ABI PRISM® GeneScan Analysis

Software
Version 3.7 for the Windows NT® Platform

User Guide
ABI PRISM ®

GeneScan Analysis
®

Software

Version 3.7 for the Windows

NT Platform
®
© Copyright 2001, Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Notice to Purchaser: Limited License
For Software License and Warranty Information, please see Appendix G, “License and Warranty,” of this manual.
Notice to Purchaser: License Disclaimer
Purchase of this software product alone does not imply any license under any process, instrument or other apparatus, system,
composition, reagent or kit rights under patent claims owned or otherwise controlled by Applied Biosystems, either expressly, implied,
or by estoppel.
ABI PRISM and its design, Applied Biosystems, BioLIMS, GeneScan, and Genotyper are registered trademarks and ABI, POP-5 and
POP-6 are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
Windows NT and Microsoft are registered trademarks of the Microsoft Corporation.
Oracle is a registered trademark and Oracle7 is a trademark of Oracle Corporation.
Sybase is a registered trademark and Sybase SQL Server and SyBooks are trademarks of Sybase, Inc.
Apple, Macintosh, and Power Macintosh are registered trademarks of Apple Computer, Inc.
All other trademarks are the sole property of their respective owners.
Applera Corporation is committed to providing the world’s leading technology and information for life scientists. Applera Corporation
consists of the Applied Biosystems and Celera Genomics businesses.
Contents
1 GeneScan Analysis Software Overview
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–1
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–1
About the GeneScan Analysis Software . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–2
What Does the Software Do . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–2
Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–2
GeneScan Software on the 310 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . .1–3
Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–3
GeneScan Analysis on the 377 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . .1–4
Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–4
GeneScan Analysis on the 3100 Instrument . . . . . . . . . . . . . . . . . . . . . . . . .1–5
Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–5
GeneScan Analysis on the 3700 Instrument . . . . . . . . . . . . . . . . . . . . . . . . .1–6
Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–6
Sequence Collector Database Option . . . . . . . . . . . . . . . . . . . . . . . . .1–7
Related Manuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–7
Registering the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–8
License and Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–8
Registering Your Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–8
Hardware and Software Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–9
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–9
Computers Connected to Applied Biosystems Instruments . . . . . . . .1–9
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–10
Minimum System Recommendations . . . . . . . . . . . . . . . . . . . . . . . .1–10
Hard Drive Partitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–11

iii
 Installing the GeneScan Analysis Software . . . . . . . . . . . . . . . . . . . . . . . . 1–12
Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1–12
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1–12
Starting the GeneScan Analysis Software for the First Time . . . . . . . . . . . 1–13
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1–13
Removing the Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1–15
If Files or Folders Have Been Moved . . . . . . . . . . . . . . . . . . . . . . . 1–15
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1–15

2 Creating a Project
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–1
In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–1
Creating Projects and Auto-Analysis on 310 Instruments . . . . . . . . . . . . . . 2–2
Process Using the 310 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–2
Creating Projects and Auto-Analysis on 377 Instruments . . . . . . . . . . . . . . 2–3
Process Using the 377 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–3
Setting Up for Automatic Analysis on 3100 and 3700 Instruments . . . . . . . 2–4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–4
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–4
What Is a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–7
Why Create a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–7
Where to Store Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–7
Working with Project Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–8
Opening an Existing Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–8
Creating a New Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–9
Unlocking Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–12
Removing Samples from a Project. . . . . . . . . . . . . . . . . . . . . . . . . . 2–13
Finding Missing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–14
When Are Files Considered Lost . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–14
When an Alert Appears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–14
Searching for Missing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . 2–14
Re-Establishing Links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–15
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–16

iv
3 Analyzing Project Files
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–1
Analyzing Project Files: About the Analysis Control Window . . . . . . . . . . .3–2
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–2
Analysis Control Window Example . . . . . . . . . . . . . . . . . . . . . . . . . .3–2
Analysis Control Window Callouts Described . . . . . . . . . . . . . . . . . .3–3
Customizing the Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–4
Using the Analysis Control Window. . . . . . . . . . . . . . . . . . . . . . . . . .3–5
Analyzing Sample Files: Using the Analysis Control Window . . . . . . . . . . .3–6
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–6
Accessing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–6
Analyzing Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–7
Specifying the Format for Printed Results . . . . . . . . . . . . . . . . . . . . .3–8
Displaying Size Standards and Analysis Parameters . . . . . . . . . . . .3–10
Displaying Sample and Dye Information . . . . . . . . . . . . . . . . . . . . .3–11
Setting Sample File Sort Order . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–13
Setting Dye Indicator Preferences . . . . . . . . . . . . . . . . . . . . . . . . . .3–14
Defining Folder Locations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–16
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–16
Storing Matrix Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3–16

4 Analyzing Sample Files

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–1
About Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–2
Sample File Generation on 377. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–2
What Files Contain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–2
Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–2
How GeneScan Analyzes Sample Files . . . . . . . . . . . . . . . . . . . . . . .4–3
Converting Macintosh Computer Sample Files. . . . . . . . . . . . . . . . . . . . . . .4–3
About Converting Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–3

v
 Converting Sample Files for Use on a Different Platform . . . . . . . . . . . . . . 4–4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–4
Converting Macintosh Files to Windows NT-Based Computer Files 4–4
Converting Windows NT-Based Computer Files to Macintosh Files 4–5
Opening Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–7
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–7
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–7
About the Sample File Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–8
What It Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–8
Five Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–8
Sample Results View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–9
What It Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–9
Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–9
Example of Sample Results View . . . . . . . . . . . . . . . . . . . . . . . . . . 4–10
Description of Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–10
Differences from the Results Display . . . . . . . . . . . . . . . . . . . . . . . 4–11
Sample Info View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–11
What It Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–11
Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–12
Example of Sample Info View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–13
Description of Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–14
Size Curve View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–20
What It Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–20
Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–20
Example of Size Curve View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–21
Curves Described . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–21
Raw Data View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–22
What It Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–22
Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–22
Example of Raw Data View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–23
What to Evaluate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–23
EPT Data View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–24
What It Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–24
Displaying the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–24

vi
 EPT Data View Example. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–25
Colored Lines Described. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–25
Analyzing a Sample File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–26
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–26
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–26
Installing a New Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4–27

5 Working with Analysis Parameters

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–1
About the Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–2
What They Are . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–2
Why They Are Necessary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–2
When to Specify a Parameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–2
Analysis Parameter Files Provided . . . . . . . . . . . . . . . . . . . . . . . . . . .5–3
When Analysis Parameters Are Used . . . . . . . . . . . . . . . . . . . . . . . . .5–3
Sizecaller Algorithm Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–4
Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–4
Setting Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–5
Default Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–5
Displaying Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–6
Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–7
Analysis Range Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–7
Data Processing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–7
Peak Detection Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–8
Sizecall Range Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–10
Sizecalling Method Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–10
Baselining Option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–12
Auto-Analysis Only Option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–12
Using Analysis Parameter Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–13
In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–13
Assigning the Same Analysis Parameters to All Files . . . . . . . . . . .5–13
Assigning Different Parameters to Single Samples . . . . . . . . . . . . .5–15
Displaying Default Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5–16

vii
 Creating Custom Analysis Parameter Files . . . . . . . . . . . . . . . . . . . 5–16
Changing an Existing Analysis Parameters File . . . . . . . . . . . . . . . 5–17
Deleting Custom Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . 5–17

6 Making a Matrix File

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
Installing a Matrix File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–4
When to Assign a Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–4
Limitations to Matrix Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–4
When to Create a New 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 Create a New Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–7
Loading and Running Dye Standards for the ABI PRISM 310 . . . . . . . . . . . 6–9
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–9
Creating a GeneScan Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . 6–9
Creating a GeneScan Injection List . . . . . . . . . . . . . . . . . . . . . . . . . 6–10
Starting the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . 6–11
If the Run was Cancelled . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–11
Run Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–11
Assigning Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–11
Loading and Running Dye Standards for the ABI PRISM 377 . . . . . . . . . . 6–12
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–12
Creating a GeneScan Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . 6–12

viii
 Loading Matrix Standards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–13
Running the Matrix Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–14
Generating Matrix Sample Files for the ABI PRISM 377 Instrument . . . . .6–15
Who Should Use This Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–15
Gel Handling on Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–15
Verify Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–15
Generating Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–15
Choosing a Scan Range for the Matrix Calculation . . . . . . . . . . . . . . . . . .6–17
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–17
Viewing the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–17
What to Look For in the Raw Data Display . . . . . . . . . . . . . . . . . . .6–18
Choosing a Scan Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–18
Eliminating Primer Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–19
Generating a New Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–20
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–20
Matrix File Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–21
Saving and Naming the Matrix File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–22
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–22
Naming Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–22
Saving the Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–22
Where to Store the Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–22
When Data Collection and Analysis Are Performed on Different
Computers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–22
Assigning the Matrix File to Sample Files . . . . . . . . . . . . . . . . . . . . . . . . .6–23
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–23
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–23
Evaluating the Matrix File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–25
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–25
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–25
Causes for Bad Matrix Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–26
If an Error Message Appears. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6–26
Two Causes of Bad Matrix Files. . . . . . . . . . . . . . . . . . . . . . . . . . . .6–26

ix
 7 Working with Size Standards
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–1
In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–1
About Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–2
What Are Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–2
Advantages of Using Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . 7–2
Size Standards Provided. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–2
When to Define Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–2
If Split Peaks Appear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–2
Defining the Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–3
Two Ways to Define the Size Standard . . . . . . . . . . . . . . . . . . . . . . . 7–3
Using the New Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–3
Using the Analysis Control Window . . . . . . . . . . . . . . . . . . . . . . . . . 7–7
Using Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–9
In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–9
Verifying Size Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7–9
Changing the Number of Peaks Detected . . . . . . . . . . . . . . . . . . . . . 7–9
Editing the Size Standard Definition . . . . . . . . . . . . . . . . . . . . . . . . . 7–9
Editing an Existing Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . 7–10
Deleting an Existing Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . 7–12
Analyzing Samples Using the Same Size Standard . . . . . . . . . . . . . 7–13
Selecting Separate Size Standards for Samples . . . . . . . . . . . . . . . . 7–14

