A bioinformatics tool to select sequences for microarray studies of mouse models of oncogenesis

One of the challenges to the effective utilization of cDNA microarray analysis in mouse models of oncogenesis is the choice of a critical set of probes that are informative for human disease. Given the thousands of genes with a potential role in human oncogenesis and the hundreds of thousands of mouse sequences available for use as probes, selection of an informative set of mouse probes can be an overwhelming task. We have developed a web based sequence mining tool using DataBase Independent (DBI) Perl to annotate publicly available sequences. The Mouse Oncochip Design Tool uses the Mouse Genome Database (MGD) developed and maintained by the Jackson Laboratories for mouse DNA sequences. There are over 380 000 sequences in their database. The output list has been ordered to present the genes more likely to be informative in a mouse model of human cancer using a candidate set of oncogenes to order the list. Mouse sequences that represent genes that are homologous with a member of a human oncogene set are listed first. In addition it provides a set of links for information on clone source gene function. Contact: http://nciarray.nci.nih.gov/cgi-bin/me/mouse_design.cgi     

Meta-analysis of gene expression data: a predictor-based approach

MOTIVATION: With the increasing availability of cancer microarray data sets there is a growing need for integrative computational methods that evaluate multiple independent microarray data sets investigating a common theme or disorder. Meta-analysis techniques are designed to overcome the low sample size typical to microarray experiments and yield more valid and informative results than each experiment separately. RESULTS: We propose a new meta-analysis technique that aims at finding a set of classifying genes, whose expression level may be used to answering the classification question in hand. Specifically, we apply our method to two independent lung cancer microarray data sets and identify a joint core subset of genes which putatively play an important role in tumor genesis of the lung. The robustness of the identified joint core set is demonstrated on a third unseen lung cancer data set, where it leads to successful classification using very few top-ranked genes. Identifying such a set of genes is of significant importance when searching for biologically meaningful biomarkers. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

A multi-array multi-SNP genotyping algorithm for Affymetrix SNP microarrays

MOTIVATION: Modern strategies for mapping disease loci require efficient genotyping of a large number of known polymorphic sites in the genome. The sensitive and high-throughput nature of hybridization-based DNA microarray technology provides an ideal platform for such an application by interrogating up to hundreds of thousands of single nucleotide polymorphisms (SNPs) in a single assay. Similar to the development of expression arrays, these genotyping arrays pose many data analytic challenges that are often platform specific. Affymetrix SNP arrays, e.g. use multiple sets of short oligonucleotide probes for each known SNP, and require effective statistical methods to combine these probe intensities in order to generate reliable and accurate genotype calls. RESULTS: We developed an integrated multi-SNP, multi-array genotype calling algorithm for Affymetrix SNP arrays, MAMS, that combines single-array multi-SNP (SAMS) and multi-array, single-SNP (MASS) calls to improve the accuracy of genotype calls, without the need for training data or computation-intensive normalization procedures as in other multi-array methods. The algorithm uses resampling techniques and model-based clustering to derive single array based genotype calls, which are subsequently refined by competitive genotype calls based on (MASS) clustering. The resampling scheme caps computation for single-array analysis and hence is readily scalable, important in view of expanding numbers of SNPs per array. The MASS update is designed to improve calls for atypical SNPs, harboring allele-imbalanced binding affinities, that are difficult to genotype without information from other arrays. Using a publicly available data set of HapMap samples from Affymetrix, and independent calls by alternative genotyping methods from the HapMap project, we show that our approach performs competitively to existing methods. AVAILABILITY: R functions are available upon request from the authors.     

