Computational comparison of common event-based differential splicing tools: practical considerations for laboratory researchers

Muller, Ittai B.; Meijers, Stijn; Kampstra, Peter; Dijk, Steven van; Elswijk, Michel van; Lin, Marry; Wojtuszkiewicz, Anna; Jansen, Gerrit; Jonge, Robert de; Cloos, Jacqueline

doi:10.1186/s12859-021-04263-9

Cited by 13 publications

(8 citation statements)

References 33 publications

(43 reference statements)

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“…a portion of transcripts from each affected gene was aberrantly spliced with the remaining transcripts displaying conventional splicing ( Figure 1E and Figure S1 ). To globally investigate alternative splicing penetrance we used rMATS, a frequently used software that performs well in independent assessments ( 36 , 37 ), to assess the inclusion level of exons across the transcriptome. For each sample we calculated an “exon commitment” score, which we define as the proportion of exons that were either completely spliced-in (i.e.…”

Section: Resultsmentioning

confidence: 99%

Reversal of splicing infidelity is a pre-activation step in B cell differentiation

et al. 2022

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IntroductionB cell activation and differentiation is central to the adaptive immune response. Changes in exon usage can have major impacts on cellular signaling and differentiation but have not been systematically explored in differentiating B cells.MethodsWe analyzed exon usage and intron retention in RNA-Seq data from subsets of human B cells at various stages of differentiation, and in an in vitro laboratory model of B cell activation and differentiation (Epstein Barr virus infection).ResultsBlood naïve B cells were found to have an unusual splicing profile, with unannotated splicing events in over 30% of expressed genes. Splicing changed substantially upon naïve B cell entry into secondary lymphoid tissue and before activation, involving significant increases in exon commitment and reductions in intron retention. These changes preferentially involved short introns with weak splice sites and were likely mediated by an overall increase in splicing efficiency induced by the lymphoid environment. The majority of transcripts affected by splicing changes showed restoration of encoded conserved protein domains and/or reduced targeting to the nonsense-mediated decay pathway. Affected genes were enriched in functionally important immune cell activation pathways such as antigen-mediated signaling, cell cycle control and mRNA processing and splicing.DiscussionFunctional observations from donor B cell subsets in progressive states of differentiation and from timecourse experiments using the in vitro model suggest that these widespread changes in mRNA splicing play a role in preparing naïve B cells for the decisive step of antigen-mediated activation and differentiation.

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Section: Resultsmentioning

confidence: 99%

Reversal of splicing infidelity is a pre-activation step in B cell differentiation

et al. 2022

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“…Five major AS event types, including intron retention, exon skipping, alternative 5′ splice site, alternative 3′ splice site, and mutually exclusive exons, were identified by AStalavista v4.0.1 according to the output files [ 49 ]. Differentially alternative splicing events (DAS) between different samples were respectively quantified using rMATS (version 4.1.2) [ 50 ]. Transcript isoforms were translated based on the longest ORF by TBtools v1.108.…”

Section: Methodsmentioning

confidence: 99%

Transcriptomic Complexity of Culm Growth and Development in Different Types of Moso Bamboo

Zhou

Liu

et al. 2023

IJMS

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Moso bamboo is capable of both sexual and asexual reproduction during natural growth, resulting in four distinct types of culms: the bamboo shoot-culm, the seedling stem, the leptomorph rhizome, and a long-ignored culm—the outward-rhizome. Sometimes, when the outward rhizomes break through the soil, they continue to grow longitudinally and develop into a new individual. However, the roles of alternative transcription start sites (aTSS) or termination sites (aTTS) as well as alternative splicing (AS) have not been comprehensively studied for their development. To re-annotate the moso bamboo genome and identify genome-wide aTSS, aTTS, and AS in growing culms, we utilized single-molecule long-read sequencing technology. In total, 169,433 non-redundant isoforms and 14,840 new gene loci were identified. Among 1311 lncRNAs, most of which showed a positive correlation with their target mRNAs, one-third of these IncRNAs were preferentially expressed in winter bamboo shoots. In addition, the predominant AS type observed in moso bamboo was intron retention, while aTSS and aTTS events occurred more frequently than AS. Notably, most genes with AS events were also accompanied by aTSS and aTTS events. Outward rhizome growth in moso bamboo was associated with a significant increase in intron retention, possibly due to changes in the growth environment. As different types of moso bamboo culms grow and develop, a significant number of isoforms undergo changes in their conserved domains due to the regulation of aTSS, aTTS, and AS. As a result, these isoforms may play different roles than their original functions. These isoforms then performed different functions from their original roles, contributing to the transcriptomic complexity of moso bamboo. Overall, this study provided a comprehensive overview of the transcriptomic changes underlying different types of moso bamboo culm growth and development.

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“…Alternative splicing (AS) analysis was performed by rMATS software, which provides a statistical method of robust and flexible detection of differential AS from replicate RNAseq data [38,39]. It identifies AS events corresponding to all major types of AS patterns and calculates the p-value and FDR.…”

Section: Alternative Splicing Analysis and Exon Skipping Events Annot...mentioning

confidence: 99%

Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects

Wang

Todorov

Cheng

et al. 2022

Cells

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Background: Gene set enrichment analysis (GSEA) was conducted on raw data, and alternative splicing (AS) events were found after mRNA sequencing of human spermatozoa. In this study, we aimed to compare unknown micro-epigenetics alternations in fresh and cryopreserved spermatozoa to evaluate the effectivity of cryopreservation protocols. Methods: Spermatozoa were divided into three groups: fresh spermatozoa (group 1), cryoprotectant-free vitrified spermatozoa (group 2), and conventionally frozen spermatozoa (group 3). Nine RNA samples (three replicates in each group) were detected and were used for library preparation with an Illumina compatible kit and sequencing by the Illumina platform. Results: Three Gene Ontology (GO) terms were found to be enriched in vitrified spermatozoa compared with fresh spermatozoa: mitochondrial tRNA aminoacylation, ATP-dependent microtubule motor activity, and male meiotic nuclear division. In alternative splicing analysis, a number of unknown AS events were found, including functional gene exon skipping (SE), alternative 5′ splice sites (A5SS), alternative 3′ splice sites (A3SS), mutually exclusive exon (MXE), and retained intron (RI). Conclusions: Cryopreservation of spermatozoa from some patients can agitate epigenetic instability, including increased alternative splicing events and changes in crucial mitochondrial functional activities. For fertilization of oocytes, for such patients, it is recommended to use fresh spermatozoa whenever possible; cryopreservation of sperm is recommended to be used only in uncontested situations.

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Computational comparison of common event-based differential splicing tools: practical considerations for laboratory researchers

Cited by 13 publications

References 33 publications

Reversal of splicing infidelity is a pre-activation step in B cell differentiation

Reversal of splicing infidelity is a pre-activation step in B cell differentiation

Transcriptomic Complexity of Culm Growth and Development in Different Types of Moso Bamboo

Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects

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