1. A primer on the analysis of high-throughput sequencing data for detection of plant virusesDenis Kutnjak, Lucie Tamisier, Ian Adams, Neil Boonham, Thierry Candresse, Michela Chiumenti, Kris De Jonghe, Jan F. Kreuze, Marie Lefebvre, Goncalo Silva, Martha Malapi-Wight, Paolo Margaria, Irena Mavrič Pleško, Sam McGreig, Laura Miozzi, Benoit Remenant, Jean-Sébastien Reynard, Johan Rollin, Mike Rott, Olivier Schumpp, Sébastien Massart, Annelies Haegeman, 2021, pregledni znanstveni članek Povzetek: High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.
Ključne besede: plant virus, high-throughput sequencing, bioinformatics, detection, discovery
Objavljeno v DiRROS: 05.08.2024; Ogledov: 53; Prenosov: 55
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2. CRISPR/Cas-mediated plant genome editing : outstanding challenges a decade after implementationTeodoro Cardi, Jana Murovec, Allah Bakhsh, Vladislava Galović, Tjaša Lukan, Kubilay Yıldırım, Milica Zlatković, Katrijn Van Laere, 2023, pregledni znanstveni članek Povzetek: The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
Ključne besede: CRISPR applications, CRISPR platforms, gene regulations, mutant detection, plant regeneration
Objavljeno v DiRROS: 05.08.2024; Ogledov: 44; Prenosov: 88
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3. BioMinerJoachim Selbig, Kristina Gruden, Michal Or-Guil, Christian Schichor, Chris Bauer, Karol Stec, Alexander Glintschert, Johannes Schuchhardt, 2015, izvirni znanstveni članek Povzetek: Personalized medicine is promising a revolution for medicine and human biology in the 21st century. The scientific foundation for this revolution is accomplished by analyzing biological high-throughput data sets from genomics, transcriptomics, proteomics, and metabolomics. Currently, access to these data has been limited to either rather simple Web-based tools, which do not grant much insight or analysis by trained specialists, without firsthand involvement of the physician. Here, we present the novel Web-based tool “BioMiner,” which was developed within the scope of an international and interdisciplinary project (SYSTHER†) and gives access to a variety of high-throughput data sets. It provides the user with convenient tools to analyze complex cross-omics data sets and grants enhanced visualization abilities. BioMiner incorporates transcriptomic and cross-omics high-throughput data sets, with a focus on cancer. A public instance of BioMiner along with the database is available at http://systherDB.microdiscovery.de/, login and password: “systher”; a tutorial detailing the usage of BioMiner can be found in the Supplementary File.
Ključne besede: data mining, multiomics data integration, biomarker detection, pathway visualization, personalized medicine, cancer
Objavljeno v DiRROS: 26.07.2024; Ogledov: 104; Prenosov: 89
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4. Survey results on nucleic acid tests of infectious diseases : present status and need for rapid and near-patient diagnosticsJörg Neukammer, Martin Hussels, Andreas Kummrow, Alison S. Devonshire, Carole A. Foy, Jim F. Huggett, Helen C. Parkes, Jana Žel, Mojca Milavec, Heinz Schimmel, Wolfgang Unger, Müslüm Akgöz, Timothy D. McHugh, Viktorija Tomič, Hans-Peter Grunert, Heinz Zeichhardt, 2015, izvirni znanstveni članek Povzetek: This survey discusses current and emerging isothermal and rapid polymerase chain reaction (PCR) based nucleic acid amplification methods for near-patient diagnostics.
To assess the clinical need of rapid diagnostics for infectious diseases based on nucleic acid tests (NATs) we performed and analysed a questionnaire among laboratories participating in corresponding INSTAND ring trials for external quality assurance. The questions concerning new amplification technologies like isothermal nucleic acid amplification, potentially suited to significantly decrease turnaround times, were complemented by questions to evaluate the present status of NATs. Besides end-users, companies were also addressed by sending out a manufacturer specific questionnaire.
