Conference Presentations by István Prazsák
In our study we investigated the genome-wide expression level in different time points during the... more In our study we investigated the genome-wide expression level in different time points during the life cycle of wild type Pseudorabies Virus (PRV). Real time RT-PCR technique was used to investigate the expression kinetic of whole viral transcriptome. We clustered genes and analyzed the total viral gene expression profile and compared two reverse transcription methods on the basis of different kinetic classes of the PrV genes. Furthermore a new reverse transcription method, called timing RT was introduced to analyze the polycistronic mRNA structure of viral genes.
Papers by István Prazsák
At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcription... more At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcriptional/translational negative feedback loops and generate a basic ~24h oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances Ihe fitness of the organism only if it corresponds to the natural day/night cycles. Light is the most effective signal in synchronizing the oscillator to environmental cycles. Light signals mediated by photoreceptors are forwarded to the oscillator and cause an acute change in the level/activity of certain clock components that eventually results in a phase shift of the oscillation (Devlin and Kay 2001). Our aim is to reveal the molecular details of this process (also called entrainment or resetting) in Arabidopsis thaliana. The plant circadian oscillator is supposed to consist of three interlocked feedback loops Locke et al. 2006). In the first loop the morningexpressed CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) transcription factors inhibit the expression of the TIMING OF CAB EXPRESSION 1 (TOC1) gene: conversely, the evening-expressed TOC1 positively regulates the transcription of CCA1/LHY. In the second loop GIGANTEA (GI) induces TOC1 expression during the afternoon/evening, while TOC1 represses GI during the night. Recent data suggested the operation of a third loop, where CCA 1/LHY up-regulate the PSEUDO RESPONSE REGULATOR 7/9 (PRR7/9) genes (homologs of TOC1) in the morning and PRR7/9 proteins down-regulate CCA1/LHY expression during the day. In Arabidopsis, CCA 1 /LHY. GI and PRR9 may represent the primary targets of resetting light signals, merely based on the fact that these clock genes are acutely light-inducible. However, the role of their light induction in phase resetting has not been tested directly.
Journal of Environmental Geography, 2008
Urban soils have generally suffered significant alteration both regarding their physical, chemica... more Urban soils have generally suffered significant alteration both regarding their physical, chemical, as well as biological properties. Soil samples were taken at 25 sites from horizons of soil profiles located in the downtown and surroundings of Szeged in order to examine diagnostic properties different from natural soils (artefacts, humus content, quality of organic matter, pH (H2O, KCl), carbonate content, nitrogen content). Furthermore, topsoils were taken nearby 9 profiles to survey some basic biological properties (i.e. abundance, taxon diversity, dominance, similarity and MGP ratios) of mezofauna elements (Oribatid mites, Collembolans) and their community structure in the three zones (city, suburban, peripheral). The high amount of artefacts, fluctuating humus and nitrogen levels, the poor quality of organic matter, the high and fluctuating carbonate content, the concomitant variance of pH and modified mechanical properties prove that the urban soils of Szeged have been modifie...
Additional file 4: Fig. S4. The transcript isoform categories used in this study and their abbrev... more Additional file 4: Fig. S4. The transcript isoform categories used in this study and their abbreviations.
Additional file 9: Table S5. Overrepresentation analysis of host gene expression levels. The firs... more Additional file 9: Table S5. Overrepresentation analysis of host gene expression levels. The first column contains clusters of host genes and numbers of genes that were characteristic of the cluster (in parentheses). GO biological processes (bold) that had the lowest false discovery rates values are presented with numbers of genes in clusters and numbers of genes in the reference dataset (in parentheses). The genes belonging to GO processes are also listed.
Additional file 7: Table S3. Introns of the host transcripts. Splice donor and acceptor positions... more Additional file 7: Table S3. Introns of the host transcripts. Splice donor and acceptor positions are shown with read counts and the sequences of the splice junctions. Letter 'M' following the sample name indicates MinION sequencing, while letter 'S' refers to Sequel sequencing. The introns were determined using the LoRTIA pipeline.
Additional file 3: Figure S3. Transcript and UTR lengths of the host (C. aethiops) (a) Length of ... more Additional file 3: Figure S3. Transcript and UTR lengths of the host (C. aethiops) (a) Length of transcripts in the uninfected and each p.i. samples. (b, c) Length of the 5' and 3' UTRs were calculated using ti.py in uninfected and each p.i. samples. Letter 'M' following the sample name indicates MinION sequencing, and the letter 'S' indicates Sequel sequencing. Horizontal lines in the box plots represent median transcript length of the given samples. Transcripts were annotated using the LoRTIA software suit.
