An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids

Abstract

BackgroundDetection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets.ResultsWe sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21–25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21–22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21–22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21–22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens.ConclusionsWe have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1428-4) contains supplementary material, which is available to authorized users.

Small RNA-Omics for Plant Virus Identification, Virome Reconstruction, and Antiviral Defense Characterization

“…Small RNA deep sequencing analysis of Citrus sp. has revealed that the Petuvirus -like EVEs representing Citrus endogenous pararetrovirus spawn predominantly 24-nt siRNAs along with less abundant 21-nt and 22-nt siRNAs ( Barrero et al, 2017 ), further supporting the involvement of RdDM. Likewise, the Petuvirus -like EVE sequences at the centromeres of Fritillaria imperialis are associated with predominantly 24-nt siRNAs and methylated cytosines at both symmetric (CG, CHG) and asymmetric (CHH) sites, which is a clear signature of RdDM ( Becher et al, 2014 ).…”

“… Xu et al (2017) have reconstructed the virome of tomatoes in China by sequencing small RNAs from 170 samples and identified 22 viruses representing 12 genera of RNA and DNA viruses and 2 viroids (see “ Xu et al, 2017 ” in Supplementary Tables S1 , S2 ): the complete genomes were reconstructed from small RNAs by de novo and reference-based assembly for 13 of the 22 viruses and near complete ones (>90% genome sequence) for another 5 viruses. Likewise, small RNA-omics has been successfully used for reconstruction of the complex viromes of vegetatively cultivated sweet potatoes ( Kreuze et al, 2009 ; Cuellar et al, 2011 ; Kashif et al, 2012 ; De Souza et al, 2013 ; Mbanzibwa et al, 2014 ; Cuellar et al, 2015 ; Untiveros et al, 2016 ; Cao et al, 2017 ; see Supplementary Tables S1 and Supplementary List S1 ) and grapevines ( Pantaleo et al, 2010 ; Zhang et al, 2011 , 2014 ; Alabi et al, 2012 ; Giampetruzzi et al, 2012 ; Wu et al, 2012 ; Glasa et al, 2014 , 2015 ; Seguin et al, 2014 ; Maliogka et al, 2015 ; Chiumenti et al, 2016a ; Eichmeier et al, 2016 ; Barrero et al, 2017 ; Czotter et al, 2018 ; see Supplementary Tables S1 , S2 and Supplementary List S1 ), which led to identification of novel RNA and DNA viruses and viroids among other virome components. Mwaipopo et al (2018) have identified 15 viral species from 10 genera of RNA and DNA viruses in the virome of common bean (see Supplementary Table S1 and Supplementary List S1 ).…”

“…In effort to improve identification and reconstruction of different types of viral genomes from various hosts, Barrero et al (2017) have developed bioinformatics algorithms for small RNA assembly using selected 21-nt and 22-nt versus 24-nt reads which represent most abundant size-classes of viral siRNAs (discussed below). This work followed earlier studies where a host genome filtering step had been employed to allow for assembling viral small RNAs in longer contigs ( Li et al, 2012 ; Seguin et al, 2014 ) that, in some cases, represented the complete genomes of both RNA and DNA viruses and viroids ( Seguin et al, 2014 ).…”

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies

“…In these cases, accurate monitoring of newly emergent strains, that can occur by introduction or recombination, can be used for timely containment actions. A second application is an eco-genomic survey in certain crops or for certain virus groups, that is, run necessarily as a pilot study in geographic areas (see the example of Australia) [ 91 ] to check the virus status already present, and avoid the introduction of unwanted agents. Pursuing the former objective, Zablocki & Pietersen [ 92 ] and Read & Pietersen [ 93 ] provided evidence through HTS analysis regarding the isolates associated with the breaking of CTV cross-protection in grapefruit industry, characterizing recombination events, and fine population mapping, even at a single gene level.…”

High-Throughput Sequencing Facilitates Characterization of a “Forgotten” Plant Virus: The Case of a Henbane Mosaic Virus Infecting Tomato

“…Since contigs assembled from sRNA reads using the above-cited pipeline were relatively short, we performed an additional assembly of sRNA reads using SPAdes ( Bankevich et al, 2012 ; Nurk et al, 2013 ). The parameter “careful” and combined k-mer sets of 15, 17, 19, and 21 were applied to produce long assembled sequences ( Barrero et al, 2017 ). For each of the two sequencing approaches, all of the contigs matching potyvirus sequences were further assembled to obtain a complete or near complete viral genome sequence using CLC Genomic Workbench 10 (Qiagen).…”