scholarly journals SUPPA2 provides fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions

2016 ◽  
Author(s):  
Juan L. Trincado ◽  
Juan C. Entizne ◽  
Gerald Hysenaj ◽  
Babita Singh ◽  
Miha Skalic ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Intron Retention ◽  
Simulated Data ◽  
Cost Effective ◽  
Sequencing Depth ◽  
Read Length ◽  
Rna Seq ◽  
Differential Splicing ◽  
The Many ◽  
Multiple Conditions

AbstractDespite the many approaches to study differential splicing from RNA-seq, many challenges remain unsolved, including computing capacity and sequencing depth requirements. Here we present SUPPA2, a new method for differential splicing analysis that addresses these challenges and enables streamlined analysis across multiple conditions taking into account biological variability. Using experimental and simulated data SUPPA2 achieves higher accuracy compared to other methods; especially at low sequencing depth and short read length, with important implications for cost-effective use of RNA-seq for splicing; and was able to identify novel Transformer2-regulated exons. We further analyzed two differentiation series to support the applicability of SUPPA2 beyond binary comparisons. This identified clusters of alternative splicing events enriched in microexons induced during differentiation of bipolar neurons, and a cluster enriched in intron retention events that are present at late stages during erythroblast differentiation. Our data suggest that SUPPA2 is a valuable tool for the robust investigation of the biological complexity of alternative splicing.

scholarly journals Plant buffering against the high-light stress-induced accumulation of CsGA2ox8 transcripts via alternative splicing to finely tune gibberellin levels and maintain hypocotyl elongation

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Bin Liu ◽  
Shuo Zhao ◽  
Pengli Li ◽  
Yilu Yin ◽  
Qingliang Niu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Light Intensity ◽  
Intron Retention ◽  
Light Stress ◽  
Photosynthetic Photon Flux Density ◽  
Photon Flux ◽  
Photon Flux Density ◽  
Rna Seq ◽  
Multiple Transcripts ◽  
Novel Transcript

AbstractIn plants, alternative splicing (AS) is markedly induced in response to environmental stresses, but it is unclear why plants generate multiple transcripts under stress conditions. In this study, RNA-seq was performed to identify AS events in cucumber seedlings grown under different light intensities. We identified a novel transcript of the gibberellin (GA)-deactivating enzyme Gibberellin 2-beta-dioxygenase 8 (CsGA2ox8). Compared with canonical CsGA2ox8.1, the CsGA2ox8.2 isoform presented intron retention between the second and third exons. Functional analysis proved that the transcript of CsGA2ox8.1 but not CsGA2ox8.2 played a role in the deactivation of bioactive GAs. Moreover, expression analysis demonstrated that both transcripts were upregulated by increased light intensity, but the expression level of CsGA2ox8.1 increased slowly when the light intensity was >400 µmol·m−2·s−1 PPFD (photosynthetic photon flux density), while the CsGA2ox8.2 transcript levels increased rapidly when the light intensity was >200 µmol·m−2·s−1 PPFD. Our findings provide evidence that plants might finely tune their GA levels by buffering against the normal transcripts of CsGA2ox8 through AS.

scholarly journals RNA-Seq Analysis Reveals Localization-Associated Alternative Splicing across 13 Cell Lines

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 820 ◽  
Author(s):  
Chao Zeng ◽  
Michiaki Hamada
Keyword(s):  
Alternative Splicing ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Biological Diversity ◽  
Rna Binding ◽  
Regulatory Mechanism ◽  
Intron Retention ◽  
Rna Seq ◽  
Motif Analysis ◽  
Structurally Stable

