Improving the diagnosis of cassava mosaic begomoviruses using Oxford Nanopore Technology sequencing.

Cassava mosaic disease (CMD), caused by begomoviruses such as African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV), poses a threat to food security in sub-Saharan Africa. Conventional PCR assays often fail to detect viral strains in symptomatic plants due to high genetic variability and recombination. In this study, we used Oxford Nanopore Technology (ONT) sequencing on 12 cassava leaf samples that had previously tested negative by PCR. We compared two strategies: direct sequencing of total plant DNA and sequencing after rolling circle amplification (RCA-MinION). Across the samples, we obtained 7,800-36,000 reads, of which 1,327-11,749 were viral reads after host filtering. While direct sequencing of total DNA detected CMD-associated reads without yielding full genomes, RCA-MinION enabled de novo assembly of complete ACMV and EACMV genomes (two to 14 contigs, N50 up to 22.2 kb). This revealed high genetic diversity, mixed infections and recombination. Building on these genomic datasets, we performed computational analyses to identify conserved genomic regions and palindromic motifs, which guided the rational design of new primers. These primers, which target the AV1, AC2, BV1 and BC1 regions, were validated in silico and by PCR. They achieved detection rates of up to 98% across diverse isolates and successfully amplified viral DNA in samples that had previously been undetected by standard primers. Palindromic motif analysis further reduced the risk of secondary structures, ensuring efficient primer binding. Sanger sequencing of the PCR products confirmed the specificity and robustness of the assays. Our findings suggest that ONT combined with RCA is a powerful tool for CMD diagnostics and surveillance, improving detection and providing the genomic insights that are critical for disease management and food security in West Africa.