Cvt2Mae Appendix

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Table of Contents








A. Cvt2Mae User's Guide


This section contains a detailed description on how to use Cvt2Mae's
sequential step by step process to convert your data to the MAExlorer
format.




 Step 1 Select Array Chip (Array Layout)


Cvt2Mae has several predefined arrays (Affymetrix, GenePix, and
Scanalyze) available from a pull down menu. These may be edited or
customized and saved under a different name. This allows the array
layout to be setup in a particular format which then can be used many
times to convert data.




One can also create, edit and save their own custom Array Layouts
using the <user-define> Array Layout. If you define other custom
arrays, they can be saved and will appear in this pulldown menu for
next time. 

User defined Array layouts are saved as a text file in a subdirectory
called "ArrayLayout" under the name used in the Edit Layout menu with
the extension ".alo".

You may also remove array layouts using the "Remove Layout" button. It
will popup a file dialog from which you can choose an Array Layout to
delete.  Downloading and installing newer versions of Cvt2Mae will not
wipe out your custom defined array layouts.






 Step 2 Select input file(s)





Depending on your data, there may be several variations on how the
data is configured. For example some array applications will save
multiple files for each sample, while others will have multiple
samples in one file. Sometimes the GIPO data is available as a
separate GIPO file. Once the files have been selected go to step 3
below.




2.1 Selecting a single data file




Click on the "Browse input file name" button to popup a file selection
window. Then select the data file for conversion.  The file name(s)
will then appear in a text area below the "Browse input file name"
button.  Some arrays (e.g. Affymetrics) may have data for multiple
samples in one file.





2.2 Selecting multiple files





You can convert multiple files. Repeatedly select files with the
"Browse input file name" button.  Each time you select a file, it will
appear in a list below the "Browse input file name" button.

Some data will consist of multiple files such as a separate GIPO.


2.3 Separate GIPO file


Sometimes array data will have a separate GIPO file. You must click on
the "Separate GIPO" check box to activate the "Browse GIPO file"
button. Then click on the "Browse GIPO file" button to choose the
separate GIPO file. Note: A detailed description of GIPO files can be
found in the MAExplorer Reference Manual under  Appendix C.







 Step 3 Edit Array Layout


The "Edit Layout", "Assign GIPO Fields", and "Assign Quant Fields"
popup wizard windows are where you specify parameters for your data
conversion. These parameters will define which fields are to be used,
what the array geometry is, field data row locations etc.


Edit Layout Button


This wizard allows you to enter the particular parameters through a
step by step process. Some parameters are optional and are listed as
"opt" in the top of the wizard window.




 W.1 Array Layout Name and Vendor  

   
 This is used as the file name for the layout - a unique name
        of the array layout designator. This is generally specified by
        the chip vendor. If it is your own chip then use your own
        designator to differentiate your chip designs. Also, you may
        choose the name of the chip vendor. If your are specifying a
        <User-defined> chip, then you can use whatever you wish
        - eg. your organization.



 W.2 Grid Geometry 

   
 These are the parameters for the orientation of the spots in
        the pseudo array displayed on the main window of
        MAExplorer. Basically a field is made up of grids, grids are
        made up of rows and columns of spots.


    


  •   Number of duplicated spot Fields in array 

   
     Some arrays have multiple or duplicate fields.This is the
	number of duplicated spot Fields in array. EVERY spot is
	duplicated. For example, each grid of spots is
	duplicated. We refer to these in MAExplorer as F1 and F2.
	If there are no duplicates, then there is 1 field.


  •   Number of Grids per Field 
     
     Number of Grids per Field. A grid contains
	  Grid Rows X Grid Columns of spots
     

  •   Number of spots per Grid Row 
     
     

  •   Number of Spots per Grid Column  
     
     


  •   Use Mol. Dynamics 'NAME-GRC' else (Grid, Row, Column) 

     
     Use the Molecular Dynamics 'NAME-GRC' specification for
	  (grid, row, column), otherwise use separate fields for (grid,
	  grid_row, grid_col)


  •   Specify array layout by Grid-geometry or by number spots per array 

    
     If you specify the array layout by Grid-geometry (ABOVE),
         then enter (#Fields, #Grids, #Grid-rows,#Grid-cols). If
         you specify the layout by the maximum number of spots in
         the array (BELOW), it will estimate a pseudo-layout that
         the spots will fit on the this array for visualization
         purposes. It does not correspond to the actual array
         layout which you do not have to enter.  


  •   Maximum number of spots in array 

     
    This is the maximum number of spots that may occur in your data.







  W.3 Input File Starting Rows Data 




    

  •  Row containing a list of sample names 

    
     Number of the row containing the names of the multiple
	 samples if the file contains multiple samples. (Row #s
	 start at row 1.) If there are no sample names, set it to 0 or
	 leave blank. If you change it from 0 to any positive row
	 number, it removes input files and re-reads the first file
	 to get the proper data field names so you may use it with the
	 Assign GIPO Fields and Assign Quant Field assignment
	 operations.


