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An Integrated Sequencing Approach for Updating the Pseudorabies Virus Transcriptome.

ABSTRACT: In the last couple of years, the implementation of long-read sequencing (LRS) technologies for transcriptome profiling has uncovered an extreme complexity of viral gene expression. In this study, we carried out a systematic analysis on the pseudorabies virus transcriptome by combining our current data obtained by using Pacific Biosciences Sequel and Oxford Nanopore Technologies MinION sequencing with our earlier data generated by other LRS and short-read sequencing techniques. As a result, we identified a number of novel genes, transcripts, and transcript isoforms, including splice and length variants, and also confirmed earlier annotated RNA molecules. One of the major findings of this study is the discovery of a large number of 5'-truncations of larger putative mRNAs being 3'-co-terminal with canonical mRNAs of PRV. A large fraction of these putative RNAs contain in-frame ATGs, which might initiate translation of N-terminally truncated polypeptides. Our analyses indicate that CTO-S, a replication origin-associated RNA molecule is expressed at an extremely high level. This study demonstrates that the PRV transcriptome is much more complex than previously appreciated.

SUBMITTER: Torma G

PROVIDER: S-EPMC7924054 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus.

Project description:Third-generation sequencing is an emerging technology that is capable of solving several problems that earlier approaches were not able to, including the identification of transcripts isoforms and overlapping transcripts. In this study, we used long-read sequencing for the analysis of pseudorabies virus (PRV) transcriptome, including Oxford Nanopore Technologies MinION, PacBio RS-II, and Illumina HiScanSQ platforms. We also used data from our previous short-read and long-read sequencing studies for the comparison of the results and in order to confirm the obtained data. Our investigations identified 19 formerly unknown putative protein-coding genes, all of which are 5' truncated forms of earlier annotated longer PRV genes. Additionally, we detected 19 non-coding RNAs, including 5' and 3' truncated transcripts without in-frame ORFs, antisense RNAs, as well as RNA molecules encoded by those parts of the viral genome where no transcription had been detected before. This study has also led to the identification of three complex transcripts and 50 distinct length isoforms, including transcription start and end variants. We also detected 121 novel transcript overlaps, and two transcripts that overlap the replication origins of PRV. Furthermore, in silico analysis revealed 145 upstream ORFs, many of which are located on the longer 5' isoforms of the transcripts.

| S-EPMC5786565 | biostudies-literature

Pseudorabies virus transcriptome sequencing

Project description:Characterization of pseudorabies virus transcriptome by Illumina sequencing

| PRJEB9526 | ENA

Characterization of pseudorabies virus transcriptome by Illumina sequencing.

Project description:BackgroundPseudorabies virus is a widely-studied model organism of the Herpesviridae family, with a compact genome arrangement of 72 known coding sequences. In order to obtain an up-to-date genetic map of the virus, a combination of RNA-sequencing approaches were applied, as recent advancements in high-throughput sequencing methods have provided a wealth of information on novel RNA species and transcript isoforms, revealing additional layers of transcriptome complexity in several viral species.ResultsThe total RNA content and polyadenylation landscape of pseudorabies virus were characterized for the first time at high coverage by Illumina high-throughput sequencing of cDNA samples collected during the lytic infectious cycle. As anticipated, nearly all of the viral genome was transcribed, with the exception of loci in the large internal and terminal repeats, and several small intergenic repetitive sequences. Our findings included a small novel polyadenylated non-coding RNA near an origin of replication, and the single-base resolution mapping of 3' UTRs across the viral genome. Alternative polyadenylation sites were found in a number of genes and a novel alternative splice site was characterized in the ep0 gene, while previously known splicing events were confirmed, yielding no alternative splice isoforms. Additionally, we detected the active polyadenylation of transcripts earlier believed to be transcribed as part of polycistronic RNAs.ConclusionTo the best of our knowledge, the present work has furnished the highest-resolution transcriptome map of an alphaherpesvirus to date, and reveals further complexities of viral gene expression, with the identification of novel transcript boundaries, alternative splicing of the key transactivator EP0, and a highly abundant, novel non-coding RNA near the lytic replication origin. These advances provide a detailed genetic map of PRV for future research.

| S-EPMC4487798 | biostudies-literature

Transcriptome-wide survey of pseudorabies virus using next- and third-generation sequencing platforms.

Project description:Pseudorabies virus (PRV) is an alphaherpesvirus of swine. PRV has a large double-stranded DNA genome and, as the latest investigations have revealed, a very complex transcriptome. Here, we present a large RNA-Seq dataset, derived from both short- and long-read sequencing. The dataset contains 1.3 million 100 bp paired-end reads that were obtained from the Illumina random-primed libraries, as well as 10 million 50 bp single-end reads generated by the Illumina polyA-seq. The Pacific Biosciences RSII non-amplified method yielded 57,021 reads of inserts (ROIs) aligned to the viral genome, the amplified method resulted in 158,396 PRV-specific ROIs, while we obtained 12,555 ROIs using the Sequel platform. The Oxford Nanopore's MinION device generated 44,006 reads using their regular cDNA-sequencing method, whereas 29,832 and 120,394 reads were produced by using the direct RNA-sequencing and the Cap-selection protocols, respectively. The raw reads were aligned to the PRV reference genome (KJ717942.1). Our provided dataset can be used to compare different sequencing approaches, library preparation methods, as well as for validation and testing bioinformatic pipelines.

