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BackgroundHigh-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount.MethodsPlant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis.ResultsThe study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols.ConclusionsThis study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

Two isolates of the T68 genotype of citrus tristeza virus (CTV) were derived from a common source, GFMS12, by single aphid transmission. These isolates, named GFMS12-8 and GFMS12-1.3, induced stem pitting with differing severity in ‘Duncan’ grapefruit (Citrus × paradisi [Macfad.]). Full-genome sequencing of these isolates showed only minor nucleotide sequence differences totaling 45 polymorphisms. Numerous nucleotide changes, in relatively close proximity, were detected in the p33 open reading frame (ORF) and the leader protease domains of ORF1a. This is the first report of full-genome characterization of CTV isolates of a single genotype, derived from the same source, but showing differences in pathogenicity. The results demonstrate the development of intragenotype heterogeneity known to occur with single-stranded RNA viruses. Identification of genetic variability between isolates showing different pathogenicity will enable interrogation of specific genome regions for potential stem pitting determinants.

Over the past 2 decades, fruit symptoms resembling a marbling pattern on the fruit skin or corking of the fruit flesh were observed on Japanese plums in South Africa, resulting in unmarketable fruit. The ability of high-throughput sequencing (HTS) to detect known and unknown pathogens was exploited by assaying affected and unaffected fruit tree accessions to identify the potential aetiological agent of marbling and/or corky flesh disease. In this study, it is shown that the disease is associated with a previously undescribed small RNA with typical viroid structural features. The potential viroid was the only pathological agent consistently detected in all symptomatic trees by HTS, and the association with the symptoms was confirmed in field surveys over two seasons. To date, this RNA was not detectable by RT-PCR in seedlings raised from seeds collected from infected trees. Although the autonomous replication of this viroid-like RNA was not proven, it was shown to be transmissible by grafting and associated with a range of symptoms that include marbling on the fruit skin, corky flesh, reduced fruit size, irregular shape, and uneven fruit surface depending on the cultivar. Moreover, the circular RNA genome, consisting of 317 nucleotides, strongly supports that this viroid-like RNA is most likely a viroid for which the name plum viroid I (PVd-I) is proposed. The primary structure of this viroid showed a less than 90% nucleotide sequence identity to viroids of the genus Apscaviroid, with which it has close phylogenetic relationships and shares conserved structural motifs.

The molecular interactions between grapevine leafroll-associated virus 3 (GLRaV-3) and three grapevine cultivars were characterised by identifying small RNAs and genes with differential expression between healthy and infected grapevine using next-generation sequencing and RT-qPCR assays. Cultivar specificity was identified in the sRNA and gene expression analyses, and both approaches identified Chenin blanc-specific responses providing the first insight into the differential symptom expression observed between cultivars. Differentially expressed genes identified in all three cultivars, provide a potential pool of genes displaying a molecular response to GLRaV-3 infection, contributing to the characterisation of this virus-plant interaction.

The economic importance of the grapevine industry creates the need for the generation of genomic resources to ensure the sustainability of the industry. These resources will extend the knowledge of grapevine physiology, cultivar-specific characteristics and understanding how diseases and environmental conditions affect the plant. Small non-coding RNAs (sRNAs) are regulator molecules that can influence normal development and/or responses to environmental stimuli. In this study, next-generation sequencing (NGS) and bioinformatic analyses were used to identify sRNA species in grapevine phloem tissue. Forty-five novel mature Vitis vinifera miRNAs were identified and the presence of ten was validated using stemloop RT-qPCRs. Both non-coding and protein-coding gene regions were identified as putative phased loci of which the majority of the protein-coding gene loci were identified as disease resistance proteins. Potential phase-initiating miRNAs were also identified. A high number of sRNAs originating from repeat sequences were identified in all cultivar groups. This study also confirmed the non-random manner in which tRNA-derived sRNAs originate. The number of tRNA halves identified in Chenin blanc was lower compared to the other cultivar groups investigated. The identification of these different sRNAs extends the grapevine sRNA knowledge base significantly and can contribute to the unravelling of sRNA regulation in plants, potentially creating tools for grapevine functional studies.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs (sRNA) that play an essential role in the regulation of target mRNAs expressed during plant development and in response to stress. MicroRNA expression profiling has helped to identify miRNAs that regulate a range of processes, including the plant's defence response to pathogens. In this study, differential miRNA expression in own-rooted Vitis vinifera cv. Cabernet Sauvignon plants infected with grapevine leafroll-associated virus 3 was investigated with microarrays and next-generation sequencing (NGS) of sRNA and mRNA. These high-throughput approaches identified several differentially expressed miRNAs. Four miRNAs, identified by both approaches, were validated by stemloop RT-PCRs. Three of the predicted targets of the differentially expressed miRNAs were also differentially expressed in the transcriptome data of infected plants, and were validated by RT-qPCR. Identification of these miRNAs and their targets can lead to a better understanding of host-pathogen interactions involved in grapevine leafroll disease and the identification of possible targets for virus resistance.

Objectives:\ud \ud To enable analysis and comparisons of different relative quantitation experiments, a web-browser application called Harbin was created that uses a quantile-based scoring system for the comparison of samples at different time points and between experiments.\ud \ud Results:\ud \ud Harbin uses the standard curve method for relative quantitation to calculate concentration ratios (CRs). To evaluate if different datasets can be combined the Harbin quantile bootstrap test is proposed. This test is more sensitive in detecting distributional differences between data sets than the Kolmogorov–Smirnov test. The utility of the test is demonstrated in a comparison of three grapevine leafroll associated virus 3 (GLRaV-3) RT-qPCR data sets.\ud \ud Conclusions:\ud \ud The quantile-based scoring system of CRs will enable the monitoring of virus titre or gene expression over different time points and be useful in other genomic applications where the combining of data sets are required.

Background The use of next-generation sequencing has become an established method for virus detection. Efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome. Findings In this study, genome coverage at different sequencing depths was determined for a number of viruses, viroids, hosts and sequencing library types, using both read-mapping and de novo assembly-based approaches. The results highlighted the strength of ribo-depleted RNA and sRNA in obtaining saturated genome coverage with the least amount of data, while even though the poly(A)-selected RNA yielded virus-derived reads, it was insufficient to cover the complete genome of a non-polyadenylated virus. The ribo-depleted RNA data also outperformed the sRNA data in terms of the percentage of coverage that could be obtained particularly with the de novo assembled contigs. Conclusion Our results suggest the use of ribo-depleted RNA in a de novo assembly-based approach for the detection of single-stranded RNA viruses. Furthermore, we suggest that sequencing one million reads will provide sufficient genome coverage specifically for closterovirus detection. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0539-x) contains supplementary material, which is available to authorized users.