GeneScan Analysis Software Version 3.1 User's Manual © Copyright 1998, The Perkin-Elmer Corporation This product is for research purposes only. GeneScan, Genotyper, and Perkin-Elmer are registered trademarks of The Perkin-Elmer Corporation. BioLIMS is a trademark of Molecular Informatics, Inc. ABI, ABI PRISM and the ABI PRISM design, Applied Biosystems, GenoPedigree, PE, and PE Applied Biosystems are trademarks of The Perkin-Elmer Corporation. Sybase is a registered trademark and Sybase SQL Server are trademarks of Sybase, Inc. Oracle is a trademark of Oracle Corporation. All other trademarks are the sole property of their respective owners. - Contents 1 GeneScan Analysis Software Overview . . . . . . . . . . 1-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 What's New in the GeneScan Analysis Software . . . . . . . . . . . . . . . . . . . . . 1-2 BioLIMS Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Fifth Dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Works with 96-Lane Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Displaying Off-Scale Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Manual Tracker Interface Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Printing from Sample File Window . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 New Neural Net Tracker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Lane Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4 About This Manual and Other Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5 ABI 373. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5 ABI 373 XL Upgrade Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5 96-lane Upgrade Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5 Manual Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5 User Attention Words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6 Using the Macintosh Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7 Macintosh Computer Vocabulary and Operations . . . . . . . . . . . . . . . 1-7 Guidelines for Optimal Performance . . . . . . . . . . . . . . . . . . . . . . . . . 1-7 Macintosh Computer Terms Used in This Manual . . . . . . . . . . . . . . . 1-8 Registering the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11 License and Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11 Registering Your Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11 Installing the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-12 System Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-12 i Turning On Virtual Memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RAM Required to Run the Software . . . . . . . . . . . . . . . . . . . . . . . . Installing the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Program Files Installed Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . Contents of Folders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GeneScan Analysis Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table of Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample File Database Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . To Reach Us On the Web . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hours for Telephone Technical Support. . . . . . . . . . . . . . . . . . . . . . To Reach Us by Telephone or Fax in North America . . . . . . . . . . . Fax-on-Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . To Reach Us by E-Mail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regional Offices Sales and Service . . . . . . . . . . . . . . . . . . . . . . . . . 1-13 1-14 1-14 1-15 1-15 1-16 1-16 1-19 1-20 1-20 1-20 1-20 1-22 1-23 1-23 2 How to Process and Edit the Gel File . . . . . . . . . . . .2-1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 Topics in This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 About Gel Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Instruments Producing Gel File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 96-Lane Gels Capability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Gel File Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 How GeneScan Tracks a Gel File . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 How to Set Gel Processing Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 Displaying the Gel Preferences Dialog Box . . . . . . . . . . . . . . . . . . . 2-3 Auto-Launch Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4 Image Generation Defaults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4 Lane Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7 Comb Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10 How to Display the Gel File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11 ii Displaying the Gel File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11 About the Gel File Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12 Differences Between Gel File Windows . . . . . . . . . . . . . . . . . . . . . . 2-12 Gel File Window Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13 Gel File Window Buttons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-14 Gel File Window Described . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15 How to Adjust the Gel Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18 Displaying/Hiding a Dye Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18 Changing Dye Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-19 Adjusting the Contrast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-20 Removing Gel Contrast Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-22 Regenerating the Gel Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23 Installing New Matrix Information . . . . . . . . . . . . . . . . . . . . . . . . . . 2-24 Verifying Gel Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26 Where the Information is Archived. . . . . . . . . . . . . . . . . . . . . . . . . . 2-26 Data Collection Run Error Log. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26 Gel Info Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26 Gel Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-27 Sample Sheet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28 Displaying the Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29 What is the Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29 Using the Copy of the Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . 2-29 Displaying the Copied Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . 2-29 Displaying a New Sample Sheet. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29 About the Sample Sheet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-31 Sample Sheet Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-31 Sample Sheet Described . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-31 Working with the Sample Sheet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-34 Editing Collection Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-34 Changing Column Widths. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-35 Editing the Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-35 Printing the Sample Sheet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-36 Installing a New Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-36 Importing Data to Sample Sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-37 iii Exporting Data From Sample Sheets . . . . . . . . . . . . . . . . . . . . . . . . About Displaying Regions of Off-Scale Data. . . . . . . . . . . . . . . . . . . . . . . Why Displaying Off-Scale Data is Important . . . . . . . . . . . . . . . . . Off-Scale Detection and GeneScan . . . . . . . . . . . . . . . . . . . . . . . . . Displaying Off-Scale Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gel File Window Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electropherogram Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Working with Lane Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Topics in This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moving Between Lanes and Channels. . . . . . . . . . . . . . . . . . . . . . . Using Control Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adding and Deleting Control Rows . . . . . . . . . . . . . . . . . . . . . . . . . What Marking Lanes as Used/Unused Means . . . . . . . . . . . . . . . . . Marking Lanes as Used or Unused . . . . . . . . . . . . . . . . . . . . . . . . . Moving Misplaced Lane Markers . . . . . . . . . . . . . . . . . . . . . . . . . . Marking and Unmarking Lanes for Extraction. . . . . . . . . . . . . . . . . . . . . . Lane Marker Rules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About Marking Lanes for Extraction . . . . . . . . . . . . . . . . . . . . . . . . Marking All Lanes for Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . Unmarking All Lanes for Extraction . . . . . . . . . . . . . . . . . . . . . . . . Marking a Single Lane for Extraction . . . . . . . . . . . . . . . . . . . . . . . Unmarking a Single Lane for Extraction . . . . . . . . . . . . . . . . . . . . . Working with Tracker Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Topics in This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hiding/Showing Tracker Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Positioning Each Lane is Important . . . . . . . . . . . . . . . . . . . . Causes to Misinterpreting Lane Positions . . . . . . . . . . . . . . . . . . . . Verifying Optimal Channel Tracking . . . . . . . . . . . . . . . . . . . . . . . . Reviewing Tracker Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moving Tracker Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deselecting a Lane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interpolating Tracker Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Use Interpolation Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv 2-37 2-38 2-38 2-38 2-38 2-40 2-41 2-43 2-43 2-43 2-44 2-45 2-46 2-47 2-47 2-48 2-50 2-50 2-50 2-51 2-51 2-52 2-52 2-53 2-53 2-53 2-54 2-54 2-55 2-55 2-56 2-56 2-57 2-57 2-57 Tracking Lanes and Extracting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-59 Ways to Track and Extract Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-59 If the Run Folder is Not Specified . . . . . . . . . . . . . . . . . . . . . . . . . . 