  •  Row containing a list of quantitative file names 

    
    Number of rows that contains the names of the data file Field names 


  •  First row containing quantitative file data 

    
     Number of first row that contains quantitative array Data in
         the file. It is assumed that this is followed by the
         rest of the array data. 


  •  First row containing optional separate GIPO file data 

    
    Number of first row that contains optional separate GIPO file array Data. 


  •  Optional comment 

    
     Leave it blank, if there are no comment lines.


  •  Initial keyword for each data row 

    
     If you specify this, it checks for it in each data row






W.4 Ratio Fluorescence Data  





    


  •  Ratio or Intensity data

    
     Data for MAExplorer is either ratio data such as Cy3/Cy5, or
	 intensity data such as P33, etc.


  •  If Ratio data use (Cy5/Cy3) else (Cy3/Cy5) 

    
     Ratio data may be presented as either (Cy5/Cy3)or (Cy3/Cy5)


  •  Fluorescent dye for intensity 1 if (ratio data) 

    
     The dye to associate with quantified data intensity 1.


  •  Fluorescent dye for intensity 2 if (ratio data) 

    
     The dye to associate with quantified data intensity 2.


  •  Have background intensity data 

    
     The input data file includes background intensity data that
	 you want to include. You do NOT have to include that data.







W.5  Optional Microarray Coordinates 
    
 



    


  •  Use microarray pseudo (X,Y) coordinates 

    
     Generate a microarray pseudo image using a representation of
	 the array based on Grids, Grid Rows, and Grid Columns.
	 Otherwise, use the (X,Y) data supplied for each spot - if it
	 exists. If this option is set, it will overide the actual
	 (X,Y) coordinates if that option is selected as well.


  •  Use actual microarray pseudo (X,Y) coordinates

    
     The actual (X,Y) coordinate data exists for each spot.  If
	 the data exists but you do NOT select this option, it will
	 use the pseudo-array option.


  •  Reuse (X,Y) coordinates of first sample for all samples 

    
     Reuse (X,Y) coordinates of first sample for all
	 samples. This is used if you want to 'Flicker' array pseudo
	 images between two samples.


  •  Swap microarray rows and columns 

    
     Reverse rows and columns in the microarray pseudo image.









 W.6 Optional Genomic Identifiers 



    


  •  Has Location data 

    
    The user data file has Location identifier data.  These could
	be 'probe_set' for Affymetrix, 'Incyte ID' for Incyte,
	etc. and are used as the gene identifier if there are no other
	IDs.


  •  Has Clone ID data 

    
    The user data file has I.M.A.G.E 'Clone ID' data.
 

  •  Has GenBank data 

    
     The user data file has 'GenBank' identifier data.  See
	 http://ncbi.nlm.nih.gov/ for more information.


  •  Has UniGene ID data 

    
     The user data file has 'UniGene' identifier data.


  •  Has dbEST data 

    
     The user data file has 'dbEST' identifier data.


  •  Has LocusLink data 

    
     The user data file has 'LocusID' identifier data.


  •  Has SwissProt data 

    
     The user data file has 'SwissProt' identifier data. 
         See http://www.expasy.ch/ for more information.


  •  Has Plate data 

    
     The user data file has user Plate well identifier data. This
	  uniquely identifies the source of the spotted clone.


  •  Get Genomic IDs from 'Description' 

    
     The Genomic IDs are encoded in the 'Description' field of
         user input file (the Affymetrix encodeing of ids).  It will
         find and generate genomic IDs for: Clone_ID if /cl=XXXX is in
         the Description, GenBank if /gb=XXXX is in the Description,
         UniGene if /ug=XXXX is in the Description.  If this switch is
         enabled, then the explicit ID options are disabled.







 W.7 Optional Gene Names (descriptions) 




    


  •    Has Gene Class user data 

    
    The user data file has Gene Class ontology data for
		        each gene. [FUTURE]


  •   Has UniGene Name user data  

    
     The user data file has UniGene Name data. This could be used
	 if the default 'GeneName' description is not available.


  •    Has separate per-spot QualCheck user data per-sample 

    
     The user data file has 'Quant' QualCheck data. This data is
	 on a per-spot basis for each array hybridization.  The code
	 (see MAExplorer Reference Manual Appendix C Table C.4.2) 
	 may be used to flag bad spots or missing spot data.


  •    Has 'GIPO' QualCheck user data for entire DB 

    
     The user data file has GIPO QualCheck data. This data is on a
         GIPO basis for the entire database.  The code (see MAExplorer
         Reference Manual Appendix C) may be used to flag bad gene
         data.