A DSRPCL-SVM approach to informative gene analysis

Microarray data based tumor diagnosis is a very interesting topic in bioinformatics. One of the key problems is the discovery and analysis of informative genes of a tumor. Although there are many elaborate approaches to this problem, it is still difficult to select a reasonable set of informative genes for tumor diagnosis only with microarray data. In this paper, we classify the genes expressed through microarray data into a number of clusters via the distance sensitive rival penalized competitive learning （DSRPCL） algorithm and then detect the informative gene cluster or set with the help of support vector machine （SVM）. Moreover, the critical or powerful informative genes can be found through further classifications and detections on the obtained informative gene clusters. It is well demonstrated by experiments on the colon, leukemia, and breast cancer datasets that our proposed DSRPCL-SVM approach leads to a reasonable selection of informative genes for tumor diagnosis.     

Statistical development and evaluation of microarray gene expression data filters.

Filtering is a common practice used to simplify the analysis of microarray data by removing from subsequent consideration probe sets believed to be unexpressed. The m/n filter, which is widely used in the analysis of Affymetrix data, removes all probe sets having fewer than m present calls among a set of n chips. The m/n filter has been widely used without considering its statistical properties. The level and power of the m/n filter are derived. Two alternative filters, the pooled p-value filter and the error-minimizing pooled p-value filter are proposed. The pooled p-value filter combines information from the present-absent p-values into a single summary p-value which is subsequently compared to a selected significance threshold. We show that pooled p-value filter is the uniformly most powerful statistical test under a reasonable beta model and that it exhibits greater power than the m/n filter in all scenarios considered in a simulation study. The error-minimizing pooled p-value filter compares the summary p-value with a threshold determined to minimize a total-error criterion based on a partition of the distribution of all probes' summary p-values. The pooled p-value and error-minimizing pooled p-value filters clearly perform better than the m/n filter in a case-study analysis. The case-study analysis also demonstrates a proposed method for estimating the number of differentially expressed probe sets excluded by filtering and subsequent impact on the final analysis. The filter impact analysis shows that the use of even the best filter may hinder, rather than enhance, the ability to discover interesting probe sets or genes. S-plus and R routines to implement the pooled p-value and error-minimizing pooled p-value filters have been developed and are available from www.stjuderesearch.org/depts/biostats/index.html.     

CSF-PR 2.0: An Interactive Literature Guide to Quantitative Cerebrospinal Fluid Mass Spectrometry Data from Neurodegenerative Disorders

The rapidly growing number of biomedical studies supported by mass spectrometry based quantitative proteomics data has made it increasingly difficult to obtain an overview of the current status of the research field. A better way of organizing the biomedical proteomics information from these studies and making it available to the research community is therefore called for. In the presented work, we have investigated scientific publications describing the analysis of the cerebrospinal fluid proteome in relation to multiple sclerosis, Parkinson's disease and Alzheimer's disease. Based on a detailed set of filtering criteria we extracted 85 data sets containing quantitative information for close to 2000 proteins. This information was made available in CSF-PR 2.0 (http://probe.uib.no/csf-pr-2.0), which includes novel approaches for filtering, visualizing and comparing quantitative proteomics information in an interactive and user-friendly environment. CSF-PR 2.0 will be an invaluable resource for anyone interested in quantitative proteomics on cerebrospinal fluid.     

HykGene: a hybrid approach for selecting marker genes for phenotype classification using microarray gene expression data

MOTIVATION: Recent studies have shown that microarray gene expression data are useful for phenotype classification of many diseases. A major problem in this classification is that the number of features (genes) greatly exceeds the number of instances (tissue samples). It has been shown that selecting a small set of informative genes can lead to improved classification accuracy. Many approaches have been proposed for this gene selection problem. Most of the previous gene ranking methods typically select 50-200 top-ranked genes and these genes are often highly correlated. Our goal is to select a small set of non-redundant marker genes that are most relevant for the classification task. RESULTS: To achieve this goal, we developed a novel hybrid approach that combines gene ranking and clustering analysis. In this approach, we first applied feature filtering algorithms to select a set of top-ranked genes, and then applied hierarchical clustering on these genes to generate a dendrogram. Finally, the dendrogram was analyzed by a sweep-line algorithm and marker genes are selected by collapsing dense clusters. Empirical study using three public datasets shows that our approach is capable of selecting relatively few marker genes while offering the same or better leave-one-out cross-validation accuracy compared with approaches that use top-ranked genes directly for classification. AVAILABILITY: The HykGene software is freely available at http://www.cs.dartmouth.edu/~wyh/software.htm CONTACT: wyh@cs.dartmouth.edu SUPPLEMENTARY INFORMATION: Supplementary material is available from http://www.cs.dartmouth.edu/~wyh/hykgene/supplement/index.htm.     

