File menu commands

The File menu offers the following commands:

New	Creates a new document.
Open	Opens an existing document.
Close	Closes an opened document.
Save	Saves an opened document using the same file name.
Save As	Saves an opened document to a specified file name.
Import	Imports additional file types: PIR, ALN, FASTA
Export	Exports additional file types: PHYLIP, PIR, ALN, FASTA
Print	Prints a document.
Print Preview	Displays the document on the screen as it would appear printed.
Print Setup	Selects a printer and printer connection.
Exit	Exits GeneDoc.


Help menu commands

The Help menu offers the following commands, which provide you assistance with this application:

Index	Offers you an index to topics on which you can get help.
Using Help	Provides general instructions on using help.
About	Displays the version number of this application.


File New command
New command (File menu)

Use this command to create a new document in GeneDoc.

You can open an existing document with the Open command.

Shortcuts

Toolbar:

Keys:	CTRL+N


File Open command
Open command (File menu)

Use this command to open an existing document in a new window.  You can open multiple documents at once.  Use the Window menu to switch among the multiple open documents.  See Window 1, 2, ... command.

You can create new documents with the New command.

Shortcuts

Toolbar:

Keys:	CTRL+O


File Open dialog box

The following options allow you to specify which file to open:

File Name

Type or select the filename you want to open.  This box lists files with the extension you select in the List Files of Type box.

List Files of Type

Select the type of file you want to open:

.MSF: Multiple Sequence Format.

Drives

Select the drive in which GeneDoc stores the file that you want to open.

Directories

Select the directory in which GeneDoc stores the file that you want to open.

Network...

Choose this button to connect to a network location, assigning it a new drive letter.


File Close command
Close command (File menu)

Use this command to close all windows containing the active document.  GeneDoc suggests that you save changes to your document before you close it.  If you close a document without saving, you lose all changes made since the last time you saved it.  Before closing an untitled document, GeneDoc displays the Save As dialog box and suggests that you name and save the document.

You can also close a document by using the Close icon on the document's window, as shown below:


File Save command
Save command (File menu)

Use this command to save the active document to its current name and directory.  When you save a document for the first time, GeneDoc displays the Save As dialog box so you can name your document.  If you want to change the name and directory of an existing document before you save it, choose the Save As command.

Shortcuts

Toolbar:

Keys:	CTRL+S


File Save As command
Save As command (File menu)

Use this command to save and name the active document.  GeneDoc displays the Save As dialog box so you can name your document.

To save a document with its existing name and directory, use the Save command.


File Save As dialog box

The following options allow you to specify the name and location of the file you're about to save:

File Name

Type a new filename to save a document with a different name.  A filename can contain up to eight characters and an extension of up to three characters.  GeneDoc adds the extension you specify in the Save File As Type box.

Drives

Select the drive in which you want to store the document.

Directories

Select the directory in which you want to store the document.

Network...

Choose this button to connect to a network location, assigning it a new drive letter.


File 1, 2, 3, 4 command
1, 2, 3, 4 command (File menu)

Use the numbers and filenames listed at the bottom of the File menu to open the last four documents you closed.  Choose the number that corresponds with the document you want to open.


Exit command
Exit command (File menu)

Use this command to end your GeneDoc session.  You can also use the Close command on the application Control menu.  GeneDoc prompts you to save documents with unsaved changes.

Shortcuts

Mouse:	Double-click the application's Control menu button.

Keys:	ALT+F4


View Toolbar command
Toolbar command (View menu)

Use this command to display and hide the Toolbar, which includes buttons for some of the most common commands in GeneDoc, such as File Open.  A check mark appears next to the menu item when the Toolbar is displayed.

See Toolbar for help on using the toolbar.


View Status Bar Command
Status Bar command (View menu)

Use this command to display and hide the Status Bar, which describes the action to be executed by the selected menu item or depressed toolbar button, and keyboard latch state. A check mark appears next to the menu item when the Status Bar is displayed.

See Status Bar for help on using the status bar.


Status Bar

The status bar is displayed at the bottom of the GeneDoc window.  To display or hide the status bar, use the Status Bar command in the View menu.

The left area of the status bar describes actions of menu items as you use the arrow keys to navigate through menus.  This area similarly shows messages that describe the actions of toolbar buttons as you depress them, before releasing them.  If after viewing the description of the toolbar button command you wish not to execute the command, then release the mouse button while the pointer is off the toolbar button.

The right areas of the status bar indicate which of the following keys are latched down:

Indicator	Description

CAP	The Caps Lock key is latched down.

NUM	The Num Lock key is latched down.

SCRL	The Scroll Lock key is latched down.


Cascade command (Window menu)

Use this command to arrange multiple opened windows in an overlapped fashion.


Tile command (Window menu)

Use this command to arrange multiple opened windows in a non-overlapped fashion.


Tile Horizontal command (Window menu)

Use this command to vertically arrange multiple opened windows in a non-overlapped fashion.


Tile Vertical command (Window menu)

Use this command to arrange multiple opened windows side by side.


Window Arrange Icons Command

Use this command to arrange the icons for minimized windows at the bottom of the main window.  If there is an open document window at the bottom of the main window, then some or all of the icons may not be visible because they will be underneath this document window.


1, 2, ... command (Window menu)

GeneDoc displays a list of currently open document windows at the bottom of the Window menu.  A check mark appears in front of the document name of the active window.  Choose a document from this list to make its window active.


Index command (Help menu)

Use this command to display the opening screen of Help.  From the opening screen, you can jump to step-by-step instructions for using GeneDoc and various types of reference information.

Once you open Help, you can click the Contents button whenever you want to return to the opening screen.


Using Help command (Help menu)

Use this command for instructions about using Help.


About command (Help menu)

Use this command to display the copyright notice and version number of your copy of GeneDoc.


Help Using Help Command
Context Help command

Use the Context Help command to obtain help on some portion of GeneDoc.  When you choose the Toolbar's Context Help button, the mouse pointer will change to an arrow and question mark.  Then click somewhere in the GeneDoc window, such as another Toolbar button.  The Help topic will be shown for the item you clicked.

Shortcut

Keys:	SHIFT+F1


Title Bar

The title bar is located along the top of a window.  It contains the name of the application and document.

To move the window, drag the title bar.  Note: You can also move dialog boxes by dragging their title bars.

A title bar may contain the following elements:

Application Control-menu button

Document Control-menu button

Maximize button

Minimize button

Name of the application

Name of the document

Restore button


The Scroll Bars
Scroll bars

Displayed at the right and bottom edges of the document window.  The scroll boxes inside the scroll bars indicate your vertical and horizontal location in the document.  You can use the mouse to scroll to other parts of the document.


Size command (System menu)

Use this command to display a four-headed arrow so you can size the active window with the arrow keys.

After the pointer changes to the four-headed arrow:

1.	Press one of the DIRECTION keys (left, right, up, or down arrow key) to move the pointer to the border you want to move.

2.	Press a DIRECTION key to move the border.

3.	Press ENTER when the window is the size you want.

Note:  This command is unavailable if you maximize the window.

Shortcut

Mouse:	Drag the size bars at the corners or edges of the window.


Move command (Control menu)

Use this command to display a four-headed arrow so you can move the active window or dialog box with the arrow keys.

Note:  This command is unavailable if you maximize the window.

Shortcut

Keys:	CTRL+F7


System Minimize Command
Minimize command (application Control menu)

Use this command to reduce the GeneDoc window to an icon.

Shortcut

Mouse:	Click the minimize icon [image] on the title bar.

Keys:	ALT+F9


Maximize command (System menu)

Use this command to enlarge the active window to fill the available space.

Shortcut

Mouse:	Click the maximize icon [image] on the title bar; or double-click the title bar.

Keys:	CTRL+F10 enlarges a document window.


Next Window command (document Control menu)

Use this command to switch to the next open document window.  GeneDoc determines which window is next according to the order in which you opened the windows.

Shortcut

Keys:	CTRL+F6


Previous Window command (document Control menu)

Use this command to switch to the previous open document window.  GeneDoc determines which window is previous according to the order in which you opened the windows.

Shortcut

Keys:	SHIFT+CTRL+F6


Close command (Control menus)

Use this command to close the active window or dialog box.

Double-clicking a Control-menu box is the same as choosing the Close command.

Note:  If you have multiple windows open for a single document, the Close command on the document Control menu closes only one window at a time.  You can close all windows at once with the Close command on the File menu.

Shortcuts

Keys: 	CTRL+F4 closes a document window

ALT+F4 closes the <<YourType>> window or dialog box


Restore command (Control menu)

Use this command to return the active window to its size and position before you chose the Maximize or Minimize command.


Switch to command (application Control menu)

Use this command to display a list of all open applications.  Use this "Task List" to switch to or close an application on the list.

Shortcut

Keys:	CTRL+ESC

Dialog Box Options

When you choose the Switch To command, you will be presented with a dialog box with the following options:

Task List

Select the application you want to switch to or close.

Switch To

Makes the selected application active.

End Task

Closes the selected application.

Cancel

Closes the Task List box.

Cascade

Arranges open applications so they overlap and you can see each title bar.  This option does not affect applications reduced to icons.

Tile

Arranges open applications into windows that do not overlap.  This option does not affect applications reduced to icons.

Arrange Icons

Arranges the icons of all minimized applications across the bottom of the screen.


Choose Font Dialog
Choose Font dialog box

Choose the font for GeneDoc to use on the GeneDoc Display.


Choose Color Dialog
Choose Color dialog box

Choose a Color for GeneDoc to use on the GeneDoc Display.


No Help Available

No help is available for this area of the window.


No Help Available

No help is available for this message box.


File Print command
Print command (File menu)

Use this command to print a document.   This command presents a Print dialog box, where you may specify the range of pages to be printed, the number of copies, the destination printer, and other printer setup options.

Shortcuts

Toolbar:

Keys:	CTRL+P


Print dialog box

The following options allow you to specify how the document should be printed:

Printer

This is the active printer and printer connection.  Choose the Setup option to change the printer and printer connection.

Setup

Displays a Print Setup dialog box, so  you can select a printer and printer connection.

Print Range

Specify the pages you want to print:

All	Prints the entire document.
Selection	Prints the currently selected text.
Pages	Prints the range of pages you specify in the From and To boxes.
Copies

Specify the number of copies you want to print for the above page range.

Collate Copies

Prints copies in page number order, instead of separated multiple copies of each page.

Print Quality

Select the quality of the printing.  Generally, lower quality printing takes less time to produce.


Print Progress Dialog

The Printing dialog box is shown during the time that <<YourApp>> is sending output to the printer.  The page number indicates the progress of the printing.

To abort printing, choose Cancel.


File Print Preview command
Print Preview command (File menu)

Use this command to display the active document as it would appear when printed.  When you choose this command, the main window will be replaced with a print preview window in which one or two pages will be displayed in their printed format.  The print preview toolbar offers you options to view either one or two pages at a time; move back and forth through the document; zoom in and out of pages; and initiate a print job.


Print Preview toolbar

The print preview toolbar offers you the following options:

Print

Bring up the print dialog box, to start a print job.

Next Page

Preview the next printed page.

Prev Page

Preview the previous printed page.

One Page / Two Page

Preview one or two printed pages at a time.

Zoom In

Take a closer look at the printed page.

Zoom Out

Take a larger look at the printed page.

Close

Return from print preview to the editing window.


File Print Setup command
Print Setup command (File menu)

Use this command to select a printer and a printer connection.  This command presents a Print Setup dialog box, where you specify the printer and its connection.


Print Setup dialog box

The following options allow you to select the destination printer and its connection.

Printer

Select the printer you want to use.  Choose the Default Printer; or choose the Specific Printer option and select one of the current installed printers shown in the box.  You install printers and configure ports using the Windows Control Panel.

Orientation

Choose Portrait or Landscape.

Paper Size

Select the size of paper that the document is to be printed on.

Paper Source

Some printers offer multiple trays for different paper sources.  Specify the tray here.

Options

Displays a dialog box where you can make additional choices about printing, specific to the type of printer you have selected.

Network...

Choose this button to connect to a network location, assigning it a new drive letter.


File Page Setup Command
Page Setup command (File menu)

<< Write application-specific help here. >>


GeneDoc Help Index
Menus: File:Project:Edit:Arrange:Shade:Groups:Score:Tree:Report:Plot:Window:Help

Running and Using GeneDoc	Arranging Alignments	Shading Alignments
Reading/Importing Data	Using the Mouse
GeneDoc Web Viewer	Using the Keyboard	Pre Configured Shading Modes
DDE support.	Tips For Arranging Alignments
		User Configured Shading Modes
GeneDoc Display Modes	Additional Arrangement Functions
Alignment View	Copy To & From Other Projects	Shading With External Files
Summary View		Search: ProSite and ReBase files
Tree View	Editing functions for DNA projects	Log Odds: Meme files
Report View	DNA Ambiguity Support	Secondary Structure Files
Plot View	DNA/Protein Translation/ReGap	User Defined Files
Gel View
	Scoring while Arranging	Super Family Group Features
Project Settings	Scoring Tables	Specialized Super Family displays
Configure Dialog	Similarity Tables
Sequence Edit Dialog		Plots
Project Titling Facility	TUTORIAL	The DStat function
Save & Load User Defaults
		Reports: Stats, Score, Composition
Find and Replace functions	Toolbars
Manual Editing	Project Toolbar	User Defined Shading.Definitions
Exporting and Copying Figures	Alignment Toolbar	User Data Files
Getting postscript file output	Tree Toolbar
RasMol Scripts	Tips on using the Toolbar	Initial Alignment of Sequences
Running and Using GeneDoc (Help Index )

GeneDoc installs as a regular windows program, into a GeneDoc program group. After starting GeneDoc, use file open and read in a MSF (Multiple Sequence File) file. GeneDoc saves configuration information for these files in the comment section of the MSF, so if the file was saved by GeneDoc, then it will be reopened with the same settings it was last saved with.

Reading/Importing Data (Help Index )

You can import non MSF files. Use the File/New menu, then select File/Import. This import dialog allows import from the Clipboard, Disk Files or manual input. Clustal, fasta and a few other types can be read this way.

GeneDoc Web Viewer (Help Index )

GeneDoc supports a command line argument as a file name. This allows GeneDoc to be run from another program, such as a web browser or database program.

DDE support (Help Index )

GeneDoc provides minimal DDE support, File Open and Print. This allows you to setup windows to have GeneDoc open or print files automatically by double clicking or right click on a MSF file icon.

GeneDoc Display Modes (Help Index )

GeneDoc has several view modes. These are accessed through the Windows menu or Project toolbar. The Alignment view, Summary View and Tree view can be opened and used at any time. The Report View and Plot view will be empty or not able to open if you have not selected a report to show or a plot to view.

Alignment View (Help Index )

When GeneDoc is first opened, you will see the Alignment view. The Alignment view should be considered the primary view of GeneDoc. In this view, you will be able to see the properties of each residue in the alignment with the various shading modes, you will be able to arrange the residues to improve your alignment. You will be able to score various sections on the alignment and add manual comments and manual shading.

Summary View (Help Index )

A useful view is the Summary view. This view shows you the presence or absence of a residue, but not its value, thus the alignment is viewed in a compressed fashion. GeneDoc will also apply shading to the Summary view. You can control the summary view settings in the Project Configuration dialog. GeneDoc can compress the Summary View up to one dot per column, so on a high resolution printer the amount of compression can be considerable.

Tree View (Help Index )

GeneDoc provides a phylogenetic tree view. This view is can be used to construct the phylogenetic tree with a point and click interface, and the Manage Expression dialog will edit, load and save phylogenetic information in Vermont syntax. GeneDoc does not attempt to compute any phylogenetic information for the alignment, though it will use it to score a selected set of columns. The view is used only to build and view phylogenetic trees.

Report View (Help Index )

GeneDoc creates several text reports, and will show them the Reports View. The Reports view holds the information, but does not save it on the disk. There is a menu entry in the reports menu for saving the report to a text file of your choice.

Plot View (Help Index )

GeneDoc displays a few Cumulative Distribution plots in the Plot view. At its simplest, the plot view can be used to show Percent Identity or Percent Favorable Substitutions for the alignment as a whole. Using the Super Family Groups of GeneDoc, the plot can be used to show that the scoring within groups is significantly better than scoring between different groups, thus demonstrating that your super family groups are well chosen. These functions are controlled through the Plot menu.

Gel View (Help Index )

GeneDoc displays a simple Enzyme Gel Simulation view. This view contains two lists, the list of sequences and the list of loaded enzymes. Select multiple sequences and multiple enzymes and click the ‘Run Gel’ button. The resulting sequence fragments are plotted on the view on a log scale.

Project Settings (Help Index )

GeneDoc has a rich set of Project Configuration settings. While some of these settings are controlled through menus, all of the settings are found in the Configuration Dialog. This dialog is accessed either through the Project menu or the Project toolbar.

Configure Dialog (Help Index )

The Configuration Dialog holds ten tabs. Each tab holds various GeneDoc settings related to each other and described by the tab title. Tab functions can be put into three groups, Project Setup, Print Control and Shading Control. The first tab, Project, controls font size, consensus lines, alignment blocking and other settings that apply to every display. The Print tab controls printer margins, page headers, footers, numbers and the like. A Shade tab mimics a lot of the entries found in the Shade menu, with a few other settings for the conserved and quantified shading style. There is a scoring tab that allows you to select which DayHoff or PAM scoring tables and substitution groups you want to use.

The rest of the tabs are for control of individual shading modes. There is a tab for Properties, Physiochemical properties, Pattern Search, Log Odds, Identities and Structure. All aspects of these display modes are controlled through these configuration tabs. Here is where you change colors, and add, edit and delete patterns or properties, load data files for display modes, whatever. The configuration dialog does not have anything to do with manual sequence arranging, though scoring settings can be controlled here.

Sequence Edit Dialog (Help Index )

The Project menu also holds the Edit Sequences dialog. In this dialog, sequences can be added or imported, deleted. You can Complement, Reverse and Duplicate Sequences here. Comments about the sequences can be entered. Weights can be changed, which are used by the Log Odds displays.

Project Titling Facility (Help Index )

The Project menu also has the Titling Facility. The titling facility gives you a convenient way to enter comments at the top of the MSF file. These comments are not saved in the usual GeneDoc encoded header, but above them in ascii text, so anyone or any program will have access to them.

Save and Load User Defaults (Help Index )

Save and Load User Defaults is a way to save the current settings as GeneDoc’s default settings. These would apply when you open a MSF file that has not been previously saved by GeneDoc. If you want to apply these settings to a file that has GeneDoc settings, then load the file and then use the Load User Defaults, these settings will replace whatever GeneDoc’s current settings are.

Arranging Alignments (Help Index )

Arranging alignments is a primary function of GeneDoc. There are many features provided to help accomplish this task. Both mouse and keyboard operations are supported. GeneDoc’s Grab and Drag arrangement mode allows you to move residues around like beads on a string. You can Slide residues, which only inserts or delete gaps immediately in front of the selected residue, preserving other gap placements. You can insert and delete a gap with either the mouse or the INS and DEL keys. You can insert gaps into every other residue from the one clicked on, and insert columns of gaps. GeneDoc also provides the ability to select groups of sequences to work on. See Tips on Arrangement and Alignment View.

Using The Mouse (Help Index )

Using the mouse for arrangements gives you quick adjustment abilities. Grab and Drag and Slide modes work by clicking on the desired residue and while holding the mouse button down moving the mouse back and forth. Other modes, such as insert single gaps or insert gaps into other sequences, use a single mouse click. You can select columns for certain alignment functions by enabling the column select mode then clicking on the start and end columns to be selected.

Moving the mouse over the alignment updates the location indicator in the lower right corner of the GeneDoc window. This will show you what sequence and residue number the mouse is positioned over.

Using the Keyboard (Help Index )

The keyboard is very useful for arrangement of alignments. The keyboard is more precise than the mouse, especially if the mouse is dirty or jumpy. The keyboard can be slower than the mouse, but every effort has been made to help keep use of the keyboard efficient. There are a few shortcut keys that are helpful as well. After you become more familiar with GeneDoc, the keyboard is often a faster way to work with GeneDoc than the mouse. See Tips on Arrangement and Alignment View.

Additional Arrangement Functions  (Help Index )

In addition to the movement of residues for arrangement purposes, there are few other helpful functions. You can select a range of columns to be deleted. As mentioned before, you can select a set of sequences to be worked upon. When you select sequences, clicking within the selected set performs work upon the selected set. When you click outside of the selected set, work is performed upon the unselected set.

Copy To & From Other Projects  (Help Index )

A rather useful function, for specific cases, is the ability to copy a range of selected columns to another project, edit just that range, then replace them back into the original project. This can be useful for a variety of purposes, from manually arranging a restricted area, to exporting an area of the alignment to be realigned or aligned with a different alignment program.

Editing functions for DNA projects (Help Index )

There are a few editing functions specific to DNA projects as well. These include complementing and reversing sequences, copying data between sequences, and creating a fasta style output of the consensus row. A specific use of the Properties mode is very helpful for DNA projects as well. If the configuration dialog has the project set to a non protein type, then Property Shading Mode level 3 has been pre setup to shade each nucleotide in a unique color.

DNA Ambiguity Support (Help Index )

GeneDoc has built in ambiguity support for DNA projects. This support is used in the realm of determining conservation for columns and where ever else is appropriate.

DNA/Protein Translation/ReGap (Help Index )

A DNA to Protein translation feature is incorporated into GeneDoc. You can choose from a set of translation tables or modify one to your own. There are several Frame Options for the translation process. If you make arrangement changes to a Protein alignment, you can ReGap the corresponding DNA project. Using the cylce of Translate/ReGap, you can keep both the DNA and Protein projects in sync.

Scoring while Arranging (Help Index )

GeneDoc provides scoring functions for the alignment view. Scores can be calculated for a selected range, presumably a range of columns you are rearranging, and then have the area recalculated conveniently after you have made arrangement changes. This is intended to give you an objective basis for determining whether your alignment changes are actually better or not. Scoring can be done with Sum of Pairs, Phylogenetic Tree, or Log Odds methods. For larger numbers of sequences, phylogenetic scoring can be rather slow.

Scoring Tables (Help Index )

Several Dayhoff and PAM similarity scoring tables are built into GeneDoc. These tables are used anywhere a score between two residues is needed. For example, while the conserved and quantify display modes depend mostly on the residue counts within a column, ties are broken by determining which of a set scores better against the rest of the column. The scoring tables are also used in the scoring features of GeneDoc.

Similarity Tables (Help Index )

Also, Similarity tables have been built for each scoring tables. These similarity tables represent selections of favorable substitutions of residues for a given scoring table. These scoring tables can be altered and saved in user defaults. They are used in the conserved and quantify display modes. When used, they show conservation of favorable substitutions as well as identities.

Find and Replace functions (Help Index )

GeneDoc also includes Find and Replace functions. The Find function is the more useful, as the replace is simple and replaces only segments of the same length. The Find function has features for mismatches and insertions and deletions, and you can select which sequences to search. There are also functions for moving the cursor to the found location or shading all found locations.

Manual Editing (Help Index )

You can enter comments, change residues and make manual shading within your alignment. These changes are saved for you. Manual comments can be entered on any non sequence line, and will over write any other characters which may be displayed on those lines, such as column number or consensus. Manual shading is done with the mouse, by selecting a color and clicking and dragging over any residues you want shaded. Comment lines may not be shaded. You can change the values of residues in your alignment as well. These changes will change the data in your MSF file, so you should have good reason for doing this.

Shading Alignments (Help Index )

Shading alignments is the other major function of GeneDoc. There are quite a few shading modes, and with super family group functions, the possibilities are extended a quite a bit. The different shading modes each represent application of different algorithms to the alignment. Each shading mode is controlled in the Configuration Dialog, as previously mentioned. Many of the toolbar buttons of the alignment view control the shading modes. You can also apply differences mode to the alignment view. This mode changes the display of residues, but not the shading. It highlights where residues are different from the consensus of residues for the column.

Pre Configured Shading Modes (Help Index )

There are three shading modes that don’t require and setup by the user, though they can be adjusted. These are Conserved, Quantified, and Physiochemical display modes. The Conserved and Quantified modes are based on the identity of residues, the similarity tables, and the score table for breaking ties in identity. These modes show percentage of conservation within a column in the alignment. Physiochemical display mode is based on the physical and chemical properties of amino acids and identifies where those properties are conserved within and alignment. These groups were originally proposed and presented as a Venn diagram by W.R. Taylor (1986), though GeneDoc has changed them somewhat.

User Configured Shading Modes (Help Index )

Another two other shading modes used by GeneDoc requires some user setup. These are Properties and Identities. Actually, Properties mode is configured for some obvious chemical properties by default, but its expected you would want to setup your own property groups. Properties mode is useful for highlighting sets of amino acids, either conserved or where ever found. For example, you could enter a set of hydrophobic amino acids and have them highlighted.  Identities mode compares the identity of one or more sequences to the rest of the alignment, so you have to choose one or more sequences to get any output from this display mode. If more than one sequence is in the chosen set, then the chosen set of sequences must be conserved before they will be compared to the rest of the alignment.

Shading With External Files  (Help Index )

There are three shading modes that require external files to be loaded into GeneDoc for them to function. These are Search, Log Odds and Structure modes. Files that need to be loaded would be ReBase or ProSite Files, Meme or similarly formatted Log Odds matrices, and Protein Structure files such as PDB or DSSP files. GeneDoc will reload structure files automatically whenever the alignment is reopened if the file is located in the same disk directory as the MSF file. Search mode is configurable by the user, but you will need to enter search strings in ReBase or ProSite syntax.

Search: ProSite and ReBase files (Help Index )

In Search mode, you load a ReBase or ProSite file and GeneDoc location enzymes or motifs found in your alignment and highlights them. The configuration dialog then allows you to delete or disable patterns, changes coloring, etc. Search mode stores the located patterns in your alignment, so you do not need to keep a copy of the ReBase or ProSite file with the alignment. You can also export a list of found Enzymes in ReBase syntax for your alignment.

Log Odds: Meme files (Help Index )

In Log Odds mode, motifs described by Log Odds arrays, such as are produced by the Meme program maintained at San Diego Supercomputing Center (www.sdsc.edu/MEME), are loaded into GeneDoc and used to highlight your alignment. Again, the Configuration Dialog contains several options for controlling usage of the motifs and shading of the alignment. You can then shade the alignment based on the motifs found in the Log Odds file.

Secondary Structure Files (Help Index )

Structure shading mode uses several different external file types. A file type can contain more than one set of information. PDB file can be read in for example, and the secondary structure information applied to the sequence in the alignment that corresponds to the PDB file. DSSP file, from EMBL can be read as well. Some EMBL sponsored programs that predict secondary structure can be read in and the prediction or other information, such as prediction probability, can be applied to the appropriate sequence as well.

User Defined Files (Help Index )

While this shading mode is called Structure mode, any information can be used, such as accessibility. GeneDoc reads in PSDB files, which are derived from the PDB information, and contain quite a large set of calculations that may be applied to a sequence in the alignment. More than one file type can be applied to each sequence, and each sequence can have file types applied to it.  There is a scheme for allowing the user to define a custom data set and read these user files into GeneDoc for shading. This allows considerable flexibility, though at the expense of some initial effort.

Super Family Group Features (Help Index )

GeneDoc supports Super Family Groups. It does this first by allowing you to define groups of sequences within the alignment as belonging to the same family through the Group Configuration Dialog. In this dialog, you can apply a subset of the the shading modes to each of the groups. For example, you could show conserved shading for each group in the alignment. The Shade Group Configuration function switches you between non group and group shading.

Specialized Super Family displays (Help Index )

Additionally, there are a number of shading functions specialized for super family groups. These functions typically contrast scores or identities between the groups or to shade across all groups before shading within groups. These specialized shading modes can be specific to either Protein or DNA alignments as well.

Plots (Help Index )

Some analysis functions are available that plot the results in graphical format. These tell about inter sequence identities or scores in graphical format. This can be convenient for alignments with a lot of sequences, since the equivalent report would be too large to be read or printed easily. GeneDoc will also use its score tables to show you favorable substitution levels as well as identity levels.

The DStat function (Help Index )

You can do more a complicated super family group analysis, using statistical functions to show the validity of your groupings. After the super family groups have been setup, then you can select a range and make a DStat plot. You will get two lines on your plot, one for scores within groups and one for score between groups. If your groups are properly selected, the will show statistical differences.

Reports: Stats, Score, Composition (Help Index )

GeneDoc provides a few reports for your alignment. A common Statistics report is given, though GeneDoc also includes favorable substitutions as determined by the current scoring table in this report. GeneDoc shows a score report, which gives scores between sequences in your alignment. There is a Base Composition report. There is a specialized enzyme report based on the sequence fragments identified by using the ReBase Search mode that sorts fragments lengths, finds unique ones, and identifies unique fragment lengths across super family groups. You can select a range of columns and compute a Log Odds matrix for it.

Exporting and Copying Figures (Help Index )

GeneDoc provides a lot of ways to write shaded (or non shaded) alignments to the clipboard or a file for use in other programs. You must first select a block within your alignment. GeneDoc typically breaks up the alignment into blocks and displays them vertically on the screen for scrolling, and after you select one or more blocks you can then copy or export them. Export types include RTF, PICT, Meta Files, HTML, Bitmap and Text. Every attempt to include all shading and other display features has been included, though of course formats like text won’t support such.

Getting postscript file output (Help Index )

There is no built in support for PostScript. You should find it convenient enough to load a postscript driver and set the output to a file for creating PostScript files.

Toolbars  (Help Index )

GeneDoc puts many common functions into Toolbars. There are a couple sets of toolbars. The two most common are the Project Toolbar and the Alignment View Toolbar.

Project Toolbar (Help Index )

The Project Toolbar is the upper toolbar and is always visible regardless of which view you are showing. This toolbar shows functions that are applicable for whatever view, such as file save and print. The Project Toolbar has buttons for the Configuration Dialog, the Edit Sequence List Dialog and the Group Configuration Dialog. The Project Toolbar also has buttons for switching between the various views of GeneDoc.

Alignment Toolbar  (Help Index )

The Alignment Toolbar controls features of the Alignment view. The Alignment view is where most of the work gets done in GeneDoc. Mainly, buttons here control the shading and arranging features of the alignment view. This toolbar is only visible when the alignment view is active.

Tree Toolbar (Help Index )

The Tree View Toolbar has a few buttons for use in constructing phylogenetic relationships with the GeneDoc interface. GeneDoc provides a GUI interface for creating and deleting nodes of the tree.

Tips on using the Toolbar (Help Index )

The toolbar can be used to access the configuration dialog in a quick fashion. If you click on a display mode, such as conserved, then GeneDoc will switch to that shading mode. If you click on the conserved toolbar button again, the configuration dialog will be opened with the conserved setup tab selected. See Toolbar Tips.

RasMol Scripts (Help Index )

GeneDoc has the ability to create simple scripts for the RasMol program. These scripts can be imported into RasMol and used to color the molecule with the shading done in GeneDoc. This is useful for coloring molecules in ways RasMol does not support, or applying the information of an alignment to the molecule for visualization purposes.


Running and Using GeneDoc
Running and Using GeneDoc. (Help Index )

Note that GeneDoc reads only GCG style  MSF files. See Common GeneDoc .msf File Read Troubles. for more information.

File Associations

After GeneDoc is run the first time, it will create an entry in the File/Associate menu of the file manager, with the default set for .MSF files. By selecting an .MSF file in the file manger and completing the association, you will then be able to launch GeneDoc by double clicking on .MSF files in the file manager.

Drag and Drop

You can drag with the mouse a .msf file and drop it on the GeneDoc icon or a running copy of Genedoc to open the data file.

Command Line

Genedoc will auto load an .MSF file if Genedoc is started with the filename as the first command line option. This feature lets you make an icon in the program manager for a particular .MSF file, and when you double click the icon in the program manager, GeneDoc runs and loads the file. This would also be useful in the context of a helper application for a web browser.


GeneDoc Web Viewer
GeneDoc Web Viewer  (Help Index )

GeneDoc can easily operate as a web viewer for your data files. A GeneDoc viewer is a convenient way to allow users to see your alignment data on your web site in a pleasing and useful manner. Data can be printed and manipulated by the user.

There is no ‘official’ mime type for GeneDoc data files. GeneDoc saves its information into a GCG style .MSF file, but the author recommends using a Application/x-Genedoc mime type.

Once you have configured your web server to support a mime type for GeneDoc, give users standard setup instructions. For example:

First, download the GeneDoc viewer software (www.cris.com/~Ketchup/genedoc.shtml)

     Before downloading the sequence files you should make Genedoc a Helper Application in your WWW Browser. If you are using the Netscape Navigator, open OPTIONS | PREFERENCES | HELPERS and click on CREATE NEW TYPE. In the window that opens enter

     MimeType:application

     Mime Subtype: x-Genedoc

     click OK

     When you return to Preferences-Helpers, under File Extensions enter: MSF

     under Action: select "Launch the Application" and in the window next to the "Browse" button

     enter C:\Genedoc\Genedoc.exe

Internet Explorer users should find a File Association for MSF files already set up once GeneDoc is run the first time, and shouldn’t have to do anything.


GeneDoc File Read Troubles
Common GeneDoc .msf File Read Troubles  (Help Index )

Previous versions of GeneDoc have had more troubles that this one, but there are still a few gotcha’s.

1: There can be no duplicate sequence names in the file.

2: Spaces in the sequence names.

3: Non Standard characters in the Sequence names. GeneDoc stores phylogenetic tree information internally as a ‘Vermont Syntax’ string. Certain characters in the names can confuse the phylogenetic tree parsing subroutine, or so I’ve heard. Its all worked pretty well when I tried it, so your mileage may vary.

GeneDoc defines a MSF file as follows.

The first important line contains a “MSF:” (or “msf:”) and a “..” (two dots). Anything before this is considered free form comments. GeneDoc saves project information for this file in this comment area. This line is also read for a “Type:” field, describing either a DNA or Protein project, and a “Check:” field containing a checksum for the entire project.

After this comes a series of rows, one for each sequence in the project. These rows must contain the field “Name:” followed by the sequence name, and should contain the fields “Weight:” and “Check:”, containing the sequence weighting and checksums.

This section of the file is terminated with a line that has a “//” (two slashes) in it.

The next section contains the sequence data. These are written in blocks of data, typically 50 residues per block for each sequence. A data line must start with the sequence name, though spaces may proceed the sequence name. At least one space must come between the sequence name and the data. The data is typically shown in chunks of ten, separated by spaces, though they don’t have to be space separated. However, since this is a possibility, spaces in the data area will be ignored. Each block must contain the same number of residues for each sequence. Note that since spaces are ignored, they can not be used to represent gaps in the alignment since this will cause inconsistent data lengths between rows in a block.

Data is read until the end of the file.

