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

ABSTRACT: BACKGROUND: 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. 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. RESULTS: In 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. CONCLUSION: We 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.

SUBMITTER: Qu Y

PROVIDER: S-EPMC2873539 | biostudies-other | 2010

REPOSITORIES: biostudies-other

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

Qu Yi Y He Fei F Chen Yuchen Y

BMC bioinformatics 20100428

<h4>Background</h4>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. 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 ...[more]

PMID: 20426803

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Project description:BACKGROUND: Alternative splicing and isoform level expression profiling is an emerging field of interest within genomics. Splicing sensitive microarrays, with probes targeted to individual exons or exon-junctions, are becoming increasingly popular as a tool capable of both expression profiling and finer scale isoform detection. Despite their intuitive appeal, relatively little is known about the performance of such tools, particularly in comparison with more traditional 3' targeted microarrays. Here, we use the well studied Microarray Quality Control (MAQC) dataset to benchmark the Affymetrix Exon Array, and compare it to two other popular platforms: Illumina, and Affymetrix U133. RESULTS: We show that at the gene expression level, the Exon Array performs comparably with the two 3' targeted platforms. However, the interplatform correlation of the results is slightly lower than between the two 3' arrays. We show that some of the discrepancies stem from the RNA amplification protocols, e.g. the Exon Array is able to detect expression of non-polyadenylated transcripts. More importantly, we show that many other differences result from the ability of the Exon Array to monitor more detailed isoform-level changes; several examples illustrate that changes detected by the 3' platforms are actually isoform variations, and that the nature of these variations can be resolved using Exon Array data. Finally, we show how the Exon Array can be used to detect alternative isoform differences, such as alternative splicing, transcript termination, and alternative promoter usage. We discuss the possible pitfalls and false positives resulting from isoform-level analysis. CONCLUSION: The Exon Array is a valuable tool that can be used to profile gene expression while providing important additional information regarding the types of gene isoforms that are expressed and variable. However, analysis of alternative splicing requires much more hands on effort and visualization of results in order to correctly interpret the data, and generally results in considerably higher false positive rates than expression analysis. One of the main sources of error in the MAQC dataset is variation in amplification efficiency across transcripts, most likely caused by joint effects of elevated GC content in the 5' ends of genes and reduced likelihood of random-primed first strand synthesis in the 3' ends of genes. These effects are currently not adequately corrected using existing statistical methods. We outline approaches to reduce such errors by filtering out potentially problematic data.

Dataset Information

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

Publications

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

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