Affymetrix® Mismatch (MM) Probes: Useful after All

Affymetrix® GeneChip® micro array design defines probe sets consisting of 11, 16, or 20 distinct 25 base pair (BP) probes for determining mRNA expression for a specific gene, which may be covered by one or more probe sets. Each probe has a corresponding perfect match (PM) and mismatch (MM) set. Traditional analytical techniques have either used the MM probes to determine the level of cross-hybridization or reliability of the PM probe, or have been completely ignored. Given the availability of reference genome sequences, we have reanalyzed the mapping of both PM and MM probes to reference genomes in transcript regions. Our results suggest that depending of the species of interest, 66%-93% of the PM probes can be used reliably in terms of single unique matches to the genome, while a small number of the MM probes (typically less than 1%) could be incorporated into the analysis. In addition, we have examined the mapping of PM and MM probes to five different human genome projects, resulting in approximately a 70% overlap of uniquely mapping PM probes, and a subset of 51 uniquely mapping MM probes commonly found in all five projects, 24 of which are found within annotated exonic regions. These results suggest that individual variation in transcriptome regions provides an additional complexity to micro array data analysis. Given these results, we conclude that the development of custom chip definition files (CDFs) should include MM probe sequences to provide the most effective means of transcriptome analysis of Affymetrix® GeneChip® arrays.