This from the GCG manual as found online:

You may find the following components in an MSF file:

•	File Type. (optional) Begins with the line (all uppercase) !!NA_MULTIPLE_ALIGNMENT 1.0 for nucleic acid sequences or !!AA_MULTIPLE_ALIGNMENT 1.0 for amino acid sequences. SeqLab uses the file type to improve performance when loading files. Do not edit or delete the file type. If present, it must appear on the first line of the file.

•	Description. (optional) Contains informative text describing what is in the file. You can add this information to the top of the MSF file using a text editor.

•	Dividing Line. (required) Must include the following attributes:

MSF. Displays the number of bases or residues in the multiple sequence alignment.

Checksum. Displays an integer value that characterizes the contents of the file.

Two periods (..). Acts as a divider between the descriptive information and the following sequence information.

•	Name/Weight. (required) Must include the name of each sequence included in the alignment, as well as its length and checksum (both non-editable) and weight (editable).

Note that the checksum of the individual sequences is important as a safety measure to ensure that you do not change the sequence data inadvertently. If this has happened, you will not be able to use the sequence(s) within the MSF file. You then can use the Reformat program to reformat the sequences and create a new checksum to reflect the file's edited contents.

•	Separating Line. (required) Must include two slashes (//) to divide the name/weight information from the sequence alignment.

•	Multiple Sequence Alignment. (required) Must include each sequence named in the above Name/Weight lines. This alignment allows you to view the relationship among sequences.


File Import
File Import (File Menu)

This menu presents you with the Import Type Dialog . When you select a file type, a default extension will be selected as well.  The file does not have to have the default file extension if the proper type is selected, but it should not have another file types extension.

Input types are:

Phylip (Interleaved): *.PHY

GCG MSF: *.MSF

PIR:  *.PIR

Clustal ALN files: *.ALN

Pearson Fasta Files: *.AA


Import Type
Import Type (File Menu)

Select which type of file you plan on importing. You can also choose to type or paste in data with the Input Button.

Select Import device.

Select whether you want the input from the Clipboard or a File.

Type of File.

Select the type of input you have. Either one of the file types or plain text.

Import.

Do the actual import. If Import from File was selected, you will get a File Open dialog.

Input

This button presents you with the Sequence Input dialog. This dialog allows you to type in residues for a single new sequence or to paste residues for a single new sequence. The sequence must be named and you cannot put ‘File Types’ in for sequence input. Any character between the range ‘A’ through ‘Z’ and the characters ‘-‘ and ‘.’ will be copied into the new sequence as residue data.

Done.

Close the dialog.


GenBank Dialog
GenBank Dialog (Import Type)

Select various parameters of the Genbank file import function.

Name

Select with field to use for the name of the sequence.

Translation or Origin

Select either the translation or the origin to use as sequence data. Protein files don’t typically have a translation …


File Export
File Export (File Menu)

This menu gives you a File Export dialog.

The Sequences to be exported, whether to a file or the clipbaord, and the type of file to be exported must be selected.

If you do not apply a file extension to the filename, one will be automatically added for you.

Export types are:

Pearson Fasta Files: *.AA

Clustal ALN files: *.ALN

Phylip (Interleaved): *.PHY

PIR:  *.PIR

Text Files: *.TXT


Export Dialog
Export Dialog (File Menu)

Select all the output options here.

Sequence Dialog.

If the select from list option is checked, then you can select the sequences you want to export here.

Select Sequences

Select either ‘Select All’ to have the all sequences exported, or select from list to be able to select sequences to be exported from the list.

Select Export device.

Select whether you want the output to go to the Clipboard or to a File.

Type of File.

Select the type of output you want. Either one of the file types or plain text.

Export.

Do the actual export.

Done.

Close the dialog.


Mouse and Keyboard Support
Mouse and Keyboard support (Help Index )

Mouse operations.

Mouse operations are fairly intuitive. If the user is in a Select Columns or Arrangement mode, then left button mouse clicks will select or arrange sequences. Right mouse clicks will generally do the exact opposite of a Left mouse click.

In the Grab and Drag arrangement mode,  you click with the left mouse button while the mouse pointer is on a character, other than a gap character, and move the character left or right while holding the mouse button down. In this mode, when the left mouse button is held down, the cursor movement is restricted to the area of the sequence characters in the row.

In Select Columns modes, a single click will select a row or column, and you can expand the selected area by holding the button down and moving the mouse. You can also expand selections by clicking once on the start of the desired selection area and once at the end of the desired selection area. If you click in an area that is already selected, then all current selections will be removed and a new start selection placed where you clicked. You can clear all current selections at any time with a double click and you can start a new selection anytime with three left mouse button clicks in a row at the same spot.

All other windows menu and toolbar operations are standard. Some of the toolbar buttons are toggle buttons and some are action buttons.

Double Clicking the left mouse button in any non-selection mode will relocate the cursor to the location of the mouse pointer.

You can update an invalidated score by double clicking in the area of the score box while in any arrangement mode. Also, updating the GeneDoc display with an autoshade function will recalculate any invalidated score. Furthermore, if the scoring table or Substitution Groups are changed, then all scores on the score line will be updated.

When the program is in residue edit mode, a block caret will be shown instead of a line caret. Any single click of the mouse button will move the caret to the new location in residue edit mode. If the caret is over a residue or gap, any proper alpha character for the Project Type will over write the current residue value and advance the caret.

Keyboard operations.

Keyboard use in GeneDoc is fairly simple and straight forward. The shift key acts in exactly the manner of the left mouse button, both for single clicks and being held down. The Enter key acts in the same manner as a double click of the left mouse button. The arrow keys will move the cursor one character or line up, down, left and right. The page up and page down will move the cursor one sequence row up or down. The home and end keys kill move the cursor to the beginning or the end of the current line. Ctrl-Enter will do the opposite as an enter.

Typically you will move the cursor to the left side of the desired location and press the shift key to either make a selection or an arrange the alignment.  In Grab and Drag mode, move the cursor to the left side of a character if you plan on moving the character right, or the right side of the character if you plan on moving the character left. Hold the shift key down and move the character with the left or right arrows. You could move the characters all the way off the line with the home or end keys. Up or down movement is prohibited when the shift key is held down in Grab and Drag mode.

You can insert or delete a gap from a sequence at any time by hitting ther INS or DEL keys.

When the program is in residue edit mode, a block caret will be shown instead of a line caret. All of the arrow keys are active and available to move the caret to the new location in residue edit mode. If the caret is over a residue or gap, any proper Alpha character for the Project Type will over write the current residue value and advance the caret.


Arrange Tips
Tips for Arranging Alignments (Help Index )

If you are working on DNA or RNA sequences you can display each base in its own color. First, set the appropriate project type on the project tab page of the configuration dialog box. Set Shade Mode to Property mode and shade levels to 4 levels (or hit F4). You can change the colors on the property tab page of the configuration dialog, For ABI users A=Green, C=Blue, TU=red, and G=Yellow (some prefer very light yellow, which gives a better black&White contrast of all 4 bases) should give good results. Use the Edit, Residue Edit mode, or U toolbar button on the right side, to edit the residues.

To align sequences, it is easiest if you are using pre aligned sequences, e.g. using ClustalX (a windows version is available under ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/ )

To correct an alignment, it is easiest to use Arrange: Slide mode first. You can slide a group of bases from left to right, with out changing the rest of the alignment for this sequence (as long as gaps are available). If too few gaps are there, you can first create a new gap all over the alignment, either by clicking the insert gap in all sequences button for a single gap, or by selecting a number of columns and insert sequences into all of them.

Since normal slide mode does not affect the rest of the sequence, it does not matter whether you work from left to right, or from right to left. The toolbar buttons insert gap in current sequence, and Insert In Other sequences are most useful, if you add a new unaligned sequence and want to manually align this with other sequences already aligned. Working from left to right. If the other sequences have extra bases, you insert a single gap into your sequence, if your sequence has extra bases, you use insert into others.

On the keyboard this has been facilitated with the Insert and Delete keys. Insert and Delete each insert or delete one gap in a sequence, sliding the residues and gaps to adjust. With Insert In Other mode enabled, the Enter key becomes insert into all other and Ctrl-Enter becomes delete from all other. Combined with the Insert and Delete keys, aligning a new sequence can be done efficiently. Some prefer to put the Sequence Block Sizing to Alignment Size while doing this.

Special case: Align two separate alignments with each other. To do this with the least effort, you should select one of the two sets using select sequences (Ctrl-T). The you can use the Insert Gap Column function clicking on either of the alignments, or sets of sequences, to insert a column of gaps into which ever set needs to be adjusted.

Example:

       1   5   10

SeqA1  ACT-ACCG--TTTAAA

SeqA2  ACT-ACGGA-TTTAAA

SeqA3  ACTTACGGATTTTAAA

SeqB1  ACTATGGGATTAAA

SeqB2  ACTA-GGGATTAAA

SeqB3  ACTA-GGGATTAAA

Select first three sequences. Select "Insert gap column into all

(selected)" Click with left mouse button at position 4 in SeqB1 (you

could just as well click into SeqB2 or SeqB3, it would not matter).

Click into SeqA1 at position 6.

The result would be:

       1   5   10

SeqA1  ACT-A-CCG--TTTAAA

SeqA2  ACT-A-CGGA-TTTAAA

SeqA3  ACTTA-CGGATTTTAAA

SeqB1  ACT-ATGGGATTAAA

SeqB2  ACT-A-GGGATTAAA

SeqB3  ACT-A-GGGATTAAA

Continue until both sequences are completely aligned. Tip: if you click once to often, you can just click with the right mouse button to remove a superfluous gap. If you detect an error in one of the alignments, you can either use the normal modes to align a single sequence within that alignment (slide mode, insert gap, insert gap into other).

Special case: one, or a group of sequences contains a long insert.

Example:

       1   5   10

Seq1  ACTATGGGGATTAAA

Seq2  ACTA-GGGGATTAAA

Seq3  ACTA-GGAATATTACGAGGCTACTGCTAATCCGGATTAAA

Seq4  ACTA-GGAATATTACGAGGCTACTGCTAATCCGGATTAAA

Select Seq 1 and 2 (If you have many sequences without insert and a few with the insert, you can select the sequences with the insert and then complement the selection with Shift-Ctrl-T). Go into column selection mode, and select the length of the insert. You can either drag the mouse, or you click on the first column, and then on the last. (Do not use the shift button, this confuses GeneDoc). Now click on the insert gaps into selected column button, and you will have inserted a gap into all sequences without the insert.

In general, at the end of a session it is often useful to Select: Edit, Clear gap columns to remove all gaps which go through the entire alignment. You would not want to do this, if you want to keep them


Project menu
Project Menu (Help Index )

The Project menu includes items that control all of Genedoc’s general user settings and project settings. You will find user settings for the current data file here, as well as saved default settings and sequence edit menu and titling facility.

You can control the user settings with the configuration dialog. The current settings can be saved as the defaults for any new project by using the Save as User Defaults option. To set the default parameters and options of an already started project to your customized set of User defaults use the Load User Defaults option.

For more information, select the Project menu item name below.

Configure	Opens the User Settings Configuration dialog.
Edit Sequences List	Bring up the Edit Sequences dialog box.
Titling Facility	Opens the Titling Facility Dialog.
Save as User Defaults	Save the current settings in the GeneDoc.ini file as the default settings for new projects.
Load User Defaults	Load the User Settings from the GeneDoc.ini file and replace the current project’s settings.
Load Structure Definition	This brings up the Load Structure Definitions menu.
Select Columns	Select a range of columns for editing with below functions.
Copy to New Project	Copy the selected range to a new project.
Replace with other project	Replaces the selected range with data from another opened project.
Trans DNA to Protein	Translation of DNA to Protein into a new project.
ReGap DNA Project	Apply Gaps from a Protein Project to a DNA project.


GeneDoc Configure dialog
GeneDoc Configure Dialog (Project menu )

The configuration dialog controls all of the User settings of GeneDoc.  All of these settings are saved in the comment section of the .MSF file, so if the file is closed and reopened the settings are restored. Any change made in the Configuration dialog can be canceled with the cancel button. The OK button checks for any changes and redisplays the GeneDoc screen and marks the document as having been changed since the last time it was permanently stored.

Many of the setting in this property sheet can be changed through menus as well. Where applicable, the setting descriptions here will point to further, probably more complete, discussion in the menu areas of the help file.

Shortcuts

Toolbar:

Keys:	CTRL+G

Configure Pages:

Project	Set GeneDoc general project parameters.
Print	Set the printer Borders and Landscape and Portrait mode.
Shade	Set GeneDoc mode and style parameters for GeneDoc shading functions. Set GeneDoc primary, secondary and tertiary level colors.
Report	Setup Option for Reports.
Property	Controls the Grouping and Shading Colors of the Chemical Properties shade mode.
Search	Controls the list of motifs for the search shading mode.
Structure	Controls the Loading of Secondary Structure or User Defined Data Files and The Selection of the Data Set to Shade by.
PhysioChemical	PhysioChemical properties shading mode setup.
Identity	Setup Identity display mode.
Log Odds	Setup LogOdds display mode.
Score Tables	Set default scoring table used by GeneDoc scoring and shading functions. Set Group of Sequence residue characters treated as equivalent by the GeneDoc shading functions.


Display page
Display Page (Configure dialog)

Current Font size

The current font size is displayed here and can be changed by typing in a new size The Size is in Points, 1 point is equal to 1/72nd of an inch. The actual font used will always be Courier New for both printing and display.

Current Font Weight

The current font weight is displayed here and can be changed by pulling down the list and selecting a new weight. Either Normal or Bold is available.

Seq Block Sizing

This controls blocking of sequence rows in the alignment view. Screen size automatically starts a new block for the alignment on the right edge of the window. Fixed means the sequence blocks are a fixed width. The user inputs the number of sequences characters, not counting the leading names titles and trailing location indicators he wants. The minimum number of characters is 10. Alignment size means there will be no blocking, and you must scroll with the horizontal scroll bar between the beginning and end of the alignment.

Consensus Line

The Consensus line above or below the sequences lines is indicated and set here. No consensus line can also be selected, in which case no consensus sequence will be displayed.

Scoring

This menu allows the user to select GeneDoc alignment scoring. This scoring method is used in the manual scoring available to the Alignment view. You would use scoring to assist in manual arrangement. You can select Pair Wise summation (sum of pairs), Phylogenetic Tree or Numbed Phylogenetic tree, or Log Odds scoring. Phylogenetic tree scoring is biologically more reasonable than sum of pairs scoring but is much slower. Log Odds scoring places less importance on gaps than Sum of Pairs scoring.

 See Score Selection Command for further information.

Sequence Location Indicator

This On/Off switch allows to turn on or off the Sequence Location indicators on the Alignment View. When on, the indicator shows you the position in the whole sequence of the last alphabetic sequence character in the sequence line. If there are no alphabetic sequence characters in the line you will see a ‘-’.

Gap Indicator

This switch allows you to select the character to be used for a Gap Indicator. The choices are the dash,  ‘-‘ and the period or dot, ‘.’.

Residues

Select whether you would like the residues to be displayed normally, as they were found in the data file, or always in upper case, or always in lower case. This switch will not affect how the residues are saved in the data file.

Project Type

When you open a new .MSF file that does not have GeneDoc Project information in the header, the project type will be automatically set to either protein or nucleic acid depending on the information in the .MSF file. GeneDoc supports four project types.

?	Protein.

?	Mixed DNA/RNA

?	DNA Only.

?	RNA Only

In Protein versus the nucleic acid, xNA, modes, the Scoring Table choices and Chemical Property Mode defaults are altered to reflect the project type.

In the Mixed DNA/RNA mode, no consensus alteration takes place.

In the DNA Only mode, Any T or U in the calculated Consensus Line is changed to a ‘T’.

In the RNA Only mode, Any T or U in the calculated Consensus Line is changed to a ‘U’.

If the project type is not protein, then the DNA Ambiguity flag will be taken into consideration. If enabled, then DNA ambiguity codes are used. Please refer to DNA Ambiguity for more information.

DNA Ambiguity

Select whether you would like the DNA Ambiguity code to be enabled or disabled. See DNA Ambiguity for more information.

Summary Disp.

Options for the Summary View are within this area. See Summary View for more information.

Sum Cols/Inch

This option controls the Columns Per Inch of the Summary Display. It was two fields, an input field and a display only field. In the input field input the desired columns per inch and the display only  field will show the actual columns per inch for the display. The actual columns per inch might be different for the printer, and you should use Print Preview to see what the printed output will look like.

2 Col 1/1, 2 Col 0/2

This option controls the display style when GeneDoc determines that two pixels are being used for each column displayed. If 1/1, then a display style that emphisizes background color is used. If the 0/2, then the display style is the same as when 1 pixel is used per column, but twice as wide.

Default Display

This option controls whether GeneDoc should automatically open the summary view when a datafile is first loaded.

Text Black

This option controls whether GeneDoc should paint the lines of the summary view as either the defined text color or always black. It is useful when the columns per inch is at a ‘higher’ value and the numbers of pixels used for each column is 1 or 2.

Pict File Adjustments.

This area controls certain aspects of the subroutine that creates MAC style Pict files. Since Pict files are defined as being 72 dots per inch resolution, conversion must take place between the screen resolution and the Pict file resolution when the Pict file is created. These calculations cannot be exact due to truncation and round off error. These entries give the user the controls to adjust the calculations results.

Width, Range (-9 to 9), add or subtract from the original calculated width of a character space in the pict file.

Height, Range (-9 to 9), add or subtract from the original calculated height of a character space in the pict file.

Char Ascent, Range (-9 to 9), add or subtract from the original calculated location of a character inside the shaded residue cell. This adjusts vertical offset of the character, or the ascent.

Marker Enable

Enable displaying marker indicators above the residue block.

Marker Symbol

In the case of non numerical markers, what symbol to use.

Marker Start Number

What number should the first column of the project be considered. This can be a negative number.

Marker Count

How may columns to be skipped before a column location indication is displayed.

Number Every

How many symbol indicators should be printed before a numeric value is displayed.

Conserved Gap Symbol

Normally spaces (‘ ‘) are placed in the conserved consensus column where the column does not meet the minimum requirements for displaying a symbol. If you would like to see some other character instead, for example a ‘-‘, enter it here.

Max Name Length

Controls the maximum number of characters of the sequence names will be displayed. If there are sequence names longer than this field, they will be truncated on the display.

Name Seperation

This field shows the string that is used to separate the sequence names from the sequence data on the display. By default is it set to “ : “, (space)(colon)(space), but can be anything you want. Don’t make it too long.

Indicator Seperation

This field shows the string that is used to separate the sequence location indicator from the sequence data on the display, though only if the sequence location indicator is enabled. By default is it set to “ : “, (space)(colon)(space), but can be anything you want. Don’t make it too long.

Show ‘~’ as ‘ ‘

Check this to show any tilde ( ‘~’ ) characters as spaces ( ‘ ‘ ).

Show Manual Shade

Check this to show any manual shading you have done. You can uncheck it to temporarily remove any manual shading that’s been done.

Show Manual Comments

Check this to show any manual comments you have done. You can uncheck it to temporarily remove any manual comments that have been done.


Printer Configuration Page
Printer Configuration. (Configure dialog)

Print Margins

These input fields control the distance in inches GeneDoc sets the borders of a GeneDoc printout to. If the Segment Sizing is fixed and the row is wider than the page minus the margin, the margin is ignored and the row truncated at the edge of the page if needed.

Portrait or Landscape mode.

This menu switch duplicates the File/Print Setup menu. This displays and allows you to edit the printer orientation.

Header Font Settings.

This shows the current font used to print the header or footer and the page line numbers, if used.

Header Font

This button changes the header/footer and page number font attributes.

Header / Footer

These radio buttons determine if there will be a header or footer displayed on a printout.

Header / Footer String

This determines the text that will be printed as a header or footer if enabled.

Page Numbers, location and offset

This determines whether there will be a page number, where the page number will be on the page, and if there will be an offset value added to the page number. You can also select to print the current date and time as well as the projects file name on the printout.

Gene Block Position

These radio buttons control the printing of residue blocks. If the option is set to ‘Next Line’, then the next block will go on the Next Line of the printout IF a block is larger than a printed page. If the blocks are smaller than the printed page, then blocks will never be printed across pages. If the option is set to next page, then a new block will always start on a new page, regardless of whether the block is larger than a page or not. If the option is set to Next Page if Bigger, then a new block will start on a new page only if blocks are larger than the length of a page.

Adding Footers/Headers and/or Page Numbers decreases the printable area of a page.

Print Shading Only

If this option is enabled, then GeneDoc will only print out the shading component of the display. No residues or names or locations will be printed out. This can useful for creating overlapping overheads.


Shade Setup page
Shade Setup Page (Configure dialog)

Shading Mode

Conserved, Quantify, Property, Search, Structure, PhysioChemical , Identity  and  Log Odds  shading modes are indicated and set here.

Shading Level

This setting corresponds to the 2-3-4 level buttons on the toolbar.

In Conserved and Quantify Display modes. 2 level means only columns that meet the Primary Conservation Setting will be shaded, thus there is non shaded and primary shading, which is 2 levels. In 3 levels, columns that meet both the primary and secondary conservation settings are shaded, and in 4 levels, columns that meet any conservation setting will be shaded.

In Chemical Properties Display mode, the 2-3-4 levels correspond to the 1-2-3 sets or levels of in Chemical property groups.

In Search mode, the 2-3-4 levels are ignored.

In Structure mode, the 2-3-4 levels are set during runtime. Each of these levels can be set to a specific data set in the Structure/User Defined shading definition. This is done by clicking on a level indicator on the toolbar. When a level button is clicked, the configuration dialog will appear with the current shade setting, if any, and a new shading for the level and be selected.

See Autoshade Sequences for more information.

Conserved Percent

These user controlled input fields accept floating point numbers and are used by the Auto Shade functions.  The numbers are interpreted as percents.  In Conserved Mode shading they determine the minimum degree of conservation that must be observed to apply the primary, secondary, and tertiary levels of shading.  For instance, we can set the primary level is set to 100., the secondary level to 80., and the tertiary level to 60, the default levels in GeneDoc.  Then all columns in the alignment that are a single residue (100 percent) will be shaded or colored in the primary shading pattern or color.  When shading groups are active this also happens where all of the residues belong to a single shading group.  Any column in the alignment with less than 100.% conservation but more than 80.% (i.e., more than 80% of the residues are a single kind or belong to a single shading group) will be shaded in the secondary shading pattern or colors.

Residue Display Mode

Determines if Differences Display Mode is enabled.

In Difference mode a master sequence is selected.  All of the sequence residues of this master sequence are displayed.  Other sequences are represented as dots in positions where they have the same residue as the master sequence.  When a sequence differs from the master sequence the differing residue is displayed.  You may select as the master sequence either the top sequence in the alignment display or the consensus sequence.  Any sequence can be moved to the top sequence in the display with the Sequence Edit dialog.

Difference Mode Style

Shows and sets the Differences Display Mode style.

Similarity Groups Enable

This toggle the Substitution Groups Enable flag used by the GeneDoc sequence Auto Shading functions.  When Substitution Groups are enabled all members of a Substitution Group subset are treated as if they are identical sequence residues for purposes of determining the degree of conservation within columns of the alignment.

See Substitution Groups Enable for more information.

Conserved Substitution Group Display Style.

Let you choose between three different ways of shading the conservation mode display. Different styles of shading columns that conserved due to favorable substitutions are chosen. The favorable substiution groups are controlled with the Score Tables  tab and the Sub Groups tab.

In Identity mode, the default, Favorable Sub columns are shaded the same as normal Identity Columns.

In Demoted mode, a Similarity Group column is is Demoted to the next lower shading level. That is, if a column is conserved due to Sub Group to the primary level, it will be demoted to the secondary level and colored with the secondary colors. If a fSub Group column is conserved to the secondary level, then it would be shaded at the tertiary level. Tertiary level columns are only shaded if they are identical.

In the Tertiary mode, All Similarity Group columns are shaded with the Tertiary color. No tertiary level columns are shaded in any color.

Display of Current Font and Colors

Each small white square serves as a background field to show the letter G in the current font.  The letter G is shown in each of the windows for each level of shading in the currently selected color and the selected background for that level.

Fore and Back buttons

These buttons bring up the normal windows color dialog for choosing colors. Custom colors can be defined.

Note however that windows cannot set a color represented by a ‘hatch pattern’ to text foreground or background. If your windows palette is set to 256, which causes windows to hatch the majority of available colors, you will not be able to use many of the colors displayed on the common color dialog. You will have to either change your systems video mode for more colors or use only the non hatched colors offered.


Configure Reports
Reports (Configure dialog)

Control various aspects of certain reports.

Stat Report

See Statistics Report

Show Calculations For

Show calculations for any combination of

Exact Matches: The number of residues that match exactly (identical residues) between the two sequences.

Juxtapostition greater than zero: The number of residues whose juxtaposition yields a greater than zero score in the current scoring table (similar residues or conservative substitutions).

Aligned With Gaps: The number of residues lined up with a gap character

Show Results as

Absolute values:

Percent values:

Ouput Style

Ouput as matrix:

Otput as list:

example for list:

Seqname1   Seqname2   Type   Score

OG_Mg      OG_Mg     Exact    109

OG_Mg      OG_Mg     Score      0

OG_Mg      OG_Mg     Gap        0

OG_Mg      OG_Mg     %Exact   100%

OG_Mg      OG_Mg     %Score     0%

OG_Mg      OG_Mg     %Gap       0%

OG_Mg      OG_Gl     Exact     82

OG_Mg      OG_Gl     Score     82

OG_Mg      OG_Gl     Gap       72

OG_Mg      OG_Gl     %Exact    48%

OG_Mg      OG_Gl     %Score    48%

OG_Mg      OG_Gl     %Gap      42%

The list format can be used to go directly into statistics or plotting programs.

Select label location

o Labels on top and on left side

o Labels on top only

Select label style

Sequence labels in a single line

Sequence labels in two lines


Chemical Property Groups page
Property Groups (Configure dialog)

Chemical Property groups are used to shade according to the chemical properties of the residues.  This menu controls the Properties Shading Mode .

Chemical Property mode shading highlights sequence residues that share a defined set of properties.  The properties groups defined as defaults in GeneDoc are based on physical and chemical properties for proteins and chemical and size properties for nucleic acids.  Users can define their own property groups based on any criteria.  Properties mode shading has two variants.  The first, shading by conserved alignment columns, assigns a common shading pattern to all columns that are composed entirely of members of a properties group. Consider using a properties group consisting of only the acidic amino acids, Glutamic acid (E) and Aspartic acid (D).  With this group all of the columns in the alignment that consist on only E or only D or a mixture of only Ds and Es, in any proportion,  will all be highlighted with the same shading pattern or color.

In the all residues style variant of properties shading every sequence residue in the alignment is colored with the character and background color of the properties group to which it is assigned.  Since the residues assigned to each properties group are under the complete control of the user, kinds of properties other than chemical properties may be used to define the properties groups.  This mode effectively highlights islands within an alignment where a few kinds of residues are highly concentrated.  In nucleic acids poly A regions or GC islands can be readily emphasized.  In proteins low complexity regions or short repeats can be effectively emphasized.

The default properties groups for proteins are defined by the charge or polarity of the amino acid side chain.  Properties groups are defined to have a 3 level hierarchical structure.  The top level, level 1, has the most inclusive classes.  For instance, there are two groups at level 1 of the default properties groups for proteins, polar and nonpolar amino acids.  At level two these two groups are subdivided into 4 groups:  Charged, polar but not charged, large nonpolar, and small nonpolar.  Finally, at level 3, the most detailed level, there are 8 distinct chemical properties groups.

Property Groups List

Shows the current set of Property Groups for the most recently chosen Select Level. This set of groups will be reset to defaults if you click the Default button for a level.

Level 1

Brings the current Level 1 set of groupings into the Property Groups List and on the display. Is indented if the current display level is at this level.

Level 2

Brings the current Level 2 set of groupings into the Property Groups List and on the display. Is indented if the current display level is at this level.

Level 3

Brings the current Level 3 set of groupings into the Property Groups List and on the display. Is indented if the current display level is at this level.

Shade Conserved, Shade All

If shade conserved, then the column is highlighted the appropriate property groups color only if 100% of the residues in the column are present. If shade all, every residue will be colored if that residue is found in one of the property groups for the current shading level.

New

Creates a new set to hold Chemical Property groupings.

Edit

Allows you to edit the grouping with the Property Group Edit dialog.

Delete

Removes the currently selected Set of groupings.

Default

Restores the current Level set of groupings to the Programmed Defaults

The Program Property Defaults

Level 1

              polar                                              non-polar

        D,E,H,K,R,N,Q,S,T                   L,I,V,M,F,Y,W,A,G,C,P

Level 2

      polar                polar            (non-polar)                          not

     charged          uncharged    hydrophobic       small        grouped

    D,E,H,K,R       N,Q,S,T       L,I,V,M,F,Y,W      A,G           C,P

Level 3

 negatively   positively                                                                          other

 charged      charged    amide  alcohol  aliphatic  aromatic  small  sulfur

  D,E          H,K,R          N,Q      S,T      L,I,V        F,Y,W     A,G    M,C    P

For nucleic acid sequences:

Grouping 1 = default

          Purine = Large            Pyrimidine = Small

              A,G                               C,T or U

Grouping 2

          Strong Hydrogen Bonding          Weak Hydrogen Bonding

                   G,C                                         A,T or U

Grouping 3

A, C, G, TU, one in each group. When combined with the property mode style setting of Shade All, this group can be used to highlight all the difference residue types of a DNA project.  This is useful in making “islands” of different types of nucleotide patterns stand out from regions that are more random in composition.


Structure/User Defined Shading Dialog
Property Group Edit Dialog (Property Groups dialog)

This dialog allows you to edit the contents of a Property Group. All letters are converted to upper case. Only alphabetic characters are allowed.


Search Configuration Page
Search (Configure dialog)

Search mode is used to highlight prosite or rebase motifs in an alignment. Motifs can either be entered manually, or a prosite.dat or GCG style rebase file can be scanned for matches in the alignment. Each motif can have its own text and background colors. Motifs can be added, removed and edited. Since the list is searched from top to bottom, motifs positions can be moved to help work with overlapping motifs in an alignment.

The list shows two columns, the name of the motif and the motif itself. The motif name shows which motif is selected in the list, and the motif itself is shaded in the colors that will be used to shade any matches found in the alignment.

When ProSite or ReBase files are scanned, any motif in the Prosite/Rebase file found in the alignment is added to the list. The colors of the located motifs start with a red background and shift to blue backgrounds. Its expected the user will change the colors after removing any unwanted matches.

GeneDoc can currently handle either ProSite or ReBase syntax, but not both at the same time.

Edit

This edits the currently selected motif. You will be presented with the Motif Edit dialog. Here you can change the name, the motif or the description.

Delete

This deletes one or more selected motifs.

New

Here you can manually create new motifs. This button will present you with the Motif Edit dialog. Enter values in at least the name and motif input fields.

Text Color

Set the text color of the currently selected motif.

Back Color

Set the background color of the currently selected motif.

Move Up

Move the currently selected motifs up one. The motifs at the top of the list are shaded before the motifs at the bottom of the list, so they can be ‘lost underneath’ another motif.

Move Down

Move the currently selected motifs down one.

Syntax

Select the syntax of the motifs. Currently ProSite and ReBase syntax’s are supported. Before is a short description of each syntax.

ProSite Syntax as taken from the ProSite database description:

2.3.5) The PA line

   The PA  (PAttern) lines  contains the definition of a PROSITE pattern. The

   patterns are described using the following conventions:

   -  The standard IUPAC one-letter codes for the amino acids are used.

   -  The symbol `x' is used for a position where any amino acid is accepted.

   -  Ambiguities are  indicated by  listing the acceptable amino acids for a

      given position,  between square  parentheses `[  ]'. For example: [ALT]

      stands for Ala or Leu or Thr.

   -  Ambiguities are  also indicated  by listing  between a  pair  of  curly

      brackets `{  }' the  amino acids  that are  not  accepted  at  a  given

      position. For  example: {AM}  stands for  any amino acid except Ala and

      Met.

   -  Each element in a pattern is separated from its neighbor by a `-'.

   -  Repetition of  an element  of the pattern can be indicated by following

      that element  with a  numerical value  or  a  numerical  range  between

      parenthesis. Examples: x(3) corresponds to x-x-x, x(2,4) corresponds to

      x-x or x-x-x or x-x-x-x.

   -  When a  pattern is  restricted to  either the  N- or  C-terminal  of  a

      sequence, that  pattern either starts with a `<' symbol or respectively

      ends with a `>' symbol.

   -  A period ends the pattern.

   Examples:

   PA   [AC]-x-V-x(4)-{ED}.

   This pattern  is translated  as: [Ala or Cys]-any-Val-any-any-any-any-{any

   but Glu or Asp}

   PA   <A-x-[ST](2)-x(0,1)-V.

   This pattern,  which must  be in  the N-terminal of the sequence (`<'), is

   translated as: Ala-any-[Ser or Thr]-[Ser or Thr]-(any or none)-Val

ReBase Syntax:

Symbol   Meaning

          ------   -------

            A       Adenine

            G       Guanine

            C       Cytosine

            T       Thymine

            U       Uracil

            Y       pYrimidine  (C or T or U)

            R       puRine      (A or G)

            W       "Weak"      (A or T or U)

            S       "Strong"    (C or G)

            K       "Keto"      (T or U or G)

            M       aMino       (C or A)

            B       not A       (C or G or T or U)

            D       not C       (A or G or T or U)

            H       not G       (A or C or T or U)

            V       not T       (A or C or G)

            N       unknown     (A or C or G or T or U)

            X       unknown     (A or C or G or T or U)

CheckFile: Prosite

This button will bring up a File Open dialog. Here you should choose a prosite.dat file. When you select the file, GeneDoc will scan the prosite file for any patterns that are found in your alignment and add any matching motifs and their names and descriptions.

CheckFile: ReBase

This button will bring up a File Open dialog. Here you should choose a GCG style ReBase file. When you select the file, GeneDoc will scan the file for any patterns that are found in your alignment and add any matching motifs and their names. See Export List below for addition information on rebase files. When this option is selected, you will be presented with a enzyme filter dialog. This will allow you to some simple descrimination on the characteristics of the enzymes.

Export List

This button will bring up a File Save dialog. Here you can save the current list of patterns that have been defined. The file will be saved in a format similar to ReBase format, so you should use the Rebase file to reload this saved list. Also, the current color information of the list will be saved to this file, and will be read back in when you load this file using the ReBase Checkfile option.

Overlap

This button will show you a color selection dialog. The color, green by default, selected here will be used in Rebase mode to color areas where the patterns found overlap each other.


Enzyme Filter Dialog
Enzyme Filter Dialog (Configure dialog)

This dialog lets you filter on the following characteristics of the enzyme patterns found in the rebase file. None of these entries need to contain any value.

Min Length: Set a minimum length of the enzyme pattern.

Max Length: Set a maximum length of the enzyme pattern.

Exclude Set: Input a set of characters that if found in the pattern will have the pattern rejected.

Include Set: Input a set of characters that must be in the pattern for it to be used.

