Using ANOVA to analyze microarray data

ANOVA provides a general approach to the analysis of single and multiple factor experiments on both one- and two-color microarray platforms. Mixed model ANOVA is important because in many microarray experiments there are multiple sources of variation that must be taken into consideration when constructing tests for differential expression of a gene. The genome is large, and the signals of expression change can be small, so we must rely on rigorous statistical methods to distinguish signal from noise. We apply statistical tests to ensure that we are not just making up stories based on seeing patterns where there may be none.     

Pathways to the analysis of microarray data

The development of microarray technology allows the simultaneous measurement of the expression of many thousands of genes. The information gained offers an unprecedented opportunity to fully characterize biological processes. However, this challenge will only be successful if new tools for the efficient integration and interpretation of large datasets are available. One of these tools, pathway analysis, involves looking for consistent but subtle changes in gene expression by incorporating either pathway or functional annotations. We review several methods of pathway analysis and compare the performance of three, the binomial distribution, z scores, and gene set enrichment analysis, on two microarray datasets. Pathway analysis is a promising tool to identify the mechanisms that underlie diseases, adaptive physiological compensatory responses and new avenues for investigation.     

Hybrid hierarchical clustering with applications to microarray data

In this paper, we propose a hybrid clustering method that combines the strengths of bottom-up hierarchical clustering with that of top-down clustering. The first method is good at identifying small clusters but not large ones; the strengths are reversed for the second method. The hybrid method is built on the new idea of a mutual cluster: a group of points closer to each other than to any other points. Theoretical connections between mutual clusters and bottom-up clustering methods are established, aiding in their interpretation and providing an algorithm for identification of mutual clusters. We illustrate the technique on simulated and real microarray datasets.     

Mining mouse microarray data

Microarrays of mouse genes are now available from several sources, and they have so far given new insights into gene expression in embryonic development, regions of the brain and during apoptosis. Microarray data posted on the internet can be reanalyzed to study a range of questions.     

Pre-processing Agilent microarray data

Background  

Pre-processing methods for two-sample long oligonucleotide arrays, specifically the Agilent technology, have not been extensively studied. The goal of this study is to quantify some of the sources of error that affect measurement of expression using Agilent arrays and to compare Agilent's Feature Extraction software with pre-processing methods that have become the standard for normalization of cDNA arrays. These include log transformation followed by loess normalization with or without background subtraction and often a between array scale normalization procedure. The larger goal is to define best study design and pre-processing practices for Agilent arrays, and we offer some suggestions.     

Maintaining data integrity in microarray data management

Gene expression microarrays are a relatively new technology, dating back just a few years, yet they have already become a very widely used tool in biology, and have evolved to a wide range of applications well beyond their original design intent. However, while the use of microarrays has expanded, and the issues of performance optimization have been intensively studied, the fundamental issue of data integrity management has largely been ignored. Now that performance has improved so greatly, the shortcomings of data integrity control methods constitute a greater percent of the stumbling blocks for investigators. Microarray data are cumbersome, and the rule up to this point has mostly been one of hands-on transformations, leading to human errors which often have dramatic consequences. We show in this review that the time lost on such mistakes is enormous and dramatically affects results; therefore, mistakes should be mitigated in any way possible. We outline the scope of the data integrity issue, to survey some of the most common and dangerous data transformations, and their shortcomings. To illustrate, we review some case studies. We then look at the work done by the research community on this issue (which admittedly is meager up to this point). Some data integrity issues are always going to be difficult, while others will become easier-one of our goals is to expedite the use of integrity control methods. Finally, we present some preliminary guidelines and some specific approaches that we believe should be the focus of future research.     

Clustering-based approaches to discovering and visualising microarray data patterns

This article focuses on clustering techniques for the analysis of microarray data and discusses contributions and applications for the implementation of intelligent diagnostic systems and therapy design studies. Approaches to validating and visualising expression clustering results and software and other relevant resources to support clustering-based analyses are reviewed. Finally, this paper addresses current limitations and problems that need to be investigated for the development of an advanced generation of pattern discovery tools.     

Mayday--a microarray data analysis workbench

Mayday is a workbench for visualization, analysis and storage of microarray data. It features a graphical user interface and supports the development and integration of existing and new analysis methods. Besides the infrastructural core functionality, Mayday offers a variety of plug-ins, such as various interactive viewers, a connection to the R statistical environment, a connection to SQL-based databases and different data mining methods, including WEKA-library based methods for classification and various clustering methods. In addition, so-called meta information objects are provided for annotation of the microarray data allowing integration of data from different sources, which is a feature that, for instance, is employed in the enhanced heatmap visualization. Supplementary information: The software and more detailed information including screenshots and a user guide as well as test data can be found on the Mayday home page http://www.zbit.uni-tuebingen.de/pas/mayday. The core is published under the GPL (GNU Public License) and the associated plug-ins under the LGPL (Lesser GNU Public License).     

