3 Contents 1 Database Getting started Introduction Software installation Quick Guide tutorial data Creating and setting up a new database Creating a new database Setting up a new database Selections of entries Manual selections Automatic search and select functions Experiments Fingerprint data Introduction Sample data Create a fingerprint type experiment Import a fingerprint gel image file Process the fingerprint gel file Define strips Define curves Normalize the gel Define bands Link fingerprint data to entries Fingerprint type experiment settings Assigning a standard pattern Calculating a calibration curve Additional practice XbaI AvrII AvrII Comparisons General comparison functions Comparison settings Compare two entries Create a new comparison Comparison window Comparison layout i
4 Add and remove entries Create groups Clustering fingerprint data Comparison window Cluster fingerprint data Matrix display functions Print a cluster analysis Additional practice Band matching tables Create a band matching table Create a composite data set Create band classes Display the band matching table Band polymorphism analysis Find discriminative band classes Sort entries by band intensity Additional practice Composite data sets Introduction Combine fingerprint experiments Create a composite data set Cluster analysis of a composite data set Additional practice Dimensioning techniques Multidimensional scaling (MDS) Calculate an MDS Change the coordinate space layout Principal components analysis (PCA) Calculate a PCA Change the PCA layout Identification Identification of unknown entries Identification of unknown entries in a comparison Identification of unknown entries in a library Create a library Identify unknown entries with a library ii
5 NOTES SUPPORT BY APPLIED MATHS While the best efforts have been made in preparing this manuscript, no liability is assumed by the authors with respect to the use of the information provided. Applied Maths will provide support to research laboratories in developing new and highly specialized applications, as well as to diagnostic laboratories where speed, efficiency and continuity are of primary importance. Our software thanks its current status for a part to the response of many customers worldwide. Please contact us if you have any problems or questions concerning the use of GelCompar II R, or suggestions for improvement, refinement or extension of the software to your specific applications: Applied Maths NV Applied Maths, Inc. Keistraat Research Boulevard, Suite Sint-Martens-Latem Austin, Texas Belgium U.S.A. PHONE: PHONE: FAX: FAX: URL: LIMITATIONS ON USE The GelCompar II R software, its plugin tools and their accompanying guides are subject to the terms and conditions outlined in the License Agreement. The support, entitlement to upgrades and the right to use the software automatically terminate if the user fails to comply with any of the statements of the License Agreement. No part of this guide may be reproduced by any means without prior written permission of the authors. Copyright c 1998, 2010, Applied Maths NV. All rights reserved. GelCompar II R and BioNumerics R are registered trademarks of Applied Maths NV. All other product names or trademarks are the property of their respective owners. GelCompar II R includes the Python R release from the Python Software Foundation (http://www.python.org/) and a library for XML input and output from Apache Software Foundation (http://www.apache.org).
6 MINIMUM HARDWARE REQUIREMENTS The minimum hardware requirements for running the GelCompar II application are the cumulative requirements needed to run the Operating System, the GelCompar II application and optional database client software and third-party software that will run concurrently (e.g. Microsoft Office). The typical minimum hardware requirements for a computer running Windows XP, Microsoft Office XP and the GelCompar II application are: Processor: 1.30 gigahertz (GHz) processor or higher Processor Type: Intel Pentium 4 or higher compatible processor Memory: 512 MB or higher Hard disk: 1 GB of free disk space (application files only) Display: XGA (1024 x 768) or higher resolution monitor, True Color (32 bit) USB port: Depending on the license type a free USB port may be required A 64-bit processor and Windows version are required for systems with more than 4 GB of RAM installed. The actual hardware requirements will largely depend on the features that will be used in Gel- Compar II, the database platform used to store the GelCompar II data and the size of the data.
