Beckman Coulter CEQ 8000 User Manual
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Beckman Coulter CEQ 8000 User Manual

Beckman Coulter CEQ 8000 User Manual

Genetic analysis system
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A16637-AA
April 2004
TM
CEQ
8000
™
Genetic
Analysis
System
User's Guide
Beckman Coulter, Inc.
4300 North Harbor Boulevard, Fullerton, CA 92834-3100
Copyright 2004 Beckman Coulter, Inc.

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  • Page 1 A16637-AA April 2004 8000 ™ Genetic Analysis System User’s Guide Beckman Coulter, Inc. 4300 North Harbor Boulevard, Fullerton, CA 92834-3100 Copyright 2004 Beckman Coulter, Inc.
  • Page 2 Copyright, Licenses and Trademarks © Beckman Coulter, Inc., 2004. All rights reserved. No part of this publication may be reproduced, transcribed, transmitted, or translated into any language in any form by any means without the written permission of Beckman Coulter, Inc.

  • Page 3: Table Of Contents

    CEQ™ 8000 Genetic Analysis System – User’s Guide Document A16637AA Table of Contents Foreword ..........................xiii About this Guide......................... xiii Symbols Used in this Guide ....................xiii Getting Help ........................xiv System Overview ........................1 Purpose of this System ......................1 Functional Description ......................1 Hardware..........................1 Sample Access Cover ....................2 Capillary Access Cover ....................2...
  • Page 4 Table of Contents Run Module.......................... 29 Main Window ......................30 Menu Bar Options....................... 31 File Menu ......................32 View Menu ......................33 Direct Control Menu .................... 34 Tools Menu......................36 Run Menu ......................38 Log Options Menu....................40 Replenish Menu....................40 Help Menu......................
  • Page 5 Menu Bar Options .......................99 File Menu ......................99 View Menu......................101 Results Menu ......................101 Fragments Menu ....................102 Analysis Menu ....................102 Reports Menu......................103 Window Menu ....................103 Help Menu ......................104 Toolbar Icons......................104 Data Manager Module ......................107 Main Window......................108 Menu Bar Options .....................109 File Menu ......................109 Edit Menu......................110 View Menu......................111 Tools Menu ......................111...
  • Page 6 Table of Contents Naming Samples and Assigning Subject IDs ............128 Turning Cell Coordinates On or Off .................. 129 Using Find.......................... 129 Using Replace ........................129 Using Property Sets......................131 Assigning Methods ......................133 Condition Method ..................... 134 Creating or Editing a Method..................... 134 Assigning Analysis Parameter Sets..................
  • Page 7 Perform Sequence-based Trimming.............164 Vector/Other Sequence Files................164 Max. # of Vector/Other Sequences ..............164 Min. Substring Length..................164 Distance from End..................164 Enable Internal Matches................164 Quality Values........................164 Preventing Alignment Problems................164 Performing Quality-based Trimming ................165 Apply Quality Trimming................167 Undo ......................167 Performing Sequence-based Trimming..............167 Apply Sequence-based Trimming ..............169 Undo ......................169 Internal Trimming ..................170 Batch Trimming......................171...
  • Page 8 Table of Contents Pinning Results ......................188 Performing a Batch Analysis ..................188 Reanalyzing a Batch ....................188 Viewing the Selected Batch Result................189 Skipping the Current Sample Analysis in a Batch............ 189 Skipping the Current Sample Set Analysis in a Batch..........189 Skipping the Current Sample Plate Analysis in a Batch...........
  • Page 9 Selecting Raw Data for the New Study ..............224 Selecting Raw Data from the List View tab ............224 Selecting Raw Data from the Plate View tab............225 Selecting an Analysis Parameter Set .................227 Selecting a Saved Analysis Parameter Set............228 Analyzing the Data ....................228 Selecting Additional Results to be added to the Study..........229 Defining Fragment Analysis Parameters..............230 Fragment Analysis Parameters - General tab............231...
  • Page 10 Table of Contents Peak Ratios........................ 274 Beginning a Peak Ratio Calculation..............274 Selecting a Reference Trace ................276 Selecting a Reference Peak ................277 Selecting a Test Peak..................277 AFLP Analysis......................278 Using the AFLP feature..................278 Setting the AFLP Analysis Parameters .............. 278 Analysis Parameters ...................
  • Page 11 Data Manager Export File Types................317 Importing Database Items....................320 Generating a Sample Run History ..................321 Administrative Tools......................321 Add Users and Groups....................321 Adding Users ......................322 Adding Groups....................323 Activating the CEQUSERS Group ..............326 Direct Control and Replenishment ..............327 Accessing the Direct Control Window ................328 Direct Control Procedures ....................328 Replacing the Wetting Tray..................328 Removing the Wetting Tray................328...
  • Page 12 Table of Contents Disposal of D.I. Water/Gel Mixture from the Wetting Tray ........360 Disposal of the Gel Waste Bottle................361 Consumable Items List....................... 362 Materials Required but not Supplied ................ 365 Diagnostics ........................367 Re-Initializing the System....................367 Homing the Plates and/or Gel Pump.................. 367 Viewing PC Settings ......................

  • Page 13: Foreword

    CEQ™ 8000 Genetic Analysis System – User’s Guide A16637-AA Foreword About this Guide This guide is intended for use with the CEQ™ 8000 Genetic Analysis System. It is divided into the following sections: • “Foreword,” this section, describes the purpose of this guide, provides a list of its contents, discusses the use of the symbols used in the document as well as providing service contact information.

  • Page 14: Getting Help

    Beckman Coulter directly using the information below. Whenever you call your local dealer or Beckman Coulter, be sure to have your registration material, instrument serial number and soft- ware version number available.

  • Page 15: System Overview

    Generation of samples for fragment length analysis is performed using dye-labeled primers. The CEQ 8000 accepts up to 96 four-color samples in a microplate. Each row of eight samples (sample set), containing labeled DNA fragments, is automatically denatured and then separated by capillary electrophoresis.

  • Page 16: Sample Access Cover

    System Overview Figure 1: Hardware Overview for the CEQ 8000 Sample Access Cover (extended) Capillary Access Cover (extended) Capillary Temperature Control Cover Rubber Latches Status Indicators Manifold Access Cover Plate Holders/ LASER Sample Transport Gel Waste Bottle Power Switch Gel Pump...

  • Page 17: Plenum

    Hardware The capillary array (Figure 2) has three components: Electrode Block (inlet), eight capillaries and the Array Fitting (outlet). • The Electrode Block is the DNA sample inlet side of the array. It holds eight hollow, stainless-steel electrodes. Each stainless-steel electrode holds a capillary in the center.

  • Page 18: Sample Transport And Plate Holders

    System Overview Refer to the caution label on the front of the plenum assembly for positive identification Figure 3: Plenum Assembly (Back View) Routing Notch Conical Depression 900696L.AI Sample Transport and Plate Holders The Sample Transport (Figure 4 ) contains the Plate Holders which are used to hold the Sample Plate, Buffer Plate w/Buffer Evaporation Cover and Wetting Tray.

  • Page 19: Sample Plate

    Hardware Sample Plate The Sample Plate (Figure 5) is used to hold the sample(s) for separation. The plate is a V-bottom, thermal cycler-compatible, polypropylene plate containing 96 wells (8-rows x 12-columns). The wells have a 200µL volume capacity. Figure 5: Sample Plate 900497e.AI Buffer Plate with Buffer Evaporation Cover...

  • Page 20: Gel Waste Bottle

    System Overview No more than one 96-well plate should be processed without CAUTION replenishing the Wetting Tray. For information on Cleaning and Filling the Wetting Tray, see page 356. Figure 7: Wetting Tray 900499e.AI Gel Waste Bottle The Gel Waste Bottle (Figure 8) is used to capture and store the waste gel that is pushed out of the manifold during the purge function.

  • Page 21: Gel Pump, Gel Cartridge And Gel Pump Plug

    Hardware Gel Pump, Gel Cartridge and Gel Pump Plug Gel Pump (Figure 9) Used to replenish the capillaries with fresh gel (from the cartridge) after each sample set and each 96-well plate runs. Gel Cartridge Contains the fresh DNA separation gel. The separation gel is used for both Sequence and Fragment Analysis.

  • Page 22: Status Indicator Lights

    System Overview Status Indicator Lights There are three status indicators: PWR (power), LASER and HV (high voltage). Green when power is supplied to the CEQ. Off when no power is supplied. LASER Green when the lasers are turned on during a run. Off when the lasers are turned off.

  • Page 23: Software

    Software Software The software provides the interface for manual or automatic (pre-programmed) control of the system and for data capture and basic data analysis. User Interface The Main Menu is shown in Figure 10. This window provides access to all modules of the CEQ Software.
  • Page 24 System Overview Table 1: Software Module Descriptions Module Description Sample Setup Module Use this module to create, save and modify sample plates. Sample plates are used to assign methods to control sample sets and determine the sequence of methods that will be used to produce data.

  • Page 25: Safety Information

    Safety Information Safety Information This section provides safety information and instructions for the hardware and accessories of the system. It is broken down into the following subsections: • Symbols • Safety Features • Chemical and Biological Safety • Electrical Safety •...
  • Page 26 System Overview BIOHAZARD Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from a biological source. In this document, the “ ” icon will accompany this symbol. WARNING LASER LIGHT Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from a laser source.

  • Page 27: Chemical And Biological Safety

    For this reason, any service requiring removal of a panel or otherwise overriding or disabling safety interlocks must be done by Beckman Coulter personnel only. Moving Parts Moving parts are limited to the sample handling system. Plate movement is safety interlocked through the Sample Access Cover.

  • Page 28: Laser Safety

    Laser Assembly. The Laser Assembly has no user serviceable parts. Service of the Laser Assembly is restricted to certified Beckman Coulter Field Engineers. During normal operation of the system, laser light is not accessible to the user.
  • Page 29 Safety Information Figure 11: Laser Label Locations Capillary Access Cover (extended) Laser Assembly Cover (cosmetic panel removed) CAUTION LASER LIGHT ACCESSIBLE WHEN COVER IS OPEN OR REMOVED. AVOID EXPOSURE *approximate location AVOID EXPOSURE LASER LIGHT IS EMITTED FROM THIS APERATURE. *approximate location rear CLASS 1 LASER PRODUCT...
  • Page 30 System Overview EMI Effect on CEQ™ (2000 Series / 8000 Series) Performance and Recommended Mitigations Under the test conditions specified by the European normative electromagnetic compatibility standard EN 61326-1, the CEQ instrument may exhibit temporary degradations in performance in accordance with the table below. Because the environmental circumstances contributing to the problem can vary, several different mitigation techniques have been provided that should help eliminate or reduce the interference.

  • Page 31: Program Description

    CEQ™ 8000 Genetic Analysis System – User’s Guide A16637-AA Program Description The following sections provide detailed descriptions of the main windows, menus, dialog boxes and software commands, in each module, that are used in controlling the system. The module titles are: •...

  • Page 32: Sample Setup Module

    Program Description Sample Setup Module The Sample Setup module is used to create, save and modify methods and sample plates. Sample plates are the means by which samples are named and organized. Methods consist of sequential events needed to perform a DNA separation. The sample plate definition contains the 96-well plate locations of the samples as well as the method assigned to each sample.

  • Page 33: Main Window

    Sample Setup Module Main Window The main window of this module is shown in Figure 13 and described in Table 2. Figure 13: Sample Setup Module, Main Window User’s Guide...
  • Page 34 Program Description Table 2: Sample Setup Module, Main Window Descriptions Item Description The Title Bar showing the module name (“Sample Setup”) and the active sample plate (“DefaultSamplePlate”). Menu Bar (See “Menu Bar Options” on page 22.) Toolbar (See “Toolbar Icons” on page 26.) View by toggles the plate view from Sample Name to Subject ID view.
  • Page 35 Sample Setup Module Table 2: Sample Setup Module, Main Window Descriptions Item Description Notes Window Used to enter explanatory text concerning the selected sample(s). The text box will display “(--multiple selection--)” when multiple samples with different descriptions are selected. Figure 14: Notes Window Property Sets Listbox Property sets are groups of properties that can be applied to a sample(s), such as Vector, Host and Primer used.

  • Page 36: Menu Bar Options

    Program Description Table 2: Sample Setup Module, Main Window Descriptions Item Description Method Window Provides a summary of the separation parameters defined for the selected method. Edit existing or create a new method by clicking on the Edit button. Figure 16: Method Window (cont'd) Analysis Window Used to:...

  • Page 37: File Menu

    Sample Setup Module File Menu Table 3: Sample Setup Module, File Menu Item Description Used to open an empty sample plate. Open Used to select an existing sample plate to open. Close Used to close the selected sample plate. Save Used to save the current sample plate.

  • Page 38: View Menu

    Program Description Item Description Paste Used to insert copied or cut items at the cursor insertion point. Find Use this dialog box to search for text within the sample name entered in a sample plate. Replace Use this dialog to search and replace text within the sample names entered in a sample plate.

  • Page 39: Run Menu

    Help file and search for information by: topic, index entry and/or keyword. About CEQ Used to access software, instrument and system System information. Beckman Used to access the Beckman Coulter Home Page on the Coulter Internet. Home Page User’s Guide...

  • Page 40: Toolbar Icons

    Sample Setup Module Toolbar Icons The toolbar (Figure 19) contains icons that correspond to common menu options. The information in Table 9 describes the function of each icon. Figure 19: Sample Setup Module, Toolbar Table 9: Sample Setup Module, Toolbar Icon Description New - Used to open an empty sample plate.
  • Page 41 Sample Setup Module Icon Description Help Topics - Used to select and print specific topics in the CEQ Help file and search for information by: topic, index entry and/or keyword. Context-Sensitive Help - Used to open the Help file related to a specific menu option. Run Sample Plate - Used to initiate the Run module and run the plate.
  • Page 42 Program Description CEQ™ 8000 Genetic Analysis System...

  • Page 43: Run Module

    Run Module Run Module The Run module provides the capability of executing pre-programmed sample plates and controlling individual functions of the instrument. Specifically, it is used for: • Running and viewing the progress of a sample plate • Viewing and changing display options •...

  • Page 44: Main Window

    Main Window The main window of the Run module is shown in Figure 22 and described in Table 10. Tables 11 through 18 provide descriptions of the menu options. Figure 22: Run Module, Main Window Table 10: Run Module, Main Window Descriptions Item Description The Title Bar displays the name of the module (CEQ Run) and the user specified system name.

