Marine bacterial, archaeal and eukaryotic microbial communities on the continental shelf of the western Antarctic Peninsula

Study Extent

Sampling was conducted on the annual Palmer LTER (western coast of the Antarctic Peninsula) midsummer research cruise (January−February 2008)

Sampling

Samples were drawn from 10 and 100 m depths from the northern and southern, inshore and offshore corners of the Palmer LTER sampling grid. We collected duplicate samples using a rosette equipped with 10 l Niskin bottles and conductivity, temperature, and depth (CTD) probes. To contrast summer and winter water, we also collected an additional Austral winter sample from 10 m depth at the northern, inshore sampling site in August 2008, using a submersible pump with silicone tubing. Water samples were filtered (1 to 2 l) through 0.2 μm SterivexTM filters (Millipore), preserved genomic DNA by flooding the 2 ml filter cartridge reservoir with sucrose lysis buffer (40 mM EDTA, 50 mM Tris-HCl, 0.75 M sucrose), and stored the filters at −80°C until processing.

Method steps

1. We extracted DNA using a Puregene DNA extraction kit (Qiagen), with modifications as described by Amaral-Zettler et al. (2009), and stored the DNA at −20°C until PCR amplification. Bacterial and archaeal V6 16S rRNA and eukaryotic V9 18S rRNA gene hypervariable regions were amplified as described previously (Huber et al. 2007, Amaral- Zettler et al. 2009), using ‘barcoded’ primers which al- lowed for multiplexed sequencing (see http://vamps. mbl.edu/resources/primers.php for details). For each sample, we pooled triplicate 50 μl PCR reaction products to minimize propagation of PCR errors and purified them using a QIAquick column-based purification kit (Qiagen). We sequenced purified amplicons on a 454 Genome Sequencer FLX (Roche) according to the manufacturer’s protocols using the LR70 kit.

Bacteria (16S ssu rRNA gene, v6 region)

1. Bacteria
   common name: Bacteria rank: domain

Archaea (16S ssu rRNA gene, v6 region)

1. Archaea
   common name: Archaea rank: domain

Eukarya (18S ssu rRNA gene, v9 region)

1. Eukarya
   common name: Eukaryotes rank: domain

Southern Ocean, Continental shelf off the Antarctic peninsula