Algorithms for large-scale genotyping microarrays

MOTIVATION: Analysis of many thousands of single nucleotide polymorphisms (SNPs) across whole genome is crucial to efficiently map disease genes and understanding susceptibility to diseases, drug efficacy and side effects for different populations and individuals. High density oligonucleotide microarrays provide the possibility for such analysis with reasonable cost. Such analysis requires accurate, reliable methods for feature extraction, classification, statistical modeling and filtering. RESULTS: We propose the modified partitioning around medoids as a classification method for relative allele signals. We use the average silhouette width, separation and other quantities as quality measures for genotyping classification. We form robust statistical models based on the classification results and use these models to make genotype calls and calculate quality measures of calls. We apply our algorithms to several different genotyping microarrays. We use reference types, informative Mendelian relationship in families, and leave-one-out cross validation to verify our results. The concordance rates with the single base extension reference types are 99.36% for the SNPs on autosomes and 99.64% for the SNPs on sex chromosomes. The concordance of the leave-one-out test is over 99.5% and is 99.9% higher for AA, AB and BB cells. We also provide a method to determine the gender of a sample based on the heterozygous call rate of SNPs on the X chromosome. See http://www.affymetrix.com for further information. The microarray data will also be available from the Affymetrix web site. AVAILABILITY: The algorithms will be available commercially in the Affymetrix software package.     

Recipe for uncovering predictive genes using support vector machines based on model population analysis

Selecting a small number of informative genes for microarray-based tumor classification is central to cancer prediction and treatment. Based on model population analysis, here we present a new approach, called Margin Influence Analysis (MIA), designed to work with support vector machines (SVM) for selecting informative genes. The rationale for performing margin influence analysis lies in the fact that the margin of support vector machines is an important factor which underlies the generalization performance of SVM models. Briefly, MIA could reveal genes which have statistically significant influence on the margin by using Mann-Whitney U test. The reason for using the Mann-Whitney U test rather than two-sample t test is that Mann-Whitney U test is a nonparametric test method without any distribution-related assumptions and is also a robust method. Using two publicly available cancerous microarray data sets, it is demonstrated that MIA could typically select a small number of margin-influencing genes and further achieves comparable classification accuracy compared to those reported in the literature. The distinguished features and outstanding performance may make MIA a good alternative for gene selection of high dimensional microarray data. (The source code in MATLAB with GNU General Public License Version 2.0 is freely available at http://code.google.com/p/mia2009/).     

Pathway analysis using random forests classification and regression

MOTIVATION: Although numerous methods have been developed to better capture biological information from microarray data, commonly used single gene-based methods neglect interactions among genes and leave room for other novel approaches. For example, most classification and regression methods for microarray data are based on the whole set of genes and have not made use of pathway information. Pathway-based analysis in microarray studies may lead to more informative and relevant knowledge for biological researchers. RESULTS: In this paper, we describe a pathway-based classification and regression method using Random Forests to analyze gene expression data. The proposed methods allow researchers to rank important pathways from externally available databases, discover important genes, find pathway-based outlying cases and make full use of a continuous outcome variable in the regression setting. We also compared Random Forests with other machine learning methods using several datasets and found that Random Forests classification error rates were either the lowest or the second-lowest. By combining pathway information and novel statistical methods, this procedure represents a promising computational strategy in dissecting pathways and can provide biological insight into the study of microarray data. AVAILABILITY: Source code written in R is available from http://bioinformatics.med.yale.edu/pathway-analysis/rf.htm.     

