Dataset Information

FIRMA: a method for detection of alternative splicing from exon array data.

ABSTRACT: Analyses of EST data show that alternative splicing is much more widespread than once thought. The advent of exon and tiling microarrays means that researchers now have the capacity to experimentally measure alternative splicing on a genome wide level. New methods are needed to analyze the data from these arrays.We present a method, finding isoforms using robust multichip analysis (FIRMA), for detecting differential alternative splicing in exon array data. FIRMA has been developed for Affymetrix exon arrays, but could in principle be extended to other exon arrays, tiling arrays or splice junction arrays. We have evaluated the method using simulated data, and have also applied it to two datasets: a panel of 11 human tissues and a set of 10 pairs of matched normal and tumor colon tissue. FIRMA is able to detect exons in several genes confirmed by reverse transcriptase PCR.R code implementing our methods is contributed to the package aroma.affymetrix.

SUBMITTER: Purdom E

PROVIDER: S-EPMC2638867 | biostudies-other | 2008 Aug

REPOSITORIES: biostudies-other

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FIRMA: a method for detection of alternative splicing from exon array data.

Purdom E E Simpson K M KM Robinson M D MD Conboy J G JG Lapuk A V AV Speed T P TP

Bioinformatics (Oxford, England) 20080623 15

<h4>Motivation</h4>Analyses of EST data show that alternative splicing is much more widespread than once thought. The advent of exon and tiling microarrays means that researchers now have the capacity to experimentally measure alternative splicing on a genome wide level. New methods are needed to analyze the data from these arrays.<h4>Results</h4>We present a method, finding isoforms using robust multichip analysis (FIRMA), for detecting differential alternative splicing in exon array data. FIRM ...[more]

PMID: 18573797

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Project description:BackgroundAlternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression.ResultsWe analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer.ConclusionDifferential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.

Project description:BackgroundNeuroblastoma (NB) tumors are well known for their pronounced clinical and molecular heterogeneity. The global gene expression and DNA copy number alterations have been shown to have profound differences in tumors of low or high stage and those with or without MYCN amplification. RNA splicing is an important regulatory mechanism of gene expression, and differential RNA splicing may be associated with the clinical behavior of a tumor.MethodsIn this study, we used exon array profiling to investigate global alternative splicing pattern of 47 neuroblastoma samples in stage 1 and stage 4 with normal or amplified MYCN copy number (stage 1-, 4- and 4+). The ratio of exon-level expression to gene-level expression was used to detect alternative splicing events, while the gene-level expression was applied to characterize whole gene expression change.ResultsPrincipal component analysis (PCA) demonstrated distinct splicing pattern in three groups of samples. Pairwise comparison identified genes with splicing changes and/or whole gene expression changes in high stage tumors. In stage 4- compared with stage 1- tumors, alternatively spliced candidate genes had little overlap with genes showing whole gene expression changes, and most of them were involved in different biological processes. In contrast, a larger number of genes exhibited either exon-level splicing, gene-level expression or both changes in stage 4+ versus stage 1- tumors. Those biological processes involved in stage 4- tumors were disrupted to a greater extent by both splicing and transcription regulations in stage 4+ tumors.ConclusionsOur results demonstrated a significant role of alternative splicing in high stage neuroblastoma, and suggested a MYCN-associated splicing regulation pathway in stage 4+ tumors. The identification of differentially spliced genes and pathways in neuroblastoma tumors of different stages and molecular subtypes may be important to the understanding of cancer biology and the discovery of diagnostic markers or therapeutic targets in neuroblastoma.

Dataset Information

FIRMA: a method for detection of alternative splicing from exon array data.

Publications

FIRMA: a method for detection of alternative splicing from exon array data.

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