8 Evaluating Analysis Results

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–1
In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–1
Process of Evaluating Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–2
Evaluating Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–2
Ways to Display Analysis Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–3
Two Ways to Display Analysis Results . . . . . . . . . . . . . . . . . . . . . . . 8–3
About the Results Display Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–4
Displaying the Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8–4

x
 Results Control Window Callouts Described . . . . . . . . . . . . . . . . . . .8–5
Using the Results Control Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–7
Selecting Display Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–7
Selecting Electropherogram Panels . . . . . . . . . . . . . . . . . . . . . . . . . .8–7
Selecting Samples to Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–8
Creating Tiled Electropherogram Displays. . . . . . . . . . . . . . . . . . . .8–10
Unselecting Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–11
Removing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–12
Displaying the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–12
Printing the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–12
Changing How the Results Are Displayed and Printed . . . . . . . . . . . . . . . .8–13
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–13
About the Sample Results View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–15
About the View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–15
Updating the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–16
Re-analyzing the Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–16
Saving and Renaming the Results Control Format . . . . . . . . . . . . . . . . . . .8–17
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–17
Important Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–17
Saving the Display Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8–17
Working with a Previously Saved Display . . . . . . . . . . . . . . . . . . . .8–18
Renaming the Current Results Display. . . . . . . . . . . . . . . . . . . . . . .8–19

9 Evaluating Electropherograms
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–1
About Electropherogram and Tabular Data Displays . . . . . . . . . . . . . . . . . .9–2
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–2
How the Window Is Divided. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–2
What Tabular Data Contains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–2
How Electropherogram Panels Are Sized . . . . . . . . . . . . . . . . . . . . . .9–2
For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–3
Displaying Electropherogram and Tabular Data . . . . . . . . . . . . . . . . . . . . . .9–4
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–4

xi
 Example of Tabular Data and Electropherogram . . . . . . . . . . . . . . . . 9–5
Table Describing Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–5
Why Some Peaks May Be Visible Only in an Electropherogram . . . 9–6
Why Some Peak Areas May Have a Negative Number . . . . . . . . . . . 9–6
Highlighting Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–6
Changing the Highlight Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–7
Adjusting Window Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–7
Hiding Selected Rows of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–7
Limiting the Rows to Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–8
Displaying Electropherogram Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–9
Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–9
Base Pairs Versus Data Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–9
Procedure to Display Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–9
Electropherogram Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–10
Electropherogram Callouts Described . . . . . . . . . . . . . . . . . . . . . . . 9–10
Working with Electropherogram Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–12
In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–12
Displaying X- and Y-Axis Positions. . . . . . . . . . . . . . . . . . . . . . . . . 9–13
Moving the Electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–13
Changing the Dye Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–14
Displaying Peak Positions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–14
Highlighting Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–15
Using Legends to Change the Display . . . . . . . . . . . . . . . . . . . . . . . 9–15
Scrolling the Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–16
Zooming In and Out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–17
Showing Off-Scale Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–18
Electropherogram Displaying Off-Scale Data . . . . . . . . . . . . . . . . . 9–19
Electropherogram Displaying the Flat-Topped Effect . . . . . . . . . . . 9–19
Showing Data by Fragment Size . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–20
Changing the Horizontal Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–21
Changing the Vertical Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–22
Assigning Standard or Custom Colors. . . . . . . . . . . . . . . . . . . . . . . 9–23
Defining Custom Colors in Electropherograms . . . . . . . . . . . . . . . . . . . . . 9–24
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–24
Why Change Colors in the Electropherogram . . . . . . . . . . . . . . . . . 9–24

xii
 Saving the Display Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–24
Defining Custom Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–25
Defining Individual Plot Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–26
Changing the Dye Scale in Electropherograms . . . . . . . . . . . . . . . . . . . . . .9–28
What the Dye Scale Defines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–28
Increasing the Dye Scale Example . . . . . . . . . . . . . . . . . . . . . . . . .9–28
Changing the Dye Scale of an Electropherogram. . . . . . . . . . . . . . .9–29
Changing the Dye Scale Preferences . . . . . . . . . . . . . . . . . . . . . . . .9–30
Process of Verifying Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–31
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–31
Steps to Verify Size Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–31
Verifying Size Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–33
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–33
Verifying for the GeneScan-350 Standard . . . . . . . . . . . . . . . . . . . .9–33
Evaluating for Multiple Size Standards . . . . . . . . . . . . . . . . . . . . . .9–35
Using the Analysis Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–36
What Is the Analysis Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–36
Displaying the Analysis Log. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–36
What to Evaluate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–37
Removing Information from the Analysis Log . . . . . . . . . . . . . . . . .9–37
Closing the Analysis Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–37
Verifying Peak Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–38
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–38
Verifying Peak Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9–38

10 Saving, Archiving, and Copying Files

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10–1
In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10–1
Why Save GeneScan Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10–2
Reasons for Saving Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10–2
Saving GeneScan Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10–3
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10–3
Saving Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10–3

xiii
 Saving Sample Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–3
Saving Results Displays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–3
Archiving Sample Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–4
When to Archive Sample Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–4
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–4
Transferring Data to Other Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . 10–5
Genotyper Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–5
Cutting and Pasting Tabular Data. . . . . . . . . . . . . . . . . . . . . . . . . . . 10–5
Creating a Text File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–5

11 Printing Results
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–1
In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–1
About Printing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–1
Ways You Can Print . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–1
If You Get Unexpected Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–1
Printing Run Results Automatically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–2
From the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . 11–2
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–2
Printing Selected Sample Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–3
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–3
Setting Printing Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–3
Printing from the Results Control Window . . . . . . . . . . . . . . . . . . . 11–3
Printing from the File Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11–4

A Creating GeneScan Analysis Modules

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A–1
In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A–1
About GeneScan Analysis Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A–2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A–2
Analysis Module Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A–2
Files Referenced by the Module . . . . . . . . . . . . . . . . . . . . . . . . . . . .A–2

xiv
 Default Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A–2
Relationship Between a Module and the Files . . . . . . . . . . . . . . . . . A–4
Never Modify the Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A–4
Creating GeneScan Analysis Modules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A–5
Summary of the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A–5
Creating a Size Standard File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A–6
Creating an Analysis Parameter File. . . . . . . . . . . . . . . . . . . . . . . . A–10

B Sizecalling Methods
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–1
In This Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–1
Least Squares Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–2
About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–2
Least Squares Sizecalling Examples. . . . . . . . . . . . . . . . . . . . . . . . . B–2
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–3
Cubic Spline Interpolation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–4
About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–4
Possible Local Sizing Inaccuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . B–4
Local Southern Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–5
About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–5
The Equation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–5
How This Method Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–6
Global Southern Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–7
About This Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–7
The Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–7
How This Method Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B–8

C GeneScan Size Standards

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–1
About the Size Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–1
Size Standards Included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–1
GeneScan 120 Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–2
About This Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–2

xv
 Special Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–2
How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–2
Fragment Lengths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–2
Denaturing Electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–2
Electropherogram of GeneScan 120 LIZ . . . . . . . . . . . . . . . . . . . . . .C–3
GeneScan 350 All Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–4
About This Size Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–4
How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–4
GeneScan 350 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . . . . . .C–4
Running Under Denaturing Conditions . . . . . . . . . . . . . . . . . . . . . . .C–5
Electropherogram of GeneScan 350 . . . . . . . . . . . . . . . . . . . . . . . . .C–6
Double-Stranded GeneScan 500 Fragments . . . . . . . . . . . . . . . . . . .C–6
GeneScan 350 377 Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–7
About This Size Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–7
GeneScan 350 377 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . .C–7
Electropherogram of GeneScan 350 377 . . . . . . . . . . . . . . . . . . . . . .C–7
GeneScan 350-250 Size Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–8
About This Size Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–8
GeneScan 350-250 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . .C–8
Electropherogram of GeneScan 350-250 . . . . . . . . . . . . . . . . . . . . . .C–8
GeneScan-400HD Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–9
About This Size Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–9
Special Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–9
How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–9
Fragment Lengths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–9
Denaturing Electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–9
Electropherogram of GeneScan-400HD . . . . . . . . . . . . . . . . . . . . .C–10
GeneScan 500 All Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–11
About This Size Standard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–11
How It Is Prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C–11
GeneScan 500 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . . . . .C–11
Running Under Denaturing Conditions . . . . . . . . . . . . . . . . . . . . . .C–11
Electropherogram of GeneScan 500 . . . . . . . . . . . . . . . . . . . . . . . .C–12
Double-Stranded GeneScan 500 Fragments . . . . . . . . . . . . . . . . . .C–12

xvi
 GeneScan 500 377 Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–13
About This Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–13
GeneScan 500 377 Molecular Lengths . . . . . . . . . . . . . . . . . . . . . . C–13
Electropherogram of GeneScan 500 377 . . . . . . . . . . . . . . . . . . . . C–13
GeneScan 500-250 Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–14
About This Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C–14
GeneScan 500-250 Molecular Lengths. . . . . . . . . . . . . . . . . . . . . . C–14
Electropherogram of GeneScan 500-250 . . . . . . . . . . . . . . . . . . . . C–15

