Next-Generation Sequencing for Diagnosis of Viruses

Abstract

Next-generation sequencing (NGS) is an unbiased approach to plant viral disease diagnosis that requires no prior knowledge of the host or the pathogen. The method involves extraction of total DNA or RNA or small RNA from the infected plant, the production of cDNA and, finally, sequencing, which produce sequences from a large range of potential pathogens. Analysis of the sequence data, development of contigs, and blasting contigs sequence against plant virus database aids in the identification of potential viruses. The results of the NGS can then be verified by designing virus-specific primers (based on the sequence of the contigs that hit viruses) and subjecting them to RT-PCR using total RNA from the infected plant, cloning and sequencing of the RT-PCR product by Sanger sequencing method. Once verified, more routine assays based on nucleic acid such as RT-PCR, real-time RT-PCR, loop-mediated isothermal amplification and recombinase polymerase amplification can be developed for quick and reliable detection of the virus in plants. The sequence data analysis is mainly based on bioinformatics software that can distinguish between host plant and viral sequences, and provides new opportunities in the areas of virus diagnosis and viral ecology.