GelBuddy Release Notes

Notes for GelBuddy v20050817 (public release): The analysis module is now included in both the public and the internal releases. The sensitivity slider now appears only when viewing an automatically generated gel markup containing unconfirmed signals. You can make it go away by using the "Analysis/Mark All Nonweak Signals As Verified" menu command. Confirmed/Unconfirmed status now appears in the tooltip text for each signal The XML file can now specify regions of the gel to be excluded from automatic analysis. See the XML data format documentation for details. 2D mode bug fix "delete all weak signals" and other slider-related bugfixes

Notes for GelBuddy v20050808 (internal release): Edit Lane Classes Mode ---------------------- You can now click on a new "Edit Lane Class" button to view and modify classes of equivalent lanes. (A "lane class" is a set lanes sharing co- migrating bands. This is equivalent to the definition of "haplotype" in eco-SQUINT, but is equivalent to the real-world definition of "haplotype" only for unpooled homozygous samples doped with a homozygous reference.) In Edit Lane Class Mode, lanes with identical patterns of recorded signals are given the same color. Lanes with lanes with a unique pattern of signals are colored red. You can also edit the markup on a lane-by-lane basis: dragging one lane onto another replaces all the signals in the destination lane with co- migrating copies of the signals in the source lane. Automatic analysis improvements ------------------------------- The "Analyze Gel" dialog has been simplified. Tooltips now appear in the Analyze Gel dialog to explain various analysis settings. A new "Background Percentile" setting allows the user to control how the background pattern is computed for EcoTilling image analysis. Values near 50 seem to construct the most accurate background pattern, but values near 20 (the default) provide better sensitivity to common polymorphisms. The band-scoring algorithm has been simplified and tuned for increased sensitivity. Sensitivity slider ------------------ GelBuddy provides a slider control to allow interactive filtering of weak signals. The user sets the slider to set the "weak signal" threshold, clicks on any weak signals that should be retained, and uses a menu command to remove the unverified signals. This procedure is discussed in more detail in the "GelBuddy Automatic Analysis" document. WARNING: The Analysis/Delete All Weak Unverified Signals command does not function correctly in this release!!! User interface improvements --------------------------- GelBuddy's menu structure has changed. Some options have been moved from the "File" menu to the "Options" menu and new "Analysis" menu has been added. The "About Gelbuddy" dialog now provides additional diagnostic information. Debug Mode ---------- The following debugging options can be enabled by the menu command "Options/Expose Debugging Options": File/Post Saved XML to server ----------------------------- This option allows the user to post an XML file directly to the server without being processed by GelBuddy. File/Analyze Test Set --------------------- This option runs the automatic analysis algorithm with given parameter settings against a set of reference markups and calculates false negative and false positive counts. Options/Increment Debug Counter ------------------------------- Has no effect in this version of GelBuddy Options/Show Extracted Data In Left Margin ------------------------------------------ Displays the following data calculated by automatic analysis algorithm: Ecotilling Mode: Green line: Common background pattern Red line: Scaled input signal Cyan line: Decorrelator output Magenta line: Decorrelator output baseline (used to separate peaks in the decorrelator output) Left virtual gel lane: Decorrelator output Second virtal gel lane: Current lane (Input signal) Right virtual gel lane: Common background pattern Mutation detection Mode: Red line: Scaled input signal Cyan line: Decorrelator output Magenta line: Decorrelator output baseline (used to separate peaks in the decorrelator output) Left virtual gel lane: Summed decorrelator output Second virtal gel lane: Left lane ("background" signal 1) Third virual gel lane: Current lane (Input signal) Right virtual gel lane: Common background pattern ("background" signal 2) All results are displayed relative to the calibration ladder for the leftmost lane. You can select the current lane by using the comma and period keys or by clicking on a lane in lane edit mode. Options/Show Scores ------------------- Displays S_signal and S_pair for each band in the markup. Disable Background Subtraction (Analysis Dialog) ------------------------------------------------ This option operates in the same manner as "common background" mode, but uses a constant gray background instead of an artificially constructed background. Select Confirmation Mode ------------------------ This allows the user to select alternative confirmation scoring methods (or no confirmation scoring at all. See the "GelBuddy Automatic Analysis" document for details. Bug Fixes --------- The following bugs have been fixed: "Inconsistent state after using delete key to delete a signal" "Extraneous files appear in the Save Gel Markup" dialog "Decorrelation code doesn't correctly handle degenerate cases"

