Analysis of microarray data - VSN International

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14Analysis of Microarray Data in GenStat5. In design 1, any two independent allocations (say A-B and C-D) will give an optimal design. Thetwo comparisons represented on the extra slides will have a higher precision that the rest. In design3, the extra slides A 1 B 1 – A 2 B 1 , and A 1 B 2 - A 2 B 2 give the best standard error for the interaction of0.535.6. The only possible connected design is a loop through all the animals, alternating between the twotreatments. The individual animals are not compared very precisely, with the SED matrix shownbelow. The contrast matrix of (-0.25, -0.25, -0.25, -0.25, 0.25, 0.25, 0.25, 0.25) for the mean of B –A has a standard error or 0.354. Adding 4 extra slides allows more accurate comparisons ofindividuals, and the 4 slides shown below reduce the between animals comparisonsaverage SED from 1.210 to 0.838 (and the maximum from 1.414 to 0.913). The Red Greentreatment standard error is reduced to 0.289, an 18% reduction.A1 B1B1 A2Target A1 A2 A3 A4 B1 B2 B3 B4A1 *A2 1.225 *A3 1.414 1.225 *A4 1.225 1.414 1.225 *B1 0.935 0.935 1.369 1.369 *B2 1.369 0.935 0.935 1.369 1.225 *B3 1.369 1.369 0.935 0.935 1.414 1.225 *B4 0.935 1.369 1.369 0.935 1.225 1.414 1.225 *A2B2A3B3A4B4B2A3B3A4B4A1Added SlidesA1 B2B3 A2A3 B4B1 A4Automatic generation of designsThe Stats | Microarrays | Design | Generate TwoChannel design menu allows you to automaticallygenerate reference, loop and balanced incompleteblock designs. You just need to specify the type ofdesign, the number of treatments, and either the loopincrement (1 always works), or the reference level.With a loop design, you can specify more than oneloop increment to get two complementary loops.Using the Treatments in columns by colour optionputs the design in a format appropriate for pastinginto the Stats | Microarrays | Design | Generate TwoChannel design menu. The spreadsheet to the rightshows the resulting balanced incomplete block design with 4 treatments.For single channel designs, the Stats | Microarrays | Design | Generate SingleChannel design menu just opens the Generate Standard Design menu, as thereare no complications with these designs, as each slide is an independent unit,as in any standard experimental design.
Analysis of Microarray Data in GenStat 15Reading microarray dataMicroarray data can be read in via the usual File | Open menu in GenStat if it is in one of the standard fileformats supported by GenStat (Excel, CSV, text etc). However, in microarray experiments you often havea file for each slide in the experiment and need these amalgamated into a single data set. The MicroarraysData menu has a dialog that is designed for this, which also supports the common formats produced byimage analysis software (GenePix, GenePix, Imagene, Spot, TIGR MEV, ScanAlyze, QuantArray,Affymetrix and generic CSV files). The Data menu contains a list of the supported file types, so chose thefile type you have. These all open the same dialog, but just pre-select type of file on this menu. You canchange between file types once the dialog is open. The selected files should all be of the same format, butsome may have extra columns, in which case missing values will be inserted in the files with thesecolumns missing. The file names may be typed in and added tothe file list using the add list, but more commonly the browsebutton is used. For example, to read in the 4 GenePix GPR filesin the Ma2Examples directory, use the menu Stats | Microarray |Data | GenePix GPR files and click the browse button as shown.Now select the 4 GPR files required in the Select Microarrayfiles dialog (as shown below, right), and click Open. To selectmultiple files, hold down the control key (Ctrl) or to select arange hold down the Shift key when clicking with the mouse.Note Windows does not enter the filenames selected in a naturalorder in the File name box, so the resulting file names in theOpen Microarray Files window are not in numerical order (asbelow). To arrange the files in the correct order, select the fileto move (in this case13-9-data.gpr) and usethe Up or Down buttonto move it within thelist (Down 3 times inthis case to put it at theend of the list).On clicking Open, the files will be opened and appended into acombined spreadsheet with an extra factor created to index the filethe data came from. You may be prompted for some extrainformation, such as which columns are factors, dates and in thecase of a GPR file, the arrangement of pins on the slide, as thisinformation is not within the data. The final spreadsheet is shown below.
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