SALDOR-Seq: a novel workflow for full-length sequencing of long dsRNA

Abstract Long double-stranded RNA (dsRNA) is a hallmark of viral replication and a prototypical pathogen-associated molecular pattern, yet it remains difficult to sequence because of its relative rarity, thermostability and poor compatibility with conventional sequencing workflows. We introduce Size-Accurate Long Double-stranded RNA-Sequencing (SALDOR-Seq), a dual-selection approach that combines stringent nuclease-based depletion of single-stranded RNA with adapter ligation and single-primer amplification to selectively recover intact dsRNA and preserve native length. SALDOR-Seq achieves high specificity and ~15,000-fold higher dsRNA recovery than short-read workflows, enabling full-length sequencing of viral dsRNA genomes exceeding six kilobases. Applied to RNA from virus-infected cells, it resolved replication-associated dsRNA intermediates, identified A-to-I editing within cytomegalovirus-derived dsRNA, and, through metagenomic analysis, revealed occult mycovirus infection in Aspergillus fumigatus. SALDOR-Seq therefore provides a dedicated, length-preserving method for high-confidence dsRNA profiling in complex samples and expands experimental access to dsRNA relevant for the study of viral replication, innate immunity and pathogen detection.