Analyzing microarray data using CLANS

Analysis of microarray experiments is complicated by the huge amount of data involved. Searching for groups of co-expressed genes is akin to searching for protein families in a database as, in both cases, small subsets of genes with similar features are to be found within vast quantities of data. CLANS was originally developed to find protein families in large sets of amino acid sequences where the amount of data involved made phylogenetic approaches overly cumbersome. We present a number of improvements that greatly extend the previous version of CLANS and show its application to microarray data as well as its ability of incorporating additional information to facilitate interactive analysis. AVAILABILITY: The program is available for download from: http://bioinfoserver.rsbs.anu.edu.au/downloads/clans/     

Computational analysis of microarray data

Microarray experiments are providing unprecedented quantities of genome-wide data on gene-expression patterns. Although this technique has been enthusiastically developed and applied in many biological contexts, the management and analysis of the millions of data points that result from these experiments has received less attention. Sophisticated computational tools are available, but the methods that are used to analyse the data can have a profound influence on the interpretation of the results. A basic understanding of these computational tools is therefore required for optimal experimental design and meaningful data analysis.     

Normalization of cDNA microarray data

Normalization means to adjust microarray data for effects which arise from variation in the technology rather than from biological differences between the RNA samples or between the printed probes. This paper describes normalization methods based on the fact that dye balance typically varies with spot intensity and with spatial position on the array. Print-tip loess normalization provides a well-tested general purpose normalization method which has given good results on a wide range of arrays. The method may be refined by using quality weights for individual spots. The method is best combined with diagnostic plots of the data which display the spatial and intensity trends. When diagnostic plots show that biases still remain in the data after normalization, further normalization steps such as plate-order normalization or scale-normalization between the arrays may be undertaken. Composite normalization may be used when control spots are available which are known to be not differentially expressed. Variations on loess normalization include global loess normalization and two-dimensional normalization. Detailed commands are given to implement the normalization techniques using freely available software.     

Between-group analysis of microarray data

MOTIVATION: Most supervised classification methods are limited by the requirement for more cases than variables. In microarray data the number of variables (genes) far exceeds the number of cases (arrays), and thus filtering and pre-selection of genes is required. We describe the application of Between Group Analysis (BGA) to the analysis of microarray data. A feature of BGA is that it can be used when the number of variables (genes) exceeds the number of cases (arrays). BGA is based on carrying out an ordination of groups of samples, using a standard method such as Correspondence Analysis (COA), rather than an ordination of the individual microarray samples. As such, it can be viewed as a method of carrying out COA with grouped data. RESULTS: We illustrate the power of the method using two cancer data sets. In both cases, we can quickly and accurately classify test samples from any number of specified a priori groups and identify the genes which characterize these groups. We obtained very high rates of correct classification, as determined by jack-knife or validation experiments with training and test sets. The results are comparable to those from other methods in terms of accuracy but the power and flexibility of BGA make it an especially attractive method for the analysis of microarray cancer data.     

Filtering genes to improve sensitivity in oligonucleotide microarray data analysis

Many recent microarrays hold an enormous number of probe sets, thus raising many practical and theoretical problems in controlling the false discovery rate (FDR). Biologically, it is likely that most probe sets are associated with un-expressed genes, so the measured values are simply noise due to non-specific binding; also many probe sets are associated with non-differentially-expressed (non-DE) genes. In an analysis to find DE genes, these probe sets contribute to the false discoveries, so it is desirable to filter out these probe sets prior to analysis. In the methodology proposed here, we first fit a robust linear model for probe-level Affymetrix data that accounts for probe and array effects. We then develop a novel procedure called FLUSH (Filtering Likely Uninformative Sets of Hybridizations), which excludes probe sets that have statistically small array-effects or large residual variance. This filtering procedure was evaluated on a publicly available data set from a controlled spiked-in experiment, as well as on a real experimental data set of a mouse model for retinal degeneration. In both cases, FLUSH filtering improves the sensitivity in the detection of DE genes compared to analyses using unfiltered, presence-filtered, intensity-filtered and variance-filtered data. A freely-available package called FLUSH implements the procedures and graphical displays described in the article.