Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification

Malboeuf, Christine M.; Yang, Xiao; Charlebois, Patrick; Qu, James; Berlin, Aaron M.; Casali, Monica; Pesko, Kendra N.; Boutwell, Christian L.; DeVincenzo, John P.; Ebel, Gregory D.; Allen, Todd M.; Zody, Michael C.; Henn, Matthew R.; Levin, Joshua Z.

Plasma samples from HIV-1 chronically infected subjects were obtained from the Ragon Institute of MGH, MIT and Harvard in Boston, MA. All subjects gave written informed consent and the study was approved by the Massachusetts General Hospital Review Board. NL4-3 (28) virus stocks were produced by transfection of HEK293T cells (ATCC, Manassas, VA) with plasmid DNA encoding a full-length infectious HIV RNA using Polyfect transfection reagent (Qiagen, Valencia, CA) according to a modified manufacturer protocol. Briefly, 1 day prior to transfection, 2.8 Â 10 6 cells were seeded in a T75 flask. For the transfection, 15 mg of plasmid DNA, at a minimum concentration of 1 mg/ml, was diluted to a 150-ml final volume in Dulbecco modified Eagle medium without supplements; 115 ml of Polyfect reagent was added, and the solution was mixed by gentle pipetting and incubated for 10 min at room temperature. During the incubation, medium was removed from the cells to be transfected followed by a single wash with cold phosphate-buffered saline (PBS), and then 7 ml of fresh medium was added. After this incubation, the transfection mixture was transferred to the flask, swirled gently to mix, and incubated for 3 hours at 37 C with 5% CO 2 . The medium was removed and discarded; the cells were washed once with cold PBS, and 7 ml of fresh medium was added before returning the cells to the incubator. Transfection supernatant was harvested after 72 hours, filtered through a 0.45-mm filter, and stored in aliquots at À80 C. NL4-3 viral titer was determined by HIV-1 p24 antigen enzyme-linked immunosorbent assay (Perkin-Elmer, Waltham, MA) per the manufacturer's protocol.