45 μL of Platinu PCR SuperMix High Fidelity (Life Technologies, USA), 10 μM of each primer, and 10–20 ng of template DNA. The reaction mix- ture was held at 94◦C for 2 min followed by 27 cycles of 94◦C for 30 s, 54◦C for 30 s, 68◦C for 45 s, with a nal extension step at 68◦C for 5 min. PCR products were puri ed (Agencour AMPur XP Kit; Beckman Coulter, USA), quanti ed (Qubit 20 Fluorometer, Life Technologies, USA), diluted to 1 ng μL−1 and submitted to New Zealand Genomics Limited (Auckland, New Zealand) for library preparation. Libraries were sequenced on a MiSeq Illumina platform (2 × 250 reads).