  • Page 45: Menu Bar Options

    Run Module Item Description Capillary Buttons - Letters A through H represent the eight capillaries in the array. Each button selected will have an associated pane shown in the Display Area. The Status Bar displays messages such as • Currently Active Database - The active database name will be displayed. •...

  • Page 46: File Menu

    Figure 23: Run Module, Menu Bar Items File Menu Table 11: Run Module, File Menu Item Description Restore Data Restores the colors of the data monitor to the original Monitor default shipped with the instrument. Defaults Save Log Used to select the folder where the system log will be stored.

  • Page 47: View Menu

    Run Module View Menu Table 12: Run Module, View Menu Item Description Toolbars Used to select the toolbars to display in the Run module window. Status Bar Toggles between displaying/not displaying the Status Bar. Status Toggles between displaying/displaying the Status Monitor Monitor (see “Status Monitor”...

  • Page 48: Direct Control Menu

    Direct Control Menu Table 13: Run Module, Direct Control Menu Item Description Unload Plates Used to move the plate holder to the Load position at the front of the instrument to load or unload plates. Plate This dialog box is used to “graphically” select the well Position location where the capillaries will be immersed.
  • Page 49 Run Module Capillary Used to change the capillary holding temperature. Temperature This temperature will be over-ridden at the end of each sample plate or when cancelled. Denature Used to initiate the denaturing of samples. Samples Optical Used to align the lasers with the detection windows of the Alignment eight capillaries.

  • Page 50: Tools Menu

    Separate Used to initiate the separation of injected samples. The data generated is not saved. Used to fill all capillaries with fresh gel. (Any gel present Capillary Fill in the capillary is forced out to the specified waste position in the buffer plate or wetting tray.) Manifold Used to clear the manifold of air bubbles and/or Purge...
  • Page 51 Run Module Table 14: Run Module, Tools Menu (Continued) Item Description Unzoom All Used to undo all zoom levels. Display Invokes the Display Options dialog box used to Options modify any or all of the following parameters: • Title • X Axis Options •...

  • Page 52: Run Menu

    Run Menu Table 15: Run Module, Run Menu Item Description Start Sample Used to open and execute a specific sample plate. Plate Pause Used to pause a running sample plate. Stop Used to stop a running sample plate or direct control System process.
  • Page 53 Run Module Item Description When stopping a direct control process: Stop Options - Specifies what process to stop: Stop All cancels all the currently running processes. stops the high Stop High Voltage Only voltage but continues to inject the sample. Monitor Baseline monitoring displays the baseline data trace.

  • Page 54: Log Options Menu

    Log Options Menu Table 16: Run Module, Log Options Menu Item Description Errors Only Causes the program to list errors only in the Log Window. All Details Causes the program to display all details in the Log Window including all messages and system activity. Sample Causes the program to only list items related to a running Plate Only...
  • Page 55 Run Module Install Used to install the capillary array. Verify or change the Capillary information requested, and then install the array. Array Release Used to remove the current capillary array. To install a Capillary new capillary array, select the Replace capillary Array array radio button.
  • Page 56 Gel/Buffer Used to view the part number, lot number for the gel Information and/or buffer, the date and time of gel cartridge installation and the number of hours the cartridge has been on the instrument. Install Gel Used to install a gel cartridge. Cartridge CEQ™...
  • Page 57 Run Module Release Gel Used to release the gel cartridge in preparation for Cartridge installing a new cartridge or preparing the system for short or long-term storage. When selected, the following confirmation dialog will open. Select OK to continue. Enter the requested information and then: •...
  • Page 58 Replace Used to replace or re-fill the wetting tray. When selected, Wetting Tray the following confirmation dialog will open. Select the position where you wish the capillaries to be immersed after the wetting tray is replaced. If you wish to select the location graphically, click on the Plates button.

  • Page 59: Help Menu

    About CEQ Used to access software, instrument and system System information. Beckman Used to access the Beckman Coulter Home Page on the Coulter Internet. Home Page Toolbar Icons There are six toolbars (Figure 24) used in the Run module: Standard, Direct Control, Data Monitor, Log, Sequence Dye Colors, and Fragment Dye Colors.

  • Page 60: Standard Toolbar

    Figure 24: Run Module, Toolbars Standard Toolbar (Table 19) Direct Control Toolbar (Table 20) Data Monitor Toolbar (Table 21) Log Toolbar (Table 22) Sequence Dye Colors Toolbar (Table 23) Fragment Dye Colors Toolbar (Table 24) (Figure 29) Standard Toolbar Figure 25: Run Module, Standard Toolbar Table 19: Run Module, Standard Toolbar Button Description...

  • Page 61: Direct Control Toolbar

    Run Module Button Description View Last Analysis - Used to execute the Sequence Analysis module and display the last sample analyzed. Help Topics - Used to select and print specific topics in the CEQ Help file and search for information by: topic, index entry and/or keyword. Context-Sensitive Help - Used to open the Help file related to a specific menu option.

  • Page 62: Data Monitor Toolbar

    Data Monitor Toolbar Figure 27: Run Module, Data Monitor Toolbar Table 21: Run Module, Data Monitor Toolbar Unzoom - Used to undo one zoom level. Unzoom All - Used to undo all zoom levels. Autoscroll On/Off- Used to scale all data to the last 10 minutes of the run on the X axis and confine the display of data to the pane on the Y axis.

  • Page 63: Log Toolbar

    Run Module Log Toolbar Figure 28: Run Module, Log Toolbar Table 22: Run Module, Log Toolbar Print - Used to print a single pane selected in the Display Area. Print Preview - Used to display a facsimile of a hardcopy printout of the active sample plate.

  • Page 64: Fragment Dye Colors Toolbar

    Fragment Dye Colors Toolbar Figure 31: Run Module, Fragment Dye Colors Toolbar Table 24: Run Module, Fragment Dye Colors Toolbar Icon Description The color red is the default color assigned to Dye #1 in the Fragment Data pane, and D1 (red) is the default name.

  • Page 65: Status Monitor

    Run Module Status Monitor The Status Monitor displays the state of the current run. Figure 32 shows the status monitor and Table 25 describes the areas. Figure 32: Run Module, Status Monitor Displays User’s Guide...
  • Page 66 Table 25: Run Module, Status Monitor Displays Item Function Progress Indicator - This area of the Status Monitor shows the Status, Event Type and Progress of a running sample plate or process. Times displayed are approximations. Information Tabs - Toggles between the tabs shown below. •...

  • Page 67: Window Selection Tabs

    Run Module Window Selection Tabs The Window Selection tabs provide access to the following windows: Data Monitor, Direct Control, Log and Instrument Data. These windows are described on the following pages. Data Monitor Window The Data Monitor window is shown in Figure 33. This window is accessed in the Display Area by selecting the Data Monitor tab.

  • Page 68: Direct Control Window

    Direct Control Window The Direct Control window is shown in Figure 34. This window is accessed in the Display Area by selecting the Direct Control tab. This window contains hot areas and function buttons that perform some of the distinct tasks listed in the “Direct Control”...
  • Page 69 Run Module Figure 34: Run Module, Direct Control Window “Right” click anywhere in window for full menu í The circled numbers shown on the window in Figure 34 represent the locations of the hot areas in the Direct Control window. Each hot area, when selected, initiates a unique Direct Control task.
  • Page 70 Table 27: Direct Control Window, Hot Areas Item Description Capillary Temperature - Used to change the capillary temperature. Gel Capillary Fill - Used to fill all capillaries with fresh gel. (Any gel present in the capillary is forced out to the specified waste position in the buffer plate or wetting tray.) Optical Alignment - Used to align the lasers with the detection windows of the eight capillaries.

  • Page 71: Log Window

    Run Module Log Window The Log window (Figure 35) is accessed in the Display Area by selecting the Log tab. The log window provides all of the messages and activity for a sample plate run. Figure 35: Run Module, Log Window Table 28: Run Module, Log Window Descriptions Item Description...

  • Page 72: Instrument Data Window

    Instrument Data Window The Instrument Data window (Figure 36) is accessed in the Display Area by selecting the Instrument Data tab. This window displays the current for the eight capillaries and the voltage level of the instrument for the current run. Figure 36: Run Module, Instrument Data Window Table 29: Run Module, Log Window Descriptions Item...

  • Page 73: Sequence Analysis Module

    Sequence Analysis Module Sequence Analysis Module The Sequence Analysis module is used to view, analyze, compare, manipulate and print data of the following types: • Raw Data* • Current Data* • Voltage Data* • Analyzed Data • Base Sequences • Optical Scan Data* •...

  • Page 74: Main Window

    Main Window The main window of the Sequence Analysis module is shown in Figure 38 and is described in Table 30. Figure 38: Sequence Analysis Module, Main Window Table 30: Sequence Analysis Module, Main Window Descriptions Item Description The Title Bar showing the module name (CEQ Sequence Analysis) and the currently active sample.
  • Page 75 Sequence Analysis Module Item Description This Display Area graphically displays the opened data. Analyzed Data Displays the data that has been analyzed for the active sample. Base Sequence Displays a text view of the bases from the analyzed data for the active sample.

  • Page 76: Menu Bar Options

    Menu Bar Options The menu bar has the options shown in Figure 39. Tables 31 through 37 provide descriptions of the menu options. Figure 39: Sequence Analysis Module, Menu Bar Options File Menu Table 31: Sequence Analysis Module, File Menu Item Description Open...
  • Page 77 Sequence Analysis Module Item Description Export from Used to open or export samples from a specific sample plate. Multiple samples can be selected using the Shift Plate key. (The Sample Plate toolbar must be open to use this menu option.) Preferences Used to: •...

  • Page 78: Edit Menu

    Item Description Properties Used to view the general properties of the currently selected item. View the item type, database and project where the item resides, as well as the date and time the item was last modified. You may also view the sample name, sample plate, sample position in the sample plate, the instrument, and the operator name.

  • Page 79: View Menu

    Sequence Analysis Module Item Description Apply Use this option to commit to the Quality-based trimming. Quality- based Trimming Apply Use this option to commit to the Sequence-based Sequence- trimming. based Trimming Audio Used to audibly announce a series of selected base Playback sequence text.
  • Page 80 Item Description Analysis Displays the Analysis Log for the active sample. Parameters Used to display the sequence analysis parameters used to Used to produce the given analyzed data. Compute The fields in this dialog box are read-only, and are not Sequence editable.

  • Page 81: Tools Menu

    Sequence Analysis Module Tools Menu Table 34: Sequence Analysis Module, Tools Menu Item Description Zoom Used to magnify a specific area of data in the display. Used to move analyzed data in the X and Y direction. Align Used to visually align bases and peaks in the analyzed data pane.
  • Page 82 Item Description Align Used to align selected points of each of the displayed Compare analyzed data sets while in the Compare mode. Views Display Invokes the Display Options dialog box used to Options modify any or all of the following parameters: •...
  • Page 83 Sequence Analysis Module Item Description Heterozygote Opens the Heterozygote Display Colors dialog. This is Display Color used to change the color of the heterozygotes in the base sequence pane. User’s Guide...

  • Page 84: Analysis Menu

    Analysis Menu Table 35: Sequence Analysis Module, Analysis MenuTrim Item Description Analyze Used to analyze active sample data or reanalyze existing sequence results. When selected, the current Working Parameters dialog box is displayed. The analysis begins when OK is pressed. Stop Terminates the executing analysis.
  • Page 85 Sequence Analysis Module Item Description Batch Used to analyze multiple Sample Data or Sample Plate Analysis Results sequentially. Invokes the following dialog box. Once files are transferred to the right-side list box and OK is selected, the analysis parameters editor dialog box is displayed.
  • Page 86 Item Description Skip While performing a batch analysis, use this item to pass Current over the sample currently being analyzed. (This item is not Sample available if Sample Plate Results was selected from the Batch Analysis Selection dialog box.) Skip While performing a batch analysis, use this item to pass Current over the sample set currently being analyzed.

  • Page 87: Window Menu

    About CEQ Used to access software, instrument and system System information. Beckman Used to access the Beckman Coulter Homepage on the Coulter Internet. Homepage Toolbar Icons There are seven toolbars (Figure 40) used in the Sequence Analysis module: (1) Standard, (2) Data, (3) Sample View, (4) Sequence Dye Colors, (5) Batch Control, (6) Sample Plate and (7) Base Sequence.
  • Page 88 Figure 40: Sequence Analysis Module Toolbars Standard Toolbar (Table 38) Data Toolbar (Table 39) Sample View Toolbar Sequence Dye Colors Toolbar (Table 41) Batch Control Toolbar (Table 42) Sample Plate Toolbar Base Sequence Toolbar (Table 40) (Figure 47) (Figure 46) CEQ™...

  • Page 89: Standard Toolbar

    Sequence Analysis Module Standard Toolbar Figure 41: Sequence Analysis Module, Standard Toolbar Table 38: Sequence Analysis Module, Standard Toolbar Icon Description Open - Used to open: Sample Data, Sequence Analysis Parameters, Optical Scan Data, Sequence Results and Sample Plate Results. Save - Saves existing data.
  • Page 90 Icon Description Stop - Terminates the executing analysis. Trim Based on Quality - Use this option to perform a Trim based on Quality. Trim Based on Sequence - Use this option to perform a Trim based on a Sequence. Restore Original Base Sequence - Used to return analyzed data display and base sequence text back to its original state (losing all edits).

  • Page 91: Data Toolbar

    Sequence Analysis Module Data Toolbar Figure 42: Sequence Analysis Module, Data Toolbar Table 39: Sequence Analysis Module, Data Toolbar Icon Description Zoom Mode - Used to magnify a specific area of data in the display. Pan Mode - Used to move analyzed data in the X and Y direction. Align Mode - Used to visually align bases and peaks in the analyzed data pane.

  • Page 92: Sample View Toolbar

    Sample View Toolbar Figure 43: Sequence Analysis Module, Sample View Toolbar Table 40: Sequence Analysis Module, Sample View Toolbar Icon Description Analyzed Data - Displays the data that has been analyzed for the active sample. Base Sequence - Displays a text view of the bases from the analyzed data for the active sample.

  • Page 93: Dye Colors Toolbar

    Sequence Analysis Module Dye Colors Toolbar Figure 44: Sequence Analysis Module, Dye Colors Toolbar Table 41: Sequence Analysis Module, Dye Colors Toolbar Icon Description The color red is the default color assigned to the Adenine (A:) nucleotide bases in the (red) Analyzed Data pane.