2-59 Tracking the Gel Without Extracting Data . . . . . . . . . . . . . . . . . . . . 2-60 Tracking and Extracting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-61 Extracting Data Without Changing the Current Tracker. . . . . . . . . . 2-64 How the GeneScan Analysis Software Names Sample Files . . . . . . . . . . . 2-67 Naming Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-67 Saving Gel Files After Editing Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-68 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-68 Saving Files After Editing Tracking . . . . . . . . . . . . . . . . . . . . . . . . . 2-68 3 Analyzing Sample Files . . . . . . . . . . . . . . . . . . . . . . 3-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1 About Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2 What Sample Files Contain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2 How GeneScan Generates Sample Files. . . . . . . . . . . . . . . . . . . . . . . 3-2 Ways to Generate Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3 How GeneScan Analyzes Sample Files . . . . . . . . . . . . . . . . . . . . . . . 3-3 Opening Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4 About The Sample File Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 What it Displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Five Views. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Sample Results View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6 What it Displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6 Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6 Sample Results View Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6 Description of Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Differences From the Results Display . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Sample Info View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8 What it Displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8 v Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8 Sample Info View Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Description of Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Size Curve View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13 What it Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13 Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13 What the Size Curve Displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14 Curves Described . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14 Raw Data View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15 What it Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15 Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15 Raw Data Example. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16 What to Evaluate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16 EPT Data View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 What it Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 EPT Data Example. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18 Colored Lines Described . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18 Analyzing a Sample File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19 Installing a New Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20 4 Creating a Project . . . . . . . . . . . . . . . . . . . . . . . . . . .4-1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Automatic Analysis and Project Creation Process . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Process Using the ABI PRISM 310 . . . . . . . . . . . . . . . . . . . . . . . . . . . Process Using the ABI 373 and the ABI PRISM 377 . . . . . . . . . . . . . Setting Up for Automatic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using a Project to Manage Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . vi 4-1 4-1 4-2 4-2 4-2 4-3 4-4 4-4 4-4 4-8 What is a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8 Why Create a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8 When GeneScan Creates Projects. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8 Where to Store Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9 Working with Project Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10 Opening an Existing Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10 Creating a New Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10 Unlocking Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12 Removing Samples from a Project . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13 Finding Missing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 When are Files Considered Lost . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 When an Alert Appears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 Searching for Missing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 Re-Establishing Links. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15 5 Analyzing Project Files . . . . . . . . . . . . . . . . . . . . . . 5-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1 Analyzing Project Files: About the Analysis Control Window . . . . . . . . . . . 5-2 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2 Analysis Control Window Example . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2 Analysis Control Window Description . . . . . . . . . . . . . . . . . . . . . . . . 5-3 Customizing the Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4 Using the Analysis Control Window. . . . . . . . . . . . . . . . . . . . . . . . . . 5-4 Analyzing Sample Files: Using the Analysis Control Window . . . . . . . . . . . 5-6 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6 Accessing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6 Analyzing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7 Specifying the Format for Printed Results . . . . . . . . . . . . . . . . . . . . . 5-9 Displaying Size Standards and Analysis Parameters . . . . . . . . . . . . 5-11 Displaying Sample and Dye Information . . . . . . . . . . . . . . . . . . . . . 5-12 Setting Sample File Sort Order . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14 Setting Dye Indicator Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15 About the Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-17 vii Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parameters Used During Automatic Analysis . . . . . . . . . . . . . . . . . Specifying Where Files are Stored. . . . . . . . . . . . . . . . . . . . . . . . . . Defining Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Six Analysis Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis Range Parameter Options . . . . . . . . . . . . . . . . . . . . . . . . . Data Processing Parameter Options . . . . . . . . . . . . . . . . . . . . . . . . . Peak Detection Parameter Options. . . . . . . . . . . . . . . . . . . . . . . . . . Size Call Range Parameter Options . . . . . . . . . . . . . . . . . . . . . . . . . Size Calling Method Parameter Options . . . . . . . . . . . . . . . . . . . . . Split Peak Correction Parameter Options . . . . . . . . . . . . . . . . . . . . Using Analysis Parameter Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analyzing Samples Using the Same Analysis Parameters. . . . . . . . Selecting Different Parameters for Analysis Samples . . . . . . . . . . . Displaying Default Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . Creating Custom Analysis Parameter Files . . . . . . . . . . . . . . . . . . . Changing an Existing Analysis Parameters File . . . . . . . . . . . . . . . Deleting Custom Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . About Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What are Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advantages of Using Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . Size Standards Provided. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . When to Define Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . If Split Peaks Appear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Specifying Where Files are Stored. . . . . . . . . . . . . . . . . . . . . . . . . . Defining the Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two Ways to Define the Size Standard . . . . . . . . . . . . . . . . . . . . . . Using the New Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using the Analysis Control Window . . . . . . . . . . . . . . . . . . . . . . . . Using Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changing the Number of Peaks Detected . . . . . . . . . . . . . . . . . . . . Editing the Size Standard Definition . . . . . . . . . . . . . . . . . . . . . . . . Editing an Existing Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii 5-17 5-17 5-17 5-18 5-18 5-19 5-19 5-20 5-21 5-21 5-22 5-24 5-24 5-24 5-25 5-26 5-26 5-27 5-28 5-29 5-29 5-29 5-29 5-29 5-29 5-30 5-31 5-31 5-31 5-34 5-36 5-36 5-36 5-36 5-37 Deleting an Existing Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-39 Analyzing Samples Using the Same Standard . . . . . . . . . . . . . . . . . 