Interactively optimizing signal-to-noise ratios in expression profiling: project-specific algorithm selection and detection p-value weighting in Affymetrix microarrays

MOTIVATION: The most commonly utilized microarrays for mRNA profiling (Affymetrix) include 'probe sets' of a series of perfect match and mismatch probes (typically 22 oligonucleotides per probe set). There are an increasing number of reported 'probe set algorithms' that differ in their interpretation of a probe set to derive a single normalized 'signal' representative of expression of each mRNA. These algorithms are known to differ in accuracy and sensitivity, and optimization has been done using a small set of standardized control microarray data. We hypothesized that different mRNA profiling projects have varying sources and degrees of confounding noise, and that these should alter the choice of a specific probe set algorithm. Also, we hypothesized that use of the Microarray Suite (MAS) 5.0 probe set detection p-value as a weighting function would improve the performance of all probe set algorithms. RESULTS: We built an interactive visual analysis software tool (HCE2W) to test and define parameters in Affymetrix analyses that optimize the ratio of signal (desired biological variable) versus noise (confounding uncontrolled variables). Five probe set algorithms were studied with and without statistical weighting of probe sets using the MAS 5.0 probe set detection p-values. The signal-to-noise ratio optimization method was tested in two large novel microarray datasets with different levels of confounding noise, a 105 sample U133A human muscle biopsy dataset (11 groups: mutation-defined, extensive noise), and a 40 sample U74A inbred mouse lung dataset (8 groups: little noise). Performance was measured by the ability of the specific probe set algorithm, with and without detection p-value weighting, to cluster samples into the appropriate biological groups (unsupervised agglomerative clustering with F-measure values). Of the total random sampling analyses, 50% showed a highly statistically significant difference between probe set algorithms by ANOVA [F(4,10) > 14, p < 0.0001], with weighting by MAS 5.0 detection p-value showing significance in the mouse data by ANOVA [F(1,10) > 9, p < 0.013] and paired t-test [t(9) = -3.675, p = 0.005]. Probe set detection p-value weighting had the greatest positive effect on performance of dChip difference model, ProbeProfiler and RMA algorithms. Importantly, probe set algorithms did indeed perform differently depending on the specific project, most probably due to the degree of confounding noise. Our data indicate that significantly improved data analysis of mRNA profile projects can be achieved by optimizing the choice of probe set algorithm with the noise levels intrinsic to a project, with dChip difference model with MAS 5.0 detection p-value continuous weighting showing the best overall performance in both projects. Furthermore, both existing and newly developed probe set algorithms should incorporate a detection p-value weighting to improve performance. AVAILABILITY: The Hierarchical Clustering Explorer 2.0 is available at http://www.cs.umd.edu/hcil/hce/ Murine arrays (40 samples) are publicly available at the PEPR resource (http://microarray.cnmcresearch.org/pgadatatable.asp http://pepr.cnmcresearch.org Chen et al., 2004).     

Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements

Cancer derived microarray data sets are routinely produced by various platforms that are either commercially available or manufactured by academic groups. The fundamental difference in their probe selection strategies holds the promise that identical observations produced by more than one platform prove to be more robust when validated by biology. However, cross-platform comparison requires matching corresponding probe sets. We are introducing here sequence-based matching of probes instead of gene identifier-based matching. We analyzed breast cancer cell line derived RNA aliquots using Agilent cDNA and Affymetrix oligonucleotide microarray platforms to assess the advantage of this method. We show, that at different levels of the analysis, including gene expression ratios and difference calls, cross-platform consistency is significantly improved by sequence- based matching. We also present evidence that sequence-based probe matching produces more consistent results when comparing similar biological data sets obtained by different microarray platforms. This strategy allowed a more efficient transfer of classification of breast cancer samples between data sets produced by cDNA microarray and Affymetrix gene-chip platforms.     

