RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS).
Ontology highlight
ABSTRACT:
SUBMITTER: Hirai M
PROVIDER: S-EPMC8209451 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don't have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.
| S-EPMC6832592 | biostudies-literature
Project description:RNA modifications are a common occurrence across all domains of life. Several chemical modifications, including N6-methyladenosine, have also been found in viral transcripts and viral RNA genomes. Some of the modifications increase the viral replication efficiency while also helping the virus to evade the host immune system. Nonetheless, there are numerous examples in which the host's RNA modification enzymes function as antiviral factors. Although established methods like MeRIP-seq and miCLIP can provide a transcriptome- wide overview of how viral RNA is modified, it is difficult to distinguish between the complex overlapping viral transcript isoforms using the short read-based techniques. Nanopore direct RNA sequencing (DRS) provides both long reads and direct signal readings, which may carry information about the modifications. Here, we describe a refined protocol for analyzing the RNA modifications in viral transcriptomes using nanopore technology.
| S-EPMC9400574 | biostudies-literature
Project description:Here we propose a new general method for directly determining RNA sequence based on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the chain terminators (RdRP sequencing). The following properties of the polymerase render it appropriate for this application: (1) the phi6 polymerase can replicate a number of single-stranded RNA templates in vitro. (2) In contrast to the primer-dependent DNA polymerases utilized in the sequencing procedure by Sanger et al. (Proc Natl Acad Sci USA, 1977, 74:5463-5467), it initiates nascent strand synthesis without a primer, starting the polymerization on the very 3'-terminus of the template. (3) The polymerase can incorporate chain-terminating nucleotide analogs into the nascent RNA chain to produce a set of base-specific termination products. Consequently, 3' proximal or even complete sequence of many target RNA molecules can be rapidly deduced without prior sequence information. The new technique proved useful for sequencing several synthetic ssRNA templates. Furthermore, using genomic segments of the bluetongue virus we show that RdRP sequencing can also be applied to naturally occurring dsRNA templates. This suggests possible uses of the method in the RNA virus research and diagnostics.
| S-EPMC1370129 | biostudies-other
Project description:Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA primer extension is a model of the copying step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that analyzes sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension.
| S-EPMC7337528 | biostudies-literature
Project description:The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximize coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infection is key to understanding the viral replication cycle and its interactions with the host cell. Traditional short-read (Illumina) sequencing approaches are problematic in this setting due to the difficulty of assigning short reads to individual RNAs in regions of transcript overlap and to the biases introduced by the required recoding and amplification steps. Additionally, different methodologies may be required to analyze the 5' and 3' ends of RNAs, which increases both cost and effort. The advent of long-read nanopore sequencing simplifies this approach by providing a single assay that captures and sequences full length RNAs, either in cDNA or native RNA form. The latter is particularly appealing as it reduces known recoding biases whilst allowing more advanced analyses such as estimation of poly(A) tail length and the detection of RNA modifications including N6 -methyladenosine. Using herpes simplex virus (HSV)-infected primary fibroblasts as a template, we provide a step-by-step guide to the production of direct RNA sequencing libraries suitable for sequencing using Oxford Nanopore Technologies platforms and provide a simple computational approach to deriving a high-quality annotation of the HSV transcriptome from the resulting sequencing data. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Productive infection of primary fibroblasts with herpes simplex virus Support Protocol: Cell passage and plating of primary fibroblasts Basic Protocol 2: Preparation and sequencing of dRNA-seq libraries from virus-infected cells Basic Protocol 3: Processing, alignment, and analysis of dRNA-seq datasets.
| S-EPMC7187905 | biostudies-literature
Project description:The characterization of microbial communities based on sequencing and analysis of their genetic information has become a popular approach also referred to as metagenomics; in particular, the recent advances in sequencing technologies have enabled researchers to study even the most complex communities. Metagenome analysis, the assignment of sequences to taxonomic and functional entities, however, remains a tedious task: large amounts of data need to be processed. There are a number of approaches addressing particular aspects, but scientific questions are often too specific to be answered by a general-purpose method.We present MGX, a flexible and extensible client/server-framework for the management and analysis of metagenomic datasets; MGX features a comprehensive set of adaptable workflows required for taxonomic and functional metagenome analysis, combined with an intuitive and easy-to-use graphical user interface offering customizable result visualizations. At the same time, MGX allows to include own data sources and devise custom analysis pipelines, thus enabling researchers to perform basic as well as highly specific analyses within a single application.With MGX, we provide a novel metagenome analysis platform giving researchers access to the most recent analysis tools. MGX covers taxonomic and functional metagenome analysis, statistical evaluation, and a wide range of visualizations easing data interpretation. Its default taxonomic classification pipeline provides equivalent or superior results in comparison to existing tools.
| S-EPMC5937802 | biostudies-other
Project description:Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids.
| S-EPMC8860579 | biostudies-literature
Project description:BackgroundIn a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally.ResultsWe have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute's (JCVI) high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates.ConclusionsAugmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus sequence that encapsulates the allelic variation of the targeted population and is a key step prior to designing degenerate primers is also formally described.
| S-EPMC3548747 | biostudies-literature
Project description: Not available
| EMPIAR-10517 | biostudies-other
Project description:BACKGROUND: Resequencing of deafness related genes using GS FLX massive parallel sequencing of PCR amplicons spanning selected genes has previously been reported as a successful strategy to discover causal variants. The amplicon lengths were designed to be smaller than the sequencing read length of GS FLX technology, but are longer than Illumina sequencing technology read lengths. Fragmentation is thus required to sequence these amplicons using high throughput Illumina technology. METHODS: We performed Illumina sequencing in 4 patients on 563 multiplexed amplicons covering the exons of 15 genes involved in the hearing process. After exploring several fragmentation strategies, the amplicons were fragmented using Covaris sonication prior to library preparation. CLC genomic workbench was used to analyze the data. RESULTS: We achieve an excellent coverage with more than 99% of the amplicons bases covered. All variants that were previously validated using Sanger sequencing, were also called in this study. Variant calling revealed less false positive and false negative results compared to the previous study. For each patient, several variants were found that are reported by ClinVar as possible hearing loss variants. CONCLUSION: Migration from GS FLX amplicon sequencing to Illumina amplicon sequencing is straightforward and leads to more accurate results.
| S-EPMC4266979 | biostudies-literature