  • Page 94: Batch Control Toolbar

    Batch Control Toolbar Figure 45: Sequence Analysis Module, Batch Control Toolbar Table 42: Sequence Analysis Module, Batch Control Toolbar Icon Description Batch Analysis - Used to analyze multiple Sample Data or Sample Plate Results sequentially. Batch Alignment - Used to select sequence results or sample plate results to align. Reanalyze Batch - After performing a batch analysis, use this option to perform a new analysis on the same data set.

  • Page 95: Sample Plate Toolbar

    Sequence Analysis Module Sample Plate Toolbar Figure 46: Sequence Analysis Module, Sample Plate Toolbar Accessed from the menu option or the icon on the Standard View | Toolbars toolbar. Used to specify the samples to open or export. Select the desired plate from the drop-down menu.

  • Page 96: Base Sequence Toolbar

    Base Sequence Toolbar Figure 47: Sequence Analysis Module, Base Sequence Toolbar Accessed from the menu option or the icon on the Standard View | Toolbars toolbar. Use this dialog box to search for specific base sequences and to view quality parameters.
  • Page 97 Sequence Analysis Module The IUB codes to enter when the Exact Match check box is unchecked are listed below. N = G, A, T, or C V = G, A, or C B = G, T, or C H = A, T, or C D = G, A, or T K = G or T S = G or C...
  • Page 98 CEQ™ 8000 Genetic Analysis System...

  • Page 99: Cequence Investigator Module

    CEQuence Investigator Module CEQuence Investigator Module CEQuence Investigator is a module used for comparing sequences to known reference sequences. For each comparison, one or two new sequence results, in either orientation, are used to form a consensus for the comparison. The software automatically determines and matches the orientation of the sequences, forms the consensus, aligns the consensus with the reference sequence, and provides a detailed report of the differences.

  • Page 100: Main Window

    Main Window Figure 49 shows the CEQuence Investigator screen layout. This consists of a title bar, menu bar, several toolbars, the display window and the status bar. A description of each screen element is described in Table 43. Figure 49: CEQuence Investigator Module, Main Window Table 43: Screen Layout and Descriptions Item Description...
  • Page 101 CEQuence Investigator Module Item Description Navigator Toolbar - Offers search and navigation tools within an alignment. Discrepancy Map Toolbar - Displays color-coded locations of any type of attributes selected. You may view the coverage of the reference and sample sequence(s). Alignment Panes - Displays the text alignment results, including the following elements: Base and codon numbering, reference and consensus amino acid translations, reference sequence, sample sequence(s), consensus sequence and the difference line.

  • Page 102: Menu Bar Options

    Menu Bar Options Table 44: CEQuence Investigator Module, File Menu Item Description Prompts for a reference file from a Windows folder, or disk, and then prompts for one or two sequence results from the current working database. Open Opens an existing compared sequence result. Close Closes the document.
  • Page 103 CEQuence Investigator Module Table 45: CEQuence Investigator Module, Edit Menu Item Description Insert Inserts a space before the currently selected position. Delete Deletes the selected position. Replace Replaces the highlighted position with a selected IUB code. Trim Removes contiguous selected bases that contain an end base in the sample sequence base-call text.
  • Page 104 Table 46: CEQuence Investigator Module, View Menu Item Description Toolbars Toggles view/hide each toolbar in a submenu. Status Bar Toggles view/hide the status bar. Unzoom All Shows each sequence in its entirety. History Opens the history log. Base Numbering Toggles on/off base numbering. Codon Numbering Toggles on/off codon numbering.

  • Page 105: Toolbar Icons

    Used to access software, instrument and System system information. Beckman Coulter Used to access the Beckman Coulter, Inc. Home Page Home Page on the internet. Toolbar Icons The toolbars provide quick and easy access to commonly used menu items. Each toolbar (Figure 50) contains buttons that correspond to commonly used menu items.
  • Page 106 Figure 50: Toolbars Standard Toolbar Dye Color Toolbar Navigator Toolbar ∆ισχρεπανχψ Μαπ Τοολβαρ Figure 51: Standard Toolbar Buttons Table 49: Standard Toolbar Button Description Button Description New - Prompts for a reference file from a Windows folder, or disk, and then prompts for one or two sequence results from the current working database.
  • Page 107 CEQuence Investigator Module Button Description Print Preview - Displays a preview of the active document as it will be printed as specified in the Report Format dialog box. Print Desktop - Prints the active desktop area as specified in the Preferences dialog box. Insert - Inserts a space before the currently selected position.
  • Page 108 Button Description Dye Color Coded Text block- Displays the base text background in the specified dye colors when selected. List Help Topics - Opens CEQ System Help contents. Display Context Sensitive Help - Click on this button first and then on a menu item, toolbar button, or window area in question for context specific help.

  • Page 109: Dye Colors Toolbar

    CEQuence Investigator Module Dye Colors Toolbar Figure 52: Dye Colors Toolbar Table 50: Dye Colors Icon Description The color red is the default color assigned to the Adenine (A) nucleotide in the Data (red) view. The color black is the default color assigned to the Cytosine (C) nucleotide in the Data (black) view.
  • Page 110 CEQ™ 8000 Genetic Analysis System...

  • Page 111: Fragment Analysis Module

    Fragment Analysis Module Fragment Analysis Module The Fragment Analysis module processes fragment data from the CEQ platform and provides size and allele information for detected peaks. The results may be viewed, exported and printed. Parameters used to identify alleles are organized as locus tags that can be customized by the user.

  • Page 112: Main Window

    Main Window The main window of the Fragment Analysis module is shown in Figure 54 and described in Table 51. Figure 54: Fragment Analysis Module, Main Window Table 51: Fragment Analysis Module, Main Window Descriptions Item Description The Title Bar showing the module name (Fragment Analysis) and the name of the currently open Study.

  • Page 113: Menu Bar Options

    Fragment Analysis Module Menu Bar Options The menu bar has the options shown in Figure 55. Tables 52 through 59 provide descriptions of the menu options. Figure 55: Fragment Analysis Module, Menu Bar Options File Menu Table 52: Fragment Analysis Module, File Menu Item Description New Study...
  • Page 114 Item Description Preferences Used to: • select for automatic display of the Study window when the Fragment Analysis module is launched. • select to graph all electropherograms by a function of either size or time. • select the number of egrams to be cached. •...

  • Page 115: View Menu

    Fragment Analysis Module View Menu Table 53: Fragment Analysis Module, View Menu Item Description Fragment Displays the Fragment List. The Fragment List contains List information for all analyzed fragments included in the Study, such as size, height, area, locus and allele ID. Results Set Displays the Results Set View.

  • Page 116: Fragments Menu

    Fragments Menu Available when the Fragment List is displayed Table 55: Fragment Analysis Module, Fragments Menu Item Description Show Used to show fragments that were excluded from the Excluded Fragment List. Manually Provides an option to manually select peaks to be included Add Peaks in the fragment list.

  • Page 117: Reports Menu

    Fragment Analysis Module Item Description New Binning Used to begin a new Binning Analysis. Analysis New Peak Used to begin a new Peak Ratio Analysis. Ratio Analysis Run LOH Used to run a Loss of Heterozygosity Analysis Analysis Reports Menu Table 57: Fragment Analysis Module, Reports Menu Item Description...

  • Page 118: Help Menu

    About CEQ Used to access software, instrument and system System information. Beckman Used to access the Beckman Coulter Homepage on the Coulter Internet. Homepage Toolbar Icons While most of the features of the Fragment Analysis module are readily available through the menu selections, there are three toolbars available with buttons for some of the most commonly used commands.
  • Page 119 Fragment Analysis Module Figure 56: Fragment Analysis Module, Toolbars Study Toolbar Dye Colors Toolbar View Toolbar Table 60: Fragment Analysis Module, Toolbars Icon Description Used to create a new Study from either raw data or result data. Open - Used to open an existing or recent Study. Save - Saves the new or edited Study.
  • Page 120 CEQ™ 8000 Genetic Analysis System...

  • Page 121: Data Manager Module

    Data Manager Module Data Manager Module The Data Manager module is used to create, save and modify databases containing: • Filter Sets • Fragment Analysis Parameters • Fragment Results • STR Locus Tags • Methods • Optical Scan Data • Sample Data •...

  • Page 122: Main Window

    Main Window The main window of the Data Manager module is shown in Figure 58 and described in Table 61. Figure 58: Data Manager Module, Main Window Table 61: Data Manager Module, Main Window Descriptions Item Description The Title Bar showing the module name (CEQ Data Manager). The Menu Bar is a list of the menu options (see “Menu Bar Options”...

  • Page 123: Menu Bar Options

    Data Manager Module Menu Bar Options The menu bar has the options shown in Figure 59. Tables 62 through 67 provide descriptions of these menu options. Figure 59: Data Manager Module, Menu Bar Items File Menu Table 62: Data Manager Module, File Menu Item Description Used to create a new Project or Standard in a database.

  • Page 124: Edit Menu

    Item Description Export Used to export a file in one of the following formats: • Standard Chromatogram Format v2.10 and v3.00 (*.scf) • CEQ (*.cq*) • ESD (*.esd) • Tab Delimited ASCII Text (*.txt) • SEQ (*.seq) • FASTA (*.fasta) •...

  • Page 125: View Menu

    Data Manager Module View Menu Table 64: Data Manager Module, View Menu Item Description Toolbar Toggles between displaying or not displaying the toolbar. Status Bar Toggles between displaying or not displaying the Status Bar. Refresh Rebuilds the window display to reflect the most recent changes.

  • Page 126: Window Menu

    Help file and search for information by: topic, index entry and/or keyword. About CEQ Used to access software, instrument and system System information. Beckman Used to access the Beckman Coulter Home Page on the Coulter Internet. Home Page CEQ™ 8000 Genetic Analysis System...

  • Page 127: Toolbar Icons

    Data Manager Module Toolbar Icons There is one toolbar (Figure 60) used in the Data Manager module. Each icon of this toolbar corresponds to a common menu item. Table 68 describes the function of each of the toolbar icons. Figure 60: Data Manager Module, Toolbars Table 68: Data Manager Module, Toolbar Icon Description...
  • Page 128 CEQ™ 8000 Genetic Analysis System...

  • Page 129: Operating The System

    CEQ™ 8000 Genetic Analysis System – User’s Guide A16637-AA Operating the System This section contains step-by-step procedures for common tasks necessary to prepare and use the system on a daily basis. It also provides procedures to perform the tasks involved in analyzing/managing samples and sample data. Set-Up &...

  • Page 130: Creating A Database

    Operating the System Creating a Database User’s Database Template A database template called USERTEMPLATE is added upon installation of the CEQ software. This database is used to store newly created methods and parameters. These methods and parameters are then included in all subsequently created databases. Use the Cut, Copy and Paste functions to add methods and parameters to the USERTEMPLATE.

  • Page 131: Running A Sample

    Running a Sample 20. Select the project you just created from the Project Name drop-down menu and click Running a Sample Starting the Run Module 21. From the CEQ Main Menu, click on the icon and verify that the CEQ Run window is displayed.
  • Page 132 Operating the System 28. In the Sample Plate Run Confimation dialog box (Figure 61), select the desired project and the side (left/right) for each prepared sample plate and click Figure 61: Sample Plate Run Confirmation Dialog 29. In the dialog box (Figure 62), verify the sample and Confirm Configuration buffer set locations.
  • Page 133 Running a Sample Figure 62: Confirm Configuration Dialog 30. Click . The Access Plates dialog in Figure 63 will appear. Load Plates Figure 63: Access Plates Dialog 31. Click Start to continue. 32. Install the plate(s) and click the check box (Figure 64) for each Plate Loaded side, if applicable, and then select the side to immerse the capillaries.
  • Page 134 Operating the System Figure 64: Capillaries Exposed Dialog 33. Click to continue. Load Refer to “Installing the Wetting Tray’ on page 328, “Loading the Sample Plate’ on page 330 and “Loading the Buffer Plate and Evap- oration Cover’ on page 331. 34.

  • Page 135: Sample Setup Procedures

    Sample Setup Procedures Sample Setup Procedures Open an Existing Sample Plate From the CEQ Main Menu, click on the icon and verify that the Sample Plate SETUP Selection dialog is displayed. To open an existing sample plate: 1. Click the radio button Open an existing sample plate 2.
  • Page 136 Scan Barcode Use the Scan Barcode option to search for a sample plate using its barcode. From the drop-down list, select the that contains the sample plate barcode. Project Name Click and type the sample plate barcode exactly as it was saved. If Scan Barcode the sample plate barcode exists, it will open the plate.

  • Page 137: Creating A New Sample Plate

    Sample Setup Procedures Creating a New Sample Plate From the CEQ Main Menu, click on the icon and verify that the Sample Plate SETUP Selection dialog is displayed. To create a new sample plate: 1. Click on the radio button. Create a new sample plate 2.

  • Page 138: Sample Selection

    b. To automatically analyze data after a sample set run, click on the Analysis tab and then on the check box in the Automatic Analysis Analysis window. The sample data will be analyzed with the parameter set selected from the drop-down menu. See “Assigning Analysis Parameter Sets” on page 135. and then return here.

  • Page 139: Selecting The Entire Row

    Sample Selection Selecting the Entire Row Place the mouse pointer over the row label on the left-hand side of the row to be selected. A small black arrow will replace the mouse pointer (see Figure 69). Click once to select the row. To remove the selection, mouse-click any sample in the grid. Figure 69: Select the entire row Selecting the Entire Column Place the mouse pointer over the column label at the top of the column to be selected.

  • Page 140: Selecting Contiguous Cells

    Selecting Contiguous Cells Contiguous cells can be selected by following the steps below. 1. Mouse-click on the first cell in the column or row to be selected. 2. Press and hold down the key. Shift 3. Mouse-click the last cell (in the column or row) to be selected. If (using the above steps) cell A1 is selected and then cell D3 is selected, all the cells between A1 and D3 will be selected.

  • Page 141: Select Non-Contiguous Cells

    Sample Selection Select Non-Contiguous Cells Non-contiguous cells can be selected using the steps below. 1. Mouse-click the first cell to be selected. 2. Press and hold down the key. Ctrl 3. Mouse-click all the remaining cells for the selection. 4. Release the key.

  • Page 142: Naming Samples And Assigning Subject Ids

    Naming Samples and Assigning Subject IDs Each Subject ID entered will be appended with its location in the plate. For instance, a sample named John at position A3 in the plate, will be named John.A3. The sample name field is automatically populated if a subject ID is entered before a sample name.

