IMIS - Marine Onderzoeksgroepen

Water samples were collected with a Van Dorn bottle. Sediments were sampled thanks to a grab-sampler, peat monoliths (approximately 30*30*30cm) were cut with a bread-knife and soil monoliths with a shovel.
Samples were collected in summer 2015, without any temporal replication. A total of 19 ecosystems were studied in Alaska, USA. The selected sites are representative of this Subantarctic region: lakes, peatlands, Nothofagus forest, pampa In each site, various samples were collected to take into account the local heterogeneity: different depths in water column and sediments, soil horizons, hollows/edges/hummocks.
After collection, samples were stored at 4°C prior to further processing. Liquid samples were filtered at 0.45µm until clogging and the filters were stored at -20°C. DNA was extracted from these filters using the PowerWater DNA isolation kit (MOBIO) while DNA was extracted from solid samples using the PowerSoil DNA isolation kit (MOBIO). DNA extracts were kept at -20°C. The V4-V5 region of 16S rRNA gene was amplified in the following conditions: 515F and 928R primers (Wang & Qian, 2009. doi:10.1371/journal.pone.0007401), 2min at 94°C, 30 cycles of 60s at 94°C, 40s at 65°C and 30s at 72°C, and 10 min at 72°C. Amplicon sequencing was carried out with Illumina MiSeq technology (2x250pb, V3). Denoising of the sequences dataset and OTU clustering was carried using the FROGS pipeline (Auer et al., 2017. doi:10.1093/bioinformatics/btx791). BLAST was used for taxonomic affiliation.