scholarly journals Alternative splicing analysis benchmark with DICAST

2022 ◽  
Author(s):  
Amit M Fenn ◽  
Olga Tsoy ◽  
Tim Faro ◽  
Fanny Roessler ◽  
Alexander Dietrich ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Event Detection ◽  
Whole Blood ◽  
Tool Development ◽  
Major Contributor ◽  
Rna Seq ◽  
Consensus Approach ◽  
Reporting Standard ◽  
Health And Disease ◽  
Isoform Quantification

Alternative splicing is a major contributor to transcriptome and proteome diversity in health and disease. A plethora of tools have been developed for studying alternative splicing in RNA-seq data. Previous benchmarks focused on isoform quantification and mapping. They neglected event detection tools, which arguably provide the most detailed insights into the alternative splicing process. DICAST offers a modular and extensible framework for the analysis of alternative splicing integrating 11 splice-aware mapping and eight event detection tools. We benchmark all tools extensively on simulated as well as whole blood RNA-seq data. STAR and HISAT2 demonstrated the best balance between performance and run time. The performance of event detection tools varies widely with no tool outperforming all others. DICAST allows researchers to employ a consensus approach to consider the most successful tools jointly for robust event detection. Furthermore, we propose the first reporting standard to unify existing formats and to guide future tool development.

scholarly journals IsoformSwitchAnalyzeR: analysis of changes in genome-wide patterns of alternative splicing and its functional consequences

2019 ◽  
Vol 35 (21) ◽  
pp. 4469-4471 ◽  
Author(s):  
Kristoffer Vitting-Seerup ◽  
Albin Sandelin
Keyword(s):  
Alternative Splicing ◽  
The Cancer Genome Atlas ◽  
Supplementary Information ◽  
Rna Seq ◽  
Genome Wide ◽  
Functional Consequences ◽  
Cancer Genome Atlas ◽  
Health And Disease ◽  
Splicing Patterns

Abstract Summary Alternative splicing is an important mechanism involved in health and disease. Recent work highlights the importance of investigating genome-wide changes in splicing patterns and the subsequent functional consequences. Current computational methods only support such analysis on a gene-by-gene basis. Therefore, we extended IsoformSwitchAnalyzeR R library to enable analysis of genome-wide changes in specific types of alternative splicing and predicted functional consequences of the resulting isoform switches. As a case study, we analyzed RNA-seq data from The Cancer Genome Atlas and found systematic changes in alternative splicing and the consequences of the associated isoform switches. Availability and implementation Windows, Linux and Mac OS: http://bioconductor.org/packages/IsoformSwitchAnalyzeR. Supplementary information Supplementary data are available at Bioinformatics online.

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 IsoformSwitchAnalyzeR: Analysis of changes in genome-wide patterns of alternative splicing and its functional consequences

2018 ◽  
Author(s):  
Kristoffer Vitting-Seerup ◽  
Albin Sandelin
Keyword(s):  
Alternative Splicing ◽  
Recent Work ◽  
The Cancer Genome Atlas ◽  
Rna Seq ◽  
Genome Wide ◽  
Functional Consequences ◽  
Cancer Genome Atlas ◽  
Health And Disease ◽  
Genome Atlas

AbstractAlternative splicing is an important mechanism involved in both health and disease. Recent work highlights the importance of investigating genome-wide changes in patters of splicing and the subsequent functional consequences. Unfortunately current computational methods only support such analysis on a gene-by-gene basis. To fill this gap, we extended IsoformSwitchAnalyzeR thereby enabling analysis of genome-wide changes in both specific types of alternative splicing as well as the predicted functional consequences of the resulting isoform switches. As a case study, we analyzed RNA-seq data from The Cancer Genome Atlas and found systematic changes in both alternative splicing and the consequences of the associated isoform switches.AvailabilityWindows, Linux and Mac OS: http://bioconductor.org/packages/IsoformSwitchAnalyzeR.ContactKVS: [email protected], AS: [email protected]

scholarly journals Obstacles to Studying Alternative Splicing Using scRNA-seq

2019 ◽  
Author(s):  
Jennifer Westoby ◽  
Pavel Artemov ◽  
Martin Hemberg ◽  
Anne Ferguson-Smith
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
High Rate ◽  
Model Organisms ◽  
Rna Seq ◽  
Simulation Based ◽  
Simulation Results ◽  
The Impact ◽  
Isoform Quantification ◽  
Low Rate