7 Part 1 Database
9 Chapter 1.1 Getting started Introduction This Quick Guide provides a general introduction to GelCompar II. Since GelCompar II is a powerful and complex application, many features are not covered in the Quick Guide. Please refer to the Manual for more detailed information on these features. A PDF version of the manual can be found in the installation directory of GelCompar II. While it is not necessary to follow the entire Quick Guide, Chapter 1.1 and Chapter 1.2 are essential for all new users. In these Chapters, you will learn how to install the software, create a new database, and add new database entries. You will then be prepared to import data in the Chapter covering fingerprints (Chapter 2.1). If you prefer to start with data analysis, you can directly go to the Comparisons and Identification Parts (Part 3 and Part 4, respectively). Your ability to use each Chapter will depend on which modules are included with your license. For example, the Identification Chapters require the Identification module; the Dimensioning techniques Chapter requires the Dimensioning and Statistics module, and so on. All modules are included when evaluating the software Software installation If you have not already installed GelCompar II, locate the CD-ROM that came with the package. The latest version of the software can also be downloaded from the Applied Maths website: go to applied-maths.com, select Download and Software. 2.1 Launch the Setup executable. During a first-time installation of GelCompar II, the Welcome dialog box will display the version number of GelCompar II that is included with the Setup package (see Figure 1.1.1). 2.2 Please verify that you are installing the correct version and click <Next> to continue. If an instance of GelCompar II 6.1 or older is already installed, the update Welcome dialog box will be displayed. This dialog box shows the version number of the installed instance of GelCompar II and the new version. The wizard will offer the choice between the installation of a new GelCompar II instance (choose a new installation directory) or to upgrade the existing instance (choose same installation directory as older version). If an instance of GelCompar II 6.5 is already installed, the Existing Installed Instances Detected dialog box will appear when launching the Setup executable. This dialog box allows you to choose between installing a new GelCompar II instance or changing an existing instance.
10 Getting started Figure 1.1.1: The Welcome dialog box. The next dialog will display the Software End User License Agreement (EULA). 2.3 Please read the EULA carefully and click the top I accept the terms of the license agreement radio button and the <Next> button to continue the installation. The user name, organization name and GelCompar II license string need to be entered in the Customer Information dialog box (see Figure 1.1.2). The license string is provided on the sleeve of the CD ROM or in case of an upgrade or an internet evaluation license, you may have obtained it electronically. 2.4 Specify the user and organization names, enter the license string and press <Next>. You must enter a valid license string to be able to continue with the installation. In addition the user and organization names cannot be empty. The installation directory for the GelCompar II application and the database home directory can be entered in the Choose Destination Location dialog box (see Figure 1.1.3). 2.5 Make sure the correct folders are specified and press <Next> once more. The GelCompar II features that you want to install on the local computer can be selected in the Select Features dialog box (see Figure 1.1.4). Clicking on a feature in the left pane will display a short description in the right pane. Install application software: In case of a standalone license, the Application software needs to be installed on each computer that you want to use to run the software. Please note that only on the computer where the dongle is attached to, you will be able to work with the software. In case of an internet license, the Application software needs to be installed on the computer that
11 Software installation 7 Figure 1.1.2: The Customer Information dialog box. you want to use to run the software. Please note that a permanent and stable internet connection is required to run the internet license. In case of a network license, the Application software needs to be installed on the computers in the network that you want to use to run the software. Install sample database and Install sample and tutorial data: The Sample database and Sample and tutorial data that are contained in the Setup package are used in the Quick Guide and in the Manual to illustrate the features of the software. Selecting these features will install the Sample database and Sample and tutorial data in the GelCompar II home directory that is specified in the Choose Destination Location dialog box (see Figure 1.1.3). Install Sentinel drivers: In case of a standalone license, the Sentinel drivers need to be installed on each computer that you want to use to run the software. In case of an internet license, you only need an internet connection to run the software. Since no USB dongle is needed to run an internet license, the Install Sentinel drivers option does not need to be checked. In case of a network license, the Sentinel drivers only need to be installed on the NetKey+ server computer in the network where the hardware security key will be connected to. Install NetKey+ server program:
12 Getting started Figure 1.1.3: The Choose Destination Location dialog box. The NetKey+ server program feature will only be visible and available for installation if a network license string has been entered in the Customer Information dialog box (see Figure 1.1.2). The NetKey+ server program feature must only be installed on the computer in the network where the hardware security key will be connected to. 2.6 Tick the appropriate check boxes for the features you want to install and press <Next>. If a network license string was entered in the Customer Information dialog box (see Figure 1.1.2), and if the GelCompar II application feature was selected for installation (see Figure 1.1.4), the NetKey+ connection settings dialog box will pop up where the NetKey+ Server name and Server port number connection parameters can be entered. 2.7 Click <Install> to start the installation. The Setup Status dialog box is displayed. If a network license string has been entered in the Customer Information dialog box (see Figure 1.1.2), and the NetKey+ server program feature was selected for installation (see Figure 1.1.4), the Setup will ask if you want to run the NetKey+ Configuration tool. This tool allows you to install and subsequently start the NetKey+ service. 2.8 Press <Finish> to close the InstallShield Wizard. 2.9 Double-click on the shortcut on your desktop to open GelCompar II. Alternatively, open the start menu and select GelCompar II under All programs. The Startup program appears (see Figure 1.1.5).
