Different effects of the probe summarization algorithms PLIER and RMA on high-level analysis of Affymetrix exon arrays

Yi Qu,Yuchen Chen,Fei He

doi:10.1186/1471-2105-11-211

Yi Qu, Yuchen Chen + Show 1 more

Open Access

https://doi.org/10.1186/1471-2105-11-211

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Abstract

BackgroundAlternative splicing is an important mechanism that increases protein diversity and functionality in higher eukaryotes. Affymetrix exon arrays are a commercialized platform used to detect alternative splicing on a genome-wide scale. Two probe summarization algorithms, PLIER (Probe Logarithmic Intensity Error) and RMA (Robust Multichip Average), are commonly used to compute gene-level and exon-level expression values. However, a systematic comparison of these two algorithms on their effects on high-level analysis of the arrays has not yet been reported.ResultsIn this study, we showed that PLIER summarization led to over-estimation of gene-level expression changes, relative to exon-level expression changes, in two-group comparisons. Consequently, it led to detection of substantially more skipped exons on up-regulated genes, as well as substantially more included (i.e., non-skipped) exons on down-regulated genes. In contrast, this bias was not observed for RMA-summarized data. By using a published human tissue dataset, we compared the tissue-specific expression and splicing detected by Affymetrix exon arrays with those detected based on expressed sequence databases. We found the tendency of PLIER was not supported by the expressed sequence data.ConclusionWe showed that the tendency of PLIER in detection of alternative splicing is likely caused by a technical bias in the approach, rather than a biological bias. Moreover, we observed abnormal summarization results when using the PLIER algorithm, indicating that mathematical problems, such as numerical instability, may affect PLIER performance.

Highlights

Alternative splicing is an important mechanism that increases protein diversity and functionality in higher eukaryotes
Due to different gene-level estimation, (Probe Logarithmic Intensity Error) (PLIER) showed a strong tendency to detect relatively skipped exons on up-regulated genes and relatively included exons on down-regulated genes, while this tendency was not observed when using (Robust Multichip Average) (RMA). To determine whether this tendency of PLIER represents a real biological situation, we used tissue-specific expression and splicing events that have been identified with sequence data and summarized in the TIssueSpecific Alternative splicing (TISA) database as references
The TISA data did not show significant enrichment of skipped exons on genes with tissue-specific expression, a finding that did not support the tendency of PLIER

Summary

Introduction

Alternative splicing is an important mechanism that increases protein diversity and functionality in higher eukaryotes. Affymetrix exon arrays are a commercialized platform used to detect alternative splicing on a genome-wide scale. Alternative splicing (AS) contributes greatly to protein diversity throughout the evolution of complex organisms. Changes in the relative expression levels of the various isoforms may have significant biological implications (for example [2]). The Affymetrix exon array is one of the microarray platforms available for this purpose. It has been applied in a number of research areas [3,4,5], especially in cancer studies [6,7,8]

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