Microarray analysis techniques are used in interpreting the data generated from experiments on DNA (Gene chip analysis), RNA, and protein microarrays, which allow researchers to investigate the expression state of a large number of genes – in many cases, an organism's entire genome – in a single experiment. Such experiments can generate very large amounts of data, allowing researchers to assess the overall state of a cell or organism. Data in such large quantities is difficult – if not impossible – to analyze without the help of computer programs.
Introduction Microarray data analysis is the final step in reading and processing data produced by a microarray chip. Samples undergo various processes including purification and scanning using the microchip, which then produces a large amount of data that requires processing via computer software. It involves several distinct steps, as outlined in the image below. Changing any one of the steps will change the outcome of the analysis, so the MAQC Project was created to identify a set of standard strategies. Companies exist that use the MAQC protocols to perform a complete analysis.
Techniques
Most microarray manufacturers, such as Affymetrix and Agilent, provide commercial data analysis software alongside their microarray products. There are also open source options that utilize a variety of methods for analyzing microarray data.
Aggregation and normalization Comparing two different arrays or two different samples hybridized to the same array generally involves making adjustments for systematic errors introduced by differences in procedures and dye intensity effects. Dye normalization for two color arrays is often achieved by local regression. LIMMA provides a set of tools for background correction and scaling, as well as an option to average on-slide duplicate spots. A common method for evaluating how well normalized an array is, is to plot an MA plot of the data. MA plots can be produced using programs and languages such as R and MATLAB. Raw Affy data contains about twenty probes for the same RNA target. Half of these are "mismatch spots", which do not precisely match the target sequence. These can theoretically measure the amount of nonspecific binding for a given target. Robust Multi-array Average (RMA) is a normalization approach that does not take advantage of these mismatch spots but still must summarize the perfect matches through median polish. The median polish algorithm, although robust, behaves differently depending on the number of samples analyzed. Quantile normalization, also part of RMA, is one sensible approach to normalize a batch of arrays in order to make further comparisons meaningful. The current Affymetrix MAS5 algorithm, which uses both perfect match and mismatch probes, continues to enjoy popularity and do well in head to head tests.
Factor analysis for Robust Microarray Summarization (FARMS) is a model-based technique for summarizing array data at perfect match probe level. It is based on a factor analysis model for which a Bayesian maximum a posteriori method optimizes the model parameters under the assumption of Gaussian measurement noise. According to the Affycomp benchmark FARMS outperformed all other summarizations methods with respect to sensitivity and specificity.
Identification of significant differential expression Many strategies exist to identify array probes that show an unusual level of over-expression or under-expression. The simplest one is to call "significant" any probe that differs by an average of at least twofold between treatment groups. More sophisticated approaches are often related to t-tests or other mechanisms that take both effect size and variability into account. Curiously, the p-values associated with particular genes do not reproduce well between replicate experiments, and lists generated by straight fold change perform much better. This represents an extremely important observation, since the point of performing experiments has to do with predicting general behavior. The MAQC group recommends using a fold change assessment plus a non-stringent p-value cutoff, further pointing out that changes in the background correction and scaling process have only a minimal impact on the rank order of fold change differences, but a substantial impact on p-values.
Clustering Clustering is a data mining technique used to group genes having similar expression patterns. Hierarchical clustering, and k-means clustering are widely used techniques in microarray analysis.
Hierarchical clustering
Hierarchical clustering is a statistical method for finding relatively homogeneous clusters. Hierarchical clustering consists of two separate phases. Initially, a distance matrix containing all the pairwise distances between the genes is calculated. Pearson's correlation and Spearman's correlation are often used as dissimilarity estimates, but other methods, like Manhattan distance or Euclidean distance, can also be applied. Given the number of distance measures available and their influence in the clustering algorithm results, several studies have compared and evaluated different distance measures for the clustering of microarray data, considering their intrinsic properties and robustness to noise. After calculation of the initial distance matrix, the hierarchical clustering algorithm either (A) joins iteratively the two closest clusters starting from single data points (agglomerative, bottom-up approach, which is fairly more commonly used), or (B) partitions clusters iteratively starting from the complete set (divisive, top-down approach). After each step, a new distance matrix between the newly formed clusters and the other clusters is recalculated. Hierarchical cluster analysis methods include:
Single linkage (minimum method, nearest neighbor) Average linkage (UPGMA) Complete linkage (maximum method, furthest neighbor) Different studies have already shown empirically that the Single linkage clustering algorithm produces poor results when employed to gene expression microarray data and thus should be avoided.
K-means clustering
… excerpt ends here. Continue reading the full article.