| S-EPMC6007087 | biostudies-literature

Integrated genome and transcriptome sequencing from the same cell

Project description:Single-cell genomics and single-cell transcriptomics have recently emerged as powerful tools to study the biology of single cells at a genome-wide scale. Here we describe a method that allows the integration of genomic DNA and mRNA sequencing from the same cell. We use this method to correlate DNA copy number variation to transcriptome variability among individual cells. First, hand-picked single cells are lysed and reverse transcribed using a poly-A primer including cell-specific barcodes, a 5' Illumina adapter and a T7 promoter overhang to convert mRNA to single stranded cDNA (ss cDNA). The gDNA and single stranded cDNA are then subjected to quasilinear whole genome amplification, as previously described, using an adapter with a defined 27 nucleotide sequence at the 5’ end followed by 8 random nucleotides. After 7 rounds of amplification, the gDNA and cDNA are copied to generate a variety of different short amplicon (0.5–2.5 kb) species, with a majority of amplicons containing adapter Ad-2 at both ends and a small fraction of cDNA derived amplicons containing Ad-2 at one end and Ad-1x at the other. Next, the sample is split into two tubes to further amplify gDNA and cDNA. The tube used to sequence gDNA is amplified using PCR. Following sonication, adapter Ad-2 removal, and cell-specific indexed Illumina library preparation, this half is used to sequence gDNA. The tube used to sequence cDNA is converted to double-stranded cDNA and amplified using in vitro transcription such that the amplified RNA (aRNA) is uniquely produced from cDNA but not gDNA. 3’ Illumina adapters are then ligated to the aRNA followed by reverse transcription and PCR, allowing quantification of mRNA.

| S-ECPF-GEOD-62952 | biostudies-other

Effects of Pseudorabies Virus Infection on the Tracheobronchial Lymph Node Transcriptome.

Project description:This study represents the first swine transcriptome hive plots created from gene set enrichment analysis (GSEA) data and provides a novel insight into the global transcriptome changes occurring in tracheobronchial lymph nodes (TBLN) and spanning the swine genome. RNA isolated from draining TBLN from 5-week-old pigs, either clinically infected with a feral isolate of Pseudorabies virus or uninfected, was interrogated using Illumina Digital Gene Expression Tag Profiling. More than 100 million tag sequences were observed, representing 4,064,189 unique 21-base sequences collected from TBLN at time points 1, 3, 6, and 14 days post-inoculation (dpi). Multidimensional statistical tests were applied to determine the significant changes in tag abundance, and then the tags were annotated. Hive plots were created to visualize the differential expression within the swine transcriptome defined by the Broad Institute's GSEA reference datasets between infected and uninfected animals, allowing us to directly compare different conditions.

| S-EPMC4725608 | biostudies-literature

Effects of Pseudorabies Virus Infection on the Tracheobronchial Lymph Node Transcriptome

2015-10-30 | GSE74473 | GEO

Effects of Pseudorabies Virus Infection on the Tracheobronchial Lymph Node Transcriptome

Project description:RNA isolated from draining tracheobronchial lymph nodes (TBLN) from 5-week old pigs, either clinically infected with a feral isolate of Pseudorabies virus or uninfected were interrogated using Illumina Digital Gene Expression Tag Profiling. Over 100 million tag sequences were observed, representing 4,064,189 unique 21-base sequences collected from TBLN at time points 1, 3, 6 and 14 days post-infection (dpi) RNA isolated from draining tracheobronchial lymph nodes (TBLN) from 5-week old pigs (% per group pooled), either clinically infected with feral isolate FS268 of Pseudorabies virus or uninfected at 1, 3, 6, and 14 days post inoculation. Over 100 million tag sequences were observed, representing 4,064,189 unique 21-base sequences.

2015-10-30 | E-GEOD-74473 | biostudies-arrayexpress

Integrated genome and transcriptome sequencing of the same cell.

Project description:Single-cell genomics and single-cell transcriptomics have emerged as powerful tools to study the biology of single cells at a genome-wide scale. However, a major challenge is to sequence both genomic DNA and mRNA from the same cell, which would allow direct comparison of genomic variation and transcriptome heterogeneity. We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification. We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA. Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells. Applications of our integrated sequencing approach could range from gaining insights into cancer evolution and heterogeneity to understanding the transcriptional consequences of copy number variations in healthy and diseased tissues.

| S-EPMC4374170 | biostudies-literature

An Unbiased Approach to Mapping the Signaling Network of the Pseudorabies Virus US3 Protein.

Project description:The US3 serine/threonine protein kinase is conserved among the alphaherpesvirus family and represents an important virulence factor. US3 plays a role in viral nuclear egress, induces dramatic alterations of the cytoskeleton, represses apoptosis, enhances gene expression and modulates the immune response. Although several substrates of US3 have been identified, an unbiased screen to identify US3 phosphorylation targets has not yet been described. Here, we perform a shotgun and phosphoproteomics analysis of cells expressing the US3 protein of pseudorabies virus (PRV) to identify US3 phosphorylation targets in an unbiased way. We identified several cellular proteins that are differentially phosphorylated upon US3 expression and validated the phosphorylation of lamin A/C at serine 404, both in US3-transfected and PRV-infected cells. These results provide new insights into the signaling network of the US3 protein kinase and may serve as a basis for future research into the role of the US3 protein in the viral replication cycle.

| S-EPMC7694389 | biostudies-literature

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