scholarly journals Applications of Deep Mutational Scanning in Virology

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1020
Author(s):  
Thomas D. Burton ◽  
Nicholas S. Eyre
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
Rna Viruses ◽  
Human Gene ◽  
Viral Gene ◽  
Epigenetic Changes ◽  
Molecular Virology ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Generation Sequencing

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. In this review, we discuss the application of this technique to RNA viruses including members of the Flaviviridae family, Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2. We also briefly discuss the reverse genetics systems which allow for analysis of viral replication cycles, next-generation sequencing technologies and the bioinformatics tools that facilitate this research.

scholarly journals MutTILL: Development of induced mutant resources for genome-wide mutations screening by using next generation sequencing technologies

2017 ◽  
Vol 1 (Special Issue-Supplement) ◽  
pp. 237-237
Author(s):  
Reddaiah Bodanapu ◽  
Krishna Lalam ◽  
Durga Khandekar ◽  
Navitha Kokkonda ◽  
Sivarama Prasad Lekkala ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Generation Sequencing ◽  
Induced Mutant

scholarly journals Upstream translation initiation expands the coding capacity of segmented negative-strand RNA viruses

2019 ◽  
Author(s):  
Elizabeth Sloan ◽  
Marta Alenquer ◽  
Liliane Chung ◽  
Sara Clohisey ◽  
Adam M. Dinan ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Influenza A ◽  
Viral Infections ◽  
Rna Viruses ◽  
Influenza Viruses ◽  
Open Reading Frames ◽  
Viral Gene ◽  
Influenza A Viruses ◽  
Negative Strand ◽  
Coding Potential

AbstractSegmented negative-strand RNA viruses (sNSVs) include the influenza viruses, the bunyaviruses, and other major pathogens of humans, other animals and plants. The genomes of these viruses are extremely short. In response to this severe genetic constraint, sNSVs use a variety of strategies to maximise their coding potential. Because the eukaryotic hosts parasitized by sNSVs can regulate gene expression through low levels of translation initiation upstream of their canonical open reading frames (ORFs), we asked whether sNSVs could use upstream translation initiation to expand their own genetic repertoires. Consistent with this hypothesis, we showed that influenza A viruses (IAVs) and bunyaviruses were capable of upstream translation initiation. Using a combination of reporter assays and viral infections, we found that upstream translation in IAVs can initiate in two unusual ways: through non-AUG initiation in virally encoded ‘untranslated’ regions, and through the appropriation of an AUG-containing leader sequence from host mRNAs through viral cap-snatching, a process we termed ‘start-snatching.’ Finally, while upstream translation of cellular genes is mainly regulatory, for sNSVs it also has the potential to create novel viral gene products. If in frame with a viral ORF, this creates N-extensions of canonical viral proteins. If not, it allows the expression of cryptic overlapping ORFs, which we found were highly conserved in IAV and widely distributed in peribunyaviruses. Thus, by exploiting their host’s capacity for upstream translation initiation, sNSVs can expand still further the coding potential of their extremely compact RNA genomes.

scholarly journals Impact of Individual Viral Gene Segments from Influenza A/H5N8 Virus on the Protective Efficacy of Inactivated Subtype-Specific Influenza Vaccine

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 368
Author(s):  
Yassmin Moatasim ◽  
Ahmed Kandeil ◽  
Ahmed Mostafa ◽  
Omnia Kutkat ◽  
Mohamed El Sayes ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Influenza A ◽  
Reverse Genetics ◽  
Protective Efficacy ◽  
Economic Losses ◽  
Viral Gene ◽  
Highly Pathogenic ◽  
H5n8 Virus ◽  
Antigenic Proteins ◽  
The Individual

Since its emergence in 2014, the highly pathogenic avian influenza H5N8 virus has continuously and rapidly spread worldwide in the poultry sector resulting in huge economic losses. A typical inactivated H5N8 vaccine is prepared using the six internal genes from A/PR8/1934 (H1N1) and the two major antigenic proteins (HA and NA) from the circulating H5N8 strain with the HA modified to a low pathogenic form (PR8HA/NA-H5N8). The contribution of the other internal proteins from H5N8, either individually or in combination, to the overall protective efficacy of PR8-based H5N8 vaccine has not been investigated. Using reverse genetics, a set of PR8-based vaccines expressing the individual proteins from an H5N8 strain were rescued and compared to the parent PR8 and low pathogenic H5N8 strains and the commonly used PR8HA/NA-H5N8. Except for the PR8-based vaccine strains expressing the HA of H5N8, none of the rescued combinations could efficiently elicit virus-neutralizing antibodies. Compared to PR8, the non-HA viral proteins provided some protection to infected chickens six days post infection. We assume that this late protection was related to cell-based immunity rather than antibody-mediated immunity. This may explain the slight advantage of using full low pathogenic H5N8 instead of PR8HA/NA-H5N8 to improve protection by both the innate and the humoral arms of the immune system.

scholarly journals Consistent Reanalysis of Genome-wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets

2017 ◽  
Author(s):  
Stefan Wyder ◽  
Michael T. Raissig ◽  
Ueli Grossniklaus
Keyword(s):  
Linear Models ◽  
Real Data ◽  
Rna Seq ◽  
Sequence Coverage ◽  
Reciprocal Crosses ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Allelic Bias ◽  
Generation Sequencing ◽  
Made In