Max Occurances/Seq: Input a maximum number of occurances that a pattern can be found in a sequence.

Exclude Ambiguous: Check this if you want to exclude enzymes with ambiguous characters.

Palindromic Only: Check this if you want only palindromic enzymes.

Exclude Duplicates: Check this is you want to exclude subsequent occurances of duplicate enzyme patterns.


Search Edit Dialog
Search Motif Edit Dialog (Configure dialog)

This dialog allows you to edit the contents of a Search Motif.


Shade Groups page
Structure (or User Defined Shading) Page (Configure dialog)

Select Master Sequence

This pulldown list is for setting the master sequence that the structure shading uses to align the shading colors. In any Structure or User Defined data file, residue data must be included. The residue data in that file will be automatically matched to the selected master sequence in order to determine where the shading values are to be applied.

There will be no Shading Data Sets if none have been loaded in the Project/Load Structure Definition menu.. However Data Sets will be loaded automatically when you Load a predefined data file, otherwise they need to be loaded from the Project/Load Structure Definition menu.

See Secondary Structure Shading for more information

Load Data File.

A master sequence must be selected before this button will function.

This buttons opens a File Open dialog box. The file selected from this menu will be passed to the Secondary Structure/User Defined shading data read routines. The extension of the file determines the file format.

After being read, the Residue Information found in the file will be attempted to be correlated with the residues of the selected master sequence.  If the master sequence and the residue information found in the Data File do not match, you will see little or no shading on the display.

See Supported Structure Files for more information.

See User Data Files for more information.

Data Set Details

This button will show all of the defined colors and their labels for the Structure Data selected in the list below. Also, double clicking on the list will do the same thing.

Secondary Structure/User Defined Shading Data List

This list will show all of the currently defined Data Choices, and whether or not any data has been read in for that choice. This list is used to select which data will be used to shade the GeneDoc display. The list will show what is currently selected. If Data is not available for the definition, the string ** No Data ** will be appended to the definition description.


PhysioChemical Properties Setup
PhysioChemical Properties Page (Configure dialog)

See PhysioChemical Properties Shade Mode for more information.

See Group PhysioChemical Properties for more information.

Chemical Properties Colors

Shows the list of possible chemical properties and the colors used to indicated presence of that property in the alignment. Selection can be made with a left click with the mouse over a property, and is indicated by a black area to the left of the name. Double clicking a property will toggle the Displayed attribute described below.

Displayed

Determines whether or not the physiochemical property is displayed. This does not disable detection of the property, its just a shortcut, saving you from having to set the colors to the same as the background color in case you don’t want to see the property.

Text Color

Shows and sets the text color for the currently selected physiochemical properties group.

Back Color

Shows and sets the background color for the currently selected physiochemical properties group.

Description

Shows the residues for the currently selection physiochemical property.

Cysteine

Set the expected Cysteine type to either Oxidized or Reduced.

See Group PhysioChemical Properties for more information.

Coloring

Indicates which shading mode the Chemical Properties shading mode uses. The Properties setting will use the colors in the Chemical Properties Colors list. The Conserved setting will use the colors in the Prop. Cons. Colors list below.

Property Conserved Colors

Shows the list of possible conservation levels and the colors for that level. Select an entry in the list with a left mouse button click.

Text Color

Shows and sets the text color for the currently selected chemical conservation levels.

Back Color

Shows and sets the background color for the currently selected chemical conservation levels.


Identity Page
Identity Mode Page (Configure dialog)

See Identities Display Mode for more information.

Sequences List

Shows the list of sequences in the alignment and which sequences are chosen to compare for identities. The chosen sequences will be highlighted in the Identity Residue Colors. You can change the selection of a sequence by double clicking on the sequence name, or by selecting it and using the sel/deselect button.

Sel/Deselect button

This button creates a new Substitution Group subset and gives you the Edit Groups dialog to enter the characters belonging to the new subset.

Text Color

Select the text color to be displayed for the residues selected as the identity set.

Back Color

Select the back color to be displayed for the residues selected as the identity set.

Show All Identities

When this style is selected, then any identity/similarity found in the other sequences in the column will be shaded with the primary conservation color.

Show Conserved Identities

When this style is selected, then the conserved level of the identities/similarities in the other sequences in the column is determined and shaded in the appropriate color. Any column that does not meet at least the level of conservation selected will not be shaded.


LogOdds Page
Log Odds page (Configure dialog)

This mode is used to read MEME style LogOdds motifs and apply various methods of shading to the alignment with these motifs. This configuration dialog is used by first selecting the MEME File button and loading a Meme file into GeneDoc. GeneDoc will only load 1 meme file at a time. Loading a new file erases the old motifs from GeneDoc.

Please see LoggOdds display mode for more information

The list shows two columns, the name of the logodds motif and the motif best consensus if available. The motif name shows which motif is selected in the list, and the motif itself is shaded in the colors that will be used to shade any matches found in the alignment.

When MEME files are scanned, any motif in the MEME file is added to the list. The colors of the located motifs start with a red background and shift to blue backgrounds. Its expected the user will change the colors after removing any unwanted matches. The motifs are Names Motif 1 though Motif N, and the names are not changeable.

Meme File

This button prompts you to load a MEME file for use in this display mode. The file must be in the format specified by the MEME program.

Edit

This edits the currently selected motif. You will be presented with the LogOdds Motif Edit dialog. Here you can change the motif or the description.

Delete

This deletes one or more selected motifs.

Text Color

Set the text color of the currently selected motif.

Back Color

Set the background color of the currently selected motif.

Overlap

This button will show you a color selection dialog. The color, green by default, selected here will be used in Rebase mode to color areas where the patterns found overlap each other.

Enabled

This check box determines whether the selected motifs are enabled or not. Disabled motifs will function the same as deleted motifs.

No Overlap

This check box determines whether motif shading will be allowed to overlap or not. If the box is no checked, ie allow overlap, no special processing is done, however, any shading that is applied where shading lready exists will be shaded as the overlap color. If the box is checked, then if ttwo areas of shading will overlap for a given motif, then the are with the highest score will be shaded. No attempt is made to disable overlaps across multiple motifs.

Display Mode

This area determines how the LogOdds motifs are displayed on the alignment. There are 4 different display modes.

Bayes

This settings uses the bayes value for the motif to determine shading. If the score for a region is equal to or higher than the bayes number for the motif, then the region is shaded. The bayes number is usually found in the MEME file, but can be input or changed by the user.

Best N

This settings shades regions with the best N scores. The number of regions to be shaded is determined by the ‘Best N’ input area.

Better Than

This settings shades regions with better than an input score. The score is input into the ‘Better Than’ input field.

Tri Level

This settings shades in three different colors. The three different colors are the Primary, Seconday and Tertiary level shading found in the Shade Tab of the Configuration Dialog. The levels are determined as a percentage of the best possible score for a given motif. The percentage values are input into the ‘Tri Level’ area input fields.

Best N Input

Input the number of regions to be shaded when in Best N display mode.

Better Than Input

Input the score to be compared with for shading in the Better Than display mode.

Tri Level Input

Input the Primary, Secondary and Tertiary shading levels as percentages. These values are used in the Tri Level display mode.

Output Settings

This section contains LogOdds Output settings. These settings control parameters used when writting logodds maxtrixs reports. These parameters can be changed in the LogOdds Output Dialog which is shown after you select the function write logodds matix.

BackGround

Determines where to obtain the background counts for the LogOdds matrix. If Static, then preprogrammed values obtained from a GeneBank search are be used. If Align, then the entire alignment is used to determine background frequencies.

Pseudo Table

Select a table to be used as the Pseudo Table.

Pseudo Multiplier

Enter a Pseudo Table multiplier.

A,C,G,T

Enter Background counts for MIXED/DNA/RNA projects.


LogOdds Edit Dialog
LogOdds Edit Dialog (Log Odds page  dialog)

This dialog allows you to edit the contents of a LogOdds motif.


Score Tables page
Scoring Tables page (Configure dialog)

Score Tables List

Shows the list of available scoring tables. The current table is shown as the selected row. Double clicking on a row will only show the current scoring tables information. You MUST use Set Current in order to change the current scoring table.

Current Table

This shows the name of the current scoring table.

Set Current

This selects the highlighted scoring table in the Score Table list to be the current scoring table used by the GeneDoc Sequence Shading functions.

Similarity Groups List

Shows the current set of Similarity Groups. This set of groups will be reset to defaults if you change the current scoring table in Scoring Table Page of this Configuration dialog. Double clicking on a list entry will give you the Group Edit dialog.

New

This button creates a new Similarity Group subset and gives you the Edit Groups dialog to enter the characters belonging to the new subset.

Delete

This button will delete the selected Similarity Group subset.

Edit

This button will give you the Edit Group dialog for the selected Similarity Group subset. The dialog allows you to enter up to 16 characters as members of a single subset.

Restore Defaults

This button will set the Similarity Group to the default subsets as determined by the current scoring table. Each table carries with it a list of default Similarity Groups.  All members of each default subset have positive similarity scores with all other members of the same subset.  Where it was necessary to choose between to possible but mutually incompatible members of a subset the potential member with the highest scores was chosen.

See Also

GeneDoc Display  and Score Selection.


Shade Group Edit Dialog
Similarity Group Edit Dialog ( Score table Page  )

This dialog allows you to edit the contents of a Similarity Group. All letters are converted to upper case. Only alphabetic characters are allowed.


GeneDoc Sequence Edit dialog
Edit Sequences (Project menu )

Brings up the Edit Sequences dialog. This dialog allows you to Add sequences, Delete sequences and Move (change the order of ) sequences. The new order of sequences, and any new sequences added to the alignment are saved in the .MSF file.  Deleted sequences are not saved in the .MSF file.  New sequences added to the alignment are read from FASTA formatted files with the file name extension .AA, (e.g., newseq.aa).

Shortcuts

Toolbar:

Keys:	CTRL+Q


GeneDoc Sequence Edit dialog
GeneDoc Edit Sequence Dialog (Project menu )

This dialog allows you to Add sequences, Delete sequences and Move (change the order of ) sequences. The new order of sequences, and any new sequences added to the alignment are saved in the .MSF file.  Deleted sequences are removed from the .MSF file if it is saved. . New sequences added to the alignment are imported from either FASTA formatted files, with the file name extension .AA, (e.g., newseq.aa), or from other .MSF files

Note, GeneDoc does NOT like multiple sequences with the same name. At this time, no error is given, except when the a file is read with sequences of the same name. Then the error you will get is ‘More residues than indicated by Len field’, because GeneDoc will be reading the two sequences into one sequence, and the result will be an oversized sequence.

Shortcuts

Toolbar:

Keys:	CTRL+Q

Current Sequences List

This displays the list of sequences currently in the .MSF file and their location in the alignment. Double clicking on a row will give you the Sequence Details dialog.

Done

Click here when finished with this dialog. Any changes can NOT be canceled.  When a sequence is deleted the deletion cannot be undone or canceled.  Thus it is a good idea to save the project before entering the Edit Sequence Dialog so that you can easily recover from a mistaken deletion.

Move Up

This will move the selected sequences up one line in the alignment, and will keep the sequences as the current selection. Thus, repeated mouse clicks will move the sequences up one line in the alignment for each mouse click.

Multiple lines or blocks of sequences can be selected and moved as well. If the multiply selected sequences are not contiguous, then sequences will be compacted as they move.

Move Down

This will move the selected sequences down one row, and will keep the sequences as the current selection.  Thus, repeated mouse clicks will move the sequence down one line in the alignment for each mouse click.

Multiple lines or blocks of sequences can be selected and moved as well. If the multiply selected sequences are not contiguous, then sequences will be compacted as they move.

Move Top

This function will move all of the selected sequences to the top of the list.

Move Bottom

This function will move all of the selected sequences to the bottom of the list.

Delete

This will prompt you for confirmation and then delete the selected sequences. The sequence will be left out of the .MSF file when the .MSF file is next saved.

Multiple selected sequences can be deleted all at once.

Details

This is allow you to edit the sequence details. The sequence details consist of the name, the weight, the starting residue number and a description of the sequence. The weight of the sequence is used in the LogOdds Matrix Output. The starting number is used on the alignment view display.

Compliment

This function does a DNA compliment of the selected sequences. DNA Compliment is defined as:

C -> G

G -> C

A -> T/U

T/U -> A

M -> K

R -> Y

W -> W

S -> S

Y -> R

K -> M

B -> V

D -> H

H -> D

V -> B

N -> N

Reverse

This function will reverse the selected sequences.

Import

This button presents you with the File Import Type dialog.

Any sequences found in the file will be added at the bottom of the current alignment.  The first new sequence in the file will be the selected sequence.  All sequences will be converted to the same length by padding the shorter sequences with gap characters.  The import function allows you to correct wrong sequence residues when combined with the delete sequence function.  It also allows you to create a common display of alignments of different sequence families.

Export

This button gives you the File Export dialog

RasMol Script.

This button creates a RasMol residue shading script for the selected sequence. The user will be presented with the RasMol Script dialog, where you can select various options and then create a RasMol coloring script..


RasMol Script Dialog
RasMol Script Dialog. (Sequence Dialog)

This dialog controls options for the RasMol coloring script routine. When you press OK, you will be presented with a File Save Dialog, asking you to save a name.scr file. This file can then be read into RasMol from the command line, and will color the protein with the colors from GeneDoc. You can also choose to show SideChains for any selection of colors.

The various options can be used to allow you to apply multiple shading/side chain scripts to a rasmol display, obtaining fairly complicated pictures. For example, I’ve shaded the backbone with structure coloring (helix, sheet, turn), shown conserved sidechains as sticks and colored them according to their conservation level, and then sidechain display option with a prosite search to display the ‘active site’ in space fill form.

To use a GeneDoc coloring script in RasMol, first open rasmol and open your protein of interest. Then, in the command window, type “script name.scr.” This will read in the script and apply the changes.

Coloring Script Options:

Clear All.

Leave this checked to include a script command to clear all previous rasmol coloring.

Safe Mode.

Leave this checked to include both the amino type and location information for the coloring script. If you uncheck the box, only the location information is written to the script file. You should have a good reason for doing this. The type and location serve as a double check to safeguard against coloring the incorrect amino acid.

Include Base Colors

This option controls whether or not to include coloring information for the base color as well as the non base colors (primary, secondary, tertiary, etc) in the coloring script. You could uncheck this option if you wanted to add GeneDoc colors to an existing protein coloring in RasMol.

PDB Offset.

By default, the GeneDoc script routine starts numbering the amino’s at location number 1. Enter here an offset to compensate for differences between the sequence data and the PDB file location

All Atoms.

This option creates coloring commands for all atoms in an amino, ie both the side chain atoms and the backbone atoms.

Backbone Only

This option creates coloring commands for only the backbone atoms in an amino. You can use this option and disable the clear all to color the backbone atoms in one scheme and the sidechain atoms in another.

Sidechain Only

This option creates coloring commands for only the sidechain atoms in an amino. You can use this option and disable the clear all to color the sidechain atoms in one scheme and the backbone atoms in another.

Show Side Chains.

Click the Box to have side chains displayed for selected GeneDoc colors.

Show Backbone As.

Select the style you would like the backbone displayed in.

Exclude Carbonyl Oxygens.

Check this to exclude the showing of Carbonyl Oxygens from the coloring script.

Show SideChains As.

Select the style you would like the side chains to be displayed in.

Select SideChains.

Select the colors you would like to see sidechains for. You can select more than one color.


Sequence Input Dialog

Name:

Input the new Sequence Name here.

Descr:

Input a sequence description here.

Paste:

Use this button to input sequence data from the clipboard.

Input Area

This is a simple text editor style input area. Standard editing functions apply. You can input multiple lines, anything found in this area will be treated as sequence data if it is a character that falls in the range of ‘A’ (or ‘a’) to ‘Z’ (or ‘z’) or the characters ‘-‘ and ‘.’.


Sequence Details dialog
Sequences Details Dialog (Sequence Edit dialog)

This dialog allows you to edit the details of an individual sequence. Currently you can edit the name or the weight of a sequence. The only effect changing these values has is in the stored .MSF file.

Name

An editable field used to change the name of the sequence.  The new name completely replaces the old name both in the current display and in any subsequently saved document or file.

Weight

This information is saved in a .MSF file, but otherwise has no effect on the project. The weight is used in the LogOdds Matrix Output Report

Description

An editable field used for a description of the sequence.


Titling Facility Menu
The Titling Facility Menu. (Project menu )

Brings up the Titling Facility Dialog.


Titling Facility Dialog
The Titling Facility Dialog. (Titling Facility Menu)

When a project is saved, four basic attributes are checked to see if you have defined them yet. If not, then the Titling Facility Dialog is presented, and the attributes that need defining are found in the bottom of the dialog box. These are:

What Program did you use to create the Initial Alignment?

What Scoring Matrix did you use to create the Initial Alignment?

What Open Gap penalty did you use to create the Initial Alignment?

What Extend Gap penalty did you use to create the Initial Alignment?

Putting any values in these box will satisfy the Titling Facility Test and the Titling Facility Dialog will not be presented when saving the file.

The Titling Facility Dialog box can be called from the GeneDoc/Project/Titling Facility menu.

The Titling Facility will also allow you to enter free form text comments in the comment area of the project .msf file.


Save User Defaults
Save as User Defaults (Project menu )

Saves the current User Setting from the Configuration Dialog into GeneDoc’s .INI file. This means that anytime you open a .msf project file without any GeneDoc settings in it, the defaults you have saved will be used. There is a set of defaults built into the program which are overridden by the saved user defined defaults stored in the genedoc.ini file. Should you need to revert to these, edit the genedoc.ini file found in your windows directory and remove the [User Defaults] section and all the values in it.


Load User Defaults
Load User Defaults  (Project menu )

Load the set of previously saved User Settings into the Configuration Dialog and changes the current projects settings. Good for changing an old set of GeneDoc settings to your new one and insuring that two different projects are formatted and highlighted consistently.


Load Structure Definitions Menu
Load Structure Definitions Menu (Project menu )

This menu allows you to load the predefined EMBL and PSDB structure definitions, load a User Defined structure/shading definition, or to clear all loaded definitions.

See Structure Shading for more Details.

EMBL	Load Definitions for some of the Secondary Structure Prediction data sets and the DSSP Secondary Structure Data set found at the European Molecular Biology Labs.
PSDB	Load definitions for Pittsburgh Supercomputing Center Secondary Structure data.
PDB	Load definitions for Brookhaven PDB protein coordinate files.
Load User Definition	Load a User Defined Secondary Structure or Shading definition.
Clear all Definitions	Clear all Structure/User Defined Definitions.


Embl Definition
EMBL Definition (Load Structure Definitions menu )

Load the Predefined EMBL Definitions. This will be automatically loaded if any EMBL Data file is read in with the Load Data File in the Structure Configuration dialog.

The EMBL Definition defines the following data sets:

Prot Pred - Structure Prediction

Prot Pred - Structure Reliability

Prot Pred - Probability Helix

Prot Pred - Probability Sheet

Prot Pred - Probability Loop

Prot Pred - Sub Structure

Prot Pred - Solvent Accessibility in 3 states

Prot Pred - Solvent Accessibility

Prot Pred - Solvent Reliability

Prot Pred - Sub Solvent Accessibility

Prot Pred - Helical Transmembrane Region

Prot Pred - Helical Trans. Reliability

Prot Pred - Helical Trans. Probability

Prot Pred - Loop Probability

Prot Pred - Sub Helical Trans.

SS Pred - Prediction

SS Pred - Clean Prediction

Predator - Prediction

Secondary Structure -- DSSP


PSDB Definition
PSDB Definition (Load Structure Definitions menu )

Load the Predefined PSDB Secondary Structure Definitions. This will be automatically loaded if any PSDB Data file is read in with the Load Data File in the Structure Configuration dialog. The EMBL DSSP Definition is included in this set as well for convenience sake.

The PSDB Definition defines the following data sets:

Chirality

Amide

Secondary Structure -- Garnier Classification

Secondary Structure -- Thornton Classification

Secondary Structure -- Scheraga Classification

Secondary Structure -- Deerfield Classification

Secondary Structure -- Deerfield4 Classification

Secondary Structure -- DSSP

Surface Area

Surface Area

Sidechain Pct

Buried Area

PFRAC

Surface Group

AMH Classification

Radial

Interchain:Surface Area

Interchain:Surface Area

Interchain:Sidechain Pct

Interchain:Buried Area

Interchain:PFRAC

Interchain:Surface Group

Interchain:AMH Classification

Ligands:Surface Area

Ligands:Surface Area

Ligands:Sidechain Pct

Ligands:Buried Area

Ligands:PFRAC

Ligands:Surface Group

Ligands:AMH Classification

Ions:Surface Area

Ions:Surface Area

Ions: Sidechain Pct

Ions:Buried Area

Ions:PFRAC

Ions:Surface Group

Ions:AMH Classification

Waters:Surface Area

Waters:Surface Area

Waters: Sidechain Pct

Waters:Buried Area

Waters:PFRAC

Waters:Surface Group

Waters:AMH Classification


PDB Definition

PDB Definition (Load Structure Definitions menu )

Load the Predefined PDB Secondary Structure Definitions. This will be automatically loaded if any PDB Data file is read in with the Load Data File in the Structure Configuration dialog.

The PDB Definition defines the following data sets:

Secondary Structure -- PDB


User Definition
Load User Definition (Load Structure Definitions menu )

This will open a file open dialog box allowing you to read and load a User Defined Secondary Structure or Shading definition.

There are example EMBL and PSDB structure definitions in the GeneDoc directory. These can be modified to change the Built In Definitions color settings or labels, or added to.

See User Defined Shading Definitions for details on the file format.


Clear Definitions
Clear Definitions (Load Structure Definitions menu )

This entry will clear all of the currently defined structure shading definitions.


Create New Window command
Copy to New Project (Project menu )

This function will copy the range of Selected Columns into a new GeneDoc document. This new document is fully functional and can be saved as a new .MSF file.

Shortcuts

Keys:	CTRL+W


Replace From Other Project
Copy From Other Project (Project menu )

This function will copy another projects data into a range of Selected Columns, replacing the data that is currently in the selected range. First select a range of columns, then this menu function. You will then be shown a list of other Projects, choose one and the data from the project will replace the currently selected data where the sequence names are the same.

This is to allow you to copy a part of a project to another project, edit and manipulate that part, and then replace it back into the original project.

Shortcuts

Keys:	CTRL+W


Replace From Other Project
Translate DNA to Protein (Project menu )

This function will translate a DNA project to a Protein project. You will be able to select from several predefined translation tables. These tables may also be modified. You will be able to select either a fixed frame or floating frame translation algorithm, each of which has options to look for a start codon before translating. In fixed frame mode, you can select a 0, 1, or 2 nucleotide frame shift for the translation. Selecting this option brings up the DNA Translation Dialog .

You can select a range of columns to be translated. If no selection has been made, then all columns are translated.

The general rules for translation are as follows:

For fixed frame translation:

If all three of the columns contain a gap character, then a gap character will be put in the corresponding position in the Protein translation, otherwise a translation will be attempted. If there are any non-nucleic characters or if what is there is not translatable then a ‘*’ will be put into the Protein project at that location.

For floating frame translation:

Characters are gathered into either the gap bucket or the translation bucket. When either bucket hold three characters, then a character is put into the Protein project. For three gaps in the gap bucket, then a gap character is put into the Protein project. For three non-gap characters in the translation bucket, a translation is attempted. If there are any non-nucleic characters or if what is there is not translatable then a ‘*’ will be put into the Protein project at that location. When a character is put into the Protein project, then the proper bucket is cleared.

Searching for a Start Codon is done in either case by ignoring non-nucleic characters and shifting a nucleic code out the left and in the right of a of a set of three until a start codon is found. After that, translation continues depending on the options selected in the DNA Translation Dialog.


DNA Translation Dialog
DNA Translation Dialog (Project menu )

This dialog controls the DNA to Protein translation options.

Translation Table.

Select which translation table you want to use, or enter your own description. Your own description will be saved, but will be lost if you use the pull down list and choose a default list entry.

Codon List

This list shows all the codons and their resulting translations. It also shows start codons where the right most column is not a ‘-‘. This list can be modified using the alter codon table area.

Translation Options

This area shows the possible options for DNA translation.

Translate Fixed Frames:

  This options makes GeneDoc translate every three columns regardless of what is in the columns. Translation can be done for all columns, or after a Start Codon. If all frames option is selected, then you can choose a frame shift of 0, 1, or 2.

Translate Float Frames:

  This option enables floating frame translation. Characters are gathered into either the gap bucket or the translation bucket. When either bucket hold three characters, then a character is put into the Protein project. For three gaps in the gap bucket, then a gap character is put into the Protein project. For three non-gap characters in the translation bucket, a translation is attempted. If there are any non-nucleic characters or if what is there is not translatable then a ‘*’ will be put into the Protein project at that location. When a character is put into the Protein project, then the proper bucket is cleared.

Searching for a Start Codon is done in either case by ignoring non-nucleic characters and shifting a nucleic code out the left and in the right of a of a set of three until a start codon is found. After that, translation continues depending on the options selected in the DNA Translation Dialog.

Alter Codon Table

This area allows you to alter the translation table. When you have selected a codon in the codon list, then the alter codon table are will be updated. You can change what the resulting amino acid is or if the codon represents a start codon.

After all the options are set, select either Translate or Cancel. Cancel does not undo any changes you made, it simply aborts the translation.


ReGap DNA
ReGap DNA Project (Project menu )

This function is used to apply the gaps from a Protein project to a DNA project. You must have a DNA project loaded into GeneDoc that contains sequences with the same names as sequences in a Protein project. GeneDoc will then remove all the gaps from the DNA sequence, then put gaps back in, putting three gaps in the DNA sequence where there is a gap in the Protein sequence. GeneDoc also will keep synchronized by checking the translation of the current three DNA nucleotides against the current Protein amino acid. If the translation fails, you will get an error message describing what location in each sequence the translation error occurred in.

In order to use this function, you should have both projects loaded into GeneDoc, with the Protein project active, or at the front. You will be shown a ReGap Select Other Project Dialog, from which you should choose the corresponding DNA project.


Select Other Project
Select Other Project Dialog (ReGap DNA Project )

Select the DNA project that corresponds to the Active Protein Project. The DNA project must have Sequences with the same names and be a proper translation with sequences in the Protein Project.


Edit Menu
Edit Menu (Help Index )

The Edit Menu contains a windows programs usual edit/copy functions as well as some GeneDoc specific functions that edit or change the project.

The Find and the Replace functions are described in more detail below.

Use select blocks to copy sections of a GeneDoc display to extern programs with format types such as Bitmap, Metafile, Pict, RTF, HTML, Text.

The functions under select columns can make significant changes to a project. Sections of the alignment can be copied to another project to be edited and copied back to the original project or saved separately.

For more information, select the Project menu item name below.

Pairwise Alignment	Open the Sequence Alignment dialog and align sequences with the Pairwise Alignment algorithm.
Find	Pattern matching function that moves the cursor or highlights strings.
Find Next	Continuation of the previous Find function.
Replace	Simple Replace function that replaces string of the same length within the alignment.
Select Blocks	Select Sequence Blocks to be copied to external programs.
Copy Selected Blocks To	Copy selected blocks for export to external programs.
Copy Alignment as Text	Copies the alignment as Text to Clipboard.
Copy Consensus as Fasta	Copies the consensus data as a fasta format file to the clipboard.
Copy Consensus as Prosite	Copies the consensus data as a Prosite format file to the clipboard.
Select Columns	Select a range of columns for editing with below functions.
Delete All Data	Delete all data in the selected range.
Copy Data Between Seqs	Copy data from one sequence to another sequence within the selected range.
Residue Edit Mode	GeneDoc Residue Edit mode allows you to change the value of residues within the current project.
Clear Gap Columns	Clears columns that have only gaps in the residues
Clear Man Comments	Clear all manually entered comments


Pairwise Alignment
Pairwise Alignment (Edit menu )

The Pairwise Alignment function. Selecting this function will give you the Align Sequences Dialog. This function is used to align all or some of the sequences to themselves or other sequences. The implementation of the algorithm has not been optimized for speed. The intented use with GeneDoc is to be able to add unaligned sequences to an alignment and align them first to themselves (if more than one) and then align to the rest of the alignment.


Align Sequences Dialog
Align Sequences Dialog (Pairwise Alignment)

The Dialog for the Pairwise Alignment function. You can align selected sequences either to themselves or to other unselected sequences. The current score table  is used by the alignment function. The current score table as determines several default values as noted below.

Align to Unselected.

This option will align the sequences that are currently selected in the sequence list to be aligned to the sequences that are unselected. You should make sure that both sets are aligned unto themselves for the best results.

Realign Selected.

This option aligns the sequences that are currently selected in the sequence list to be realigned as a set independent of the rest of the alignment. It is likely that you would want to have the ‘Degap Sequences’ option selected if you do this. Your likely to be doing this because your going to align the selected set to the rest of the alignment next.

DeGap Sequences.

This option will move all gaps found in each of the selected sequences to the end of the row, so that all the residues come first followed by a length of gaps. This helps improve the result of the pairwise alignment function when doing ‘realign selected’

Score Table.

Displays the score table currently selected in the score table dialog

Const Cost Length.

Value passed to the constant cost length parameter of the alignment function.

Gap Open Cost.

Value passed to the gap open cost parameter of the alignment function.

Gap Extend Cost.

Value passed to the gap extend cost parameter of the alignment function.


Find
Find (Edit menu )

The find function. Selecting this function will give you the Find Dialog. This function is used to do quick location of strings within selected sequences within the alignment. You can either have the cursor moved to the found location, or have all the found locations temporarily shaded.

The find function uses the simple syntax of a string, with options for mismatches and insertions/deletions included. GeneDoc provides additional Search functions using ReBase and ProSite files.

Please see the Find Dialog for information on the Find Function options.


Find Dialog
Find Dialog (Find Function)

You can select a set of sequences to perform the search on with the sequence list on the left of the Find Function dialog. By default, the entire set of sequences is selected, or if you have already selected a subset of sequences, then that set will be remembered.

The find function uses the simple syntax of a string, with options for mismatches and insertions/deletions included. Gaps are ignored and not shaded by the find function. Found strings can overlap each other.

You can select to find from the current cursor location either forward or backward. You can start a search from the beginning of the alignment as well. The cursor will be set at the first character of what is considered to be the found string.

The Shade all option allows you to color found strings in a selected color. Use the Text and Back buttons to set the color of the text and background respectively. Gaps are not colored and found strings can overlap.

The mismatch option allows parts of the search string to different from the string found. If you enter one mismatch, then the search string will match any string that is the same as the search string except for one character. The mismatch can be in any position of the search string. If you enter two for mismatches, then any two locations in the found string can be different from the search string, and so on.

Insertions and deletions allows for varying lengths between the search string and the found string. If you enter one for insertions and deletions, then the found string can be the same length, one character shorter or one character longer than the found string. In the case the found string is one character longer, then it will match the search string except for the insertion (or appendage) of an extra character. In the case of the found string being one character shorter, then the found string will be missing one of the characters in the search string, at any position.

A Missing (deletion) character is not the same as a mismatch. You can enter values for both mismatches and insertions/deletions for the find function.

The Find Function is not case sensitive.


Find Next
Find Next (Edit menu )

The find next function repeats the previous find function, moving the cursor to the beginning of the next found string.

Please see the Find Dialog for information on the Find Function options.


Substitute String
Replace (Edit menu )

This function will prompt you for a search string and a replace string. The strings will be checked to insure that they are the same length. Then the string matching the search string in the Selected Range will be replaced with the replace string.


Substitute String Dialog
Replace Dialog (Edit menu )

Enter the string to find and the string to replace here. The string must be the same number of characters.


Select Rows Command
Select Blocks for Copy (Edit menu )

Use this command to select sequences by rows. Selected rows can be used for the Copy or Copy Inverted commands.

Copying data to the clipboard replaces the contents previously stored there.

Shortcuts

Toolbar:

Keys:	CTRL+E


Copy menu
Copy Menu (Edit menu )

The Copy menu has items that copy Genedoc displays to the clipboard or to files. Included here is the ability to copy to Pict Files, Meta Files, Bitmaps and Text. Further discussion on using these functions to publish GeneDoc displays can be found in Getting Useful GeneDoc Output..

For more information, select the copy menu item name below.

Copy to Metafile	Copies selected Blocks to the Clipboard as a Windows style Enhanced MetaFile.
Copy to Metafile File	Copies selected Blocks to a file as a Windows style Enhanced MetaFile (*.emf) file.
Copy to Pictfile	Copies selected Blocks as a MAC style Pict (*.pct) file.
Copy to HTML file	Copies selected block as HTML file. Gives options.
Copy to RTF file	Copies selected blocks as a RTF file.
Copy Selection as Bitmap	Copies selection as bitmap.
Copy Selection Inverted	Copies data from the document to the clipboard inverted.


Copy MetaFile
Copy Selection to MetaFile (Copy menu )

Use this command to copy selected data to the clipboard in the Windows Enhanced MetaFile format. This command is unavailable if there is no data currently selected.

Once copied to the clipboard, the copied GeneDoc display can then be pasted into other programs. Typically, the Metafile paste will not have the same dimensions as the original GeneDoc selection, but in most cases the pasted display can be resized to the desirable size.

Using a Meta file for copying GeneDoc displays to other programs is preferable to a bitmap because it will print at normal printer quality.

This function is not available in the 16 bit version of GeneDoc.

Shortcuts

Toolbar:

Keys:	CTRL+C


Copy Pict File
Copy as MetaFile File (Copy menu )

Use this command to copy selected data to the clipboard in the Windows Enhanced MetaFile format. This command is unavailable if there is no data currently selected.

Once copied to a file, the Meta file can be read into some other programs on Windows or MAC platforms.

Using a Meta file for copying GeneDoc displays to other programs is preferable to a bitmap because it will print at normal printer quality.

This function is not available in the 16 bit version of GeneDoc.


Copy Pict File
Copy to Pict File (Copy menu )

Use this command to copy selected data into a MAC Style Pict File. This command is unavailable if there is no data currently selected.

You will be prompted for a filename to save the .pct file as, with the current project name as the default. This Pict file can then be read into most Windows publishing programs and most MAC publishing programs as well. Using a Pict file for copying GeneDoc displays to other programs is preferable to a bitmap because it will print at normal printer quality.

See also Pict File Adjustments for additional information.


Copy Html File
Copy to HTML File (Copy menu )

Use this command to copy selected data into a HTML Style File. This command is unavailable if there is no data currently selected.

You will first be prompted with the HTML Output dialog. Here you can choose between HTML output styles and options.

You will be prompted for a filename to save the .htm file as, with the current project name as the default. This HTML file can then be put in any web page, either as a standalone page or inserted into an existing page, depending out html output options. Large alignments would create rather large HTML files in the case of the TABLE option, but it can be broken up by rows. Note that printing from the NetScape Navigator works rather well, being paginated. Printing from MicroSofts Internet Explorer does not work at all.


