Identification and genetic characterization of a novel Orthobunyavirus species by a straightforward high-throughput sequencing-based approach

Shifman, Ohad; Cohen-Gihon, Inbar; Beth-Din, Adi; Zvi, Anat; Laskar, Orly; Paran, Nir; Epstein, Eyal; Stein, Dana; Dorozko, Marina; Wolf, Dana; Yitzhaki, Shmuel; Shapira, Shmuel C.; Melamed, Sharon; Israeli, Ofir

In the protocol described in this study, the clinical sample was first grown in cell culture until a high viral titer was obtained, and the growth medium was then harvested. Previous experiments conducted by our group demonstrated that although the growth medium fraction contained less RNA than the cell pellet, the viral RNA was enriched in that fraction (data not shown). We used the QIAamp Viral Mini Kit for extraction and purification of the RNA since this is most suitable kit for the subnanogram amounts of RNA commonly present in the growth medium fraction. We found that carrier RNA, which was added to improve the yield of the extraction, normalized the amount of initial RNA needed for library preparation and did not interfere with downstream processes, improving upon previous protocols that included a carrier RNA depletion step [32] [33] [34] . We chose to prepare the HTS libraries using the SMARTer Pico RNA Kit for several reasons. The initial RNA amount requirements for this kit are among the lowest for commercial kits, and the RNA concentrations obtained from the RNA extraction step are within the required range. This kit uses random (and not oligo-dT) priming for first-strand synthesis and proprietary SMART technology for template switching and second-strand synthesis. These unique features favor the probability of obtaining full-length transcripts, even from nonpolyadenylated viruses, as in our case. In addition, depletion of the ribosomal cDNA originating from host cells is integrated into the kit and allows whole library preparation to be performed in a single tube and in a relatively short amount of time.