Vaccinia virus transcriptome sequencing usign PacBIO RSII, Sequel platforms and Oxford Nanopore M... more Vaccinia virus transcriptome sequencing usign PacBIO RSII, Sequel platforms and Oxford Nanopore MinION device.
In this paper a new velvet spider species from Morocco is described from an overgrazed former cor... more In this paper a new velvet spider species from Morocco is described from an overgrazed former cork oak [Quercus suber (Linné 1753)] forest. It is the second known species of the hitherto monotypic genus Loureedia. Loureedia maroccana sp. n. is distinguished from L. annulipes (Lucas, 1857) by the morphology of the conductor, the anteriorly widening cephalic region of the prosoma and opisthosoma decorated with a lobed, bright red marking on the dorsal side. Furthermore, three partial gene fragment sequences (histone 3, 28S ribosomal and cytochrome c oxidase) are also given, supporting the establishment of the new species.
The secondary structure of NTO3 (a. and b.) as well as the hybrid formed by the ORF62–5′ fragment... more The secondary structure of NTO3 (a. and b.) as well as the hybrid formed by the ORF62–5′ fragment and the NTO3 (c and d). a. The secondary structure of the unedited NTO3 with a free energy of − 143.2 kcal/mol. The adenines in the editing sites are colored in green. b. The secondary structure of the edited NTO3 with a free energy of − 169.4 kcal/mol. The inosines in the editing sites are colored in orange. c. The secondary structure of the sense-antisense hybrid composed of the first 467 bases of ORF62 labeled ORF62–5′ (gray) and the full sequence of NTO3 (blue), the latter is its unedited form. The free energy of the structure formed by the two molecules is − 822.9 kcal/mol. d. The secondary structure of the sense-antisense hybrid composed of the first 467 bases of ORF62 labeled ORF62–5′ (gray) and the full sequence of NTO3 (blue), the later in its edited form. The free energy of the structure formed by the two molecules is − 818.7 kcal/mol. The position of I·U base pairs is marked ...
Scientific Reports, 2022
In this study, two long-read sequencing (LRS) techniques, MinION from Oxford Nanopore Technologie... more In this study, two long-read sequencing (LRS) techniques, MinION from Oxford Nanopore Technologies and Sequel from the Pacific Biosciences, were used for the transcriptional characterization of a prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcript species, of which 759 were novel and 116 were annotated previously. These RNA molecules include 41 novel putative protein coding transcripts [each containing 5′-truncated in-frame open reading frames (ORFs), 14 monocistronic transcripts, 99 polygenic RNAs, 101 non-coding RNAs, and 504 untranslated region isoforms. This work also identified novel replication origin-associated transcripts, upstream ORFs, cis-regulatory sequences and poly(A) sites. We also detected RNA methylation in 99 viral genes and RNA hyper-editing in ...
Frontiers in Genetics, 2020
In this study, we used two long-read sequencing (LRS) techniques, Sequel from the Pacific Bioscie... more In this study, we used two long-read sequencing (LRS) techniques, Sequel from the Pacific Biosciences and MinION from Oxford Nanopore Technologies, for the transcriptional characterization of a prototype baculovirus, Autographacalifornica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby to distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcripts, of which 759 are novel and 116 have been annotated previously. These RNA molecules include 41 novel putative protein coding transcript (each containing 5’-truncated in-frame ORFs), 14 monocistronic transcripts, 99 multicistronic RNAs, 101 non-coding RNA, and 504 length isoforms. We also detected RNA methylation in 12 viral genes and RNA hyper-editing in the longer 5’-UTR transcript isoform of ORF 19 gene.
Pathogens, 2021
Viral transcriptomes that are determined using first- and second-generation sequencing techniques... more Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse tra...