ABSTRACTGenomic imprinting leads to different expression levels of maternally and paternally derived alleles. Over the last years, major progress has been made in identifying novel imprinted candidate genes in plants, owing to affordable next-generation sequencing technologies. However, reports on sequencing the transcriptome of hybrid F1 seed tissues strongly disagree about how many and which genes are imprinted. This raises questions about the relative impact of biological, environmental, technical, and analytic differences or biases. Here, we adopt a statistical approach, frequently used in RNA-seq data analysis, which properly models count overdispersion and considers replicate information of reciprocal crosses. We show that our statistical pipeline outperforms other methods in identifying imprinted genes in simulated and real data. Accordingly, reanalysis of genome-wide imprinting studies in Arabidopsis and maize shows that, at least for the Arabidopsis dataset, an increased agreement across datasets can be observed. For maize, however, consistent reanalysis did not yield in a larger overlap between the datasets. This suggests that the discrepancy across publications might be partially due to different analysis pipelines but that technical, biological, and environmental factors underlie much of the discrepancy between datasets. Finally, we show that the set of genes that can be characterized regarding allelic bias by all studies with minimal confidence is small (~8,000/27,416 genes for Arabidopsis and ~12,000/39,469 for maize). In conclusion, we propose to use biologically replicated reciprocal crosses, high sequence coverage, and a generalized linear model approach to identify differentially expressed alleles in developing seeds.

scholarly journals Targeted resequencing identifies genes with recurrent variation in cerebral palsy

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
C. L. van Eyk ◽  
M. A. Corbett ◽  
M. S. B. Frank ◽  
D. L. Webber ◽  
M. Newman ◽  
...  
Keyword(s):  
Cerebral Palsy ◽  
Rare Variants ◽  
Neurodevelopmental Disorder ◽  
Genetic Determinants ◽  
Zebrafish Model ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
A Genome ◽  
Significant Burden ◽  
Generation Sequencing

Abstract A growing body of evidence points to a considerable and heterogeneous genetic aetiology of cerebral palsy (CP). To identify recurrently variant CP genes, we designed a custom gene panel of 112 candidate genes. We tested 366 clinically unselected singleton cases with CP, including 271 cases not previously examined using next-generation sequencing technologies. Overall, 5.2% of the naïve cases (14/271) harboured a genetic variant of clinical significance in a known disease gene, with a further 4.8% of individuals (13/271) having a variant in a candidate gene classified as intolerant to variation. In the aggregate cohort of individuals from this study and our previous genomic investigations, six recurrently hit genes contributed at least 4% of disease burden to CP: COL4A1, TUBA1A, AGAP1, L1CAM, MAOB and KIF1A. Significance of Rare VAriants (SORVA) burden analysis identified four genes with a genome-wide significant burden of variants, AGAP1, ERLIN1, ZDHHC9 and PROC, of which we functionally assessed AGAP1 using a zebrafish model. Our investigations reinforce that CP is a heterogeneous neurodevelopmental disorder with known as well as novel genetic determinants.

scholarly journals Minireview: Applications of Next-Generation Sequencing on Studies of Nuclear Receptor Regulation and Function

2012 ◽  
Vol 26 (10) ◽  
pp. 1651-1659 ◽  
Author(s):  
Clifford A. Meyer ◽  
Qianzi Tang ◽  
X. Shirley Liu
Keyword(s):  
Next Generation Sequencing ◽  
Nuclear Receptors ◽  
Sequence Information ◽  
Regulation Of Transcription ◽  
Next Generation ◽  
Single Experiment ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
And Function ◽  
Generation Sequencing

Abstract Next-generation sequencing technologies have expanded the experimental possibilities for studying the genome-wide regulation of transcription by nuclear receptors, their collaborating transcription factors, and coregulators. These technologies allow investigators to obtain abundance and DNA sequence information in a single experiment. In this review, we highlight proven and potential uses of next-generation sequencing in the study of gene regulation by nuclear receptors. We also provide suggestions on how to effectively leverage this technology in a collaborative environment.

scholarly journals ASpli2: Integrative analysis of splicing landscapes through RNA-Seq assays

2020 ◽  
Author(s):  
Estefania Mancini ◽  
Andres Rabinovich ◽  
Javier Iserte ◽  
Marcelo Yanovsky ◽  
Ariel Chernomoretz
Keyword(s):  
Alternative Splicing ◽  
Real Data ◽  
Data Availability ◽  
Rna Seq ◽  
Genome Wide Analysis ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Analysis Workflow ◽  
Sophisticated Analysis ◽  
Generation Sequencing

AbstractGenome-wide analysis of alternative splicing has been a very active field of research since the early days of NGS (Next generation sequencing) technologies. Since then, ever-growing data availability and the development of increasingly sophisticated analysis methods have uncovered the complexity of the general splicing repertoire. However, independently of the considered quantification methodology, very often changes in variant concentration profiles can be hard to disentangle. In order to tackle this problem we present ASpli2, a computational suite implemented in R, that allows the identification of changes in both, annotated and novel alternative splicing events, and can deal with complex experimental designs.Our analysis workflow relies on the analysis of differential usage of subgenic features in combination with a junction-based description of local splicing changes. Analyzing simulated and real data we found that the consolidation of these signals resulted in a robust proxy of the occurrence of splicing alterations. While junction-based signals allowed us to uncover annotated as well and non-annotated events, bin-associated signals notably increased recall capabilities at a very competitive performance in terms of precision.

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