Alternative splicing, a ubiquitous phenomenon in eukaryotes, is a regulatory mechanism for the biological diversity of individual genes. Most studies have focused on the effects of alternative splicing for protein synthesis. However, the transcriptome-wide influence of alternative splicing on RNA subcellular localization has rarely been studied. By analyzing RNA-seq data obtained from subcellular fractions across 13 human cell lines, we identified 8720 switching genes between the cytoplasm and the nucleus. Consistent with previous reports, intron retention was observed to be enriched in the nuclear transcript variants. Interestingly, we found that short and structurally stable introns were positively correlated with nuclear localization. Motif analysis reveals that fourteen RNA-binding protein (RBPs) are prone to be preferentially bound with such introns. To our knowledge, this is the first transcriptome-wide study to analyze and evaluate the effect of alternative splicing on RNA subcellular localization. Our findings reveal that alternative splicing plays a promising role in regulating RNA subcellular localization.

scholarly journals Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis

2015 ◽  
Vol 28 (3) ◽  
pp. 298-309 ◽  
Author(s):  
Alyssa Burkhardt ◽  
Alex Buchanan ◽  
Jason S. Cumbie ◽  
Elizabeth A. Savory ◽  
Jeff H. Chang ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Intron Retention ◽  
Draft Genome ◽  
Pseudoperonospora Cubensis ◽  
Rna Seq ◽  
Transcriptome Regulation ◽  
Oomycete Pathogen ◽  
Alternatively Spliced ◽  
Genome Annotations ◽  
Obligate Pathogen

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5′- and alternative 3′-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes.

scholarly journals EmpiReS: Differential Analysis of Gene Expression and Alternative Splicing

2020 ◽  
Author(s):  
Gergely Csaba ◽  
Evi Berchtold ◽  
Armin Hadziahmetovic ◽  
Markus Gruber ◽  
Constantin Ammar ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
High Reliability ◽  
Simulated Data ◽  
Absolute Quantification ◽  
Differential Analysis ◽  
Rna Seq ◽  
Error Distributions ◽  
Alternatively Spliced ◽  
Replicate Measurements ◽  
Recall Level

ABSTRACTWhile absolute quantification is challenging in high-throughput measurements, changes of features between conditions can often be determined with high precision. Therefore, analysis of fold changes is the standard method, but often, a doubly differential analysis of changes of changes is required. Differential alternative splicing is an example of a doubly differential analysis, i.e. fold changes between conditions for different isoforms of a gene. EmpiRe is a quantitative approach for various kinds of omics data based on fold changes for appropriate features of biological objects. Empirical error distributions for these fold changes are estimated from Replicate measurements and used to quantify feature fold changes and their directions. We assess the performance of EmpiRe to detect differentially expressed genes applied to RNA-Seq using simulated data. It achieved higher precision than established tools at nearly the same recall level. Furthermore, we assess the detection of alternatively Spliced genes via changes of isoform fold changes (EmpiReS) on distribution-free simulations and experimentally validated splicing events. EmpiReS achieves the best precision-recall values for simulations based on different biological datasets. We propose EmpiRe(S) as a general, quantitative and fast approach with high reliability and an excellent trade-off between sensitivity and precision in (doubly) differential analyses.

scholarly journals Genetics of alternative splicing evolution during sunflower domestication

2018 ◽  
Vol 115 (26) ◽  
pp. 6768-6773 ◽  
Author(s):  
Chris C. R. Smith ◽  
Silas Tittes ◽  
J. Paul Mendieta ◽  
Erin Collier-zans ◽  
Heather C. Rowe ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Intron Retention ◽  
Population Divergence ◽  
Evolutionary Transition ◽  
Differential Splicing ◽  
Evolutionary Transitions ◽  
Protein Coding ◽  
Standing Variation ◽  
Splice Forms ◽  
Splicing Patterns