D Troubleshooting the GeneScan Software

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–1
In This Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–1
Troubleshooting Projects and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–2
Table Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–2
Troubleshooting Gel Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–4
Table Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–4
Troubleshooting Genotyping Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–7
Table Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–7
GeneScan Analysis Software Error Messages . . . . . . . . . . . . . . . . . . . . . . . D–8
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–8
Analysis Log Error Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D–8
Error Messages When Defining Size Standards . . . . . . . . . . . . . . . . D–9

E GeneScan Analysis Software Files

Table of Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1

F Technical Support
Contacting Technical Support. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F–1
To Contact Technical Support by E-mail . . . . . . . . . . . . . . . . . . . . . F–1
Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . F–2
To Contact Technical Support by Telephone or Fax . . . . . . . . . . . . . F–2

xvii
 To Reach Technical Support Through the Internet . . . . . . . . . . . . . . F–6
To Obtain Documents on Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . F–6

G License and Warranty

Applera Corporation Software License and Limited Product Warranty . . . . G-1
Warranty Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1
Copyright . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1
License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1
Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2
Limited Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2
Limitation of Liability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2
Term . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2
Miscellaneous. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-3

Glossary

Index

xviii
GeneScan Analysis
Software Overview 1
Overview
1
Introduction This chapter provides a general introduction to the ABI PRISM ®
GeneScan ® Analysis Software, information about the organization of
this manual, and instructions on how to get help from Applied
Biosystems.

In This Chapter Topics in this chapter include the following:

Topic See Page

About the GeneScan Analysis Software 1-2
GeneScan Software on the 310 Instrument 1-3
GeneScan Analysis on the 377 Instrument 1-4
GeneScan Analysis on the 3100 Instrument 1-5
GeneScan Analysis on the 3700 Instrument 1-6
Registering the Software 1-8
Hardware and Software Requirements 1-9
Installing the GeneScan Analysis Software 1-12
Starting the GeneScan Analysis Software for the First Time 1-13
Removing the Software 1-15

GeneScan Analysis Software Overview 1-1

About the GeneScan Analysis Software

What Does the The GeneScan Analysis Software performs DNA fragment analysis,
Software Do which separates a mixture of DNA fragments according to their lengths,
provides a profile of the separation, and estimates the lengths and sizes
of the fragments.

Instruments GeneScan Analysis Software Version 3.7 works on the following

instruments:
♦ ABI PRISM ¤ 310 Genetic Analyzer
♦ ABI PRISM ¤ 377 DNA Sequencer
♦ ABI PRISM ¤ 3100 Genetic Analyzer
♦ ABI PRISM ¤ 3700 DNA Analyzer

1-2 GeneScan Analysis Software Overview

GeneScan Software on the 310 Instrument

Flowchart User starts instrument run with

automatic analysis

Data Collection software

automatically captures raw data
and creates sample files

Data Collection automatically

launches GeneScan Analysis
for sizecalling of sample files

If set in GeneScan Analysis, the

software automatically submits
files for printing

User reviews GeneScan

Analysis software Error Logs

User makes changes required

by problems in Error Log
No
OK?

Yes
User adjusts parameters and
User reviews sizecalling results starts analysis

No
OK?
User adjusts parameters and
Yes reanalyzes samples files
User may open analyzed
sample files to edit fragment
sizecalls

DNA fragment ready for further

processing

GeneScan Analysis Software Overview 1-3

GeneScan Analysis on the 377 Instrument

Flowchart User starts instrument run with

automatic analysis

Data Collection software

automatically captures raw data into
gel files

Data Collection automatically

launches Gel Processor, which
tracks lanes and extracts data into
sample files

Gel Processor launches GeneScan

Analysis, which performs sizecalling

If set in GeneScan Analysis, the

software automatically submits files
for printing

User reviews GeneScan Analysis

software Error Logs

User makes changes required

No by problems in Error Log
OK?

Yes User adjusts parameters and

User reviews sizecalling results starts analysis

No
OK?
User adjusts parameters and
Yes re-analyzes samples files
User may open analyzed sample files
to edit fragment sizecalls

DNA fragment ready for further

processing

1-4 GeneScan Analysis Software Overview

GeneScan Analysis on the 3100 Instrument

Flowchart

External Computers
3100 Instrument

LIMS database Detector

3100 NT
External computer Import Workstation
data 3100 Data Collection
with sample records
to create software
in a spreadsheet
plate
records

Sequence Export
Collector unanalyzed Instrument
database data database

ABIF sample files

External computer with: Export unanalyzed

• GeneScan Analysis software or analyzed data
• DNA Sequencing Analysis software

External computer with customized

GeneScan Analysis DNA Sequencing Analysis
software prepared with ABIF
software software
Sample File Toolkit

GeneScan Analysis Software Overview 1-5

GeneScan Analysis on the 3700 Instrument

Flowchart

External Computers
3700 Instrument

LIMS database Detector

3700 NT
External computer Import Workstation
3700 Data Collection
with sample records to create software with database
in a spreadsheet plate
records
Unanalyzed data

Sequence Export
Collector unanalyzed Data Extractor
database data
Decision point

Auto-analysis feature ABIF sample files

External computer with: Export unanalyzed

• GeneScan Analysis software or analyzed data
• DNA Sequencing Analysis software

External computer with customized GeneScan Analysis DNA Sequencing Analysis

software prepared with ABIF software software
Sample File Toolkit

1-6 GeneScan Analysis Software Overview

Sequence Collector With the Sequence Collector database system, data is collected by the
Database Option Windows NT-based computer and exported to a Sequence Collector
database on a networked server.

The data can later be viewed, edited, and analyzed using the
GeneScan Analysis Software version 3.7 for Windows NT ®, either using
the same Windows NT-based computer used to collect the data, or a
different Windows NT-based computer with access to the Sequence
Collector database. The data can also be viewed and edited (but not
analyzed) using the GeneScan Analysis Software on a Macintosh ®
computer.

Related Manuals GeneScan Analysis Software is part of a suite of Applied Biosystems

hardware and software products.

If the information you need is not in this manual, it may be in one of the
other manuals listed in the table below.

For more information about… See… Part Number

ABI PRISM ® 3700 DNA Analyzer ABI PRISM 3700 DNA Analyzer User’s 4325941
Manual
ABI PRISM ® 3100 Genetic Analyzer ABI PRISM ® 3100 Genetic Analyzer User’s 4315834
Manual
ABI PRISM ® 377 DNA Sequencer ABI PRISM ® 377 DNA Analyzer User’s 4325703
Manual
ABI PRISM ® 310 Genetic Analyzer ABI PRISM ® 310 Genetic Analyzer User’s 4317588
Manual
specific GeneScan chemistry ♦ GeneScan® Reference Guide, 4303189
protocols, designing experiments, and Chemistry Reference for the ABI
preparing samples PRISM 310 Genetic Analyzer, or
♦ The protocols that accompany Applied
Biosystems reagent kits.
attaching the new matrix to an Gel Processor User’s Manual
ABI PRISM 377 gel file
accessing and managing a Sequence Sequence Collector User Guide NT 4319527
Collector database
installation and administration of Sequence Collector Installation and 4319526
Sequence Collector System Administration Guide

GeneScan Analysis Software Overview 1-7

Registering the Software

License and Before you begin, read Appendix G, “License and Warranty.” This
Warranty 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
Applied Biosystems.
Note 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:

1-8 GeneScan Analysis Software Overview

Hardware and Software Requirements

Introduction The GeneScan Analysis Software can be installed on the Windows

NT ®-based computer connected to your ABI PRISM ® instrument or on
any other Windows NT-based computer that meets the minimum
requirements stated below. The software can be installed on a
computer used for analysis only, and on one used for both data
collection and analysis.

Computers The following table provides important information about computers

Connected to connected to Applied Biosystems instruments:
Applied
Biosystems If... Then...
Instruments you received this software with a the GeneScan Analysis Software
newly purchased instrument was installed by your Applied
Biosystems Customer Support
Engineer as part of the installation
and setup of the instrument.
The system requirements for that
computer are described in the
instrument user’s manual.
you are updating the GeneScan ensure your computer meets the
Analysis Software, or if you are minimum requirements provided
installing the software on a computer below before loading the software.
other than the one supplied with
your instrument IMPORTANT Your computer
MUST meet the requirements listed
to run the new GeneScan Analysis
Software.

GeneScan Analysis Software Overview 1-9

 System Below are the system requirements to run the GeneScan Analysis
Requirements Software v. 3.7 for Windows NT ® platform on your instrument or
analysis computer.
IMPORTANT Applied Biosystems strongly recommends using the computer
supplied with your instrument for running GeneScan Analysis software. The
software was optimized and tested on systems similar to that shipped with your
instrument. Running the software on systems that do not meet the following
requirements may cause data loss or other significant problems.

Note These are the minimum requirements. In general, the more memory, the
larger the screen size, and the more processing power you have, the better.

System Requirements
System For use with 310 and 377 For use with 3100 and
Component Instruments 3700 Instruments
Model Dell 733 GX 100 Medium Dell WorkStation 220
Desktop
Processor 733 MHz/133 MHz 733 MHz/133 MHz
Speed/Bus
CD-ROM drive Any Any
Operating Windows NT v. 4. with Windows NT v. 4. with
system Service Pack 5 Service Pack 5
RAM The minimum memory 256 MB RAM
requirement is 128 MB,
although 256 MB is
recommended.

