Viral Genome Sequencing Protocol

The extracted RNA was processed for random reverse transcription as previously described [1 (link),2 (link)] using the FR26RV-N primer (5' GCC GGA GCT CTG CAG ATA TCN NNN NN 3') at a concentration of 1 μM. In addition, FR40RV-T (5' GCC GGA GCT CTG CAG ATA TC (T)₂₀ 3') was added at a concentration of 5 nM to specifically amplify the 3' end of positive strand viruses. After the first cDNA synthesis, the double stranded cDNA was synthesized by Klenow reaction the presence of random primers. In order to amplify 5' ends of rhinoviruses the following primer was added to the Klenow reaction at a concentration of 10–20 nM (5'GCC GGA GCT CTG CAG ATA TC TTA AAA CTG G 3'). PCR amplification used high fidelity Taq Gold DNA polymerase (ABI) with the FR20RV primer (5' GCC GGA GCT CTG CAG ATA TC 3'). PCR amplicons were A-tailed with dATP and 5 units of low fidelity DNA polymerase (Invitrogen) at 72°C for 30 minutes. A-tailed PCR amplicons were analyzed in a 1% agarose gel and fragments between 500 and 1000 nt were gel purified. Amplicons were ligated en masse into the Topo TA cloning vector (Invitrogen) and transformed into competent one shot Topo top 10 bacterial cells (Invitrogen). For DNA viruses, the purified viral DNA was denatured and complementary strands synthesized by Klenow reaction as indicated for ds-cDNA from first strand cDNA. Clones were plated on LB/Amp/XGal agar, and individual colonies were picked for sequencing. The clones were sequenced bidirectionally using the M13 primers from the topo TA vector. We routinely sequenced a total of 192 or more per library. Sequencing reactions were performed at the Joint Technology Center (an affiliate of the J Craig Venter Institute: JCVI) on an ABI 3730 xl sequencing system using Big Dye Terminator chemistry (Applied Biosystems).