  • Page 143: Turning Cell Coordinates On Or Off

    Turning Cell Coordinates On or Off Turning Cell Coordinates On or Off To view or hide cell coordinates, select from the View | Cell Coordinates menu. (When checked, coordinates are displayed. When unchecked, coordinates are not displayed.) Using Find To search for text in the sample plate, select and enter the desired text in Edit | Find text field.
  • Page 144 Check Match whole word only to locate and replace text that is flanked by spaces. If you do not select “Match whole word only,” the system will find the text, even if it is part of another word. Check to find and replace the entered text exactly the way you type it into the Match case field.

  • Page 145: Using Property Sets

    Using Property Sets Using Property Sets Property sets are groups of information (properties) that apply to a sample. To access the Sample Property Sets, select or select a sample View | Sample Property Sets cell in the Sample Plate Screen and refer to the Note Tab. In the Property Set Table, use the Tab or arrow keys to move from cell to cell.
  • Page 146 To delete the contents of the row(s), select the desired row(s) and click the Delete button . The selected row will be removed from the property set. In order to prevent accidental deletions, a warning message will appear before allowing the user to delete one or more rows. Press to delete the contents or return to the editor.

  • Page 147: Assigning Methods

    Assigning Methods Assigning Methods The method is the program of events the system uses to collect the data. The method controls the hardware, i.e. the temperatures, voltages and times, which work together to gather data. The system comes with several methods that are optimized for the different software applications.

  • Page 148: Condition Method

    Condition Method The Condition method (see Figure 78) should be used before a run if the system has not been used for a long period of time. Performing this method will fill the capillaries with gel 6 times. Then, after separation, thoroughly purge the manifold with fresh gel followed by a capillary fill.

  • Page 149: Assigning Analysis Parameter Sets

    Assigning Analysis Parameter Sets The first phase is to “coax” the sample into the capillaries. The second phase is to perform the normal separation of the samples. d. Click to exit the Method dialog box or continue with step 8 to make additional changes to the method.

  • Page 150: Specifying Sample Plate Print Options

    Specifying Sample Plate Print Options To define the print options and report format of the currently open sample plate, perform the following steps. 1. With a cell highlighted, select the tab at the bottom of the window. Analysis 2. Select the check box to print a report immediately after completion Print Report of the run.

  • Page 151: Export Sample Plate

    Reports Export Sample Plate The sample plate can be exported for use in compatible equipment or to manually edit the plate information. To export the sample plate follow the steps below. 1. Open the sample plate that will be exported. If it is already open, make sure it has been saved before proceeding.
  • Page 152 Figure 80: Report Format dialog CEQ™ 8000 Genetic Analysis System...

  • Page 153: Lock

    Lock Figure 81: Trimming Log Lock To prevent editing of the currently displayed plate, select . The File | Lock Notes tabs are disabled when the sample plate is locked. Method Analysis View Summary A read-only window showing the attributes of the currently selected plate in tabular format is displayed by selecting View | View Summary User’s Guide...
  • Page 154 CEQ™ 8000 Genetic Analysis System...

  • Page 155: Run Procedures

    Opening the Run Module Run Procedures Opening the Run Module From the CEQ Toolbar, click on the Run icon and verify that the Run window is displayed. Defining System Preferences To define the system preferences, perform the following steps. 1. Select from the menu.

  • Page 156: Gel Re-Estimation Feature

    Run Procedures Figure 82: 500 MB Warning Dialog If you do not create and select a new database, the system will use the default database (CEQ.mdb) to store your data. If you have created a sample plate in a database that is too large and are presented with the warning, perform the following set of procedures to transfer your sample plate to a new database and process the plate for data storage in a new database.

  • Page 157: Gel Re-Estimation Procedure

    Running a Sample Plate You will be notified and the system will perform a gel volume re-estimation if you attempt to run a plate when the total cartridge gel volume is less than 1.5 mL more than the minimum amount needed to process the plate. If the system determines that there might not be enough gel to run the plate, it will perform a 2-step re-estimation process automatically.

  • Page 158: Gel Volume Notification

    Run Procedures The system will measure the gel volume accurately and will determine whether or not to proceed with the run. 4. If the actual volume (re-estimated by the system) is greater than 1.5 mL more than the total volume needed to run the plate, the system will automatically start the plate.

  • Page 159: Gel Plug Warning

    Running a Sample Plate You will also be presented with the dialog box in Figure 87 if you attempt to run a sample plate and the gel cartridge does not have enough gel to process any sample sets in that plate. Figure 87: Gel Usage Confirmation Dialog You must click to end the run and return to the Run main window.

  • Page 160: Pausing A Sample Plate Run

    Run Procedures Figure 89: Device Tab – Gel Plug Figure 90: Device Tab – Gel Cartridge installed installed Pausing a Sample Plate Run To pause the currently executing sample plate, click the button. The Pause system will pause at the next executable step of the sample plate. To resume the run, click the button again.

  • Page 161: Viewing The Last Analysis Performed

    Viewing the Last Analysis Performed b. Select the Save Collected Data option (if desired). c. Select the Perform Shutdown Method (to purge the manifold and capillaries and set capillary chamber temperature to 35°C), then select Viewing the Last Analysis Performed To view the last analysis performed on the CEQ System, perform the following steps.
  • Page 162 Run Procedures CEQ™ 8000 Genetic Analysis System...

  • Page 163: Sequence Analysis Procedures

    Viewing Sample Data Sequence Analysis Procedures Viewing Sample Data To view sample data, perform the following steps. 1. Select from the menu. File | Open Figure 92: Open Dialog 2. In the dialog box, select the appropriate tab ( Open Sample Data Sample Plate Results...
  • Page 164 Sequence Analysis Procedures Sequence Results: This tab displays the list of analyzed data that resides in the selected project. On the right-hand side of the list box you may view the date and time the sample was run as well as the raw data from which the sequence result was derived.

  • Page 165: Sample View Toolbar

    Analyzing Sequence Data Sample View Toolbar Icon Description Analyzed Data - Displays the data that has been analyzed for the active sample. Base Sequence - Displays a text view of the bases from the analyzed data for the active sample. Raw Data - Displays the raw data for the active sample.

  • Page 166: General Tab

    Sequence Analysis Procedures Figure 93: Sequence Analysis Parameters Editor Dialog 4. Make appropriate changes and click to create a new Sequence Analysis Save As Parameter name or click to save and exit the editor. General Tab The general tab is displayed by default. N Threshold This value is the threshold for the probability of correctness, below which a called base will be changed to an “N.”...
  • Page 167 Analyzing Sequence Data To specify the threshold, enter the desired value, between 0.00 and 1.00, in the N Threshold text box. A value of 0.99 will call all peaks as indeterminate (N’s) except the high quality peaks. A value of 0.50 will call only the lowest quality peaks as ‘N’s. A value of 0.00 will not call any peaks as ‘N’s.
  • Page 168 Sequence Analysis Procedures Use Default Mobility Calibration The analysis algorithm determines the mobility corrections (Mobility Calibration) to be applied to each of the dye channels for every sample analyzed; stored mobility calibrations are not used. When processing short PCR samples, the software has too little data to determine an accurate mobility calibration from the data itself.

  • Page 169: Initial Data Detection

    Analyzing Sequence Data Check the Use Final Values as Initial Values check box to use the final values to re-compute the color calibration the next time the data is analyzed. Otherwise the system will use the initial values to re-compute the color calibration. (This option is not available if accessed from the Parameters Used to Compute Sequence menu option under the View menu.) Initial Data Detection...
  • Page 170 Sequence Analysis Procedures Delay The start of data Delay is the delay, in minutes, between the detected start of data and the data that will be analyzed. The range is 0.00 to 180.00 and the default is 0.75 minutes. The default value will be 0.20 minutes if <200 Bases is selected on the General tab.

  • Page 171: Heterozygote Detection Tab

    Analyzing Sequence Data Heterozygote Detection Tab Select the tab as shown in Figure 95 to define the Heterozygote Detection parameters the system will use to detect heterozygotes. These parameters will only be used if the option is selected in the General tab. Detect Heterozygote Figure 95: Heterozygote Detection Tab Detect Heterozygotes...
  • Page 172 Sequence Analysis Procedures Height Ratio Use this parameter to define the minimum percent of the primary peak’s height that a second peak falling within the defined range must be to be considered a heterozygous peak. The range is 0 to 100% and the default is 30%. Sensitivity Use this parameter to define the sensitivity of the system when looking at the sharpness of the peak.

  • Page 173: Alignment Template Tab

    Analyzing Sequence Data The system will use the IUB ambiguity codes in Table 69 to represent heterozygous bases. Table 69: IUB Ambiguity Codes Code Definition Mnemonic Adenine Cytosine Guanine Thymine puRine pYrimidine Keto aMino Strong 3 H bonds Weak 2 H bonds Not A Not C Not G...
  • Page 174 Sequence Analysis Procedures Figure 96: Alignment Reference Tab Reference The user can choose from three templates that are stored as part of the software or choose a reference file from a disk. If the radio button is not selected, then the text box and the Reference File Browse button below it are disabled.

  • Page 175: Advanced Alignment Parameters

    Analyzing Sequence Data Advanced Alignment Parameters When the checkbox is selected, the Perform Alignment Advanced Alignment button becomes active. Click the Parameters Advanced Alignment button to access the feature described below. Parameters Figure 97: Advanced Alignmant Parameters The information on this tab is used to define the parameters used to perform alignments with the active sequence.

  • Page 176: Quality-Based Trimming Tab

    Sequence Analysis Procedures Check Find matching substrings to optimize the alignment. When the alignments are optimized, the system will find matching substrings first, then run the alignment algorithm on the bases remaining outside of the substrings, reducing the execution time and storage space. Check the Find Local alignment to use only the substrings with the highest score,...

  • Page 177: Ends To Be Trimmed

    Analyzing Sequence Data Ends To Be Trimmed Click the radio button to select the end(s) to be trimmed. Select to trim Both Ends outside the 5' and 3' coding region. Select to trim to the left of the 5' end Left (5') End or select to trim to the right of the 3' end.

  • Page 178: Perform Sequence-Based Trimming

    Sequence Analysis Procedures Perform Sequence-based Trimming Select this check box to enable Sequence-based trimming. Vector/Other Sequence Files Press to select a reference sequence. It must be in the .fasta format and stored in an accessible folder. Press to remove a previously selected reference sequence. There is no practical limit on the number of .fasta reference sequences that can be used.

  • Page 179: Performing Quality-Based Trimming

    Quality Values • the average Call Score/Quality Value in a window of 25 bases falls below 0.75 - 0.8 on a linear scale and 6 - 7 on a log scale, • there are at least 5 bases within that 25 base window whose Call Score/Quality Value are less than 0.75 on a linear scale and 6 on a log scale.
  • Page 180 Sequence Analysis Procedures Figure 100:Base Sequence and Trace Views after Quality-based Trimming CEQ™ 8000 Genetic Analysis System...

  • Page 181: Apply Quality Trimming

    Quality Values Apply Quality Trimming If the trimming is acceptable, select the menu Edit | Apply Quality-based Trimming item. The resulting Base Sequence and Trace view after a Quality-based trimming has been applied is shown in Figure 101. Figure 101:Base Sequence and Trace Views after Quality-based Trimming – Quality-based Trimming Applied Undo The original sequence can be recovered after...
  • Page 182 Sequence Analysis Procedures 3. Select the Sequence-based Trimming tab. 4. Click the Perform Sequence-based Trimming check box. And make all the needed selections discussed above. 5. Click and save the new parameters. Save As 6. Click At this point, the result can be trimmed. 1.

  • Page 183: Apply Sequence-Based Trimming

    Quality Values Apply Sequence-based Trimming If the trimming is acceptable, select the Edit | Apply Sequence-based Trimming menu item to remove the trims from the sequence. The resulting Base Sequence and Trace view after a Sequence-based trimming has been applied is shown in Figure 103. When selecting applying sequence-based trimming, quality-based trimming will automatically be applied first if quality-based trimming was performed.

  • Page 184: Internal Trimming

    Sequence Analysis Procedures Internal Trimming Select the checkbox from the Sequence-based Trimming Enable Internal Matches tab to trim subsequences from within the sequence. To exclude a subsequence from final removal, right-click on the subsequence and select Vector/Other and select the subsequence. Only those subsequences marked with a Subsequences check (Figure 104) will be removed from the sequence when trimming is applied.

  • Page 185: Batch Trimming

    Quality Values The internal matches in the resulting Base Sequence and Trace view, after a Sequence-based trimming has been applied, are replaced by the letter “x”, as shown in Figure 105. Figure 105:Base Sequence and Trace Views after Sequence-based Trimming – Enable Internal Matches Batch Trimming Batch trimming has been added to enable the automatic trimming of a large number of...
  • Page 186 Sequence Analysis Procedures 3. Select the results to be trimmed and click the right-arrow to move them to the “selected” list box. Figure 106:Batch Trimming Result Selection 4. Click 5. Verify that the required Analysis Parameters are selected in the Working Parameters dialog.

  • Page 187: Print Grid Data

    Quality Values Figure 107:Export Options 7. Click is created that can be viewed and printed. To view the Trimming Trimming Report Report select the menu item. The new pane will open at the View | Trimming Report bottom of the screen. Items displayed include: •...

  • Page 188: Export Grid Data

    Sequence Analysis Procedures Export Grid Data Select to save the grid data as a comma separated (*.csv) or tab Export Grid Data delimited (*.txt) text file. Format Grid Format Grid allows the user to customize the look of the data grid. If rows or columns are hidden, Print Grid Data will omit them.

  • Page 189: Export Options

    Quality Values Figure 109:Grid Properties Export Options Two tools are available in the export dialog: Apply Trimming and Export Only If Sequence > X nt. Apply Trimming If the Export option was selected during sample setup or batch analysis, selecting the option (Figure 110) will remove the trims from the sequence before Apply Trimming it is exported.

  • Page 190: Inserting Bases In The Base Sequence Pane

    Sequence Analysis Procedures 3. From the Insert Base dialog box, select the desired base and then click . (The lower-case base is inserted in both the Analyzed Data and the Base Sequence pane.) Inserting Bases in the Base Sequence Pane To insert bases in the Base Sequence pane, perform the following steps.