HTML Options
HTML Output Options (Copy menu )

This dialog gives you a couple of choices for HTML output.

Table/Background Colors

This option gives a HTML TABLE description of the alignment. This gives HTML that is very close to the GeneDoc display. The description can be rather large however, so that large alignments may present a download or display delay for a browser.

Text Colors Only.

This option sets only the Text color in the HTML output. This mode of HTML is compact and loads and displays quickly.

Full Web Page.

This option includes HTML header and end tags in the output file.

Alignment HTML Only

This option does NOT include HTML header and end tags in the output file. Therefore it would be easier to concatenate several displays or include into another HTML file.


Copy RTF File
Copy to RTF File (Copy menu )

Use this command to copy selected data into a RTF Style File. This command is unavailable if there is no data currently selected.

You will be prompted for a filename to save the .rtf file as, with the current project name as the default. This RTF file can then be input into many word processors or graphics programs, and is platform independent.


Copy Command
Copy selection as Bitmap (Copy menu )

Use this command to copy selected data onto the clipboard.  This command is unavailable if there is no data currently selected.

Copying data to the clipboard replaces the contents previously stored there.

GeneDoc currently copies a bitmap into the clipboard. Future versions may Create a MetaFile for the clipboard so as to be able to import GeneDoc rows into Graphics Programs.


Copy Inverted Command
Copy Selection Inverted (Copy menu )

Use this command to copy selected data onto the clipboard.  This command is unavailable if there is no data currently selected. The bits copied onto the clipboard will appear as they were when they were selected instead of the normal GeneDoc display.

Copying data to the clipboard replaces the contents previously stored there.

GeneDoc currently copies a bitmap into the clipboard. Future versions may Create a MetaFile for the clipboard so as to be able to import GeneDoc rows into Graphics Programs.

Shortcuts

Keys:	CTRL+I


Copy All as Text
Copy Alignment as Text (Edit menu )

This option copies the entire alignment to the clipboard without any shading.


Copy Consensus as Fasta
Copy Consensus as Fasta (Edit menu )

This option copies the current consensus sequence as a fasta format file to the clipboard.


Copy Consensus as Prosite
Copy Consensus as Prosite (Edit menu )

This option copies the current consensus sequence as a Prosite format file to the clipboard.


Select Columns Command
Select Columns (Edit menu )

Use this command to select sequences by columns. Selected columns can be used for Score Selection , Create New Window, and Fill Range and Delete Filler from Range commands.

Shortcuts

Toolbar:

Keys:	CTRL+L


Delete All Data
Delete All Data (Edit menu )

This function will prompt you for two sequences. Data will be copied from the sequence on the left to the sequence on the right, but only for the selected range


Copy Sequence Data
Copy Data Between Seqs (Edit menu )

This function will prompt you for two sequences. Data will be copied from the sequence on the left to the sequence on the right, but only for the selected range


Residue Edit mode
Residue Edit Mode (Edit menu )

Residue Edit mode allows you to change individual residue values or gaps to other residue values or a gap. Any changes you make will be stored in the data in the projects .MSF file if you save the project and as such, this mode should be used with great care. Get in, make you changes and get out.

When GeneDoc is in residue edit mode, a block caret will be shown instead of a line caret. If the caret is then over a residue or gap character on the screen, pressing any legal residue value or gap character will replace the current residues value and advance the caret. Legal residue values are determined by the project type, either protein or dna. A wide set of dna values can be entered. A legal gap character is either a ‘.’ or ‘ ‘-‘.

When is residue edit mode, clicking the mouse at any location will move the caret to that location. You can also make normal annotation while in Residue Edit mode.

Shortcuts

Toolbar:

Keys:	CTRL+U


Clear Gap Columns
Clear Gap Cols (Edit menu )

This function clears columns that are 100% gaps in the residues of the blocks. It can be convenient after extensive alignment editing.


Clear Manual Comments
Clear Manual Comments (Edit menu )

This function clears any manual comments that have been made. Manual comments are made by putting the caret on any non sequence location in the alignment and typing on the keyboard.


Arrange Sequences menu
Arrange Menu (Help Index )

The menu entries under Arrange Sequences control GeneDoc’s sequence arrangement modes. The current mode will be indicated by the check mark in front of the menu item for that mode. The current mode can be changed by selecting the desired menu item. All GeneDoc arrangement modes are available on the toolbar. Any change in arrangement will be saved in the .MSF file, and thus the GeneDoc document is marked as ”modified since the last save operation” anytime you make an arrangement change.

Grab and Drag	Toggle grab and drag arrangement mode.
Slide Sequences	Toggle the slide sequences mode.
Insert Dash	Toggle insert dash arrangement mode.
Delete Dash	Toggle delete dash arrangement mode.
Select Sequences to work on	Select the set of sequences that arrangements apply to.
Complement Selection	Complement the set of selected sequences.
Insert Gaps into Other	Toggle insert Gaps into other sequences arrangement mode.
Delete Gaps from Other	Toggle delete Gaps from other sequences arrangement mode.
Insert Gap Column	Insert a single column in all selected sequences.
Delete Gap Column	Delete a single column in all selected sequences.
Select Columns	Select a range of columns for editing with below functions.
Fill Selection with Gaps	Execute fill selection with Gaps function.
Delete Selection if Gaps	Execute delete selection if Gaps function.


Grab and Drag mode
Grab and Drag (Arrange menu)

This menu item toggles the ‘Grab and Drag’ arrangement mode of GeneDoc. With the left mouse button or shift key, you ‘Grab’ alphabetic characters and ‘Drag’ them back and forth across the current line.

When you drag a character, all other alphabetic characters will be moved to the left or right to make room for the movement of the grabbed character. Gap characters are removed from between the other characters as they are moved, so as to minimize impact on the whole sequence line.  Gap characters removed from the path of the dragged sequence characters will be reinserted in the wake of the dragged characters. In simpler terms, the algorithm of ‘Grab and Drag’ could be likened to beads on a string. If they are all packed together, moving one will move them all. If they are spread out, moving one will only effect contiguous groups of residues uninterrupted by gaps.

As characters move off a row to make room, they are moved to the previous or following row, and if needed the characters of that row are moved as well. If there are no gaps between the grabbed character and the beginning of the sequence, no movement left will be possible.  The equivalent rearrangement can be achieved by moving all of the other sequences to the right.  See the section Insert Gaps in Other Sequences for how to do this easily.  Movement right is always possible as GeneDoc will grow the length of the sequences to accommodate character movement. New rows will be added to accommodate the growth of the sequences as needed when you let go of the left mouse button or the shift key.

When you select a sequence character and drag it to the right all of the sequence characters between the selected sequence character and the first gap to the right of the selected sequence character will be moved to the right.  As these characters are moved to the right they displace gap characters which are removed from their current location in the sequence and are reinserted immediately to the left of the selected sequence character.  When all of the gap characters in a gap have been displaced the sequence characters to the right of the now filled gap and to the left of the next gap in the sequence are added to the moving group of sequence characters.  Moving the selected sequence character to the left provides an exactly analogous, symmetrical effect.

The sequence location indicator at the end of any sequence row effected the rearrangement will be updated. Any shading on characters will be retained and move with the character.  This helps you keep track of the movements.  It also means that when you are satisfied with the rearrangement you will need to clear the shading and reapply any desired automatic shading operations.  The consensus or score lines can be arranged as well.

Additional information in the Mouse and Keyboard support section.

Shortcuts

Toolbar:

Keys:	CTRL+A


Slide Sequences mode
Slide Sequences (Arrange menu)

This menu item toggles the Slide arrangement mode of GeneDoc. With the left mouse button or shift key, you ‘Grab’ alphabetic characters and ‘Slide’ them back and forth across the current line.

When you slide a character, gaps are inserted or deleted to the left of the character only. No other formatting is done. Where there are no gaps to the immediate left of the character you have grabbed, then no movement left is possible.

Shortcuts

Toolbar:

Keys:	F5


Insert Dash mode
Insert Dash into Sequence (Arrange menu)

This menu item toggles the Insert Dash arrange mode. This mode will insert a dash into the sequence at the location of the cursor (to the left of the character on which the cursor is placed) when the left mouse button is clicked or the location of the caret if the shift key is pressed. The sequence characters and Gaps to the right of the insert location will be shifted one position right, and the sequence rows will be added if needed. The consensus and score lines will insert a space. No other rearranging is done.


Delete Dash mode
Delete Dash from Sequence (Arrange menu)

This menu item toggles the Delete Dash arrange mode. This mode will delete a dash from the sequence at the location of the cursor when the left mouse button is clicked or the location of the caret if the shift key is pressed. The sequence characters and Gaps to the right of the delete location will be shifted one position left. Only spaces can be deleted from consensus or score lines. No other rearranging is done.


Select Arrangement Sequences
Select Sequences to work on (Arrange menu)

This menu opens the Select Arrangement Sequences dialog. From this dialog you select sequences that will be affected by certain arrangement functions. Only Insert Gaps in Other, and Insert Gap Column arrangement modes are affected by this selection. Also, insert gaps into selected area and delete if gaps are affected as well. Since these latter two functions respond to both the select sequences and the select columns, you can define a square area within your alignment for inserting or deleting gaps.

Shortcuts

Toolbar:

Keys:	F8


Select Arrangement Sequences
Select Arrangement Sequences (Select Sequences to work on)

This dialog allows you to select a set of sequences for some of the arrangement functions. You can also complement the selection and deselect all, which means that all sequences would be affected my arrangement operations.


Complement Selection
Complement Selection (Arrange menu)

This menu item complements the set of Selected Sequences.

Shortcuts

Keys:	Ctrl+F8


Insert Gaps in Other mode
Insert Gaps in Other Sequences (Arrange menu)

This menu item toggles the Insert Gaps in Other Sequences arrange mode. This mode will insert a dash into all sequence lines other than the one selected. See insert dash into sequence for further information. Consensus and score lines will also have a space inserted.

If the right mouse button is hit or the Ctrl-Enter key is pressed, then gaps will be deleted.

Shortcuts

Toolbar:

Keys:	F6


Delete Gaps from Other mode
Delete Gaps from Other Sequences (Arrange menu)

This menu item toggles the Delete Dash from Other Sequences arrange mode. This function will delete a dash from all sequence lines other than the one selected. See delete dash from sequence for further information. Comment and score lines have a space deleted.


Insert Gap Coumn
Insert Gap Column (Arrange menu)

This menu item toggles the insert a single gap column. A single gap column will be inserted in the selected sequences when the left mouse button is clicked or the return key hit. If the right mouse button is clicked or the Ctrl-Enter key is hit, then a single gap column is deleted.

Shortcuts

Toolbar:

Keys:	F7


Delete Gap Coumn
Delete Gap Column (Arrange menu)

This menu item deletes a single gap column when the mouse is clicked or the return key hit.


Fill Selection with Gaps function
Fill Selection with Gaps (Arrange menu)

This function will shift all of the sequence and comment and scoring lines to the left and fill the selected area with Gaps and spaces. This is a function and not a arrangement mode.  That is this function does not change the alignment.  It provides a spacer region between two parts of the alignment.  This allows you to do ‘Grab and Drag’ operations on either part of the alignment without disturbing the other part.

Shortcuts

Toolbar:

Keys:	CTRL+F


Delete Selection if Gaps function
Delete Selection if Gaps (Arrange menu)

This function will remove an entire range of alignment columns of gap characters. If there is any alphabetic sequence character in the selected area or any non space character in the comment or score lines, the function will do nothing and an error dialog displayed. This is a function and not a arrangement mode.  That is, it does not change the alignment but removes the effects of previous changes that resulted in an overall shorter alignment.

Shortcuts

Mouse Right Click.


Shade menu
Shade Menu (Help Index )

This menu controls GeneDoc’s shading functions. The Quantify mode and the Enable Similarity Groups Flag are controllable from the toolbar. The current shading mode is saved in the .MSF file.

Conserved Mode	Conservation mode produces a display that emphasizes the degree of conservation in each column in the alignment.
Quantify Mode	Quantify Shading mode focuses attention on the pattern of changes in the columns of the alignment
Properties Mode	Chemical Property groups are used to shade according to the chemical properties of the residues
Search Mode	Search mode is used to highlight areas where motifs can be found.
Structure Mode	Used for Predefined or User Secondary Structure shading display or used for Generalized User Defined Shading.
PhysioChem Mode	PhysioChemical mode displays areas of conserved physiochemical properties.
Identity Mode	Identity Mode displays areas that contain identities or similarity to a chosen set of sequences.
LogOdds Mode	LogOdds mode displays LogOdds motifs as found in a MEME file in several different display modes.
Enable Similarity Groups	If this flag is set, a Similarity Group will be used if at least any two of the characters found in the Similarity Group are found in the alignment column
Differences Mode	If this flag is set, show all residues using Differences mode functions. Note that the differences mode can either be consensus differences or difference against the top sequence.
Shade to 4 Levels	Shade all sequences to primary, secondary and tertiary levels.
Shade to 3 Levels	Shade all sequences to primary, and secondary levels.
Shade to 2 Levels	Shade all sequences to primary levels.
Clear All Shading	No shading is shown in the alignment view
Manual Shade Mode	GeneDoc Manual Shading modes.
Clear All Man Shade	Clear all manually made shading.


Conservation Mode
Conservation Mode (Shade  menu)

This menu item toggles the Conservation Shading mode.  As its name implies Conservation mode produces a display that emphasizes the degree of conservation in each column in the alignment.  This focuses attention on which parts of the sequences are least tolerant of change, or where a change in the sequence is most likely to change the structure or function of the molecule represented by the sequence.  Up to four different Shading Levels of conservation can be distinguished.  The degree of conservation at each level can be set by the user in the Configuration dialog.  Default levels are set to complete or 100 percent conserved, 80 percent or greater conserved, 60 percent or greater conserved, and less than 60 percent conserved.  All four levels can be represented by different colors or Shading.  The top two levels are also distinguished by either upper or lower case characters on the consensus line.

In conservation mode, consensus residues are assigned based on the number of occurrences of the character in the column. If the Similarity Groups Enabled flag is set, then the Similarity Group list of equivalent residues (i.e., conservative substitutions) will be used to find the consensus residue.  The counts of all of the residues in the set of equivalent residues will be added together and treated as the count of a single residue type.  If the count for one of the sets of equivalent residues is the highest count for a column the consensus residue for that column is that set of equivalent residues.  The set of equivalent residues is represented by a numerical character rather than an alphabetical character.  The numerical character is an arbitrary choice based on the number of sets of equivalent residues that are currently in effect and the order in which they were defined..

If the number of occurrences of the most abundant character or set of equivalent residues meets or exceeds the primary, secondary or tertiary levels then the characters are assigned one of the respective shading colors.  All other characters are set to the base color.  If there is a tie for the most abundant character or group of characters in a column, then the current scoring table is used to determine which set of characters score better against all other characters.  The better scoring residue is given the higher ranking. The primary, secondary and tertiary levels are set in the Configuration dialog on the Shade Setup page.  A consensus line is built and displayed on either the upper or lower consensus line.  On the consensus line, if the alignment column was assigned a primary or secondary level from single characters, the character is displayed in upper or lower case respectively.  If the number of occurrences of characters in a Similarity Group meets or exceeds the primary level, then the group number is built into the consensus line. All other consensus line locations are cleared.


Quantify Mode
Quantify Mode (Shade  menu)

This menu item toggles the Quantify Shading mode.  Quantify Shading mode focuses attention on the pattern of changes in the columns of the alignment.  Columns that show the same Shading have the same pattern of changes even though the actual residues present in those columns may be different.  The Quantify Shading mode ranks the residues in each column of the alignment in terms of its prevalence in the column.  That is the residue with the highest, second highest, and third highest counts in each column are identified.  The most common residue in each column (the residue with the highest count) is assigned to the first shading level and the second most common residue in each column is assigned to the next shading level.  The matching of shading levels with the rank of the counts is continued until the selected number of shading levels has been reached.

Different patterns of change or evolution among the sequence would give rise to different shading patterns.  Thus the consistency of the shading patterns is an indication of how consistent the sequences are with respect to particular patterns of evolution.

In quantify mode, consensus residues are assigned based on the relative number of occurrences.  This is the same as in Conservation mode, however unlike conservation mode a consensus residue is displayed for every column in the alignment.  The most abundant character or set of equivalent residues in a Similarity Group is set to the primary level color.  The next most abundant character or set of equivalent residues is set to the secondary color and the third most abundant to the tertiary color.  Ties between equal numbers of occurrences are broken by comparing the score of all characters in the set against all other characters in the column using the current scoring table. This is again the same as in Conservation Shading mode.  A consensus line is built and displayed and contains the most abundant character of the column.

Shortcuts

Toolbar:


Property Mode
Property Mode (Shade  menu)

This menu item enables the Chemical Properties shading mode. This mode is controlled by the Properties Tab in the Configuration dialog.

Chemical Property groups are used to shade according to the chemical properties of the residues. There are program defined sets of groupings in the various levels, but you can change these as you want and save them in your User Defaults. This dialog allows you to manage three hierarchical levels or sets of groups. Level 1 corresponds to the Auto Shade 2 Levels function, Level 2 to the Shade 3 levels function, Level 3 to the Shade 4 levels function. In other words, if you are in Properties Shade mode and you execute the Shade to 4 levels function (either in the menu or on the toolbar), then you will get shading according to the Level 3 set of groupings.

The use of levels allows you to develop an hierarchical description of the desired properties.  As an example consider the default chemical properties groups for proteins.  At level 1 the amino acids are divided into two properties groups, polar and nonpolar.  At level 2 the polar group is further divided into charged and uncharged properties group while the nonpolar group is subdivided into large and small.  Finally, at level 3 the charged group from level 2 is further divided into positively and negatively charged properties groups.  The complete definition of the default properties hierarchy and groups is given below.

There are two styles of Properties Shading. The Conserved style of properties shading checks all the residues in a column. If each residue is found in a single properties group, then every residue in that column is shaded to the color of that properties group. The Shade All style looks at individual residues.  Every residue in the alignment is shaded to the pattern or color associated with the properties group to which it belongs.

The groups are not currently checked for duplicates, however, if duplicates characters exits across sets, then the character will only count in the set with the highest number.

See Property Setup in the Configuration Dialog for more information.

Shortcuts

Toolbar:


Search Mode
Search Mode (Shade  menu)

This menu item enables the Search Shading mode, controlled by the Search Tab in the Configuration dialog.

Typically a user will search a Prosite.dat file for matches found in the alignment. The matches found are saved in the GeneDoc header information and can be recalled at any time by selecting this shading mode.


Structure Mode
Structure Mode (Shade  menu)

This menu item enables the Secondary Structure or User Defined shading mode. This mode is controlled by the Structure Tab in the Configuration dialog.

The structure groups and shading facility provide an extremely powerful and flexible set of tools for integrating sequence information with structural information.  The facility is flexible enough to allow the user to display almost any kind of information as color codes along the sequence.  Such states can include the obvious secondary structure state of the residue in the three dimensional structure.  Less obvious properties like the solvent accessible surface area of the residue or its side chain can also be displayed.  The fraction of the side chain in a polar environment is another characteristic that is sometime informative.

One of the most powerful kinds of integration of structure and sequence information allows the user to visually examine the variation in structure or some structural property that occurs as the sequence varies in a series of homologous proteins.  This same display allows the user to adjust the alignment based on information derived from the varying structures of a series of homologous proteins.

Alternatively, you can have several copies of the same sequence in the alignment by adding additional copies with different names through the sequence import facility.  Remember that each copy must have its own distinct name.  These copies of the same sequence can all be highlighted using a different property.  This allows you to easily visualize possible correlation of properties or of properties with sequence or structure.

Another use for multiple copies of a single sequence in the alignment is to contrast predictions of structure and properties with that observed in an experimentally determined three dimensional structure.

A wide spread use of the structure shading is to project the structure or properties from a sequence of known structure onto sequences whose structures have not be experimentally determined.  This is accomplished by combining the structure shading facility with the group facility.  At the same time you use the structure shading dialogue to associate a structure file with a specific sequence in the alignment you can designate that sequence as the master sequence of a structural group.  This allows you to associate several sequences with a single structure file.  These associated sequences will be shaded with the same colors in the same column of the alignment as the master sequence regardless of the sequence residue present in that position of the sequence.  Thus this is essentially a low resolution homology modeling facility.

All of these features can be combined into a very informative display for studying structure-function relationships in the following procedure.  First, associate a structure file with each sequence in your alignment whose structure has been determined by X-ray crystallography or NMR.  Make these sequences the master sequence for a group of the most closely related sequences in the alignment.  Ideally the sequences within each group should have common biochemical properties such as substrate specificity, while different groups can have different substrate specificity’s.  Use the sequence editing facility to put sequences in the same group on adjacent rows of the alignment.  Put the group master sequence at the top of each group.  Then set the display mode to differences mode and make sure this is applied to all of the groups.

This yields a display with all of the group master sequences, that is the sequences with known structures, displayed with all of their residues.  The other sequences in each group have a dot displayed where they have the same sequence residue as the group master.  Sequence residues that are different from the group master sequence are shown.  This highlights substitutions within each group that are presumably successful site directed mutagenesis experiments performed by nature.  Differences between groups may be associated with the change in substrate specificity or other property.

It can be very helpful to have exactly the same alignment in a second window highlighted in group contrast mode.  This combination of displays can be a very powerful tool for examining structure-function relationships by integrating a large amount of information in an easy to comprehend format and presentation.


PhyioChemical Mode
PhysioChem Mode (Shade  menu)

This menu item enables the PhysioChemical shading mode. This mode is controlled by the PhysioChem Tab in the Configuration dialog.

The PhysioChemical Properties display mode is a Single Group adaptation of the Group PhysioChemical Properties Shading algorithm.


Identity Mode
Identity Mode (Shade  menu)

This menu item enables the Identity mode. This mode shows you areas in the alignment that are identical or similar to a selected set of sequences.

This mode uses the Similarity Groups Enabled flag: If not enabled, only strict identities will be found, if enabled, then identities and favorable substitutions (similarities) will be found.

First you will need to select a sequence or sequences as the chosen set. This set will first be examined for conservation. If all the residues in the current column of the chosen set are identical or similar, ie 100% conserved, then the other sequences will be inspected for identities or similarities. Obviously if the chosen set contains only one sequence, then it will always be considered conserved.

The set of chosen seqeuences will be shaded in the color defined in the Identity tab in the Configuration dialog, but only if the column is found to be 100% conserved.

The Identity mode has two styles, All and Conserved identities.

In the All style, any Identity or Similarity found in the other sequences will be shaded. The color will be the color of the primary level used for conservation mode. There is no minimum level of conservation required.

In the conserved style, then the identities/similarities found in the other sequences will be counted and a conservation percentage calculated. The other sequences will then be shaded in either the primary, secondary or tertiary levels used for the conserved display mode. The level of conservation must be at least the minimum level of conservation being displayed, or no shading will take place.

The consensus line of the Identity mode is also unique. If the consensus of the other sequences is different than the consensus of the chosen set, then the other sequences’ consensus value will be shown in the consensus line. Otherwise the consensus line will be blank, indicating that the chosen set and the other set share the same consensus result.


LogOdds Mode
LogOdds Mode (Shade  menu)

This menu item enables the LogOdds mode, controlled by the LogOdds Tab in the Configuration dialog.

Typically a user will load a MEME data file for motifs found in the alignment.

The MEME system allows you to discover motifs (highly conserved regions) in groups of related DNA or protein sequences using meme and, search sequence databases using motifs using MAST.

Please visit http://www.sdsc.edu/meme for complete information.

GeneDoc offers several helpful display modes in the LogOdds display, as well as easy creating of a logodds matrix for a selected area and a matrix vs. Location score report for easy visualization of repeats in an alignment.


Shade to 4 Levels
Shade to 4 Levels (Shade  menu)

This function will compute shading values for all the sequence characters in the alignment and update the screen. The shading values are represented by Shade Colors as selected in the Configuration dialog. The value assigned to a character is dependent on the shading mode in effect when the shading is applied.

Similarity Groups as set in the Configuration dialog are used if the Enable Flag is checked. A Similarity Group will be used if at least any two of the characters found in the Similarity Group are found in the alignment column.

See the Shading menu for a detailed description of each mode.

Shortcuts

Toolbar:

Keys:	F4


Shade to 3 Levels
Shade to 3 Levels (Shade  menu)

The same as Shading to 4 Levels except the tertiary level and color is not used.

Shortcuts

Toolbar:

Keys:	F3


Shade to 2 Levels
Shade to 2 Levels (Shade  menu)

The same as Shading to 4 Levels except the secondary and tertiary levels and colors are not used.

Shortcuts

Toolbar:

Keys:	F2


Clear all Shading
Clear All Shading (Shade  menu)

This function sets the shading of all sequence and consensus and score lines to the base color as selected in the Configuration dialog on the Shade Colors page.

Shortcuts

Toolbar:

Keys:	CTRL+B


Shade Group Enable
Enable Similarity Groups (Shade  menu)

This menu item toggles the ‘Enable Similarity Groups’ flag, also found in the Configuration dialog on the Similarity Groups page and as a button on the tool bar. If this flag is set, a Similarity Group will be used if at least any two of the characters found in the Similarity Group are found in the alignment column.  A Similarity Group is a set of sequence residues that are to be treated as if they are equivalent.  One consistent basis for selecting the members of a Similarity Group is to select a set of residues where the score for any pair of residues in the set is greater than zero in the current scoring table.  The default Similarity Groups for each scoring table have been selected this way. Thus, these Similarity Groups represent what are usually called conservative substitutions, evolutionary substitutions that are evidence in favor of the sequences being homologous (related through a common evolutionary ancestor).

Shortcuts

Toolbar:


Residue Mode Difference
Differences Mode (Shade  menu)

This menu item toggles the Residue Differences Display mode.  In Differences mode the only residues displayed in each column are the “master” residue that is displayed at the top of the column and the residues that differ from this master residue.  All residues that match the master residue are displayed as dots, “.”.  This focuses attention on the specifics of changes in each column.  This is especially true where a group of sequences may have changed in concert to a particular residue that is conserved within that group.  Such changes can mark the adaptation of a group of sequences to a new evolutionary or environmental niche.  Such changes can also mark the residues critical to the evolution of a new biochemical function or change in substrate specificity in an enzyme.

The master sequence can be specified in one of two ways.  It can be one of the sequences in the alignment.  In this case the sequence must first be moved to the top sequence in the list of sequences.  This can be done in the Sequence Dialog List dialog. The other choice for the master sequence is to use the consensus sequence.  This would somewhat complicate the interpretation of the differences mode display but may be useful in a protein engineering context.

In Differences mode, consensus residues are assigned based on the relative number of occurrences.  This is the same as in both Conservation and Quantify modes.  Like Quantify mode a consensus residue is displayed for every column in the alignment.  The consensus sequence in Differences mode is always displayed in the top consensus line.  Also the gap character in Differences mode is always a dash because the dot is reserved for other purposes.

In Differences mode with the consensus sequence used as the master sequence, the most abundant character or most abundant members of a set of equivalent residues in a Similarity Group are displayed as dots (‘.’).  Ties for most abundant residue or set of equivalent residues are again resolved by selecting the residue (or set of equivalent residues) that scores lowest (least distant) against the other residues using the current scoring table.  A consensus line is built and displays the most abundant character or in the case of a set of equivalent set of residues being the most abundant, the number of the set of equivalent residues.  All color shading is cleared except in the consensus line where the characters are colored as columns would be colored in conserved mode.  If the number of occurrences of a character or set of characters in the column meets or exceeds the primary level, then the character in the consensus line is colored with the primary level color, and so forth.

Shortcuts

Toolbar:


Manual Shading
Manual Shade Mode (Shade  menu)

GeneDoc allows manual shading of the individual characters in an alignment column. This shading is not saved in the .MSF file and if the file is closed and reopened, any manual shading will be lost. Manual shading will also be cleared if any of the automatic sequence shading functions are executed.  Any manual shading present in an alignment will be printed or copied to a bitmap or PICT file.

When manual shading is selected, you will be presented with the Choose Shading  dialog box. Select the desired shading colors here and either double click or select the OK button. You can shade the residues, but not the consensus or comment lines by holding down the left button or pressing the shift key and moving the cursor or caret.

If you first select a residue whose current color does not match the selected shading color, all shading will be set to the selected color. If you select a residue with the same color as your selected shading color, all shading will be done in the base color.

Shortcuts

Toolbar:

Keys:	CTRL+M


Manual Shading Color
Choose Manual Shading Color (Manual Shading)

Select the desired shading colors here and either double click or select the OK button. You can shade the residues, but not the consensus or comment lines, by holding down the left button or pressing the shift key and moving the cursor or caret.


Clear Manual Shading
Clear Manual Shade (Shade  menu)

Removes all manual shading thats been done.


Group menu
Groups Menu (Help Index )

The Group menu controls Group Feature specific commands.

See also SuperFamily Group Features discussion for additional information.

For more information, select the Tree menu item name below.

Configure Groups	This open the Group Configuration Dialog.
Shade Group Configuration	Sets the GeneDoc display to shade with the group configuration information.
Shade Group Contrast	Sets the GeneDoc display to Shade the groups using the Group Contrast algorithm.
Shade Group Contrast/No Gaps	Sets the GeneDoc display to Shade the groups using the Group Contrast algorithm, however columns with gaps in it are not shaded.
Shade Group Conserved	Set the GeneDoc display to Shade the groups using the Group Conserved shading algorithm.
Shade Group Cons. W/Xshade	Shade in Group Conserved Shading with Cross Shading Mode.
Shade Group PCR Contrast	Set the GeneDoc display to Shade the groups using the Group PCR Contrast shading algorithm.
Shade PCR W/Similarities	Set the GeneDoc display to Shade the groups using the Group PCR Contrast shading algorithm using the similarity array on the Group Setup Dialog.
Shade PhysioChemical Prop	Shade in Group PhysioChemical Properties mode.
Shade DNA Group Set	Shade in Group DNA Set analysis mode.


Group Configure
Group Configuration (Group menu )

Use this menu item opens the group configuration dialog

See SuperFamily Group Features for additional discussion on Groups.

Shortcuts

Toolbar:


Group Configure Dialog
Group Configuration Property Sheet (Group menu )

This is the main group control dialog. Here groups are created and deleted, and sequences are added and removed from the groups. The list on the left shows whether specific sequences are part of a group or not. The current group is shown in the group control listbox and work done from this dialog will be done to the current group. On the bottom right are a few specific controls relating to some of the group shading modes.

Group shading comes in two fashions. There is a group configuration shading and multiple group contrast/conserved shading modes.

When at least one group is defined, you will see additional tabs next to the group tab. These tabs open the shading, property and structure dialog pages, which are exactly the same configuration dialogs as in the Project Configuration property sheet. The difference is that the shading setups here apply only to the currently selected group and are only shown when the Shade Group Configuration mode is selected.

The Group contrast and conserved modes use the grouping information here, but do not use any information from the other shading/property/structure tabs. The few variables for those shading algorithm’s are displayed on the bottom right in the Group Contrast/Cons settings area.

The most obvious use of the Shade Group configuration mode is to display secondary structure information. In this case, it is likely you will want to define a group of sequences for each of your secondary structure data files. Then you will, for each group, select a master sequence and load a data file for that sequence. Secondary Structure or User defined shading will be applied to all of the sequences within a group. As mentioned in the group features discussion, another useful as feature is to apply residues differences display, against the top sequence of the group, to each group. This will help to remind you that the Secondary Structure or User Defined shading is specific to only one sequence within a group, and where there are differences the shading may no longer be accurate.

Applying a change, such as a different structure data set, to all of the groups can be done in two ways from the here. One would be to select each group and in turn change the desired parameter for each group. The other is to use the ‘Modify All’ option found on this dialog. If that option is enabled, then any change made from any of the other configuration dialogs will be applied to all groups.

If the GeneDoc display is in Shade Group Configuration mode, then changes made from the toolbar will be applied to all groups as well. This is discussed in more detail in Toolbar Tips.

Sequences for Group list.

This list shows you all of the sequence names in the project. If any sequences belong to any group, then the sequence name will be colored in the groups colors. This is a multiple selection list. More than one sequence can be selection either by dragging the mouse with the button pressed down or by multiple clicks with the shift or control key pressed. Standard windows stuff. You select sequences here in order to add or remove sequences from the groups.

Group control Group list.

This pulldown list holds all of the currently defined groups names. The current group that is being worked on is shown in this listbox. Pull down the box’s list to select another group to work on.

Double clicking on this list will either add the sequence to the current group, or remove the sequence from the group its already in.

Modify All Groups.

This radio button is used to apply changes made in the other configuration dialogs to all groups, as discussed above.

New Group.

This button creates a new Group. First you will be asked for a group name. This must be a unique group name. Second you will be asked for a group background color. This color must not be white. The new group will be placed at the bottom of the group list and made the current group.

Delete Group.

This button deletes the current group.

Text Color

This button gives you a common color dialog box from which to chose the current groups text color.

Back Color

This button gives you a common color dialog box from which to chose the current groups background color.

Color Display Box

This area shows you a ‘G’ in the current groups Text and Background colors.

Group Sequence Add

This button will add any selected sequences to the current group. Any sequences that are in another group will be removed from that group.

Group Sequence Remove

This button will remove any selected sequences to the current group.

Color Sequence Names

This option will causes the sequences names on the GeneDoc display to be colored by their group colors.

Group Cons Level

This value is used by the Shade Group Contrast Mode and the Shade Group PCR modes. This is the amount of conservation required before the column of the group will have shading. The default is 100%

PCR Similarities

This array or characters is used by the Shade PCR W/Similarities mode. The default is blank, and if it is blank then there will be no difference between the Shade Group PCR and Shade PCR W/Similarities modes. Putting values in here is much the same as the similarities groups found in the Project Configuration/Sub groups, it allows for more than one type of residue to be allowed in determining the conservation level of a group. You can either enter values of your own or select values from the pulldown list.

PCR Maximum Level

This value used by the Group Shade PCR modes. The value is sort of the compliment of the group cons level, it determines how many characters outside of a group that is a candidate for shading can be allowed. For example, if the value is 0%, the default, then no characters that have been found to have the group conservation level (as set above) can be allowed outside the group if it is to be shaded.


Group Name
Group Name (Group menu )

Enter the name of the new group here.


Group Shade Configuration
Shade Group Configuration (Group menu )

Use this menu item to set the GeneDoc display to shade in the Group Configuration mode. This mode will shade like the regular conserved, quantify, properties, structure modes, except each groups shading parameters are individually controlled by the group configuration dialog.

See SuperFamily Group Features for additional discussion on Groups.

Shortcuts

Toolbar:


Group Shade Contrast
Shade Group Contrast (Group menu )

Use this menu item to set the GeneDoc display to shade in the Group Contrast mode.

See SuperFamily Group Features for additional discussion on Groups.


Group Shade Contrast No Gaps
Shade Group Contrast/No Gaps (Group menu )

Use this menu item to set the GeneDoc display to shade in the Group Contrast mode, except that within groups, columns that have gaps in them will not be shaded.