13 Software installation 9 Figure 1.1.4: The Select Features dialog box. Figure 1.1.5: The GelCompar II Startup screen.
14 Getting started Quick Guide tutorial data The data used in the first two Parts of the Quick Guide (Database and Experiments) can be installed with the software (check Install sample and tutorial data in the Select Features dialog box, see Figure 1.1.4). Alternatively, the data can be downloaded from the Applied Maths website: go to com, select Download, Sample data, and GelCompar II Tutorial Data. The Comparisons and Identification Parts use the DemoBase Connected database. This database can be installed with the application software (check Install sample database in the Select Features dialog box, see Figure 1.1.4).
15 Chapter 1.2 Creating and setting up a new database Creating a new database A GelCompar II database is a collection of samples which are potentially comparable to each other. Although a single database can be organized into subsets, very large databases can be unwieldy, so it is preferable to maintain unrelated samples in separate databases. As an example, we will create a new database containing closely related E. coli samples. 1.1 In the GelCompar II Startup screen (see Figure 1.1.5), press the button to enter the New database wizard. 1.2 Specify E. coli as the new database name and press <Next> twice. 1.3 Press <Finish> and press <Proceed> to set up the new database as a connected Access database. 1.4 Press <Proceed> in the Plugin installation toolbox. Plugins will be installed later. A new, empty database opens (see Figure 1.2.1) Setting up a new database As an exercise, we will import data from the text file Ecoli-info.txt (see Figure 1.2.2) into our E. coli database. If the Install sample and tutorial data feature was checked in the Install wizard (see Figure 1.1.4), this text file can be found in the Sample and Tutorial data folder in the database home directory. Alternatively, this text file can be downloaded from our website: go to select Download, Sample data, and GelCompar II Tutorial Data. 2.1 Select File > Import or press to call the Import tree. 2.2 In the Import tree, expand Entry information data, highlight Import fields (text file) and press <Import>. 2.3 In the Select file step, browse for the Ecoli-info.txt file in the GelCompar II Tutorial data \Database folder. Select the file, select the TAB separator from the Field separator list and press <Next> (see Figure 1.2.3). The way the entry information should be imported from the selected file into the database needs to be specified with an import template. 2.4 Press the <Create new> button to create a new import template.
16 Creating and setting up a new database Figure 1.2.1: The GelCompar II main window. Figure 1.2.2: Import entries from a text file. This brings up a new dialog box (see Figure 1.2.4). Each column in the selected file corresponds to a row in the grid (column 1 in the file corresponds to row 1 in the grid, column 2 corresponds to row 2, etc.). The text File field is specified in the Source type column and the column names are displayed in the Source column. The last row in the grid holds the name of the file. 2.5 Select the second row in the grid, press the <Edit destination> button and select the GelCompar II Key field from the list. Press <OK>. The grid is updated (see Figure 1.2.4). 2.6 Highlight the five other external fields in the grid panel using the Shift-key (make sure the last row in the grid is not selected). Press the <Edit destination> button and select the Entry info field option from the list. Press <OK>. 2.7 Press <OK> once more to accept the default suggested names and press <Yes>. The grid is updated (see Figure 1.2.4).
17 Setting up a new database 13 Figure 1.2.3: Select the file to import and the separator. Figure 1.2.4: Define a new import template. 2.8 Optionally, change the default suggested template Name and press <OK>. The import template is added to the list and is automatically selected. 2.9 Press <Next> twice. The E. coli database now contains twelve new entries, each with six database information fields filled in
18 Creating and setting up a new database (see Figure 1.2.5). Figure 1.2.5: The GelCompar II main window after import Double-click on database entry CL001 to open the Entry edit window. Information can be edited in the Entry edit window and saved (see Figure 1.2.6). Figure 1.2.6: The Entry edit window Close the Entry edit window. Alternative to using the Entry edit window, information in the information fields can be edited directly by clicking twice (not double-click) on an information field in the database. The information will appear highlighted and can be edited Click twice on one of the information fields (not a Key information field) or select Ctrl+Enter. The information appears selected blue against a bright colored background and can be modified (see Figure 1.2.7).