Minimum System System

Recommendations Component Recommendations
Monitor A 17-inch monitor or larger with 1024 x 768 resolution
Disk Space Storage requirements depend primarily on the quantity of
data to be generated and stored. It is common to store
many sample files on the analysis computer.
Each sample file is approximately 150–250 KB.
Printer A PostScript-compatible color printer

1-10 GeneScan Analysis Software Overview

Hard Drive Use the following information to determine on which drive to install
Partitions software. During installation, the hard drive of the Windows NT-based
computer was partitioned to create the following drives:

When installing... Use drive...

programs for general use C.
♦ Data Collection program D.
database software
♦ GeneScan Analysis software

GeneScan Analysis Software Overview 1-11

Installing the GeneScan Analysis Software

Preparation To prepare for the installation:

Step Action
1 Check that you have at least 40 MB of free disk space to
accommodate the GeneScan Analysis Software.
2 Exit all programs that are running.
3 Turn off any virus protection software that you may have running.

Procedure To install the GeneScan Analysis Software from a CD-ROM:

Step Action
1 Insert the GeneScan v. 3.7 software CD-ROM into the computer’s
CD-ROM drive. It will automatically launch the setup when you
insert the CD and install the software.
2 If it does not run the setup automatically, you can either:
♦ Double-click Setup (.exe) or
♦ Click Start, point to Run, then browse to the CD-ROM drive and
click OK
Follow the instructions to install the software.
The Setup Complete window opens when the setup has finished
installing the software on your computer.

1-12 GeneScan Analysis Software Overview

Starting the GeneScan Analysis Software for the First Time

Procedure Follow this procedure when you start the GeneScan Analysis Software
the first time or when you start the software after moving the application
from the Applied Biosystems folder.

To start the GeneScan Analysis Software for the first time:

Step Action
1 Before opening the GeneScan Analysis Software for the first time:
a. Click Start, point to Settings, and click Printers.
b. Right-click the printer you expect to use for GeneScan Analysis
Software data.
c. Click the Close button in the Printers window.
2 Go to the D:\AppliedBio\GeneScan\Bin folder and double-click the
GeneScan icon to start the software.
The GeneScan Analysis Software startup screen opens.

GeneScan Analysis Software Overview 1-13

 To start the GeneScan Analysis Software for the first time: (continued)

Step Action
3 The Product Registration dialog box opens.
Enter your registration information into the three fields and click OK.
Note The registration code is the number you recorded on
page 1-8.

This dialog box opens the first time you start the GeneScan
Analysis Software, and any time that you move the software to a
different disk or partition.
4 When the Product Registration dialog box closes, the GeneScan
Analysis Software menu bar opens.
For information about... See Page
Creating a Project 2-9
Opening Sample Files 4-7

1-14 GeneScan Analysis Software Overview

Removing the Software

This section describes how to remove the GeneScan Analysis Software

v3.7 from your Windows NT-based computer. The uninstall process
deletes all folders and files installed by the GeneScan Analysis
Software Installer.

If Files or Folders If you have moved the GeneScan Analysis Software files or folders from
Have Been Moved their original installed locations they may not be found and deleted by
the uninstall operation.
Any files that have been added to the application folders, such as those
created when the applications are run, are not deleted by the uninstall
operation.

Procedure To remove installed GeneScan Analysis Software:

Step Action
1 Click Start, point to Settings, click Control Panel, and then
double-click Add/Remove Programs.
The following dialog box opens:

2 On the Install/Uninstall tab, select GeneScan and click Add/Remove.

GeneScan Analysis Software Overview 1-15

 To remove installed GeneScan Analysis Software: (continued)

Step Action
3 At the conclusion of the remove operation, an alert box opens with
the message whether or not the remove was successful.
4 If files have been moved or added to the GeneScan Analysis
Software or AppliedBio folders, the remove operation will be
reported as unsuccessful.
If this happens do one of the following:
• Examine and delete the remaining files yourself, or
• On the Start menu point to Remove GeneScan Analysis v3.7,
then select Programs, and Applied Biosystems.

1-16 GeneScan Analysis Software Overview

Creating a Project
Overview
2 2
In This Chapter Topics in this chapter include the following:

Topics See Page

Creating Projects and Auto-Analysis on 310 Instruments 2-2
Creating Projects and Auto-Analysis on 377 Instruments 2-3
Setting Up for Automatic Analysis on 3100 and 3700 2-4
Instruments
Working with Project Files 2-8
Finding Missing Sample Files 2-14

Creating a Project 2-1

Creating Projects and Auto-Analysis on 310 Instruments

Process Using the The following diagram illustrates data analysis using the
310 Instrument ABI PRISM ® 310 Genetic Analyzer.
Run folder
Add Sample file Add Sample file with
#1 to Run folder #2 to Run folder analyzed
Sample files
•••
Collecting
injection #1

Add Sample
New Project, Add
Start file, Analyze & Data-
Sample file,
GeneScan Print Collection
Analyze & Print
Idle and Analysis
completed
Analyzing
Sample file #1

•••

Create project and Add reference to Project with

add reference to Sample file #2 all
Sample file #1 to project references

2-2 Creating a Project

Creating Projects and Auto-Analysis on 377 Instruments

Process Using the The following diagram illustrates the process of automatic analysis and
377 Instrument project creation for data from the ABI PRISM ® 377 DNA Sequencer.
Run folder
Add Sample file with
#1 to Run folder analyzed
Sample files
Extract
sample

Add Sample
New Project, Add
Start file, Analyze & Data-
Sample file,
GeneScan Print Collection
Analyze & Print
and Analysis
completed
Analyzing
Sample file #1

•••

Create project and Add reference to Project with

add reference to Sample file #2 all
Sample file #1 to project references

Creating a Project 2-3

Setting Up for Automatic Analysis on 3100 and 3700 Instruments

Introduction To set up the GeneScan ® Analysis Software for automatic analysis after
data-collection, you must have previously defined analysis parameters
and size standards.

For more information refer to the following sections:

♦ “Chapter 5, “Working with Analysis Parameters.”.
♦ “Defining the Size Standard” on page 7-3.

Procedure To set up for automatic analysis after using the 3100 and 3700 Data
Collection software:
Step Action
Complete the following steps in the 3100 and 3700 Data Collection software.
1 Set the GeneScan Run default preferences to auto-analyze and
use the pop-up menu to locate and select the GeneScan Analysis
Software.
2 In the GeneScan Sample Sheet, for each sample to be analyzed,
do the following:
♦ Enter the sample name.
This field must be completed for the samples to be active in the
Injection or Run Sheet.
♦ Indicate which dye is the standard.
♦ Select the check box labeled Pres (Present) for each
dye/sample you want auto-analyzed.
♦ Select any additional check boxes.

IMPORTANT If you plan to use the Genotyper ® software, you

must complete the Sample Info box correctly. For more information,
refer to the Genotyper User’s Manual.

2-4 Creating a Project

To set up for automatic analysis after using the 3100 and 3700 Data
Collection software: (continued)
Step Action
3 In the Injection List or Run Sheet for each applicable sample, select
AutoAnalyze, and choose one of the following from one of the
appropriate pop-up menus:
♦ Matrix file
♦ Analysis parameters
♦ Size standard
The following table lists considerations when choosing a size
standard.
If you choose a... Then GeneScan...
Size Standard definition file in performs analysis using the
the Collection Injection List or dye specified in the
Run Sheet, but you do not Auto-Analysis defaults.
specify which dye is the
standard in the Sample Sheet
dye as the standard in the does not perform size calling.
Sample Sheet, but do not
specify a size standard
definition file in the Collection
Injection List or Run Sheet

4 Select Auto Print to print automatically.

Complete the following steps:
1 If you are using the Auto-Analysis defaults, choose Auto-Analysis
Defaults from the Settings menu.
The Auto-Analysis Defaults dialog box opens.
The parameters set in the Auto-Analysis Defaults dialog box apply
when you have:
♦ Specified <Analysis Defaults> in the Data Collection software.
♦ Selected the check box labeled Always Override Collection
Settings.

Creating a Project 2-5

 To set up for automatic analysis after using the 3100 and 3700 Data
Collection software: (continued)
Step Action
2 Select the check box labeled Always Override Collection Settings to
have the new parameters take precedence over the following files
specified in the 3100 and 3700 Data Collection programs:
♦ Size standard
♦ Analysis parameters
♦ Dye standard
3 Select a new Size Standard if:
♦ Analysis Defaults is specified for the size standard in the data
collection settings, or
♦ To override the specified size standard.
4 Select the Dye pop-up menu to specify the dye that represents the
internal size standard you are running with the samples.
5 Select the analysis parameters you want to use from the
Parameters pop-up menu.
To use the default parameters, select Analysis Parameters.
6 In the Auto-Print section, there are the following options:
Choose... To print...
Show Electropherograms electropherograms.
Select the appropriate radio
button to specify whether the
electropherograms for the four
dyes appear:
♦ Together in one panel
(overlaid), or
♦ In separate panels (tiled).
Show Tabular data tabular data.

7 Click OK.
The GeneScan Analysis Software is now prepared to perform
automatic analysis.

Note The settings that you specify are initial settings. Make two
or three trial runs, fine tuning the parameters with each run, to
determine which parameters work the best for a particular protocol.

