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Results for “Oligonucleotide Array Sequence Analysis”

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PubMed2006

OligoSpawn: a software tool for the design of overgo probes from large unigene datasets.

BACKGROUND: Expressed sequence tag (EST) datasets represent perhaps the largest collection of genetic information. ESTs can be exploited in a variety of biological experiments and analysis. Here we are interested in the design of overlapping oligonucleotide (overgo) probes from large unigene (EST-contigs) datasets. RESULTS: OLIGOSPAWN is a suite of software tools that offers two complementary services, namely (1) the selection of "unique" oligos each of which appears in one unigene but does not occur (exactly or approximately) in any other and (2) the selection of "popular" oligos each of which occurs (exactly or approximately) in as many unigenes as possible. In this paper, we describe the functionalities of OLIGOSPAWN and the computational methods it employs, and we report on experimental results for the overgo probes designed with it. CONCLUSION: The algorithms we designed are highly efficient and capable of processing unigene datasets of sizes on the order of several tens of Mb in a few hours on a regular PC. The software has been used to design overgo probes employed to screen a barley BAC library (Hordeum vulgare). OLIGOSPAWN is freely available at http://oligospawn.ucr.edu/.

Base Sequence↗
PubMed2004

[Dynamic pattern of gene expression in rat hearts responding to transient ischemia/reperfusion detected by cDNA microarray and cluster analysis].

OBJECTIVE: To analyze the pattern of gene expression programs during rat myocardial ischemia/reperfusion at multiple time points. METHODS: Rat model of myocardial ischemia/reperfusion was established by repeating the occlusion and relaxation of left coronary artery. cDNA microarray was used to analyze the pattern of gene expression programs in rat myocardium at 1, 3, 6, 12, 24 hours after reperfusion. SOM cluster analysis was used to identify different cluster of genes in which each cluster had similar expression pattern. RESULTS: Altogether 75, 779, 205, 155, and 166 genes were differentially expressed at 1, 3, 6, 12, 24 hours after the reperfusion respectively. Clusters analysis identified 12 clusters of genes in which each cluster had similar expression pattern. CONCLUSION: Analysis of gene expression pattern revealed sequential induction of subsets of genes that characterize each response.

Animals↗
PubMed2006

Normalization of microarray data: single-labeled and dual-labeled arrays.

DNA microarray is a powerful tool for high-throughput analysis of biological systems. Various computational tools have been created to facilitate the analysis of the large volume of data produced in DNA microarray experiments. Normalization is a critical step for obtaining data that are reliable and usable for subsequent analysis such as identification of differentially expressed genes and clustering. A variety of normalization methods have been proposed over the past few years, but no methods are still perfect. Various assumptions are often taken in the process of normalization. Therefore, the knowledge of underlying assumption and principle of normalization would be helpful for the correct analysis of microarray data. We present a review of normalization techniques from single-labeled platforms such as the Affymetrix GeneChip array to dual-labeled platforms like spotted array focusing on their principles and assumptions.

Cluster Analysis↗
PubMed2004

Rapid and sensitive p53 alteration analysis in biopsies from lung cancer patients using a functional assay and a universal oligonucleotide array: a prospective study.

PURPOSE: Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. EXPERIMENTAL DESIGN: p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. RESULTS: Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. CONCLUSIONS: The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.

Aged↗
PubMed2005

Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays.

Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

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 approximately 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.

Alleles↗
PubMed2003

Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation.

We have developed a methodology we call ROMA (representational oligonucleotide microarray analysis), for the detection of the genomic aberrations in cancer and normal humans. By arraying oligonucleotide probes designed from the human genome sequence, and hybridizing with "representations" from cancer and normal cells, we detect regions of the genome with altered "copy number." We achieve an average resolution of 30 kb throughout the genome, and resolutions as high as a probe every 15 kb are practical. We illustrate the characteristics of probes on the array and accuracy of measurements obtained using ROMA. Using this methodology, we identify variation between cancer and normal genomes, as well as between normal human genomes. In cancer genomes, we readily detect amplifications and large and small homozygous and hemizygous deletions. Between normal human genomes, we frequently detect large (100 kb to 1 Mb) deletions or duplications. Many of these changes encompass known genes. ROMA will assist in the discovery of genes and markers important in cancer, and the discovery of loci that may be important in inherited predispositions to disease.

Aneuploidy↗
PubMed1999

Molecular interactions on microarrays.

The structural features of nucleic acid probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implication for the hybridization process. We discuss how arrays of oligonucleotides provide powerful tools to study the molecular basis of these interactions on a scale which is impossible using conventional analysis.

Base Composition↗
PubMed2005

The 18S rDNA sequence of Synchytrium endobioticum and its utility in microarrays for the simultaneous detection of fungal and viral pathogens of potato.

