MADS+: discovery of differential splicing events from Affymetrix exon junction array data

Abstract

Motivation: The Affymetrix Human Exon Junction Array is a newly designed high-density exon-sensitive microarray for global analysis of alternative splicing. Contrary to the Affymetrix exon 1.0 array, which only contains four probes per exon and no probes for exon-exon junctions, this new junction array averages eight probes per probeset targeting all exons and exon-exon junctions observed in the human mRNA/EST transcripts, representing a significant increase in the probe density for alternative splicing events. Here, we present MADS+, a computational pipeline to detect differential splicing events from the Affymetrix exon junction array data. For each alternative splicing event, MADS+ evaluates the signals of probes targeting competing transcript isoforms to identify exons or splice sites with different levels of transcript inclusion between two sample groups. MADS+ is used routinely in our analysis of Affymetrix exon junction arrays and has a high accuracy in detecting differential splicing events. For example, in a study of the novel epithelial-specific splicing regulator ESRP1, MADS+ detects hundreds of exons whose inclusion levels are dependent on ESRP1, with a RT-PCR validation rate of 88.5% (153 validated out of 173 tested).

Availability: MADS+ scripts, documentations and annotation files are available at http://www.medicine.uiowa.edu/Labs/Xing/MADSplus/.

Fig. 1.

Detection of a differentially spliced cassette exon using MADS+. (A) The HJAY probe design for cassette exons. Each probeset has eight probes. (B) Exon 13 of SLK is positively regulated by ESRP1. (C) The HJAY data indicate significantly elevated exon inclusion of SLK exon 13 in response to ESRP1 overexpression. In each panel, the dashed red line indicates the average gene expression levels of SLK in four replicate samples transduced with EV or ESRP1. The individual blue lines indicate the average background-corrected intensities of individual probes in a probeset. MADS+ P-value is shown below the name of each probeset. The ‘+’ and ‘−’ signs indicate the direction of change in splicing indexes from EV to ESRP1.