Empirical evaluation of oligonucleotide probe selection for DNA microarrays.
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ABSTRACT:
SUBMITTER: Mulle JG
PROVIDER: S-EPMC2847945 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of < or =85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (< or =15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than -30 kcal/mol for 50-mer probes or -40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.
| S-EPMC1169010 | biostudies-literature
Project description:Oligonucleotide-based DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms. Here we report a systematic study of the sensitivity, specificity and dynamic range of microarray signals and their dependence on the labeling and hybridization conditions as well as on the length, concentration, attachment moiety and purity of the oligonucleotides. Both a controlled set of in vitro synthesized transcripts and RNAs from biological samples were used in these experiments. An algorithm is presented that allows the efficient selection of oligonucleotides able to discriminate a single nucleotide mismatch. Critical parameters for various applications are discussed based on statistical analysis of the results. These data will facilitate the design and standardization of custom-made microarrays applicable to gene expression profiling and sequencing analyses.
| S-EPMC117213 | biostudies-literature
Project description:BACKGROUND: When analyzing microarray data a primary objective is often to find differentially expressed genes. With empirical Bayes and penalized t-tests the sample variances are adjusted towards a global estimate, producing more stable results compared to ordinary t-tests. However, for Affymetrix type data a clear dependency between variability and intensity-level generally exists, even for logged intensities, most clearly for data at the probe level but also for probe-set summarizes such as the MAS5 expression index. As a consequence, adjustment towards a global estimate results in an intensity-level dependent false positive rate. RESULTS: We propose two new methods for finding differentially expressed genes, Probe level Locally moderated Weighted median-t (PLW) and Locally Moderated Weighted-t (LMW). Both methods use an empirical Bayes model taking the dependency between variability and intensity-level into account. A global covariance matrix is also used allowing for differing variances between arrays as well as array-to-array correlations. PLW is specially designed for Affymetrix type arrays (or other multiple-probe arrays). Instead of making inference on probe-set summaries, comparisons are made separately for each perfect-match probe and are then summarized into one score for the probe-set. CONCLUSION: The proposed methods are compared to 14 existing methods using five spike-in data sets. For RMA and GCRMA processed data, PLW has the most accurate ranking of regulated genes in four out of the five data sets, and LMW consistently performs better than all examined moderated t-tests when used on RMA, GCRMA, and MAS5 expression indexes.
| S-EPMC2358895 | biostudies-other
Project description:BACKGROUND:Long oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear. RESULTS:Unmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 microm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR. CONCLUSIONS:Microarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.
| S-EPMC151289 | biostudies-literature
Project description: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.
| S-EPMC208741 | biostudies-literature
Project description:High density oligonucleotide microarrays present a big challenge for statistical data processing methods which aim to separate changes induced by experimental factors from those caused by artifacts and measurement inaccuracies. Despite huge advances in the field of microarray probe design methods, the signal variation between probes that target a single transcript is substantially larger than their between-replicate array variability, suggesting a large influence of various probe-specific effects that introduce bias to the data. In this work we present the influence of probe-related design variations on the expression intensities of individual probes, focusing on five potential sources of high probe signal variance: the GC composition of the probe, the distance between individual probe target sites, G-quadruplex formation in the probe sequence, the occurrence of sequence motifs complementary to the oligo(dT) primer, and the specificity of unrecognized alternative splicing probeset assignment. By focusing on two high quality microarray datasets based on two distinct array designs we show the extent of variance between probes that target a specific transcript providing guidelines for the future design of microarrays and data processing methods.
| S-EPMC3926573 | biostudies-literature
Project description:BackgroundArray comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome.ResultsThis paper presents a new probe selection algorithm (PanArray) that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage.ConclusionPanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on a single microarray chip. These unique pan-genome tiling arrays provide maximum flexibility for the analysis of both known and uncharacterized strains.
| S-EPMC2753849 | biostudies-literature
Project description:To optimize oligonucleotide probe design criteria, PCR products with different similarities to probes were hybridized to a functional gene microarray designed to detect homologous genes from different organisms. In contrast to more restrictive probe designs based on a single criterion, simultaneous consideration of the percent similarity (< or =90%), the length of identical sequence stretches (< or =20 bases), and the binding free energy (> or =-35 kcal mol(-1)) was found to be predictive of probe specificity.
| S-EPMC1392891 | biostudies-other
Project description:DNA/DNA duplex formation is the basic mechanism that is used in genome tiling arrays and SNP arrays manufactured by Affymetrix. However, detailed knowledge of the physical process is still lacking. In this study, we show a free energy analysis of DNA/DNA duplex formation these arrays based on the positional-dependent nearest-neighbor (PDNN) model, which was developed previously for describing DNA/RNA duplex formation on expression microarrays. Our results showed that the two ends of a probe contribute less to the stability of the duplexes and that there is a microarray surface effect on binding affinities. We also showed that free energy cost of a single mismatch depends on the bases adjacent to the mismatch site and obtained a comprehensive table of the cost of a single mismatch under all possible combination of adjacent bases. The mismatch costs were found to be correlated with those determined in aqueous solution. We further demonstrate that the DNA copy number estimated from the SNP array correlates negatively with the target length; this is presumably caused by inefficient PCR amplification for long fragments. These results provide important insights into the molecular mechanisms of microarray technology and have implications for microarray design and the interpretation of observed data.
| S-EPMC1807971 | biostudies-literature
Project description:Array-based comparative genomic hybridization (CGH) measures copy-number variations at multiple loci simultaneously, providing an important tool for studying cancer and developmental disorders and for developing diagnostic and therapeutic targets. Arrays for CGH based on PCR products representing assemblies of BAC or cDNA clones typically require maintenance, propagation, replication, and verification of large clone sets. Furthermore, it is difficult to control the specificity of the hybridization to the complex sequences that are present in each feature of such arrays. To develop a more robust and flexible platform, we created probe-design methods and assay protocols that make oligonucleotide microarrays synthesized in situ by inkjet technology compatible with array-based comparative genomic hybridization applications employing samples of total genomic DNA. Hybridization of a series of cell lines with variable numbers of X chromosomes to arrays designed for CGH measurements gave median ratios for X-chromosome probes within 6% of the theoretical values (0.5 for XY/XX, 1.0 for XX/XX, 1.4 for XXX/XX, 2.1 for XXXX/XX, and 2.6 for XXXXX/XX). Furthermore, these arrays detected and mapped regions of single-copy losses, homozygous deletions, and amplicons of various sizes in different model systems, including diploid cells with a chromosomal breakpoint that has been mapped and sequenced to a precise nucleotide and tumor cell lines with highly variable regions of gains and losses. Our results demonstrate that oligonucleotide arrays designed for CGH provide a robust and precise platform for detecting chromosomal alterations throughout a genome with high sensitivity even when using full-complexity genomic samples.
| S-EPMC535426 | biostudies-literature