  • Page 191: Deleting Bases In The Base Sequence Pane

    Quality Values Deleting Bases in the Base Sequence Pane To delete bases in the Base Sequence pane, perform the following steps. 1. Select from the menu. Tools | Edit 2. Highlight the base(s) in the base sequence text to delete and then press the Delete key (or use the Cut icon).

  • Page 192: Analysis Log

    Sequence Analysis Procedures Analysis Log The system will log all the steps used in performing the base-calling routine on the raw data in the Analysis Log. For example: the system will log a message when it is unable to find the end of PCR product data. The Analysis Log may be viewed to see if the system was unable to find the end of PCR data.

  • Page 193: Sequence Result Properties

    Quality Values Sequence Result Properties The sequence result properties are used to view Method, Analysis, Alignment, Consumable, General information, Notes and Property set information. Open the Properties dialog box by selecting when a sequence result File | Properties is open. See Figure 112. If no alignment has been performed on the open sequence result, the Align- ment tab will not be shown.

  • Page 194: Notes Tab

    Sequence Analysis Procedures Notes Tab Any notes associated with the sample well for the open sequence result can be viewed. These notes are entered in the Sample Setup module on the Notes tab at the bottom of the main window. See Figure 113. Figure 113:Note Tab, Properties Dialog CEQ™...

  • Page 195: Property Set Tab

    Quality Values Property Set Tab The user can view any property sets associated with the sample well for the open sequence result. These Property Sets are defined in the Sample Setup module on the Notes tab at the bottom of the main window. See Figure 114. Figure 114:Property Set Tab, Properties Dialog User’s Guide...

  • Page 196: Method Tab

    Sequence Analysis Procedures Method Tab The method event parameters used to collect the raw data for the open sequence result are shown here. These parameters are defined on the Method tab in the Sample Setup module, prior to running the sample plate. See Figure 115. Figure 115:Method Tab, Properties Dialog CEQ™...

  • Page 197: Analysis Tab

    Quality Values Analysis Tab The user can view the sequence analysis parameter set used to analyze the open results. The Analysis tab lists the Analysis Parameter Set name and the defined values for that parameter set, as shown in Figure 116. Figure 116:Analysis Tab, Properties Dialog User’s Guide...

  • Page 198: Alignment Tab

    Sequence Analysis Procedures Alignment Tab The user can view the alignment template information and the alignment parameters used to create the alignment of the currently open result. See Figure 117. Figure 117:Analysis Tab, Properties Dialog This tab will only be visible if an alignment has been performed on the sequence result.

  • Page 199: Consumables Tab

    Setting or Changing Display Options Consumables Tab Information for all of the consumable products used to collect raw data associated with the currently open sequence result are presented in the tab. The capillary array information, the gel information and the buffer information are shown here, as defined in the Run module.

  • Page 200: Setting Or Changing X Axis Options

    Sequence Analysis Procedures Setting or Changing X Axis Options To define the X Axis properties of the displayed data panel(s), perform the following steps. 1. Double-click the mouse button in the desired pane. 2. In the dialog box, select the tab.

  • Page 201: Setting Or Changing Dye Trace Displays

    Setting or Changing Display Options Setting or Changing Dye Trace Displays To set or change the dye trace properties, perform the following steps. 1. Double-click the mouse button in the Raw or Analyzed data panes. 2. In the dialog box, select the tab.

  • Page 202: Changing Heterozygote Display Color

    Sequence Analysis Procedures Changing Heterozygote Display Color The heterozygous bases will be bold and red, by default. You can change the color of the heterozygotes in the base sequence pane by selecting Tools | Heterozygotes . Select a new color and click .

  • Page 203: Viewing The Selected Batch Result

    Setting or Changing Display Options 3. Select to start the reanalysis. To keep the results from the previous analysis, “pin” the results tab by clicking on the pin icon . Otherwise, the results will be overwritten by any subsequent reanalyses. Viewing the Selected Batch Result To view the selected batch result after performing a batch analysis, click the desired sample in the upper pane of the Batch Analysis window and then select...

  • Page 204: Compare Mode

    Sequence Analysis Procedures Compare Mode In the Sequence Analysis module, you can select up to 15 results to compare against the open result, enabling the comparison of a maximum of 16 sequence results at one time. If you select more than 16 results from the Compare dialog box you will be presented with the following warning.
  • Page 205 Setting or Changing Display Options 12. Pan through the data in a result other than the original one and all sixteen results will pan together. 13. Use the horizontal scroll bar to move the data along the X-axis. 14. The data panes will scroll synchronously. Aligning Results 15.

  • Page 206: Sequence Result Report

    Sequence Analysis Procedures Sequence Result Report The sequence result report includes additional information when certain Sample Elements are selected in the Report Format dialog box. The following table describes what each sample element includes. The header contains summary information for the sample such as sample name, position, the method under which it was run, the sample plate name, the analysis parameter set name, and capillary serial number.
  • Page 207 Setting or Changing Display Options The Analysis Parameters are the parameters used to produce result data and output for a given sample, including heterozygote detection and alignment. If the data has been analyzed using a sequence analysis parameter set, the sequence analysis parameter set will be printed. In addition, the alignment template and the alignment parameters information Analysis will be included in this section, when applicable.
  • Page 208 Sequence Analysis Procedures Figure 119:Sequence Results Report CEQ™ 8000 Genetic Analysis System...

  • Page 209: Displaying Quality Parameters In A Report

    Setting or Changing Display Options Displaying Quality Parameters in a Report 1. To display the Quality Parameters in a report, go to the menu and select File Report Format 2. In the section enable then click Sample Elements Quality Parameters Figure 120:Report Format Dialog Box 3.
  • Page 210 Sequence Analysis Procedures Figure 121:Quality Parameters Report The categories on the Quality Parameters Report are described in Table 70: Table 70: Quality Parameters Report Categories the number of the called base Number the single letter designation of the called base (A, C, G, T or Base other IUB code) base identity score assigned by the base-caller, ranging...

  • Page 211: Magnifying (Zooming) Data

    Setting or Changing Display Options the actual number of the data points in the analyzed data Data Point corresponding to the called base(s) indicates a base that has been manually added to the base Insert sequence indicates that the base call has been manually changed Edit Magnifying (Zooming) Data To magnify data, perform the following steps.

  • Page 212: Specifying The Base Call Location

    Sequence Analysis Procedures 3. To visually align the peak with the base call, click the Align Mode icon, then click down on the apex of the peak for which you wish to view the alignment. (A hairline cursor is displayed through the apex of the peak to its corresponding base.) 4.

  • Page 213: Computing A New Color Calibration

    Setting or Changing Display Options This is a read-only dialog box. To make changes to the color calibrations, select Analysis | Working Analysis Parameters | Edit | Color 5. To add a successful color calibration to the list of available color calibrations, click in the dialog box.

  • Page 214: Sequence Result Export

    Sequence Analysis Procedures Sequence Result Export To export from the Sequence Analysis Module, follow the steps below: 1. Launch the Sequence Analysis application and open a sequence result. 2. Select to open the Export dialog box. File | Export 3. Select a target folder and file name. 4.

  • Page 215: Sequence Export File Types

    Setting or Changing Display Options Sequence Export File Types Listed in Table 71 are the available export file types and options. Table 71: Sequence Export Options Format Description The SCF (Standard Chromatogram Format) format is used to store analyzed DNA sequence data for a single sample. The format describes both "public" and "private"...
  • Page 216 Sequence Analysis Procedures Table 71: Sequence Export Options (Continued) Format Description The FASTA format only applies to the sequence results portion (e.g., text bases) of analyzed data, and contains a comment line that is used to identify the data. Along with the text file, a quality values file is also generated (*.FASTA.QUAL) which lists the quality value for each base.
  • Page 217 Setting or Changing Display Options Table 71: Sequence Export Options (Continued) Format Description If you select the CEQ format, the format native to our database, you will export sample data, sequence or fragment results, sample plate results, sequence or fragment analysis parameters, sample plates, methods, locus tags (fragment data only), standards (fragment data only), and optical scan data.

  • Page 218: Customizing Filename Extension

    Sequence Analysis Procedures Customizing Filename Extension To supply your own custom filename extension (or none at all) to exported files, modify the CEQ.ini file as follows: If there is no Export section in the CEQ.ini file, export any sample file and the Export section will be generated. Then modify as needed. 1.
  • Page 219 Setting or Changing Display Options b. Select the appropriate Export Data Format radio button and then click 3. Select from the menu. Analysis | Third Party Analysis 4. In the dialog box, enter the filename and then click . The data will be Export exported and the third party package launched with the exported data open.
  • Page 220 Sequence Analysis Procedures CEQ™ 8000 Genetic Analysis System...

  • Page 221: Cequence Investigator Procedures

    Creating a Reference File CEQuence Investigator Procedures Creating a Reference File The reference file is a text file that can be created using notepad or any other text editor. It may be composed of one or two sections; the sequence and, optionally, specific nucleotide or amino acid mutations.

  • Page 222: Selecting A Reference And Sequence Results

    CEQuence Investigator Procedures A default Reference folder is created upon installation of the software. You may store your reference files in that location or in any other folder. When you create a new comparison, navigate to the appropriate folder to select your reference.
  • Page 223 Creating a Reference File The Open Sequence Results dialog will then be displayed. Select one or two results and press To select two results, highlight the first result, hold the button down and Ctrl use the left mouse button to highlight second result. Figure 124:Open Sequence Results Dialog If two results are in different projects, select “(none)”...

  • Page 224: Numbering

    CEQuence Investigator Procedures A new window will appear as shown in Figure 125. Figure 125:CEQuence Investigator Main Screen By default the Standard, Navigator, Dye Color, and Discrepancy Map toolbars are displayed and docked. If they are not displayed, select and enable the View | Toolbars desired toolbar.

  • Page 225: Viewing Reference Amino Acid Translations

    Creating a Reference File Viewing Reference Amino Acid Translations When the system generates the comparison, it translates the nucleotide sequence of the reference file into an amino acid sequence and displays it above the reference base sequence line. From the beginning of the reference, the bases are separated into groups of three nucleotides (codons).

  • Page 226: Viewing Sequence Traces

    CEQuence Investigator Procedures Viewing Sequence Traces When you first perform a comparison, you select one or two sequences to form the consensus. Once the comparison is performed, the traces for these sequences are displayed. When you click on any position in alignment view, the corresponding peak(s) in the traces will move to the center of the trace view and will be framed by two vertical lines.

  • Page 227: Quality Values

    Creating a Reference File Quality Values If you are using a single sequence, the quality value assigned to each base in the consensus will match each base in the sequence. If you are using two sequences and the bases agree, one of two calculations will occur: If the orientation of the two sequences is opposing, the two quality values will be added.

  • Page 228: Navigation And Editing

    CEQuence Investigator Procedures Navigation and Editing The Navigator toolbar allows you to search for locations of interest in the consensus based on attributes. You may also search for specific base(s) and view their associated quality values and difference code(s). To use the Navigator toolbar, click menu option, then check the View | Toolbars option.

  • Page 229: Complementing The Sequence

    Navigation and Editing Complementing the Sequence When the system performs a comparison it will determine which orientation, forward or reverse, provides a better fit with the reference. If the system determines that the sequence(s) will generate a better comparison in the opposite orientation, it will automatically reverse complement the sequence(s).

  • Page 230: Searching The Consensus For Specific Bases

    CEQuence Investigator Procedures Searching the Consensus for Specific Bases To search for a specific base or contiguous multiple bases, click on the Consensus radio button and enter the bases in the text box. Text Search To search for the exact match for text, check the check box.

  • Page 231: Displaying The Discrepancy Map

    Navigation and Editing Displaying the Discrepancy Map The Discrepancy Map toolbar allows you to look for areas of interest in relationship to the reference. You may view the coverage of the consensus and sample sequence(s). You may also view the color-coded locations in the experiment of any type of attributes of interest that you specify.

  • Page 232: Printing And Reports

    CEQuence Investigator Procedures Printing and Reports You may print a report for the active compared sequence result. To set up your report click and select the default printer to use by clicking on the File | Report Format down-arrow and selecting the desired printer. Set your preferred page Printer Name layout by clicking on and select the following option elements...

  • Page 233: Exporting

    Exporting Exporting You may export the active compared sequence in either of three formats; text (*.txt), tab delimited text (*.txt) and protein (*.pro). The text file will export the base sequence of the consensus. In tab delimited text files, data appears in a single line and all carriage returns (↵) are replaced with tabs (→).

  • Page 234: Saving

    CEQuence Investigator Procedures Saving To save a new document, select from the menu toolbar. A dialog File | Save Save As will be displayed prompting you to enter a file name and folder location. Press Save and the data is saved to the flat file for later retrieval. If the document has previously been saved, select from the menu toolbar File | Save...

  • Page 235: Fragment Analysis Procedures

    Fragment Analysis Procedures The CEQ System Fragment Analysis module estimates DNA fragment sizes and amounts and identifies alleles represented by specific DNA fragments. Parameters used to identify alleles are organized as locus tags that can be customized by the user. The results of the analyses may be viewed, exported and printed.

  • Page 236: Performance Safeguards

    Fragment Analysis Procedures Performance Safeguards Available controller resources are checked before result processing begins. If it is determined that the resources needed to complete the analysis are not available, a warning dialog (see Figure 129) will open. If this occurs, it is recommended to close all applications, re-start the Fragment Analysis module and attempt the analysis again.

  • Page 237: Selecting The Components Of The New Study

    Figure 130:Study window - Creating or Opening a Study A new can be created from either or previously Study Raw Data Analyzed . Make your selection using one of the option buttons and click Results continue. The Study Wizard will be launched to guide you through the steps of creating a Study.

  • Page 238: Selecting Raw Data For The New Study

    Fragment Analysis Procedures Selecting Raw Data for the New Study When you select the option button and click , the will Raw Data Study Wizard be launched and the window will be displayed. Select Raw Data Figure 131:Select Raw Data window - Selecting the Components of the New Study This window is used to select the raw data to be analyzed from a database of samples that have been “run”...

  • Page 239: Selecting Raw Data From The Plate View Tab

    • If the project you are looking for is not displayed, it may be located in a different database. • To enter a different database, all applications must be closed. See the topic “Set Working Database” in the Online Help for more information. 3.
  • Page 240 Fragment Analysis Procedures Figure 132:Selecting Raw Data from the Plate View tab 1. Select the Project folder that contains the sample data that you wish to include in the Study from the drop-down list box. Project • Click on the down-arrow located on the right side of the list box Project to reveal the available projects in the working database.