Analysis of the answers from 48 laboratories in 14 European countries revealed that a much shorter turnaround time is requested for selected pathogens compared to about 2 h or longer when applying temperature cycling amplification, i.e. PCR. In this context, most frequently mentioned were methicillin-resistant Staphylococcus aureus (MRSA), norovirus, influenza A and B viruses, cytomegalovirus (CMV) as well as hepatitis B virus (HBV) and hepatitis C virus (HCV). At present, 8% of the laboratories having participated in this survey apply isothermal amplification of nucleic acids to identify infectious pathogens.
Ključne besede: nucleic acid tests, infectious diseases, virus detection, bacteria detection, isothermal nucleic acid amplification, status report, questionnaire, NAT, PCR
Objavljeno v DiRROS: 26.07.2024; Ogledov: 103; Prenosov: 261
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5. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detectionDavid Dobnik, Dejan Štebih, Andrej Blejec, Dany Morisset, Jana Žel, 2016, izvirni znanstveni članek Povzetek: The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.
Ključne besede: digital PCR, DNA targets, GMO detection
Objavljeno v DiRROS: 25.07.2024; Ogledov: 137; Prenosov: 73
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6. Next generation sequencing for detection and discovery of plant viruses and viroids : comparison of two approachesAnja Pecman, Denis Kutnjak, Ion Gutiérrez-Aguirre, Ian Adams, Adrian Fox, Neil Boonham, Maja Ravnikar, 2017, izvirni znanstveni članek Povzetek: Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).
Ključne besede: next generation sequencing, small RNA, ribosomal RNA depleted total RNA, detection, plant viruses, plant viroids
Objavljeno v DiRROS: 25.07.2024; Ogledov: 98; Prenosov: 59
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7. Loop-mediated isothermal amplification : rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultryPolona Kogovšek, Jerneja Ambrožič, Alenka Dovč, Tanja Dreo, Hristo Hristov, Uroš Krapež, Maja Ravnikar, Brigita Slavec, Marjetka Lotrič, Jana Žel, Olga Zorman-Rojs, 2018, izvirni znanstveni članek Povzetek: Infections with pathogenic Escherichia colican lead to different animal- and human-associated diseases. E. coliinfections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli(APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeatable, and reproducible. High sensitivities of the assays are shown, where as few as 1,000 bacterial cells/mL can be detected in different matrices. On-site applicability of this LAMP method is demonstrated through testing of different sample types, from animal swabs and tissues, and from environmental samples collected from 6 commercial poultry farms. All 3 virulence genes were detected at high rates (above 85%) in samples from layer and broiler chickens with clinical signs and, interestingly, high prevalence of those genes was detected also in samples collected from clinically healthy broiler flock (above 75%) while lower prevalence was observed in remaining 3 clinically healthy chicken flocks (less than 75%). Importantly, these virulence genes were detected in almost all of the air samples from 11 randomly selected poultry houses, indicating air as an important route of E. colispread. Three LAMP assays that target APEC-associated virulence genes are shown to be sensitive and robust and are therefore applicable for rapid on-site testing of various sample types, from animal swabs to air. This on-site LAMP testing protocol offers rapid diagnostics, with results obtained in <35 min, and it can be applied to other important microorganisms to allow the required prompt measures to be taken.