Notes for GelBuddy v20050628 (internal release): Analysis Features ----------------- GelBuddy can automatically detect mutations -- sometimes correctly! This work is incomplete, but you're welcome to play with it. Clicking on the "brain" button will initiate automatic analysis by displaying a window with analysis settings. Several buttons at the bottom of the window allow you select presets for various applications (e.g. 1D and 2D pooled mutation detection, 1D and 2D ecotilling). The menu option "Options/Show Extracted Data in Left Margin" is included to facilitate debugging of analysis features. The internal release is identical to the public release of GelBuddy except for the inclusion of the automatic analysis module (tgat/Analyzer.class in the .JAR file). The "brain button" will appear on the toolbar to indicate the presence of the analysis module.

Notes for GelBuddy v20050628 (public release): All-in-one installer for Microsoft Windows ------------------------------------------ This is the first version of GelBuddy to be available as a self-extracting all-in-one installer executable. Running GelBuddyInstaller20050628.exe will install GelBuddy, shortcut icons, and a private copy of Java JRE version 1.4.2_08 and Java Advanced Imaging library version v1.1.2. We thank the software development team at LI-COR Biosciences (http://www.licor.com) for their assistance in preparation of the installer package. Improved calibration accuracy above 700bp ----------------------------------------- GelBuddy now uses the known fragment length (in bp, from the Gel Information dialog) and position (from the 0% marker at the left margin of the image) of the full-length product to construct a separate calibration curve between the 700bp (upper) marker and the full-length product. This means IT IS NOW IMPORTANT TO ENTER THE PROPER FULL-LENGTH PRODUCT SIZE IN THE GEL INFORMATION DIALOG AND TO ALIGN THE 0% MARKER TO THE POSITION OF THE FULL-LENGTH PRODUCT IN THE GEL IMAGE. Since SQUINT takes relative mobility (%) instead of fragment length (bp) as input, GelBuddy reports an "effective" relative mobility for signals between 700bp and the full length product so that SQUINT will calculate the correct fragment length using the old calibration formula. There is still a small (~2%) difference between the fragment length reported by GelBuddy and the fragment length reported by SQUINT, probably due to roundoff error. This feature is turned off for old gel markups but turned on by default when you generate a new gel markup. The menu option "Options/Force 0% Marker To Full Length Product Size" controls this behavior. Repositioning of 0% and 100% markers ------------------------------------ By default, the new version of GelBuddy leaves 0%/100% marker positions unchanged. (Previous versions of GelBuddy would change the location of the 0% and 100% markers whenever the user adjusted the 200bp and 700bp markers.) The menu option "Options/Reposition 0%/100% Markers When Adjusting Upper/Lower Standards" controls this behavior. Reporting changes ----------------- In previous versions of GelBuddy, reports were sent to the log window for review by the user. Reports are now separate from log output. The "View Log" button is now the "Generate Report" button. Click this button to generate a new report, or shift-click to view log output. Changes in group edit mode -------------------------- * In group edit mode, previous versions of GelBuddy would display signals from both channels simultaneously. The current version shows only the data for the current channel. * If "Show Pool Boundaries" is selected, GelBuddy now omits connecting lines between signals of different row/column pools. Changes in lane edit mode ------------------------- * The comma/< and period/> keys now select the current lane. * The boundaries of the currently selected lane are now displayed in yellow. * The "delete" key now deletes the currently selected lane. (In previous versions, the "delete" key deleted the lane under the cursor. The new behavior allows convenient deletion of several consecutive lanes.) Markup (.XML) file changes -------------------------- * Additional properties in the <calibrationoptions> section * A new <analysisoptions> section * <comment> fields * Automatically generated signals now include <detectionscore> and <confirmationscore> fields Other changes ------------- * Edit/Clear All Signals deletes all signals from an existing markup * Help/Java Runtime Environment License Information displays license information for the Java VM (windows only) * Calibration bug fixes ("spikes" were sporadically generated when constructing de-smiling curves) * Lane numbers at the top of gel image are now vertically oriented for improved legibility. * Updated icon and startup/about screen artwork.