Alternative splicing enables organisms to produce the diversity of proteins necessary for multicellular life by using relatively few protein-coding genes. Although differences in splicing have been identified among divergent taxa, the shorter-term evolution of splicing is understudied. The origins of novel splice forms, and the contributions of alternative splicing to major evolutionary transitions, are largely unknown. This study used transcriptomes of wild and domesticated sunflowers to examine splice differentiation and regulation during domestication. We identified substantial splicing divergence between wild and domesticated sunflowers, mainly in the form of intron retention. Transcripts with divergent splicing were enriched for seed-development functions, suggesting that artificial selection impacted splicing patterns. Mapping of quantitative trait loci (QTLs) associated with 144 differential splicing cases revealed primarilytrans-acting variation affecting splicing patterns. A large proportion of identified QTLs contain known spliceosome proteins and are associated with splicing variation in multiple genes. Examining a broader set of wild and domesticated sunflower genotypes revealed that most differential splicing patterns in domesticated sunflowers likely arose from standing variation in wildHelianthus annuusand gained frequency during the domestication process. However, several domesticate-associated splicing patterns appear to be introgressed from otherHelianthusspecies. These results suggest that sunflower domestication involved selection on pleiotropic regulatory alleles. More generally, our findings indicate that substantial differences in isoform abundances arose rapidly during a recent evolutionary transition and appear to contribute to adaptation and population divergence.

scholarly journals LIQA: long-read isoform quantification and analysis

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yu Hu ◽  
Li Fang ◽  
Xuelian Chen ◽  
Jiang F. Zhong ◽  
Mingyao Li ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Myeloid Leukemia ◽  
Expectation Maximization Algorithm ◽  
Read Length ◽  
Specific Gene ◽  
Rna Seq ◽  
Sequencing Data ◽  
Long Read ◽  
Isoform Quantification ◽  
Universal Human Reference

AbstractLong-read RNA sequencing (RNA-seq) technologies can sequence full-length transcripts, facilitating the exploration of isoform-specific gene expression over short-read RNA-seq. We present LIQA to quantify isoform expression and detect differential alternative splicing (DAS) events using long-read direct mRNA sequencing or cDNA sequencing data. LIQA incorporates base pair quality score and isoform-specific read length information in a survival model to assign different weights across reads, and uses an expectation-maximization algorithm for parameter estimation. We apply LIQA to long-read RNA-seq data from the Universal Human Reference, acute myeloid leukemia, and esophageal squamous epithelial cells and demonstrate its high accuracy in profiling alternative splicing events.

scholarly journals spliceR: An R package for classification of alternative splicing and prediction of coding potential from RNA-seq data

2013 ◽  
Author(s):  
Kristoffer Vitting-Seerup ◽  
Bo T Porse ◽  
Albin Sandelin ◽  
Johannes E Waage
Keyword(s):  
Alternative Splicing ◽  
Intron Retention ◽  
Exon Skipping ◽  
R Package ◽  
Full Length ◽  
Alternative Transcript ◽  
Rna Seq ◽  
Donor And Acceptor ◽  
Coding Potential

Background: With the increasing depth and decreasing costs of RNA-sequencing researchers are now able to profile the transcriptome with unprecedented detail. These advances not only allow for precise approximation of gene expression levels, but also for the characterization of alternative transcript usage/switching between conditions. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing and prediction of coding potential. Results: spliceR uses the full-length transcripts output from RNA-seq assemblers, to detect single- and multiple exon skipping, alternative donor and acceptor sites, intron retention, alternative first or last exon usage, and mutually exclusive exon events. For each of these events spliceR also annotates the genomic coordinates of the differentially spliced elements facilitating downstream sequence analysis. Furthermore, isoform fraction values are calculated for effective post-filtering, i.e. identification of transcript switching between conditions. Lastly spliceR predicts the coding potential, as well as the potential nonsense mediated decay (NMD) sensitivity of each transcript. Conclusions: spliceR is a easy-to-use tool that allows detection of alternative splicing, transcript switching and NMD sensitivity from RNA-seq data, extending the usability of RNA-seq and assembly technologies. spliceR is implemented as an R package and is freely available from the Bioconductor repository (http://www.bioconductor.org/packages/2.13/bioc/html/spliceR.html).