AbstractBackgroundEarly single-cell RNA-seq (scRNA-seq) studies suggested that it was unusual to see more than one isoform being produced from a gene in a single cell, even when multiple isoforms were detected in matched bulk RNA-seq samples. However, these studies generally did not consider the impact of dropouts or isoform quantification errors, potentially confounding the results of these analyses.ResultsIn this study, we take a simulation based approach in which we explicitly account for dropouts and isoform quantification errors. We use our simulations to ask to what extent it is possible to study alternative splicing using scRNA-seq. Additionally, we ask what limitations must be overcome to make splicing analysis feasible. We find that the high rate of dropouts associated with scRNA-seq is a major obstacle to studying alternative splicing. In mice and other well established model organisms, the relatively low rate of isoform quantification errors poses a lesser obstacle to splicing analysis. We find that different models of isoform choice meaningfully change our simulation results.ConclusionsTo accurately study alternative splicing with single-cell RNA-seq, a better understanding of isoform choice and the errors associated with scRNA-seq is required. An increase in the capture efficiency of scRNA-seq would also be beneficial. Until some or all of the above are achieved, we do not recommend attempting to resolve isoforms in individual cells using scRNA-seq.

scholarly journals OP0021 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN EARLY RHEUMATOID ARTHRITIS PATIENTS RESPONDING TO TOCILIZUMAB

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 12.2-12
Author(s):  
I. Muller ◽  
M. Verhoeven ◽  
H. Gosselt ◽  
M. Lin ◽  
T. De Jong ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
T Cells ◽  
Gene Ontology ◽  
Regulatory T Cells ◽  
Early Rheumatoid Arthritis ◽  
Whole Blood ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Double Blind

Background:Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhibiting IL-6R signal transduction to downstream inflammatory mediators. TCZ has shown to be effective as monotherapy in early rheumatoid arthritis (RA) patients (1). However, approximately one third of patients inadequately respond to therapy and the biological mechanisms underlying lack of efficacy for TCZ remain elusive (1). Here we report gene expression differences, in both whole blood and peripheral blood mononuclear cells (PBMC) RNA samples between early RA patients, categorized by clinical TCZ response (reaching DAS28 < 3.2 at 6 months). These findings could lead to identification of predictive biomarkers for TCZ response and improve RA treatment strategies.Objectives:To identify potential baseline gene expression markers for TCZ response in early RA patients using an RNA-sequencing approach.Methods:Two cohorts of RA patients were included and blood was collected at baseline, before initiating TCZ treatment (8 mg/kg every 4 weeks, intravenously). DAS28-ESR scores were calculated at baseline and clinical response to TCZ was defined as DAS28 < 3.2 at 6 months of treatment. In the first cohort (n=21 patients, previously treated with DMARDs), RNA-sequencing (RNA-seq) was performed on baseline whole blood PAXgene RNA (Illumina TruSeq mRNA Stranded) and differential gene expression (DGE) profiles were measured between responders (n=14) and non-responders (n=7). For external replication, in a second cohort (n=95 therapy-naïve patients receiving TCZ monotherapy), RNA-seq was conducted on baseline PBMC RNA (SMARTer Stranded Total RNA-Seq Kit, Takara Bio) from the 2-year, multicenter, double-blind, placebo-controlled, randomized U-Act-Early trial (ClinicalTrials.gov identifier: NCT01034137) and DGE was analyzed between 84 responders and 11 non-responders.Results:Whole blood DGE analysis showed two significantly higher expressed genes in TCZ non-responders (False Discovery Rate, FDR < 0.05): urotensin 2 (UTS2) and caveolin-1 (CAV1). Subsequent analysis of U-Act-Early PBMC DGE showed nine differentially expressed genes (FDR < 0.05) of which expression in clinical TCZ non-responders was significantly higher for eight genes (MTCOP12, ZNF774, UTS2, SLC4A1, FECH, IFIT1B, AHSP, and SPTB) and significantly lower for one gene (TND2P28M). Both analyses were corrected for baseline DAS28-ESR, age and gender. Expression of UTS2, with a proposed function in regulatory T-cells (2), was significantly higher in TCZ non-responders in both cohorts. Furthermore, gene ontology enrichment analysis revealed no distinct gene ontology or IL-6 related pathway(s) that were significantly different between TCZ-responders and non-responders.Conclusion:Several genes are differentially expressed at baseline between responders and non-responders to TCZ therapy at 6 months. Most notably, UTS2 expression is significantly higher in TCZ non-responders in both whole blood as well as PBMC cohorts. UTS2 could be a promising target for further analyses as a potential predictive biomarker for TCZ response in RA patients in combination with clinical parameters (3).References:[1]Bijlsma JWJ, Welsing PMJ, Woodworth TG, et al. Early rheumatoid arthritis treated with tocilizumab, methotrexate, or their combination (U-Act-Early): a multicentre, randomised, double-blind, double-dummy, strategy trial. Lancet. 2016;388(10042):343-55.[2]Bhairavabhotla R, Kim YC, Glass DD, et al. Transcriptome profiling of human FoxP3+ regulatory T cells. Human Immunology. 2016;77(2):201-13.[3]Gosselt HR, Verhoeven MMA, Bulatovic-Calasan M, et al. Complex machine-learning algorithms and multivariable logistic regression on par in the prediction of insufficient clinical response to methotrexate in rheumatoid arthritis. Journal of Personalized Medicine. 2021;11(1).Disclosure of Interests:None declared