Notes for GelBuddy v20050418b: First public release of GelBuddy. This is the version of GelBuddy described in the NAR paper (Zerr T, Henikoff S. (2005) Nucleic Acids Res. 33(9):2806-2812) with additional user-interface features and improvements in calibration accuracy. Calibration Improvements ------------------------ * By default, this version of GelBuddy constructs three independent de-smiling curves from the image data, at the location of the lower (200bp) marker, the location of the upper (700bp) marker, and the location of the 0% marker (full length product). You can change this behavior on a per-markup basis by using the menu commands: Options/Calibrate Lower Size Standard Using Image Data Options/Calibrate Upper Size Standard Using Image Data and Options/Calibrate Full Length Product Using Image Data respectively. * The calibration algorithm has been tuned to provide more accurate tracking of the 200bp marker. * The tolerance level used for automatic grouping has been reduced significantly. Suggestions: * It is now important to adjust the 0% marker to track the full-length PCR product signal. If you cannot achieve this (if the top of the image is clipped or if GelBuddy just can't track the signal), turn off this feature by un-selecting "Calibrate Full-Length Product" in the Options menu. * Zoom to 50% or 100% to check the alignment of the size ladder (the green calibration lines) with the background bands of the image. You may find a combination of Options settings that gives more accurate calibration results. It may also be useful to displace the size standards slightly to get better tracking of background bands. * Don't worry if the full length-product size indicated at the left margin of the gel doesn't match the amplicon size. Variation in running conditions is known to result in inaccurate determination of fragment sizes > 1000 bp. * For darker gels, you may get more accurate results by using the 800 channel signal for calibration (using the Options/Use 800 Channel... command), as this can reduce calibration inaccuracy caused by detector saturation. New user interface for editing signals -------------------------------------- A new and more versatile user interface is provided for adding, moving, and deleting signals from the gel. You can now drag a single signal or all signals in a lane onto another lane, or onto a range of lanes. The basic rules are: (1) Dragging a signal to a different position within the same lane alters the recorded position of the signal. (2) Dragging a signal to another lane creates a copy of that signal with the same mobility. (3) Holding down the SHIFT key while dragging a signal to another lane creates a copy of that signal in all lanes between where you depressed the mouse button and where you released it. (4) Holding down the CONTROL key while dragging a signal to another lane creates a copy of each signal in the lane where you depressed the mouse button. (5) Holding down the ALT key while clicking the mouse deletes a signal. (6) Holding down the ALT key while dragging the mouse deletes the signal you clicked on and all signals of similar mobility between where you depressed the mouse button and when you released it. (7) Pressing ESC while dragging the mouse prevents changes from being made to the gel. Modifier keys can be used in combination: for example, depressing both the SHIFT and CONTROL keys while dragging the mouse lets you copy a haplotype/genotype to a range of lanes. As with drag-and-drop modifier keys in most applications, modifier keys must remain depressed while the mouse button is released to obtain the desired effect. Keyboard interface ------------------ The DELETE key deletes the signal (in signal-edit mode) or lane (in lane-edit mode) under the current mouse position without confirmation. There are now shortcuts for several menu commands. Mac users: use the apple key instead of the control key. Control-letter-oh : open gel images Control-M : open gel markup Control-S : Save Control-plus (really control-equals): Zoom in Control-minus : zoom out Control-zero : fit image to window Straight lanes -------------- An option in the "find lanes" dialog forces GelBuddy to generate straight vertical lane tracks. Tunable parameters ------------------ Several parameters for lane-finding and signal calibration can be altered on a per-machine basis using the "Edit/Alter Global Configuration" command. These settings are provided for use with different protocols (especially running conditions and gel-loading procedures) and should not be altered for use with the current TILLING protocol.

Release notes for old internal releases of GelBuddy. This information is reconstructed from e-mail and is most certainly incomplete.