  • Page 241: Selecting An Analysis Parameter Set

    • The sample ID will be moved to the Current Plate / Selected Wells area of the window, indicating that the sample has been removed from the Study. 5. To select or remove entire rows or columns of wells, click on the corresponding row letters or column numbers.

  • Page 242: Selecting A Saved Analysis Parameter Set

    Fragment Analysis Procedures The CEQ System Software is equipped with its own set of default analysis parameters. These are used to estimate unknown fragment lengths from a calibration curve based on the migration times of the size standard fragments. The default set of analysis parameters cannot be edited.

  • Page 243: Selecting Additional Results To Be Added To The Study

    Figure 134:Analyze Data window - Analysis in Progress (shown) 4. When the analysis is complete, the status column will indicate if the analysis succeeded or failed for each sample included in the Study. 5. To review the analysis log, highlight a sample in the upper samples list and scroll through the analysis information in the lower analysis log area of the window.

  • Page 244: Defining Fragment Analysis Parameters

    Fragment Analysis Procedures Defining Fragment Analysis Parameters The previous sections on described the application of saved Creating a Study parameter sets to analyze raw sample data. The following sections will describe how to edit a parameter set or create an entirely new set of analysis parameters to meet the specific requirements of your samples.

  • Page 245: Fragment Analysis Parameters - General Tab

    Figure 135:Fragment Analysis Parameters window - Creating or Editing a Parameter Set Fragment Analysis Parameters - General tab By default, the tab is the first tab to be displayed when opening the General window. This tab contains general parameter set Fragment Analysis Parameters information and peak identification properties.
  • Page 246 Fragment Analysis Procedures Peak Criteria 4. The field allows you to specify the minimum rate of the signal Slope Threshold increase on the leading edge of peaks in order for a peak (standard or unknown) to be detected. • Enter a value between 1 - 1000. The default value is 10. •...

  • Page 247: Fragment Analysis Parameters - Analysis Method Tab

    Fragment Analysis Parameters - Analysis Method tab This tab allows you to specify the size standard and model to be applied to the calibration curve. The model selected should be the best fit for the standard, as it is used to generate the calibration curve, which estimates sizes for the unknown fragments.
  • Page 248 Fragment Analysis Procedures • For example, in the SizeStandard-400, the peaks in the larger half of the size range are spaced 20 nucleotides apart, while some in the smaller half of the size range are spaced 10 nucleotides apart. Setting the Analysis Method 1.

  • Page 249: Fragment Analysis Parameters - Quantitation Tab

    • For calibrations that span a long size-range or extend into size regions where the size selectivity of the separation system is very non-linear, a fourth degree polynomial ( ) may result in more accurate size estimates than a Quartic Curve cubic curve.
  • Page 250 Fragment Analysis Procedures Figure 137:Fragment Analysis Parameters window - Quantitation tab Calculate Using 2. Within the area of the window, use the option buttons to select Calculate Using the method which the software will use to calculate the quantity of your reference peak.

  • Page 251: Fragment Analysis Parameters - Str Locus Tags Tab

    6. The field allows you to input a variable quantity to be considered +/- Window when identifying the reference peak. • For example, if you listed the size of your reference peak as 90 nucleotides, you may enter a value of '1' in the +/- Window field. •...
  • Page 252 Fragment Analysis Procedures Fragment Analysis Parameters window - Selecting STR Locus Tags Selecting Locus Tags 1. To select a locus tag to be included in your parameter set, click to highlight the locus in the left “available” frame, then click the right-arrow button to move the selection to the right “selected”...

  • Page 253: Fragment Analysis Parameters - Snp Locus Tags Tab

    Fragment Analysis Parameters - SNP Locus Tags tab This tab allows you to select the locus tags that will be used for SNP analysis with the parameter set being configured. Selections can be made from a list of available SNP locus tags previously configured for analysis.
  • Page 254 Fragment Analysis Procedures Selecting Locus Tags 1. To select a locus tag to be included in your parameter set, click to highlight the locus in the left “available” frame, then click the right-arrow button to move the selection to the right “selected” frame. 2.

  • Page 255: Fragment Analysis Parameters - Advanced Tab

    Fragment Analysis Parameters - Advanced Tab The Advanced tab is used to set additional fragment analysis parameters. From this window you can assign dye mobility correction values to compensate for the varying effects of different dyes on fragment mobility, select a standard mobility reference and indicate whether dye spectra used should be estimated from sample data or from saved system spectra.
  • Page 256 Fragment Analysis Procedures • AE ver.1 is the calibration used for the CEQ Fragment Analysis test sample. • is used when performing a . The SNP analysis SNP ver. 1 SNP analysis will still proceed if the mobility correction is not selected, but the values obtained may vary slightly.
  • Page 257 Only one set of system spectra is stored. This set of system spectra is automatically used if one or more of the needed spectra cannot be estimated from the sample data. • option button is the default setting. This Use calculated dye spectra selection uses the set of dye spectra obtained from the sample data.

  • Page 258: Str Locus Tag Editor

    Fragment Analysis Procedures STR Locus Tag Editor allows you to create a new STR Locus Tag or edit an STR Locus Tag Editor existing one. It can be accessed from the tab of the STR Locus Tag Analysis window (see previous section). Parameters Figure 139:STR Locus Tag Editor window - Creating a New Locus Tag Locus Tag Editor...
  • Page 259 Locus Information area of the window contains parameters that are used to Locus Information generate a list of expected alleles in your analysis. 1. You may input the identification of the locus in the field. Locus Description This is an optional field. 2.

  • Page 260: Generating And Viewing The Allele List

    Fragment Analysis Procedures Primer Label (optional) area of the window allows you to indicate (for supplementary Primer Label reporting purposes) on which strand the primer is labeled with the dye. • Select the option button if the primer is dye labeled on the forward forward strand.

  • Page 261: Allele List Generation Using Interpolation And Linear Regression

    Refer to the following pages for more information regarding generation of the allele list using interpolation or linear regression. columns describe the number of points Standard Deviation and # of Points included in each bin and the sample standard deviation. Both of these columns are generated as a result of the automatic binning feature (see the “Bin Analysis”...

  • Page 262: Str Locus Tag Editor - Allele Id Criteria

    Fragment Analysis Procedures STR Locus Tag Editor - Allele ID Criteria tab contains the parameters used to accurately identify your Allele ID Criteria alleles and to set these “true” alleles apart from various other anomalies identified in the fragment list. A sample fragment is identified as representing a particular allele if its apparent size is found to be the very similar to the size of a particular allele fragment in the allele list.
  • Page 263 Figure 140:Locus Tag Editor window - Allele ID Criteria tab Stutter Definition Stutter are usually small peaks, generally DNA amplification artifacts that form as a result of halted polymerase activity. When this happens, allele fragments are formed that differ in length from the true allele by an integral number of repeats (i.e., n-1, n-2, etc.).
  • Page 264 Fragment Analysis Procedures 5. Selecting this check box enables the system to detect stutters longer than (occurring after) the allele Spurious Peak Detection Spurious peaks are generally small trace artifacts that are detected as a result of sample contaminants. These peaks tend to have a different shape than fragment peaks but often fall within the size range of a locus tag and are detected.
  • Page 265 If the box is not checked, the software will identify one of the two Detect +/- A alleles as the true allele based on the check box. Other Use +A peak to call allele peaks will be included in the fragment list as unknown alleles Use +A peak to call allele Select this check box if the +A form is expected to be the higher peak and leave it...
  • Page 266 Fragment Analysis Procedures Use +A Peak to Call Allele All fragments assumed not to be +A. All fragments assumed not to be +A. Any fragments without +A are Any fragments without +A are Not Checked identified as the allele in the allele identified as the allele in the allele table.

  • Page 267: Snp Locus Tag Editor

    SNP Locus Tag Editor allows you to create a new SNP Locus Tag or edit an SNP Locus Tag Editor existing one to be used for SNP analysis. It can be accessed from the SNP Locus tab of the window (see previous section). Analysis Parameters Figure 141:SNP Locus Tag Editor window - Creating a New Locus Tag Input or edit the name of the locus tag in the...
  • Page 268 Fragment Analysis Procedures Apparent sizes of SNP probe fragments as estimated with SNP reference fragments will often differ more from actual sizes than is the case with STR allele fragments. This is due to the effects of primary structure on fragment mobilities. Therefore, it is important that the apparent size entered into the SNP locus tag definition is a known value.

  • Page 269: Result Set Filtering

    Result Set Filtering The first steps in the creation of a Study involve the application of a set of fragment analysis parameters to analyze the raw sample data. These parameters are specifically configured to process the fragment data for a particular sample or set of samples. The outcome of this analysis is a result set of data consisting of characterized fragments and identified alleles.

  • Page 270: Applying Exclusion Filters To The Result Set Data

    Fragment Analysis Procedures The Filter Set area is where the user-defined exclusion filters are selected and applied to the data. Each column contains a menu of parameters to be applied as exclusion filters. The system comes equipped with two pre-defined sets of exclusion filters.

  • Page 271: Available Exclusion Filters

    The end result of this “second level” filtering is to leave only results that you are certain of and to save you the time of “weeding out” unwanted fragment data later. Available Exclusion Filters All of the exclusion filters can be found in the drop-down list box available in the Name column of the Filter Set...

  • Page 272: Viewing Trace Data

    Fragment Analysis Procedures Viewing Trace Data Individual sample traces can be viewed at any time while working within the fragment analysis module. They can be viewed individually or in selected groups along with the parameters used or collected during the separation and analysis steps. Sample traces can also be stacked or overlaid on top of one another for comparison purposes.
  • Page 273 Refer to the Online Help for detailed descriptions of each of the available tabs in the Single Result View. Table 72: Information available in the Single Result View Description Fragment Data Displays a plot of the dye signal traces. Raw Data Displays a plot of the raw, four-channel electropherogram.

  • Page 274: Copying A Trace

    Fragment Analysis Procedures Table 72: Information available in the Single Result View Description General/Notes Displays a list of system and user-generated notes specific to the selected result (i.e., sample name, dates, database location, hardware information, etc.) Property Set Displays a list of user-defined property fields and the values associated with the selected result.

  • Page 275: Stacked Graph

    Subtitles are copied with the image, as shown in Figure 145. To paste the image in other software, use its Paste command. 34 - D11S1984 - 0.1pmol, 0.25ulDNA.B05_031030135P 55000 50000 45000 186.0 2 40000 35000 30000 25000 14 0 20000 90 1 00 15000 197 .87...

  • Page 276: Excluding Results

    Fragment Analysis Procedures Excluding Results Results may be excluded automatically through the use of filter sets or manually through the command. To remove results through the application Exclude Results of filter sets, refer to the Filter Set area of this manual or the Online Help. If selected for display, results that have been excluded through the application of filter sets will be shaded in a teal color.

  • Page 277: Re-Analyze Results

    Re-Analyze Results The Fragment Analysis module contains a Re-Analysis feature that allows you to select one or more samples from the Result Set view to be re-analyzed. This provides a useful method of applying a different set of fragment analysis parameters to a previously analyzed set of data to pick up missed peaks or to avoid picking up unwanted peaks.
  • Page 278 Fragment Analysis Procedures 5. When an Analysis Parameter Set has been selected, click Next to proceed to the Analyze Data window. Figure 147:Re-Analyzing the Data (re-analysis shown in progress) Refer to the Analyzing Data area of this manual or the Online Help for information on this window and the data analysis process.

  • Page 279: Fragment Lists

    Fragment Lists The Fragment List window allows you to view and modify the results of the fragment data identified in the Study. This fragment list can be subjected to a “3rd Level” of filtering through application of additional exclusion filters as defined in the filter set. Accessing the Fragment List View •...

  • Page 280: Applying Exclusion Filters To The Fragment List

    Fragment Analysis Procedures The Filter Sets applied to the Result Set data (2nd Level Filtering) are different from the Filter Sets applied to the Fragment List data (3rd Level Filtering). To the Result Set you apply filters to an entire sample which may contain numerous fragments.

  • Page 281: Manually Selecting Peaks For The Fragment List

    Manually Selecting Peaks for the Fragment List The system provides the option of manually selecting peaks to be added to a fragment list, or clearing the fragment list completely and building a new one “from scratch”. To access this function, right-click within the fragment list and select the Manually Select Peak command from the menu displayed.

  • Page 282: Bin Analysis

    Fragment Analysis Procedures Bin Analysis “Binning” is a one-dimensional clustering, by size, of fragments compiled from multiple samples. Bin analysis is used to generate an allele list that is representative of the population being studied. This is particularly useful when establishing a database of information on alleles and possible anomalies that are associated with them (such as imperfect repeats).
  • Page 283 Figure 149:New Binning Analysis window - Bin Parameters A minimum of 10 samples is required to begin a Bin Analysis. Bin Analysis Parameters The first step in creating a new bin analysis is to set the bin analysis parameters. These parameters identify which data in the fragment list will be included in the bin analysis and also define the appearance of the view generated.
  • Page 284 Fragment Analysis Procedures When finished setting the bin analysis parameters, click Next . The new parameters will be applied to the scatter plot and the bins will be assigned as specified. Figure 150:New Binning Analysis window - Bin Analysis View Bin Analysis View Once the bin analysis parameters have been applied to the selected data, the system displays the results of the bin analysis.

  • Page 285: Updating The Locus Tag And Allele List

    value can be adjusted to exclude any data Minimum Relative Peak Height points generated from small “noise” peaks. The relative peak height is calculated in the color and size range specified in the initial binning parameters window. The system also generates Phantom Bins at all intervening positions for whole number nominal sizes not already assigned to a bin.

  • Page 286: Reviewing The Source Data

    Fragment Analysis Procedures Figure 151:New Binning Analysis window - Updating Locus Tag You may select an existing Locus Tag from the drop down menu by clicking on it and may also add any ancillary information to the selected locus tag at this time. When you have finished editing, save the new data to the selected locus tag so that the information updates the allele list with regards to Dye, Size and Repeat Unit Length.
  • Page 287 The Fragment Results table and Summary area list all of the data samples included in the Bin Analysis. You may view and compare individual samples (see Single Result View, Stacked View & Overlay), manually include or exclude individual samples or groups of samples from the analysis (see Add or Remove Results) and Re-Analyze selected samples (see Re-Analyzing Results).

