Experimental comparison and cross-validation of the Affymetrix and Illumina gene expression analysis platforms - PubMed (original) (raw)

Comparative Study

. 2005 Oct 19;33(18):5914-23.

doi: 10.1093/nar/gki890. Print 2005.

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Comparative Study

Experimental comparison and cross-validation of the Affymetrix and Illumina gene expression analysis platforms

Michael Barnes et al. Nucleic Acids Res. 2005.

Abstract

The growth in popularity of RNA expression microarrays has been accompanied by concerns about the reliability of the data especially when comparing between different platforms. Here, we present an evaluation of the reproducibility of microarray results using two platforms, Affymetrix GeneChips and Illumina BeadArrays. The study design is based on a dilution series of two human tissues (blood and placenta), tested in duplicate on each platform. The results of a comparison between the platforms indicate very high agreement, particularly for genes which are predicted to be differentially expressed between the two tissues. Agreement was strongly correlated with the level of expression of a gene. Concordance was also improved when probes on the two platforms could be identified as being likely to target the same set of transcripts of a given gene. These results shed light on the causes or failures of agreement across microarray platforms. The set of probes we found to be most highly reproducible can be used by others to help increase confidence in analyses of other data sets using these platforms.

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Figures

Figure 1

Figure 1

Schematic representation of the experimental design. See Materials and Methods for details. The key features of the design are the use of a single pair of RNA samples for all analyses, mixed together in varying proportions and analyzed in technical replicates on each platform. Unlike the Affymetrix platform, each Illumina BeadArray slide contains multiple arrays, allowing us to analyze a complete dilution series on one slide. Note that the BeadArray slides actually contain eight arrays per slide, but we only used six for the data described here.

Figure 2

Figure 2

Hierarchical clustering results of all 36 024 comparable pairs of probes. The dilution step is shown as a graph at the top of the figure (Blood/Placenta). Black bars at the side indicate large clusters of genes that appear to show clear dilution effects in both platforms. Gray bars indicate examples of clusters that appear to show dilution effects in one platform but not consistently in the other. Lighter colors indicate higher relative levels of expression on an arbitrary scale. Note that in this figure, if a gene occurs multiple times on one platform, it is shown in all possible valid comparisons with matching probes on the other platform.

Figure 3

Figure 3

Distributions of correlations for the Illumina HumanRef-8 BeadArrays (A) and the Affymetrix HG-U133 Plus 2 arrays (B). Correlations near −1 and 1 reflect probes whose targets are differentially expressed between the two samples. Correlations near zero reflect probes which do not show a dilution effect. The dilution effect is more pronounced for probes targeted at ‘known’ genes. This effect is stronger for Illumina (C) than for Affymetrix (D), though the Illumina platform has fewer probes which cannot be assigned to known genes (Table 1). For complete data see the Supplementary Data.

Figure 4

Figure 4

Distributions of correlations stratified by high and low expression levels (log2) for the Illumina HumanRef-8 BeadArrays (A) and the Affymetrix HG-U133 Plus 2.0 arrays (B). On both platforms, the probes not showing dilution effects tend to express at low levels, whereas highly expressed probes show strong dilution effects. For complete data see the Supplementary Data.

Figure 5

Figure 5

Cross-platform agreement for all ‘known’ genes (A), stratified by differential expression (B) and for placenta/blood specific genes (C). For complete data see the Supplementary Data.

Figure 6

Figure 6

Cross-platform agreement measured by the rank correlation of expression levels as a function of expression level (log2) (A) and distance between probes in base pairs (B). For complete data see the Supplementary Data.

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