BE viral metagenome from Cuatro Cienegas Basin
Approximately ten-liter water samples were collected in polypropylene bottles previously sterilized. The concentration process was performed within twelve hours from the collection by the tangential ultrafiltration method (Polaczyk et al., 2008). Each sample  was separately filtered through a polysulfone filter F80A (Fresenius) with a 15 to 20 KDa cut-off point which was previously washed with a solution of NaPP 0.1%  and using a peristaltic pump Masterflex (Cole-Parmer) recirculating water at flow rate 1700 mL/min. The samples were concentrated about 100 times and preserved frozen. To purify nucleic acids from the concentrated samples, these were subjected to series of centrifugations. First, salt crystals and other debris were removed by low speed centrifugation (1800 x g for 30 minutes), followed by repeated 12-ml ultracentrifugation at 200,000 x g for 2 hours in a SW40 rotor (Beckman), keeping the pellet after each step. The final pellet was resuspended in filtered PBS, and subjected to additional centrifugation at 250,000 x g for 2 h in a SW55 rotor (Beckman). The supernatant was decanted and the pellet was resuspended in 450  l of PBS overnight at 4oC. Before extraction, purified samples were treated with Turbo DNAse (Ambion, USA) and RNAse (Sigma, USA) for 30 minutes at 37 C and immediately chilled on ice. Nucleic acids were then extracted from 200  l of material using PureLink Viral RNA/DNA kit according to manufacturer s instructions (Invitrogen, USA), employing linear  acrylamide in place of yeast tRNA as carrier. Nucleic acids were eluted in nuclease-free water, aliquoted, quantified in NanoDrop ND-1000 (NanoDrop Technologies, DE) and stored at -70 C until further use.  The random amplification of nucleic acids was performed essentially as described previously (MA Mercado). Reverse transcription was done using SuperScript III reverse transcriptase (Invitrogen, USA) and primer-A (5'-GTTTCCCAGTAGGTCTCN9-3'). The complementary DNA (cDNA) strand was generated by two rounds of synthesis with Sequenase 2.0 (USB, USA), followed by amplification with Phusion DNA polymerase (Finnzymes) using primer-B (5'-GTTTCCCAGTAGGTCTC-3'). The amplified products were purified with DNA Clean & Concentrator-5 kit (Zymo Research, USA), and digested with GsuI to remove 16 nucleotides from 5  and 3  ends generated by PCR. The resulting DNA was once more purified by DNA Clean & Concentrator-5 kit and used as input material for Illumina library construction.