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Estimating Gene Function With LS-NMF 37the Euclidean distance. The inclusion of the gene and array specific standarddeviation (σ ij) improves the recovery of functional information (9).2. Materials1. The source code for LS-NMF including a graphical user interface for loadinginput files and visualizing output files can be downloaded from the Fox ChaseCancer Center Bioinformatics Group at http://bioinformatics.fccc.edu/software/OpenSource/LS-NMF/java/LS-NMF.shtml (see Note 1).2. The ClutrFree visualization and gene oncology analysis tool is available fromhttp://bioinformatics.fccc.edu/software/OpenSource/ClutrFree/clutrFree.shtml.3. The sample data set and associated gene ontology (GO) annotations can bedownloaded from http://bioinformatics.fccc.edu/papers/methodsLS-NMF/data_GO.zip.4. An updated version of the automated sequence annotation pipeline (ASAP II) is availableat http://bioinformatics.fccc.edu/software/OpenSource/ASAP/ASAP.shtml;however, it does require considerable systems administration skills to implement.Users might instead gather GO and other annotations for ClutrFree using differentsystems, such as OntoExpress (10).5. It is often useful to access the organism specific database for the particular dataset. Here GeneDB and the Schizosaccharomyces pombe database is used, http://www.genedb.org/genedb/pombe/.3. MethodsThe procedure for LS-NMF simulation on microarray data sets involvesthree steps:1. Preprocessing microarray data into proper format for LS-NMF analysis (seeNote 2).2. Setting parameters for LS-NMF simulation, and running the simulation with theset of the parameters.3. Interpreting the simulation results.In order to go through the whole procedure in detail, a sample microarray dataset that is a reduced version of the S. pombe cell cycle experiment is provided(11). Every step in the implementation is applied specifically on this sample dataset, so readers can follow the description below step-by-step. For different data,the steps are the same. It is recommended that, users new to bioinformatics toolsapply the process first to the sample data set to learn the procedures.3.1. Downloading Files and Preparing for AnalysisEach of the files noted in the Subheading 2. should be downloaded (with theexception of the ASAP system). This can be done with a typical web browseron any system. The files should be handled in the following manner.