Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).

The diagnosis of novel unidentified viral plant diseases can be problematic, as the conventional methods such as real‐time PCR or ELISA may be too specific to a particular species or even strain of a virus, whilst alternatives such as electron microscopy (EM) or sap inoculation of indicator species do not usually give species level diagnosis. Next‐generation sequencing (NGS) offers an alternative solution where sequence is generated in a non‐specific fashion and identification is based on similarity searching against GenBank. The conventional and NGS techniques were applied to a damaging and apparently new disease of maize, which was first identified in Kenya in 2011. ELISA and TEM provided negative results, whilst inoculation of other cereal species identified the presence of an unidentified sap transmissible virus. RNA was purified from material showing symptoms and sequenced using a Roche 454 GS‐FLX+. Database searching of the resulting sequence identified the presence of Maize chlorotic mottle virus and Sugarcane mosaic virus, a combination previously reported to cause maize lethal necrosis disease. Over 90% of both viral genome sequences were obtained, allowing strain characterization and the development of specific real‐time PCR assays which were used to confirm the presence of the virus in material with symptoms from six different fields in two different regions of Kenya. The availability of these assays should aid the assessment of the disease and may be used for routine diagnosis. The work shows that next‐generation sequencing is a valuable investigational technique for rapidly identifying potential disease‐causing agents such as viruses.

No abstract

No abstract

No abstract