Cross-hybridization modeling on Affymetrix exon arrays

Abstract

Motivation: Microarray designs have become increasingly probe-rich, enabling targeting of specific features, such as individual exons or single nucleotide polymorphisms. These arrays have the potential to achieve quantitative high-throughput estimates of transcript abundances, but currently these estimates are affected by biases due to cross-hybridization, in which probes hybridize to off-target transcripts.

Results: To study cross-hybridization, we map Affymetrix exon array probes to a set of annotated mRNA transcripts, allowing a small number of mismatches or insertion/deletions between the two sequences. Based on a systematic study of the degree to which probes with a given match type to a transcript are affected by cross-hybridization, we developed a strategy to correct for cross-hybridization biases of gene-level expression estimates. Comparison with Solexa ultra high-throughput sequencing data demonstrates that correction for cross-hybridization leads to a significant improvement of gene expression estimates.

Availability: We provide mappings between human and mouse exon array probes and off-target transcripts and provide software extending the GeneBASE program for generating gene-level expression estimates including the cross-hybridization correction http://biogibbs.stanford.edu/~kkapur/GeneBase/.

Fig. 1.

Shown here is an illustration of a probe (top sequence) matching a transcript (bottom sequence) with a 1-bp mismatch and a 1-bp insertion/deletion.

Fig. 2.

The empirical cdf of gene standardized residuals, separated by paralog classification.

Fig. 3.

R² statistics modeling full probe intensities by corresponding matching off-target gene expression levels, separated by the number of mismatches and insertion/deletions.

Fig. 4.

Plots of probe intensities from genes Scd3 (transcript cluster 6869918) and Scd1 (transcript cluster 6873271), separated by those which uniquely match the target transcripts and those which match the corresponding off-target transcript. Unique probes from Scd3 show a different intensity patterns from those which match Scd1, suggesting a cross-hybridization bias.

Fig. 5.

Histogram of T-values to compare the concordance of the two sets of exon array expression estimates, GeneBASE and GeneBASE-xhyb, with sequencing in liver. Histogram of (a) all genes with altered estimates between GeneBASE and GeneBASE-xhyb and (b) genes with absolute log moderated fold-change between GeneBASE and GeneBASE-xhyb estimates above 1.

Fig. 6.

Histogram of T-values to compare the concordance of the two sets of exon array expression estimates, GeneBASE and GeneBASE-xhyb-Filter, with sequencing. Histogram of (a) all genes with altered estimates between GeneBASE and GeneBASE-xhyb-Filter and (b) genes with absolute log moderated fold-change between GeneBASE and GeneBASE-xhyb-Filter estimates above 1.