IMIS - Marine Research Groups

Integrated samples were collected within the euphotic zone from the surface down to 5 m in deep lakes and from about 30 cm below the surface in shallow lakes.
Study Extent: Samples were collected once in 14 freshwater bodies (ranging from oligotrophic to eutrophic) from Chubut Province, Argentinean Patagonia, to Hope Bay, Antarctic Peninsula (45º22′ to 63º24′ S of latitude) (Fig. 1). In Antarctic lakes, samples were taken during the austral summer 2004, whereas in Patagonian lakes, samples were collected in late spring 2007 and 2008.
Method step description:

Water samples were pre‐filtered in situ through a 50 µm net to remove zooplankton, then filtered with a vacuum pump first through a 20 µm pore‐size polycarbonate filter and then through a 3 µm and 0.2 µm pore‐size polycarbonate filters (diameter 47 mm; Millipore, Cork, Ireland). The filters were placed in cryovials with 1.8 ml of lysis buffer (40 mM EDTA, 50 mM Tris‐HCl, 0.75 M sucrose) and stored at −80°C until DNA extraction. The 0.2–3 µm size fraction was used for this study. The procedures followed for DNA extraction (phenol/chloroform extraction) and touchdown polymerase chain reaction (PCR) amplifications were previously described in detail (Unrein et al., 2005).

We amplified extracted DNA using primers specific to the V9 variable region of the 18S rRNA gene using the protocol as in Amaral‐Zettler et al. (2009), and adapted after Lara et al. (2015). Sequencing was performed by the company Fasteris (Geneva, Switzerland) using Illumina HiSeq 2500 technology; paired end reads were around 200 bp in length.