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

Pico Input Mammalian (TaKaRa Bio) was used for library construction prior to sequencing on a MiSeq instrument (Illumina). This kit combines three technologies-SMART (switching mechanism at the 5′ end of RNA template) technology, LNA (locked nucleic acid), and a ribosomal cDNA depletion method-and the procedure can be completed in 5 hours. Five nanograms of the extracted RNA was used for fragmentation at 94 °C for 4 min. First-strand synthesis was performed using SMARTer oligonucleotides, a template-switching oligo mix, partial Illumina adaptor sequences, and SMARTScribe reverse transcriptase at 42 °C for 90 min and then at 70 °C for 10 min. The cDNA was then amplified by 5 cycles of PCR with SeqAmp DNA polymerase and adapters for Illumina sequencing (with specific barcodes) and purified by two rounds of clean-up with 1 × AMPure XP beads (Agencourt). Ribosomal cDNA was depleted by a ZapR-mediated process, in which the library fragments originating from rRNA (18S and 28S) and mitochondrial rRNA (m12S and m16S) are cleaved by ZapR in the presence of R-Probes (which are mammalian-specific). These R-Probes were hybridized to ribosomal RNA and mitochondrial rRNA sequences for 60 min at 37 °C and then for 10 min at 72 °C. The depleted cDNA fragments were further amplified with universal Illumina primers for 16 PCR cycles. Lastly, the PCR products were purified once again by a single round of clean-up with 1 × AMPure XP beads to yield the final cDNA library. The library was sequenced in single-read mode (60 nucleotides) on a MiSeq instrument using the V2 Kit.