Resting spores extracted from wart (Synchytrium endobioticum)-infected potato tubers were used for DNA extraction and amplification of 18S rDNA. Analysis of the cloned, sequenced fragment revealed high similarity to members of the Chytridiomycota. Using this information, specific oligonucleotide probes were designed and arrayed onto glass slides for detection of the pathogen. Viral sequence information available in the databank was retrieved, or new viral sequences were generated, and used to design probes for specific detection of important quarantine viruses of potato. To determine the sensitivity and specificity of the oligonucleotide probes, total RNA from infected plants was reverse transcribed, labelled with Cyanine 5, and hybridised with the microarray. A significant number of the oligonucleotide probes exhibited high specificity to S. endobioticum, Andean potato latent virus, Andean potato mottle virus, Potato black ringspot virus, and Potato spindle tuber viroid. Hybridisation signals of sub-arrays within slides were reproducible (r = 0.79) with a high correlation coefficient of hybridisation repetitions (0.73). Our results demonstrate the potential of microarray-based hybridisation for identification of multiple pathogen targets, which will find application in quarantine laboratories, where parallel testing for diverse pathogens is essential.

Chytridiomycota↗
PubMed2006

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films.

Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.

Alleles↗
PubMed2005

Sequence dependence of cross-hybridization on short oligo microarrays.

One of the critical problems in the short oligo microarray technology is how to deal with cross-hybridization that produces spurious data. Little is known about the details of cross-hybridization effect at molecular level. Here, we report a free energy analysis of cross-hybridization on short oligo microarrays using data from a spike-in study. Our analysis revealed that cross-hybridization on the arrays is mostly caused by oligo fragments with a run of 10-16 nt complementary to the probes. Mismatches were estimated to be energetically much more costly in cross-hybridization than that in gene-specific hybridization, implying that the sources of cross-hybridization must be very different between a PM-MM probe pair. Consequently, it is unreliable to use MM probe signal to track cross-hybridizing signal on a corresponding PM probe. Our results also showed that the oligo fragments tend to bind to the 5' ends of the probes, and are rarely seen at the 3' ends. These results are useful for microarray design and data analysis.

Base Pair Mismatch↗
PubMed2003

Advanced computational techniques for re-sequencing DNA with polymerase signaling assay arrays.

Re-sequencing, the identification of the specific variants in a sequence of interest compared with a known genomic sequence, is a ubiquitous task in today's biology. Universal arrays, which interrogate all possible oligonucleotides of a certain length in a target sequence, have been suggested for computationally determining a polynucleotide sequence from its oligonucleotide content. We present here new methods that use such arrays for re-sequencing. Our methods are applied to data obtained by the polymerase signaling assay, which arrays single-based primer extension reactions for either universal or partial arrays of pentanucleotides. The computational analysis uses the spectrum alignment algorithm, which is refined and enhanced here in order to overcome noise incurred by the use of such short primers. We present accurate re-sequencing results for both synthetic and amplified DNA molecules.

Algorithms↗
PubMed2004

Effect of normalization on significance testing for oligonucleotide microarrays.

MOTIVATION: Normalization techniques are used to reduce variation among gene expression measurements in oligonucleotide microarrays in an effort to improve the quality of the data and the power of significance tests for detecting differential expression. Of several such proposed methods, two that have commonly been employed include median-interquartile range normalization and quantile normalization. The median-IQR method applied directly to fold-changes for paired data also was considered. Two methods for calculating gene expression values include the MAS 5.0 algorithm [Affymetrix. (2002). Statistical Algorithms Description Document. Santa Clara, CA: Affymetrix, Inc. http://www.affymetrix.com/support/technical/whitepapers/sadd-whitepaper.pdf] and the RMA method [Irizarry, R. A., Bolstad, B. M., Collin, F., Cope, L. M., Hobbs, B., Speed, T. P. (2003a). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31(4,e15); Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., Scherf, U., Speed, T. P. (2003b). Exploration, normalization, and summaries of high density oligonucleotide array probe-level data. Biostatistics 4(2):249-264; Irizarry, R. A., Gautier, L., Cope, L. (2003c). An R package for analysis of Affymetrix oligonucleotide arrays. In: Parmigiani, R. I. G., Garrett, E. S., Ziegler, S., eds. The Analysis of Gene Expression Data: Methods and Software. Berlin: Springer, pp. 102-119]. RESULTS: In considering these methods applied to a prostate cancer data set derived from paired samples on normal and tumor tissue, it is shown that normalization methods may lead to substantial inflation of the number of genes identified by paired-t significance tests even after adjustment for multiple testing. This is shown to be due primarily to an unintended effect that normalization has on the experimental error variance. The impact appears to be greater in the RMA method compared to the MAS 5.0 algorithm and for quantile normalization compared to median-IQR normalization.

