Improved methods for RNAseq-based alternative splicing analysis.
Ontology highlight
ABSTRACT:
SUBMITTER: Halperin RF
PROVIDER: S-EPMC8144374 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Genome-wide analyses estimate that more than 90% of multi exonic human genes produce at least two transcripts through alternative splicing (AS). Various bioinformatics methods are available to analyze AS from RNAseq data. Most methods start by mapping the reads to an annotated reference genome, but some start by a de novo assembly of the reads. In this paper, we present a systematic comparison of a mapping-first approach (FARLINE) and an assembly-first approach (KISSPLICE). We applied these methods to two independent RNAseq datasets and found that the predictions of the two pipelines overlapped (70% of exon skipping events were common), but with noticeable differences. The assembly-first approach allowed to find more novel variants, including novel unannotated exons and splice sites. It also predicted AS in recently duplicated genes. The mapping-first approach allowed to find more lowly expressed splicing variants, and splice variants overlapping repeats. This work demonstrates that annotating AS with a single approach leads to missing out a large number of candidates, many of which are differentially regulated across conditions and can be validated experimentally. We therefore advocate for the combined use of both mapping-first and assembly-first approaches for the annotation and differential analysis of AS from RNAseq datasets.
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Project description:The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation. The splicing regulatory proteins PTBP1 and PTBP2 show distinct temporal expression profiles in the developing brain. Neuronal progenitor cells predominantly express PTBP1, whereas developing neurons express high levels of PTBP2, which are subsequently reduced late in neuronal maturation. We show here that PTBP2 and the program of splicing it controls are essential to proper neuronal maturation and survival. To investigate its in vivo function, we generated conditional PTBP2 null alleles in mice. Loss of PTBP2 in neuronal progenitor cells leads to neonatal death without gross defects in brain architecture. Mice with specific depletion of PTBP2 in the cortex and forebrain are viable. However over the first three postnatal weeks, when the normal cortex expands and develops mature circuits, the PTBP2 null cortices degenerate. We find that PTBP2-/- neurons cultured from embryonic brain show the same initial viability as wild type cells with proper early marker expression and neurite outgrowth. Strikingly, between 10 and 20 days in culture PTBP2 null neurons undergo a catastrophic failure to mature and die. To assess the target transcripts leading to these phenotypes, we examined the genomewide splicing changes in the PTBP2 null brains. This identified a large number of mis-regulated exons that share a temporal pattern of regulation; in the absence of PTBP2 many isoforms normally found in adults are precociously expressed in the developing brain. Transcripts following this pattern encode essential neuronal proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program essential for neuronal survival and maturation, where PTBP2 acts to temporarily repress expression of protein isoforms until the final maturation of the neuron. Mice carrying a conditional "floxed" PTBP2 allele of PTBP2 were crossed to mice carrying Cre recombinase driven by either the nestin or Emx1 promoters. The resulting knockout mutant mouse brains were analyzed for changes in gene expression and alternative splicing. Knockout mice were compared to wildtype littermates. Whole mouse brain polyA plus RNA was isolated from two Nestin-cre knockout embryos at embryonic day 18 and compared to two wildtype littermates. Total polyA plus RNA was isolated from the cortices of an Emx-cre knockout and its wildtype littermate at postnatal day 1. This RNA was converted to standard Illumina paired end libraries using the Truseq kit. The Emx sample and control libraries were strand specific through elimination of the second strand of cDNA using the USER enzyme. Libaries were sequenced on an Illumina HiSeq using the standard paired end protocol. Data was analyzed using the Cufflinks pipeline. Splicing analysis was carried out using the SpliceTrap program.
