Metagenome assembly of an Amundsen Sea (Antarctica, Southern Ocean) water sample

Study Extent

Water samples were taken with a CTD Rosette equipped during a phytoplankton bloom event in the ASP in 2010–2011.

Sampling

A sample was taken from the surface water layer in the center of a bloom (073° 34′243S 112° 40′080W, chlorophyll a > 17 μg/L, temperature of −1.2°C, phosphate: 1.31 μM, nitrite: 0.02 μM, ammonium: 0.05 μM, silicate: 77.8 μM) on 19 December 2010. This sample (6 l, 10 m depth) was passed over a 20 μm mesh, collected onto a 0.2 μm Sterivex membrane filter cartridge by pressure filtration, quickly frozen in the headspace of a LN2 dewar and stored at −80°C.

Method steps

1. DNA extraction was performed using the Puregene kit (Gentra) after disruption of the cells with lytic enzyme coupled to proteinase K (Sinigalliano et al., 2007). DNA was quantified using a Nanodrop 2000 instrument (Thermo Fisher Scientific, Wilmington, DE).
2. Metagenomic libraries were generated with the OVATION ultralow kit (NuGen) using 100 ng of DNA and 8 amplification cycles. We constructed overlapping (2X100 nt with ~40 nt of overlap) and gapped (2X108 nt with an insert size of ~600 nt) metagenomic DNA libraries using a Pippin prep electrophoresis platform to precisely select the desired length for DNA fragments to be used for sequencing on a Hiseq platform (Illumina).

Amundsen Sea, Southern Ocean