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Abstract: Viroidne bolezni predstavljajo nenehno grožnjo svetovnemu kmetijstvu, saj lahko ti majhni in strukturno preprosti patogeni okužijo širok spekter gospodarsko pomembnih rastlin. Učinkovito preprečevanje in obvladovanje viroidnih okužb zato zahteva diagnostične pristope, ki omogočajo zanesljivo, občutljivo in specifično identifikacijo povzročiteljev bolezni. Z razvojem biokemije in molekularne biologije se je v zadnjih letih izboljšala občutljivost in specifičnost številnih diagnostičnih metod. Z zniževanjem stroškov sekvenciranja in večjo dostopnostjo se zelo hitro povečuje zanimanje za uporabo tehnologij visokopretočnega sekvenciranja (HTS) v rutinski rastlinski diagnostiki. Kljub velikemu potencialu HTS pa uvedba teh tehnologij v laboratorijsko prakso ostaja zahtevna. Med ključne izzive sodijo izbira ustrezne sekvenčne platforme in laboratorijskih protokolov, uporaba primernih bioinformacijskih orodij, zagotavljanje strokovno usposobljenega kadra ter vzpostavitev ustrezne računalniške infrastrukture. Čeprav so že oblikovani določeni standardi in smernice za uporabo HTS, ti zaradi specifičnih potreb laboratorijev niso univerzalno uporabni. Pregledni članek obravnava ključne vidike različnih pristopov HTS, njihove prednosti in omejitve ter prikazuje njihovo uporabnost na primeru diagnostike viroidnih bolezni.
Keywords: diagnostika, visoko-pretočno sekvenciranje (HTS), nanopore tehnologija (ONT), bioinformatika, viroidi
Published in DiRROS: 26.02.2026; Views: 65; Downloads: 58
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Abstract: Phytoplasmas are non-culturable obligate intracellular bacteria that cause considerable economic losses in agriculture. Genome sequencing provides crucial insights into their biology and vector dependence. However, genome studies on phytoplasmas are often hampered by their low abundance in naturally infected plants. Propagation in test plants is usually necessary but time-consuming and resource-intensive, especially for quarantine phytoplasmas such as the phytoplasma causing Flavescence dorée (FD), a serious threat to European viticulture. To overcome these challenges, we aimed to develop a protocol for efficient enrichment of phytoplasma DNA directly from field-collected samples, enabling genome sequencing using both Illumina and Oxford Nanopore Technologies platforms. We evaluated six sample preparation protocols that included stepwise enrichment steps to improve phytoplasma genome coverage and assembly quality. The most effective approach combined differential centrifugation, CTAB extraction and removal of CpG-methylated host DNA and resulted in a notable increase in the relative abundance of phytoplasma reads compared to other protocols. Rarefaction analysis of the dataset generated using this protocol demonstrated that the entire phytoplasma genome was covered by reads in a dataset comprising 3 billion nucleotides. We also evaluated and compared de novo phytoplasma genome assemblies generated from short Illumina reads and long nanopore sequencing reads. While Illumina sequencing yielded more accurate assemblies with longer total lengths, the assemblies derived from nanopore sequencing data contained longer individual contigs. This advantage was reflected in hybrid assemblies that combined both technologies, yielding longer phytoplasma contigs than assemblies from Illumina datasets and lower mismatch rates compared to assemblies from nanopore sequencing datasets. A hybrid de novo assembled genome of the Slovenian FD phytoplasma isolate achieved 96% reference genome coverage, with high contiguity and low error rates. This streamlined and accessible protocol enables high-quality genome sequencing of phytoplasma-infected grapevines without the need for propagation in test plants. This facilitates broader phytoplasma research and can potentially be extended to other naturally infected phytoplasma hosts or organisms infected with other non-culturable microbes.
Keywords: plant pathogen, nanopore, grapevine, enrichment, HTS, Flavescence dorée, phytoplasma
Published in DiRROS: 08.10.2025; Views: 378; Downloads: 197
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Keywords: high throughput sequencing, HTS, viruses, metagenomic analysis, doctoral dissertations
Published in DiRROS: 05.09.2025; Views: 471; Downloads: 203
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Abstract: The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.
Keywords: plant viruses and viroids, high throughput sequencing (HTS), biological characterization, plant health, regulatory agencies, Pest Risk Analysis (PRA), virus disease
Published in DiRROS: 12.07.2024; Views: 1497; Downloads: 700
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