organism: freshwater metagenome
description: The sample was collected at a site nearly 400km upstream from Manaus, Brazil at the end of the dry season (September, average rainfall for the month 50 mm). The sample was collected from a depth of 20 m from the Solimões-Amazon River (3°56'11,08" S, 63°10'14,45" W) with a 10 liter Niskin bottle on 17 September, 2008, at 2:00 pm. River depth and width at this location were 15 m and 2100 m respectively, and the sample was taken 600 m away from the shore. Four liters of water were filtered in 3 independent Sterivex filters. Each sample was sequentially filtered through a AP20 glass fiber filter (142 mm of diameter); 5 µm pore size polycarbonate filter (142 mm of diameter) and 0.22 µm-pore size Sterivex GV filters (Durapore, Millipore) using a peristaltic pump. Sterivex filters (retaining the 0.2-5µm-diameter planktonic cells) were filled up with lysis buffer (40 mM EDTA, 50 mM Tris/ HCl, 0.75 M sucrose) and stored at -20 ºC until DNA extraction. The solution was harvested from the filters and DNA was extracted using the Metagenomic DNA from Environmental Water Samples Kit (EPICENTRE, WI, USA). DNA integrity was checked by agarose gel electrophoresis and quantified spectrophotometrically in a NanoDrop ND 1000 instrument (Thermo Scientific, DE, USA). Five micrograms was used for sequencing in the 454 Sequencing GS FLX Titanium platform (Laboratório Nacional de Computação Científica, Petrópolis, Rio de Janeiro, Brazil).