5-39 Selecting Separate Standards for Samples . . . . . . . . . . . . . . . . . . . . 5-40 Defining Folder Locations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-41 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-41 Storing Matrix Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-41 Specifying File Locations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-42 6 Making a Matrix File . . . . . . . . . . . . . . . . . . . . . . . . 6-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1 About Matrix Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Matrix File Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Multicomponent Definition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Why is a Matrix File Necessary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3 When to Create a Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3 Sample Files Using Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 Applying a Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 When to Assign a Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 Limitations to Matrix Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5 When to Create New a Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5 Considerations Before Making a Matrix File . . . . . . . . . . . . . . . . . . . 6-6 Where to Store Matrix Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6 Process of Creating a New Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7 Process Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7 Steps to Creating a New Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8 The Dye Matrix Standard Kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8 Table of Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8 Preparing Matrix Standards for the ABI PRISM 310 . . . . . . . . . . . . . . . . . . . 6-9 About Formamide and Samples in Formamide . . . . . . . . . . . . . . . . . 6-9 Deionizing Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9 Preparing the Formamide-Size Standard Mix. . . . . . . . . . . . . . . . . . 6-10 Preparing Matrix Standard Samples . . . . . . . . . . . . . . . . . . . . . . . . . 6-10 ix ..................................................... Preparing Matrix Standards for the ABI 373 and ABI PRISM 377 . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table of Matrix Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Loading and Running Dye Standards for the ABI PRISM 310 . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Creating a GeneScan Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . Creating a GeneScan Injection List . . . . . . . . . . . . . . . . . . . . . . . . . Starting the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . 6-11 6-12 6-12 6-12 6-12 6-13 6-13 6-13 6-14 6-15 If the Run was Cancelled . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15 Run Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16 Assigning Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16 Loading and Running Dye Standards for the ABI PRISM 377 . . . . . . . . . . 6-17 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17 Creating a GeneScan Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . 6-17 Loading Matrix Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-18 Running the Matrix Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19 Loading and Running Matrix Samples on the ABI 373 (non XL) . . . . . . . 6-20 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20 Loading the Matrix Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20 Completing the Sample Sheet in the 672 Software . . . . . . . . . . . . . 6-21 Running the Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22 Completing the GeneScan Sample Sheet. . . . . . . . . . . . . . . . . . . . . 6-23 Loading and Running Matrix Standards on the ABI 373 with XL Upgrade6-24 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-24 Completing the GeneScan Sample Sheet. . . . . . . . . . . . . . . . . . . . . 6-24 Loading the Matrix Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-25 Running the Matrix Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26 Generating Matrix Sample Files for the ABI 373 and ABI PRISM 377 . . . 6-27 Who Should Use This Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Verify Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Generating Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choosing a Scan Range for the Matrix Calculation . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x 6-27 6-27 6-27 6-29 6-29 Viewing the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-29 What to Look For in the Raw Data Display . . . . . . . . . . . . . . . . . . . 6-30 Choosing a Scan Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-30 Eliminating Primer Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31 Generating a New Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-32 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-32 Matrix File Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-33 Saving and Naming the Matrix File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-34 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-34 Naming Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-34 Saving the Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-34 Where to Store the Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-34 When to Make Copies of a Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . 6-34 Assigning the Matrix File to Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . 6-35 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35 Evaluating the Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37 Causes for Bad Matrix Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38 If an Error Message Appears. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38 Two Causes of Bad Matrix Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38 7 Evaluating Analysis Results. . . . . . . . . . . . . . . . . . . 7-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1 Process of Evaluating Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2 Steps to Evaluating Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . 7-2 Ways to Display Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3 Two Ways to Display Analysis Results. . . . . . . . . . . . . . . . . . . . . . . . 7-3 The Results Display Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4 Displaying the Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4 Results Control Window Description . . . . . . . . . . . . . . . . . . . . . . . . . 7-5 xi Using the Results Control Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-7 Selecting Display Format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-7 Selecting Electropherogram Panels . . . . . . . . . . . . . . . . . . . . . . . . . . 7-7 Selecting Samples to Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-8 Creating Tiled Electropherogram Displays . . . . . . . . . . . . . . . . . . . 7-10 Deselecting Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-11 Removing Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12 Displaying the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12 Printing the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12 Changing How the Results are Displayed and Printed . . . . . . . . . . . . . . . . 7-13 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-13 The Sample Results View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16 About the View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16 For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16 Updating the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16 Re-Analyzing the Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16 Saving and Renaming the Results Control Format. . . . . . . . . . . . . . . . . . . 7-17 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-17 Important Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-17 Saving the Display Format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-17 Working with a Previously Saved Displays . . . . . . . . . . . . . . . . . . . 7-18 Renaming the Current Results Display . . . . . . . . . . . . . . . . . . . . . . 7-19 About Electropherogram and Tabular Data Displays . . . . . . . . . . . . . . . . . 7-20 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20 How the Window is Divided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20 What Tabular Data Contains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20 How Electropherogram Panels are Sized . . . . . . . . . . . . . . . . . . . . . 7-20 Peaks Visible in Electropherogram Not Tabular Data . . . . . . . . . . . 7-21 For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-21 Displaying Electropherogram and Tabular Data. . . . . . . . . . . . . . . . . . . . . 7-22 Displaying Electropherogram and Tabular Data . . . . . . . . . . . . . . . 7-22 Tabular Data and Electropherogram Example . . . . . . . . . . . . . . . . . 