19 Selections of entries 15 Figure 1.2.7: Clicking twice on an information field enables direct editing Use the ArrowUp and ArrowDown keys on the keyboard to jump to the previous/next row To jump to the next column, use the Tab key To jump to the previous column, select Shift+Tab on the keyboard Selections of entries Manual selections 3.1 To select an entry in the database, hold the Ctrl key and click on the entry in the Database entries panel. Alternatively, use the space bar to select entries. The selected entry is marked by a colored arrow (see Figure 1.2.8). Figure 1.2.8: A selected entry. 3.2 Selected entries are unselected in the same way (Ctrl+click). 3.3 In order to select a group of entries, click on an entry, hold the Shift key and click on another entry. All entries that are listed between these two entries are selected, including the two entries. 3.4 To select all entries in the database, use Edit > Select all entries (Ctrl+A). 3.5 To clear a selection in the database, use Edit > Unselect all entries (, F4) Automatic search and select functions Simple and intuitive search functions can be used to search and select entries. 3.6 Select Edit > Search entries... (, F3). 3.7 In the Entry search window, enter for example Dallas in the Origin field (see Figure 1.2.9) and press <Search>. All entries having the name Dallas in their Origin field are selected. 3.8 To clear the selection, use Edit > Unselect all entries (, F4).
20 Creating and setting up a new database Figure 1.2.9: The Entry search window.
21 Part 2 Experiments
23 Chapter 2.1 Fingerprint data Introduction In this Chapter we will: Create a fingerprint type experiment Import a fingerprint gel image file Process the fingerprint gel file Link fingerprint data to entries Sample data As an exercise, we will import a 8-bit TIFF gel image which was generated by PFGE with the restriction enzyme Xba-I in our E. coli database. The gel image can be downloaded from the Applied Maths website: go to select Download, Sample data, and GelCompar II Tutorial Data. If the Install sample and tutorial data feature was checked in the Install wizard (see Figure 1.1.4), the gel image can be found in the Sample and Tutorial data folder in the database home directory Create a fingerprint type experiment First we need to create a new fingerprint type experiment before we can import a gel image file in our E. coli database. 3.1 In the GelCompar II startup screen, double-click on the E. coli database created in to open it. 3.2 In the GelCompar II main window, select Experiments > Create new fingerprint type from the main menu, or press the button from the Experiments panel toolbar and select New fingerprint type. 3.3 Enter a name, for example PFGE-XbaI and press <Next>. 3.4 In the next window, select Two dimensional TIFF files and 8 bit OD depth (256 gray values). Press <Next> to proceed. 3.5 In the next window, select Yes for fingerprints with inverted densitometric values.
24 Fingerprint data 3.6 Press <Next> to proceed. 3.7 In the final step, leave No selected for applying a background subtraction. 3.8 Press <Finish>. The Experiments panel now lists the fingerprint type PFGE XbaI (see Figure 2.1.1). Figure 2.1.1: The Experiments panel. You can still change the resolution, inversion, and background subtraction settings later when processing a gel Import a fingerprint gel image file 4.1 Select File > Add new experiment file... ( ) in the GelCompar II main window. 4.2 Select the file ec-xbai-001.tif in the GelCompar II Tutorial data \PFGE TIFFS folder. A box appears asking if you want to edit the image. Press <No> if you are sure the file is an uncompressed gray scale TIFF image. For the conversion to an uncompressed gray scale TIFF file press <Yes>. 4.3 Since the example file is an uncompressed gray scale TIFF file, press <No>. The gel image is now available in the Files panel (see Figure 2.1.2). The file is marked with a red N ( ) indicating that the image has not been processed yet. Figure 2.1.2: The Experiments panel and Files panel Process the fingerprint gel file Before linking our fingerprint lanes to entries in our database, we must define and process the lanes in the Fingerprint data window. 5.1 In the Files panel, double click on the filename ec-xbai-001 to open the Fingerprint file window.