Notes for GelBuddy v20050307: The version of GelBuddy submitted for review to the NAR editorial staff. Contains bug fixes, improvements in lane finding and signal grouping, and is generally just chock-full of goodness: * Lane finding improvements * If GelBuddy is zoomed to fit the image to the current window size, resizing the window resizes the gel image * When GelBuddy loads a gel markup file, it searches for source images in the current directory automatically. * A "recalibrate signals" option updates the lane numbers, molecular weights, etc. of recorded signals based on the current calibration and lane tracking information. * Preferences are stored as a per-user option rather than a per-machine option * Options to show and hide the status bar and lane numbers * Option to edit run name in the gel info dialog (really useful if you're editing "700.tif" and "800.tif")

Notes for GelBuddy v20041119 ---------------------------- This release includes features that are very useful for analyzing ecotilling and 2D-pooled tilling gels (and are also useful for other tilling gel images): ---------------------------------------------------- GelBuddy can Save and Restore Gel Markup information ---------------------------------------------------- Once you've loaded an image pair, you can save a Gel Markup file that contains Names of the source images Lane location information Calibration information User-entered signals Grouping information by using the "File/Save Gel Markup" command. The Gel Markup file does not contain the source image data, so it's relatively small (~60K). To review or change a previously saved Gel Markup file, use the "File/Load Gel Markup" command. GelBuddy will attempt to automatically load the source images. If the image files are not found in the folder in which they were located when the markup file was saved, GelBuddy prompts the user to locate the source images. --------------------------------------------------------------- GelBuddy can post Gel Markup information to the SQUINT and PICK --------------------------------------------------------------- (Thanks to Samson for his help in making this work) You can now automatically post gel markup information from GelBuddy to SQUINT. Once you've entered all the signals on the gel, and reviewed GelBuddy's analysis with the View Report command, you can send locations and mobilities of mutations/polymorphisms directly to SQUINT instead of entering the information on a form. To do this: (1) Use the "File/Post Gel Markup to Server" command (2) Verify that the run name is correct (3) Enter the following URL in the "Post To URL" box: http://*************************/squint.pl (4) Enter the user name and password that you usually use for SQUINT. (5) Click OK. In a few seconds, a window containing the response from SQUINT will appear. (6) Go to the SQUINT page for the gel to enter gel and signal quality information or to make any changes. GelBuddy saves the URL and user name between sessions. (GelBuddy doesn't save the password.) The "File/Reset Preference Information" command can be used to reset this information. -------------------------------------------------------------------------- GelBuddy needs for squinter's initials and full-length product information -------------------------------------------------------------------------- When you begin picking out signals in the gel, GelBuddy will ask for the squinter's initials, the size of the full length product, and whether you're doing 8x8 pooling. _Please_ fill in this information! The full-length product size is now needed to correctly match 700nm channel signals with their 800nm channel counterparts. It usually works best to enter the actual full- length product size, even if this differes from the apparent size of the full-length product in the gel image. ------------------------------------------------------- GelBuddy can handle signals present in only one channel ------------------------------------------------------- In previous releases, GelBuddy could analyze a signal only if there was a confirming signal in the other channel. The latest version of GelBuddy can analyze signals that appear in only one channel. You can verify that signals have been properly paired (or not paired) by viewing the GelBuddy report. For example, here's what you'll see in the report for a 2D-arrayed gel if the 800 channel signal drops out in a row pool lane: Signals sorted by lane ---------------------- Lane 700% ( MW) 800% ( MW) (Total) 6 12.8 (1115) ( ) 15 13.3 (1105) 67.0 ( 347) ( 1452) Grouped signal pairs sorted by molecular weight ----------------------------------------------- 700% ( MW range) 800% ( MW range) Lanes 13.0 (1105-1115) 67.0 ( 347- 347)* 6* 15 * Contains unpaired signals --------------------------------------- GelBuddy lets the user tag failed lanes --------------------------------------- You can tag a lane as "failed": Enter Lane Edit mode Click on the failed lane Use the "Edit/Mark Lane as Failed" command A red triangle will appear above the marked lane. A lane may be "failed" due to absence of signal, excessive background signal, smearing, or anything else that you think may prevent detection of a mutation/polymorphism. This information isn't used by GelBuddy's signal analysis but is forwarded to SQUINT and PICK. ------------------------ Other minor improvements ------------------------ -- Reporting functions are on a separate menu -- Marks denoting "edited" lanes appear at the top of the gel. -- GelBuddy remembers the most recently opened folder between sessions.