  • Page 288: Peak Ratios

    Fragment Analysis Procedures Peak Ratios Once a Study is created, you can begin Peak Ratio calculations. Peak Ratio calculations involve the analysis of a result from a study that quantifies the amount of DNA present in a fragment. The quantity of DNA is calculated by the system by relative comparison to a user defined reference peak.
  • Page 289 Peak Ratio Parameters • Select the option button to perform a quantitation based on Use Peak Height peak height (Relative Fluorescence Units (RFU)). • Select the option button to perform a quantitation based on peak Use Peak Area area (mm x RFU). •...

  • Page 290: Selecting A Reference Trace

    Fragment Analysis Procedures Mean, Data Points & Standard Deviation The lower left area of the Peak Ratio window displays system generated values for each of the calculated columns in the Peak Ratio Result Table. Based on the reference and test peaks selected, the system will calculate the , the Mean # of Data Points...

  • Page 291: Selecting A Reference Peak

    Selecting a Reference Peak • A reference peak is a trace peak selected by the user from the reference result trace. The reference peak is compared to the test peak and a relative length, height and area are then calculated by the system. These results are added to the Peak Ratio Results Table.

  • Page 292: Aflp Analysis

    Fragment Analysis Procedures AFLP Analysis AFLP (Amplified Fragment Length Polymorphism) Analysis is a highly sensitive DNA fingerprinting technique that relies on the selective amplification of restriction fragments from a total digest of genomic DNA. The CEQ System includes AFLP Fingerprint analysis, which allows you to rapidly score AFLP fingerprints with output as a text format which is compatible with most statistical and pattern matching software.

  • Page 293: Analysis Parameters

    Figure 154:New AFLP window Analysis Parameters The analysis parameters area allows you to set filtering parameters for including or excluding fragment results in your AFLP analysis. field is used to set the limiting value given to the width of Maximum Bin Width the individual bins.
  • Page 294 Fragment Analysis Procedures • If the results list contains samples with fragments with more than one dye (either multiple dye-labeled fragments pooled in a sample or multiple samples with single dye-labeled fragments in each sample), you can select the appropriate dyes to be exported.

  • Page 295: Starting A New Aflp Analysis

    Starting a New AFLP Analysis When you have finished setting the analysis parameters, click OK to generate the New AFLP Analysis Figure 155:New AFLP Analysis window Viewing the Cluster Analysis When the clustering operation is completed, a table will be displayed showing the results of the AFLP analysis.

  • Page 296: Cluster Analysis Parameters And Display Settings

    Fragment Analysis Procedures Cluster Analysis Parameters and Display Settings The selections available at the bottom of the AFLP Cluster Analysis window allow you to make additional settings that affect the values and output format of the fields displayed. • Selecting the option button will display the Sample Bin: Fragment Count number of components within each sample bin as either zero components, one...

  • Page 297: Exporting Your Results

    Exporting your Results Exporting the AFLP Analysis • Open the menu and select the command. File Export AFLP • window will be displayed. Export AFLP • Select a file name and destination folder for the data and click Save Some 3 party software may require that you export the sample information in a specific format.
  • Page 298 Fragment Analysis Procedures Figure 156:Run LOH Analysis Pairs of samples to be compared must have the same “root” names followed by characters that allow the distinction between reference and test samples or loci. All data to be analyzed will reside in a temporary file created by the CEQ System Fragment Analysis application.
  • Page 299 The LOH Analysis results must be saved as a “Microsoft Excel Workbook (*.xls)” file type in order for the data to be saved correctly. Additionally, the file name must be changed from the default name displayed in the Save As... dialog; otherwise, the result will be saved as a temporary file and will be overwritten in the next LOH Analysis.

  • Page 300: Customizing The Results List

    Fragment Analysis Procedures Customizing the Results List The components of the results lists are user-defined and can be modified through commands available in the right-click menus. These commands allow you to select specific columns for display in the results list, customize the arrangement of information, add or remove columns and modify several other parameters of the results displayed.
  • Page 301 Selecting Columns for Display • Highlight the column selection in the area of the window Possible Columns and click the right-arrow button to move the selection to the Selected Columns area of the window. • To select all columns for display in the results list, click the right-double-arrow button.

  • Page 302: Sorting The Results List

    Fragment Analysis Procedures • Columns can also be locked and unlocked directly from the fragment result table by right-clicking on the column header and selecting the Lock Column command or the command from the pop-up menu displayed. Unlock Column Sorting the Results List The components of the results list can be sorted in ascending or descending order for any of the column parameters displayed.
  • Page 303 More information on these and other display modifications can be found in the Online Help. User’s Guide...

  • Page 304: Reporting Results

    Fragment Analysis Procedures Reporting Results The CEQ software includes a powerful suite of utilities for creating reports. It also allows you to export data and results to other 3rd party software packages. All of the reporting features available in the Fragment Analysis module are located under the Reports tab of the Study Explorer.

  • Page 305: Report Templates

    • Click OK to display the new report containing the selected information. Creating a New Report from the Study Explorer: • Select the Reports tab available at the bottom of the Study Explorer frame of the window. • Right-click to highlight the type of report you would like to generate from the list of those available in the Reports tab and select the New command from the menu displayed.
  • Page 306 Fragment Analysis Procedures 4. Table Delimiters: TableLoop: Allele = Context.Locus.Alleles Standard Nominal Size (nt) Apparent Size (nt) Deviation (nt) Num. Points Comments Allele.ID FormatNumber FormatNumber FormatNumber Allele.Fragment- Allele.Comment (Allele.TrueSize, (Allele.Apparent- (Sqr(Allele.Frag- Count Size, 2) mentSizeVari- ance)) 5. Graph Objects E.g. Graph: Service.BinGraph (Context, Site, DisplayOptions) 6.

  • Page 307: Editing Graph Displays

    Iterations of similar data can be achieved using Loop delimiters or Tables. While the data object names may appear squashed in the report template, if the data fits in the space provided, the tables should look normal in the report. Graph objects and Graph Placeholders must remain together for proper graph display.

  • Page 308: Editing The Y-Axis

    Fragment Analysis Procedures In the DisplayOptions section of the Alternative text: box, enter minimum and maximum values as follows: <DisplayOptions XScaleType="BySize" MinX="100" MaxX="300" /> Editing the Y-axis In the DisplayOptions section of the Alternative text: box, enter minimum and maximum values as follows: <DisplayOptions XScaleType="BySize"...

  • Page 309: Editing The Number Of Dyes Displayed On The Graph

    Editing the number of dyes displayed on the Graph SelectedDyes is dye selection options. It allows Egram graph to select which dye to report. The FA view dye visibility will not affect this option. The possible value for selectedDyes are “all”, “1”, “2”, “3”, “4” or combination of “1”, “2”, “3”, “4”. “All” means display all four dyes.

  • Page 310: Exporting Results

    Fragment Analysis Procedures Exporting Results You are able to include all of the property information when exporting a fragment result in text format. You may open the exported file in any text editor and/or in MS Excel and Preview the file prior to printing. Text File Format Follow these steps to export the Properties information with a fragment result: •...

  • Page 311: Features Of The Export Options Window

    The Export Options window allows you to define the export parameters and options, including the file name and location. Figure 161:Export Options Features of the Export Options window: • field displays the selected export format (file name Export result as... extension), the file name and the directory path.

  • Page 312: Text File Format

    Fragment Analysis Procedures Text File Format The text file format is an export of the results file. The data in this file are tab delimited and the export format has the extension *.txt. Figure 162:Fragment Analysis Export Dialog • Select a target folder and file name. •...

  • Page 313: Exporting Fragment Lists Or Genotypes From A Study

    Figure 163:Fragment Result Export Dialog To export fragment results in CEQ (*.cq*) format, perform the following steps. • Launch Fragment Analysis and open a fragment result. • Select to invoke the Export dialog box. File | Export • Navigate to a folder to save the data file and enter a file name. •...

  • Page 314: Format Descriptions And Exporting Requirements

    Fragment Analysis Procedures Figure 164:Exporting Fragment Lists and Genotypes • Input the desired export format, the file name and the directory path. • There are four export formats in the Save as type: drop-down list: • CSV (Comma) *.csv • Pedin (Linkage) *.pre •...
  • Page 315 The Pedin export requires a list of locus names, in a file named “LocusList.txt”. This file must be stored in the CEQ application’s ...\export or root directory. This text file must have only one locus name per line. The order of the loci included in this file will determine the order of the loci in the Pedin file.

  • Page 316: Locus List Editor

    Fragment Analysis Procedures • When an allele is not found, the empty cell is replaced with None found. Figure 165:Genotype Summary Report Locus List Editor The locus list (LocusList.txt) is required to create the Genotype Summary Report (GSV) and to perform some exports. The locus list editor is used to customize the LocusList.txt file.
  • Page 317 Table 73: Locus List Editor - Legend Study Loci The loci from the database that have not yet been moved to the Selected Loci list box. Other Loci The loci that will be removed from the LocusList.txt file. Selected Loci The loci that will be added to the LocusList.txt file.
  • Page 318 Fragment Analysis Procedures CEQ™ 8000 Genetic Analysis System...

  • Page 319: Data Manager Procedures

    User’s Database Template Data Manager Procedures User’s Database Template A database template called USERTEMPLATE is added upon installation of the CEQ software. The USERTEMPLATE database is used to store newly created methods and parameters. These methods and parameters are then included in all subsequently created databases.

  • Page 320: Renaming A Project Folder Or Database

    Data Manager Procedures When a folder is deleted, all of the data contained within that folder CAUTION will be deleted. You will be warned by the system. Renaming a Project Folder or Database To rename an existing folder, perform the following steps. 1.

  • Page 321: Shrink Database

    Shrink Database Shrink Database Use this menu option to reduce the size of the currently selected database. Often, when studies or results are removed from the database, the size of the database does not immediately reduce in size. Use this option to manually shrink the database to its new (smaller) size.

  • Page 322: Backup Database

    Data Manager Procedures 3. Select the menu item. Tools | Shrink Database Figure 169:Shrink Database menu item Backup Database Periodic backups are essential in reducing the risk of losing data. If a database is deleted or damaged, only the data created since the last backup will be lost. Backups can be saved to any folder on a local drive.
  • Page 323 Backup Database 2. Mouse-click the database to be backed up as shown in Figure 170. Figure 170:Database selection 3. Select the menu item. Tools | Backup Database Figure 171:Backup Database menu item 4. Select the location, on the local disk, to save the backup file. 5.

  • Page 324: Restore Database

    Data Manager Procedures 6. Click Save Figure 172:Backup Database dialog Restore Database Restore Database allows for the recovery of a database that was previously saved using the backup procedure. If you are restoring a database that has the same name as one already residing in the Database Manager, you must either restore it with a new name or delete the existing database before restoring it (NOT Recommended).
  • Page 325 Restore Database 2. Mouse-click in any window. Figure 173:Data Manager 3. Select the menu item Tools | Restore Database Figure 174:Restore Database menu item User’s Guide...
  • Page 326 Data Manager Procedures 4. Select the location, on the local disk, where the backup file was saved. Figure 175:Restore Database dialog 5. Select the file to restore and click Restore If a database with the same name exists, a warning will appear (Figure 176). Figure 176: If this occurs, click and either:...

  • Page 327: Convert Individual Ids To Subject Ids

    Restore Database Convert Individual IDs to Subject IDs This menu option (Figure 177) is used to convert all sample data records that use Individual IDs to records that use Subject IDs. This applies only to the working database only and is active only when the working database is selected. All other modules must be closed prior to the start of conversion.

  • Page 328: Exporting Database Items

    Data Manager Procedures Exporting Database Items Sample Plate Result Export When exporting a sample plate from the Data Manager the dialog in Figure 179 will appear if any of the sample results are missing. This dialog box provides a list of the sample results that were not exported.
  • Page 329 Exporting Database Items Figure 180: User’s Guide...

  • Page 330: Exporting From The Data Manager

    Data Manager Procedures Exporting from the Data Manager To export from the Data Manager Module, follow the steps below: 1. Launch the Data Manager and open a sequence result. 2. Select to open the Export dialog box. File | Export 3.

  • Page 331: Data Manager Export File Types

    Exporting Database Items Data Manager Export File Types Listed in Table 74 are the available export file types. Table 74: Data Manager Export Options Format Description The SCF (Standard Chromatogram Format) format is used to store analyzed DNA sequence or fragment data for a single sample. The format describes both “public”...
  • Page 332 Data Manager Procedures Table 74: Data Manager Export Options (Continued) Format Description The FASTA format only applies to the sequence results portion (e.g., text bases) of analyzed data, and contains a comment line that is used to identify the data. Along with the text file, a quality values file is also generated (*.FASTA.QUAL) which lists the quality value for each base.
  • Page 333 Exporting Database Items Table 74: Data Manager Export Options (Continued) Format Description If you select the CEQ format, the format native to our database, you will export sample data, sequence or fragment results, sample plate results, sequence or fragment analysis parameters, sample plates, methods, locus tags (fragment data only), standards (fragment data only), and optical scan data.

  • Page 334: Importing Database Items

    Data Manager Procedures Table 74: Data Manager Export Options (Continued) Format Description The CRV export option from the Data Manager module exports the four colors (dyes) as four separate files. When batches are exported, the resulting four files will contain separate dye colors for all the samples. You can select *.CRV as an export file type for exporting fragment results from the Fragment Analysis module.

  • Page 335: Generating A Sample Run History

    Generating a Sample Run History Generating a Sample Run History To generate a sample run history, perform the following steps. 1. Select from the menu. View | Sample Run History 2. In the dialog box: Sample Run History a. Select a name from the drop-down menu.

  • Page 336: Adding Users

    Data Manager Procedures Adding Users Add Users by selecting, from the computer desktop, Start | Programs | . The Computer Management Administrative Tools | Computer Management console shown in Figure 182 will be displayed. Add New Users following the steps below: Figure 182:Windows 2000 Computer Management console 1.

  • Page 337: Adding Groups

    Administrative Tools 3. Right-click on Users and select (Figure 182). New User Figure 183:Add New User menu 4. In the New User dialog (Figure 184), enter the new User name (required) and edit the other fields as necessary. 5. Click Create Figure 184:New User dialog Adding Groups...
  • Page 338 Data Manager Procedures 3. Right-click on Groups and select (Figure 185). New Group Figure 185:Add New Group menu 4. In the New Group dialog (Figure 186), enter the new Group name CEQUSERS and a description if necessary. Figure 186:New Group dialog 5.
  • Page 339 Administrative Tools 7. Scroll down the list and select the user names created in the Adding Users Name section above. If more than one User was created, mouse-click on the first name, press and hold the key on the keyboard and select any additional names to Ctrl add to the group.