scholarly journals OP0286 PROTEOMICS AND RNA SEQUENCING APPROACHES HIGHLIGHT THE ROLE OF ENDOTHELIAL CELL DYSREGULATION IN IL-1 AND IFN MEDIATED AUTOINFLAMMATORY DISEASES, NOMID AND CANDLE

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 178-179
Author(s):  
S. Alehashemi ◽  
M. Garg ◽  
B. Sellers ◽  
A. De Jesus ◽  
A. Biancotto ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Whole Blood ◽  
Cell Activation ◽  
Immune Cell ◽  
Differentially Expressed ◽  
Neutrophilic Dermatosis ◽  
Healthy Controls ◽  
Differentially Expressed Proteins ◽  
Rna Seq ◽  
Before And After

Background:Systemic Autoinflammatory diseases present with sterile inflammation. NOMID (Neonatal-Onset Multisystem Inflammatory Disease) is caused by gain-of-function mutations inNLRP3and excess IL-1 production, presents with fever, neutrophilic dermatosis, aseptic meningitis, hearing loss and eye inflammation; CANDLE (Chronic Atypical Neutrophilic Dermatosis, Lipodystrophy and Elevated Temperature) is caused by loss-of-function mutations in proteasome genes that lead to type-1 interferon signaling, characterized by fever, panniculitis, lipodystrophy, cytopenia, systemic and pulmonary hypertension and basal ganglia calcification. IL-1 blockers are approved for NOMID and JAK-inhibitors show efficacy in CANDLE treatment.Objectives:We used proteomic analysis to compare differentially expressed proteins in active NOMID and CANDLE compared to healthy controls before and after treatment, and whole blood bulk RNA seq to identify the immune cell signatures.Methods:Serum samples from active NOMID (n=12) and CANDLE (n=7) before and after treatment (table 1) and age matched healthy controls (HC) (n=7) were profiled using the SomaLogic platform (n=1125 proteins). Differentially expressed proteins in NOMID and CANDLE were ranked after non-parametric tests for unpaired (NOMIDp<0.05, CANDLE,p<0.1) and paired (p<0.05) analysis and assessed by enriched Gene Ontology pathways and network visualization. Whole blood RNA seq was performed (NOMID=7, CANDLE=7, Controls =5) and RPKM values were used to assess immune cells signatures.Table 1.Patient’s characteristicsNOMIDN=12, Male =6CANDLEN=7, Male =6AgeMedian (range)12 (2, 28)16 (3, 20)Ethnicity%White (Hispanic)80 (20)100 (30)GeneticsNLRP3mutation(2 Somatic, 10 Germline)mutations in proteasome component genes(1 digenic, 6 Homozygous/compound Heterozygous)Before treatmentAfter treatmentBefore treatmentAfter treatmentCRPMedian (range) mg/L52 (16-110)5 (0-23)5 (0-101)1 (0-4)IFN scoremedian (range)0NA328 (211-1135)3 (0-548)Results:Compared to control, 205 proteins (127 upregulated, 78 downregulated) were significantly different at baseline in NOMID, compared to 163 proteins (101 upregulated, and 62 downregulated) in CANDLE. 134 dysregulated proteins (85 upregulated, 49 downregulated) overlapped in NOMID and CANDLE (Figure 1). Pathway analysis identified neutrophil and monocyte chemotaxis signature in both NOMID and CANDLE. NOMID patients had neutrophilia and active neutrophils. CANDLE patients exhibited active neutrophils in whole blood RNA. Endothelial cell activation was the most prominent non-hematopoietic signature and suggest distinct endothelial cell dysregulation in NOMID and CANDLE. In NOMID, the signature included neutrophil transmigration (SELE) endothelial cell motility in response to angiogenesis (HGF, VEGF), while in CANDLE the endothelial signatures included extracellular matrix protein deposition (COL8A) suggesting increased vascular stiffness. CANDLE patients had higher expression of Renin, 4 out of 7 had hypertension, NOMID patients did not have hypertension. Treatment with anakinra and baricitinib normalized 143 and 142 of dysregulated proteins in NOMID and CANDLE respectively.Conclusion:Differentially expressed proteins in NOMID and CANDLE are consistent with innate immune cell activation. Distinct endothelial cell signatures in NOMID and CANDLE may provide mechanistic insight into differences in vascular phenotypes. Treatment with anakinra and Baricitinib in NOMID and CANDLE leaves 30% and 13% of the dysregulated proteins unchanged.Acknowledgments:This work was supported by Intramural Research atNational Institute of Allergy Immunology and Infectious Diseases of National Institutes of Health, Bethesda, Maryland, the Center of Human Immunology and was approved by the IRB.Disclosure of Interests:None declared

scholarly journals Comparative Transcriptome Analysis Reveals Sex-Based Differences during the Development of the Adult Parasitic Wasp Cotesia vestalis (Hymenoptera: Braconidae)