7-22 Table Describing Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-23 Highlighting Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-23 Adjusting Window Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-24 xii Hiding Selected Rows of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-24 Limited the Rows Displayed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-24 Displaying Electropherogram Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25 Base Pairs Versus Data Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25 Electropherogram Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-26 Descriptions of the Electropherogram . . . . . . . . . . . . . . . . . . . . . . . 7-26 Working with Electropherogram Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27 In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27 Displaying X-and Y-Axis Positions . . . . . . . . . . . . . . . . . . . . . . . . . 7-27 Moving the Electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-28 Changing the Dye Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-28 Displaying Peak Positions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-29 Highlighting Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-29 Using Legends to Change the Display . . . . . . . . . . . . . . . . . . . . . . . 7-30 Scrolling the Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30 Zooming In and Out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-31 Showing Off-Scale Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-32 Electropherogram Displaying Off-Scale Data . . . . . . . . . . . . . . . . . 7-34 Electropherogram Displaying the Flat Topped Effect . . . . . . . . . . . 7-35 Showing Data by Fragment Size. . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-35 Changing the Horizontal Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-37 Changing the Vertical Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-38 Assigning Standard or Custom Colors . . . . . . . . . . . . . . . . . . . . . . . 7-39 How to Define Custom Colors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-40 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-40 Why Change Colors in the Electropherogram . . . . . . . . . . . . . . . . . 7-40 Saving the Display Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-40 Defining Custom Color for All Electropherogram . . . . . . . . . . . . . . 7-41 Defining Individual Plot Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-42 How to Change the Dye Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-44 What the Dye Scale Defines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-44 Increasing the Dye Scale Example . . . . . . . . . . . . . . . . . . . . . . . . . 7-44 Changing the Dye Scale of an Electropherogram. . . . . . . . . . . . . . . 7-44 xiii Changing the Dye Scale Preferences . . . . . . . . . . . . . . . . . . . . . . . . Process of Verifying Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Steps to Verifying Size Calculation . . . . . . . . . . . . . . . . . . . . . . . . . How to Verify Size Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Verifying for the GeneScan-350 Standard . . . . . . . . . . . . . . . . . . . . Evaluating for Multiple Internal Size Standards . . . . . . . . . . . . . . . Using the Analysis Log. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What is the Analysis Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Displaying the Analysis Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What to Evaluate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Removing Information from the Analysis Log . . . . . . . . . . . . . . . . Closing the Analysis Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How to Verify Peak Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Verifying Peak Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-45 7-46 7-46 7-46 7-48 7-48 7-48 7-50 7-51 7-51 7-51 7-52 7-52 7-52 7-53 7-53 7-53 8 Saving, Archiving and Copying Files . . . . . . . . . . . .8-1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saving GeneScan File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Save . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saving Projects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saving Sample Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saving Gel Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saving Results Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How to Archive Sample and Gel Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Archiving Sample and Gel Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transferring Data to Other Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cutting and Pasting Tabular Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . Creating a Text File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv 8-1 8-1 8-2 8-2 8-3 8-3 8-4 8-5 8-6 8-6 8-6 8-7 8-7 8-7 8-8 9 Printing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1 In this Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1 About Printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-2 Ways You Can Print . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-2 If You Get Unexpected Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-2 Automatically Printing Run Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-3 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-3 From the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . . 9-3 From the GeneScan Analysis Software. . . . . . . . . . . . . . . . . . . . . . . . 9-3 Printing the Gel Image. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-4 About Printing the Gel Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-4 Printing Selected Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5 Setting Printing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5 From the Results Control Window . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5 From the File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-6 Printing Supporting Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-6 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-6 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-6 A Using GeneScan with the ABI 373 . . . . . . . . . . . . . A-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Processing Using the ABI 373. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ABI 373 Versus ABI PRISM 377 Data. . . . . . . . . . . . . . . . . . . . . . . . Using the GeneScan Analysis Software Before Version 2.0. . . . . . . Using the GeneScan Analysis Software Version 2.0 or Later. . . . . . Processing Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison of GeneScan Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . Setting Gel Processing Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1 A-1 A-2 A-2 A-2 A-2 A-2 A-3 A-3 A-5 xv Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5 Opening the Collection File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7 Completing the Sample Sheet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-9 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-9 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-9 Tracking Lanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-11 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-11 Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-11 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-11 Generating Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-12 When to Generate Sample Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . A-12 B GeneScan Size Standards . . . . . . . . . . . . . . . . . . . . B-1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1 In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1 GeneScan 350 Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2 What To Use It For. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2 How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2 GeneScan 350 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2 Running Under Denaturing Conditions . . . . . . . . . . . . . . . . . . . . . . . B-2 Double-Stranded GeneScan 500 Fragments . . . . . . . . . . . . . . . . . . . B-3 GeneScan-400HD Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4 What To Use It For. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4 Special Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4 How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4 Fragment Lengths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4 Denaturing Electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5 Non-denaturing Electropherogram. . . . . . . . . . . . . . . . . . . . . . . . . . . B-5 GeneScan 500 Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-6 What To It Use For. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-6 How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-6 xvi GeneScan 500 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . . . . . .B-6 Running Under Denaturing Conditions . . . . . . . . . . . . . . . . . . . . . . .B-6 Double-Stranded GeneScan 500 Fragments . . . . . . . . . . . . . . . . . . . .B-7 GeneScan 1000 Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-8 How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-8 GeneScan 1000 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . . . . .B-8 Running Under Native Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . .B-9 GeneScan 2500 Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-10 How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-10 What To Use It For . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-10 Running Under Native Conditions . . . . . . . . . . . . . . . . . . . . . . . . . .B-10 GeneScan 2500 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . . . .B-11 C Size Calling Methods . . . . . . . . . . . . . . . . . . . . . . . . C-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1 In This Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1 Least Square Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-2 About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-2 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-2 Cubic Spline Interpolation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-4 About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-4 Possible Local Sizing Inaccuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-4 Local Southern Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-5 About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-5 The Equation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-5 How This Method Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-6 Global Southern Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-7 About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-7 The Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-7 How This Method Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-8 D Troubleshooting the GeneScan Software . . . . . . . . D-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1 xvii In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1 Troubleshooting Projects and Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2 Table Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2 Troubleshooting Gel Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5 Table Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5 Troubleshooting Genotyping Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-8 Table Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-8 GeneScan Error Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-9 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-9 GeneScan Analysis Software Crashes with BioLIMS . . . . . . . . . . . . D-9 Analysis Log Error Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-10 Error Messages When Defining Size Standards. . . . . . . . . . . . . . . . D-11 E Using the BioLIMS Database . . . . . . . . . . . . . . . . . E-1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1 In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1 About the GeneScan Analysis Software and the BioLIMS Database. . . . . . E-2 What is the BioLIMS Database? . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-2 Accessing the BioLIMS Database . . . . . . . . . . . . . . . . . . . . . . . . . . . E-2 Before Using the BioLIMS Database . . . . . . . . . . . . . . . . . . . . . . . . E-2 Modes in GeneScan Analysis Software . . . . . . . . . . . . . . . . . . . . . . . E-2 Comparing Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3 Configuring the BioLIMS Database Server . . . . . . . . . . . . . . . . . . . . . . . . . E-4 Sybase or Oracle?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-4 Configuring for Sybase SQL Server Connection . . . . . . . . . . . . . . . . E-4 Configuring for Oracle Server Connection . . . . . . . . . . . . . . . . . . . . E-7 Switching Between Sample File and BioLIMS Mode . . . . . . . . . . . . . . . . E-10 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-10 Switching Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-10 How to Access the BioLIMS Database. . . . . . . . . . . . . . . . . . . . . . . . . . . . E-12 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-12 Before Accessing a Sybase Database. . . . . . . . . . . . . . . . . . . . . . . . E-12 Accessing the BioLIMS Database . . . . . . . . . . . . . . . . . . . . . . . . . . E-13 About Server Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 xviii Sybase or Oracle? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 How Names are Recognized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 Sybase SQL Server Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 Oracle Server Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-18 Using the Collection Browser Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-20 Applications that Use the Collection Browser Window . . . . . . . . . . E-20 Ways to Search the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-20 In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-20 Displaying the Collection Browser Window. . . . . . . . . . . . . . . . . . . E-21 Collection Browser Window Example . . . . . . . . . . . . . . . . . . . . . . . E-22 Parts of the Collection Browser Window . . . . . . . . . . . . . . . . . . . . . E-22 Collection Search Criteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-24 Fragment Search Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-25 Searching the BioLIMS Database. . . . . . . . . . . . . . . . . . . . . . . . . . . E-28 F Troubleshooting the BioLIMS Database. . . . . . . . . F-1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1 In This Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1 If the BioLIMSTM Preference Page Does Not Appear . . . . . . . . . . . . . . . . . . F-2 Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-2 Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-2 About Troubleshooting the Client to Sybase Connection . . . . . . . . . . . . . . . F-3 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-3 SybPing and Telnet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-3 Troubleshooting Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-4 Troubleshooting Flow Chart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-4 Procedures for Troubleshooting the Client to Sybase Connection . . . . . . . . F-6 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-6 Troubleshooting from the Macintosh Client. . . . . . . . . . . . . . . . . . . . F-6 Required Sybase Extension Files . . . . . . . . . . . . . . . . . . . . . . . . . . . F-11 Required Oracle Extension Files . . . . . . . . . . . . . . . . . . . . . . . . . . . F-11 Troubleshooting from the Unix (Sybase) Server . . . . . . . . . . . . . . . F-12 About Troubleshooting the Client to Oracle Connection . . . . . . . . . . . . . . F-14 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-14 xix Telnet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting Flow Chart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedures for Troubleshooting the Client to Oracle Connection . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting from the Macintosh Client . . . . . . . . . . . . . . . . . . Troubleshooting from the Unix (Oracle) Server . . . . . . . . . . . . . . . F-14 F-15 F-15 F-17 F-17 F-17 F-22 G License and Warranty . . . . . . . . . . . . . . . . . . . . . . . G-1 Software License and Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1 Perkin-Elmer Software License and Limited Product Warranty . . . . G-1 Copyright . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1 License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1 Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2 Limited Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2 Limitation Of Liability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2 Term . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-3 Miscellaneous. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-3 Glossary Index xx GeneScan Analysis Software Overview 1 Introduction In This Chapter This chapter provides a general introduction to the ABI PRISM® 1 See page 1-2 1-5 1-7 1-11 1-12 1-16 1-20 GeneScan® Analysis Software. It provides information about the organization of this manual and instructions on how to get help from PE Applied Biosystems. Topics in this chapter include the following: Topic What's New in the GeneScan Analysis Software About This Manual and Other Instruments Using the Macintosh Computer Registering the Software Installing the Software GeneScan Analysis Software Files Technical Support Note Already familiar with previous versions of the GeneScan Analysis Software and want to know what is new and different in this version? Turn to "What's New in the GeneScan Analysis Software" on page 1-2. GeneScan Analysis Software Overview 1-1 What's New in the GeneScan Analysis Software BioLIMS Support GeneScan Analysis Software version 3.1 supports the BioLIMSTM Genetic Information Management System v. 2.0. In the BioLIMS mode, the GeneScan Analysis Software extracts sample data from gel files and writes it to the database. The contents of the gel file are not saved to the database.The GeneScan Analysis Software can read and write sequence-fragment to the database just as it writes sample files to the Macintosh® hard disks. Both Oracle® and Sybase® databases are supported. For information about using the GeneScan Analysis Software in BioLIMS mode, see "Using the BioLIMS Database" on page E-1. Fifth Dye The GeneScan Analysis Software version 3.1 can display and analyze raw data from up to five dye colors. This feature has been implemented in the software to support future developments in the dye chemistries. The current maximum number of dyes available is four. The availability of a fifth dye for the GeneScan Analysis Software is dependent on the progress made in the dye development chemistry and can not be accurately projected at this point. You can receive more information on the availability of additional dyes for the GeneScan Analysis Software when they become available through our web site (see "To Reach Us On the Web" on page 1-20). Works with The GeneScan Analysis Software version 3.1 can open and analyze 96-Lane Gels 96-lane gels. For more information, refer to the ABI PRISM 377 DNA Sequencer 96-Lane Upgrade User's Manual (P/N 4305423). 