  • Page 340: Activating The Cequsers Group

    Data Manager Procedures 9. Click to save the new group. Create Figure 188:New Group dialog Activating the CEQUSERS Group Once the CEQUSERS group has been created and the users added, it must be activated within the Data Manager module. This is done only once and users can be added/removed at any time thereafter.

  • Page 341: Direct Control And Replenishment

    Direct Control and Replenishment The following procedures apply to the Direct Control functions of the Run module. Direct Control functions can be accessed via the menu, the Direct Control menu and the of the Direct Control window. Replenish Hot Areas Use Figure 190, User Accessible Hardware Components, to locate hardware components referenced in this section.

  • Page 342: Accessing The Direct Control Window

    Direct Control and Replenishment Accessing the Direct Control Window From the CEQ Toolbar, click on the icon. Verify that the Run window is displayed and then click on the tab. Direct Control Direct Control Procedures Replacing the Wetting Tray Removing the Wetting Tray 1.
  • Page 343 Direct Control Procedures Figure 191:Replacing the Wetting Tray Wetting Tray Lid Wetting Tray Sample Plate Holder Wetting Tray Retainers Wetting Tray Well (front) 901002L.A 6. Close the Sample Access Cover and then click the button in the Done Replace dialog box (Figure 192). Wetting Tray Figure 192:Replace Wetting Tray Dialog User’s Guide...

  • Page 344: Loading The Sample Plate Or Buffer Plate

    Direct Control and Replenishment Loading the Sample Plate or Buffer Plate 1. Select from the menu. Direct Control | Access Plates Figure 193:Access Plates Dialog 2. Click . The Capillaries Exposed dialog (Figure 194) will appear. Start Figure 194:Capillaries Exposed Dialog 3.

  • Page 345: Loading The Buffer Plate And Evaporation Cover

    Direct Control Procedures Figure 195:Loading the Sample Plate Sample Plate Sample Notched Edge Plate Wetting Tray Retainers 900497e.AI Sample Pate Sample Plate align Holder Guide Pin 900497e.A1 900500e.AI 900500L AI 3. When finished positioning the plate, close the Sample Access Cover and then click button of the dialog box (Figure 196).

  • Page 346: Specifying Capillary Temperature

    Direct Control and Replenishment 3. At the rear of the Buffer Plate Holder, align the Buffer Evaporation Cover Guide Pin with the Buffer Evaporation Cover Alignment Notch and then gently lower the cover over the Buffer Plate. Figure 197:Loading the Buffer Plate and Buffer Evaporation Cover Buffer Evaporation Cover Buffer Plate...

  • Page 347: Injecting A Sample

    Direct Control Procedures Injecting a Sample 1. Select from the menu. Direct Control | Inject 2. In the dialog box: Inject a. Enter a value for the voltage in kV. b. Enter a time duration in seconds. c. Identify the position of the sample using the spin controls and Sample Set then select...

  • Page 348: Viewing Or Changing Capillary Information

    Direct Control and Replenishment b. Enter a name in the Name field. c. Select a Project from the drop-down menu. d. Select Align Prior to performing an Optical Alignment, it is advisable to purge the Array Manifold and fill the capillaries with fresh gel (in that order). See “Purging the Manifold”...
  • Page 349 Direct Control Procedures a. The system automatically updates the new buffer part number. b. The system automatically updates the date and time installed. c. Use the spin control to change the if you wish to Hours on Instrument change the system’s alert mechanism. 4.

  • Page 350: Removing And Replacing The Capillary Array

    Direct Control and Replenishment Removing and Replacing the Capillary Array Care must be exercised when installing/removing the capil- CAUTION lary array to prevent damage and maintain low background signals. Clean all gel residues thoroughly. This procedure assumes that an expended capillary array is being replaced with a new capillary array.
  • Page 351 Direct Control Procedures Figure 201:Manifold Access Cover Capillary Temperature Control Cover (open) Short Plenum Assembly LASER Manifold Access Cover captive screw 900487e .AI 900485e .AI Manifold Access 900485e.AI 900487e.AI Cover (removed) 900488e.AI 900488e .AI 6. Loosen the two Plenum Assembly captive screws (Figure 202), pull the Plenum Assembly straight back and away from the instrument and set it aside.
  • Page 352 Direct Control and Replenishment 8. Grasp the Array Fitting tab (Figure 203) and then: a. Pull the fitting approximately one inch out of the manifold. b. Touch the tip of the fitting to the bottom of the Optics Base Plate. c.
  • Page 353 Direct Control Procedures Figure 203:Removing/Replacing the Array Fitting Guide Pins Eject Lever Array Fitting gel strand Optics Base Plate (bottom) Array Fitting 900489e .AI 900489e.AI 900507e .AI 900507e.AI 6. Grasp the Electrode Block tab (Figure 204) with your left hand and pull the block out and away from the instrument.
  • Page 354 Direct Control and Replenishment Always grip the capillary array fitting near the end during removal CAUTION or installation of the tip cap to prevent flexing and possible breakage of the capillary array. While holding the Array Fitting tab (of the new capillary array), align the Array Fitting (Figure 203) with the manifold opening and guide pins.
  • Page 355 Direct Control Procedures Figure 205:Plenum Assembly - Front View CAUTION: USE THIS FRONT PLENUM ONLY WITH A 33 CM CAPILLARY ARRAY. PRINED IN U.S.A. 608542-AA 900697L.AI Figure 206:Capillary Array Routing 900694L.AI When reinstalling the Plenum Assembly, gently gather the capillaries between your thumb and forefinger to make sure they pass through the hole in the Plenum Assembly without any constriction.
  • Page 356 Direct Control and Replenishment 15. If you are installing a new capillary array, in the Install Capillary Array dialog box (Figure 207) select the correct part number, enter the serial number, click the button then . The number of runs and days on instrument will Set to New Done revert to “0.”...

  • Page 357: Removing And Replacing A Gel Cartridge/Gel Pump Plug

    Direct Control Procedures Removing and Replacing a Gel Cartridge/Gel Pump Plug Care must be exercised when installing/removing a gel car- CAUTION tridge due to the viscosity of the gel mixture. Do not allow gel to remain on the instrument. This procedure assumes that an expended gel cartridge is being replaced with a fresh gel cartridge or that a used gel cartridge is being removed (for storage purposes) and being replaced with the gel pump plug.
  • Page 358 Direct Control and Replenishment Figure 208:Removing/Replacing the Gel Cartridge Cartridge Locking Lever (closed) Cartridge Barrel LASER 900463.AI Cartridge Locking Gel Pump/ Lever (open) Gel Cartridge Access Cover 900491e .AI Cartridge Barrel 900463e.AI 900491e.AI 900492e.AI 900492e .AI 4. Grasp the wings of the gel cartridge/gel pump plug and pull it out of the barrel. Always note the lot number and the hours on the instrument for a gel cartridge if you are planning to use it for more than one session.
  • Page 359 Direct Control Procedures Figure 209:Removing/Replacing the Gel Cartridge - Continued Cartridge Locking Cartridge Lever (open) Wings 900494e.AI Gel Cartridge OR Cartridge Barrel Gel Pump Plug 900494e.AI 900493e.AI 6. Remove any air pockets from the gel cartridge by grasping the cartridge wings between your first two fingers, and depressing the plunger with your thumb until a small amount of gel is pushed out of the cartridge tip.
  • Page 360 Direct Control and Replenishment Figure 210:Remove Gel Cartridge Dialog 11. If was selected in step 10, proceed to step 5. Install Plug was selected in step 10, use the Install Cartridge Install Gel Cartridge dialog box (Figure 211) to enter the following information: a.

  • Page 361: Removing The Manifold Plug

    Direct Control Procedures Figure 211:Install Gel Cartridge Dialog Dispose of the used tissue and spent gel cartridge in accordance with the procedure, “Disposal of the Gel Cartridge’ on page 360. Removing the Manifold Plug The Manifold Plug is installed in the CEQ instrument during shipping and when the instrument is not is use to prevent drying of gel.
  • Page 362 Direct Control and Replenishment Figure 212:Manifold Access Cover Capillary Temperature Control Cover (open) Plenum Assembly LASER Manifold Access Cover captive screw 900487e .AI 900485e .AI Manifold Access 900485e.AI 900487e.AI Cover (removed) 900488e.AI 900488e .AI 6. Lift the Eject Lever to release the Manifold Plug. 7.
  • Page 363 Direct Control Procedures Figure 213:Removing the Manifold Plug Guide Pins Eject Lever Array Fitting gel strand Optics Base Plate (bottom) Array Fitting 901290L.AI 6. Click from the dialog box (Figure 214). Remove Manifold Plug Figure 214:Remove Manifold Plug Dialog 7. To install the capillary array, see “Removing and Replacing the Capillary Array” on page 336.
  • Page 364 Direct Control and Replenishment CEQ™ 8000 Genetic Analysis System...

  • Page 365: Routine Maintenance

    CEQ™ 8000 Genetic Analysis System – User’s Guide A16637-AA Routine Maintenance Overview This section provides routine maintenance and biological waste disposal procedures. It also provides a list of the consumable materials used in the system. Routine Maintenance Use Figure 215 to locate hardware components referenced in this section.

  • Page 366: Cleaning The Capillary Array

    Routine Maintenance Cleaning the Capillary Array The capillary array windows must kept free of any contaminants. CAUTION Otherwise, high backgrounds and/or drifting baselines will occur. All background levels should be below 6000 counts. Water used during this procedure must be fresh, distilled, deionized water (18 Mohm/cm water).
  • Page 367 Routine Maintenance d. Pull the fitting away from the instrument. e. Use a tissue to wipe gel strands off of the instrument. Figure 217:Removing/Replacing the Array Fitting Guide Pins Eject Lever Array Fitting gel strand Optics Base Plate (bottom) Array Fitting 900489e .AI 900489e.AI 900507e .AI...
  • Page 368 Routine Maintenance Figure 218:Cleaning the Detection Window Detection Window Stroke in one direction ONLY! 900501e.AI 900501e.AI 900502e.AI Array Fitting 900502e.AI 10. With a new water-moistened swab, repeat wiping on the other side of the window. 11. With a dry swab, gently wipe the windows to remove excess water, again repeating on the backside with a new, dry swab.
  • Page 369 Routine Maintenance Figure 219:Remove Capillary Dialog 15. While holding the Array Fitting tab (of the clean capillary array), align the Array Fitting with the manifold opening and guide pins. Push the fitting into the manifold until it is completely seated against the bases of the Guide Pins. See Figure 217. 16.

  • Page 370: Replacing The Gel Waste Bottle

    Routine Maintenance After cleaning the Detection Windows, perform the Optical CAUTION Alignment procedure and monitor the baseline. If background levels are above 6000 counts, repeat the cleaning process. Replacing the Gel Waste Bottle This procedure assumes that a used (full) waste bottle is being replaced with an empty waste bottle.

  • Page 371: Installing The Wetting Tray

    Routine Maintenance No more than one 96-well plate should be processed, for each CAUTION wetting tray, without replenishing the Wetting Tray. Periodically check the liquid level in the wetting tray. Liquid level should NEVER be allowed to rise into the eight recesses of the wetting tray lid, nor drop below the fill level indicator line (9 mL minimum).

  • Page 372: Biological Waste Disposal

    Routine Maintenance Figure 222:Replace Wetting Tray Dialog Biological Waste Disposal WARNING The CEQ System has been designed to minimize exposure to hazardous chemicals and biological waste. However, care must still be exercised when removing used chemicals and biological samples from the instrument. The information below provides the minimum protocols to use when handling hazardous chemicals and biological waste.

  • Page 373: Bulk Disposal

    Biological Waste Disposal Bulk Disposal WARNING When performing this procedure, use an exhaust ventilation unit that meets TLV requirements. 1. Using a 1L side arm flask as a trap, connect the trap to a vacuum. 2. Attached a pipette to the trap using chemical resistant tubing. 3.

  • Page 374: Bulk Disposal

    Routine Maintenance Bulk Disposal WARNING When performing this procedure, use an exhaust ventilation unit that meets TLV requirements. 1. Using a 1L side arm flask as a trap, connect the trap to a vacuum. 2. Attached a pipette to the trap using chemical resistant tubing. 3.

  • Page 375: Disposal Of The Gel Waste Bottle

    Biological Waste Disposal Dispose of D.I. water/gel mixture in accordance with all applicable WARNING federal, state and local environmental regulations concerning hazardous liquid waste. Disposal of the Gel Waste Bottle WARNING When performing this procedure, use an exhaust ventilation unit that meets TLV requirements.

  • Page 376: Consumable Items List

    • Mineral Oil • Sample Loading Solution (SLS) Sequencing Test Samples 608070 CEQ Separation Gel 608010 11.5 mL of gel in CEQ 8000 compatible container. LPA 1 Sufficient for 12 fills of the Capillary Array (96 sequencing runs). CEQ Separation Buffer 608012 Each container has a screw top and pour tip.
  • Page 377 Required for use as the CEQ 8000 sample plate. 25 Plates/Box a. Contact your local Beckman Coulter representative for specific consumable material requirements based on your application and frequency of use. Table 76 provides a list of the required consumable items for the Fragment Analysis system.
  • Page 378 200 µL volume capacity. Required for use as the CEQ 8000 sample plate. 25 Plates/Box. a. Contact your local Beckman Coulter representative for specific consumable material requirements based on your application and frequency of use. CEQ™ 8000 Genetic Analysis System...

  • Page 379: Materials Required But Not Supplied

    100mM Na -EDTA pH8.0 (500 mM Sigma Cat. # 7889) — ✔ PCR enzyme and buffer — Labeled primers (contact your local Beckman Coulter representative for ✔ — information on vendors carrying the appropriate primers) ✔ Thermal cycling plates and caps —...
  • Page 380 Routine Maintenance CEQ™ 8000 Genetic Analysis System...

  • Page 381: Diagnostics

    Re-Initializing the System Diagnostics Re-Initializing the System To re-initialize the system, select from the Run menu. Run | Reset Homing the Plates and/or Gel Pump To re-establish the position of the plates and/or gel pump, perform the following steps. 1. Select from the Run menu.

  • Page 382: Monitoring The Baseline

    Diagnostics Monitoring the Baseline You must perform the optical alignment procedures prior to monitor- ing the baseline (“Performing an Optical Alignment’ on page 333). To monitor the baseline, perform the following steps. 1. Select from the Run Module main menu. Run | Monitor Baseline 2.

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