METAGENOMIC DEEP-SEQUENCING: A PROMISING TOOL TO ELUCIDATE ETIOLOGY

Next-generation high-throughput sequencing is fast gaining credibility and popularity as a tool to determine the genetic composition of environmental samples, mainly because of the all inclusive and unbiased nature of analyzing completely unknown samples. We describe elucidating the etiology of virus diseases of grapevine by implementing a bioinformatic pipeline to analyze metagenomic sequence data. Double stranded RNA was isolated from 44 pooled, randomly selected vines from a leafroll-diseased vineyard in South Africa, and used in a deep sequencing analysis to build a census of the RNA virus population. The dsRNA was sequenced using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II, and yielded 837 megabases of metagenomic sequence data. Sequence reads were assembled into scaffolds using Velvet de novo assembler. Multiple de novo assemblies were performed with a range of parameter settings to determine the optimal assembly conditions for this data set. Scaffolds from the optimized de novo assemblies, were subjected to BLAST searches against the NCBI databases and the top hit scores used for virus identification. Based on the BLAST results, full-length genome sequences were selected from the NCBI database and used as reference sequences in MAQ re-assembly analysis. Four known grapevine viral pathogens were identified; as expected Grapevine leafroll-associated virus 3 was found to be the most prevalent, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was also identified in the study. Moreover, novel sequences aligning to mycoviruses not previously identified in grapevine, were also detected. The identification of all the viruses present in the leafroll-diseased vineyard will assist in the elucidation of the viral disease etiology. This methodology is not restricted to viral diseases and can be applied to investigate the etiology of diseases caused by other pathogens.