Accurate and sensitive diagnosis of geminiviruses through enrichment, high-throughput sequencing and automated sequence identification

Existing diagnostic techniques used to identify plant-infecting DNA viruses and their associated molecules are often limited in their specificity and can be challenged by samples containing multiple viruses. We adapted a simple method of amplifying circular viral DNA and, in combination with high-throughput sequencing and bioinformatic analysis, used it as a virus diagnostic method. We validated this diagnostic method with a plant sample infected with a tomato yellow leaf curl geminivirus infectious clone and also compared PCR- and high-throughput-sequencing diagnostics on a geminivirus-infected field sample, showing that both methods gave similar results. Finally, we analyzed infected field samples of pepper from Mexico and tomato from India using this approach, demonstrating that it is both sensitive and capable of simultaneously identifying multiple discrete DNA viruses and subviral DNA elements in densely infected samples.