See SuperFamily Group Features for additional discussion on Groups.


Group Shade Conserved
Shade Group Conserved (Group menu )

Use this menu item to set the GeneDoc display to shade in the Group Conserved mode. In the Group conserved mode, any column that has 100% conservation across all sequences in the alignment will be shaded with the 100% conserved color from the Project Configuration/Shade Colors dialog. Then for each group, any column that has enough identical residues to meet the Group Cons Level setting will be shaded with that groups color.

It should be noted that in the Group Conserved Shading mode, similarity enabled is controlled by both the Project Configuration/Shade tab and for each group the Group Configuration/Shade tab. This comes into play typically when attempting to switch the Similarity enabled setting for this display.

See SuperFamily Group Features for additional discussion on Groups.


Group Shade Conserved
Shade Group Conserved With Cross Shading (Group menu )

Use this menu item to set the GeneDoc display to shade in the Group Conserved mode. In the Group conserved mode, any column that has 100% conservation across all sequences in the alignment will be shaded with the 100% conserved color from the Project Configuration/Shade Colors dialog. Then for each group, any column that has enough identical residues to meet the Group Cons Level setting will be shaded with that groups color.

In addition, if a column in a group has no shading, then shading for chars found in the other groups will be applied.

Group cross shading is useful with a small number of groups where the sequences are relatively well conserved within the groups.  In this display alignment columns that are completely conserved within the group are highlighted in the color assigned to the group.  When an alignment column is not conserved within the group individual residues within that group are colored when they match a residue that is completely conserved within another group in the same alignment column.  These colored residues in groups without complete conservation are colored with the color assigned to the group in which that residue is completely conserved.  These colored residues are then indicative that even though the residue is completely conserved within some group  it is less likely to be essential to maintaining the unique features of that group than if it did not occur outside of the group.

It should be noted that in the Group Conserved Shading mode, similarity enabled is controlled by both the Project Configuration/Shade tab and for each group the Group Configuration/Shade tab. This comes into play typically when attempting to switch the Similarity enabled setting for this display.

See SuperFamily Group Features for additional discussion on Groups.


Group Shade PCR Contast
Shade Group PCR Contrast (Group menu )

Use this menu item to set the GeneDoc display to shade in the Group PCR contrast mode.

See SuperFamily Group Features for additional discussion on Groups.


Group Shade PCR Contast W/Similarities
Shade PCR W/Similarities (Group menu )

Use this menu item to set the GeneDoc display to shade in the Group PCR Contrast mode, using the residue similarities array found in the Group Configuration Dialog.

See SuperFamily Group Features for additional discussion on Groups.


Group Shade PhysioChemical Properties
Shade PhysioChemical Properties (Group menu )

The parameters of this display are controlled from the Project Configuration dialog and the PhysioChem tab.

See SuperFamily Group Features for additional discussion on Groups.

The physiochemical properties group analysis is an analytical function that assigns each column in your alignment to one of the twelve predefined groups of physiochemical properties.  These groups were originally proposed and presented as a Venn diagram by W.R. Taylor (1986) and have been widely used since then.  We have modified the original classification by removing Lysine from the hydrophobic group and by putting Glycine into its own group as they did with Proline.  The reason for putting Glycine into its own group is the observation that a rather high proportion of the amino acids that are uniquely conserved in alignments of divergent families of proteins are Glycine (Nicholas et al., 1995).  We excluded Lysine from the hydrophobic, or non-polar, group because while it does sometimes participate in hydrophobic interactions with the four methylene groups in its side chain, these interactions are constrained by the necessity of the usually charged amine group at the end of the methylene chain being in a polar environment.  For instance, when a Lysine (or sometimes Arginine) participates in helix to helix packing it is generally at the end of the helix packing interface rather than at one of the interior positions (Chothia et al., 1981).

Another sizable difference between the physiochemical properties groups used in GeneDoc and those of Taylor is that we have added an Amphoteric group that is a proper subset of the Polar group.  The Amphoteric group contains only those amino acids whose side chains have more than one hetero atom (Nitrogen, Oxygen, or Sulfur).  Further, the Amphoteric group members have the ability for one of the hetero atoms of the side chain to act as a hydrogen bond donor while another hetero atom of the side chain acts as a hydrogen bond acceptor.  Also note that Cysteine is assigned differently depending on whether it is expected to be in the oxidized or reduced state (disulfide bonds or free sulfhydrals, respectively).

The twelve groups represent three overlapping hierarchies.  The first hierarchy is based on size, the second is based electrical charge for the polar amino acids, and the third, for non-polar amino acids, is based on aromaticity.

This analysis allows the user to distinguish between proteins with free sulfhydrals and those with disulfide bonds by choosing either oxidized or reduced Cysteine.  Free sulfhydral groups (reduced Cysteine) are generally found in intracellular proteins while the disulfide bonds of oxidized Cysteine are usually found in extracellular proteins.

The analysis proceeds by first creating a set representation, called a composite residue (McClain and Nicholas, 1987), for each column in the alignment.  The composite residue has a composite identity of all of the individual kinds of residues present in the column.  For instance, if a six sequence alignment had a column with three Alanines, two Glycines, and a Proline the composite residue would be the set {Alanine, Glycine, Proline} or {A,G,P} in the single letter code.  This composite residue is then compared with the physicochemical composite residue for each of the groups in the appropriate table below.  The first physicochemical composite residue that contains all of the amino acids in the alignment composite residue is assigned as the physiochemical group for that alignment column.  In the example above, the composite {A,G,P} belongs to the Small group.  That is, the Small group contains all of these amino acids and there is no other group that contains all of these amino acids and that has fewer total different kinds amino acid.  The alignment columns are then color coded to identify the physiochemical group to which they belong.

A second, simpler, shading is also available as part of this analysis, which is the ‘Shade Conserved’ selection of the configuration dialog.  This analysis also allows the user to distinguish between proteins with free sulfhydrals and those with disulfide bonds by choosing either oxidized or reduces Cysteine.  In the full analysis shading described above each of the fourteen physiochemical hierarchy groups is a unique color code which is applied to appropriate alignment columns.  While this conveys a lot of information it can be overwhelming in its detail.  Because of this GeneDoc provides an optional three state shading based on the same analysis.  A black background with white text is used to indicate columns in the alignment where a single residue is conserved across the entire alignment (i.e., every residue in the column is identical).  The second level of shading is a dark gray background with white text.  It is applied to columns of the alignment within a user defined group of sequences.  It identifies columns in the alignment where a single residue is conserved in all of the sequences within one of the user defined sequence groups.  The third shading, a light gray background with black text, is again is applied to columns of the alignment within a user defined group of sequences. It identifies columns in the alignment where a single physiochemical properties group is conserved in all of the sequences within one of the user defined sequence groups

Physiochemical properties groups assignments used by GeneDoc for extracellular proteins or other proteins where the Cysteine residues are expected to be in the oxidized state and form disulfide bonds.

                                                              Color

                                               Text       Background

             **ILVCAGMFYWHKREQDNSTP BZX**

     Proline:                     P   X -      Blue          Red

     Glycine:       G                 X -      Green         Red

        Tiny:      AG           S     X -      Blue          Yellow

       Small:    VCAG         DNSTP   X -      Green         Yellow

    Positive:            HKR          X -      Red           Blue

    Negative:               E D       X -      Green         Blue

     Charged:            HKRE D       X -      White         Blue

  Amphoteric:            H REQDN    BZX -      Red           Green

       Polar:          YWHKREQDNST  BZX -      Black         Green

   Aliphatic:  ILV A                  X -      Red           Gray

    Aromatic:         FYWH            X -      Blue          Gray

 Hydrophobic:  ILVCAGMFYWH       TP   X -      White         Black

Physiochemical properties groups assignments used by GeneDoc for intracellular proteins or other proteins where the Cysteine residues are expected to be in the reduced state and form free sulfhydral groups.  The color coding for the groups is the same as above.

                    Amino Acid

             **ILVCAGMFYWHKREQDNSTP BZX**

     Proline:                     P   X -

     Glycine:       G                 X -

        Tiny:     CAG           S     X -

       Small:    V AG         DNSTP   X -

    Positive:            HKR          X -

    Negative:               E D       X -

     Charged:            HKRE D       X -

  Amphoteric:            H REQDN    BZX -

       Polar:     C    YWHKREQDNST  BZX -

   Aliphatic:  ILV A                  X -

    Aromatic:         FYWH            X -

 Hydrophobic:  ILVCAGMFYWH       TP   X -

Physiochemical properties groups assignments originally proposed (Taylor, 1986) for extracellular proteins or other proteins where the Cysteine residues are expected to be in the oxidized state and form disulfide bonds.

                    Amino Acid

             **ILVCAGMFYWHKREQDNSTP BZX**

     Proline:                     P   X

        Tiny:      AG           S     X

       Small:    VCAG         DNSTP   X

    Positive:            HKR          X

    Negative:               E D       X

     Charged:            HKRE D       X

       Polar:          YWHKREQDNST  BZX

    Aromatic:         FYWH            X

   Aliphatic:  ILV                    X

 Hydrophobic:  ILVCAGMFYWHK      T    X

References

Chothia, C., Levitt, M. and Richardson, D.  1981.  Helix to Helix Packing in Proteins.  J. Mol. Biol.  vol. 145, pp. 215 - 250.

Taylor, W.R.  1986.  The Classification of Amino Acid Conservation.  J. Theor. Biol., vol. 119, pp. 205 - 218.


Group Shade DNA Set
Shade DNA Group Set (Group menu )

Representing nucleotide (DNA and RNA) sequences as sets

Symbol	Nucleotides Present	Set Representation
	none:  Null set	{0,0,0,0}
A	A	{0,0,0,1}
C	C	{0,0,1,0}
G	G	{0,1,0,0}
T or U	T or U	{1,0,0,0}
M	A, C:  aMine	{0,0,1,1}
R	A, G:  puRine	{0,1,0,1}
W	A, T:  Weak	{1,0,0,1}
S	C, G:  Strong	{0,1,1,0}
Y	C, T or U:  pYrimidine	{1,0,1,0}
K	G, T or U:  Ketone	{1,1,0,0}
V	A, C, G:  not U	{0,1,1,1}
H	A, C, T or U:  not G	{1,0,1,1}
D	A, G, T or U:  not C	{1,1,0,1}
B	C, G, T or U:  not A	{1,1,1,0}
N (or X)	A, C, G, T or U:  any	{1,1,1,1}

Set Operations on Nucleotides

Operation	Set Notation	Result
Union of A and G:	A{0,0,0,1} .or. G{0,1,0,0}	R{0,1,0,1}
Union of R and S:	R{0,1,0,1} .or. S{0,1,1,0}	V{0,1,1,1}
Intersection of A and G:	A{0,0,0,1} .and. G{0,1,0,0}	0{0,0,0,0}
Intersection of R and S:	R{0,1,0,1} .and. S{0,1,1,0}	G{0,1,0,0}

The use of this set representation allows us to create a more informative kind of consensus sequence that we call a composite sequence.  A composite sequence is formed by the union of all of the nucleotides in a group of sequence at a given position.  When all of the sequences within the group are equally effective at carrying out the same biochemical reaction or physiological role, as is the case for multiple isoaccepting tRNA sequences within an organism, the composite sequence allows us to carry out a very powerful contrast analysis.

The contrast analysis takes the intersection of the composite sequences of two or more groups.  The intersection nucleotides of these composite sequences should be a valid member of all of the groups included in the intersection.  Positions where the intersection is empty (the null set) are interesting because they completely discriminate groups.  Thus these null set positions are candidates for the positions that are critical for the unique biochemical or physiological of the discriminated groups of sequences.

Example:  tRNA Sequences

ARG_acg_Ec  	GCAUCCG

ARG_ccg_Ec  	GCGCCCG

ARG_ccg_St  	GCGCCCG

ARG_ccu_Ec  	GUCCUCU

ARG_ucu_Ec  	GCGCCCU

ARG_ucu_T4  	GUCCCGC

Arg Composite	GYVYYSB

GLN_uug_Ec  	UGGGGUA

GLN_cug_Ec  	UGGGGUA

GLN_uug_T4  	UGGGAAU

GLN_uug_T5  	UGGGGAU

Gln Composite	UGGGRWW

Arg Composite	GYVYYSB

Gln Composite	UGGGRWW

Intersection	GU

Each of the above sequences is identified by  the three letter code for its amino acid acceptor activity, the three nucleotides of the anticodon, and a two letter code denoting the organism.  Ec denotes Escherichia coli, St denotes Salmonella typhimurium, and T4 and T5 denote the bacteriophage T4 and T5 respectively.  The nucleotides are the first seven from the mature tRNA, the amino acid acceptor stem.

This kind of contrast analysis was developed as a formal model of the discrimination of tRNA sequences by the amino acyl synthetase enzymes that attach a specific amino acid to each tRNA molecule.  It was presented in the first three papers cited below.  The fourth paper is a review of a number of experiments designed using this contrast analysis.  These experiments successfully mapped the nucleotides in various tRNAs responsible for the observed specificity in tRNA amino acid accepting activity.

Nicholas, H.B. Jr. and McClain, W.H.  1987.  An algorithm for discriminating transfer RNA sequences.  Computer Applications in the Biosciences, 3, pp. 177 - 181.

Nicholas, H.B., Jr., Chen, Y-M., and McClain, W.H.  1987.  Comparisons of transfer RNA sequences.  Computer Applications in the Biosciences, vol. 3, p. 53.

McClain, W.H. and Nicholas, H.B,Jr.  1987.  Discrimination between transfer RNA molecules.  Journal of Molecular Biology, vol.  194,  pp 635 - 642.

McClain WH, 1993. Rules that govern tRNA identity in protein synthesis.  Journal of  Molecular Biology, vol. 234, pp. 257-280

The set representation of nucleic acid sequence has a long history in molecular phylogenetics.  See, in particular the parsimony analyses of W.M. Fitch who introduced us to the set representation.  Fitch, W.M. 1971. Toward defining the course of evolution: minimum change for a specific tree topology. Systematic Zoology 20: 406-416. Williams, P.L. and Fitch, W.F., Phylogeny Determination Using a Dynamically-Weigh ted Parsimony Method. Methods in Enzymology, 183, 615-625 (1990).


Group Features
SuperFamily Group Features  (Help Index )

Increasingly, families of genes and proteins are organized into superfamilies.  The percentage of residue identity is often used as the criterion for distinguishing whether a pair of sequences are assigned to the same family or different families within a superfamily.  Perhaps a more useful criterion is whether or not a gene duplication has taken place in the common evolutionary history of the two sequences.  Evolutionary biologists use this classification and refer to homologous sequences that  have only speciation events and not have gene duplication events in their common evolutionary history as being orthologous.  Homologous sequences that have a gene duplication event in their common evolutionary history are referred to as paralogous.

Orthologous genes or proteins generally carry out the same biochemical and physiological functions while paralogous proteins generally carry out similar but related functions such as different substrate specificity.  For instance, mammalian myoglobins, which carry oxygen within cells are a orthologous family.  They are part of a superfamily that includes the alpha hemoglobins and the beta hemoglobins, both of which are also orthologous families.  These three homologous families are part of the same paralogous superfamily, as are other globin genes and proteins.

Families and superfamilies can be organized around functional criteria as well as evolutionary criteria and sequence divergence.  Although all sixty plus transfer RNA sequences in E. coli are paralogous with each other they can be grouped together by their twenty amino acid acceptor activities.

The group functions in GeneDoc allow users to work with and analyze groups based on any of the above criteria.  Groups can also be defined for any criteria the user deems important divisions among the sequences.  The first step in working with groups is to access the groups configuration dialog. The group configuration dialog can be accessed either by selecting the “edit sequence groups” item on the groups menu or by clicking the groups button.  The groups button is the button marked with an upper case G on the upper toolbar.  The group configuration dialog allows the user to allocate the sequences to groups and to select a color for each group.  In most of the GeneDoc group analyses, sequences that are not explicitly assigned to a group will be treated as if each unassigned sequence constitutes the only member of its own group.  These implicit groups will not be analyzed but the sequences will be used in the analyses of the defined groups as “other“ groups and thus they will contribute to the analysis.

The group analyses result in different shading for individual groups.  These shadings highlight different degrees of different kinds of conservation of residues or properties within and between groups.  One analysis, referred to as the Dstat analysis, measures how different the groups are from one another and whether this difference is statistically significant..  The Dstat analysis presents its results as a graph and numerical values.

The simplest analysis is performed by the “shade group conserved” entry on the Groups menu.  This analysis highlights positions within each group that are completely conserved, that is there is only one residue at that position within the group.  This highlighting is done in the color assigned to the group in the group definition dialog.  This measurement of conservation within the groups does not take into account any equivalency groups, even if they are active.  The second thing this analysis does is to highlight the positions that are completely conserved across all of the groups, that is there is only one residue at that position for all of the sequences in the alignment.  This part of the analysis does take the equivalency groups into account if they are in effect.  The final action is to compute a consensus sequence based on the entire alignment.

The most useful information derived from this analysis is to identify for the user the regions of the alignment where structural or functional requirements may have been relaxed or eliminated for some groups relative to others.  Alternatively the structural or functional requirements may have been added as the group evolved a new function.  For this kind of information to be reliable the conserved groups need to be both large and from a diverse range of organisms.  Otherwise the observed conservation may simply be the result of a small data set with highly dependent observations.

A more stringent analysis is performed by the “shade group PCR contrast” entry on the Groups menu.  Sites highlighted by this analysis meet two criteria.  First is that a single residue is completely conserved within the group.  Second, this conserved residue does not appear, at that position, in any sequence outside of the group in which it is conserved.  This analysis marks unique sequence features of the group that can be useful in defining a group.

The “shade group contrast” entry on the Groups menu performs an analysis similar to that of the “shade group PCR contrast” entry.  This analysis makes use of the scoring table designated for alignment scoring to divide scores for pairs of amino acids into three classes, positive, negative, and neutral.  The positive scores are those that are positive numbers in the similarity scores form of the table.  Similarly, the negative scores are those that are negative numbers in the similarity scores form of the table, while the neutral scores have a zero score.  The scores are stored in GeneDoc as distance or dissimilarity scores and hence must be converted to the similarity form.  This is done by subtracting the score for a pair of sequence residues from a constant called the zero cost distance.  The zero cost distance is stored with the table.  Thus the largest distances become negative similarities and small distances become positive similarities.  The interpretation of the scoring tables is that positive similarities are conservative substitutions and are favored over random substitutions in the evolutionary process relating the sequences.

The analysis performed by the “shade group contrast” entry on the Groups menu is less restrictive about the degree of conservation within the group than is the “shade group PCR contrast” analysis.  All of the sequence residues found at a position within the group are required to have a positive similarity score with each other, and thus to be conservative substitutions.  This analysis is, however, more restrictive than is the analysis performed by the “shade group PCR contrast” analysis when dealing with residues outside the group.  The residues outside of the group must have a negative similarity score with every residue from within the group.  Thus they are not allowed to be either conservative or neutral substitutions.  An example of using this kind of analysis to study the recognition of transfer RNAs by aminoacyl tRNA synthetase enzyme can be found in McClain and Nicholas, 1987.  Nicholas et al., 1987 describes using the contrasts to plan site directed mutagenesis experiments to confirm the analysis of the tRNAs.

The analysis called the Dstat analysis is the Kolmogorov-Smirnov test for the equality of two distributions.  The Dstat analysis is accomplished by first selecting a region (or all) of the alignment for use in the test calculations.  You can select the analysis under the Dstat menu.  As noted above, the Dstat analysis is a statistical test of whether the groups defined by the user are significantly different from each other.

The first step in the test is to compute an alignment score for each pair of sequences over the region selected by the user.  These scores are the partitioned into two distributions.  The first distribution is composed entirely from scores where both of the sequences used to compute the score are members of different user defined groups.  This is called the between groups distribution.  The second distribution is composed entirely from scores where both of the sequences used to compute the score are members of the same user defined group.  Note that this includes scores from every group with two or more sequences.  This distribution is called the within groups distribution.

These two distributions are plotted as cumulative distributions.  That is  the score is plotted versus the fraction of the scores in the distribution that are less than or equal to the score being plotted.  The Kolmogorov-Smirnov D statistic (Dstat) is the maximum difference between the two distributions (along the fractional axis).  Recent advances in the understanding of  the distribution of values taken on by Dstat allow us to compute its one-sided significance probability.  The one-sided significance probability is used rather than the two-sided significance probability because we are only interested in the case where the between groups distribution is composed of larger scores than the within groups distribution.  The other situation, where the within groups distribution is composed of larger scores than the between groups distribution corresponds to either convergent evolution or some sort of selection in favor of divergence.  These situations are not usually part of the hypothesis we want to test.

The Kolmogorov-Smirnov test was selected instead of the more common Student’s t test or the F test because it is sensitive to both the location of the distributions along the scores axis and to the shape of the distribution.  Student’s t test is sensitive to only the location of the distributions and the F test is sensitive only to differences in the variance of the distribution, only one of several aspects affecting the shape of the distributions.  Thus the Kolmogorov-Smirnov test can find the distributions to be different when either Student’s t test or the F test might have failed.  Because of this it is necessary for the user to examine the plot carefully to determine the exact nature of the differences in the two distributions being tested.  The user should exercise care that the biological hypothesis being examined should lead to the type of difference actually observed.

Examples of testing biological hypotheses with sequence data and the Kolmogorov-Smirnov test can be found in Nicholas and Graves, 1983 and in Nicholas and McClain, 1995.  The Nicholas and Graves paper contains an extended discussion of formulating Kolmogorov-Smirnov tests that correspond to different kinds of biological hypotheses.

Nicholas, H.B. Jr., and Graves, S.B.  1983.  Clustering of transfer RNA by cell type and amino acid specificity.  Journal of Molecular Biology, vol. 171, pp. 111 - 118.

Nicholas, H.B., Jr., Chen, Y-M., and McClain, W.H.  1987.  Comparisons of transfer RNA sequences.  Computer Applications in the Biosciences, vol. 3, p. 53.

McClain, W.H. and Nicholas, H.B,Jr.  1987.  Discrimination between transfer RNA molecules.  Journal of Molecular Biology, vol.  194, pp. 635 - 642.

Nicholas, H.B. Jr. and McClain, W.H.  1987.  An algorithm for discriminating transfer RNA sequences.  Computer Applications in the Biosciences, 3, pp. 177 - 181.

Nicholas, H.B. Jr. and McClain, W.H.  1995.  Searching tRNA Sequences for Relatedness to Aminoacyl tRNA Synthetase Families.  Journal of Molecular Evolution, vol. 40, pp. 482-486.


Score menu
Scoring Menu (Help Index )

The Scoring menu includes items that control all of GeneDoc’s alignment scoring functions. These functions are useful when manual arranging the alignment and you want to check that you are improving the score of the area that you are working on. This provides a more scientific justification for your changes than arrangements that look better in various shading modes. Your goal would be to make arrangement changes that result in a lower score for an area, lower being better, representing less evolutionary cost.

For more information, select the Score menu item name below.

Score by Sum of Pairs	Set scoring functions for score line to score using PairWise summations algorithm.
Score by Phylogenetic Tree	Set Scoring functions for score line to score using the Phylogenetic tree layout and phylogenetic tree scoring algorithm.
Score by Numbed Tree	Set Scoring functions for score line to score using the Phylogenetic tree layout and phylogenetic tree scoring algorithm, except in a numbed mode.
Score by LogOdds	Set Scoring functions for score line to scoring using the Log Odds algorithm. The log odds method
Select Columns	Select Sequence range by Columns.
Score Selection	Scores the currently selected range of alignment columns.
Write LogOdds Matrix	Calculate and write a LogOdds matrix for the selected region.
ReScore All Scores	ReCalcs all defined scoring ranges.
Clear All Scores	Clear all scores.
Show Score Table	Shows the values in Distance of the current scoring table.
Show Scoring Info	Used to Produce Ascii Phylogenetic tree scoring output for debugging and investigation purposes.


Score PairWise
Score by Sum of Pairs (Scoring menu )

This is the only mode currently supported. See Scoring Page in the User Settings dialog for more information.

See Score Selection for more information.


Score by Phylogenetic Tree
Score by Phylogenetic Tree (Scoring menu )

Scores the alignment using weighted parsimony on a user defined tree.  There is an option to compute the scores with numbed nodes.  See the scores selection section for more details about scoring. . Phylogenetic tree scoring is weighted parsimony scoring which assigns residues to internal nodes in the user specified tree.  The residues are assigned to the internal nodes to create the shortest total length of the branches in the tree.  The length of the branches is determined from the distance scoring tables.

See Score Selection for more information.


Score by Numbed Phylogenetic Tree
Score by Numbed Tree (Scoring menu )

Scores the alignment using weighted parsimony on a user defined tree.  There is an option to compute the scores with numbed nodes.  See the scores selection section for more details about scoring. . Phylogenetic tree scoring is weighted parsimony scoring which assigns residues to internal nodes in the user specified tree.  The residues are assigned to the internal nodes to create the shortest total length of the branches in the tree.  The length of the branches is determined from the distance scoring tables.

See Score Selection for more information.


Score by Log Odds
Score by Log Odds (Scoring menu )

Log Odds, or Minimum entropy scoring is one of the advances derived from the application of probability theory to multiple sequence alignments.  It is closely related to the scoring functions used to guide the creation of Hidden Markov Models (HMMs) and multiple sequence alignments derived from the HHMs.  Additional information can be found in chapter 6.2 of Durbin, R., Eddy, S., Krogh, A., and Mitchison, G.  1998. "Biological Sequence Analysis: Probabilistic models of proteins and nucleic acids."  Cambridge University Press.

See Score Selection for more information.


Gene Score command
Score Selection (Scoring menu )

This function computes a score for the alignment over the selected range of columns. The score is displayed on the score line.  Scores are not currently saved in the .MSF file along with the configuration settings which are saved. Thus, if you save and reload a .MSF file, the scores are cleared.

Scores are computed by either of three methods:  Sum of pairs (Pair Wise) scoring, Phylogenetic Tree scoring based on weighted parsimony, or Log Odds - Minimum Entropy Scoring.  Phylogenetic scoring is much slower than sum of pairs scoring but is generally preferable since it explicitly includes a model of evolution.  Phylogenetic scoring may be carried out either with either the nodes numbed or not numbed.  Numbing the nodes, in GeneDoc, is both defined as and carried out by requiring that the sequence residue at each node be the same residue that is found at one of the node’s two descendant nodes.  This definition is applied recursively to all the nodes in the tree.  This results in a tree where any residue assigned to an internal node has a continuous path of nodes assigned the same residue to a terminal or leaf node also assigned the same residue.

All scores are computed using distances rather than similarities.  Distances allow more reliable conclusions to be drawn from the comparison of several scores computed for alternative alignments of the same set of sequences.  Any scores that are to be compared should be computed by the same method and using the same scoring table as well as the same sequences.

Score comparison can be used either to compare different alignments or to compare different trees.  It is also legitimate to compare combinations that include both different alignments and different trees, that is, compare different evolutionary hypotheses.

Phylogenetic tree scoring is biologically more reasonable than sum of pairs scoring but is much slower.  Phylogenetic tree scoring uses the weighted parsimony scoring described by D. D. Sankoff.  This scoring procedure assigns sequence residues to all internal nodes of a user supplied bifurcating tree.  The assignments made are those that minimize the scores (weights) associated with the pairs of residues on adjacent nodes of the tree. This involves testing a large number of allowable combinations and is hence very time consuming.  However, this can be worthwhile, especially when the placement of gaps is involved.  Sum of pairs scoring has a strong tendency to force gaps to be in the same locations in the sequences that require gaps.  This happens even when it is clear that there must have been multiple insertion or deletion events in the evolutionary history of the sequences.  Phylogenetic tree scoring will score the separate insertion or deletion events as separate events even if they are assigned to the same location in the sequences.

The sum of the pairs score for a designated block of a multiple sequence alignment of NS sequences the same score that would be computed by summing the scores for each of the ((NS-1)*NS) / 2 independent pairs of aligned sequences.  Thus the name, sum of pairs scores.  Gaps are scored using Stephen Altschul's Natural gap cost for sum of pairs scoring and an afine gap penalty function.

( Gap Penalty = NewGap + Length_of_Gap * GapLen ).

The scoring table used for scoring is selected in the Configuration dialog on the Scoring tables page.  The built in scoring tables include a selection of PAM (Point Accepted Mutation) tables, and BLOSUM (BLOck SUM) tables for protein sequences.  For nucleic acid sequences there are PAM tables for both an equal number of transitions and transversions and for a 3 to 1 ratio.  The scores are computed as distances (sometimes referred to as costs).  This makes the comparison of the scores from several different alternative alignments of the same sequences more informative.  The same scoring table should be used for all scores in a comparison.

Each scoring table has associated with it default values for the NewGap and Length_of_Gap penalties.  It also has a default group or set of residues that can be treated as equivalent when shading alignment columns.  All of the members in each set of equivalent residues have a positive (greater than zero) score with all other members of the set of equivalent residues in the similarity scoring table.  This is a useful and objective definition of conservative substitutions for a family of sequences.

Log Odds, or Minimum entropy scoring is one of the advances derived from the application of probability theory to multiple sequence alignments.  It is closely related to the scoring functions used to guide the creation of Hidden Markov Models (HMMs) and multiple sequence alignments derived from the HHMs.  Additional information can be found in chapter 6.2 of Durbin, R., Eddy, S., Krogh, A., and Mitchison, G.  1998. "Biological Sequence Analysis: Probabilistic models of proteins and nucleic acids."  Cambridge University Press.

The minimum entropy score is derived by computing an entropy measure for each selected column of the alignment and then summing the entropies for all of the selected columns.  Lower scores correspond to better alignments, with a score of zero being the limiting score for an alignment made up entirely of columns that contain only a single, completely conserved, sequence residue.  Since GeneDoc uses pseudocounts to adjust for small sample sizes, GeneDoc will never actually report a score of zero.

The entropy for each column is computed with the following formula.

  Entropy = Sum over i of { f(i) * -log( f(i) ) }.

Where i denotes the different kinds of that can be present in the sequence, 20 amino acids or 4 nucleotides, and f(i) is the fraction of amino acids in a particular column of the alignment that are of kind i. As can be seen from this formula, the highest scores will be reported for columns that have all possible residues present in equal numbers.

In order to use minimum entropy scoring the user must specify parameters for computing pseudocounts.  GeneDoc uses Henikoff (Henikoff & Henikoff, 1996) style pseudocounts in calculating minimum entropy scores and other log-odds measures on the alignment.  These pseudocounts are computed using the matrix of observed exchange frequencies, the Q(ij) matrix, that underlies substitution or similarity scoring matrices.  Thus the Q(ij) matrix is the first item the user must specify.  For proteins, the Q(ij) matrix is a square 20 by 20 matrix with an entry for every pair of amino acids, i and j.  Each column of the matrix contains the 20 frequencies that tell how often a particular amino acid is observed to be converted to one of the other 19 amino acids or to remain the same. Like similarity matrices the observed exchange frequencies are different for different degrees of evolutionary divergence between the sequences.

The basic idea behind probabilistic pseudocounts is to add to the alignment the effects that are likely to be observed as more new sequences are added to the alignment.  Thus for alignments with many sequences from widely divergent species and functional subfamilies new amino acids (or nucleotides) that are not already present at a particular column of the alignment are not very likely to be observed in any new sequence.  Thus the appropriate choice for the Q(ij) matrix in these circumstances, is one for a low degree of evolutionary divergence since you would have a low expectation of seeing new amino acids or nucleotides.

In the circumstances where you have few sequences from closely related species and little or no subfamily variation it would be appropriate to choose a Q(ij) matrix for high evolutionary divergence.  This would reflect your expectation that if you gathered many new sequence from divergent species that you would discover many amino acids not previously seen at each column of the alignment.

The second item that the user must select is the pseudocount multiplier, PCM.  This multiplier determines the total number of pseudocounts added to each column of the alignment.  The total number of pseudocounts added to a specific column of the alignment is given by the product of the PCM multiplied by the total number of different kinds of sequence residues present in that column.  Thus column that is a completely conserved, with only a single amino acid present will have many fewer pseudocounts added than will a column that has several different kinds of amino acids (or nucleotides).  This accurately reflects our expectation that over even very long evolutionary periods, sequence positions with highly conserved residues will not vary as much as sequence positions that show appreciable variation over even short evolutionary periods.

The emerging consensus is that a relatively high number of pseudocounts is appropriate.  These will be largely overwhelmed by large alignments with many diverse sequences but will exert a noticeable effect on small alignments with few sequences.  This is basically saying that with alignments of only a few sequences we are unsure about the reliability or stability of the degree of observed sequence invariability - that new sequences may change it drastically.  Thus the Henikoff's suggest a value of 4 or 5 for PCM.

The suggestion of a PCM of 4 or 5 was based of the results of searching sequence databases with profiles made using this kind of pseudocounts rather than the traditional Gribskov style profiles.  The Henikoff's found that profiles constructed this way were more effective than the traditional Gribskov style profiles.  GeneDoc computes the Henikoff style profiles under its log-odds functions and exports them in the MEME format.  These can be easily edited into the GCG profile format and used with the GCG ProfileSearch program.  Also, programs for calculating the Henikoff style profiles and using them to search sequence databases are available at the Pittsburgh Supercomputing Center.

The third thing to note in computing the minimum entropy scores is that the sequence weights, which can be set in the sequence dialogue box, are used.  The weights are also used in computing other log-odds scores.

Henikoff, J.G., and Henikoff, S.  1996  Using substitution probabilities

to improve position-specific scoring matrices.  CABIOS, vol. 12, pp. 135

- 143.  (CABIOS has been renamed to Bioinformatics).

Shortcuts

Toolbar:

Keys:	CTRL+R


Write LogOdds Matrix
Write LogOdds Matrix (Scoring menu )

This option is activated when you select a range of residues. This option will first give you the Write Log Odds Matrix dialog. Here you can make any adjustments to the LogOdds matrix variables or select any sequences to be EXCLUDED from the matrix calculation.

After that, you will be given a file Save As dialog to chose a file name and path.

The LogOdds matrix is written in MEME/MAST format. It can be imported into MAST for database searching. It could also be re-imported into GeneDoc for use in shading or producing a LogOdds Report

The Sequence Weights found in the Sequence Dialog are used when creating and writing the LogOdds Matrix.


Write Log Odds Matrix Dialog
Write Log Odds Matrix Dialog (Scoring menu )

Change any of the LogOut output parameters and/or select and sequence to EXCLUDE from the Log Odds Matrix output.

BackGround

Determines where to obtain the background counts for the LogOdds matrix. If Static, then preprogrammed values obtained from a GeneBank search are be used. If Align, then the entire alignment is used to determine background frequencies.

Pseudo Table

Select a table to be used as the Pseudo Table.

Pseudo Multiplier

Enter a Pseudo Table multiplier.

A,C,G,T

Enter Background counts for MIXED/DNA/RNA projects.


