Microbial composition in permafrost-affected soils of the NW Canadian Arctic - Archea 16S
Site description and sample collection Active layer samples were collected from four low-center polygons in the Western Canadian Arctic, on Herschel Island and the Yukon Coast: Drained Lake Polygon (DP, N 69 deg 34M-bM-^@M-^Y43, W 138 deg 57M-bM-^@M-^Y25), Lake Polygon (LP, N 69 deg 36 00.6, W 139 deg 03 56.8), King Point Polygon (KP, N 69 deg 05 26, W 137 deg 56 45) and Mainland Polygon (MP, N 69 deg 28 23.7, W 139 deg 11 06.3 (Figure 2). The soil at all four sites were characterized as hemic glacistel classified according to the U.S. Soil Taxonomy (Soil Survey Staff, 1998) with poor drainage and a loamy soil texture. The vegetation period on Herschel Island and the Yukon Coast spans yearly from mid-June to end of September. Average air temperatures vary annually between -26.3 deg C in February to 8.7 deg C in July with temperatures at the surface of the active layer ranging from -35 deg C in the winter to 25 deg C in the summer (Burn and Zhang, 2009). The sampling sites were characterized by an active layer (the layer of ground that is subject to annual thawing and freezing) consisting of a large peat horizon, with a depth varying between 25 cm and 36 cm as measured using a permafrost probe. Replicated sampling was conducted as follows: a large pit was dug in the center of each polygon down to the permafrost table, exposing a clean vertical profile of over a meter in width. At every 5 cm depth, soil subsamples were taken with a sharp sterile knife to cover small-scale horizontal heterogeneity. Subsamples obtained from each layer were then homogenized into sterile 125 mL Nalgene screw-cap containers (Thermo Fischer Scientific Inc., Waltham, MA). The knife was wiped and sterilized with ethanol between different samples. Soil samples were frozen immediately after sampling and stored at M-bM-^HM-^R20 deg C upon arrival in the laboratory. All subsequent subsampling was performed under sterile and anaerobic conditions in an atmosphere-controlled glove box. DNA extraction and PCR amplification Total genomic DNA was extracted in duplicate from 0.6 g of soil using the PowerSoilM-bM-^DM-" DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA). Duplicates were then pooled for downstream analyses. The obtained genomic DNA extracts were quantified using the PicoGreen dsDNA quantitation assay (Invitrogen, Carlsbad, CA). DNA was then stored at -20 deg C for further use. 16S rRNA gene amplification was carried out using multiplex identifiers as described in Bell et al. (2013) for bacteria and Yergeau et al. (2012) for archaea. Reactions were carried out in 25 M-BM-5l volumes containing 2 M-BM-5l of template DNA, 0.5 M-BM-5M each primer and 12.5 M-BM-5l of KAPA2G Robust HotStart ReadyMix (KAPA Biosystems, Woburn, MA). Cycling conditions involved an initial 5 min denaturing step at 95 deg C, followed by 35 cycles of 30 s at 95 deg C, 30 s at 57 deg C, and 45 s at 72 deg C, and a final elongation step of 10 min at 72 deg C. PCR products were purified on 2.5% w.v-1 agarose gels using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) and quantified using the PicoGreen dsDNA quantitation assay. All 20 amplification products from the different samples were pooled in an equimolar ratio (one pool for Bacteria and one pool for Archaea) and sequenced together. A total of 3.50x107 molecules were used in an emulsion PCR using the Ion OneTouchTM 200 Template Kit (Life Technologies, Carlsbad, CA) and the OneTouchTM instruments (Life Technologies) according to the manufacturerM-bM-^@M-^Ys protocol. Sequencing of the pooled library was done using the Personal Genome MachineTM (PGM) system and a 314 chip with the Ion Sequencing 200 kit according to manufacturerM-bM-^@M-^Ys protocol.