Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X<sup>+</sup> Genome Contains a 3.7 kb Internal Duplication
2023
Hailun Ma | Trent J. Bosma | Arifa S. Khan
We previously reported a novel rhabdovirus produced from the <i>Spodoptera frugiperda</i> Sf9 cell line, designated as Sf-rhabdovirus X<sup>+</sup> since it contained a unique accessory gene X. The Sf-rhabdovirus X<sup>+</sup> genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio’s single-molecule real-time sequencing and Oxford’s Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X<sup>+</sup> cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767–8523 in the X<sup>+</sup> virus. Thus, the X<sup>+</sup> genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X<sup>+3.7</sup>. The 3.7 kb duplication was found in all Sf9 cell clones producing the X<sup>+</sup> variant virus. Furthermore, the Sf-rhabdovirus X<sup>+3.7</sup> genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.
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