 W.8 Optional DNA Calibration and user plate names, Unigene speices name 



    


  •  Name of calibration DNA (if in database) 

    
    This is the default name given to calibration DNA spotted on
	the array for calibration purposes and indicated in the GIPO
	file 'Clone ID' or 'GenBank ID' field.



  • Name of researcher's special clones (if in database) 

    
    This is the default name given in place of a I.M.A.G.E.  Clone
	ID when the researcher's clones have not yet been placed im
	the I.M.A.G.E. respository and thus have no ID.  It indicated
	in the GIPO file 'Clone ID' or 'GenBank ID' field.



  • Name of empty wells 

    
    If you have empty or blank rows of spot data, type in the name
        (i.e. 'empty') if any.



  • Name species (opt) 

    
    Species name (Mouse, Human, ...). It is used to document the
        Array Layout



  •  Name UniGene Species prefix (opt)

    
     UniGene species prefix (Mouse Mm, Human Hs, etc.). This is
	 used in querying Genomic Web databases. If you do not see the
	 prefix you want in the choice menu, type it in.








 W.9 Optional Database and Data Quantifcation program 



    

  •  Your name of the created database (opt) 

    
     The name you want to give to the created database name.




  •  Your name of the database subset (opt) 

    
    Your name for the database subset used in the initial .mae startup file. 


  •  Generic project name for all samples (opt) 

    
    Generic name of the project to be used for all samples in the
	database. If no name is specified, it uses the input data
	files folder.


  •  Name of spot quantification program (opt) 

    
    Name of the program used to quantitate the spot data
        from the sample images. 









 W.10 Optional Hybridized Sample "set" class names 



    


  •  Default name of X samples 'set' 

    
     This is the name for the samples assign to the 'X set'.



  •  Default name of Y samples 'set' 

    
     This is the name for the samples assign to the 'Y set'.








 W.11 Optional Default Data Filtering Thresholds 




    

  •  Default cluster similarity threshold [0 : 1000] 

    
    Default cluster similarity threshold used in some of the 
	      clustering methods. This is the initial value shown in 
	      popup sliders. 


  •  Default # genes in highest/lowest 

    
     The number of genes reported in gene Reports or in the 
	      data Filter when this restriction is invoked.


  •  Default # clusters for K-means clustering [1 : 1000] 

    
     Default # of clusters used in the K-means clustering method. 
	      This is the initial value shown in popup sliders.


  •  Default p-value threshold (for t-tests) [0.0 : 1.0] 

    
     Default p-Value used in the t-Test data Filter. 
	      This is the initial value shown in popup sliders.


  •  Default Coeff. Of Variation threshold [0.0 : 1.0] 

    
    Default Coefficient Of Variation used in the data Filters. This 
	      is the initial value shown in popup sliders. 


  •  Default absolute difference threshold [0.0 : 4.0] 

    
     Default absolute difference threshold used in the data Filters. 
	      This is the initial value shown in popup sliders.







3.2 "Assign GIPO Fields" Button



MAExplorer extracts data from the gene-in-plate-order (GIPO) gene
coordinate table. This links spots in a microarray to these Genomic
"gene ID"s and gene names. This table may also contain Clone ID,
GenBank, dbEST, UniGene IDs, LocusID corresponding to these Master
Gene IDs. An optional table of Clone IDs and Gene Classes the gene
belongs to may also be defined. The "Assign GIPO Fields Button" will
allows the user to associate or customize their fields. They may have
different names than what MAExplorer uses. A detailed description of
the MAExplorer GIPO file can be found in the MAExplorer manual under
 Appendix C.




Often GIPO files supplied by array vendors have additional fields not
currently used by MAExplorer. You can leave them in (they will be
ignored) or take them out (loading a database is faster).





3.3 "Assign Quant Fields" Button


Quantified hybridized sample array spot data (Quant files) from each
array is put into a separate data file.  Essentially, a data file
contains one or more spot intensity values per gene in each row of the
data file.  A spot location is specified by a GIPO (field#, grid#,
grid column#, grid row#) 4-tuple with the field value optional. If the
field specification is omitted and there are duplicate spots in
multiple fields of grids, then it is defined implicitly. In that case,
the corresponding spot intensity data for each field for a gene is
specified as separate columns going from left to right.
Similar to the GIPO fields the "Assign Quant Fields Button" will
allows the user to associate or customize their fields. They may have
different names than what MAExplorer uses. A detailed description of
the MAExplorer Quant files can be found in the MAExplorer manual under
 Appendix C.






 Step 4 Choose output folder/directory


This allows you to pick the location of where the converted data will
be stored. We recommend using the first one, "Create New project Folder".




Select Output Folder Options: 




A. "Create New project Folder"


 This will create a new project folder anywhere you want .



B. "Merge with Exisiting Project Folder"
 


 This will put the new files into an existing folder. This will
not merge data.