1-2 GeneScan Analysis Software Overview Displaying A new feature has been added in version 3.1 to display offscale regions. Off-Scale Data By clicking the OS button on the tool bar when the gel image is displayed, if the gel contains offscale data, then this data appears as white bands on the gel image.Off-scale data can also be viewed while in Results Display by selecting Show Offscale Regions from the View menu. Note To see off-scale portions in the gel image, click the Vertical Expand button. The resolution of the full display is low and does not show the off-scale data. Manual Tracker In version 3.1, the new tracker lines consist of control points. The new Interface Changes interface allows the selection and movement of multiple control points simultaneously, interpolation of tracker line shapes between two lanes, multiple lane selection, and movement. Printing from In version 2.1, only the electropherogram display is printed when Sample File printing from the Sample File window. Window The following displays can now be printed also: o o o o Sample Info View Size Curve View Raw Data View EPT Data View New Neural Net About the Neural Net Tracker Tracker This was the most significant improvement in the version 3.1 release of GeneScan Analysis Software. o The Neural Net Tracker program uses a neural net-based algorithm to automatically track gel lanes. The Neural Net Tracker has been taught how to recognize bands and how to track curved lanes. IMPORTANT Before auto-tracking the first time, you need to verify that the comb type is set correctly. The default comb type is Square. o The Neural Net Tracker program exists as a separate program within the GeneScan Analysis Software folder. Also associated with the Neural Net Tracker program are a set of Tracker settings files that have been optimized for number of lanes and comb-types. GeneScan Analysis Software Overview 1-3 o The Neural Net Tracker program is "headless." This means that although it stands as a separate program file, it does not have a user interface. The Tracker program is opened automatically from within the GeneScan Analysis Software. IMPORTANT The gel file must be multicomponented using the correct matrix in order to be auto-tracked. Table of Tracking Times Tracking times depend upon the number of lanes, channels, and scans in the gel file. Consult the table below to estimate gel tracking times for your GeneScan Analysis Software. Number of Number of Number of Size of Gel Lanes Channels Scans (MB) 36 36 48 48 64 64 96 194 194 388 388 388 388 480 9152 11424 4516 7768 4565 5708 6840 21.6 26.8 21.5 36.1 21.7 26.8 39.5 Time (min) for CPU/Speed 7200/90a 15 20 26 38 26 30 46 4400/200a 9 11 10 17 10 13 24 9500/200b c c c c c c c G3/266a 5 6 5 8 6 6 13 a. 7200, 4400, G3:32MB + 10MB VM b. 9500: 64MB + 10MB VM c. Could not test this system Lane Extraction In version 3.1, you can extract lanes using weighted averaging or pre-averaging offscale detection. These options are available in the Lane Extraction section of the Gel Preferences dialog box. For more information, see "Lane Extraction" on page 2-7. 1-4 GeneScan Analysis Software Overview About This Manual and Other Instruments ABI 373 GeneScan Analysis Software works version 3.1 with ABITM 373 data in the same way as the it works with ABI PRISM® 377 data. If you use the GeneScan® 672 Software to collect data on the ABI 373, you must transfer your data to a Power Macintosh® computer, and perform certain extra steps. These extra steps are described in Appendix A, "Using GeneScan with the ABI 373." ABI 373 XL The ABITM 373 DNA Sequencer with XL upgrade updates the data Upgrade Users collection interface to match the ABI PRISM 377, increases the maximum image resolution to 388 channels, and increases the maximum number of lanes to 66. Note ABI 373 with XL upgrade users do not need to follow the procedures described in this Appendix A, "Using GeneScan with the ABI 373." 96-lane Upgrade The ABI PRISM® 377 DNA Sequencer 96-lane upgrade kit enhances Kit both the capabilities of the 377 DNA sequencer to support up to 96 lanes for using the GeneScan Analysis Software. Manual Text Manual Text Applies to other ABI PRISM Genetic Analysis Instruments The GeneScan Analysis Software and this manual apply to three different genetic analysis instruments supported by PE Applied Biosystems. Instrument 310 ABI Genetic Analyzer PRISM® Description Analyzes one sample at a time using capillary electrophoresis technology. This instrument provides automatic sample loading while using a minimal amount of sample. Performs slab gel electrophoresis, allowing the user to analyze multiple samples on a gel. A high-throughput slab gel electrophoresis instrument, created to meet the needs of high-volume DNA sequencing or genetic analysis laboratories.Throughput is up to than four times that of the ABI 373. ABI 373 DNA Sequencer ABI PRISM 377 DNA Sequencer GeneScan Analysis Software Overview 1-5 User Attention The text of this manual includes the following user attention words to Words draw your attention to specific details of the information presented in this manual. User Attention word Note IMPORTANT Description Used to call attention to information. Indicates information that is necessary for proper operation of the software. 1-6 GeneScan Analysis Software Overview Using the Macintosh Computer Macintosh To use the GeneScan Analysis Software, you should be familiar with the Computer following basic Macintosh® computer vocabulary and operations: Vocabulary and Description Operations Vocabulary and operations Using the mouse Choosing commands Clicking and double-clicking, selecting and dragging. Using pull-down and pop-up menus, dialog boxes, radio buttons and checkboxes. Opening and closing, resizing and repositioning, scrolling, understanding the active window. Finding files and creating folders. Working with windows Using the Macintosh computer hierarchical file system If you do not understand some or all of these, refer to the Macintosh® System Software User Guide or the Owner's Guide for more information. Guidelines for Computers require regular attention and maintenance to operate Optimal efficiently and consistently. The ABI PRISM® software works with large Performance files and accesses the hard disk often, so it is especially important that you follow maintenance procedures to minimize the occurrence of errors during operation. Follow these general guidelines for optimal performance: o o o o o Install only one Macintosh® computer system per hard disk. Back up all programs and files regularly. Remove nonessential files from the hard disk. Use a hard disk maintenance program regularly. Use discretion when adding software programs, especially Control Panel (CDEV) and Extension (INIT) files. Refer to the ABI PRISM Instrument User Manual for more detail. GeneScan Analysis Software Overview 1-7 Macintosh The following Macintosh® computer terms are used frequently in this Computer Terms manual to describe how to use GeneScan Analysis Software. Used in This Macintosh computer terms used in this manual. Manual Item Dialog Boxes Description Appear when you must make a decision or enter information. All other actions on the monitor screen are suspended until you close the dialog box by clicking a button, such as Cancel, OK, or Done. Menus Provide access to various functions you can perform with the software. A heavy arrow ( ) after a menu item indicates that a submenu appears when you click that choice and hold down the mouse button. When you choose a menu command followed by an ellipsis (...) a dialog box appears. Menu Submenu Pop-up menus Display a heavy arrow ( ) and are found in dialog boxes. When you click a pop-up menu and hold down the mouse button, a submenu appears. These menus allow you to choose dialog box entries from specific lists of items. 