Notes for GelBuddy v20041018: ----------------------------- The really cool thing about this release is that it can "de-smile" (or "de-frown") images by following the pattern of background bands in the region of the 700 bp marker. Having more accurate calibration is especially important for ecotilling, since it's important to correctly group bands of equal mobility to distinguish haplotypes. You almost don't need "group edit" mode anymore! GelBuddy can also follow background bands at the 200 bp marker, but since many gels have a lot of junk down there that can throw off the calibration algorithm, this is turned off by default. A new "Options" menu allows you to "de-smile" either the 200 bp calibration line, the 700 bp calibration line, both, or neither. You can also select which channel to use for desmiling (700 or 800). The "show pool boundaries" option also lives on this menu. There are also fixes for lane-finding errors that occur when lanes run off the end of the gel and some other problems. INSTALLATION ------------ For Mac users, GelBuddy now comes as an application bundle, so you no longer have to deal with properly installing separate .JAR and script files. (0) Remove any copies of the GelBuddy script or TGAT.JAR from your machine. (1) Extract and open the attached file "GelBuddy20041018.dmg". (2) You should see an icon with the FHCRC double-helix logo. Drag the icon to make a copy on your applications folder (_not_ the toolbar dock). (3) There should be a disk-drive-shaped-icon marked "GelBuddy20041018" on your desktop. Drag it to the trash can. (4) Drag the GelBuddy icon (double-helix logo) from the applications folder to the dock. (This doesn't actually move the file, it just creates a link from the dock to the applications folder.) You should now be able to run GelBuddy by clicking on the double-helix logo on the dock.

Notes for GelBuddy v20040924: ----------------------------- The changes in this release are oriented toward improving usability, allowing the user to determine signal grouping (i.e. sets of signals that appear to have the same mobility) for ecotilling and 2D-pooling, and posting GelBuddy data directly to a server. Here's a short description of each new feature: Changes in signal recording --------------------------- * The "record a signal" cursor shape has changed so that the center of the cursor no longer obscures the signal to be recorded. * The signal boxes for the current channel are now bold (to distinguish them from the signal boxes for the background channel). * You now must double-click a signal to delete it. * It is now possible to display or save a report even if there are no signals recorded. Control of signal grouping (for 2D pooling and ecotilling) ---------------------------------------------------------- * The user now can control which signals are grouped (i.e. are considered to correspond to a fragment of the same length). * When the user begins recording signals, a dialog will appear to ask the user whether to group signals across all lanes (for ecotilling) or whether signals should be grouped only within sets of 16 adjacent lanes (for 2D pooling). * Recording signals is the same as before, but after recording the user can click on the "Edit Signal Groups mode" button (the fourth button from the left) to see how GelBuddy has grouped signals. In Edit Signal Groups mode: -- Grouped signals appear as boxes of the same color connected by horizontal lines. Different signal groups have different colors. -- Ungrouped signals appear in green. -- Clicking on a signal selects the signal. Selected signals appear as a solid rectangle. -- Shift-clicking on a signal allows selection of multiple signals. -- Double-clicking (or double-shift-clicking) selects all the signals in a group. -- Three new menu options allow the user to change grouping: Group selected signals: creates a new group for all currently selected signals. Merge selected signal groups: merges all groups containing at least one selected signal Remove selected signals from group: removes all selected signals from any group Show/hide signals button ------------------------ Clicking on the Show/hide signals button allows you to temporarily hide all signal information. Zoom to window button --------------------- Clicking on the Zoom To Window button lets you view the entire gel. Show Pool Boundaries -------------------- Select Edit/Show Pool boundaries to show a red vertical stripe every eighth lane (this is useful for squinting 2-D pooled and multiple individual gels) Posting data to the server -------------------------- There are three new menu commands ("Save XML data", "Post XML data to server", and "Post Saved XML data to server") to allow GelBuddy to communicate with the server. The server isn't yet ready to accept GelBuddy input -- I'll send out more mail when Samson and I have this ready for testing.

