IMIS - Marine Onderzoeksgroepen

University Valley permafrost core samples were collected with a SIPRE corer (USA Cold Regions Research and Engineering Laboratory, Hanover, NH, USA). The cores were 0.3 km apart from each other. Samples were shipped to McGill University in a thermally insulated box and maintained at −20 °C until processing. All sample handling and processing were carried out according to protocols developed for low biomass permafrost soils to minimize contamination. Initial core processing took place in a walk-in freezer held at −5 °C in a dedicated laminar flow hood where 1 cm of the outside of the core was removed with a sterilized chisel. An additional 1 cm of the outside core was removed in a biological safety cabinet at room temperature immediately before samples being weighed and aliquoted for analysis. Dedicated biological safety cabinets, which had been pretreated to remove DNA/RNA or cells, were used for sample processing, nucleic acid extraction and PCR preparation.
Study Extent: Samples were taken in December 2009 from permafrost soils in University Valley (1650–1800 m above sea level (a.s.l.)), located 450 m above Beacon Valley in the Quartermain Range. Samples analysed in this study were located near the head of the valley close to the glacier, a shadowed region where the soil surface experiences few thaw hours, and where the uppermost 50 cm of ice content in the ice-cemented permafrost formed from water vapour diffusion into the cryotic soils rather than from liquid water.
Quality Control: Negative controls (H2O in place of soil) underwent identical handling during the extraction procedure and were used as templates for PCR using 16S rRNA gene primers (27 F and 1492 R) to ensure no contamination during extraction.
Method step description:

1. Community DNA was extracted from 2 g of permafrost soil using the UltraClean Soil DNA Isolation kit (MoBio Laboratories Inc., Carlsbad, CA, USA), as described in the alternative protocol for maximum yield, and a bead beating step was added to aid lysis. For each sample, five extractions were performed and the resulting DNA was pooled and concentrated.
2. DNA was sent for small subunit rDNA pyrosequencing analyses at the Research and Testing Laboratory (Lubbock, TX, USA) using the Roche 454 GS-FLX platform (Roche 454, Branford, CT, USA). Sample libraries were prepared with the following primers for bacterial 16S rRNA gene (28F-5′-GAGTTTGATCNTGGCTCAG-3′, 519R-5′-GTNTTACNGCGGCKGCTG-3′), archaeal 16S rRNA gene (349F-5′-GYGCASCAGKCGMGAAW-3′, 806R-5′-GGACTACVSGGGTATCTAAT-3′), Eukaryal/fungal internal transcribed region (ITS) (ITS1F-5′-CTTGGTCATTTAGAGGAAGTAA-3′, ITS4R-5′-TCCTCCGCTTATTGATATGC-3′) genes.