2-6 Creating a Project

What Is a Project A project is a file containing references to a set of sample files that you
want to analyze and display together. The project contains Analysis
Control (Ctrl+1) and Results Control (Ctrl+2) windows that allow you to
analyze specific dye/samples and display the results of analysis.

Why Create a You can create a new project and add any combination of sample files,
Project allowing you to analyze and display samples from different runs.
Adding a sample file to the project sets up a link between the project
and the sample file. The file itself is not imported into the project.

For more information, see “Creating a New Project” on page 2-9.

Where to Store Keep the sample files in the same location on the hard disk relative to
Projects the project file so the GeneScan ® Analysis Software can locate them
when the project is opened.

If... Then...
sample files are moved and the see “Finding Missing Sample Files”
software does not find them when on page 2-14.
you open the project

Creating a Project 2-7

Working with Project Files

Opening an To open an existing project:

Existing Project
Step Action
1 Select Open (Ctrl+O) from the File menu.
The Open Existing dialog box opens.

2 Click the Project icon.

An Open dialog box opens.

3 In the dialog box, find and select the project you want to open.
4 Click Open.
The project opens in an Analysis Control window.

2-8 Creating a Project

Creating a New To create a new project:
Project
Step Action
1 Select New (Ctrl+N) from the File menu.
The Create New dialog box opens.

2 Click the Project icon.

An untitled Analysis Control window opens.

Note For more information on using the Analysis Control window,

refer to page 3-6.
3 There are two ways to add files to a project:

To add... Select...
sample files you select to the Add Sample Files (Ctrl+B) from
open project the Project menu.
The Add Sample dialog box
opens (refer to “Using the Add
Sample Dialog Box” below).
a sample currently open to the Add “file name” from the
open project Project menu.

Creating a Project 2-9

 Using the Add Sample Dialog Box

To use the Add Sample dialog box:

Step Action
1 When the Add Sample Files dialog box opens, find the folder
containing the samples that you want to add from the Look in
pop-up menu.

2-10 Creating a Project

To use the Add Sample dialog box: (continued)

Step Action
2 You can take the following action:

If you want to... Then...

select a single sample file double-click the file or select
the file and click Add.
select all the sample files click Add All.
add a random selection of click each file name while
sample files pressing the Ctrl key.
add a continuous list of a. Click the first sample that
sample files you want to add.
b. Press the Shift key and
click the last sample you
want to add.
All the files between the first
and last file are selected.

3 Click Finish when you have added all the sample files.

If the... Then...
sample files appear in the refer to “Analyzing Sample
Analysis Control window Files: Using the Analysis
Control Window” on page 3-6.
locked files alert appears (see go to “Unlocking Sample Files”
below) below.

Creating a Project 2-11

 Unlocking Sample Adding Locked Sample Files to a Project
Files
If... Then...
the sample files added to a project the GeneScan Analysis Software
are locked does not allow changes to them.
You cannot analyze locked files.
When you add locked files, an alert
appears.

Procedure

To unlock files:
Step Action
1 Select Save Project (Ctrl+S) from the File menu and close the
project.
2 Click the Start button, and then point to Programs.
3 Click Windows NT Explorer.
4 Select the applicable sample files and select Properties from the
File menu.

The Properties dialog box opens.

5 In the Attributes section, unselect the Read-only check box and
click OK.

6 Close the window and reopen the project.

If... Then...
you did not close the project once the files are unlocked,
before unlocking the files close and open the project so
the GeneScan Analysis
Software recognizes that the
sample files are unlocked.

2-12 Creating a Project

Removing Samples Note A removed sample file is not deleted from the hard disk. The reference
from a Project is removed from the project.
To remove sample files from a project:
Step Action
1 Select the file or files in the Analysis Control window (Ctrl+1) or the
Results Control window (Ctrl+2) that you want to remove.

Note Ctrl+click to select multiple files.

2 Select Remove Sample Files from the Project menu, or press the
Delete key.

A warning dialog box opens.

3 Click Remove.

Creating a Project 2-13

Finding Missing Sample Files

When Are Files The project and related sample files are usually located in the Run
Considered Lost folder created by the Data Collection software.
If... Then...
the sample files or the project are the GeneScan Analysis Software
moved so they are no longer in the might not be able to locate the
same relative position sample files when the project is
opened.
Usually, this occurs only if the
sample files are moved to another
disk drive, another server on a
network, or another disk partition on
the hard drive.

When an Alert The following table describes what happens when the software does
Appears not locate the sample files associated with a project:
If... Then...
the GeneScan Analysis Software an alert box opens and the sample
does not locate the sample files file names appear dimmed when the
associated with a project project opens.

Searching for You can re-establish the links between the sample files and the project
Missing Sample by choosing Find Missing Sample Files from the Project menu and
Files choosing one of the following options from the submenu:
To find missing files:
Choose... If you... Description
Fast Search suspect that the Once the volume is
GeneScan Analysis mounted, or the
Software could not find diskette containing the
the missing sample files is inserted, Fast
files because they are Search finds them
located on an immediately.
unmounted external
storage device, or
diskette.

2-14 Creating a Project

 To find missing files: (continued)

Choose... If you... Description

Search a Folder know what folder Specify a folder in
contains the missing which to search for
sample files. missing sample files.
The GeneScan
Analysis Software then
immediately locates
the sample files and
re-establishes links to
the project.
Exhaustive Search do not know where any The GeneScan
of the specified missing Analysis Software
sample files are searches all mounted
located. disk drives, and
available servers.
When the files are
found, the software
re-establishes links to
the project.

Re-Establishing To re-establish the links with sample files:

Links
Step Action
1 Click the dimmed file name to select it.
When the missing file is selected the Find “file name” command
becomes active.
The name of the missing file appears inside the quotation marks.
2 Select Find “file name”.
A file dialog box opens.
3 In the file dialog box, locate the proper folder and file.
4 Click Open or double-click the file name.

Creating a Project 2-15

 Procedure To set up for automatic analysis after data collection, complete the
following steps in the data collection software:
Step Action
1 Set the GeneScan Run default preferences to auto-analyze and
use the pop-up menu to locate and select the GeneScan Analysis
Software.
2 In the GeneScan Sample Sheet, for each sample to be analyzed,
do the following:
♦ Enter the sample name.
This field must be completed for the samples to be active in the
Injection or Run Sheet.
♦ Indicate which dye is the standard.
♦ Select the check box labeled Pres (Present) for each
dye/sample you want auto-analyzed.
♦ Select any additional check boxes.

IMPORTANT If you plan to use the Genotyper ® software, then

you must complete the Sample Info box correctly. For more
information, refer to the Genotyper User’s Manual.
3 In the Injection List or Run Sheet for each applicable sample, select
AutoAnalyze, and choose from the appropriate pop-up menus one
of the following:
♦ Matrix file or dye set name
♦ Analysis parameters
♦ Size standard
The following table lists considerations when choosing a size
standard.

If you choose a... Then GeneScan...

Size Standard definition file in performs analysis using the
the Collection Injection List or dye specified in the
Run Sheet, but you do not Auto-Analysis defaults.
specify which dye is the
standard in the Sample Sheet
dye as the standard in the does not perform size calling.
Sample Sheet, but do not
specify a size standard
definition file in the Collection
Injection List or Run Sheet

4 Select Auto Print to print automatically.

2-16 Creating a Project

Analyzing Project
Files
Overview
3 3
In This Chapter Topics in this chapter include the following:

Topics See Page

Analyzing Project Files: About the Analysis Control Window 3-2
Analyzing Sample Files: Using the Analysis Control Window 3-6
Defining Folder Locations 3-16

Analyzing Project Files 3-1

Analyzing Project Files: About the Analysis Control Window

Introduction When you open a project, the Analysis Control window opens. The
Analysis Control window is the main window of a project. You can use
this window to specify the following for each sample in the project:
♦ Dye that represents the size standard you ran with the sample
♦ Size standard
♦ Analysis parameters
♦ Specific dyes to be analyzed
♦ Format the document and print the results automatically

For more information, refer to “Analyzing Sample Files: Using the

Analysis Control Window” on page 3-6.

Analysis Control The following is an example of the Analysis Control window:

Window Example

4 5

7 6

3-2 Analyzing Project Files

Analysis Control The following table describes the callouts shown for the Analysis
Window Callouts Control window in the above figure:
Described Analysis Control window callouts:

Callout Description
1 Use these columns to choose the dye colors to analyze and to
specify which is the size standard.
2 Diamonds mark the standards.
For more information, refer to “What Are Size Standards” on
page 7-2.
3 Ctrl+click a dye/sample field to specify that dye/sample as the
standard.
4 From the arrow pop-up menu , you can:
♦ Choose Collection Setting.
♦ Choose a user-defined size standard.
♦ Define a new size standard for that sample.
For more information, refer to:

Topic See Page

Defining the Size Standard 7-3
Using Size Standards 7-9

5 From the arrow pop-up menu , you can:

♦ Choose Collection Setting.
♦ Choose an analysis parameters file.
For more information, refer to:

Topic See Page

About the Analysis Parameters 5-2
Using Analysis Parameter Files 5-13

Analyzing Project Files 3-3

 Analysis Control window callouts: (continued)

Callout Description
6 Double-click the size standard text field or the analysis
parameters text field to edit the size standard or the analysis
parameters.
For more information, refer to:
Topic See Page
Editing an Existing Size Standard 7-10
Changing an Existing Analysis Parameters File 5-17

7 A notation appears in this Information Display field when you

move the cursor over a sample file name or over a dye color field.