Ključne besede: APEC virulence genes, isothermal amplification LAMP, on-site detection, colibacillosis
Objavljeno v DiRROS: 24.07.2024; Ogledov: 105; Prenosov: 39
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8. Detection of four new tomato viruses in Serbia using post hoc high-throughput sequencing analysis of samples from a large-scale field surveyAna Vučurović, Denis Kutnjak, Nataša Mehle, Ivana Stanković, Anja Pecman, Aleksandra Bulajić, Branka Krstić, Maja Ravnikar, 2021, izvirni znanstveni članek Povzetek: Tomato production worldwide is affected by numerous plant virus species. The early and accurate detection of viruses is a critical step for disease control. However, the simultaneous detection of the most known tomato viruses can be difficult because of the high number and diversity of tomato-infecting viruses. Here, we have identified four new viruses in Serbia by applying target-independent small RNA high-throughput sequencing (HTS). HTS was applied on pools of samples and separate samples, in total comprising 30 tomato samples that exhibited (severe) virus-like symptoms and were collected in Serbia during three annual surveys (2011 to 2013). These samples had previously tested negative for the presence of 16 tomato viruses using targeted detection methods. Three divergent complete genome sequences of Physostegia chlorotic mottled virus were obtained from different localities, indicating for the first time that this virus is widespread in Serbia and might represent an emergent viral pathogen of tomato. The tomato torrado virus was detected at one locality with devastating yield losses. The southern tomato virus was detected at two localities, and the spinach latent virus was detected at one locality. In addition, we detected the presence of one already-known virus in Serbia, the tomato spotted wilt orthotospovirus. All the HTS results were subsequently confirmed by targeted detection methods. In this study, the successful application of post hoc HTS testing of a limited number of pooled samples resulted in the discovery of new viruses. Thus, our results encourage the use of HTS in research and diagnostic laboratories, including laboratories that have limited resources to resolve disease etiology.
Ključne besede: viruses, tomato, detection, identification
Objavljeno v DiRROS: 19.07.2024; Ogledov: 118; Prenosov: 152
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9. Genome-informed design of a LAMP assay for the specific detection of the strain of ‘Candidatus Phytoplasma asteris’ phytoplasma occurring in grapevines in South AfricaŠpela Alič, Marina Dermastia, Johan Burger, Matthew Dickinson, Gerhard Pietersen, Gert Pietersen, Tanja Dreo, 2022, izvirni znanstveni članek Povzetek: Grapevine yellows is one of the most damaging phytoplasma-associated diseases worldwide. It is linked to several phytoplasma species, which can vary regionally due to phytoplasma and insect-vector diversity. Specific, rapid, and reliable detection of the grapevine yellows pathogen has an important role in phytoplasma control. The purpose of this study was to develop and validate a specific loop-mediated isothermal amplification (LAMP) assay for detection of a distinct strain of grapevine ‘Candidatus Phytoplasma asteris’ that is present in South Africa, through implementation of a genome-informed test design approach. Several freely available, user-friendly, web-based tools were coupled to design the specific LAMP assays. The criteria for selection of the assays were set for each step of the process, which resulted in four experimentally operative LAMP assays that targeted the ftsH/hflB gene region, specific to the aster yellows phytoplasma strain from South Africa. A real-time PCR was developed, targeting the same genetic region, to provide extensive validation of the LAMP assay. The validated molecular assays are highly specific to the targeted aster yellows phytoplasma strain from South Africa, with good sensitivity and reproducibility. We show a genome-informed molecular test design and an efficient validation approach for molecular tests if reference and sample materials are sparse and hard to obtain.
Ključne besede: aster yellows, fruit, LAMP, molecular detection, pathogendetection, phytoplasma, Prokaryotes
Objavljeno v DiRROS: 17.07.2024; Ogledov: 113; Prenosov: 68
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10. Evaluation of methods and processes for robust monitoring of SARS-CoV-2 in wastewaterOlivera Maksimović, Živa Lengar, Zala Kogej Zwitter, Katarina Bačnik, Irena Bajde, Mojca Milavec, Anže Županič, Nataša Mehle, Denis Kutnjak, Maja Ravnikar, Ion Gutiérrez-Aguirre, 2022, izvirni znanstveni članek Povzetek: The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic.
Ključne besede: waste water, method evolution, virus detection, SARS-CoV-2
Objavljeno v DiRROS: 17.07.2024; Ogledov: 97; Prenosov: 74
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