Notes for GelBuddy v20040823 ---------------------------- Calibration changes * Molecular weight calibration is now determined by the position of the 200 bp and 700 bp markers. Samson has updated the SQUINT pages to accept "200" as a lower MW marker. (700 was already supported by SQUINT). * There are now two red markers on the left margin (marked "0%" and "100%") that specify the range of reported mobility percentages. By default, 0% is the 50 bp position and 100% is the 1500 bp position, but the user can drag "0%" and "100%" to any desired location. NOTE: The "0%" and "100%" markers will follow the calibration ladder when you adjust the "200" and "700" marker positions. The calibration ladder remains unchanged when you adjust the "0%" and "100%" positions. Other changes: * Calibration lines are turned off by default. * GelBuddy now handles overlapping 700 channel and 800 channel signals properly * In lane-edit mode you can now drag an entire lane horizontally. * Haplotype information is omitted from the concise report.

Notes for GelBuddy v20040813.03 ------------------------------- Fixes a rather annoying error in the "verbose report" function. -------------------------------------------------------------------------------- Notes for GelBuddy v20040813.01 ------------------------------- (1) Better lane calling: You can call lanes based on the 700 channel image, the 800 channel image, or both. Lane pruning is much improved, so you're much less likely to see bogus lanes on the left and right sides of the gel. Missing lane interpolation method has also changed (improved, I hope)! (2) Lane editing: The button with the arrow and vertical stripes puts you in lane edit mode. Once in lane edit mode, you can: -- Select a lane by clicking on it. -- Edit control points in the currently selected lane (drag the little squares) -- Delete the currently selected lane (Edit/Delete Lane). -- Insert one or more lanes to the left or the right of the currently selected lane (Edit/Insert Lane). If you want to re-find lanes with different parameters (or just undo all your edits) use Edit/Find Lanes to regenerate the lane data. (3) Lane numbers appear above the displayed image: Useful when you're zoomed in on a particular signal. (4) Better reporting: There are now two flavors of reports: "verbose" and "concise" (which looks more like a squint form). Each report contains -- lone signals -- signal pairs (with summed molecular weights) -- aggregate signal pairs (sets of signal pairs in multiple lanes that are "nearly the same"; a range of molecular weights is reported) -- haplotypes (sets of lanes where all the signal pairs are "nearly the same") Some caveats: -- GelBuddy doesn't include "lone signals" in the other reports -- GelBuddy doesn't know the size of the full-length product, so may mispair signals -- The definition of "nearly the same" is pretty loose at this time, since GelBuddy doesn't do any per-lane calibration (yet) Reports can be sent to the log window or saved to a file. You can also process multiple gel pairs, send a report for each pair to the log window, and save the contents of the log window by using the File/Save Log command. (5) GelBuddy now prompts you to adjust the calibration data before accepting signal input. (6) Increased gamma adjustment range (this can be useful for faint gel image pairs)

Notes for GelBuddy v20040730 ---------------------------- New features include: * Clicking the right mouse button on the image toggles between the 700 channel and 800 channel. (What? You have a one button mouse on your mac? Go out and buy a two-button mouse! It's worth it just for this!) * Molecular weight standards -- Drag the "50" and the "700" on the left side of the window to put the molecular weight standard where you think they belong. The green stripes between the 50 and the 700 should match pretty well to the standard LI-COR ladder. The toolbar icon with the horizontal lines turnes the calibration lines on and off. The status bar at the bottom of the screen now reports calibration information for the current mouse cursor position. * Gel markup. Select the crosshair icon on the left side of the toolbar. Now you can add markers by clicking on a feature on the gel. Clicking on an existing marker allows you to delete it. Molecular weight data is recorded for each mark based on the current position of the weight markers. The markup data is _not_ updated if you change the weight marker positions. * Reporting. Once you've marked up your gel, select File/Send Report To Log Window. A report of your markers will appear, sorted by lane and channel number. Some "Save annotated JPEG" bugs have been fixed and new ones introduced.

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