title: Sharp-spined notothen associated microviruses
design: Approximately 1 g of stomach from sharp-spined notothen (Trematomus pennellii) caught using hook and line in McMurdo Sound during the summer field season of 20122013seven fish were grouped and each sample type was homogenized in 20 ml of SM buffer (0.1 M NaCl, 50 mM Tris/HClpH 7.4, 10 mM MgSO4) using a mortar and pestle. The homogenate was centrifuged at 10000g for 20 min. The resulting supernatant was sequentially filtered through 0.45 m and 0.2 m (pore size) syringe filters. 1.5 g of PEG 8000 (Sigma, USA) was added to 10 mls of the filtrate and mixed gently until dissolved, before being incubated overnight at 4 C. The solution was centrifuged at 10000xg for 20 mins and the resulting pellet was resuspended in 1ml of SM buffer. 200 l of this was used for viral DNA extraction using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, USA). Circular viral DNA was then enriched using rolling-circle amplification (RCA) with the TempliPhiTM kit (GE Healthcare, USA). The RCA products were sequenced on an Illumina HiSeq 2000 sequencer at Macrogen Inc. (South Korea).