title: Titanium9 Barcelona raw sewage
design: 100 µL of virion concentrate was treated with DNase to remove non-virion associated DNA. Virion nucleic acid was purified from the DNase treated sample and 100 µL of untreated virus prep using the Qiagen DNeasy Blood and Tissue Kit (69504) using the “Animal Blood (Spin-Column Protocol)” except that elution was performed with 30 µl of dH2O. To enable subsequent detection of both RNA and DNA viruses, total virion-associated nucleic acid from each sample was reverse-transcribed and amplified as follows. Briefly, RNA templates were reverse transcribed using PrimerA (5’-GTTTCCCAGTCACGATANNNNNNNNN) containing a 17-nucleotide specific sequence followed by 9 random nucleotides for random priming.  Sequenase (United States Biochemical) was used for second-strand cDNA synthesis and for random-primed amplification of DNA templates using PrimerA. Each sample was then subjected to 40 cycles of PCR amplification using a barcoded PrimerB (5'-XXXXXXGTTTCCCAGTCACGATA) using the following program: 30 s at 94°C, 30 s at 40°C, 30 s at 50°C, and 60 s at 72°C. The amplified material was visualized on an agarose gel as a final quality control step and was sequenced at the Washington University Genome Sequencing Center on the 454 GS FLX Titanium platform (454 Life Sciences) according to manufacturer’s instructions.