Dataset Information

Direct integration of intensity-level data from Affymetrix and Illumina microarrays improves statistical power for robust reanalysis.

ABSTRACT:

Background

Affymetrix GeneChips and Illumina BeadArrays are the most widely used commercial single channel gene expression microarrays. Public data repositories are an extremely valuable resource, providing array-derived gene expression measurements from many thousands of experiments. Unfortunately many of these studies are underpowered and it is desirable to improve power by combining data from more than one study; we sought to determine whether platform-specific bias precludes direct integration of probe intensity signals for combined reanalysis.

Results

Using Affymetrix and Illumina data from the microarray quality control project, from our own clinical samples, and from additional publicly available datasets we evaluated several approaches to directly integrate intensity level expression data from the two platforms. After mapping probe sequences to Ensembl genes we demonstrate that, ComBat and cross platform normalisation (XPN), significantly outperform mean-centering and distance-weighted discrimination (DWD) in terms of minimising inter-platform variance. In particular we observed that DWD, a popular method used in a number of previous studies, removed systematic bias at the expense of genuine biological variability, potentially reducing legitimate biological differences from integrated datasets.

Conclusion

Normalised and batch-corrected intensity-level data from Affymetrix and Illumina microarrays can be directly combined to generate biologically meaningful results with improved statistical power for robust, integrated reanalysis.

SUBMITTER: Turnbull AK

PROVIDER: S-EPMC3443058 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Publications

Direct integration of intensity-level data from Affymetrix and Illumina microarrays improves statistical power for robust reanalysis.

Turnbull Arran K AK Kitchen Robert R RR Larionov Alexey A AA Renshaw Lorna L Dixon J Michael JM Sims Andrew H AH

BMC medical genomics 20120821

<h4>Background</h4>Affymetrix GeneChips and Illumina BeadArrays are the most widely used commercial single channel gene expression microarrays. Public data repositories are an extremely valuable resource, providing array-derived gene expression measurements from many thousands of experiments. Unfortunately many of these studies are underpowered and it is desirable to improve power by combining data from more than one study; we sought to determine whether platform-specific bias precludes direct i ...[more]

PMID: 22909195

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Project description:Gene expression microarrays measure the levels of messenger ribonucleic acid (mRNA) in a sample using probe sequences that hybridize with transcribed regions. These probe sequences are designed using a reference genome for the relevant species. However, most model organisms and all humans have genomes that deviate from their reference. These variations, which include single nucleotide polymorphisms, insertions of additional nucleotides, and nucleotide deletions, can affect the microarray's performance. Genetic experiments comparing individuals bearing different population-associated single nucleotide polymorphisms that intersect microarray probes are therefore subject to systemic bias, as the reduction in binding efficiency due to a technical artifact is confounded with genetic differences between parental strains. This problem has been recognized for some time, and earlier methods of compensation have attempted to identify probes affected by genome variants using statistical models. These methods may require replicate microarray measurement of gene expression in the relevant tissue in inbred parental samples, which are not always available in model organisms and are never available in humans.By using sequence information for the genomes of organisms under investigation, potentially problematic probes can now be identified a priori. However, there is no published software tool that makes it easy to eliminate these probes from an annotation. I present equalizer, a software package that uses genome variant data to modify annotation files for the commonly used Affymetrix IVT and Gene/Exon platforms. These files can be used by any microarray normalization method for subsequent analysis. I demonstrate how use of equalizer on experiments mapping germline influence on gene expression in a genetic cross between two divergent mouse species and in human samples significantly reduces probe hybridization-induced bias, reducing false positive and false negative findings.The equalizer package reduces probe hybridization bias from experiments performed on the Affymetrix microarray platform, allowing accurate assessment of germline influence on gene expression.

Project description:BackgroundThe original spotted array technology with competitive hybridization of two experimental samples and measuring relative expression levels is increasingly displaced by more accurate platforms that allow determining absolute expression values for a single sample (for example, Affymetrix GeneChip and Illumina BeadChip). Unfortunately, cross-platform comparisons show a disappointingly low concordance between lists of regulated genes between the latter two platforms.ResultsWhereas expression values determined with a single Affymetrix GeneChip represent single measurements, the expression results obtained with Illumina BeadChip are essentially statistical means from several dozens of identical probes. In the case of multiple technical replicates, the data require, therefore, different stistical treatment depending on the platform. The key is the computation of the squared standard deviation within replicates in the case of the Illumina data as weighted mean of the square of the standard deviations of the individual experiments. With an Illumina spike experiment, we demonstrate dramatically improved significance of spiked genes over all relevant concentration ranges. The re-evaluation of two published Illumina datasets (membrane type-1 matrix metalloproteinase expression in mammary epithelial cells by Golubkov et al. Cancer Research (2006) 66, 10460; spermatogenesis in normal and teratozoospermic men, Platts et al. Human Molecular Genetics (2007) 16, 763) significantly identified more biologically relevant genes as transcriptionally regulated targets and, thus, additional biological pathways involved.ConclusionThe results in this work show that it is important to process Illumina BeadChip data in a modified statistical procedure and to compute the standard deviation in experiments with technical replicates from the standard errors of individual BeadChips. This change leads also to an improved concordance with Affymetrix GeneChip results as the spermatogenesis dataset re-evaluation demonstrates.ReviewersThis article was reviewed by I. King Jordan, Mark J. Dunning and Shamil Sunyaev.

Dataset Information

Direct integration of intensity-level data from Affymetrix and Illumina microarrays improves statistical power for robust reanalysis.

Background

Results

Conclusion

Publications

Direct integration of intensity-level data from Affymetrix and Illumina microarrays improves statistical power for robust reanalysis.

Similar Datasets

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