Increased measurement accuracy for sequence-verified microarray probes

1 Division of Pulmonary and Critical Care Medicine, Department of Medicine and Pulmonary Bioinformatics, the Lung Biology Center, Brigham and Women’s Hospital, Boston, Massachusetts 02115 2 Children’s Hospital Informatics Program, Harvard Medical School, Boston, Massachusetts 02115 3 Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110 Microarrays have been extensively used to investigate genome-wide expression patterns. Although this technology has been tremendously successful, it has suffered from suboptimal individual measurement precision. Significant improvements in this respect have been recently made. In an effort to further explore the underlying variability, we have attempted to globally assess the accuracy of individual probe sequences used to query gene expression. For mammalian Affymetrix microarrays, we identify an unexpectedly large number of probes (greater than 19% of the probes on each platform) that do not correspond to their appropriate mRNA reference sequence (RefSeq). Compared with data derived from inaccurate probes, we find that data derived from sequence-verified probes show 1 ) increased precision in technical replicates, 2 ) increased accuracy translating data from one generation microarray to another, 3 ) increased accuracy translating data from oligonucleotide to cDNA microarrays, and 4 ) improved capture of biological information in human clinical specimens. The logical conclusion of this work is that probes containing the most reliable sequence information provide the most accurate results. Our data reveal that the identification and removal of inaccurate probes can significantly improve this technology. Affymetrix; Agilent; reference sequence; RefSeq