Note For more information on how to customize this field, refer

to “Displaying Sample and Dye Information” on page 3-11.

Customizing the The following table explains how you can customize the display by
Display changing the settings:
Note These preferences also apply to the Results Control window. Refer to
Chapter 8, “Evaluating Analysis Results.”

To change... Choose... For more information

information Project Options from the “Displaying Sample and
displayed in the Settings menu and Dye Information” on
information display Sample Info Display from page 3-11.
field the submenu.
the sorting of Project Options from the “Setting Sample File Sort
sample files Settings menu and Order” on page 3-13.
Sample File Sorting from
the submenu.
dye indicator code Preferences from the “Setting Dye Indicator
dye color Settings menu and Dye Preferences” on
Indicators from the page 3-14.
submenu.

3-4 Analyzing Project Files

Using the Analysis To... Then... Result
Control Window
select all the samples click the upper-left cell. All the columns in the
Click here Analysis Control
window are selected.

select all of one dye click the column The column is

color heading for that dye highlighted for the color
color. selected.
select all dyes for one click the row number. All the colors in the row
sample are selected.
change all the a. Click the arrow in The same size
standards in the size the column heading. standard is displayed
standards column to b. Select a file from for all the samples.
the same setting the pop-up menu.
change a size standard a. Click the arrow in The size standard for
in one of the rows the row. the selected row
b. Select a file from changes.
the pop-up menu.
change all the a. Click the arrow in The same parameter is
parameters in the the row heading. displayed for all the
parameters column to b. Select a file from samples.
the same setting the pop-up menu.
change a parameter in a. Click the arrow in The parameter for the
one of the rows the row. selected row changes.
b. Select a file from
the pop-up menu.
apply a choice to a. Click the row in the The value in the
selected fields in the column containing selected rows changes
size standards or the information you to the value in the first
parameters column want to apply and row selected.
drag down.
b. Select Fill Down
(Ctrl+D) from the
Edit menu.

Analyzing Project Files 3-5

Analyzing Sample Files: Using the Analysis Control Window

Introduction This section describes using the Analysis Control window to perform
the following tasks:

Topic See Page

Accessing Sample Files 3-6
Analyzing Sample Files 3-7
Specifying the Format for Printed Results 3-8
Displaying Size Standards and Analysis Parameters 3-10
Displaying Sample and Dye Information 3-11
Setting Sample File Sort Order 3-13
Setting Dye Indicator Preferences 3-14

Accessing Sample There are two ways to access sample files contained in a project from
Files the Analysis Control window.
Note Sample files that are dimmed can not be found by the project. To find
missing files, refer to “Finding Missing Sample Files” on page 2-14.

You can... Then...

double-click a sample file name
If the sample
file is... Then that...
open Sample File
window
becomes
active.
not open sample file
opens to its
Sample Results
view.

select a sample file and select one the Sample File window appears in
of the five display modes from the the display mode selected.
Sample menu

3-6 Analyzing Project Files

Analyzing Sample The Analysis Control window (Ctrl+1) allows you to analyze multiple
Files samples easily. You choose dyes to analyze, dye standard, size
standard, and analysis parameters for each sample file, and then
analyze the sample files using these settings.

To analyze sample files:

Step Action
1 Click the dye color fields for each sample you want to analyze as
follows:
To... Click the...
select a dye for all samples colored column header for that
dye.
select all dyes for a single index number at the left end of
sample file the row in which the sample file
appears.
all dyes for all samples area above the row index
numbers.

2 Identify the sample containing the standard as follows:

To... Ctrl+click the...

identify each sample that colored field that represents
contains a size standard the size standard.
select the same dye as the colored column heading for
size standard for all samples that dye.

A diamond appears in the field to identify the dye color as the size
standard.
3 Select a defined size standard setting from the pop-up menu in the
Size Standard column as follows:

To... Refer to...

define a new size standard “Using Size Standards” on
page 7-9.
edit a size standard “Editing an Existing Size
Standard” on page 7-10.

4 To install a new matrix, select a set or all of the samples in the

Sample File column and choose Install New Matrix from the Sample
menu.

Analyzing Project Files 3-7

 To analyze sample files: (continued)

Step Action
5 Select a parameter setting from the pop-up menu in the Parameters
column as follows:
To... See...
use the default analysis “About the Analysis
parameters to specify different Parameters” on page 5-2.
parameters

6 Select the Print Results check box to print the results automatically.
For information on print set-up, refer to “Specifying the Format for
Printed Results” on page 3-8.
7 Click Analyze.

8 To verify the results, refer to “Process of Verifying Results” on

page 9-31.

Specifying the To specify the format for printed results:

Format for Printed
Step Action
Results
1 In the Analysis Control window (Ctrl+1), select the check box
labeled Print Results.
When this check box is selected, the Print Setup button becomes
active.

3-8 Analyzing Project Files

To specify the format for printed results: (continued)

Step Action
2 Click the Print Setup button.
The Auto Print Setup dialog box opens (see below).
All the dyes selected for analysis are also selected for printing.
Sample Dye color fields

Sample Information Display field

3 Moving the cursor over a Sample File name or over a dye color
field, a notation appears in the Sample Information Display field.
4 Click the sample dye color fields to specify any sample you do not
want to print as shown above.

Analyzing Project Files 3-9

 To specify the format for printed results: (continued)

Step Action
5 Select the format by clicking either or both of the buttons at the right
of the window.
Click this button... To print...
electropherograms for the
samples and dyes selected for
analysis.
Select the appropriate radio
button to specify whether the
electropherograms for all the
dyes appear:
♦ Together in one panel
(overlaid), or
♦ In separate panels (tiled).
tabular data.

6 Click OK.

Displaying Size Use the Analysis Control window to open, review, or change size
Standards and standard and analysis parameters.
Analysis To display these files:
Parameters
For more information
You can... Then... see...
double-click the field the Size Standard or ♦ “Using Analysis
containing the size Analysis Parameter Parameter Files” on
standard or the window opens. page 5-13.
analysis parameters
♦ “Defining the Size
file
Standard” on
page 7-3.

3-10 Analyzing Project Files

Displaying Sample How to Display Sample and Dye Information
and Dye Move the cursor over a dye color field or over a sample file name field to
Information display information about the samples and dyes.

How to Specify the Information Displayed

The following procedure describes how to specify the information
displayed when moving the cursor over a dye color field or a sample file
name field:

To specify the information displayed:

Step Action
1 Select Project Options from the Settings menu and Sample Info
Display from the submenu.

The Sample Info Display dialog box opens.

2 Select the check boxes in the Dye/Sample Info & Legend section to
control what appears when you move the cursor over the dye color
fields in the Control windows.
The following table describes the check boxes:
If you select this check box... This appears...
File Name Sample file name.
Sample Name Name of the sample file from
the sample file.
Sample Info Sample information from the
sample file.
Comment Comment from the sample file.

Analyzing Project Files 3-11

 To specify the information displayed: (continued)

Step Action
3 In the Sample File Info heading, select the check boxes for the
information that you want to display when you move the cursor over
the Sample File name field.
The following table describes the buttons:

Select this button... To display...

Show User Name user name from the sample file.
Show Instrument Name instrument name from the
sample file.
Show Path Name path and name of the folder
where the file is located.
Show Run Date run date and start time from the
sample file.
Show Creation Date date and time the sample file
was created.

4 Select the check box labeled Save as Defaults to have the options
you choose saved as the default settings.
5 Click OK.

3-12 Analyzing Project Files

Setting Sample Use this option to specify how sample files are sorted using three
File Sort Order criteria. If no sorting option is specified, then the program sorts by
sample number.

To set sample file sort order:

Step Action
1 Select Project Options from the Settings menu and Sample File
Sorting from the submenu.

The Sample File Sorting dialog box opens.

2 From the pop-up menus, select from the following items:

♦ File Name
♦ Directory
♦ Sample Number
♦ User Name
♦ Instrument Name
♦ Run Date
♦ Creation Date
♦ As Added (sorts the files in the order that they were added)
The precedence indicates the sorting level.
3 Select a button for each item to indicate whether to sort in
ascending or descending order.
4 Select the check box labeled Save as Defaults to have the options
you choose saved as the default settings.
5 Click OK.

Analyzing Project Files 3-13

 Setting Dye The following procedure describes how to change the defaults that
Indicator determine what dye colors appear on the screen and on printed results.
Preferences Setting default dye and plot colors sets the colors used for both the
Control windows and the Results displays:

To set default dye and plot colors:

Step Action
1 Select Preferences from the Settings menu and Dye Indicators from
the submenu.
The Preferences window opens.
If the Preferences window is already displayed, select Dye
Indicators from the pop-up menu.
Vertical scroll bar

Note Use the scroll bar to see the orange dye color.
2 The following table describes the Dye Color and Plot Color columns:

Item Description
Dye Color Shows the colors that represent the dyes in the
column Control window list.

The dye color is also identified in the left color

legend in the Results display.
Plot Color Shows the colors used for plotting the data in
column the electropherograms.

3 Use the vertical scroll bar to change the dye color and plot color for
a fifth dye.

3-14 Analyzing Project Files

To set default dye and plot colors: (continued)

Step Action
4 To change a code, type a different character in the appropriate
entry field in the Code column.
5 Take the following action to change a color:

To... Then...
change a color select a new color from the pop-up
menu.
define a new color Select Other from the pop-up menu.
A color picker opens.

