SpliceGrapher: detecting patterns of alternative splicing from RNA-Seq data in the context of gene models and EST data

We propose a method for predicting splice graphs that enhances curated gene models using evidence from RNA-Seq and EST alignments. Results obtained using RNA-Seq experiments in Arabidopsis thaliana show that predictions made by our SpliceGrapher method are more consistent with current gene models than predictions made by TAU and Cufflinks. Furthermore, analysis of plant and human data indicates that the machine learning approach used by SpliceGrapher is useful for discriminating between real and spurious splice sites, and can improve the reliability of detection of alternative splicing. SpliceGrapher is available for download at .     

Computational identification of tissue-specific alternative splicing elements in mouse genes from RNA-Seq

Tissue-specific alternative splicing is a key mechanism for generating tissue-specific proteomic diversity in eukaryotes. Splicing regulatory elements (SREs) in pre-mature messenger RNA play a very important role in regulating alternative splicing. In this article, we use mouse RNA-Seq data to determine a positive data set where SREs are over-represented and a reliable negative data set where the same SREs are most likely under-represented for a specific tissue and then employ a powerful discriminative approach to identify SREs. We identified 456 putative splicing enhancers or silencers, of which 221 were predicted to be tissue-specific. Most of our tissue-specific SREs are likely different from constitutive SREs, since only 18% of our exonic splicing enhancers (ESEs) are contained in constitutive RESCUE-ESEs. A relatively small portion (20%) of our SREs is included in tissue-specific SREs in human identified in two recent studies. In the analysis of position distribution of SREs, we found that a dozen of SREs were biased to a specific region. We also identified two very interesting SREs that can function as an enhancer in one tissue but a silencer in another tissue from the same intronic region. These findings provide insight into the mechanism of tissue-specific alternative splicing and give a set of valuable putative SREs for further experimental investigations.     

SpliceNet: recovering splicing isoform-specific differential gene networks from RNA-Seq data of normal and diseased samples

Conventionally, overall gene expressions from microarrays are used to infer gene networks, but it is challenging to account splicing isoforms. High-throughput RNA Sequencing has made splice variant profiling practical. However, its true merit in quantifying splicing isoforms and isoform-specific exon expressions is not well explored in inferring gene networks. This study demonstrates SpliceNet, a method to infer isoform-specific co-expression networks from exon-level RNA-Seq data, using large dimensional trace. It goes beyond differentially expressed genes and infers splicing isoform network changes between normal and diseased samples. It eases the sample size bottleneck; evaluations on simulated data and lung cancer-specific ERBB2 and MAPK signaling pathways, with varying number of samples, evince the merit in handling high exon to sample size ratio datasets. Inferred network rewiring of well established Bcl-x and EGFR centered networks from lung adenocarcinoma expression data is in good agreement with literature. Gene level evaluations demonstrate a substantial performance of SpliceNet over canonical correlation analysis, a method that is currently applied to exon level RNA-Seq data. SpliceNet can also be applied to exon array data. SpliceNet is distributed as an R package available at http://www.jjwanglab.org/SpliceNet.     

Detection and measurement of alternative splicing using splicing-sensitive microarrays

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.     

Extent and diversity of human alternative splicing established by complementary database annotation and microarray analysis

Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor, and acceptor sites. In large-scale microarray studies in humans and the mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across six pair of diverse cell lines and validated the database annotation process. Results suggest that splicing in humans is more complex than simple exon-skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology.     

GandrKB--ontological microarray annotation and visualization

SUMMARY: The Gandr (gene annotation data representation) knowledgebase is an ontological framework for laboratory-specific gene annotation. Gandr uses Protege 2000 for editing, querying and visualizing microarray data and annotations. Genes can be annotated with provided, newly created or imported ontological concepts. Annotated genes can inherit assigned concept properties and can be related to each other. The resulting knowledgebase can be visualized as interactive network of nodes and edges representing genes and their functional relationships. This allows for immediate and associative gene context exploration. Ontological query techniques allow for powerful data access.     

可变剪接的生物信息数据分析综述

前体mRNA的可变剪接是扩大真核生物蛋白质组多样性的重要基因调控机制。可变剪接的错误调节可以引起多种人类疾病。由于高通量技术的发展,生物信息学成为可变剪接研究的主要手段。本文总结了可变剪接在生物信息学领域的研究方法,同时也分析并预测了可变剪接的发展方向。     

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation

Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.     

Artemis: sequence visualization and annotation

SUMMARY: Artemis is a DNA sequence visualization and annotation tool that allows the results of any analysis or sets of analyses to be viewed in the context of the sequence and its six-frame translation. Artemis is especially useful in analysing the compact genomes of bacteria, archaea and lower eukaryotes, and will cope with sequences of any size from small genes to whole genomes. It is implemented in Java, and can be run on any suitable platform. Sequences and annotation can be read and written directly in EMBL, GenBank and GFF format. AVAILABITLTY: Artemis is available under the GNU General Public License from http://www.sanger.ac.uk/Software/Artemis