Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data

Genome-wide expression profiling is a powerful tool for implicating novel gene ensembles in cellular mechanisms of health and disease.The most popular platform for genome-wide expression profiling is the Affymetrix GeneChip.However, its selection of probes relied on earlier genome and transcriptome annotation which is significantly different from current knowledge.The resultant informatics problems have a profound impact on analysis and interpretation the data.Here, we address these critical issues and offer a solution.We identified several classes of problems at the individual probe level in the existing annotation, under the assumption that current genome and transcriptome databases are more accurate than those used for GeneChip design.We then reorganized probes on more than a dozen popular GeneChips into gene-, transcript-and exon-specific probe sets in light of up-to-date genome, cDNA/EST clustering and single nucleotide polymorphism information.Comparing analysis results between the original and the redefined probe sets reveals 30-50% discrepancy in the genes previously identified as differentially expressed, regardless of analysis method.Our results demonstrate that the original Affymetrix probe set definitions are inaccurate, and many conclusions derived from past GeneChip analyses may be significantly flawed.It will be beneficial to re-analyze existing GeneChip data with updated probe set definitions.It should be pointed out that three recent papers also investigated some of the problems for the HG-U133A, HG-U95A and HG-U133 Plus 2.0 GeneChips but no systematic solution was provided (7-9).For example, Harbig et al. re-annotated 37% of the probes on the HG-U133 Plus 2.0 array based on