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Reverse transcriptase template switching: a method for without a priori amplification of full-length genome of positive-sense single-strand polyadenylated RNA plant viruses. [P.51]

Claude L., Fernandez E., Roumagnac P., Filloux D.. 2019. In : Livre des résumés des 17 ème Rencontres de virologie végétale. Aussois : INRA, p. 110-110. Rencontres de Virologie Végétale (RVV 2019). 17, 2019-01-27/2019-01-31, Aussois (France).

In the absence of sequence information, obtaining the full-length sequence of RNA plant virus genomes is a real challenge. These viruses have linear genomes, sometimes relatively long (up to ~ 20 kb), and variable sequence ends which makes any PCR amplification impossible without a priori. While 3 'ends of polyadenylated viruses can be obtained by exploiting the presence of the poly-A tail, obtaining the 3' ends of non-polyadenylated viruses and 5 'ends requires artificial polyadenylation using, for example, Poly (U) Polymerase (3'RACE) and Terminal Transferase (5'RACE), respectively. We present here a method that can potentially amplify the full-length sequence of RNA plant virus genomes. We first focus towards obtaining the complete genome of positive-sense single-strand polyadenylated RNA plant viruses. The method called reverse transcriptase template switching, simultaneously uses the reverse transcriptase and template switching activities of the Moloney murine leukemia virus Reverse Transcriptase to synthesize in one step cDNAs flanked at each end by arbitrary sequences called anchors. These anchors then allow long-range PCR amplification of the cDNAs. The product of this amplification has been particularly adapted to be sequenced by the Nanopore technology which allows the sequencing of long fragments (100 bp to >20 kb). We here show that the nearly full-length sequences of both yam mild mosaic virus (Potyvirus genus) and yam chlorotic necrosis virus (Macluravirus genus), members of the large and socio-economically relevant Potyviridae family, could be obtained using the reverse transcriptase template switching approach followed by Nanopore sequencing. Studies aiming at obtaining full-length sequence of non-polyadenylated RNA plant virus genomes are underway.

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