Probe selection for high-density oligonucleotide arrays

Abstract

High-density oligonucleotide microarrays enable simultaneous monitoring of expression levels of tens of thousands of transcripts. For accurate detection and quantitation of transcripts in the presence of cellular mRNA, it is essential to design microarrays whose oligonucleotide probes produce hybridization intensities that accurately reflect the concentration of original mRNA. We present a model-based approach that predicts optimal probes by using sequence and empirical information. We constructed a thermodynamic model for hybridization behavior and determined the influence of empirical factors on the effective fitting parameters. We designed Affymetrix GeneChip probe arrays that contained all 25-mer probes for hundreds of human and yeast transcripts and collected data over a 4,000-fold concentration range. Multiple linear regression models were built to predict hybridization intensities of each probe at given target concentrations, and each intensity profile is summarized by a probe response metric. We selected probe sets to represent each transcript that were optimized with respect to responsiveness, independence (degree to which probe sequences are nonoverlapping), and uniqueness (lack of similarity to sequences in the expressed genomic background). We show that this approach is capable of selecting probes with high sensitivity and specificity for high-density oligonucleotide arrays.design ͉ modeling ͉ microarray design H igh-density oligonucleotide arrays (1, 2) have revolutionized the study of gene expression. The technology enables researchers to detect and quantify tens of thousands of transcripts in a single experiment and has become a standard for the discovery of gene functions, drug evaluation, pathway dissection, and classification of clinical samples (3). With the availability of mRNA sequences for a large subset of the draft of the human genome (4, 5) microarrays provide the potential to simultaneously monitor the whole expressed human genome. The expression profile of the whole human genome will allow a detailed and comprehensive view of cellular processes, responses, and their functional consequences.Quantitative detection of transcripts requires that microarray probes exhibit a sensitive and predictable response to concentrations of specific targets of the probes. This response must occur in the presence of a complex mixture of nonspecific targets. The previous probe selection method for GeneChip expression microarrays was to select candidate probes based on a set of heuristic rules (1). The rules act as filters to remove extreme sequence features that were known to degrade probe performance. However, probes passing the filters were treated as being of equal quality. To select optimal probe sets, it is essential to establish a continuous metric that distinguishes superior probes from merely good probes. Several theoretical studies of microarray probe selection (6, 7) were based on solution thermodynamics. No experimental data were produced to demonstrate that the theoretical predictions for hybr...

Base Pair Interactions and Hybridization Isotherms of Matched and Mismatched Oligonucleotide Probes on Microarrays

“…It can be described as the sum of positional and base dependent terms, σ k P (B) (see Eq. 8), in accordance with previous models 20,21,28,29 .…”

“…Positional dependent SB models were recently used to predict microarray probe intensities 20,21 . In our notation the SB model decomposes the sensitivity of each probe into a sum of sensitivity contributions σ k P (ξ P k ), depending on the base at position k = 1…N b of the probe sequence, ξ P k , …”

Application of a High-Density Oligonucleotide Microarray Approach To Study Bacterial Population Dynamics during Uranium Reduction and Reoxidation

“…In the second step, a subset of the prevalent targets was selected and reverse complemented into probe orientation, avoiding those capable of mis-hybridization to an unintended amplicon. Probes presumed to have the capacity to mis-hybridize were those 25-mers that contained a central 17-mer matching sequences in more than one OTU (34,56). Thus, probes that were unique to an OTU solely due to a distinctive base in one of the outer four bases were avoided.…”

“…Probes complementary to target sequences that were selected for fabrication are termed PM probes. As each PM probe was chosen, it was paired with a control 25-mer (mismatching [MM] probe), identical in all positions except the 13th base (34). The MM probe did not contain a central 17-mer complementary to sequences in any OTU.…”

A Strategy to Rapidly Re-Sequence the NF1 Genomic Loci Using Microarrays and Bioinformatics for Molecular Classification of the Disease

“…Quantitative detection of DNA requires that microarray probes exhibit a sensitive and predictable response to concentrations of specific targets of the probes. This response must occur in the presence of a complex mixture of nonspecific targets [7]. This presents an additional problem that needs to be overcome for our method to work.…”