Effect of polymorphisms within probe-target sequences on olignonucleotide microarray experiments.
Hybridization-based technologies, such as microarrays, rely on precise probe-target interactions to ensure specific and accurate measurement of RNA expression. Polymorphisms present in the probe-target sequences have been shown to alter probe- hybridization affinities, leading to reduced signal intensity measurements and resulting in false-positive results. Here, we characterize this effect on exon and gene expression estimates derived from the Affymetrix Exon Array. We conducted an association analysis between expression levels of probes, exons and transcripts and the genotypes of neighboring SNPs in 57 CEU HapMap individuals. We quantified the dependence of the effect of genotype on signal intensity with respect to the number of polymorphisms within target sequences, number of affected probes and position of the polymorphism within each probe. The effect of SNPs is quite severe and leads to considerable false-positive rates, particularly when the analysis is performed at the exon level and aimed at detecting alternative splicing events. Finally, we propose simple solutions, based on 'masking' probes, which are putatively affected by polymorphisms and show that such strategy results in a large decrease in false-positive rates, with a very modest reduction in coverage of the transcriptome.
Benovoy
David
D
Department of Human Genetics, McGill University, Montreal, QC, Canada. davidbenovoy@gmail.com
Kwan
Tony
T
Majewski
Jacek
J
D020137:Base Pair Mismatch; D005091:Exons; D020869:Gene Expression Profiling / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D015341:Nucleic Acid Probes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D015341:Nucleic Acid Probes