454 gs flx titanium technology

Filtered water samples were PCR-amplified as described in Herlemann et al. (2011) (link). In brief, 30 ng of the extracted DNA was amplified using the primers Bakt_341F and Bakt_805R, complemented with 454 adapters and sample-specific 5-bp barcodes. The PCR conditions consisted of a denaturing step of 95° for 5 min, 25 cycles of 40 s at 95°C, 40 s at 53°C, and 60 s at 72°C, and a final extension step of 5 min at 72°C. The resulting amplicons were purified using Agencourt^© AMPure^® XP (Becker Coulter), quantified with the Picogreen assay (Molecular Probes), mixed in equimolar amounts, and sequenced from the reverse primer direction by MWG Eurofins using Roche/454 GS FLX Titanium technology. The raw sequences from the February cruise were deposited in the ENA Sequence Read Archive under accession number PRJEB14590 (July data are deposited under ENA accession number PRJEB1245).

Water (Table 1) was filtered over 0.22 µm polycarbonate membrane filters (293 mm diameter; Nuclepore; Whatman) to remove cellular organisms. Subsequently, viruses in the filtrate were concentrated successively using a spiral-wound ultra-filtration device suitable for large volumes (PTHK Prep/Scale-TFF; Millipore; Atlantic Ocean: 30 kDa molecular weight cutoff [MWCO]; Mediterranean Sea: 100 kDa MWCO) followed by a smaller ultra-filtration device (Vivaflow 200; 30 kDa MWCO; PES; Sartorius Stedim Biotech) until a final volume of ∼50 mL was reached. The virus concentrates were flash-frozen in liquid nitrogen and stored at −80°C until processed. Subsequently, virus concentrates were thawed and further concentrated to a final volume of 200 µL using Amicon Ultra-15 centrifugal ultra-filtration devices (Ultracel; 30 kDa MWCO; Millipore) by repeatedly loading ∼15 mL of concentrate onto the filters until the entire volume of each concentrate was passed through one filter. DNA extraction was performed using a QIAmp MinElute Virus Spin Kit (Qiagen) following the manufacturer's instructions. Viral metagenomic data sets were obtained from at least 100 ng of DNA per sample by pyro-sequencing on 1/4 of a pico-titer plate using Roche-454 GS FLX Titanium technology performed at the Broad Institute of MIT and Harvard (Cambridge, USA).