25 Process the fingerprint gel file In the Fingerprint file window, select File > Edit fingerprint data to open the Fingerprint data window. 5.3 In the next dialog box, select PFGE-XbaI and press <OK>. The Fingerprint data window opens and the imported gel image is surrounded by a green rectangle. Since GelCompar II recognizes the darkness as the intensity of a band, make sure the bands appear as dark bands on a white background (see Figure 2.1.3). In case the bands appear as white bands on a black background, invert the values: press to open the Fingerprint conversion settings window, check/uncheck the Inverted values check box, and press <OK> to apply the changes Define strips The first step in processing a gel is to crop the image to remove empty space, and to define the lanes. 5.4 Delineate the area of the gel lanes by clicking and dragging the nodes of the rectangle to adjust it. Exclude the wells from the rectangle (see Figure 2.1.3). Figure 2.1.3: Area of gel lanes. 5.5 In the toolbar, press to let the software search for the individual lanes. 5.6 Enter 10 as the approximate number of lanes and press <OK>. 5.7 In the toolbar, press to open the Fingerprint conversion settings window. When opened in the Strips panel the Raw data tab is selected by default. 5.8 Adjust the Thickness of the image strips so the splines border the edges of the bands (e.g. 33 points). 5.9 Press <OK> Adjust the horizontal position of each spline as necessary by clicking and dragging a blue node. Hold down the Shift key while dragging a node to adjust the spline locally.
26 Fingerprint data 5.11 Decrease the thickness of the spline in lane 10 by clicking one of its nodes and repeatedly pressing or F8. Then move the spline over to the good portion of the lane to the left (see Figure 2.1.4). Figure 2.1.4: Adjusting the thickness and positions of the splines. Next, we will edit the tone curve to improve the band visibility From the pull down menu, select Edit > Edit tone curve Press <Linear>. The visible gray scale interval now ranges between the minimum and maximum values in the image (see Figure 2.1.5). Figure 2.1.5: The Gel tone curve window Press the other editing buttons such as <Enhance weak bands> a few times until you have a clear and sharp image Press <OK> to save changes to the tone curve settings. The gel tone curve does not change the image s densitometric values, only the way they appear in the Fingerprint data window Press to save the work already done Press to proceed to the Curves step or press the Curves tab.
27 Process the fingerprint gel file Define curves Now that the lanes have been defined, the software can generate densitometric curves describing the optical density across the spline along each lane. The left panel shows the strips extracted from the image file, the right panel shows the densitometric curve of the selected pattern (see Figure 2.1.6). The area between the blue lines of each lane will be used to calculate the densitometric curve Select lane 3. Near the top of the lane in the spline there is a pinpoint spot. As a result, a thin peak appears on the densitometric curve shown on the right side of the window It might help to zoom in on the image using the zoom slider Open the Fingerprint conversion settings window again (click on the button) Change the Averaging thickness for curve extraction to 15 in the Densitometric curves tab Check Median filter and press <OK>. In lane 3, the thin peak resulting from the pinpoint spot has disappeared in the densitometric curve shown on the right side of the window (see Figure 2.1.6). Figure 2.1.6: Increased splines and median filtering. GelCompar II can analyze the gel image to determine the optimal settings for removing background noise from the densitometric curves Select Curves > Spectral analysis. The following settings are shown in the Spectral analysis window: 1. Wiener cutoff scale: Determines the optimal setting for least square filtering. 2. Background scale: Estimation of the disk size for background subtraction.
28 Fingerprint data 5.24 Close the Spectral analysis window Open the Fingerprint conversion settings window again (click on the button) Check Apply least square filtering and specify a Least square filtering Cut off as indicated by the Wiener cutoff scale in the Spectral analysis window (use the percentage value, e.g. 1). Least square filtering removes very small peaks from the curves Check Apply in the Background subtraction panel and specify a Background subtraction disk size as indicated by the background scale in the Spectral analysis window (use the percentage value, e.g. 14). Background subtraction removes large background trends from the curves Press <OK>. The background noise has been removed from the curves (see Figure 2.1.7). Figure 2.1.7: Curves after filtering Press to save the work already done Press to proceed to the Normalization step or press the Normalization tab Normalize the gel Every fingerprint type experiment needs at least one reference system to normalize its gels. Since this is the first Xba-I gel we have imported, we need to create a reference system based on the standard pattern in this gel. We will use the standard s molecular weights to name the reference positions. Subsequent Xba-I gels, provided they contain the same standard, will be normalized with the same reference system Press to enter the normalized view. For now, the normalized view looks the same as the original view. In a first step we will define the reference lanes and the reference bands Select lane 1 and press to assign it as a reference lane or select References > Use as reference lane Repeat this for lane 6 and lane Choose the most suitable standard lane for creating the reference system. For this exercise select lane 6 by clicking anywhere in the lane so that the lane number turns orange.
BioNumerics Tutorial: Importing and processing gel images 1 Aim Comprehensive tools for the processing of electrophoresis fingerprints, both from slab gels and capillary sequencers are incorporated into
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