Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

Stenglein, Mark D.; Jacobson, Elliott R.; Wozniak, Edward J.; Wellehan, James F. X.; Kincaid, Anne; Gordon, Marcus; Porter, Brian F.; Baumgartner, Wes; Stahl, Scott; Kelley, Karen; Towner, Jonathan S.; DeRisi, Joseph L.

Sanger sequencing and RACE. The sequencing-based virus genome assembly was validated using RACE and Sanger sequencing. PCR mixtures contained 1ϫ iProof High-Fidelity DNA polymerase mix (Bio-Rad), 0.5 M (each) primer, and 2 l diluted cDNA, prepared as described above. Thermocycling conditions were 98°C for 30 s and then 40 cycles of 98°C for 10 s, 60°C for 20 s, and 72°C for 1 min, with a final 5-min 72°C extension. Primers were designed to tile across the assembly in over-lapping~1,200-bp amplicons (Fig. 4B) (78) . Amplicons were verified on agarose gels and sequenced directly. In some cases, amplicons were gel purified with the Zymoclean kit (Zymo Research), cloned into the pCRBlunt vector (Invitrogen) according to the manufacturer's protocols, and sequenced. 5= and 3= RACE was used to validate end sequences, essentially as described previously (24, 25) , with primers listed in Table S1 in the supplemental material. Multiple RACE amplicons were cloned and sequenced as described above. The complete and validated genome se-quence has been deposited with the NCBI (see below). Subgenomic RNA junctions were amplified and sequenced similarly.