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 896
Author(s):  
Yuenan Zhou ◽  
Pei Yang ◽  
Shuang Xie ◽  
Min Shi ◽  
Jianhua Huang ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Adult Development ◽  
Sexual Development ◽  
Molecular Mechanisms ◽  
Parasitic Wasp ◽  
Differentially Expressed Gene ◽  
Regulation Mechanism ◽  
Rna Seq ◽  
Male And Female ◽  
Cotesia Vestalis

The endoparasitic wasp Cotesia vestalis is an important biological agent for controlling the population of Plutella xylostella, a major pest of cruciferous crops worldwide. Though the genome of C. vestalis has recently been reported, molecular mechanisms associated with sexual development have not been comprehensively studied. Here, we combined PacBio Iso-Seq and Illumina RNA-Seq to perform genome-wide profiling of pharate adult and adult development of male and female C. vestalis. Taking advantage of Iso-Seq full-length reads, we identified 14,466 novel transcripts as well as 8770 lncRNAs, with many lncRNAs showing a sex- and stage-specific expression pattern. The differentially expressed gene (DEG) analyses showed 2125 stage-specific and 326 sex-specific expressed genes. We also found that 4819 genes showed 11,856 alternative splicing events through combining the Iso-Seq and RNA-Seq data. The results of comparative analyses showed that most genes were alternatively spliced across developmental stages, and alternative splicing (AS) events were more prevalent in females than in males. Furthermore, we identified six sex-determining genes in this parasitic wasp and verified their sex-specific alternative splicing profiles. Specifically, the characterization of feminizer and doublesex splicing between male and female implies a conserved regulation mechanism of sexual development in parasitic wasps.

scholarly journals Characterization of alternative splicing events and prognostic signatures in breast cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Pihua Han ◽  
Jingjun Zhu ◽  
Guang Feng ◽  
Zizhang Wang ◽  
Yanni Ding
Keyword(s):  
Breast Cancer ◽  
Alternative Splicing ◽  
Proportional Hazards ◽  
Pearson Correlation ◽  
Original Data ◽  
Cox Proportional Hazards ◽  
Prognostic Indicators ◽  
Splicing Factors ◽  
Rna Seq

Abstract Background Breast cancer (BRCA) is one of the most common cancers worldwide. Abnormal alternative splicing (AS) frequently observed in cancers. This study aims to demonstrate AS events and signatures that might serve as prognostic indicators for BRCA. Methods Original data for all seven types of splice events were obtained from TCGA SpliceSeq database. RNA-seq and clinical data of BRCA cohorts were downloaded from TCGA database. Survival-associated AS events in BRCA were analyzed by univariate COX proportional hazards regression model. Prognostic signatures were constructed for prognosis prediction in patients with BRCA based on survival-associated AS events. Pearson correlation analysis was performed to measure the correlation between the expression of splicing factors (SFs) and the percent spliced in (PSI) values of AS events. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted to demonstrate pathways in which survival-associated AS event is enriched. Results A total of 45,421 AS events in 21,232 genes were identified. Among them, 1121 AS events in 931 genes significantly correlated with survival for BRCA. The established AS prognostic signatures of seven types could accurately predict BRCA prognosis. The comprehensive AS signature could serve as independent prognostic factor for BRCA. A SF-AS regulatory network was therefore established based on the correlation between the expression levels of SFs and PSI values of AS events. Conclusions This study revealed survival-associated AS events and signatures that may help predict the survival outcomes of patients with BRCA. Additionally, the constructed SF-AS networks in BRCA can reveal the underlying regulatory mechanisms in BRCA.

scholarly journals GLiMMPS: Robust statistical model for regulatory variation of alternative splicing using RNA-seq data

2013 ◽  
Vol 14 (7) ◽  
pp. R74 ◽  
Author(s):  
Keyan Zhao ◽  
Zhi-xiang Lu ◽  
Juw Park ◽  
Qing Zhou ◽  
Yi Xing
Keyword(s):  
Alternative Splicing ◽  
Statistical Model ◽  
Rna Seq ◽  
Regulatory Variation

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.

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