6 Click OK when finished changing dye indicator preferences.

Analyzing Project Files 3-15

Defining Folder Locations

Introduction The GeneScan Analysis Software looks in the designated folders for
the:
♦ Size Standard file
♦ Analysis Parameter file
♦ Matrix file

When saving one of these files for the first time, the default folder
locations for saving the files are those same designated folders.

Storing Matrix Store matrix files, that are intended for use by Data Collection software
Files to assign to collection runs, in the AppliedBio folder. The AppliedBio
folder is located on the computer on which the Data Collection software
is installed.

If Data Collection and Analysis are Performed on Different Computers

Make a copy of a matrix and store it as follows. This is useful when data
collection and analysis are performed on different computers.

Store a copy in the... For use by the...

AppliedBio folder Data Collection software.
GS Matrix Folder GeneScan Analysis Software.

Note The ABI PRISM® instrument Data Collection software uses the files
installed by the GeneScan Analysis Software in the AppliedBio folder. When
you run the analysis software, the program also creates several files (such as a
Preference file) and an Analysis Log.

3-16 Analyzing Project Files

Analyzing Sample
Files
Overview
4 4
In This Chapter Topics in this chapter include the following:

Topics See Page

About Sample Files 4-2
Converting Macintosh Computer Sample Files 4-3
Converting Sample Files for Use on a Different Platform 4-4
Opening Sample Files 4-7
About the Sample File Window 4-8
Sample Results View 4-9
Sample Info View 4-11
Size Curve View 4-20
Raw Data View 4-22
EPT Data View 4-24
Analyzing a Sample File 4-26

Analyzing Sample Files 4-1

About Sample Files

Sample File The Gel Processor bundled with 377 Data Collection software creates
Generation on 377 Sample files after extracting lanes. The information from each lane in
the gel file is tracked and extracted, and the resulting Sample files are
placed in their respective sample folder. If you change tracking, lane
assignment, or Sample Sheet information, you have to regenerate the
Sample files.

The software consults Sample Sheet information to determine whether

a lane is used (contains sample). The lane tracker uses this information
to assign lane numbers to the tracker lines. In addition, the Gel
Processor Software only extracts those lanes identified as Used.

What Files Sample files contain electrophoresis data collected on ABI PRISM ®
Contain instruments. Unanalyzed data contain raw data.

Sample Files Refer to the following table on how Sample files are generated:

Software That Generates

Instrument Sample Files Notes
ABI PRISM 310 310 Data Collection Sample file is created after
each injection.
Data Collection software
invokes GeneScan software for
auto-analysis
ABI PRISM ® 377 DNA Gel Processor software Gel file is created by 377 Data
Sequencer Collection software. Gel
Processor tracks gels and
extracts data into Sample files.
Gel Processor invokes
GeneScan for auto-analysis.
ABI PRISM ® 3100 Genetic Data Extractor Sample files are extracted by
Analyzer Data Extractor after each run is
completed. If set for
ABI PRISM ® 3700 DNA
auto-analysis, the software is
Analyzer
invoked to analyze the data.

GeneScan software is used to manually analyze, edit and view sample

files generated on any ABI PRISM instrument.

4-2 Analyzing Sample Files

 How GeneScan The GeneScan ® Analysis Software performs the following steps in
Analyzes Sample analyzing sample files:
Files
Step Action
1 Processes the raw data signals to generate analyzed data signal
and then uses the analyzed signals to detect the signal peaks
associated with DNA fragments.
2 Performs size calling by identifying the peaks of the in-lane size
standard found in each sample.
3 Determines the fragment size of each experimental peak within the
sample based on the size calling curve generated using the size
standard peaks, the selected size calling method, and by
comparing it to the pre-defined size standard file.
The algorithmic steps of the process from raw data to analyzed
data are as follows:
♦ Baselining
♦ Smoothing, if any
♦ Peak detection

Converting Macintosh Computer Sample Files

About Converting When you insert the GeneScan Analysis Software version 3.7 for
Files Windows NT ® platform CD-ROM into a Macintosh ® computer’s
CD-ROM drive, a folder appears that contains two applications, Mac to
Win and Win to Mac.
Use these applications to change sample files created on the
Macintosh computer to files that can be read by a Windows NT-based
computer, and to change files that were created on a Windows
NT-based computer so that they can be read by a Macintosh computer.

Analyzing Sample Files 4-3

Converting Sample Files for Use on a Different Platform

Introduction Use the conversion utilities to change sample files created on the
¤
Macintosh ® computer to files that can be read by a Windows NT -based
computer, and to change files that were created on a Windows
NT-based computer so that they can be read by a Macintosh computer.
IMPORTANT This utility will run only on a Macintosh computer.

Converting To convert Macintosh sample files for use on the Windows NT-based
Macintosh Files to computer:
Windows
Step Action
NT-Based
Computer Files 1 Insert the ABI PRISM ® GeneScan Analysis Software v. 3.7 for
®
Windows NT platform CD-ROM into the Macintosh computer
where your Macintosh sample files are stored.
2 Double-click the CD-ROM icon.
3 Double-click the Sample File Mac to Win icon to start the application.

The following dialog box is displayed:

4-4 Analyzing Sample Files

 To convert Macintosh sample files for use on the Windows NT-based
computer: (continued)
Step Action
4 Click Run.
The following directory dialog box is displayed:

5 Navigate to the folder that contains the sample files that you want to
convert and click Choose.
The program will perform the task and automatically quit. The
converted sample files will have the extension .ab1.

Converting To convert Windows NT-based computer sample files for use on the
Windows Macintosh computer:
NT-Based
Step Action
Computer Files to
Macintosh Files 1 Insert the GeneScan Analysis Software v. 3.7 for Windows NT
platform CD-ROM into the Macintosh computer where your
Windows NT-based sample files are stored.
2 Double-click the CD-ROM icon.

Analyzing Sample Files 4-5

 To convert Windows NT-based computer sample files for use on the
Macintosh computer: (continued)
Step Action
3 Click the Sample File Win to Mac icon to start the application.

The following directory dialog box is displayed:

4 Navigate to the folder that contains the sample files that you want to
convert and click the Choose button.
If... Then...
there are no problems, When you open the folder, you can
the program will perform double-click the sample files to open
the task and them using GeneScan Analysis
automatically quit Software on a Macintosh computer.

4-6 Analyzing Sample Files

Opening Sample Files

Introduction Sample files can be opened as separate files outside of projects, and
display related information about each sample file.

To open sample files:

If you are interested in... Then...
one or two sample files it is often more convenient to open sample
files individually and analyze or view the
data without opening an entire project.
multiple sample files use a project.
For information on opening Sample Files
from within a Project, see “Accessing
Sample Files” on page 3-6

Procedure To open a sample file as a separate file:

Step Action
1 Select Open (Ctrl+O) from the File menu. The Open Existing dialog
box appears.

Note You can also double-click the sample file name in the folder
containing the files. If the GeneScan Analysis Software is not
running, the software starts and opens the sample file.

Analyzing Sample Files 4-7

 To open a sample file as a separate file: (continued)

Step Action
2 Click the Sample icon. An Open dialog box appears.

3 In the dialog box, navigate to the folder and select the sample file
that you want to open.
4 Click Open. The Sample File window appears.
For more information on the Sample File window, refer to page 4-8.

About the Sample File Window

What It Displays You can use the display modes in the Sample File window to review the
analyzed and raw data, and all pertinent data collection, sizing and
sample description information from a single window. The Sample
Results view appears as the default.

Five Views The five views of the Sample File window are:

View See Page

Sample Results View 4-9
Sample Info View 4-11
Size Curve View 4-20
Raw Data View 4-22
EPT Data View 4-24

4-8 Analyzing Sample Files

Sample Results View

What It Displays The Sample Results View displays the sample file’s analyzed data in
both electropherogram and tabular data form.

Displaying the The following table describes ways to display the Sample Results View:
View
To display the view... Do this...
from the Sample File
window You can either Result
click the button for the The Sample Results
Sample Results view View appears.
at the bottom left of
Refer to “Example of
the Sample File
Sample Results View”
window.
on page 4-10.

select Sample Results

(Ctrl+E) from the
Sample menu.

from a project window a. Select a sample or Ctrl+click to select multiple

samples in the Analysis Control (Ctrl+1) or the
Results Control (Ctrl+2) window.
b. Select Sample Results (Ctrl+E) from the
Sample menu.

Analyzing Sample Files 4-9

 Example of The following is an example of the Sample Results view:
Sample Results
View

Description of The following table describes the columns in the above figure:
Columns
This column Identifies
Dye/Sample Peak Dye color and Peak number.
Minutes The time, in minutes, from the start of the run to the
time the fragment was detected.
Size The number of base pairs in the fragment.
This value is calculated automatically only if you:
♦ Run the size standard in the same lane or injection
as the sample, and
♦ Perform size calling.
Peak Height Signal size.
Peak Area Area of the detected peak.
Data point Data point of the fragment at its maximum peak
height.

4-10 Analyzing Sample Files

 Differences from The Sample Results view displays the same electropherogram and
the Results Display tabular data as the Results Display, with the following differences:
♦ One sample file is displayed.
♦ Show or hide dye/sample data by clicking the buttons below the
electropherogram.
♦ Cannot display legends.
♦ Cannot use custom plot colors.

