Enhanced recovery of terminal sequences of viral RNAs via fine-tuning of the high-throughput sequencing library preparation and evidence of a non-templated nucleotide addition at the 3' end of the minus strand in members of the genus Ilarvirus.

High-throughput sequencing (HTS) technologies have transformed life sciences by enabling rapid, large-scale analysis of nucleic acids, providing unprecedented insights into genomics and transcriptomics. Accurate determination of RNA termini remains, however, a major challenge. We have addressed this limitation by fine-tuning a commercial HTS library preparation protocol. The optimized strategy was validated on plant virus isolates representing diverse taxonomic groups and genome architectures and benchmarked against conventional RACE, demonstrating its effectiveness and robustness. Investigations in members of the genus Ilarvirus allowed to prove the occurrence of non-templated nucleotide additions at the 3' end of the minus-strand viral RNAs in vivo, expanding previous results obtained in vitro in related members in the Bromoviridae and supporting that this feature may be common across the family.