A verification protocol for the probe sequences of Affymetrix genome arrays reveals high probe accuracy for studies in mouse, human and rat

BackgroundThe Affymetrix GeneChip technology uses multiple probes per gene to measure its expression level. Individual probe signals can vary widely, which hampers proper interpretation. This variation can be caused by probes that do not properly match their target gene or that match multiple genes. To determine the accuracy of Affymetrix arrays, we developed an extensive verification protocol, for mouse arrays incorporating the NCBI RefSeq, NCBI UniGene Unique, NIA Mouse Gene Index, and UCSC mouse genome databases.ResultsApplying this protocol to Affymetrix Mouse Genome arrays (the earlier U74Av2 and the newer 430 2.0 array), the number of sequence-verified probes with perfect matches was no less than 85% and 95%, respectively; and for 74% and 85% of the probe sets all probes were sequence verified. The latter percentages increased to 80% and 94% after discarding one or two unverifiable probes per probe set, and even further to 84% and 97% when, in addition, allowing for one or two mismatches between probe and target gene. Similar results were obtained for other mouse arrays, as well as for human and rat arrays. Based on these data, refined chip definition files for all arrays are provided online. Researchers can choose the version appropriate for their study to (re)analyze expression data.ConclusionThe accuracy of Affymetrix probe sequences is higher than previously reported, particularly on newer arrays. Yet, refined probe set definitions have clear effects on the detection of differentially expressed genes. We demonstrate that the interpretation of the results of Affymetrix arrays is improved when the new chip definition files are used.

[1]  D. Lockhart,et al.  Expression monitoring by hybridization to high-density oligonucleotide arrays , 1996, Nature Biotechnology.

[2]  Zhijin Wu,et al.  Preprocessing of oligonucleotide array data , 2004, Nature Biotechnology.

[3]  Andrew I Su,et al.  Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics' , 2005, Nature Genetics.

[4]  Lukas Wagner,et al.  A Greedy Algorithm for Aligning DNA Sequences , 2000, J. Comput. Biol..

[5]  Guoying Liu,et al.  NetAffx: Affymetrix probesets and annotations , 2003, Nucleic Acids Res..

[6]  Kenneth H Buetow,et al.  Detecting false expression signals in high-density oligonucleotide arrays by an in silico approach. , 2005, Genomics.

[7]  L. Wagner,et al.  21. UniGene: A Unified View of the Transcriptome , 2003 .

[8]  L. Wagner,et al.  21. UniGene: A Unified View of the Transcriptome , 2003 .

[9]  L Helmuth,et al.  A Genome Glossary , 2001, Science.

[10]  J. Nap,et al.  Genetical genomics: the added value from segregation. , 2001, Trends in genetics : TIG.

[11]  Rainer Breitling,et al.  Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments , 2004, FEBS letters.

[12]  Steen Knudsen,et al.  Alternative mapping of probes to genes for Affymetrix chips , 2004, BMC Bioinformatics.

[13]  Kim D. Pruitt,et al.  18. The Reference Sequence (RefSeq) Project , 2007 .

[14]  K. Aldape,et al.  A model of molecular interactions on short oligonucleotide microarrays , 2003, Nature Biotechnology.

[15]  S. Salzberg,et al.  The Transcriptional Landscape of the Mammalian Genome , 2005, Science.

[16]  Rudi Alberts,et al.  A Statistical Multiprobe Model for Analyzing cis and trans Genes in Genetical Genomics Experiments With Short-Oligonucleotide Arrays , 2005, Genetics.

[17]  Jo McEntyre,et al.  The NCBI Handbook , 2002 .

[18]  Zoltan Szallasi,et al.  Increased measurement accuracy for sequence-verified microarray probes. , 2004, Physiological genomics.

[19]  Dawood B. Dudekula,et al.  Genome-wide assembly and analysis of alternative transcripts in mouse. , 2005, Genome research.

[20]  Felix Naef,et al.  Solving the riddle of the bright mismatches: labeling and effective binding in oligonucleotide arrays. , 2002, Physical review. E, Statistical, nonlinear, and soft matter physics.

[21]  T. Speed,et al.  Summaries of Affymetrix GeneChip probe level data. , 2003, Nucleic acids research.

[22]  Tero Aittokallio,et al.  Integrating probe-level expression changes across generations of Affymetrix arrays , 2005, Nucleic acids research.