A web-based platform for rice microarray annotation and data analysis

Rice (Oryza sativa) feeds over half of the global population. A web-based integrated platform for rice microarray annotation and data analysis in various biological contexts is presented, which provides a convenient query for comprehensive annotation compared with similar databases. Coupled with existing rice microarray data, it provides online analysis methods from the perspective of bioinformatics. This comprehensive bioinformatics analysis platform is composed of five modules, including data retrieval, microarray annotation, sequence analysis, results visualization and data analysis. The BioChip module facilitates the retrieval of microarray data information via identifiers of “Probe Set ID”, “Locus ID” and “Analysis Name”. The BioAnno module is used to annotate the gene or probe set based on the gene function, the domain information, the KEGG biochemical and regulatory pathways and the potential microRNA which regulates the genes. The BioSeq module lists all of the related sequence information by a microarray probe set. The BioView module provides various visual results for the microarray data. The BioAnaly module is used to analyze the rice microarray’s data set.     

Analysis of repeatability in spotted cDNA microarrays

We report a strategy for analysis of data quality in cDNA microarrays based on the repeatability of repeatedly spotted clones. We describe how repeatability can be used to control data quality by developing adaptive filtering criteria for microarray data containing clones spotted in multiple spots. We have applied the method on five publicly available cDNA microarray data sets and one previously unpublished data set from our own laboratory. The results demonstrate the feasibility of the approach as a foundation for data filtering, and indicate a high degree of variation in data quality, both across the data sets and between arrays within data sets.     

Inferential Clustering Approach for Microarray Experiments with Replicated Measurements

Cluster analysis has proven to be a useful tool for investigating the association structure among genes in a microarray data set. There is a rich literature on cluster analysis and various techniques have been developed. Such analyses heavily depend on an appropriate (dis)similarity measure. In this paper, we introduce a general clustering approach based on the confidence interval inferential methodology, which is applied to gene expression data of microarray experiments. Emphasis is placed on data with low replication (three or five replicates). The proposed method makes more efficient use of the measured data and avoids the subjective choice of a dissimilarity measure. This new methodology, when applied to real data, provides an easy-to-use bioinformatics solution for the cluster analysis of microarray experiments with replicates (see the Appendix). Even though the method is presented under the framework of microarray experiments, it is a general algorithm that can be used to identify clusters in any situation. The method's performance is evaluated using simulated and publicly available data set. Our results also clearly show that our method is not an extension of the conventional clustering method based on correlation or euclidean distance.     

The latent process decomposition of cDNA microarray data sets

We present a new computational technique (a software implementation, data sets, and supplementary information are available at http://www.enm.bris.ac.uk/lpd/) which enables the probabilistic analysis of cDNA microarray data and we demonstrate its effectiveness in identifying features of biomedical importance. A hierarchical Bayesian model, called Latent Process Decomposition (LPD), is introduced in which each sample in the data set is represented as a combinatorial mixture over a finite set of latent processes, which are expected to correspond to biological processes. Parameters in the model are estimated using efficient variational methods. This type of probabilistic model is most appropriate for the interpretation of measurement data generated by cDNA microarray technology. For determining informative substructure in such data sets, the proposed model has several important advantages over the standard use of dendrograms. First, the ability to objectively assess the optimal number of sample clusters. Second, the ability to represent samples and gene expression levels using a common set of latent variables (dendrograms cluster samples and gene expression values separately which amounts to two distinct reduced space representations). Third, in constrast to standard cluster models, observations are not assigned to a single cluster and, thus, for example, gene expression levels are modeled via combinations of the latent processes identified by the algorithm. We show this new method compares favorably with alternative cluster analysis methods. To illustrate its potential, we apply the proposed technique to several microarray data sets for cancer. For these data sets it successfully decomposes the data into known subtypes and indicates possible further taxonomic subdivision in addition to highlighting, in a wholly unsupervised manner, the importance of certain genes which are known to be medically significant. To illustrate its wider applicability, we also illustrate its performance on a microarray data set for yeast.