Re-Score All
Re-Score all Scores (Scoring menu )

This function re calculates and updates all score areas in the project.

You can get a re score of a specific area by double clicking on it, or using the Score Accelerator key


Score Key
Re Score Key (Scoring menu )

Use this key when you want to re score the area the caret is in.


Clear all Shading
Clear all Scores (Scoring menu )

This function clears all the scores created.


Show Score Table
Show Score Table (Scoring menu )

This menu entry shows the table of scoring values in Distance form. The Gap and Extend Gap distances are shown as well. The Zero Cost Distance is shown.

See Score Selection for more information.


Score Table
Score Table. (Show Score Table )

This table shows the current values of the scoring array. It can be used to help understand the scoring, scoring tables, or debug output.


Show Score Info
Show Scoring Info (Scoring menu )

This function enables Acsii debugging output from the Phylogenetic scoring routine. If there is an area already scored, the score will be recalculated so the scoring output can be generated, otherwise scoring output will be generated next time a selection is scored. The output is shown on the screen, if it fits, and is saved to a file. The filename will be the same as the current projects name with a .tsd extension.

See Score Selection for more information.


Score Cancel
Score Cancel Dialog (Scoring menu )

This dialog lets you cancel the Phylogenetic scoring function. The numbers in the dialog, columns and trees, show some indication of status. The columns show the number of columns left to be scored, and the trees shows the total number of trees scored so far.


Tree menu
Tree Menu (Help Index )

The tree menu controls Tree View specific commands.

For more information, select the Tree menu item name below.

Copy to Clipboard	Copy the tree image to the clipboard as a bitmap.
Create Node	Combine two selected tree components and make a new node.
Delete Node	Delete all selected tree nodes.
Move Up	Move the currently selected node up if possible.
Move Down	Move the currently selected node down if possible.
Clear Tree	Clear all nodes on tree.
Display Tree Weights	Toggles between displaying node numbers and node weights.
Manage Expression	Open Phylogenetic tree expression dialog box.


Copy tree to clipboard
Copy to Clipboard (Tree menu )

Use this command to copy an image of the tree to the clipboard in monochrome DIB format.

Shortcuts

Toolbar:


Create Node
Create Node (Tree menu )

Combine two selections into a Node. A shortcut for this with the mouse is to click to select the first branch for the new node and then double click the second branch. This will create a new node automatically. The first branch will always be the uppermost branch in the new node.

Shortcuts

Toolbar:


Delete Node
Delete Node (Tree menu )

Delete selected nodes. A shortcut for this with the mouse is to double click the node to be deleted.

Shortcuts

Toolbar:


Move Node Up
Move Up (Tree menu )

Move the selected Node or Leaf up one notch. It will remain the selected Node/Leaf.

Shortcuts

Toolbar:


Move Node Up
Move Down (Tree menu )

Move the selected Node or Leaf down one notch. I will remain the selected Node/Leaf.

Shortcuts

Toolbar:


Clear Tree
Clear Tree (Tree menu )

Delete all nodes in the tree after confirmation.

Shortcuts

Toolbar:


Display Tree Weight
Display Tree Weight (Tree menu )

This toggles between displaying the tree branch and node numbers and the tree branch and node weights. The weights are usually imported from a tree file, though they can be manually input through the Manage Tree Expression dialog


Manage Tree Expression
Manage Tree Expression (Tree menu )

This menu item brings up the Manage Tree expression dialog. Here GeneDoc will show you the expression for the current state of the tree.  The expression uses the New Hampshire nested parentheses notation to describe a tree.  This is the same notation used to describe a tree in the Phylip suite of phylogenetic analysis programs and in the Nexus file format used by several other phylogenetic programs.  There are buttons for import, export, copy and paste for the expression.

Shortcuts

Toolbar:


Manage Tree Expression
Manage Tree Expression Dialog (Tree menu )

Tree Expression Edit Window.

This input field allows you to manually modify the expression. You can put in extra CR’s and SPACES for more comprehensible formatting, but they will be discarded when you click the OK button.

OK Button.

This button will cause GeneDoc to parse the expression in the Expression Edit window and use it for the Current Project’s phylogenetic tree if it parses successfully. If the tree does not parse properly, an error message will be displayed and the Manage Tree Expression dialog box will be reopened, allowing you to correct the tree expression.  The expression must conform to the New Hampshire nested parentheses notation for describing trees.

Cancel Button

Ignore the current tree expression and close the dialog box.

Export Button

This button allows you to export the current tree expression to a file. You will be prompted for a file name.

Import Button

This button allows you to import the current tree expression from a file. You will be prompted for a file name.  The imported expression must conform to the New Hampshire nested parentheses notation for describing trees.  The file may contain just the bare tree (Phylip format) or a limited amount of additional information in the Nexus format.

Copy Button

This button allows you to export the current tree expression to the clipboard as text in the New Hampshire nested parentheses notation for describing trees.

Paste Button

This button allows you to import the current tree expression from the clipboard as text.


Report menu
Reports Menu (Help Index )

The Views menu contains items that select the various views of GeneDoc.

For more information, select the Views menu item name below.

Statistics Report	Creates a Identities and Similarities Statistics report.
Score Report	Creates a Score between sequences report.
Base Composition Report	Creates a Base Composition Report.
Column Composition Report	Create a Report showing the composition of columns in the alignment.
Column Composition W/Weights	Create a Report showing the composition of columns in the alignment, using the weights in the Sequence Dialog
Enzyme Fragments	Create an Enzyme Fragments report. You must have a DNA project loaded and a ReBase strings in the search configuration for this function to work.
LogOdds Report	Creates a file LogOdds scores for selected sequences and a selected motif.
Shading Report	Creates a report detailing the locations of the current shading mode.
Manual Shading Rep	Creates a report detailing the locations of all the manually added shading locations.
Configure Reports	Opens the configuration dialog to the reports tab.
Save Report File	Saves the current report as a text file.


Create Statistics File
Statistics report (Reports menu )

This function creates a project.sta file that holds statistics describing the relationships between pairs of sequences. Each sequence is compared to every other sequence and three numbers are generated.

1: The number of residues that match exactly (identical residues) between the two sequences.

2: The number of residues whose juxtaposition yields a greater than zero score in the current scoring table (similar residues or conservative substitutions).

3: The number of residues lined up with a gap character.

Each number is expressed as a count and as percentage on different sides of the matrix diagonal. The diagonal shows how many locations have at least one residue for the single sequence, i.e., the sequence length.

The statistics reports are only updated on command, so if you make any residue arrangements, you will have to regenerate the statistics report.

Shortcuts

Toolbar:


Create Score File
Score Report (Reports menu )

This function creates a report that holds a square two-way table of the scores between individual sequences. Each sequence is compared to every other sequence and one number is generated, the score between the two complete sequences.

Each number is expressed in absolute value. The diagonal shows the score of the sequence against itself.

The score report are only updated on command, so if you make any residue arrangements, you will have to regenerate the score file for those changes to be reflected in the scores.

Shortcuts

Toolbar:


Base Composition Report
Base Composition Report (Reports menu )

This function creates a report that gives the relative percentage of each of the base types in the alignment. You can select a range for this report.

Shortcuts

Toolbar:


Column Composition Report
Column Composition Report (Reports menu )

Creates a report showing the composition of all the selected columns. All possible AA’s are shown, each one with a fraction of the total, all fractions summing to 1.0. Gaps are ignored, each value in the column counts for 1 unit of its type.


Column Composition W/Weights Rep
Column Composition W/Weights Rep (Reports menu )

Creates a report showing the composition of all the selected columns. All possible AA’s are shown, each one with a fraction of the total, all fractions summing to 1.0. Gaps are ignored, each value in the column counts for Weight units of its type.


Enzyme Fragment Report
Enzyme Fragment Report (Reports menu )

This runs the enzyme fragment report.

You have to have the search display mode in Rebase syntax mode and at least one motif entered. If these criteria are met, then you will get the Search Report dialog. This allows you to select or deselect the various reports.

For each sequence list, the total sequence length is listed afterwards enclosed in square brackets, [999].

The following reports are currently generated.

1: Cut Sites.

For each enzyme, the location where it cuts sequences. The  motif name and pattern is listed, then a list of sequences. For each sequence listed, a list of cut sites follows.

AciI C'CGC

------------------- CUT SITES -------------------------------------

CESNU41   [143] 83

CESNU42   [145] 8 84

CESNU43   [145] 84

2: Sorted Fragment Lengths.

For each motif, a sorted list of fragment lengths for each sequence the enzyme cuts.  The fragments lengths are sorted from longest to shortest.

AciI C'CGC

---------------- SORTED FRAGMENT LENGTHS --------------------------

CESNU41   [143] 82 60

CESNU42   [145] 76 61 7

CESNU43   [145] 83 61

3: Unique Fragment Lengths.

For each motif, the fragment lengths are check for uniqueness within a user set percentage. The percentage defaults to 20 percent, so that if a cut sequence creates a fragment that is unique in size within + or - 20 percent (or whatever user value), then it will be listed in this report.

AciI C'CGC

---------------- UNIQUE FRAGMENT LENGTHS --------------------------

CESNU41   [143] 60 82

CESNU42   [145] 7 61

CESNU43   [145] 61 83

4: Group Specific Fragment Lengths

To be unique to a group then the fragment length should be present in all sequences of the group to within plus or minus 5% and should not be present outside the group within the previous plus or minus 20% range. Actually the values are user adjustable. This makes for easy test to identify whether a new kind of bean belongs to one family (group)  or another.  Sort of like paternity testing.

AluI AG'CT

---------------- GROUP SPECIFIC FRAGMENT LENGTHS ----------------

g1: 72 72

g1: 54 55

g4: 123 125

5: Sorted Cut Sites Report.

All cut sites are sorted and printed out one per row. First column is the cut site, second column is the enzyme name, third column is the sequence name and total length.

---------------- SORTED CUT SITES ---------------------------------

    8 AciI       CESNU42[145]

    8 AciI       CHKURU4A[146]

    8 Hin6I      ATU41SN[155]

    8 Hin6I      CHKURU4C[142]

    9 DdeI       HUMURU4B[145]

    9 DdeI       HUMURU4C[142]

    9 DdeI       RATURU4A[146]

    9 DdeI       RATURU4B[145]

    9 DdeI       RATURU4C[142]

    9 SfcI       TTU41[136]

   10 CfoI       ATU41SN[155]

   10 CfoI       CHKURU4C[142]

Shortcuts

Toolbar:


Fragment Report Dialog
Fragment Report Dialog (Help Index )

Select which reports you want calculated.

See Enzyne Fragment Reports  for more information.


LogOdds Report
LogOdds Report (Reports menu )

This runs the Logodds report. This options first brings up the LogOdds Report Dialog which enables you to select a motif and a set of sequences. Motifs are setup in the LogOdds Tab. The LogOdds scores for the chosen motif for each sequence will then be shown in the report view.

It is expected that this report will be imported to a graphing program.


LogOdds Report Dialog
LogOdds Report Dialog ( LogOdds Report )

Select a single motif and one or more sequences.

See LogOdds Reports  for more information.


Shading Report
Shading Report (Reports menu )

Creates a report detailing the locations of shading produced by GeneDoc.


Manual Shading Report
Manual Shading Report (Reports menu )

Creates a report detailing the locations of manual shading entered by users of this alignment.


Configure Reports
Configure Report (Reports menu )

Jump to the Configuration Dialog and open the Report Tab.


Save Report File
Save Report File (Reports menu )

This saves the currently viewed report as a text file. You will be given a File Save dialog from which to set the file name and save location.


Dstat menu
Plot Menu (Help Index )

The Dstat menu controls DStat View specific commands.

See SuperFamily Group Features for more information.

See Percent Identity Plots for more information.

For more information on the menu item, select the DStat menu item name below.

Plot % Identity	Shows a C.D.F. plot for the Alignments Percentage of Identities.
Plot % Fav. Subs.	Shows a C.D.F. plot for the Alignments Percentage of Favorable Substitutions.
Plot Group % Ident.	Shows C.D.F. plots and Dstat analysis between a group and all other sequences or between two groups.
Plot Group % Fav Subs.	Shows C.D.F. plots and Dstat analysis between and group and all other sequences or between two groups.
Plot Unique Fragments	Plots unique fragments as generated by the Enzyme Unique Framents report.
Score Selection (Groups)	Score the Highlighted selection. Use only residues that are within defined groups
Score Selection (All)	Score the highlighted selection Use all residues, though there must be at least one group.
Copy to Clipboard	Copy the displayed graphic to the clipboard as a metafile..
Setup Group	Open the DStat graph setup dialog.
Write Plot data.	Write the data used to display the graph to a data file. This would represent the post calculation results.
Write Score Data	Write the scoring data used as an input to the DStat calculations to a file. Mostly useful for debugging purposes.
Read Set 1	Reads an external Data file into the Within group.
Read Set 2	Reads an external Data file into the Between group.
Read Both Sets	Reads an external Data file into the both the Within and Between group.


Dstat Plot Identities
DStat Plot % Identities (DStat menu )

Create a CDF plot of the Percentage of Identities for the alignment. See Percent Identities Plots for more information.


Dstat Plot Identities
DStat Plot % Favorable Substitutions (DStat menu )

Create a CDF plot of the Percentage of Favorable Substitutions for the alignment. See Percent Identities Plots for more information.


Dstat Plot Identities
DStat Plot Group % Identities (DStat menu )

Create a CDF plot of the Percentage of Identities between a group and all other sequences or between two groups in the alignment. You will be presented with the group selection dialog, pick a group on the right and <all other> on the left or another group. See Percent Identities Plots for more information.


Dstat Plot Identities
DStat Plot Group % Favorable Substitutions (DStat menu )

Create a CDF plot of the Percentage of Identities between a group and all other sequences or between two groups in the alignment. You will be presented with the group selection dialog, pick a group on the right and <all other> on the left or another group. See Percent Identities Plots for more information.


Dstat Plot Unique Fragments
DStat Plot Unique Fragments (DStat menu )

Create a CDF plot of the unique fragments as generated by the Enzyme Fragment Report.


Dstat Score Selection (Groups)
DStat Score Selection (Groups) (DStat menu )

This command will score the current selected columns and open the DStat graph display. There must be at least two groups defined for there to be a Within group and a Between group.

See SuperFamily Group Features for more information.


Dstat Score Selection (All)
DStat Score Selection (All) (DStat menu )

This command will score the current selected columns and open the DStat graph display. There must be at least one group defined. The sequences within a defined group will be scored in the within set, any other sequences, including those not in groups, will be scored in the between set.

See SuperFamily Group Features for more information.


Dstat Copy to Clipboard
DStat Copy to Clipboard (DStat menu )

This command will copy the graph to the clipboard as a metafile. This can then be pasted into word or whatever.


Dstat Setup Graph
DStat Graph Setup (DStat menu )

This menu item opens the DStat graph setup dialog.


Dstat Graph Setup Dialog
DStat Graph Setup Dialog (DStat menu )

Page Width

The width in inches of the graph.

Page Height

The height in inches of the graph.

X Border %

The distance from the bottom of the X Axis on the display.

Y Border %

The distance from the bottom of the X Axis on the display.

DStat X %

The X distance from the top of the page of the DStat displayed value.

DStat Y %

The Y distance from the top of the page of the DStat displayed value.

KSpr X %

The X distance from the top of the page of the KS probability displayed value.

KSpr Y %

The Y distance from the top of the page of the KS probability displayed value.

Within Count

The number of scores included in the within group.

Between Count

The number of score included in the between group.

Within Set Larger

Check if the score values of the within group are larger than the between group. Not typically the case..

Between Set Larger

Check if the score values of the between group are larger than the within group. Typically the case..

Either Set Larger

Check if the score values of the between and the within group are of unknown relationship.

Chart Title

A string that will be displayed on the top of the graph.

Y Axis Title

A string that will be displayed vertically on the Y-axis of the graph.

X Axis Title

A string that will be displayed on the X-axis of the graph.

Chart Title %

Location of the Chart title away from the left hand side of the page.

Y Axis Title %

Location of the Y Axis title away from the bottom of the page.

X Axis Title %

Location of the X Axis title away from the left hand side of the page.

Line Width

Number of pixels used to draw the graph lines.

X Axis Rounding

The rounding factor used to determine the values of the x axis scale.

Text Font button

Button brings up the font selection dialog to select the font characteristics of the text displayed on the graph.


Group Select
Group Select (DStat menu )

This dialog is used for two purposes.

If the case of a DStat option, select which group will be the first group in the plot and which group will be the second group in the plot.

In the case of a Copy Sequences Data option, select which sequence to copy from on the left list and which sequence to copy to on the right list.


Dstat Write Plot Data
Write Plot Data (DStat menu )

This menu item opens a save file dialog so that you can write the values used to draw the graph to an Ascii file. This would be useful for importing in another graphing program.


Dstat Write Scores
Write Score Data (DStat menu )

This menu item opens a save file dialog so that you can write the values input to the Dstat calculation subroutines in Ascii format. This may be useful for debugging purposes.


Dstat Read Set 1
Read Set 1 (DStat menu )

This menu item opens a open file dialog so that you can read Ascii data into the within set. One floating point value is expected per row in the file. This is here in case you feel like doing this.


Dstat Read Set 2
Read Set 2 (DStat menu )

This menu item opens a open file dialog so that you can read Ascii data into the between set. One floating point value is expected per row in the file. This is here in case you feel like doing this.


Dstat Read Both Sets
Read Both Sets (DStat menu )

This menu item opens a open file dialog so that you can read Ascii data into both the within and between data sets. One floating point value is expected per row in the file. The first values will go into the within set. When a row is encountered with the character ‘X’ or ‘x’ in it, then subsequent data will be read into the between set. This is here in case you feel like doing this.


Percent Identity Plots
Percent Identity Plots (Help Index )

The percent identity plots in GeneDoc are an alternative to tables showing the percentage of sequence residues that are identical in every pair of sequences in the alignment.  Such tables have often been published as a way to describe the degree of divergence among the sequences in the alignment.  This has been a useful guide to the probable amount of information available in the alignment and robustness of the alignment.  More divergent sequences make the alignments more informative but also less robust and reliable.

The data in these plots can be either strict identities or the count conservative or favorable substitutions as if they were also identical residues.  These are the same values that GeneDoc computes for the “Stats” or statistics view where they are shown in the upper right triangular part of the table.

This type of plot is called a cumulative distribution function (c.d.f.) or sometimes an empirical cumulative distribution function.  In this context empirical refers to the fact that the plot is experimental, or empirical, data rather than derived from a mathematical equation describing a theoretical distribution such as the gaussian distribution.  The plot is created by first sorting the data to be plotted into ascending order.  Then for each data point you compute the fraction of data points that have the same or a smaller value.  The data is then compressed to eliminate multiple points that have the same data value.  The point with the highest value for the fraction of data items with the same or lower value is retained during compression.  These compressed data points are the plotted as a step function.

The step function has as its horizontal axis the data values being plotted.  The vertical axis is the fraction of data points with as small or smaller a data value.  Thus it always ranges from 0.0 to 1.0.  Plotting the data as a step function requires drawing two line segments for all of the data point except the first.  For the first data point we draw a single vertical line from the horizontal axis up to the first data point.  For all subsequent points we draw two line segments.  The first line segment is a horizontal segment from the data value of the previous point to the data value of the current point.  This line segment is drawn at the fractional value for the previous data point.  This indicates the function retains this value until the next observed data value is reached.  The second line segment is a vertical segment from the fractional value of the previous data point to the fractional value of the current data point.  This gives the plot its characteristic appearance of a set of irregular steps.

The plot is related to the more commonly used histogram plot in a straight forward manner.  As the name, c.d.f., implies it is the cumulative plot of the same data plotted in the histogram.  The c.d.f. can be thought of as the integral of the distribution plotted in the histogram.  This leads many users to ask “Well, why can’t I have the histogram, it is easier to look at?”  Our response is that c.d.f.s are also easy to look at, you just have not looked at them enough to be comfortable looking at them yet.  More importantly, because drawing a histogram of experimental data, an empirical histogram, always requires putting the data into bins.  This process of  putting the data into bins always results in the loss of some information that is not lost in the c.d.f..  One piece of information that is immediately obvious from the c.d.f. of any distribution is its median.  With a histogram the approximate location of the median is easily seen only for smooth, symmetrical, unimodal, and unskewed distributions, such as the normal distribution.

Another important reason for using c.d.f.s rather than histograms is the Kolmogorov-Smirnov (K-S) test for the difference between distributions.  The K-S test determines whether two distributions are different from each other and is performed on their c.d.f.s.  Thus it can easily be presented in terms of the c.d.f.s, but not in terms of the histograms.  GeneDoc uses this test in several places.  It is used in conjunction with alignment scores to test whether the groups you have defined are statistically distinct.  It does this by comparing the distribution of scores between pairs of sequences within groups with the distribution of scores between pairs of sequence in different groups.  If the groups are distinct the scores for pairs of sequences in different groups are larger than the scores for pairs of sequences within the same group.

GeneDoc also uses the K-S test to tell you if the sequences in one group are more diverged from each other than are the sequences in a second group.  The K-S test allows GeneDoc to compute a significance probability to tell you how likely the observed difference in the c.d.f.s is to have occurred by chance in groups of the same size.  The K-S test is a useful general purpose test for differences between two groups because it measures all aspects of the differences between groups.  That is, the K-S test will pick up differences in the shape of the c.d.f.s of the two groups as well as differences in the average of two groups.

More commonly used test such as Students t test and the F test detect differences only in the average or the variance of the groups respectively.

The percent identity plot is also useful for selecting a scoring matrix to use with the alignment.  Particularly for protein sequence alignments the scoring matrix is important both for computing alignment scores and for determining which amino acids are included in the equivalence groups defined and used in some GeneDoc functions.  The PAM matrices for both proteins and nucleic acids and the Blosum matrices for proteins are tuned for use with groups of sequences that have diverged to a different degree.  The right hand column in the table below shows the degree of divergence appropriate for different PAM matrices shown in the middle column.  The left hand column identifies the Blosum matrix that supplies the same amount of entropy per position in an alignment.  Matrices that show the same amount of entropy per position in an alignment are suitable for sequences of the same degree of divergence.

Comparable Blosum and PAM Scoring Matrices based on Equivalent Entropy

Blosum Table Degree of Identity	Entropy of the Blosum Table	PAM Value  of the Table	Entropy of the PAM Table	Percent Sequence Identity of the PAM Table
Blosum 90	1.18	PAM 100	1.18	43
Blosum 80	0.99	PAM 120	0.98	38
Blosum 60	0.66	PAM 160	0.70	30
Blosum 52	0.52	PAM 200	0.51	25
Blosum 45	0.38	PAM 250	0.36	20


Window menu commands (Help Index )

The Window menu offers the following commands, which enable you to arrange multiple views of multiple documents in the application window:

Alignment View	Activates the GeneDoc Alignment View.
Summary View	Activates the GeneDoc Summary View.
Report View
Phylogenetic Tree	Activates the GeneDoc Phylogenetic Tree view.
DStat View	ReOpens the DStat View if one has been created. Does NOT create a rescoring of a selection.
Cascade	Arranges windows in an overlapped fashion.
Tile	Arranges windows in non-overlapped tiles.
Arrange Icons	Arranges icons of closed windows.
Toolbar	Shows or hides the toolbar.
Status Bar	Shows or hides the status bar.
Window 1, 2, ...	Goes to specified window.


Score by Phylogenetic Tree
Alignment View (Windows menu )

This is the main view which shows the aligned sequences, the consensus sequence, scored blocks, and any highlighting.  Alignment editing is carried out in this view.  See the Alignment View section for more information.

Shortcuts

Toolbar:


The GeneDoc Display
The Alignment View (Windows menu )

See Table of Contents for more information.

Multiple Document interface

GeneDoc runs as a standard windows Multiple Document program. This means that more than one .MSF file can be open at the same time. Also, subsections of an alignment may be selected, put into their own window, worked on separately and saved as new .MSF files. GeneDoc has a ‘Windows’ menu where windows can be tiled, cascaded and tracked.

Sequence Blocks

GeneDoc presents sequence information in the form of multiple blocks.  Each of these blocks is composed of several lines, one for each sequence in the alignment plus three annotation lines.  The annotation lines are the upper and lower consensus sequence lines and the scores line.  The user can enter custom annotation on any of the annotation lines. The width of these blocks can be set to either adjust automatically to the current window width, or be a fixed width based on the number of characters desired per row. This is done in the Display page of the Configuration dialog.  There are enough blocks in the document to accommodate the entire length of the alignment.

Each block is composed of the following lines or rows.

The score line

The first line of a sequence row is the score line. When you select a range of sequence columns and use the Score function, a score is calculated for the selected columns using the current scoring table and Substitution Groups and  displayed on the score line. A typical score might look like this ““[___2845___]””.  If the sequence columns of a scoring region are modified by an editing operation the score is marked as no longer valid by striking through the characters of the score.  Multiple ranges of scores can be built, but the sequence columns for each range of scores cannot overlap. If you score over a previously scored area, the remainder of the old score will be removed. Users can manually add comments on this line by moving the cursor on the line and typing characters on the keyboard. These manually entered comments are not saved and will be wiped out if they fall within the range of a scoring selection.

You can update an invalidated score by double clicking in the area of the score box while in any arrangement mode. Also, Updating the GeneDoc display with an autoshade function will recalculate any invalidated score. Furthermore, if the scoring table or Substitution Groups are changed, then all scores on the score line will be updated.

Upper consensus line

The second line of a sequence row is the upper consensus line. If the Configuration dialog setting, Consensus/Line Above is set, then any consensus information built by a shading action will be displayed on this line. If the shading clear function is used, the consensus line will be cleared. Users can manually add comments on this line by putting the cursor on the line and typing characters on the keyboard, but these are not saved and any subsequent shading action will remove them.

A consensus line will have asterisks ( ‘*’ ) in it if any part of it has been invalidated by residue rearrangement. Only the invalidated parts of the consensus line will have asterisks in it and they will be cleared by using one of the autoshade functions.

Sequence Lines

After the Score Line and the Upper Consensus Line the Sequence lines begin.  Immediately below the Sequence Lines there is a Lower Consensus Line, the last line in the alignment row.

The first eleven characters of the line are the sequences name and a colon, ‘:’. The name can be edited in the sequence dialog.

The next N characters are the single letter codes for the sequence residues, either nucleic acid or protein. The letters coding the sequence residues are not generally editable to prevent accidental change of the sequence by adding, deleting, or changing sequence residues.  GeneDoc will insert a gap  character (either a dash ‘-’ or a dot “.”) into any gaps created while arranging the sequences. Placing the cursor over any position in these lines and pressing keys (other than function or accelerator keys) will have no effect unless GeneDoc is in Residue Edit mode.  In Residue Edit mode the sequence residues can be added, deleted, or changed.

When the program is in residue edit mode, a block caret will be shown instead of a line caret. Any single click of the mouse button will move the caret to the new location in residue edit mode. If the caret is over a residue or gap, any proper Alpha character for the Project Type will over write the current residue value and advance the caret.  Inserting new residues is accomplished by inserting gap characters in alignment editing mode and then changing them to sequence residues in residue editing mode.  The reverse operation is used to delete residues.

The last six characters of this line show a colon, ‘:’, and a number.  This number gives the sequence location of the last sequence residue in the line.  Only sequence residues are counted, gaps are ignored in this count.  This number is automatically updated when you are arranging the sequence characters on this line, though it will not change unless the last character on the line is changed by the editing operation.

The Sequence Location indicator can be toggled On and Off in the GeneDoc Configuration Dialog.

Lower consensus line

The last line of a sequence row is the lower consensus line. It works the same as the upper consensus line.  You can select which of the two consensus lines is used by GeneDoc to display consensus sequence information in the Configuration Dialog.

Shortcuts

Toolbar:


Summary View Menu
Summary View (Windows menu )

The Summary view displays only an indication of either a residue or a gap. See Summary View for complete information.

Shortcuts

Toolbar:


The Summary View
The Summary View (Windows menu )

See Project Configuration for more information.

The Summary view displays only an indication of either a residue or a gap. The views blocks are the same height as the Alignment View, but their width can be greatly reduced. All shading information can be applied to the view as well.

For each sequence, a a summary line is displayed. In the summary line, if there are residues, two lines are displayed. If there is a gap in the position, a single line though the middle is displayed. Background shading information for the column is added to the background of the column, and the foreground coloring is used to draw the residue lines, unless overridden. The residue lines can be always displayed in black with the ‘Text Black’ option.

The summary view uses a configuation parameter of Columns Per Inch when drawing. The input columns per inch is compared with the output resolution (out can be either display or printer), and a closest actual value is determined. For example, the typical CRT display is 96 dots/inch. If you input 50 columns per inch, you will actually get 96 columns per inch. In you input 40 col/inch, you would get an actual 48 col/inch. In the former case, 1 pixel would be used to draw a column, thus giving you 96 col/inch. In the later, 2 pixels would be used to draw each column resulting in 48 co/inch. The configuration dialog shows both the input and actual values for columns per inch for the display.

The summary view draws in either 1, 2, or 3 or more pixels per column. 3 pixels or more per column gives the best results. If your printer prints at 300dpi, then a setting of 100 columns per inch will give you 96 columns per inch on the display, being drawn at 1 pixel per column, but will give you 100 columns per inch on the printer being drawn at 3 pixels per column, and will look much better when printed.

A Mac pict file is defined as being a resolution of 72 dots per inch, and when you copy the summary view selection into a pict file, this should be taken into consideration. In order to get your columns draw as 3 pixels per column in a pict file, the columns per inch value should be set to 72 / 3 = 24. Of course 48 columns per inch would give 2 pixels per column in a pict file and 72 or more will give 72 columns per inch drawn with 1 pixel per column. There are cases were what you see on the screen will not be the same in your pict file since the resolutions are different. 32 columns per inch for example, will give you 32 col/inch with 3 pixels per column on the display, but 48 col/inch with 2 pixels per column in a pict file.

When the summary view is drawn with 2 pixels per column, you can choose either the 2/0 or 1/1 mode in the Configuration Dialog. The 2/0 mode will display columns the same as with 1 pixel per column logic only at twice the width. This display would look best with the ‘’Text Black’ option checked, which forces the lines dispalyed to always be in black. This setup would be good for pict files. The 1/1 mode displays columns in a fashion that emphasizes the background shading more.

The Text Black option forces the residue lines to be drawn always in black. This looks better for higher values in the columns per inch value.

The Def Disp option causes GeneDoc to automatically switch to the Summary View when this data file is loaded.

Shortcuts

Toolbar:


Report View Menu
Report View (Windows menu )

The Report view display the most recent report to have been generated. You can save and print these reports with the Save Report File function

Shortcuts

Toolbar:


Report View
Report View (Windows menu )

The Report view is used to display any textual information, typically created by functions under the Report Menu.

You can save and print these reports with the Save Report File function when the reports view is active.

Shortcuts

Toolbar:


The GeneDoc Display
The Phylogenetic Tree View (Windows menu )

The Phylogenetic tree view displays only manually loaded or entered information. The interface uses Vermont Style tree syntax. You can load or edit in information. See Tree View for more information.

Shortcuts

Toolbar:


Phylogenetic tree view
The Phylogenetic Tree View (Windows menu )

This view enables the user to create and edit the Phylogenetic tree for this project.  The tree can be created by clicking on a pair of nodes that are to be joined as the descendant nodes of an ancestral node created in the process.  The original nodes are the individual sequences in the alignment.  A pre-computed tree can be imported into GeneDoc by reading a file that specifies the tree in either Phylip or Nexus format.

Phylogenetic tree scoring is weighted parsimony scoring which assigns residues to internal nodes in the user specified tree.  The residues are assigned to the internal nodes to create the shortest total length of the branches in the tree.  The length of the branches is determined from the distance scoring tables.  See the tree menu for instructions on using this view.

Shortcuts

Toolbar:


Dstat View
Plot View (Windows menu )

The DStat view shows various alignment data in graphical format. You create a plots in the Plot Menu. This menu item and toolbar just reopens the Plot view. It does not cause a re-calculation of the current plot.

Shortcuts

Toolbar:


Dstat View
Plot View (Plot View )

The DStat view shows various alignment data in graphical format. You create a plots in the Plot Menu. This menu item and toolbar just reopens the Plot view. It does not cause a re-calculation of the current plot.

Shortcuts

Toolbar:


Gel View
Gel View (Windows menu )

The Gel View displays a simple Enzyme Gel Simulation display. You will need to load enzymes into GeneDoc through the Search Configuration Dialog. This view displays two lists, the list of sequences and the list of enzymes. Select multiple sequences and select multiple enzymes and click the Run Gel button. The fragments of the sequences selected, as determined by the cut locations of the enzymes selected, will be displayed in this view on a logarithmic scale. The minimum value of the Y axis will always be 1 and the maximum value will be the largest uncut sequence length. You can print the Gel display or use the Copy button to copy it as a MetaFile to the clipboard.

Shortcuts

Toolbar:


Enzyme Gel Simulation
Enzyme Gel Simulation (Gel View )

The Gel View displays a simple Enzyme Gel Simulation display. You will need to load enzymes into GeneDoc through the Search Configuration Dialog. This view displays two lists, the list of sequences and the list of enzymes. Select multiple sequences and select multiple enzymes and click the Run Gel button. The fragments of the sequences selected, as determined by the cut locations of the enzymes selected, will be displayed in this view on a logarithmic scale. The minimum value of the Y axis will always be 1 and the maximum value will be the largest uncut sequence length. You can print the Gel display or use the Copy button to copy it as a MetaFile to the clipboard.

Shortcuts

Toolbar:


Gel View
Gel View (Plot View )

The Gel View displays a simple Enzyme Gel Simulation display. You will need to load enzymes into GeneDoc through the Search Configuration Dialog. This view displays two lists, the list of sequences and the list of enzymes. Select multiple sequences and select multiple enzymes and click the Run Gel button. The fragments of the sequences selected, as determined by the cut locations of the enzymes selected, will be displayed in this view on a logarithmic scale. The minimum value of the Y axis will always be 1 and the maximum value will be the largest uncut sequence length. You can print the Gel display or use the Copy button to copy it as a MetaFile to the clipboard.

Shortcuts

Toolbar:


toolbars
Toolbars (Help Index )

The Project toolbar is displayed across the top of the application window, below the menu bar.  The toolbar provides quick mouse access to many tools used in GeneDoc,

There is also a toolbar for the Alignment View and the Tree view. These toolbars provide access to function specific to each of the views.

To hide or display the Toolbar, choose Toolbar from the View menu (ALT, V, T).

See Toolbar Tips for more information.

Project Toolbar

Click	To

Open a new document.

Open an existing document.

Save the active document or template with its current name.

Print the active document.

Show GeneDoc version and copyright with about dialog.

Context sensitive help

GeneDoc Configure dialog

Edit Sequences dialog

Alignment View

Summary View

Report View

Tree View

Plot View

Statistics Report

Score Report

Base Composition Report

Display Manual Shading

Display Comments

Alignment View Toolbar

Click	To

Select Blocks select sequences by rows.