scholarly journals Shark: fishing in a sample to discard useless RNA-Seq reads

2019 ◽  
Author(s):  
Paola Bonizzoni ◽  
Tamara Ceccato ◽  
Gianluca Della Vedova ◽  
Luca Denti ◽  
Yuri Pirola ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
High Throughput ◽  
Rna Seq ◽  
Differential Splicing ◽  
Massive Datasets ◽  
Computational Problem ◽  
Gene Assignment ◽  
Alignment Free ◽  
Input Dataset ◽  
Alternative Splicing Events

Recent advances in high throughput RNA-Seq technologies allow to produce massive datasets. When a study focuses only on a handful of genes, most reads are not relevant and degrade the performance of the tools used to analyze the data. Removing such useless reads from the input dataset leads to improved efficiency without compromising the results of the study.To this aim, in this paper we introduce a novel computational problem, called gene assignment and we propose an efficient alignment-free approach to solve it. Given a RNA-Seq sample and a panel of genes, a gene assignment consists in extracting from the sample the reads that most probably were sequenced from those genes. The problem becomes more complicated when the sample exhibits evidence of novel alternative splicing events.We implemented our approach in a tool called Shark and assessed its effectiveness in speeding up differential splicing analysis pipelines. This evaluation shows that Shark is able to significantly improve the performance of RNA-Seq analysis tools without having any impact on the final results.The tool is distributed as a stand-alone module and the software is freely available at https://github.com/AlgoLab/shark.

scholarly journals Normalization of generalized transcript degradation improves accuracy in RNA-seq analysis

2018 ◽  
Author(s):  
Bin Xiong ◽  
Yiben Yang ◽  
Frank R. Fineis ◽  
Ji-Ping Wang
Keyword(s):  
High Throughput ◽  
Simulated Data ◽  
Sequencing Depth ◽  
Rna Seq ◽  
Effective Performance ◽  
High Throughput Assay ◽  
In Cells

AbstractRNA-seq is a high-throughput assay to profile transcriptional activities in cells. Here we show that transcript degradation is gene-/sample-specific and presents a common and major source that may substantially bias the results in RNA-seq analysis. Most existing global normalization approaches are ineffective to correct for the degradation bias. We propose a novel pipeline named DegNorm (stands for degradation normalization) to adjust read counts for transcript degradation heterogeneity on a gene-by-gene basis while simultaneously controlling the sequencing depth. The robust and effective performance of this method is demonstrated in an extensive set of real RNA-seq data and simulated data.

scholarly journals PacBio and Illumina RNA Sequencing Identify Alternative Splicing Events in Response to Cold Stress in Two Poplar Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Jingli Yang ◽  
Wanqiu Lv ◽  
Liying Shao ◽  
Yanrui Fu ◽  
Haimei Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Stress Response ◽  
Cold Stress ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Intron Retention ◽  
Global Analysis ◽  
Populus Trichocarpa ◽  
Rna Seq ◽  
Long Read

In eukaryotes, alternative splicing (AS) is a crucial regulatory mechanism that modulates mRNA diversity and stability. The contribution of AS to stress is known in many species related to stress, but the posttranscriptional mechanism in poplar under cold stress is still unclear. Recent studies have utilized the advantages of single molecular real-time (SMRT) sequencing technology from Pacific Bioscience (PacBio) to identify full-length transcripts. We, therefore, used a combination of single-molecule long-read sequencing and Illumina RNA sequencing (RNA-Seq) for a global analysis of AS in two poplar species (Populus trichocarpa and P. ussuriensis) under cold stress. We further identified 1,261 AS events in P. trichocarpa and 2,101 in P. ussuriensis among which intron retention, with a frequency of more than 30%, was the most prominent type under cold stress. RNA-Seq data analysis and annotation revealed the importance of calcium, abscisic acid, and reactive oxygen species signaling in cold stress response. Besides, the low temperature rapidly induced multiple splicing factors, transcription factors, and differentially expressed genes through AS. In P. ussuriensis, there was a rapid occurrence of AS events, which provided a new insight into the complexity and regulation of AS during cold stress response in different poplar species for the first time.

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