Analysis of Variance↗
PubMed2002

Expression analysis of pediatric solid tumor cell lines using oligonucleotide microarrays.

We identified patterns of differentially-expressed genes in cell lines derived from several pediatric solid tumors. Affymetrix Human Cancer G110 Arrays, carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell lines originating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of soft parts. Hierarchical clustering clearly differentiated these 3 entities and revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific up-regulation in these cell lines, respectively. Whereas ET lines demonstrated increased expression of microtubule-associated protein tau (MAPT), protein phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples exhibited high expression of wingless-type mouse mammary tumor virus integration site family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and adenomatous polyposis coli (APC) which are involved in regulating free beta-catenin levels. These genes likely maintain tumor-specific characteristics and participate in key downstream regulatory mechanisms. We also correlated the expression levels of up-regulated genes in ETs with their chromosomal localization and compared these data to the comparative genomic hybridization profiles of the cell lines. We demonstrate that gains of genetic material contribute essentially to differential gene expression.

Child↗
PubMed2005

Selection of choriocarcinoma-associated genes using bioinformatics.

OBJECTIVE: To analyze the data of gene expression profiles of choriocarcinoma and screen for choriocarcinoma- related genes. METHODS: Human cDNA expression microarray containing 4 096 genes was used to study the gene expression profiles in specimens of complete hydatidiform moles (n=3) and choriocarcinomas (n=3), with normal placental villi serving as the control group. The candidate genes with similar expression profiles were identified by hierarchical cluster analysis, and their expressions in normal and neoplastic tissues analyzed by electronic Northern analysis and other bioinformatics methods. Three selected genes were analyzed by beta-actin semiquantitative reverse transcriptase-PCR to confirm the data. RESULTS: A total of 52 coexpressed candidate genes were identified from the gene expression data derived from the choriocarcinoma specimens, of which 19 genes were selected as choriocarcinoma-related genes by further cluster analysis, such as dynamin (L07807), katanin p60 (AF056022), zinc finger protein ZNF184 (U6656), calmodulin (U12022), carboxypeptidase M (BC022276), calcineurin-binding protein cabin 1 (NM_012295) and transducin-like enhancer protein TLE1(M99435). CONCLUSION: The identification of choriocarcinoma-related genes by cluster analysis provides new clues for seeking the key genes associated with the progression and metastasis of choriocarcinoma.

Choriocarcinoma↗
PubMed2002

Match-only integral distribution (MOID) algorithm for high-density oligonucleotide array analysis.

BACKGROUND: High-density oligonucleotide arrays have become a valuable tool for high-throughput gene expression profiling. Increasing the array information density and improving the analysis algorithms are two important computational research topics. RESULTS: A new algorithm, Match-Only Integral Distribution (MOID), was developed to analyze high-density oligonucleotide arrays. Using known data from both spiking experiments and no-change experiments performed with Affymetrix GeneChip arrays, MOID and the Affymetrix algorithm implemented in Microarray Suite 4.0 (MAS4) were compared. While MOID gave similar performance to MAS4 in the spiking experiments, better performance was observed in the no-change experiments.MOID also provides a set of alternative statistical analysis tools to MAS4. There are two main features that distinguish MOID from MAS4. First, MOID uses continuous P values for the likelihood of gene presence, while MAS4 resorts to discrete absolute calls. Secondly, MOID uses heuristic confidence intervals for both gene expression levels and fold change values, while MAS4 categorizes the significance of gene expression level changes into discrete fold change calls. CONCLUSIONS: The results show that by using MOID, Affymetrix GeneChip arrays may need as little as ten probes per gene without compromising analysis accuracy.

Algorithms↗
PubMed2004

High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides.

BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as probes. The production of BAC/PAC and cDNA arrays is time consuming and expensive. AIM: To evaluate the use of spotted 60 mer oligonucleotides (oligos) for array CGH. METHODS: The hybridisation of tumour cell lines with known chromosomal aberrations on to either BAC or oligoarrrays that are mapped to the human genome. RESULTS: Oligo CGH was able to detect amplifications with high accuracy and greater spatial resolution than other currently used array CGH platforms. In addition, single copy number changes could be detected with a resolution comparable to conventional CGH. CONCLUSIONS: Oligos are easy to handle and flexible, because they can be designed for any part of the genome without the need for laborious amplification procedures. The full genome array, containing around 30000 oligos of all genes in the human genome, will represent a big step forward in the analysis of chromosomal copy number changes. Finally, oligoarray CGH can easily be used for any organism with a fully sequenced genome.

Breast Neoplasms↗