Copy the selection to the clipboard as a windows metafile.

Copy Inverted the selection to the clipboard inverted as a 16 Color DIB.

Clear All Shading function

Shade at 2 Levels function

Shade at 3 Levels function

Shade at 4 Levels function

Conserved mode

Quantify  mode

Property  mode

Search  mode

Structure mode

PhysioChem mode

Identity mode

LogOdds mode

Residue Difference function

Enable Substitution Groups mode

Enable Group Display mode

Select Columns select sequences by columns.

Score Selection function

Insert Filler function

Arrange Sequences mode

Slide Sequences mode

Insert Gaps mode

Insert Other mode

Insert Gap Column function

Select Sequences function

Manual Shading function

Edit Residues mode

Phylogenetic Tree View

Click	To

Copy Tree to the clipboard as bitmap

Create Node from currently selection

Delete Nodes currently selected

Move Node Up

Move Node Down

Clear all Nodes

Weight

Manage Tree Expression


Toolbar Tips
Toolbar Tips (Help Index )

The C, Q, P and S buttons show you which mode your displaying, as you know. If you click on a button that is already depressed, you will get the configuration dialog that pertains to that shading style.

If the 'G' button on the lower toolbar is depressed, then you are shading with groups configuration mode, ie conserved, quant, prop, or structure. If you push one of the C,Q,P,S buttons, the shading mode will be automatically applied to all groups. Also, as above, if you click on an already depressed button, the GROUP configuration dialog for that shading mode will be shown. Any changes made will be applied to all groups. This also applies to the 'D' (differences display) and the 'DEST' enable similarity groups buttons.

For example, this can be real convenient when changing between structure shading. The S will be depressed, and if you click on the S again, you will get the list of possible structure shading data you have defined. Select one, click OK, and all groups (if data is available) will be shaded with the new data set.

Another trick that is specific to structure shading is the level buttons. When secondary structure shading mode is enabled, click on any level (2,3,4) button. Likely no buttons will be depressed by default. When you click on one, for example 4, then you will get the list of secondary structure data sets, as in when you re-press the 'S' button. Select a data set. Then press a different level, for example 3, and you will get the list again. This time select a different data set. The display will now be shaded with this new data set. Now go back and press the 4 again. The display will change back to the data set you selected for the level 4 button. You have now 'defined' a different data set for both of the level 3 and 4 buttons and can switch shading between these data sets quickly by using these level buttons. This can be done for the level 2 button as well, of course. These assignments are saved in the .msf file header.


Copy Screen Functions

GeneDoc Copy Screen Functions. (Help Index )

GeneDoc supports copying selected blocks to HTML files. See Copy Selection as HTML File for further information.

GeneDoc supports copying selected blocks to MAC style Pict files. See Copy Selection as Pict File for further information.

GeneDoc supports copying selected blocks to Windows Enhanced Meta files. See Copy Selection as MetaFile for further information.

GeneDoc supports copying selected blocks to Rich Text Format (RTF) files. See Copy Selection as RTF for further information.

GeneDoc supports copying all of the blocks of text to the clipboard as text, without any shading. See Copy All as Text for further information.

The copy function supports both Device Independent Bitmaps (DIB’s) and Old Style Windows Bitmaps. This type of copy is the least desirable because the pasted image will look OK on the screen, but when printed will be very pixelated. For best printing results, paste a meta file or save as a pict file and import that into documentation programs. Supported DIB’s are:

 16 Colors. This is the default, created when you use the copy buttons on the toolbar to copy the window.

 256 Colors

 Display Resolution. This creates a DIB defined with as many colors as the current display.

Windows Bitmap. This creates an Old style Windows Bitmap. Some 16 bit programs, PaintBrush for example, can only read Windows Bitmaps, not the newer preferred DIB’s.

Using the Toolbar buttons or the 16 color DIB copy is generally the best all around choice because it requires the least memory and provides sufficient colors.


GeneDoc Output
Getting Useful GeneDoc Output  (Help Index )

GeneDoc support a wide variety of output options. Printing your output is an obvious one, but not one that is always satisfactory for publications.

Often, users of GeneDoc will select a one or more blocks and wish to paste them into a word processing program. The worst results are obtained with Bitmaps. The best results are obtained with either Pict files or with metafiles. This is because the Bitmaps will look OK on the screen, but will become pixelated when printed.

Pict files often are the most convenient in terms of simplicity of use, though you will have to write them to a file and read them in again. Pict can be troublesome to edit afterwards because of a large number of ‘objects’ in the file.

Metafile can be copied and pasted directly through the clipboard, but in the case of Win/95 will often have to be resized to the original size.

RTF files show the best promise of platform independence. Beware though that the number of colors in the file can be compressed by the word processor, thus loosing distinction of color levels in complicated shadings.

In MS Word, a GeneDoc block is often ‘Framed’ so that it can be moved around the page easily, and so that borders and a Figure number can be added.

GeneDoc supports HTML file output. This may be useful in publishing research on the web, though these HTML ‘tables’ require a lot of text to describe when background shading is included.


Getting PostScript Output
Getting PostScript Output. (Help Index )

GeneDoc does not directly support postscript output, but you can easily make a postscript file. You will need to load a suitable postscript driver onto your system, even though there may not be a postscript printer attached directly to it. Once a postscript driver is loaded, configure it to save the output to a file. Every time you print to this ‘printer’, the driver will ask you what filename to save the postscript output to.


User Defined Shading Definitions.
User Defined Shading Definitions  (Help Index )

User Defined Shading Definitions are read in from an Ascii file. This file will be expected to have the extension of .SDU, but is not required to. This file will contain rows that describe how to interpret Shading Data read in from a file. The shading data does not have to be read in with a User Data file, though that will probably be the usual case. User Data files are indicated by files with the extension .stu.

A shading definition contains two types of rows. The first maps colors to symbols. The second maps data sets or data types to color/symbol sets. More than one data type can point to the same color/symbol set. The two row types are referred to as COLORMAP rows and DATATYPE rows.

Each row type contains several fields. All fields are comma separated. Each row is separated by a CR/LF (or LF only).

In a definition file, all the COLORMAP rows should be entered before and DATATYPE rows that refer to them.

COLORMAP rows take the following format:

Field1: COLORMAP

Field2: Colormap Set Name, case sensitive.

Field3: "Colormap Label"

Field4: Colormap symbol (single char), case sensitive.

Field5: Backgroup color Red component

Field6: Backgroup color Green component

Field7: Backgroup color Blue component

Field8: Text color Back/White indicator: OPTIONAL or

Field8: Text color Red component: OPTIONAL

Field9: Text color Green component: OPTIONAL

Field10: Text color Blue component: OPTIONAL

Spaces or tabs between fields will be ignored.

As and example:

COLORMAP,EMBLESH,"sheet",E,255,255,0,0

COLORMAP,EMBLESH,"no pred", ,255,255,255,0

COLORMAP,EMBLESH,"helix",H,240,0,128,0,0,0

Field 1 must always be the string COLORMAP, for a colormap row.

Field 2 will be a string that identifies which set the color row is to be assigned to.

Field 3, which must be enclosed in double quotes, is a short label for this color.

Field 4, which must be a single character, is the symbol that will be used from the data file to shade a residue this color.

Field 5,6, and 7, specify a RGB triplet that determines the color assigned to the specified symbol. The values must be in the range 0-255, 0 being darkest, 255 being brightest.

Field 8, 9, and 10 are optional, and also may vary in definition.

If there are no fields after field 7, GeneDoc will assign the text color to be back or white depending on what it thinks will be the most visible.

If there is only a field 8, and no fields 9 and 10, then it must be either 0 or non 0. If 0, then the text color will be black, else white.

If there is a field 8,9, and 10, then they will specify a RGB triplet for the Text color. The triplet follows the same rules as the background color fields.

The other type of rows in a User Definition file is DATATYPE rows. These rows determine the entries found in the Sequence Configuration Dialog. As was stated, many DATATYPE rows may point to the same COLORMAP set. Since DATATYPE rows reference colormap sets, the colormap rows for the set referenced must proceed the datatype rows that makes the reference.

As with the COLORMAP rows, all fields will be comma separated, and many fields must be enclosed in quotes.

DATATYPE rows take the following format.

Field 1: DATATYPE

Field 2: “DataFile Search String”.

Field 3: DataFile Start Data Location.

Field 4: DataFile Data Maximum Length.

Field 5: “Data Type Short Label”.

Field 6: “Data Type Long Label”.

Field 7: ColorMap set (COLORMAP Field2)

As an Example:

DATATYPE,"PHD sec |",0,0,"PP PHD SEC","Prot Pred - Structure Prediction",EMBLESH

Field 1: Must Always be the string DATATYPE for a datatype row.

Field 2: Data File Search String. When the String specified in this field is found in a Data File, then the rest of the row in the Data File will be copied and used for the Data Values in this Data Set. This field must take on specific values if the data is to be read from a predefined Data File Type. This field is completely configurable if the data will be read from a User Data File (*.stu)

Field 3: This field will only be used when reading a User Data File. User Data files are indicated by files with the extension .stu. When a row is found that contains the string in Field 2, then symbols starting at the column in this field will be copied and used to determine coloring.

Field 4: This field is the second part of Field 3. When reading a User Data file, this field describes that maximum amount of symbols to copy from the current row.

Field 5: This field is a Short label description of the data set. This field is not currently used. In a future version, this field will be used to building legends.

Field 6: This field is the long description of the Data set. This field is the text that is used to populate the Data Set List in the Structure Configuration Dialog.

Field 7: This field maps this DATATYPE row to a COLORMAP set. More than one DATATYPE row can refer to the same COLORMAP set.

Note that in User Data Files, it is stated that Fields 3 and 4 are expected to contain the same values for all DATATYPE rows, at least in this version.

Thus, a complete example of a User Shading Definition:

COLORMAP,EMBLESH,"sheet",E,255,255,0,0

COLORMAP,EMBLESH,"no pred", ,255,255,255,0

COLORMAP,EMBLESH,"helix",H,240,0,128,0,0,0

DATATYPE,"PHD sec |",0,0,"PP PHD SEC","Prot Pred - Structure Prediction",EMBLESH

See also User Data Files for information on how to create a User Data File.


User Data Files.
User Data Files  (*.stu) (Help Index )

The following shows an example of a User Data File. The example definition in User Defined Shading Definitions would work for this example User Data File.

          .........|.........|.........|.........|.........|.........|

Sequence| HSNIGPFPVELLQDIMNYIVPILVLPRVNEKLQKGFPLPTPARVQLYNVVLQPHQNFLLF

PHD sec |               EEEEEEEEEEEE  HHHHHHEEEE     EEEEEEEEHHHHHHHEE

          .......

Sequence| GADVVYK

PHD sec | EEE

The first important feature of a Structure or User Data file is that it must contain the residue information to be matched with the master sequence. The residue information in a Data File may contain gaps, though they will be discarded. Any Shading data (symbols) in the same column as a residue gap will be discarded as well.

Rows that do not have significant

It is preferable to have a row of residue data proceed the corresponding rows of shading data.

A row of residue data is detected by searching for the hard coded string ‘Sequence|’. Note the upper case ‘S’ and the vertical bar ‘|’. Once a row with this string is found, residue data will be copied from the row in as described by fields 3 and 4 of one of the DATATYPE rows. Which one is undetermined, there for it is appropriate to have all the DATATYPE rows have the same values in field 3 and 4. The Author of GeneDoc realizes this is poor design, but is waiting for some user to request an improvement before expending more time on the issue. In the meantime, its enough to get started.

As rows are read in from the Data Files. They will be searched for the strings found in Field 2 of the DATATYPE rows in the Shading Definitions. If any of these strings are found, then the symbols in the row found in the columns described by Fields 3 and 4 of the DATATYPE records will be copied and used to determine shading.

As additional rows of ‘Sequence|’ data or Shading data are read, they will be appended to data that has already been. If at any time during reading the Data, a Shading row is read that result in less shading symbols than the current amount of residues that have been read so far, the shading data rows will be appended with spaces. This is why it is preferable to have Sequence Residue information proceed Shading Symbol information.

Once all the data has been read, the residue data will be searched for non residue characters. If any are found, such as would be the case if information is in an ‘aligned’ format, then the data this column will be discarded for both the residue data and the shading symbol data.


Structure Data Details

This dialog shows the values and names of the colors in the structure display.


Support Structure Files
Supported Structure Files  (Help Index )

Currently supported file formats are:

PDB: Standard PDB structure files (*.pdb)

EMBL: Protein Predictor (*.phd)

EMBL: Preditor (*.prd)

EMBL: SS Prediction (*.ssp)

EMBL: DSSP Files. (*.dss)

PSC: PSDB Files. (.psd)

User Defined Files. (*.stu)

Support for these file types has been included by the author only as a matter of convenience. Absolutely no implication of support for the methods used to create these data files can be assumed. There are plenty of other methods that produce equally useful data. EMBL labs support was included because it is a convenient starting point for lots of this kind of data. Any other data could be worked to be readable by the User Defined shading routines. There is no guarantee that these files types will be supported or available in the future. The data reading routines may not be accurate for any or all cases. The data should be carefully checked against what GeneDoc displays. There are known problems with the whole business. The routines have been only minimally tested.

Pretty much any kind of shading can be applied to GeneDoc displays with the User Defined Shading Definitions and User Defined Data files.


GeneDoc Use
Initial Alignment of Sequences  (Help Index )

The Uses of Multiple Sequence Alignments

Multiple sequence alignments are a powerful tool for investigating the relationship between structure and function in biomolecules. Such alignments represent the evolutionary history of the group of sequences (Nicholas and Graves, 1983; Zukerkandl and Pauling, 1965).  This evolutionary history is a record of successful mutagenesis experiments carried out by nature on a family of macromolecules.  The multiple sequence alignment demonstrates the extent to which specific residues may be changed without destroying the essential structure and function of the macromolecule.  At the same time they can identify which residues must be changed to create a new and different function within a similar structural framework (Nicholas et al., 1987).  As such, a multiple sequence alignment is the logical starting point for laboratory mutagenesis experiments used to study the relationship between structure and function in families of macromolecules (Eckstein et al., 1996).  GeneDoc’s analysis functions are designed to help users derive these kinds of information from multiple sequence alignments and apply it to experimental situations like those described below.

Multiple sequence alignments have been the essential representation of the data in investigations that successfully identified the sequence residues necessary for the correct functioning of different families of transfer RNAs (Nicholas and McClain, 1987; McClain and Nicholas, 1987) and for determining the correct secondary structure of many families of structural RNAs (Gutell et al., 1994).  Such alignments also provide a means of testing hypotheses about gene duplication events and the origins of regulatory genes (Nicholas et al., 1995).  Multiple sequence alignments also form the basis for databases of motifs, patterns that are diagnostic for membership in particular families of biomolecules (Bairoch et al., 1995) or for identifying the sequence features defining the sites for posttranslational modifications (Rosenquist and Nicholas, 1993; Nicholas and Rosenquist, 1997) as well as predicting the outcome of RNA selection experiments (Schmidt et al., 1996).  The carefully constructed alignment of all of the aldehyde dehydrogenases sequences known at the time (Hempel et al., 1993) is still being used to guide site directed mutagenesis studies (Sheikh et al., 1997), as a guide to examining important features in the recently determined crystal structure of the enzyme (Liu et al. 1997) and modeling paralogous members of the same superfamily (Hempel et al., 1997).

Programs for Multiple Sequence Alignments

Thus multiple sequence alignments are a valuable source of information that can be brought to bear in many ways while investigating the properties, characteristics, and functions of macromolecules.  However, multiple sequence alignments created with today’s programs suffer from both theoretical and practical shortcomings (Nicholas et al., 1995b; McClure et al., 1994).  The ability to easily create multiple sequence alignments that more accurately reflect the evolution of the sequences in the alignment should make multiple sequence alignments an even more useful and powerful guide for experimental studies.  GeneDoc’s alignment editing and analysis features are designed to help users improve their alignment and overcome the current limitations in multiple sequence alignment programs.  Other GeneDoc analysis features are intended to help you extract information from the alignment to use in planning and intrepreting experimental work.

 There are currently a variety of approaches being used to create multiple sequence alignments.  Each has both theoretical and practical shortcomings.  Currently used programs fall into three categories.

1?	Multi-dimensional dynamic programming methods, which are able to guarantee that a solution is optimal within the volume of solution space examined.  However, memory and computational time requirements for this method scale exponentially, as the product of the lengths of the sequences.  This limits the lengths and number of sequences that can be considered, thus limiting a rigorous multiple sequence alignment to relatively small problems.

1?	In contrast, heuristic methods, the progressive pairwise programs, require less memory and computational time than the dynamic programming methods. Heuristic methods cannot guarantee that the alignment obtained  is optimal in any rigorous sense because they are sensitive to local  extrema which effects even alignments among closely related sequences.  The multiple sequence alignments produced by these programs are also dependent upon the order in which the sequences are aligned.

2?	A new program, SAGA (Sequence Alignment by Genetic Algorithm – Notredame and Higgens, 1996) is proving useful as a means of refining alignments produced by the progressive pairwise programs.  The resulting alignments generally match, as they were intended to, MSA alignments.  This sets an upper bound to the improvements.

The best current program for creating multiple sequence alignments is the MSA program (Lipman et al., 1989).  MSA is superior to the more widely used progressive pairwise methods (Feng and Doolittle, 1987) found in programs such as PileUp (Genetics Computer Group) and CLUSTAL W (Thompson et al., 1994) because MSA rigorously optimizes a well defined alignment criteria.  MSA minimizes the weighted sum-of-pairs alignment scores with quasi-natural gap costs (Lipman et al., 1989, Altschul, 1989).  Although MSA is generally superior to other programs available today it still has shortcomings

MSA is severely limited in the number of sequences that it can consider, because it requires memory and processor time proportional to the product of the lengths of the sequences being aligned.  On a Cray C-90 where 256 MWords of memory were available as many as 20 phospohlipases A2 of about 125 amino acids have been aligned.  For proteins in the 350 to 450 amino acid length range (aspartyl proteases and lipid binding protein superfamily members) the limit was in the range of 6 to 8 sequences.  To make alignment of more than three or four short sequences possible and to achieve alignments of the size described above MSA combines information from pairwise sequence alignments and a progressive pairwise multiple sequence alignment to restrict the volume of alignment space explored in such a way that the alignment is no longer guaranteed to be the global (absolute) minimum sum-of-pairs cost alignment for the set of sequences.  In some cases no alignment can be found within the restricted volume of the alignment space.

Another limitation of MSA and of the less capable progressive pairwise programs is that they are forced to use biologically unrealistic alignment criteria because computing biologically realistic alignment criteria is too time consuming for the large number of evaluations required during the alignment computation (Lipman et al., 1989, Altschul, 1989).

The problems of current interest can require the alignment of hundreds of sequences simultaneously, making the use of unrestricted multidimensional dynamic programming methods impossible.  The MSA program (Lipman, et al., 1989) utilizes the Carrillo-Lipman strategy (Carrillo and Lipman 1988), a multidimensional dynamic programming scheme, to search for an optimal solution within a K-dimensional subset of the alignment space.  The particular subset of solution space that MSA searches is determined using an heuristic pairwise progressive algorithm and individually computed alignments of each pair of sequences.

 Heuristic progressive pairwise procedures have been developed to create multiple sequence alignments of large numbers of sequences.  These are implemented in the widely used Clustal series of programs and the Genetics Computing Group’s PileUp program.  The heuristic procedures perform adequately on sets of sequences that are minimally diverged from each other but are virtually sure to produce misleading alignments with more diverged sets of sequences.  The main limitations in the heuristic procedures are described below.

The multiple sequence alignments are far from robust and the alignment  is dependent on the order in which the sequences are entered into the alignment process.  The addition of even one new sequence into the     alignment process at an early stage can result in a quite different alignment than would result from aligning the new sequence with an already completed alignment of the other sequences.

?	No explicit score is being optimized over the entire alignment.  Rather, the alignment is generated in a cumulative fashion by first aligning the most similar sequences, and then aligning each subsequent sequence relative to the current cumulative alignment.  Even though the score at each step is optimized, that process can be interpreted as the assumption that new data does not improve our understanding of  the relationships among the sequences already aligned, since the new data is not allowed to modify the previously generated incomplete alignment.

?	The alignment process is not guided by biologically reasonable alignment criteria.  Because of this, phylogenetic analyses bases on these alignments virtually always find phylogenetic trees with a topology identical to the order of joining used in creating the alignment.

?	The alignments are strongly problem dependent making it difficult to develop guidelines about how much reliance can be placed in the multiple sequence alignment.

 The SAGA (Notredame and Higgins, 1996) program computes sum-of-pairs alignments using a maximization method, the genetic algorithm (Holland, 1975), developed by computer scientists.  The basic strategy is to generate arbitrary alignments and to evaluate them by some well defined criteria.  The initial alignments can be generated by a variety of methods.  In SAGA the initial alignment is generally imported from the Clustal W program.  The initial alignment is then modified by introducing random changes.  These modified alignments reevaluated and further operations are carried out on a selected subset of the alignments.  This includes replacing regions from one alignment by the comparable region from another alignment and then introducing more random changes.

0	These changes generate a large sample of the possible alignments.  Eventually this sampling process finds good alignments and refine them.  This leads to high quality alignments, as defined by the criteria used to evaluate the sampled alignments.  None the less, there are disadvantages to the SAGA method.

?	One result of the complicated customized mutation operators used in the SAGA genetic algorithm is that it is very computationally intensive compared to a simple genetic algorithm or the heuristic pairwise methods.  As a program running on a work station, only alignment refinements are practical.

?	So far, only sum-of-pairs scoring is available as an alignment criterion.

?	SAGA uses a very biased sampling routine to select the alignments to be generated and evaluated.  This sampling is dependent on a user input phylogenetic tree.  Alignments that are not a good fit to the user specified tree are not likely to be generated and evaluated.  Thus the best alignment may be missed.

Editing Sequence Alignments

GeneDoc provides a combination of alignment editing and alignment analysis capabilities intended to help users refine their alignments.  These refinements should be directed toward the goal creating an alignment that accurately reflects the evolutionary history of the sequences being aligned.  The editing can be directed toward aligning specific sequence residues to reflect structural or biochemical information that could not be incorporated in the initial alignment procedure.  Changes can also be made to compensate for the limitations of multiple sequence alignment procedures discussed above.  A very powerful guide to alignment editing is to determine whether or not the changes have improved the score of the alignment.  The scores are an important guide because they are based on the accumulated knowledge of evolutionary processes incorporated in the empirical log-odds scoring matrices available in GeneDoc (Dayhoff et al., 1978; Henikoff and Henikoff, 1992; States et al., 1991, Altschul, 1991).  GeneDoc offers two different ways to compute a score for your alignment or any section of it.  These allow you evaluate the changes you make in an alignment using GeneDoc's alignment editing capabilities.

The first is sum-of-pairs scoring of the alignment.  Sum-of-pairs scoring is much faster than weighted parsimony scoring.  Sum-of-pairs scoring involves scoring all of the alignments between the independent pairs of sequences and adding these scores together to yield the total alignment score.  While sum-of-pairs scoring is less than ideal, it results in alignments that are closer to those produced by superposition of three dimensional structures than do alignments produced by the heuristic methods.  Sum-of-pairs scoring may be particularly appropriate in some protein engineering context or in the core regions of globular proteins where few if any alignment gaps are required.

Sum-of-pairs scoring is also useful in correcting some of the deficiencies of alignments produced by the progressive pairwise programs.  These deficiencies are caused by the failure of the progressive pairwise methods to optimize a well defined alignment criterion and are generally referred to as local minima or local extrema.  The most common deficiency, or local minima, is to fail to align a residue that is in fact identical in all of the sequences in the alignment.  It is important to use objective criteria for changing an alignment.  Ideally, there will be biochemical or structural data that indicates that a sequence residue is conserved during evolution and that the alignment should therefore have a corresponding column of identical residues.  If such evidence is not available it is desirable that the changes in the alignment should improve the score for all of the effected alignment positions.

While the most common kind of local minima is failure to align a residue that is in fact identical in all of the sequences in the alignment, it is not the only kind.  The user should keep in mind that spurious columns of identical residues can also result from local minima.  It is difficult to identify these in the absence of external information about the biochemistry or structure of the molecules whose sequences are being aligned.  Once again, comparing the scores of alternative alignments can provide useful guidance.  One useful strategy to examine your alignment for artifacts of this kind is to perform the alignment several times wile varying the gap penalties, the scoring matrices, the order in which the sequences are aligned, and even the program used to create the alignment.  Most often a number of features in the alignment will recur in all of the alignments.  These robust features can serve as anchor points in the absence of contradictory structural or biochemical information.  The less robust a feature is the more carefully it should be evaluated before it is accepted as a part of the final alignment.

It is worth noting that there are strategies for refining progressive pairwise alignments that eventually converge to the sum-of-pairs alignment (Gotoh, 1996; Hirosawa et al., 1995).  These strategies involve removing groups of sequences from the completed alignment and then realigning them to the remaining alignment.  A simple form of this strategy was initially proposed by Barton and Sternberg (1987).  The current implementation of the Clustal series, Clustal X, has rudimentary capabilities for alignment refinement.

The second is weighted parsimony scoring, an alignment criteria that has long been considered desirable but has been avoided because of its computational requirements (Sankoff and Cedegren, 1983).  Weighted parsimony will result in an alignment that is most congruent with a user specified phylogenetic tree relating the sequences.  The tree can be derived from whatever source the user prefers such as morphological data or another set of sequences.

Weighted parsimony alignments should alleviate some of the theoretical deficiencies of sum-of-pairs scoring.  One obvious artifact and deficiency in alignments produced by rigorous sum-of-pairs scoring is forcing the first residue before a gap and the first residue after the gap to coincide, that is to be in the same column of the alignment.  This is more or less reasonable if the phylogeny of the sequences indicates that only one insertion or deletion event is necessary to explain the presence of a gap in the sequences where it is observed in the alignment.  However, it seems very unlikely when the identical gap is present in rodent sequences and in bacterial sequences while the gap is completely missing from primate sequences and from invertebrate sequences.  It is unlikely because these gaps most likely require at least two insertion and deletion events in the evolutionary history of the sequences.  Unfortunately this is a common feature of sum-of-pairs alignments.  Sometimes this problem is obvious and can be alleviated either by visual inspection and manual adjustment or by recomputing the alignment with a lower penalty for gaps.  Manual adjustment carries the risk that the subjective criteria on which it is based are, in fact, incorrect in this instance.  Lowering the gap penalty may create an even more inaccurate alignment in other parts of the sequences.  Thus using a more sophisticated, evolution based alignment criteria, such as weighted parsimony, is a much better solution.  These considerations apply equally to nucleic acid or protein alignments.

Weighted parsimony also alleviates some cases of the extreme prejudice that sum-of-pairs scoring has against including very dissimilar amino acids in the same position of an alignment.  For example Aspartic acid and Tryptophan are among the very most dissimilar amino acids according to the Dayhoff PAM 250 similarity matrix (Dayhoff et al., 1978) or the Blosum series of matrices (Henikoff and Henikoff, 1992).  Attempts to put them into the same column of an alignment are heavily penalized in sum-of-pairs scoring.  However, if the phylogeny of the sequences indicate that the Aspartic acid containing sequences are connected to the Tryptophan containing sequences through nodes that could plausibly have been Histidine and Tyrosine the mutations are unexceptionable since all are observed at least as often as we would expect from random mutations.  Weighted parsimony scoring would take into account favorable mutational pathways in the phylogenetic tree relating the sequences while still penalizing dissimilar amino acids not related by a plausible mutational pathway.  Such prejudices can lead to the alignment of sequence positions which are not in fact homologous.  These misalignments can propagate into adjacent parts of the overall alignment.  These problems cannot be alleviated by alterations in the scores and are virtually impossible to detect and correct by visual inspection.  The only practical solution is improved alignment criteria. Similar benefits accrue to weighted parsimony scoring for nucleic acid sequences by allowing different weights to be assigned to transitions and transversions.

References

 Altschul, S.F.  1991.  Amino acid substitution matrices from an information theoretic perspective.  J. Mol. Biol., vol. 219, pp. 555 - 565.

Altschul, S.F.  1989.  Gap Costs for Multiple Sequence Alignment.  J. Theor. Biol., vol. 138, pp. 297 - 309.

Bairoch, A., Bucher, P. and Hofmann K.,  1995.  The PROSITE database, its status in 1995.  Nuc. Acids Res.  vol. 24, pp. 189 - 196.

Barton, G.J. and Sternberg, M.J.  1987.  A strategy for the rapid multiple alignment of protein sequences. Confidence levels from tertiary structure comparisons. J. Mol. Biol., vol. 198, pp. 327 - 337.

Carrillo, H., and Lipman, D. 1988, SIAM J. Appl. Math., vol. 48, pp. 1073.

Dayhoff, M.O., Schwartz, R.M., Orcutt, B.C.  1978.  A model of evolutionary change in proteins. In "Atlas of Protein Sequence and Structure" vol. 5(3)  M.O. Dayhoff (ed.),  National Biomedical Research Foundation, Washington.  pp. 345 - 352.

Eckstein, J.W., Beer-Romero, P. and Berdo, I.  1996.  Identification of an essential acid residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases.  Prot.  Sci. vol. 5, pp. 5 - 12.

Feng, D.F. and Doolittle, R.F.  1987.  Progressive sequence alignment as a prerequisite to correct phylogenetic trees.  J. Mol. Evol.  vol. 25, pp. 351 - 360.

Gotoh,  O.  1996.  Significant improvement in accuracy of multiple protein sequence alignments by iterative refinement as assessed by reference to structural alignments.  J. Mol. Biol., vol. 264, pp.  823 - 838.

Gutell, R.R., Larsen, N. and Woese, C.R.  1994.  Lessons from an Evolving rRNA: 16S and 23S rRNA Structures from a Comparative Perspective.  Microbiological Review, vol. 58, pp. 10 - 26.

Hempel, J., Nicholas, H., and Lindahl, R.  1993.  Aldehyde dehydrogenases: Widespread structural and functional diversity within a shared framework.  Protein Science, vol. 2, pp. 1890 - 1900.

Hempel, J., Perozich, J., Rose, J., Liu, Z.J., Lindahl, R., Wang, B.C. 1997. Aldehyde Dehydrogenase:  Catalytic Mechanism and Structural Model for the Entire Family.  FEBS Letters (submitted).

Henikoff S. and Henikoff, J.G.  1992.  Amino acid substitution matrices from protein blocks.  Proc. Natl. Acad. Sci. USA. vol. 89, pp. 10915 - 10919.

Hirosawa, M., Totoki, Y, Hoshida, M., and Ishikawa, M.  1995.  Compreshensive study on iterative algorithms of multiple sequence alignment.  Comput. Appl. Biosci., vol. 11, pp. 13 - 18.

Holland, J.H. (ed.), Adaptation in Natural and Artificial Systems, University of Michigan Press, 1975.

Lipman, D.J., Altschul, S.F., and Kececioglu, J.D. 1989.  A tool for multiple sequence alignment, Proc. Natl. Acad. Sci. USA.,  vol. 86, pp. 4412 - 4415.

Liu, Z.J., Hempel, J., Sun, J. 1997.  The First Structure of an Aldehyde Dehydrogenase Reveals Novel Interactions between NAD and the Rossmann Fold.  Nature Struct. Biol., vol. 4, pp. 317 - 326.

McClain, W.H. and Nicholas, H.B. Jr.  1987.  Discrimination between transfer RNA molecules.  J. Mol. Biol., vol.  194, pp 635 - 642.

McClure, M.A., Vasi, T.K., and Fitch, W.M.  1994.  Comparative analysis of multiple protein-sequence alignment methods.  Mol. Biol. Evol. vol. 11, pp. 571 - 592.

Nicholas, H.B. Jr., and Graves, S.B.  1983.  Clustering of transfer RNA by cell type and amino acid specificity.  J. Mol. Biol., vol. 171, pp. 111 - 118.

Nicholas, H.B. Jr. and McClain, W.H.  1987.  An algorithm for discriminating transfer RNA sequences.  Computer Applications in the Biosciences, vol. 3, pp. 177 - 181.

Nicholas, H.B. Jr., Chen, Y-M., and McClain, W.H.  1987.  Comparisons of transfer RNA sequences.  Computer Applications in the Biosciences, vol. 3, p. 53.

Nicholas, H.B. Jr., Persson, B., Jornvall, H. and Hempel, J.  1995.  Ethanol utilization regulatory protein:  Profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.  Prot. Sci. vol.  4, pp. 2621 - 2624.

Nicholas, H.B. Jr., Ropelewski, A.J., Deerfield, D.W. II., and Behrmann, J.G.  1995b.  A Metric Measure for Comparing Sequence Alignments.  Proceedings of the 10th International Conference on Methods in Protein Structure,  Eds. M.Z. Atassi and E. Appella, Plenum Press, New York  pp. 515 - 525.

Nicholas, H.B. Jr. and Rosenquist, G.L.  1997.  A position specific scoring matrix for identifying sites of protein tyrosine sulfation.  Manuscript in preparation.

Notredame, C. and Higgins, D.G.  1996.  SAGA: sequence alignment by genetic algorithm.  Nucleic Acids Res ., vol. 24, pp. 1515 - 1524.

Rosenquist, G.L. and Nicholas, H.B. Jr.  1993.  Analysis of Sequence Requirements for Protein Tyrosine Sulfation.  Protein Science, vol. 2, pp.215 - 222.

Sankoff, D. and Cedegren, R.J.  1983.  Simultaneous comparisons of three or more sequences related by a tree.  In "Time Warps, String Edits, and Macromolecules:  The Theory and Practice of Sequence Comparison."  D. Sankoff and J.B. Kruskal (eds.) pp. 253 - 263.  Addison-Wesley, Reading, MA

Schmidt, F. J., Cho, B., and Nicholas, H.B., Jr. 1996.  RNA Libraries and RNA Recognition, Ann. NY Acad Sci. vol. 782, pp. 526 - 533.

Sheikh, S., Ni, L., Weiner, H.  1997.  Mutation of the conserved amino acids of mitochondria aldehyde dehydorgenase.  Role of the conserved residues in the mechanism of reaction.  Adv. Exp. Med. Biol., vol. 414, pp. 195 - 200.

States, D.J., Gish, W., and Altschul, S.F.  1991.  Improved sensitivity of nucleic acid database searches using application-specific scoring matrices.  Methods, vol. 3, pp. 66 - 70.

Thompson, J.D., Higgins, D.G. and Gibson, T.J. 1994  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.  Nucleic Acids Research, vol. 22, pp. 4673 - 4680.

Zuckerkandl, E. and Pauling, L.  1965  Molecules as documents of evolutionary history.  J. Theor. Biol.  vol. 8, pp. 357 - 366.