C. "Use Input folder for output files"


 Use the current input project directory.









 Step 5 Convert Data


Once you have setup all of the essential parameters you can now
convert your data by clicking on the green "Run" button. The status
lines will show what the converter is doing. Once it is done, the red
Abort button will change into a green "Done" button which you press to
exit the program. This means that your data has been successfully
converted and you can now go to the MAE folder to click on the
Startup.mae file to run MAExplorer on your converted data.





 

B. Generation of a pseudoarray geometry if no array geometry is specified


MAExplorer requires the data in the GIPO and Quant files be specified
by a spot position. This is indicated by the array spot geometry of
(#fields, #grids, #rows/grid, #columns/grid).  The #fields is the
number of duplicated sets of grids if available - it is 1
otherwise. This 4-tuple must be specified in the Configuration file.
However, some array data does not have spot geometry position data
available. The alternative is to generate a pseudoarray geometry. This
is possible since the pseudoarray image in MAExplorer is used simply
to indicate success of the data filter or relative differences
depending on the "(Plot | Show Microarray)" menu option. The algorithm
presented below will generate a geometry
(nGrids,nGridRows,nGridCols) that is compatible with the
visual use of the pseudoarray. The only assumption is the
nRowsExpected, the number of spots in the microarray (rows in
the database input file). The number of spots in the array is computed
automatically and the option to use the pseudoarray instead of the
actual array geometry is selected in the 
Edit Layout Wizard for Grid Geometry.









Pseudoarray Geometry Algorithm 



    OPT_GRID_SIZE = 1200;                /* Optimal grid size for MAExplorer viewing */
    ROWS_TO_COLS_ASPECT_RATIO = 3.0/4.0; /* desired rows/cols aspect aspect for a grid */ 
    extra = 0;                           /* # of extra grid cols required */ 
           
    /* Estimate # of grids. Assume a square aspect ratio */ 
    if(n <= OPT_GRID_SIZE)
      nGrids = 1;
    else
      nGrids = (n / OPT_GRID_SIZE)+1;
          
   /* Estimate rows (r) and columns (c) from a rectangular grid 
    * where cols = (4/3) rows.
    * Then, c = (4/3)r and r*c= area. 
    * Then (4/3)*r*r = area or
    * r = sqrt((3/4)*area).
    */ 
   if(nRowsExpected > 0)
     while(true)
       { /* iterate to optimal size */
         gridSize = n/nGrids;
         nGridRows = sqrt( ROWS_TO_COLS_ASPECT_RATIO * gridSize );
         nGridCols = (nGridRows / ROWS_TO_COLS_ASPECT_RATIO);
         nGridCols += extra;
         estTotSize = (nGrids * nGridRows * nGridCols);
        if(estTotSize > nRowsExpected)
          break;
        else
          extra++;                       /* keep trying until meet criteria */
       } /* iterate to optimal size */
                 














C. Examples of some typical input files


A typical sample database table might look like:



    

  1.  Single spot/gene intensity data.


      grid    grid col    grid row   RawIntensity  Background
  1       1           1          2226.8        32.6
  1       1           2          1234.8        25.6
      . . .
  10      25          28         3333.8        23.6

      
    


  2.  Double spots/gene intensity data contained in two fields of duplicate spots.


      grid   grid col  grid row  RawIntensity1 Background1 RawIntensity2 Background2
  1      1         1         2226.8        32.6        2345.9        39.4
  1      1         2         1234.8        25.6        1245.9        39.4
      . . .
  10     25        28        3333.8        23.6        3345.9        25.4

      
    



  3.  Double spots/gene intensity data contained in two fields of duplicate spots.


      field grid   grid col  grid row  RawIntensity  Background
  1     1      1         1         2226.8        32.6
  1     1      1         2         1234.8        25.6
      . . .
  1     10     25        28        3333.8        23.6
      . . .
  2     1      1         1         2226.8        39.4
  2     1      1         2         1234.8        39.4
      . . .
  2     10     25        28        3333.8        25.4

      
    



  4.  Double spots/gene intensity data using the Molecular Dynamics' NAME_GRC notation.


      NAME_GRC      RawIntensity1   RawIntensity2
  GRID- 1-R1C1  2126.500        3662.350
  GRID- 1-R2C1  2311.430        3306.290
  GRID- 1-R3C1  3696.470        5780.310
  GRID- 1-R4C1  3167.450        5245.440
  . . .

      
    


  5.  Cy3/Cy5 spot/gene ratio data.


      grid   grid col  grid row  Cy3     Cy3Bkgd   Cy5      Cy5Bkgd
  1      1         1         2226.8  32.6      2345.9   39.4
  1      1         2         1234.8  25.6      1245.9   39.4
      . . .
  10     25        28        3333.8  23.6      3345.9   25.4

      
    






 








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