title: HIGH ARCHAEAL DIVERSITY IN ANTARCTIC CIRCUMPOLAR DEEP WATERS
design: Archaeal sequences were amplified by the use of domain-specific archaeal primers targeting a portion of the 16S rRNA (340F and 1000R, Gantner et al., 2011) complemented with pyrosequencing adapters at their 5’ ends, and a 5 bp sample-specific barcode sequence on the forward primer. The forward primer anneals to E. coli 16S rRNA positions 341-357 and the reverse primer anneals to positions 1000-1017. These were designed such that two barcodes differ in at least two positions and homo-polymers were lacking. The forward primer (340F) was slightly modified for increased coverage (e.g. a single base was removed from the 5´end of the primer, i.e. 5’-CCTAYGGGGYGCASCAG -3’; see Gantner et al., 2011). The resulting partial 16S rRNA gene sequences start at E. coli position 358 and include both more conserved and variable regions, the latter represented by hypervariable region V3 (Van de Peer et al., 1996) known to resolve closely related archaeal populations (Yu et al., 2008; Gantner et al., 2011).   PCR reactions (20 µL) contained a final conc. of 0.4 mg l-1 Bovine Serum Albumin, 0.5 mM MgCl2, 0,5 µM of each primer, 0.8 mM of each dNTP and 0,8 Units of KAPA2G Robust Hot Start polymerase (KAPABiosystems, Boston, USA). The template DNA concentration varied between 1 and 10 ng per reaction. The PCR cycles started with a 2 min initial denaturation at 98°C, followed by 30 cycles of 98°C for 30 sec., 57°C for 30 sec, and 72°C for 90 sec and a final 7 min elongation at 72°C. Triplicate samples were pooled and gel-purified using the Qiaquick gel purification kit as described by the manufacturer (Quiagen, Hilden, Germany). The purified amplicons were subject to pyrosequencing from the forward primer using a 454 FLX system (454 Life Sciences, Branford, CT, USA) at the Royal Institute of Technology (KTH, Stockholm, Sweden).
identifiers.namespace: Uppsala University
identifiers.id: Southern Ocean Archaea Survey