GAAS - Tutorial

FIRST TIME USE (here for an extended guide)

• To use GAAS, a user must be registered in the MasterDB system database. For first use, login in the GAAS logon window with:

  	User Name: demoUser
  	Password:  demoUser 

  During first use of the Analyzer program, select the "MasterDB Management" command in the "Tools" menu of the Analyzer main window. A new window (MasterDB management window) appears: in the "Users" section of this window register a new user specifying its user name and password.
  After first use, start the Analyzer program and login in the GAAS logon window as master user using:  User Name: masterUser;   Password: masterUser.  Change the master user identifiers through the "Users" section in the MasterDB management window, which can be opened by selecting the "MasterDB Management" command in the "Tools" menu of the Analyzer main window.
  
  
• To perform analyses, any input data database must be provided associated with a label database specifying identifiers and location on the spotted array of the clones to be analyzed. Label databases must be registered in the MasterDB system database. For first use, the label databases "LabelFilters.mdb" and "LabelMicro.mdb" of the provided testing data have already been registered. However, if asked search in the appropriate location of the local path for the adequate label database file to use.
  To register a new label database in MasterDB, open the Analyzer program, go to the "Tools" menu of the Analyzer main window and choose the "MasterDB Management" command. In the "Labels" section of the new opened MasterDB management window, load the new label database.

 

ASSEMBLER PROGRAM USE (here for an extended guide)

1. Using one of the following procedures, check if the input data structures and label data structures stored in MasterDB define adequately the data to be processed:
   • Open the Analyzer program, go to the "Tools" menu and choose the "MasterDB Management" command.
   • Open the MasterDB database and check the contents of the "InputStructure" and "LabelStructure" tables (for more details see here and here, respectively).
2. Start the Assembler program and login through the GAAS logon window.
3. In the Assembler main window, click "Open data files ..." and select the data files to be processed.
4. Choose the corresponding input file structure if prompted to do so.
5. Choose one of the available matching label databases through the Labels "Name" pull-down button.
   • If no suitable label database type has been previously registered in MasterDB, the user can select and register a new label database by clicking the "Select Database" button.
   • If the data under processing refer to a subsection of a label database (i.e. only a fraction of the plates of a library), the user must first generate a MS-Excel data file containing the list of plates in use, and select this file using the "Load Plate List" button.
6. Click "Process..." and define the name of the MS-Access database to be created.

 

ANALYZER PROGRAM USE (here for an extended guide)

8. Start the Analyzer program and login through the GAAS logon window.
9. Choose the input database containing the data to be analyzed by:
   • selecting the appropriate data file from the "Input Database" panel on the upper left side of the Analyzer main window, or
   • selecting the "Database" menu, choosing the "Open ..." command, searching for the database file in the appropriate location, and providing data type, structure and label information. Structure and label database of the input database must be registered in MasterDB. If not, in the "Tools" menu of the Analyzer main window choose the "MasterDB Management" command and register them in the "InputStructure" and "Labels" sections of the new opened MasterDB management window, respectively. Alternatively, open the MasterDB database and check the contents of the "InputStructure" and "LabelStructure" tables (for more details see here and here, respectively).
10. Selecting a data file in the "Input Database" panel on the upper left side of the Analyzer main window a new window (Input array data type assignment window) appears. In this window, choose the experiment types (Control or Test), and the experimental replica each data set belongs to. Click "OK"; the name and status of the data sets will be displayed in the "Tables" panel on the bottom left side of the Analyzer main window. Select in the "Tables" panel one of the loaded data sets, to visualize in the Input data window the loaded spot expression data set.
11. Open the Parameter setting window by selecting the "Parameter Settings" command in the "Tools" menu of the Analyzer main window and choose a set of values for the analysis parameters and whether to apply background correction. The parameter setup can be stored in MasterDB by clicking the "Save" button in the Parameter setting window. Conversely, choose a previously saved parameter set already stored in MasterDB by selecting it in the bottom right corner of the Parameter setting window.
12. Open the Gene expression bound window by selecting the "Expression Bounds" command in the "Tools" menu of the Analyzer main window and define upper and lower thresholds of expression levels.
13. Choose the type of normalization factor by selecting the "Normalization Types" command in the "Tools" menu of the Analyzer main window.
    • If only high quality signals are to be included in the analysis, check the "Valid clones" check box in the Normalization option window.
    • If only a subset of array clone intensities is to be used for data normalization, select in the bottom of the Normalization option window the correspondent clone filter. See here details on how building a clone filter.
14. Run the data analysis by subsequently selecting the following commands in the "Analysis" menu of the Analyzer main window:
    • "Background": background quality labels are automatically assigned to exclude array regions altered by stains.
    • "Single": analysis of expression levels within a single array is performed (spot quality labels automatically determined to reject expression values affected by spotting errors; background correction, to eliminate eventual topological hybridization differences; expression level normalization to make comparable expression values from distinct arrays or experimental conditions).
    • "Pair": statistically significant differential expressions in a single experiment is evaluated on the basis of expression intensity ratios (test vs. control condition).
    • "Replica": significant differential expressions are determined across multiple replica experiments.
    Conversely, select the "All Analyses" command to run subsequently all previous analysis steps.
    When a data analysis is run, the used parameter set is automatically associated to the input database to avoid repeating setup of analysis parameter values in future analyses of the same input data. However, the parameter set to be use can be always modified.
15. View the analysis results in tabular form inside the Background, Single, Pair, and Replica panels, and in graphical form in the Histogram and Scatter Plot panels of the Analyzer main window.
    For scatter plot, in the Plotbar that automatically appears above the panels when the Scatter Plot is selected (or can be shown by selecting the "Plotbar" command in the "View" menu of the Analyzer main window), select the datasets to plot on each scatter plot axis.
    Select the "Show Columns" command in the "Tools" menu of the Analyzer main window to open the Show column window and customize visualization in each data panel. Click with the mouse right button on a graphical panel to show an option menu for customizing the graph visualization.
    
16. To search and interactively navigated the analysis results, select the "Clone Search" command in the "Tools" menu of the Analyzer main window. A new window (Clone search window) appears: specify either Location (in the array), Accession number, or Clone ID of the clone to search, and click the correspondent "Find" button. The rows in all tabular panels (Background, Single, Pair, and Replica) containing the searched clone data are highlighted and in the Scatter Plot the searched clone is identified with a green cross.
17. Open the Saving Options window by selecting the "Saving Options" command in the "Database" menu of the Analyzer main window and customize the output data structure. The defined output data structure can be stored in MasterDB by clicking the "Save" button in the Saving Options window. Conversely, choose a previously saved output data structure already stored in MasterDB by selecting it in the bottom right corner of the Saving Options window.
18. To store in an output database the analysis results, select the "Save" command in the "Database" menu of the Analyzer main window and define the name of the MS-Access database to be created.
    When analysis results are saved, the used output data structure is automatically associated to the input database to avoid repeating output data structure customization in future analyses of the same input data. However, the output structure to be used can be always modified.