1. Expression Analysis of the Sphingolipid Metabolism Gene Extraction: Pathway Modification: Branch Addition: Gene Addition: Data Formatting Download GenMAPP v2.1 Download Pathways Data Application: Ι Π ΙΙΙ ΙVΙV V VΙVΙ

2. I. GenMAPP v2.1: GenMAPP v2.1, a Windows operating program, provides a technique for conducting a genomic analysis through the visualization of gene expression data within a metabolic pathway. Expression data derived from microarray and other similar genomic experiments can be imported and recognized by GenMAPP using multiple gene identifiers such as Entrez Gene, Ensembl, and Affy ID's. This program allows for the genes within a biological pathway, such as the sphingolipid metabolism, to be color-coded based on a profile established by the user. In addition, tools provided within GenMAPP allow such metabolic pathways to be created or edited by the user. The figure shown below demonstrates an example of what one would obtain after importing expression data into GenMAPP. www.genmapp.org

3. II. How to Extract Sphingolipid Related Genes: Perl program instructions The perl script accepts a text file with the list of gene symbols or the tag ID’s of interest. It opens the microarray file with normalized gene expression values and searches for rows that match with the gene symbol or Tag ID’s.The matched rows are copied to a new test file that may be opened using excel and further data analysis can be performed. Example of a text file with gene listExample of excel file with normalized gene expression values Requirement: Perl 5.8 must be down loaded and installed on the windows system from www.activestate.com withoutwww.activestate.com charge. Mac ‘s have built in perl in their X11 command line. USE: Make sure to have program file (affyfilter.pl), genelist file and normalized microarray file in the same folder where perl is installed. At the command line type: C:\ perl > perl affyfilter.pl microarray.txt genelist.txt > output.txt The results of the filtering will be stored in a output file within the same folder (Note that the names of the file can are user defined) Program Name: Affyfilter.pl Function: Script for filtering gene expression values of interest from Normalized gene expression file ** If desired genes have been previously extracted, proceed to step ΙΙΙ.

4. III. How to Format Data in GenMAPP GenMAPP recognizes data to be imported in a specific format as illustrated below. Column headings: GeneID, System Code, and a user given data heading (e.g. Control), must be included for proper identification. The system code is a code provided by GenMAPP corresponding to the gene database used for identifying the GeneID. The data sheet should be saved as a text document (.txt), tab-delimited file (.tab), or comma-separated values (.csv). Gene Identification *Code Corresponding Data 1 to Gene Database GeneIDSystem CodeControl 54218L1.43 14421L0.51 14421L0.47 12091L2.07 -.txt,.tab, or.csv file * System Codes: EntrezGene: LMGI: M Ensemble: EnUniProt: S Unigene: UAffy: X

5. IV. Download Sphingolipid Pathways: Download Sphingolipid pathways (.mapp file) from www.sphingomap.com → Sphingo- GeneMap and open within GenMAPP.www.sphingomap.com Available Pathways Include: De novo Lacto Ganglio Neolacto IsoGlobo Globo

6. V. How to Modify Provided Sphingolipid Pathways: - Gene Addition a)Select species specific database, See Gene Database SelectionGene Database Selection b)Click on gene box located in the tool bar of the draftboard c)Right click gene to open “Gene Finder” d)Enter the gene ID and the corresponding gene ID system e)Within “Gene Label” enter gene name

7. V. How to Modify Provided Sphingolipid Pathways: - Branch Addition

8. VI. How to Apply Data to Pathway Using GenMAPP: 1. Gene Database Selection a)Data → “Download Data from GenMAPP.org” → expand “Gene Database” and select species used b)Within “Database File Name”, select database to be used and click “start” c)Return to draftboard d)Data → “Choose Gene Database” → select database downloaded

9. VI. How to Apply Data to Pathway Using GenMAPP: 2. Creating a Color-Criteria a)Data → “Expression Dataset Manager” b)Expression Datasets → “New Dataset” → select saved data file c)Color Sets → New d)Using Boolean expression values provided, establish color-criteria based on data e)Save Expression File and exit “Expression Dataset Manager” ** Values in GenMAPP represent Fold Changes

10. Sphingolipid Metabolism *Detailed Instructions, list of additional functions available, and downloads are available at http://www.genmapp.org/http://www.genmapp.org/