US: unique short (region of the genome) UTR: untranslated region (of a mRNA) VACV: Vaccinia virus... more US: unique short (region of the genome) UTR: untranslated region (of a mRNA) VACV: Vaccinia virus VF: viral factory VZV: Varicella zoster virus WR: Western Reserve (strain of VACV) almost 6 years to puzzle them together with 2-dimensional chromatography 55. By 1977, Sanger and Coulson had used the 'plus and minus' method to sequence the first DNA genome of phiX174 bacteriophage using radioactive labeled P-isotopes 17,56. This method allowed a 50-80 base sequence s to be resolved in a single experiment, however it was laborious and time-consuming and was suitable for only single-stranded DNA 52. Inspired by Kornberg's experiments, Sanger and Coulson improved their sequencing method, which relied on stopping the polynucleotide synthesis with a nucleosideanalogue and they could read almost 500 bp of DNA at once 52,57,58. Sanger's method has become the most commonly used technology to sequence DNA. Many improvements have been made in the intervening years e.g. fluorescently-labeled terminators instead of radioactively-labeled nucleotides made the detection of fragments safer, introduction of capillary gel electrophoresis provided highconfidence base calling (. Figure 1A), recombinant new enzymes enabled sequencing up to 1000 bps 59. This technique was the basis for sequencing the entire human genome and transcriptome and has made the genomic revolution of medicine over the last four decades a possibility 60,61. Sanger sequencing is still in use today for sequencing individual fragments of DNA/cDNA. Despite its many advantages, it is not able to achieve low-cost, high-throughput sequencing, partly because detection and synthesis are separated during the sequencing process and it cannot handle millions of bases simultaneously. Short read sequencing techniques Following the terminology in the history of sequencing, next-generation sequencing (NGS) refers to second, third and fourth generation sequencing techniques. Nonetheless, other authors and I prefer to distinguish NGS based on the length of reads, namely short-and long-read sequencing techniques, because the latter nomenclature better depicts the essence of NGS 62. Short-read sequencing (SRS) techniques have dramatically changed the market of whole genome sequencing in the last decade due to their extremely high cost-efficiency. The common principle of these techniques is sequencing-bysynthesis (except in the case of SOLiD-sequencing-by-ligation-system). The sequencing reaction takes place on the surface of a chip to which many thousands of samples can be attached, thus achieving massive parallel sample handling. SRS-techniques differ from first generation (or chain termination) sequencing techniques in that they do not need gel electrophoresis, because signal detection and sequence reading occur simultaneously, not separately, which results in highly efficient throughput. The leading NGS-technique has been pyrosequencing. Its principles have been known since 1993, briefly: during the synthesis of the complement strand, pyrophosphate (PPi) is released and transformed into ATP. The ATP then activates the enzyme luciferase (. Figure 1C), which produces a light signal indicating specific base incorporation 63. The technique utilizes the advantages of emulsion PCR, all reactions take place on beads in picoliter volumes. Pyrosequencing was commercialized by J. Rothberg's company, 454, in 2003 and made it possible to sequence parts of the Neanderthal genome for the first time 64. Although pyrosequencing can read only 300-400 bp, the technique was well implemented in several genotyping assays and genome methylation analyses of Herpesviruses 65-68 and Vaccinia viruses 69. Marketing of the pyrosequencing platform was discontinued in 2013 when the technology became noncompetitive against other SRS platforms. Illumina Researchers struggled with the automated Sanger method to sequence the entire human genome, there was a need for a fast, high-throughput, but cheap sequencing technique. Partway through the Human Genome Project a new sequencing idea-invented by two Cambridge scientists, Balasubramanian and Klenermanand-brought the Solexa company into being 70. Their technique uses adapter molecules instead of emulsion PCR. The adapters are passed over a lawn of complementary oligonucleotides bound to a solid phase flow cell. PCR produces a population of clusters from neighboring templates 59. The company's machine resequenced the PhiX174 genome in 2005. It was a landmark in the history of NGS platforms, therefore Solexa was purchased by Illumina, which has invested in the development of personalized genome sequencers 71. Soon after, Illumina announced that they had succeeded at the $1000 genome challenge in 2014, which meant a remarkably drastic fall in the cost of whole genome sequencing compared to the 13-year-long Human Genome Project with its $1 billion bill 72. The GenBank currently contains more than 36 petabytes (PB) of data and is expected to grow to 43 PB by 2023 73. The vast majority of the data have been derived from Illumina sequencing. The popularity of this method can be explained by its low-cost efficacy coupled with extreme sequence accuracy and powerful marketing. Sanger sequencing determines the sequence of any DNA by measuring the mass of the growing strand using chain terminating modified ddNTP-s. One limitation of this method is that it cannot read more than 800 bp long chains, it can only handle a limited number of parallel sequences and evaluation of the results is time consuming. Contrarily, Illumina produces only short reads in a massively parallel manner, so it is able to sequence whole genomes in a defined time period. The principle of this method is sequencing-by-synthesis by reversible chain termination 74,75. The DNA or cDNA population needs to be fragmented into 35-75 bp long portions prior to library preparation, then adapters are ligated to bind them to the surface of the so-called flow-cell, where bridge amplification is done. During sequencing the DNA polymerase incorporates one of the four fluorescently labeled modified nucleotides (the reversible terminators, which are protected at their 3' ends) then an image is taken to catch the light signals of the growing strand 74. The protection group is cleaved to allow incorporation of the next base; thereafter unincorporated nucleotides are washed away (. Figure 1B). The flow cell is imaged in each cycle resulting in a quasi-error-free base-by-base determination of the complement strand. Although Illumina platforms provide diverse solutions for different, large scale applications, including genomics, metagenomics, whole genome transcriptomics, small RNA transcriptomics, methylation arrays, ribosome profiling, clinical diagnostics, they generate millions of reads and they are not ideal technologies in some ways: amplification bias, high GC-content, homopolymers, difficult sample preparation, long run time, expensive instruments and the generated short reads all hamper their future distribution 76 .
