Project description:Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provides the most comprehensive set of annotated splice variants in C. elegans to date, and is therefore expected to faciliate focused, high resolution in vivo functional assays of AS function.
Project description:High-throughput sequencing of mixed-stage Caenorhabditis elegans small RNAs. total RNA, ~18-26nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH Keywords: high-throughput 454 sequencing
Project description:Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provides the most comprehensive set of annotated splice variants in C. elegans to date, and is therefore expected to faciliate focused, high resolution in vivo functional assays of AS function. Alternative splicing events were identified from alignments of C. elegans mRNA/EST sequences (UniGene Build #26) to C. elegans genomic sequence (NCBI timestamp: Sept. 25, 2006), essentially as previously described (Pan et al. 2005; Pan et al. 2004). In total, 499 cassette type AS events were identified. For each AS event, 3 exon probes and 3 exon junction probes were designed to profile the AS event on the microarray, essentially as previously described (Pan et al. 2004). This submission represents the expression microarray component of the study.
Project description:The use of RNA-seq as the preferred method for the discovery and validation of small RNA biomarkers is hindered by high variability and biased sequence counts. In this paper we develop a statistical model for sequence counts that accounts for ligase bias and stochastic variation in library amplification steps and sequencing depth variation. Our analytical contributions are the description of the Linear Quadratic (LQ) relation between the mean and variance of the sequence counts in an RNA-seq experiment and the derivation of the Poisson truncated mixture as the underlying probability distribution for RNA-seq data. Using a large number of sequencing datasets, we demonstrate here how one can use this modeling framework to calculate empirical correction factors for ligase bias, while accounting for random variation in sequence counts. Bias correction may remove the majority of bias in the absence of differential expression and more than 40% of the bias in the presence of variable expression of miRNAs. Empirical bias correction factors appear to be nearly constant over at least one and up to four orders of magnitude of total RNA input and independent of sample composition.
Project description:Small RNA and mRNA high-throughput sequencing was done to explore the roles of henn-1 and small RNA methylation in regulating gene expression and small RNA stability in C. elegans. Our results point to a requirement for henn-1 in methylating piRNAs, primary siRNAs, and a subset of miRNAs. In the absence of henn-1, modest changes in gene expression were observed in animals grown under optimal condition.
Project description:This project aims to identify novel RNA binding proteins in the nematode, Caenorhabditis elegans. Since interactions between RNAs and proteins may be transient, these animals were crosslinked with UV light at 254 nm which promotes the covalent link between proteins and RNAs. After this, polyadenylated mRNAs were purified via oligo(dT) coupled to magentic beads under stringent conditions. Finally, samples were subjected to mass spectrometry analysis. To rule out the possibility of RNA-independent binding we also analysed other samples: i) samples digested with RNase one; ii) samples where we performed competition assays with polyadenylic acid