1-8 GeneScan Analysis Software Overview Macintosh computer terms used in this manual. (continued) Item Windows Description Display information, and, in some cases, allow you to edit or enter additional information. The top border of an active window always has six horizontal lines and usually has a Close box in the upper-left corner. If many windows are open, click one window to make it active. When a window is active, you can click the top border, hold down the mouse button, and drag the window to another location on the screen. When you are finished working with a window, click the Close box to remove the window from the screen, or click another window. Close box Window GeneScan Analysis Software Overview 1-9 Macintosh computer terms used in this manual. (continued) Item Entry fields Description Rectangular areas in which you can enter information. Click in an entry field to display a cursor, and use the keyboard to enter the information. Checkboxes Boxes that you click to select certain options in a dialog box. When you click an empty checkbox, an "x" appears in it, indicating that you have selected that option. You can usually select multiple checkboxes. Radio buttons Small circles that appear in front of choices. When you click a radio button with the cursor, a black dot appears in the center of the circle to indicate your choice. You can only select one at a time. Buttons Rectangles with rounded corners that allow you to accept or cancel the contents of a dialog box or perform functions (such as printing) within the dialog box. A button with a heavy outline is the default button that applies if you press the Return key. 1-10 GeneScan Analysis Software Overview Registering the Software License and Before you begin, please read Appendix G, "License and Warranty." Warranty This appendix explains your rights and responsibilities regarding the software. Registering Your To register your copy of the GeneScan Analysis Software, complete the Software registration card (included in this software package) and return it to PE Applied Biosystems. Registering the software enables us to send you notification of software updates and any other future information that may be specific to GeneScan Analysis Software owners. IMPORTANT Your product registration number is located on the Registration card. Be sure to record this number here before you return the Registration card. Registration Number: GeneScan Analysis Software Overview 1-11 Installing the Software System The following table lists the system specifications1. Specifications Item CPU Specifications A Power Macintosh® computer (PowerPC CPU). (You will benefit from using the fastest computer available.) CD-ROM Drive Operating System Disk Space Any Mac OS version 8.0 with Open Transport 1.1 or later. A minimum of 15 MB free disk space. Storage requirements depend primarily on the quantity of data to be generated and stored. Sample files are approximately 150--250 KB each and gel files are 20--70 MB each. It is common to store many Sample files on the analysis computer. Gel files are usually stored on the computer that is connected to the instrument and are removed or archived frequently. Memory (RAM) The minimum memory requirement is 32 MB total with at least 20 MB of this available to run the GeneScan Analysis Software. Virtual memory MUST be turned on and set to 16 MB. Set the Memory control panel so that the memory available after restart is at least 48M. For information on how, see "Turning On Virtual Memory" on page 1-13. If your computer has 48 MB of physical RAM, virtual memory does not need to be turned on. Monitor A 17-inch monitor or larger is recommended. Although a monitor of 640 x 480 resolution can be used. you will benefit from having a monitor of higher resolution. disk drive Hard disk with a minimum of 250 MB storage. Virtual Memory 1. Call technical support for the latest system specifications. 1-12 GeneScan Analysis Software Overview Item Printer Specifications The following printers are supported: o o HP DeskJet 1200C/PS HP DeskJet 1600C/PS System software Macintosh computer system software version 8.0. Turning On To turn on Virtual Memory: Virtual Memory Step 1 Action From the Apple menu (ð), choose Control Panels and from the list choose Memory. The memory dialog box appears. 2 Ensure that Virtual Memory is On and that the memory available is at least 48M. GeneScan Analysis Software Overview 1-13 RAM Required to The system requires approximately 32 MB of RAM available to have Run the Software both the Data Collection software and the analysis programs open simultaneously. This does not include the RAM required by the Macintosh computer operating system. The RAM required by each program is as follows: Program Data Collection software GeneScan Analysis Software Approximate RAM required 5.5 MB 32 MB How to Check the RAM Available Choose About this Macintosh® from the Apple (ð) menu in the Finder. Installing the The following procedure describes how to install the GeneScan Software Analysis Software. Before beginning the installation, backup the copies of the older versions of the GeneScan Analysis Software. IMPORTANT After installation, be sure to register your software. To install the GeneScan Analysis Software: Step 3 4 5 6 7 Action Quit all currently running applications. Hold down the Shift key and choose Restart from the Special menu to turn off all extensions. Continue to hold down the shift key until the message "Extension Off" appears. Insert the GeneScan Analysis Software install disk. Double-click the Installer icon. The GeneScan Analysis Software installer appears. 8 9 10 11 Choose a folder in which to install the software. Choose either 310 GeneScan Analysis 3.0 or 373/377/XL GeneScan Analysis 3.0, depending on your instrument. Click Install. Insert other disks as the Installer asks for them. Click Quit at the end of the installation. 1-14 GeneScan Analysis Software Overview Program Files The following diagram shows the program files installed on the hard Installed Diagram disk. ABI PRISM GeneScan® 3.0 Folder Contents ABI PRISM GeneScan 3.0 GS Gel Tracker folder GeneScan GS Parameters folder GS Standards folder GS Matrix folder Contents of The installation program creates an ABI PRISM GeneScan 3.0 folder on Folders the hard disk containing the following. Folder GS Parameters folder GS Standards folder GS GelTracker folder Contents Use to store user-defined analysis parameters files if you create your own files. Use for storing size standard files when you define them. GS Gel Tracker program, tracker settings file, and extensions. You may also see a set of 20 to 30 matlab text files. You can ignore these files; if you throw them away new ones are created when you next track a gel. Note Advise keeping the files because they are useful for troubleshooting auto-tracking problems. Note Do not change the name of the folder, the location of the folder, or the files in the folder. GS Matrix folder Use for storing the matrix file generated using the GeneScan Analysis Software. For more information about matrix files, see Chapter 6, "Making a Matrix File." GeneScan Analysis Software Overview 1-15 GeneScan Analysis Software Files Table of Files The following table lists the files that the GeneScan Analysis Software reads, writes, and, in most cases, creates. The software does not create gel files, and creates Sample files only through lane extraction from a gel. For information on the files installed by the GeneScan Analysis Software, see "Program Files Installed Diagram" on page 1-15. GeneScan Analysis Software files Location on File type 373XL, ABI PRISM 377, 377XL, 377 96-Lanes, or ABI PRISM 310 GeneScan 3.0 folder Description ABI 373 GeneScan 3.0 folder The GeneScan Analysis Software analyzes data sent from the ABI PRISM instrument after a run. A large file created by the Data Collection software. The gel file contains the raw data collected during the instrument run. It also contains the Sample Sheet and run information. For a typical run, this file can be very large. More than 20 MB for 194 channels, more than 40 MB for 388 channels, and more than 50 MB for 96 lanes (up to 70 MB for long runs). GeneScan Analysis Software The gel file (373 or 377 only) Run folder created by ABI PRISM 377 Data-Collection during run Run folder created by ABI 373 Data-Collection during run 1-16 GeneScan Analysis Software Overview GeneScan Analysis Software files (continued) Location on File type 373XL, ABI PRISM 377, 377XL, 377 96-Lanes, or ABI PRISM 310 Run folder created by the Data Collection software during run Description ABI 373 Sample file folder created by GeneScan during run Organizes, displays, and analyzes Sample files and results. Projects contain references to Sample files. Project files can contain references to Sample files from more than one run. Sample Files Run folder created by data-collection software during run Sample file folder created by GeneScan during run Created by ABI PRISM 310. For ABI 373 and ABI PRISM 377, GeneScan extracts sample information from the gel file to create Sample files. Each file contains data for a single lane or injection. Size Standard Files GS Standards folder or other specified folder GS Standards folder or other specified folder Identify peak sizes for specified size standards run under certain conditions. Define the standards after running them on the instrument. Tracker files and extensions GS GelTracker folder in the GS 3.0 folder (ABI PRISM 377 and ABI 373 XL) GS Tracker folder in the GS 3.0 folder The Project File GeneScan Analysis Software Overview 1-17 GeneScan Analysis Software files (continued) Location on File type 373XL, ABI PRISM 377, 377XL, 377 96-Lanes, or ABI PRISM 310 GS Parameters folder or other specified folder Description ABI 373 GS Parameters folder or other specified folder Specify certain ranges and methods used during GeneScan analysis. You can also create and save custom parameters files for future use. Matrix files ABI folder, GS Matrix folder or other specified folder ABI folder, GS Matrix folder or other specified folder Contains mathematical matrices that correct for spectral overlap. They are applied to data based on the chemistry run. Analysis Log GeneScan folder GeneScan folder Contains a running record of analysis performed by the GeneScan Analysis Software. Sample information in spreadsheet form. Used for attaching to gel file. Analysis Parameters Sample Sheet GeneScan or specified folder GeneScan or specified folder 1-18 GeneScan Analysis Software Overview Sample File GeneScan Analysis Software Sample files can be written to and read Database Support from the BioLIMS database. The BioLIMS Genetic Information Management System (BioLIMS