Indiana Vesiculovirus (IVV; formerly as Vesicular stomatitis virus and Vesicular stomatitis India... more Indiana Vesiculovirus (IVV; formerly as Vesicular stomatitis virus and Vesicular stomatitis Indiana virus) causes a disease in livestock that is very similar to the foot and mouth disease thereby an outbreak may lead to significant economic loss. Long-read sequencing (LRS) -based approaches revealed a hidden complexity of the transcriptomes in several viruses already. This technique was utilized already for the sequencing of the IVV genome, but our study is the first for the application of this technique for the profiling of IVV transcriptome. Since LRS is able to sequence full-length RNA molecules, and thereby providing more accurate annotation of the transcriptomes than the traditional short-read sequencing methods. The objectives of this study were to assemble the complete transcriptome of using nanopore sequencing, to ascertain cell-type specificity and dynamics of viral gene expression and to evaluate host gene expression changes induced by the viral infection. We carried out a...
Pathogens, 2021
Vesicular stomatitis Indiana virus (VSIV) of genus Vesiculovirus, species IndianaVesiculovirus (f... more Vesicular stomatitis Indiana virus (VSIV) of genus Vesiculovirus, species IndianaVesiculovirus (formerly as Vesicular stomatitis virus, VSV) causes a disease in livestock that is very similar to the foot and mouth disease, thereby an outbreak may lead to significant economic loss. Long-read sequencing (LRS) -based approaches already reveal a hidden complexity of the transcriptomes in several viruses. This technique has been utilized for the sequencing of the VSIV genome, but our study is the first for the application of this technique for the profiling of the VSIV transcriptome. Since LRS is able to sequence full-length RNA molecules, it thereby provides more accurate annotation of the transcriptomes than the traditional short-read sequencing methods. The objectives of this study were to assemble the complete transcriptome of using nanopore sequencing, to ascertain cell-type specificity and dynamics of viral gene expression, and to evaluate host gene expression changes induced by th...
BMC Research Notes, 2021
Objective In this study, we applied two long-read sequencing (LRS) approaches, including single-m... more Objective In this study, we applied two long-read sequencing (LRS) approaches, including single-molecule real-time and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of host gene expression as a response to Vaccinia virus infection. Transcriptomes determined using short-read sequencing approaches are incomplete because these platforms are inefficient or fail to distinguish between polycistronic RNAs, transcript isoforms, transcriptional start sites, as well as transcriptional readthroughs and overlaps. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Results In this work, we identified a number of novel transcripts and transcript isoforms of Chlorocebus sabaeus. Additionally, analysis of the most abundant 768 host transcripts revealed a significant overrepresentation of the class of genes in the “regulation of signaling receptor activity” Gene Ontology annotati...
Scientific Reports, 2020
Characterization of global transcriptomes using conventional short-read sequencing is challenging... more Characterization of global transcriptomes using conventional short-read sequencing is challenging due to the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps. Long-read sequencing (LRS) can overcome these limitations by reading full-length transcripts. Employment of these technologies has led to the redefinition of transcriptional complexities in reported organisms. In this study, we applied LRS platforms from Pacific Biosciences and Oxford Nanopore Technologies to profile the vaccinia virus (VACV) transcriptome. We performed cDNA and direct RNA sequencing analyses and revealed an extremely complex transcriptional landscape of this virus. In particular, VACV genes produce large numbers of transcript isoforms that vary in their start and termination sites. A significant fraction of VACV transcripts start or end within coding regions of neighbouring genes. This study provides new insights into the transcriptomic profile o...
African swine fever virus (ASFV) is an important animal pathogen causing substantial economic los... more African swine fever virus (ASFV) is an important animal pathogen causing substantial economic losses in the swine industry globally. At present, little is known about the molecular biology of ASFV, including its transcriptome organization. In this study, we applied cutting-edge sequencing approaches, namely the Illumina short-read sequencing (SRS) and the Oxford Nanopore Technologies long-read sequencing (LRS) techniques, together with several library preparation chemistries to analyze the ASFV dynamic transcriptome. SRS can generate a large amount of high-precision sequencing reads, but it is inefficient for identifying long RNA molecules, transcript isoforms and overlapping transcripts. LRS can overcome these limitations, but this approach also has shortcomings, such as its high error rate and the low coverage. Amplification-based LRS techniques produce relatively high read counts but also high levels of spurious transcripts, whereas the non-amplified cDNA and direct RNA sequencin...
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Conference Presentations by István Prazsák
Papers by István Prazsák