Similarity Tables
The Similarity Tables  (Help Index )

When Similarity Groups are enabled, GeneDoc assigns amino acids to substitution groups, groups of amino acids that are treated as if they are equivalent to each other, for the purpose of measuring the degree conservation in each column of the alignment.  We have attempted to place the selection of members of each equivalence group on an objective and rational basis.  Thus the members of each equivalence group are a set of amino acids that have mutually positive scores in the similarity representation of the scoring matrix selected for use with an alignment.  The default scoring matrix is the Blosum 62 matrix and thus the default equivalence groups reflect the scores in this matrix.  Note that GeneDoc, in order to properly deal with multiple sequence alignments, uses the similarity matrices in their distance representation.  The discussion below is in terms of the similarity representation and the matrices below are shown in the similarity form while those displayed GeneDoc will be in their distance representation.  The two forms can be interconverted with the equation:

  Distance(i,j) = Maximum Similarity - Similarity(i,j).

Where (i,j) designates the score for a specific pair of amino acids.

The choice of similarity matrix determines both the pattern and the extent of substitutions that will be considered as favorable in evaluating the alignment.

In order to objectively evaluate an alignment one must have a quantification of whether the substitution of one amino acid for another is likely to conserve the physical and chemical properties necessary to maintain the structure and function of the protein or is more likely to disrupt essential structural and functional features of the protein.  Numerous bases have been used in creating similarity tables:  explicit or implicit (empirical) evolutionary models, structural properties such as Chou-Fasman propensities, chemical properties such as charge, polarity, and shape, as well as combinations like those used in the Structural-Genetics Matrix. Regardless of the underlying bases, all similarity tables are attempts to quantify whether a mutation preserves or disrupts the function of a protein.

Similarity scores used in GeneDoc are based on observed substitutions of one amino acid (or nucleotide) for another in homologous proteins or genes.  Similarity scores organize the observations into scores that contrast the observed pattern of substitutions in homologous proteins with the random pattern of substitutions we would expect to observe in unrelated proteins.  Modern similarity scores, computed as log-odds scores, have been shown to be the most efficient way to use the observed substitution data to detect homologous sequences.

If the replacements are favored during evolution (i.e. a conservative replacement) the similarity score will be greater than zero and if there is selection against the replacement (i.e., a nonconservative replacement) the similarity score will be less than zero.  Thus similarity scores above zero indicate that two amino acids replace each other more often during evolution than we would expect if the replacements were random.  Likewise, similarity scores below zero indicate that amino acids replace each other less often than we would expect if the replacements were random.

Differences in the way replacements are counted is one of the biggest differences between the two most widely used families of similarity matrices, the PAM matrices and the more recently developed Blosum matrices.  The PAM matrices use counts derived from an explicitly tree like, branching evolutionary model.  The Blosum matrices use counts directly derived from highly conserved blocks within an alignment.

Margaret Dayhoff and her co-workers performed the first careful, systematic study to create the first amino acid similarity matrix, the Point Accepted Mutation (PAM) similarity matrix.  In computing the PAM matrices the alignment was created from a limited set of closely related sequences.  The alignment was a global alignment, that is, it encompassed the entire length of the sequences.  Thus both highly conserved regions and highly variable regions are included in the alignments and used in counting replacements.

The PAM 250 matrix, originally created by Margaret Dayhoff is shown below.  This matrix is appropriate for searching for alignments of sequence that have diverged by 250 PAMs, 250 mutations per 100 amino acids of sequence. Because of back mutations and silent mutations this corresponds to sequences that are about 20 percent identical.

PAM 250 Amino Acid Similarity Matrix

C 12

G -3   5

P -3  -1   6

S  0   1   1   1

A -2   1   1   1   2

T -2   0   0   1   1   3

D -5   1  -1   0   0   0   4

E -5   0  -1   0   0   0   3   4

N -4   0  -1   1   0   0   2   1   2

Q -5  -1   0  -1   0  -1   2   2   1   4

H -3  -2   0  -1  -1  -1   1   1   2   3   6

K -5  -2  -1   0  -1   0   0   0   1   1   0   5

R -4  -3   0   0  -2  -1  -1  -1   0   1   2   3   6

V -2  -1  -1  -1   0   0  -2  -2  -2  -2  -2  -2  -2   4

M -5  -3  -2  -2  -1  -1  -3  -2   0  -1  -2   0   0   2   6

I -2  -3  -2  -1  -1   0  -2  -2  -2  -2  -2  -2  -2   4   2   5

L -6  -4  -3  -3  -2  -2  -4  -3  -3  -2  -2  -3  -3   2   4   2   6

F -4  -5  -5  -3  -4  -3  -6  -5  -4  -5  -2  -5  -4  -1   0   1   2   9

Y  0  -5  -5  -3  -3  -3  -4  -4  -2  -4   0  -4  -5  -2  -2  -1  -1   7  10

W -8  -7  -6  -2  -6  -5  -7  -7  -4  -5  -3  -3   2  -6  -4  -5  -2   0   0  17

   C   G   P   S   A   T   D   E   N   Q   H   K   R   V   M   I   L   F   Y   W

The PAM 250 matrix above has been arranged so that similar amino acids are close to each other.  This gives rise to regions along the diagonal of the matrix that contain only positive scores.  These regions provide an objective basis for defining conservative substitutions, namely as amino acids that replace each other more frequently than would be expected from random replacements.  Note that the amino acids that make up these regions can change at different levels of sequence divergence, that is, different similarity scores matrices correspond to different sets of conservative substitutions. The diagonal terms of the matrix vary appreciably.  This variation reflects both how often an amino acid is found in protein sequences and how often it is observed to be replaced by other amino acids. Thus rare amino acids which are replaced infrequently have the highest scores.

The Blosum Family of Matrices

There are three principal differences between the Blosum and PAM matrices. The first difference is that the PAM matrices are based on an explicit evolutionary model (that is, replacements are counted on the branches of a phylogenetic tree), whereas the Blosum matrices are based on an implicit rather than explicit model of evolution.  The second difference is the sequence variability in the alignments used to count replacements.  The PAM matrices are based on mutations observed throughout a global alignment, this includes both highly conserved and highly mutable regions.  The Blosum matrices are based only on highly conserved regions in series of alignments forbidden to contain gaps.  The last difference is in the method used to count the replacements.  The Blosum procedure uses groups of sequences within which not all mutations are counted the same.

Blosum 45 Amino Acid Similarity Matrix

G  7

P -2   9

D -1  -1   7

E -2   0   2   6

N  0  -2   2   0   6

H -2  -2   0   0   1  10

Q -2  -1   0   2   0   1   6

K -2  -1   0   1   0  -1   1   5

R -2  -2  -1   0   0   0   1   3   7

S  0  -1   0   0   1  -1   0  -1  -1   4

T -2  -1  -1  -1   0  -2  -1  -1  -1   2   5

A  0  -1  -2  -1  -1  -2  -1  -1  -2   1   0   5

M -2  -2  -3  -2  -2   0   0  -1  -1  -2  -1  -1   6

V -3  -3  -3  -3  -3  -3  -3  -2  -2  -1   0   0   1   5

I -4  -2  -4  -3  -2  -3  -2  -3  -3  -2  -1  -1   2   3   5

L -3  -3  -3  -2  -3  -2  -2  -3  -2  -3  -1  -1   2   1   2   5

F -3  -3  -4  -3  -2  -2  -4  -3  -2  -2  -1  -2   0   0   0   1   8

Y -3  -3  -2  -2  -2   2  -1  -1  -1  -2  -1  -2   0  -1   0   0   3   8

W -2  -3  -4  -3  -4  -3  -2  -2  -2  -4  -3  -2  -2  -3  -2  -2   1   3  15

C -3  -4  -3  -3  -2  -3  -3  -3  -3  -1  -1  -1  -2  -1  -3  -2  -2  -3  -5  12

   G   P   D   E   N   H   Q   K   R   S   T   A   M   V   I   L   F   Y   W   C

Summary:  PAM and Blosum Matrices

The Blosum and PAM matrices are the most widely used amino acids similarity matrices for database searching and sequence alignment.  In empirical tests of the effectiveness of the matrices both generally perform well.  However, the Blosum matrices have most often been the better performers.  This likely reflects the fact that the Blosum matrices are based on the replacement patterns found in more highly conserved regions of the sequences.  This appears to be an advantage because these more highly conserved regions are those discovered in database searches and they serve as anchor points in alignments involving complete sequences.  It is reasonable to expect that the replacements that occur in highly conserved regions will be more restricted than those that occur in highly variable regions of the sequence.  This is supported by the different pattern of positive and negative scores in the two families of matrices.  These different patterns of positive and negative scores reflect different estimates of what constitute conservative and nonconservative substitutions in the evolution of proteins.  These differences reflect the differences in constructing the two families af matrices.  Some of the difference is also likely to be because the Blosum matrices are based on much more data than the PAM matrices.

The PAM matrices still perform relatively well despite the small amount of data underlying them.  The most likely reasons for this are the care used in constructing the alignments and phylogenetic trees used in counting replacements and the fact that they are explicitly based on a simple model of evolution. Thus they still perform better than some of the more modern matrices that are less carefully constructed.  Both the PAM and Blosum matrices generally perform better than matrices explicitly based on criteria other than observed replacement frequencies.

We can see the concrete result of these differences in the PAM 250 and Blosum 45 matrices shown above.  These two similarity tables are directly comparable and are suitable for alignments of sequences the same degree of divergence from each other.  They have the same amount of information per alignment position (entropy) for determining whether or not sequences are homologous.  Thus differences in these scores should reflect differences in the data and model used in counting substitutions rather than any other effects.

One striking difference is among the amino acids with carboxylate and amide side chains.  At physiological pH the carboxylate side chains can only act as a proton acceptor while the amide side chain can simultaneously accept and donate a proton.   In the PAM 250 table these four amino acids, Asp, Asn, Glu, and Gln (along with His) form a single conservative substitution group, that is the similarity score for any pair is greater than zero.  His, has two nitrogen atoms in its side chain and, like the amide side chains of Asn and Gln, can simultaneously both donate and accept a hydrogen bond.  In the Blosum 45 similarity table this single group must be split into two groups:  One with the carboxylate amino acids Asp and Glu, and the other with Asn, Gln, and His.

Another noticeable difference is the relationship between Ser and Thr, the alcoholic side chain amino acids, and other small amino acids.  In the PAM 250 table any of the three amino acids Gly, Pro, or Ala can be added to the Ser, Thr pair to form a three member conservative substitution group.  In the Blosum 45 table Ser and Thr form a two member conservative substitution group with no other possible members.  The last difference we will mention is that only Phe and Tyr are members of an aromatic conservative group in the PAM 250 table while Trp is also a member in the Blosum 45 table.

Which Similarity Scores to Use

Similarity scores for sequence alignments perform much better if the similarity scores are based on replacement patterns that correspond to the degree of divergence of the aligned sequences.  More information about similarity scores is available in the references below or in the on-line tutorial on sequence databasse searching at the Pittsburgh Supercomputing Center.  The URL for this tutorial is:

 http://www.psc.edu/biomed/TUTORIALS/SEQUENCE/DBSEARCH/tutorial.html

Altschul, S.F.  1991.  "Amino acid substitution matrices from an information theoretic perspective." Journal of Molecular Biology, 219: 555-665. This paper looks at the PAM and Blosum scoring matrices in the context of information theory and develops guidelines making effective use of the information encapsulated in scoring matrices.

Dayhoff, M.O., Schwartz, R.M., Orcutt, B.C.  1978.  "A model of evolutionary change in proteins."   In "Atlas of Protein Sequence and Structure" 5(3)  M.O. Dayhoff (ed.), 345 - 352, National Biomedical Research Foundation, Washington. This paper describes the development of the PAM family of protein scoring matrices.

States, D.J., Gish, W., Altschul, S.F.  1991.  "Improved Sensitivity of Nucleic Acid Database Search Using Application-Specific Scoring Matrices"    Methods: A companion to Methods in Enzymology 3(1): 66 - 77. Scoring matrices for nucleic acid sequence that take into account different levels of sequence divergence and different rates of transversions and transitions.

Steven Henikoff and Jorja G. Henikoff.  1992  "Amino acid substitution matrices from protein blocks."  Proc. Natl. Acad. Sci. USA. 89(biochemistry): 10915 - 10919. This paper describes the calculation of the Blosum family of protein scoring matrices.

M.S. Johnson and J.P. Overington.  1993.  "A Structural Basis of Sequence Comparisons:  An evaluation of scoring methodologies."  Journal of Molecular Biology.  233: 716 - 738. Comparison of Amino Acid substitution matrices with visual representation of the the important features of the matrices for protein similarity and the differences between them.

Steven Henikoff and Jorja G. Henikoff.  1993.  "Performance Evaluation of Amino Acid Substitution Matrices."  Proteins: Structure, Function, and Genetics.  17: 49 - 61. Comparison of Amino Acid substitution matrices.

Karlin, S. and Altschul, S.F.  1990.  "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes"  Proc. Natl. Acad. Sci. USA.  87: 2264 - 2268.


DNA Ambiguity
DNA Ambiguity Support  (Help Index )

GeneDoc supports DNA/RNA ambiguity codes and incorporates them into a few appropriate display modes.  The GeneDoc DNA/RNA ambiguity support allows users to specify nucleic acid sequence residues whose identity is not unambiguously known.  The most widely used ambiguity codes are R, which can be either A or G (purine) and Y, which can be either C or T/U (pyrimidine).  GeneDoc supports the complete IUB set of nucleic acid ambiguous residues that encompass all possible combinations of the four primary nucleic residues (A, C, G, and T/U).  The complete set of ambiguous nucleotides is shown below in the table titled: Representing nucleotide (DNA and RNA) sequences as sets.

GeneDoc display modes with DNA/RNA Ambiguity support are the Conservation Mode, the Quantify Mode, the Identity Mode, and the Group PCR Contrast display.  As well, the Differences Residue Display Style supports DNA/RNA Ambiguity codes.  GeneDoc supports DNA Ambiguity codes primarily when determining the conservation level of a column in the alignment.  Occurrences of the four DNA types, C, G, A, T/U are counted, as well the ambiguous codes.  When an ambiguous code is encountered in a column, the count for each of the nucleotides that the ambiguous code represents is incremented.  The count for the ambiguous nucleotide symbol is also incremented.  For example, when GeneDoc encounters an 'R' it increments the counts for A, G, and R.  When all symbols in the column are counted, the symbol with the highest count determines the level of conservation.

Once GeneDoc determines the conservation level GeneDoc shades all of characters that contributed to the conserved symbol.

For example, if GeneDoc determines that A has the highest count, then an A will be put into the consensus and all occurrences of A, will be shaded along with any occurrences of, M, R, W, V, H, D, N, and X.

Another example, if a column consists of all R’s, then the consensus will be R code and it will be shaded at 100%. If a column consists of all R’s and one A, then the consensus will be A and the column will be shaded at 100%

A more exact set of rules:

For each column,

1: Build a count of the different letters in the column. In the case of

ambiguous letters, say R for example, I would increment the appropriate

other letters as well, or in this example, increment the count for R, G,

and A.

2: Sort the counts

3: If an ambiguous letter ('R') is in the highest count, I would put that

in the consensus.

4: If there is more than one non-ambiguous letter with the highest count,

create an ambiguous letter for the consensus based on which ones they are,

for example if both A and G have a tie for highest count, then a 'R' goes

into the consensus.

5: If a single non-ambiguous letter has the highest count, it will go into

the consensus.

6: If the count is greater or equal to the primary level, capitalized the

consensus letter.

7: If the count is between the primary and secondary percentage levels,

make the consensus lower case.


GeneDoc Tutorial
Starting GeneDoc

Find the file inside the GeneDoc install directory, inside the tutorial directory, that is named aldh-start.  This file will either have the three letter extension .msf or will be described as a “GeneDoc Document”.  Double click on the icon for this file, which should be the GeneDoc  icon.  The GeneDoc program should start up and load the aldh-start.msf file into its working window.  You may want to position and size the window for your own convenience.

Scroll to a point where the consensus line, which is not labeled with a sequence name, at the bottom of each block of the alignment is visible.  Select the Shade / Enable Similarity Groups menu item, or the Toggle Similarity Groups toolbar button.  This toggles whether or not conservative substitutions are  counted in determining the level of conservation for each column of the alignment.  Notice the highlighting and the consensus line change as you toggle the switch.

Using HELP

In order to learn how GeneDoc determines which substitutions should be considered conservative substitutions and which should not be considered conservative substitutions open the help menu and select INDEX.  In the Help Index select the entry for similarity tables and read the entry.

Close the help facility and toggle off the use of conservative substitutions or similarity groups.  You may find it useful to make use of the help facility at this point to discover the meaning of some of the buttons on the GeneDoc tool bars and to look over the description of the GeneDoc display.

Conservation Highlighting

Near the left end of the second or lower tool bar are four buttons that have the numbers 2, 3, and 4 on three separate buttons and one with a “No Smoking” symbol.  The buttons with the letters on them immediately to the right of these numbered buttons represents different highlighting or shading modes in GeneDoc.  The “C” button, which should be depressed at this point, turns on highlighting by the level of conservation in each column. .  Beginning with the button with the “No Smoking” symbol or “Do Not Enter” symbol click on each of these buttons in left to right order.  This should initially remove all highlighting and then restore highlighting to successive less stringent levels of conservation.

Click on the “C” button on the top row of tool bar buttons (the configure button, not the conservation highlighting button described in the preceding paragraph).  This open a dialog box that has several labeled tabs.  Each tab has controls and setting for different aspects of GeneDoc.  Bring up the dialog box for specifying the details of the shading and highlighting scheme by clicking on the tab labeled “Shade”. You may want to change the “Conserved and Quantity Mode Colors” at the bottom of the box.  Click on the “OK” button of the dialog box to activate any colors you have changed and to leave the dialog box.  You can also change the minimum level of conservation that is required to cause a column in the alignment to be highlighted.  You may set up to three levels which will be highlighted in the first three (three leftmost) color schemes that you are allowed to define.  The fourth color scheme is used for all of the unhighlighted columns and other areas of the alignment.  This is convenient if you want to create a transparency with a totally black, or some other dark colored background.

Highlighting Conserved Hydrophobic Residues in Proteins – or other groups of residues conserving specific properties in either proteins or nucleic acids.

There are at least three ways in GeneDoc to indicate hydrophobic positions in a protein alignment.  Two make use of GeneDoc's shading facilities and the third makes use of the "Consensus Sequence" facility.  These procedures can be adapted to highlighting other desired groups of amino acids or nucleotides in an alignment.  However, since hydrophobic amino acids are a very widely examined feature in protein alignments I will present the examples in those terms.

The simplest method is to use the automatic Physiochemical properties shading mode.  Simply click the "H" button on the second row of tool bar buttons and the alignment should be shaded according to the built-in Physiochemical properties scheme.  This scheme is based on the nested, hierarchical Venn diagram of amino acid properties first published by Dickerson and Geis in 1969.  It has recently been brought back into wide spread use by W. R. Taylor and by Livingstone and Barton.

The Venn diagram can be seen on line at:

   http://www.sander.embl-heidelberg.de/future/aainfo/pchem.htm

Aliphatic amino acids are shaded in a Grey or Silver background with Red letters and Aromatic amino acids are shaded in the same Grey or Silver background with Blue letters.  Both of these sets of amino acids are nested within the non-polar set which is shaded in a Black background with white letters.  It should be noted that the non-polar set includes Lysine.

These assignment may be examined within the GeneDoc program by first clicking on the "C" button on the upper row of the tool bar.  In the "configuration" dialog box that opens you should select the tab labeled "PhysioChem".  A list of the hierarchical sets will be shown with its assigned shading.  Select the set whose member list you want to examine by clicking on it.  Then click on the "Description" button at the bottom of the dialog box.  A small box will pop-up and display the list of amino acids that are allowed to be present in an alignment column given the associated shading.  Each column in the alignment is shaded in the colors that indicate the most restrictive (smallest) set to which the column can be assigned.

The second method of identifying hydrophobic amino acids involves the GeneDoc "Property" shading facility.  This is invoked by clicking on the "P" button on the second row of tool bar buttons.  This facility has three built-in hierarchical levels of sets of amino acids.  The amino acids assigned to each of these sets can be edited and changed by the users or the user can create a completely new family of sets of amino acids to use in shading the alignment.  The default sets and levels in the Property shading facility are derived from Smith and Smith, 1990.

To highlight hydrophobic amino acids using the "Property" shading facility you should first configure it so that it correctly meets your precise needs.  Start by clicking on the "C" button on the upper row of the tool bar.  In the "configuration" dialog box that opens you should select the tab labeled "Properties".  The first thing is to look at the three levels of discrimination among amino acids that are built-in to the function.  Level one has the broadest sets; the amino acids are divided into two disjoint sets at this level.  These two sets correspond to polar and non-polar.  In the second level each of the two first level sets have be divided into two sets for a total of four sets.  The second level sets have again been subdivided to create the third level sets.

Any of the sets can be edited and redefined by selecting the set you want to edit in the list of sets.  Then click on the "EDIT" button and revise the list of amino acids.  I suggest that it is generally a good idea to maintain disjoint sets.  Failure to do this can lead to ambiguities in the display.  A set assignment later in the list will over-ride an assignment from earlier in the list.  Thus, the ordering of sets in the list can make a difference in what is displayed if the sets are not disjoint.

The default second level sets are useful for marking hydrophobic amino acids.  At this level the hydrophobic amino acids are split into two sets, tiny and larger.  Tiny contains only Glycine and Alanine, you may want to move Proline into this group as well.  Well conserved positions of tiny and small amino acids often mark critical regions in the folding and packing of a structure.  Columns of well conserved Glycines, in particular, are found much more often in protein alignments than would be expected from their frequency of occurrence in proteins (Nicholas et al., 1995 and Brocchieri and Karlin, 1998) or from their mutability (Dayhoff et al., 1978) and generally mark critical regions in the packing or folding of a structure.

Next you need to decide if you want to highlight columns that consist entirely of amino acids in one of the defined sets or if you wish to highlight every amino acid in the alignment with the shading assigned to the set to which it belongs.  To highlight only the conserved columns, click on the "Conserved" button in the dialog box.

After you have configured the Property shading to reflect your needs, you can revert to that shading by clicking on the "P" button at any time. This configuration can be made the default by selecting the menu item Project / Save User Defaults.

The third method of identifying conserved columns of hydrophobic amino acids makes use of GeneDoc's built-in scoring tables and the Information Theory interpretation of these tables developed by Altschul, 1991.

Again begin by clicking on the "C" button on the upper row of the tool bar.  In the "configuration" dialog box that opens you should select the tab labeled "Score Table".  You will see a list of built-in scoring tables with the currently active scoring table highlighted.  For protein alignments this list should consist entirely of Blosum (35, 45, 62, 80, and 100) (Henikoff and Henikoff, 1992) and PAM (120, 160, 200, and 250) (Dayhoff et al., 1978) tables.  If there are nucleic acid or DNA tables in the list the project type is set incorrectly.  To correct this select the "Project" tab and set the alignment type to protein.

Under the "Score Tables" tab there is a second list labeled "Similarity Groups".  Each element in the list consist of a number followed by a colon and a set of single letter amino acid codes.  The amino acids in each element of the list identify amino acids whose similarity score with all of the other amino acids in that list element is positive.  This means that according to the current scoring table these amino acids replace each other during evolution more often than would be expected at random.  This provides an objective definition for the concept of a conservative substitution, which allows us to group these amino acids into "equivalence groups".

If you have clicked the "Similarity Groups enabled" check box on the "Shade" tab these groups will be used in computing the degree of conservation in each column of the alignment.  Namely, each member of a similarity group will be treated as the same amino acid.  Thus if one similarity group consists of L, I, V, and M, then any column of the alignment that has only these four amino acids present will be shaded as if it were 100% conserved in the "Conserved" shading mode.  That location of the consensus sequence will display the number associated with the similarity group, and this number can be visually searched for to indicate hydrophobic areas of the alignment.

Different scoring tables have different similarity groups.  You can examine these by highlighting the desired scoring table in the list and then clicking on the "Set Current" button just below the list.  This will cause the "Similarity Groups" list to change to reflect the newly selected scoring table.

The Wu and Brutlag (1996) paper is a recent systematic effort to find groups of amino acids that are conserved in a wide range of aligned protein families.  It also reviews some of the earlier work referred to above.  Their results are summarized in the following diagram taken from the paper.  The details of the paper are well worth the time spent finding a copy and reading it.

Subsumed Substitution Groups:  Wu and Brutlag

            fivlmy

           /      \

        fivlm    filvy

       /   |      /

    ilvm   |     /                  rkqe

   /   \   \    /                   /  \

  /    ivl  \  lfy  fwy           rkq  kqe                tsa

 /     / \   \/   \ |             |  \/  \                /  \

|     iv  \  /\   /-+fy   yh     rk  /\   qe  ed  dn     ts   sa

|    / \   \/  \ /  |  \ / \     / \/  \ / \ / \ / \    /  \ / \

m   i   v   l   f   w   y   h   r   k   q   e   d   n   t   s   a  c  g  p

Note that these groups are neither all encompassing nor mutually exclusive.  This reflects the fact that amino acids express overlapping sets of physical properties.  Also note that Cys, Gly, and Pro are not members of any of the conservation groups.

Saving your work

Click on the file menu and select “SAVE”.  The GeneDoc titling facility will open.  The four text boxes at the bottom must be filled in – they represent essential data you will need if you ever want to reproduce your work.  Fill these in with

The Program:	3D ALI database

The Scoring Matrix:	Structural Superposition

The Open Gap Penalty:	None

The Extended Gap Penalty:	None

Include any additional comments that you want in the box at the top.  Click “OK” to save the file and return to work.  The saved file included any changes you have made in highlighting colors.

Defining Groups of Sequences in GeneDoc

Now we will organize the sequences into three groups that reflect the differences in substrate specificity: Class 2, Class 3 and Betaine. First click on the “G” (for groups) button on the top tool bar.  A dialog box will open.  Next click on the button marked “New Group”.  Enter  “Class 2” for the name of the group.  Pick red for the group color and click “OK”.  Now click the “Text Color” button and select white.

In the list of sequences, click on the name of the top sequence – this should be the name Cow-2.  Hold the shift key down and click on the last class 2 sequence in the list.  This is Human-5.  This will select all of the class 2 sequences.

Now click the “Add” button in the “Group Sequences” box.  Note that the Class 2 sequences are colored in Red, indicating that they have been assigned to the Class 2 group.

Repeat the procedure to create a second and third group that contain Class 2 sequences: Rat-3 to Synec-aldh and Betaine sequences: Cod_beta to Ecoli-bet2. Pick your own unique colors for each group.

In the lower right of the Group Dialog is a click box labeled “Color Sequence Names” – click in this box. Then click the OK button to close the Group Dialog.

Notice that the sequence names in the alignment view are colored by the group colors.

Then click the “G” button for “Shade per Group Configuration” button on the second tool bar.  By default, this highlights the alignment by the degree of conservation within each group, as indicated by the “C” – “Conserved” button on the lower toolbar. Click the “G” - “Shade per Group Configuration” button a few times. Notice that this toggles the display of groups based shading.

With the “G” – Groups shading off, select the “H” – Physiochemical button on the lower toolbar. This shades the alignment by physiochemical properties. Now toggle the “G”, groups shading button again. Notice that the groups shading mode retains the Conserved display setting, and the non group shading retains the physiochemical shading setting.

Associating structural information with specific sequences and groups of sequences in GeneDoc

Now click on the “G”(groups) button on the top tool bar.  In the group control select Class 2.  Select the “Structure” tab.  Open the list labeled “Select Master Sequence” by clicking on the small arrowhead at the right.  Select the sequence name Cow-2. Click on the “load data file” button. Open the 1a4z.pdb file, which is the PDB file for the Cow-2 sequence.

You will return to the structure dialog and see a list of the kinds of data found in PDB files, which is only Secondary Structure. Other types of files contain more data sets. Select the secondary structure – PDB list entry.   Now click on the tab marked “Shade”.  Select the shading mode labeled “Structure”.  Also select the differences mode style labeled “Diff/Top Sequence”.

Repeat these steps for the other two groups, using and 1ad3 for the Rat-3 sequence in the Class 3 group and 1a4s.pdb for the Cod-beta sequence in the Betaine group.

Click the OK button to close the Group Dialog. You will need to switch to Groups and Structure shading to show the PDB shading. Set the “G” – “Shade per Group Configuration” button to depressed and after that set the “S” – Structure Shading button to depressed. You will now see the structure of each master sequence applied to each group. The rest of the sequences in a group will show periods “.” at residues that are identical to the master sequence of that group.

Other kinds of files containing structural data can be read by GeneDoc and used to highlight your alignment with a similar procedure.  This includes the PSdb (Protein Structure database) files.  This facility also allows the user to create a personal set of data files that can specify highlighting by almost any criteria and to incorporate that information into the GeneDoc highlighting Scheme.

Highlighting Sequence Motifs

GeneDoc can highlight motifs or patterns in the sequences.  The motifs can be described either by consensus patterns expressed in either the ProSite or ReBase pattern description syntax or as a log-odds matrix from a MEME output file.

Be sure the “G” – “Shade per Group Configuration” button is not depressed.

The first step of this section is using the logodds matrix from a MEME output file.  Begin by clicking on the “C” - “configuration” button on the top tool bar and selecting the tab labeled “Log-Odds”.  (If you wanted to use a ProSite or ReBase pattern you would select the “Search” tab.)

In the Log-Odds setup table select the button marked “MEME file” and click it.  A file open dialog should appear and you want to select the file named spzoops3.out.  You may want to select different colors with which to highlight the three different motifs.

Next select the tab marked “Shade” and on that form select shading by “Log-Odds” from the list of shading styles.  Now click the “OK” button and the motifs should be shaded.

Note that you can Enable Group Shading, the “G” - “Shade per Group Configuration” button on the lower toolbar and it will remember the structure shading. You can also now select Log Odds shading for Groups, but there is no distinction between showing log odds shading in groups or non groups mode.

You should repeat this exercise selecting the ‘Search” tab instead of the “Log-Odds” tab.  Click the “ProSite file” button and load the file PS00135.dat.  Again open the “Shade” form by selecting the “Shade” tab.  This time, however, select the “Search” highlighting mode and then click “OK”. Now click on the “G” button on the top tool bar and open the “Shade” form.  Again select “Search” highlighting and click “OK”.  Any instance of the ProSite motif present in the alignment should now be highlighted.

Creating Rasmol scripts with GeneDoc

To create a script file that can be used to transfer the highlighting in a GeneDoc alignment to a Rasmol visualization of the three dimensional structure of a specific sequence it is necessary that the sequence in the GeneDoc alignment exactly match the sequence in the Brookhaven PDB file that contains the structural data.  It is also necessary that the residue numbers in the BrookhavenPDB file be contiguous.  This sometimes means that you need to manually edit a PDB file that has residue numbers such as 125 – 125a – 126 …

Begin by getting the alignment highlighted in the way you want.  Then click on the “S” (for sequence) button on the top tool bar.  Select the sequence in your alignment that exactly matches the sequence in the PDB file you are going to use to create the display.  Then click on the button at the bottom of the sequence dialog that is marked “Rasmol Script”.  Select from the list of toggle switches the display features you want, including which highlighting colors you want to show side chains for.  Then click on “OK” to write and save the script file.

The most common problems at in this feature are that the PDB file may be missing residues, particularly at the beginning of the sequence, and that the residues may not be numbered contiguously.  Missing residues may be accommodated by using the “OffSet” box in GeneDoc.  Also note that if the PDB file has multiple chains all of them will be highlighted.  For a cleared view you may want to remove all but one chain from the PDB file.

Schematic view of the alignment

Under the “Reports” menu at the top of the screen you can select the schematic view of the alignment.  This view is often helpful as an abstraction since it retains all of the highlighting of the alignment display but condenses the alignment so that it can all be seen on one screen or printed on one page of paper.  The summary view reflect what ever highlighting and colors you have selected for the main sequence view.  The dimensions of the summary are controlled in the configuration dialog “Project” tab.  The font size determines the vertical space taken by each sequence in the alignment.  The horizontal length of the alignment is determined by numeric value found in the Sum Cols./inch box within the Summary Display area of the “Project” tab.

Further Capabilities of GeneDoc

The GeneDoc web site contains additional useful examples and information on how to use GeneDoc in your research.  The URL is:  http://www.cris.com/~Ketchup/genedoc.shtml

The help files are also fairly extensive and contain discussions of additional analyses and features found in GeneDoc and how they might be applied to specific problems.  Also you should feel free to send the author, Karl Nicholas, e-mail at “Ketchup@cris.com”.  Both questions about the use of GeneDoc and suggestion for additional features are welcome.  Many of the current features are the result of previous suggestions by laboratory scientists who were using the program.

References

Altschul, S.F., 1991.  Amino acid substitution matrices from an information theoretic perspective.  J. Mol. Biol.  vol. 219, pp. 555-565.

Brocchieri, L. and Karlin, S.  1998.  A Symmetric-iterated Multiple Alignment of Protein Sequences.  J. Mol. Biol.  vol. 276, pp. 249-264.

Dayhoff, M.O., Schwartz, R.M., Orcutt, B.C.  1978.  A model of evolutionary change in proteins.  In "Atlas of Protein Sequence and Structure "  5(3)  M.O.  Dayhoff (ed.), pp. 345-352, National Biomedical Research Foundation, Washington.

Dickerson, R.E. and Geis, I.  1969.  The Structure and Action of Proteins.  Harper and Row, New York.

Henikoff S. and Henikoff, J.G.  1992.  Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA. vol. 89, pp. 10915 - 10919.

Livingstone, C.D. and Barton, G.J.  1993.  Protein sequence alignments: A strategy for the hierarchical analysis of residue conservation.  Computer Applications in the Biosciences.  vol. 9, pp. 745 - 756.

Nicholas, H.B. Jr., Persson, B., Jornvall, H., and Hempel, J.  1995.  Ethanol utilization regulatory protein:  Profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.  Protein Science.  vol. 4, pp. 2621-2624.

Smith, R.F., Smith, T.F.  1990.  Automatic generation of primary sequence patterns from sets of related protein sequences.  Proc.  Nat. Acad. of Sci. USA  vol. 87,  pp. 118-122.

States, D.J., Gish, W., Altschul, S.F.  1991 Improved Sensitivity of Nucleic Acid Database Search Using Application-Specific Scoring Matrices.  Methods: A companion to Methods in Enzymology vol. 3, pp. 66-77.

Taylor, W.R.  1986.  The Classification of Amino Acid Conservation.  J. Theor. Biol. vol. 119, pp. 205-218.

Wu, T.D. and Brutlag, D.L.  1996.  Discovering Empirically Conserved Substitution Groups in a Database of Protein Families.  In:  Proceedings Fourth International conference on Intelligent Systems for Molecular Biology.  Edited:  States, D.J., Agarwal, P., Gaasterland, T., Hunter, L., and Smith, R.F.   AAAI Press, Menlo Park, CA. USA.