2013-12-01 | E-GEOD-51733 | biostudies-arrayexpress
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Project description:We describe a method, microarray analysis of differential splicing (MADS), for discovery of differential alternative splicing from exon-tiling microarray data. MADS incorporates a series of low-level analysis algorithms motivated by the "probe-rich" design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data on a mouse neuroblastoma cell line after shRNA-mediated knockdown of the splicing factor polypyrimidine tract binding protein (PTB). From a list of exons with predetermined inclusion/exclusion profiles in response to PTB depletion, MADS recognized all exons known to have large changes in transcript inclusion levels and offered improvement over Affymetrix's analysis procedure. We also identified numerous novel PTB-dependent splicing events. Thirty novel events were tested by RT-PCR and 27 were confirmed. This work demonstrates that the exon-tiling microarray design is an efficient and powerful approach for global, unbiased analysis of pre-mRNA splicing.
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Project description:BackgroundUltra-fast pseudo-alignment approaches are the tool of choice in transcript-level RNA sequencing (RNA-seq) analyses. Unfortunately, these methods couple the tasks of pseudo-alignment and transcript quantification. This coupling precludes the direct usage of pseudo-alignment to other expression analyses, including alternative splicing or differential gene expression analysis, without including a non-essential transcript quantification step.ResultsIn this paper, we introduce a transcriptome segmentation approach to decouple these two tasks. We propose an efficient algorithm to generate maximal disjoint segments given a transcriptome reference library on which ultra-fast pseudo-alignment can be used to produce per-sample segment counts. We show how to apply these maximally unambiguous count statistics in two specific expression analyses - alternative splicing and gene differential expression - without the need of a transcript quantification step. Our experiments based on simulated and experimental data showed that the use of segment counts, like other methods that rely on local coverage statistics, provides an advantage over approaches that rely on transcript quantification in detecting and correctly estimating local splicing in the case of incomplete transcript annotations.ConclusionsThe transcriptome segmentation approach implemented in Yanagi exploits the computational and space efficiency of pseudo-alignment approaches. It significantly expands their applicability and interpretability in a variety of RNA-seq analyses by providing the means to model and capture local coverage variation in these analyses.
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Project description: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 analysing alternative splicing integrating eleven 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.
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Project description:BACKGROUND: The use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. The aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism Corynebacterium glutamicum by RNAseq in order to improve its genome annotation and to describe important features for transcription and translation. RESULTS: RNAseq data sets were obtained by two methods, one that focuses on 5'-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3'-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5'-UTRs (untranslated regions) for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. Interestingly, a high number of mRNAs (33%) is transcribed as leaderless transcripts. From the data, consensus promoter and ribosome binding site (RBS) motifs were identified and it was shown that the majority of genes in C. glutamicum are transcribed monocistronically, but operons containing up to 16 genes are also present. CONCLUSIONS: The comprehensive transcriptome map of C. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements (e.g. promoter regions, gene starts and stops, 5'-UTRs, RBSs, transcript starts and ends) and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. The methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future.
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Project description:The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation. The splicing regulatory proteins PTBP1 and PTBP2 show distinct temporal expression profiles in the developing brain. Neuronal progenitor cells predominantly express PTBP1, whereas developing neurons express high levels of PTBP2, which are subsequently reduced late in neuronal maturation. We show here that PTBP2 and the program of splicing it controls are essential to proper neuronal maturation and survival. To investigate its in vivo function, we generated conditional PTBP2 null alleles in mice. Loss of PTBP2 in neuronal progenitor cells leads to neonatal death without gross defects in brain architecture. Mice with specific depletion of PTBP2 in the cortex and forebrain are viable. However over the first three postnatal weeks, when the normal cortex expands and develops mature circuits, the PTBP2 null cortices degenerate. We find that PTBP2-/- neurons cultured from embryonic brain show the same initial viability as wild type cells with proper early marker expression and neurite outgrowth. Strikingly, between 10 and 20 days in culture PTBP2 null neurons undergo a catastrophic failure to mature and die. To assess the target transcripts leading to these phenotypes, we examined the genomewide splicing changes in the PTBP2 null brains. This identified a large number of mis-regulated exons that share a temporal pattern of regulation; in the absence of PTBP2 many isoforms normally found in adults are precociously expressed in the developing brain. Transcripts following this pattern encode essential neuronal proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program essential for neuronal survival and maturation, where PTBP2 acts to temporarily repress expression of protein isoforms until the final maturation of the neuron.
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