database) provides a relational database for storage and retrieval of DNA fragment data. If you have obtained the GeneScan Analysis Software as part of the BioLIMS Client package, you can use the program in either of two modes: Mode Sample File mode Description Fragment data extracted from gel files is written to individual sample files. Extracted fragment data viewed and processed within the GeneScan Analysis Software is read from and saved to sample files. BioLIMS mode Fragment data extracted from gel files is written directly to a BioLIMS database that resides on a UNIX Server. Extracted fragment data viewed and processed within the GeneScan Analysis Software is read from and written back to the same BioLIMS database. For more information on using the BioLIMS database, see Appendix E, "Using the BioLIMS Database." GeneScan Analysis Software Overview 1-19 Technical Support To Reach Us On Our web site address is http://www.perkin-elmer.com/ab the Web We strongly encourage you to visit our web site for answers to frequently asked questions, and to learn more about our products. You can also order technical documents and/or an index of available documents and have them faxed to you through our site (see the "Fax-on-Demand" section below). Hours for In the United States and Canada, technical support is available Telephone between 5:30 a.m. and 5:00 p.m. Pacific Time. Technical Support These hours are for all products except the following: Product Chemiluminescence LC/MS Hours 9:00 a.m. to 5:00 p.m. Eastern Time 9:00 a.m. to 5:00 p.m. Pacific Time See the "Regional Offices Sales and Service" section below for how to contact local service representatives outside of the United States and Canada. To Reach Us by Call Technical Support at 1-800-831-6844, and select the appropriate Telephone or Fax option (below) for support on the product of your choice at any time in North America during the call. (To open a service call for other support needs, or in case of an emergency, press 1 after dialing 1-800-831-6844.) For Support On This Product DNA Synthesis Press 21 Fluorescent DNA Sequencing Press 22 Fluorescent Fragment Analysis (includes GeneScan applications) Press 23 FAX 650-638-5981 FAX 650-638-5891 FAX 650-638-5891 Dial 1-800-831-6844, and... 1-20 GeneScan Analysis Software Overview For Support On This Product BioLIMS Dial 1-800-831-6844, and... Press 25 FAX 650-638-5891 FAX 650-638-5891 FAX 203-761-2542 Integrated Thermal Cyclers Press 24 PCR and Sequence Detection Press 5, or call 1-800-762-4001, and press 1 for PCR, or 2 for Sequence Detection Peptide Synthesis Press 31 Protein Sequencing Press 32 Chemiluminescence Telephone 1-800-542-2369 (U.S. only), or 1-617-271-0045 (Tropix) LC/MS Telephone 1-800-952-4716 FAX 617-275-8581 (Tropix) 9:00 a.m. to 5:00 p.m. ET FAX 650-638-6223 9:00 a.m. to 5:00 p.m. PT FAX 650-638-5981 FAX 650-638-5981 GeneScan Analysis Software Overview 1-21 Fax-on-Demand Free 24-hour access to PE Applied Biosystems technical documents is available by fax. You can access Fax-on-Demand documents through the internet or by telephone: If you want to order... through the internet Then... Use http://www.perkin-elmer.com/ab/techsupp You can search for documents to order using keywords. Up to five documents can be faxed to you if you already know the titles. by phone from the United States or Canada a. b. Call 1-800-487-6809 from a touch-tone phone. Have your fax number ready. Press 1 to order an index of available documents and have it faxed to you. Each document in the index has an ID number. (Use this as your order number in step "d" below.) Call 1-800-487-6809 from a touch-tone phone a second time. Press 2 to order up to five documents and have them faxed to you. Dial your international access code, then 1-650-596-4419, from a touch-tone phone. Have your complete fax number and country code ready (011 precedes the country code). c. d. by phone from outside the United States or Canada a. b. Press 1 to order an index of available documents and have it faxed to you. Each document in the index has an ID number. (Use this as your order number in step "d" below.) Call 1-650-596-4419 from a touch-tone phone a second time. Press 2 to order up to five documents and have them faxed to you. c. d. 1-22 GeneScan Analysis Software Overview To Reach Us by Contact technical support by e-mail for help in the following product E-Mail areas. For this product area Chemiluminescence Genetic Analysis LC/MS PCR and Sequence Detection Protein Sequencing, Peptide and DNA Synthesis Use this e-mail address info@tropix.com galab@perkin-elmer.com apisupport@sciex.com pcrlab@perkin-elmer.com corelab@perkin-elmer.com Regional Offices If you are outside the United States and Canada, you should contact Sales and Service your local PE Applied Biosystems service representative. The Americas United States PE Applied Biosystems 850 Lincoln Centre Drive Foster City, California 94404 Tel: Fax: (650) 570-6667 (800) 345-5224 (650) 572-2743 Canada (Mississauga, Ontario) Tel: Fax: (905) 821-8183 (800) 668-6913 (905) 821-8246 Latin America (Del.A. 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(Praha) Tel: Fax: 2 61 22 21 64 2 61 22 21 68 Norway (Oslo) Tel: Fax: (0) 22 02 1500 (0) 22 02 1501 GeneScan Analysis Software Overview 1-23 Europe (continued) Denmark (Allerød) Tel: Fax: 48 100 400 48 100 401 Poland (Warszawa) Tel: Fax: (48 22) 866 40 10 (48 22) 866 40 20 Finland (Espoo) Tel: Fax: 09 751 72 700 09 751 72 701 Russia (Moskva) Tel: Fax: 095 935 8888 095 564 8787 France (Paris) Tel: Fax: (1) 69 59 85 85 (1) 69 59 85 00 South Africa (Johannesburg) Tel: Fax: 27 11 478 0411 27 11 478 0349 Germany (Weiterstadt) Tel: Fax: (0) 6150/ 101 0 (0) 6150/ 101 101 Spain (Madrid) Tel: Fax: (1) 806 1200 (1) 804 0414 Sweden (Sundbyberg) Tel: Fax: (0)8 619 4400 (0)8 619 4401 United Kingdom (Warrington, Cheshire) Tel: Fax: (01925) 825650 (01925) 282502 Switzerland (Rotkreuz) Tel: Fax: (0) 41 799 7708 (0) 41 790 0676 All Other European Countries, Middle East/West Asia, Africa (Langen, Germany) Tel: Fax: 49 6103 708 301 49 6103 708 310 Japan Japan (Chiba) Tel: Fax: (0473) 80 8500 (0473) 80 8505 Eastern Asia, China, Oceania Australia (Scoresby, Victoria) Tel: Fax: (03) 9212 8585 (03) 9212 8502 Malaysia (Kuala Lumpur) Tel: Fax: 60 3 758 1118 60 3 754 9043 China (Beijing) Tel: Fax: 86 10 6238 1156 86 10 6238 1162 Singapore Tel: Fax: 65 336 0322 65 338 3991 1-24 GeneScan Analysis Software Overview Eastern Asia, China, Oceania Hong Kong Tel: Fax: 852 2756 6928 852 2756 6968 Taiwan (Taipei Hsisn) Tel: Fax: 886 2 698 3505 886 2 698 3405 Korea (Seoul) Tel: Fax: 822 592 7238 822 532 4908 Thailand (Bangkok) Tel: Fax: 662 719 6406 662 319 9788 GeneScan Analysis Software Overview 1-25 How to Process and Edit the Gel File 2 Introduction 2 See page 2-2 2-3 2-11 2-12 2-18 2-26 2-29 2-31 2-34 2-38 2-43 2-50 2-53 2-57 2-59 In This Chapter This chapter contains information about how to view and edit the gel file, and how to generate Sample files or BioLIMSTM database records after editing the gel file. Note This section does not apply if you are analyzing Sample files generated on the ABI PRISM® 310 instrument. IMPORTANT Refer to Appendix A, "Using GeneScan with the ABI 373," before using the GeneScan program to analyze data collected on the ABI 373. Topics in This Topics in this chapter include the following: Chapter Topic About Gel Files How to Set Gel Processing Preferences How to Display the Gel File About the Gel File Window How to Adjust the Gel Image Verifying Gel Information Displaying the Sample Sheet About the Sample Sheet Working with the Sample Sheet About Displaying Regions of Off-Scale Data Working with Lane Markers Marking and Unmarking Lanes for Extraction Working with Tracker Lines Interpolating Tracker Lines Tracking Lanes and Extracting Data How to Process and Edit the Gel File 2-1 Topic How the GeneScan Analysis Software Names Sample Files Saving Gel Files After Editing Tracking See page 2-67 2-68 About Gel Files Instruments The gel file, which is generated only by the ABITM 373 DNA Sequencer Producing Gel File instrument, the ABI 373 with XL upgrade, the ABI PRISM® 377 DNA Sequencer instrument, and the ABI PRISM® 377 with XL upgrade, stores the raw data collected during the entire run of the instrument. A gel file can be 10 MB to 60 MB. 96-Lane Gels The GeneScan® Analysis Software can open and analyze 96-lane gels. Capability Gel File Contents Initially, the file contains the following: o o o o Raw data collected during the run. Gel image, which is similar to an autoradiogram, but in color. Copy of the Data Collection software sample sheet. Copy of the matrix file. After lane tracking and editing, the file also contains the lane tracking information and any changes you make to the original information in the file. How GeneScan When the GeneScan Analysis Software tracks the gel file, it creates a Tracks a Gel File tracker line for each lane in the gel. Each tracker line is trying to find the center of the bands, not the brightest part of the band. The tracker will only track if a matrix is attached to the gel file. During data extraction, the software generates a Sample file for each tracked lane by averaging the data from the tracked channel and the number of channels you specify on either side of it. The software also copies to each new Sample file all the information required to identify and analyze the sample. 2-2 How to Process and Edit the Gel File