Sample Info View

What It Displays Displays the following sample file information:

Information displayed See Page

Run Information 4-14
Data Collection Settings 4-14
Sample Information 4-14
Gel Information 4-15
Analysis Records 4-15
Dyes Within Analysis Records 4-16
Data Collection Settings 4-17
Gel Information (Polymer) 4-17
Sample Information 4-18
Analysis Records 4-18
Dyes Within Analysis Records 4-19

Analyzing Sample Files 4-11

 Displaying the The following table describes ways to display the Sample Info View:
View
To display the view... Do this...
from the Sample File
window You can either Result
click the button for the The Sample Info view
Sample Info view at appears.
the bottom left of the
Refer to “Example of
Sample File window.
Sample Info View” on
page 4-13.

select Sample Info

(Ctrl+I) from the
Sample menu.

from a project window a. Select a sample or Ctrl+click to select multiple

samples in the Analysis Control (Ctrl+1) or the
Results Control (Ctrl+2) window.
b. Select Sample Info (Ctrl+I) from the Sample
menu.
The information is organized in four panels.
Click the triangles to expand or collapse the
panels to display specific information.

4-12 Analyzing Sample Files

 Example of The following is an example of the Sample File Information window in
Sample Info View Sample Info View:

Analyzing Sample Files 4-13

 Description of This information is generally applied to Data Collection software. The
Information following tables list the information in the Sample Info View:

Run Information

Information found under the

header Information entered in
♦ User Name Data Collection software Run file
and run information
♦ Instrument
♦ Data Collection software version
♦ Run date and start time
♦ Tube
♦ Run Date
♦ Start Time
♦ Run Duration
♦ Total Points

Data Collection Settings

Information found under the

header Information is entered in
♦ Module File Data Collection software Run file
and run information
♦ Matrix
♦ Analysis Parameters
♦ Size Standard
♦ Run Voltage, Injection Voltage,
and Injection Duration
♦ Temperature
♦ Laser power

Sample Information

Information found under header Information is entered in

Sample Name Data Collection software Plate Setup
Sample data and comment for each Record
dye color

4-14 Analyzing Sample Files

Gel Information
This information applies only to samples from the ABI PRISM 377
instrument.

Information found under header Information is entered in

♦ Gel Type Data Collection software
♦ Gel File name
♦ Gel Percent
♦ Gel Thickness
♦ Well-To-Read Distance
♦ Number of Channels
♦ Lane Number
♦ Channel Averaging
♦ Lane Extraction Range

Analysis Records

Information found under header Information is entered in

Date and time each color was Analysis information
analyzed, and more panel-display
arrows

Analyzing Sample Files 4-15

 Dyes Within Analysis Records

Information found under header Information is entered

Analysis parameters file and range Analysis Settings
analyzed
Whether baselined or
multicomponented
Data smoothing
Peak detection threshold and
minimum half-width
Polynomial degree
Peak window
Size standard file Analysis Control window
The dye color used for the standard,
Sizing method, and range
Standard peak detection threshold Analysis Settings
Baseline window size
Slope threshold for peak start
Value for end
Total number of peaks: found in Analysis results
sample and dye standard, defined in
standard matched with standard
peaks

4-16 Analyzing Sample Files

Data Collection Settings

Information found under the

header Information is inserted from
♦ Module File ABI 373 or ABI PRISM 377 gel file
(data collection Run File and run
♦ Matrix File
information).
♦ Analysis Parameters
This information is embedded in the
♦ Size Standard gel file.
♦ Electrophoresis voltage, current, ABI PRISM 310 data collection Run
and power (ABI 373 and file and run information.
ABI PRISM 377 only)
♦ Run Voltage, Injection Voltage,
and Injection Duration
(ABI PRISM 310 only)
♦ Temperature
♦ Laser power

Gel Information (Polymer)

Information found under the

header Information is inserted from
ABI PRISM 310
♦ Gel type Optional (user entered)
♦ Length to detector Data Collection information
♦ Lot # and expiration date
ABI 373 and ABI PRISM 377
♦ Gel type Optional (user entered).
♦ Name of gel file Gel file (data collection Run file)
This information is embedded in the
gel file.
♦ Gel percentage and thickness Optional (user entered)
♦ Well-to-read (separation)
distance
♦ Number of channels Gel file (data collection Run file)
♦ Number of Lanes This information is embedded in the
gel file.

Analyzing Sample Files 4-17

 Information found under the
header Information is inserted from
♦ Channel Averaging Gel processing parameters

Note Zero (0) indicates use of

pre-averaging offscale data.

The following is an example of how

pre-averaging offscale data appears:

Extraction method Displayed

3 channel averaging 3
3 channel averaging 30
with pre-averaging
offscale
3 channel -3
averaging-weighted
3 channel - 30
averaging-weighted,
pre-averaging
offscale

♦ Range of data points extracted

Sample Information

Information found under header Information is inserted from

Sample data and comment for each ♦ ABI PRISM 310 Sample Sheet
dye color
♦ ABI 373 or ABI PRISM 377 gel file
(Sample Sheet)
This information is embedded in the
gel file.

Analysis Records

Information found under header Information is inserted from

Date and time each color was Analysis information
analyzed, and more panel-display
arrows

4-18 Analyzing Sample Files

Dyes Within Analysis Records

Information found under header Information is inserted from

♦ Analysis parameters file and Analysis Settings
range analyzed
♦ Whether baselined or
multicomponented
♦ Data smoothing
♦ Peak detection threshold and
minimum half-width
♦ Size standard file Analysis Control window
♦ The dye color used for the
standard, Sizing method, and
range
♦ Standard peak detection Analysis Settings
threshold
♦ Split Peak correction
♦ Total number of peaks: found in Analysis results
sample and dye standard,
defined in standard matched with
standard peaks

Analyzing Sample Files 4-19

Size Curve View

What It Displays Displays sizing curves for sample files. The size curve is a measure of
how well the internal size standard matches the standard definition, and
whether or not it is linear.

Displaying the The following table describes ways to display the Size Curve View:
View
To display the view... Do this...
from the Sample File
window You can either Result
click the button for the The Size Curve view
Size Curve view at the appears.
bottom left of the
Refer to “Example of
Sample File window.
Size Curve View” on
page 4-21.

select Size Curve

(Ctrl+U) from the
Sample menu.

from a project window a. Select a sample or Ctrl+click to select multiple

samples in the Analysis Control (Ctrl+1) or the
Results Control (Ctrl+2) window.
b. Select Size Curve (Ctrl+U) from the Sample
menu.
For each selected sample, a Sample File window
opens and displays its Size Curve view.

4-20 Analyzing Sample Files

 Example of Size The Size Curve view displays two curves, as shown in the figure below.
Curve View For a description of the curves, refer to “Curves Described” below.

Curves Described The following table describes the curves in the above figure:
Note Sizing errors due to anomalous mobilities may be displayed as
nonlinear.

This curve Represents

Red curve The sizecalling curve, based on the sizecalling method
used to analyze the data.
Black curve The best-fit least squares curve, which the GeneScan
Analysis Software calculates for all samples, regardless of
the size calling method.
This curve is provided to help evaluate the linearity of the
sizing curve.
When the sizing curve and best-fit curve match, they
overlap so you see only the size curve.

Analyzing Sample Files 4-21

Raw Data View

What It Displays It displays the raw data collected for a sample. This information is stored
in the sample file.

Displaying the The following table describes ways to display the Raw Data View:
View
To display the view... Do this...
from the Sample File
window You can either Result
click the button for the The Raw Data View
Raw Data view at the appears.
bottom left of the
Refer to “Example of
Sample File window.
Raw Data View” on
page 4-23.

select Raw Data

(Ctrl+R) from the
Sample menu.

from a project window a. Select a sample or Ctrl+click to select multiple

samples in the Analysis Control (Ctrl+1) or the
Results Control (Ctrl+2) window.
b. Select Raw Data (Ctrl+R) from the Sample
menu.

4-22 Analyzing Sample Files

Example of Raw The following is an example of the Sample File window in Raw Data
Data View View:

Relative peak amplitude (signal intensity) Data points

What to Evaluate Use the Raw Data view to evaluate:

♦ Problems or noise in the baseline that could result in poor size
calling.
♦ Start and stop points for analysis.

For information on changing the horizontal (refer to page 9-21) or

vertical scale of the data (refer to page 9-22).

Analyzing Sample Files 4-23

EPT Data View

What It Displays The EPT Data View (electrophoresis power and temperature) displays
this information collected for a sample, and it is stored in the sample file.

Displaying the The following table describes ways to display the EPT Data View:
View
To display the view.... Do this...
from a Sample File
window You can either Result
click the button for the The EPT Data view
EPT Data view at the appears.
bottom left of the
Refer to “EPT Data
Sample File window.
View Example” on
page 4-25.

select EPT Data

(Ctrl+M) from the
Sample menu.

from a project window a. Select a sample or Ctrl+click to select multiple

samples in the Analysis Control (Ctrl+1) or the
Results Control (Ctrl+2) window.
b. Select EPT Data (Ctrl+M) from the Sample
menu, or click the EPT data button.

4-24 Analyzing Sample Files

EPT Data View The following is an example of the Sample File window in EPT Data
Example View:

Green line

Red line

Black line

Blue line

Colored Lines The following table describes the lines in the above figure:
Described
Line Description
Blue Electric voltage in volts/10
Black Electric power in watts
Red Run temperature in °C
Green Electric current in mA (milliamps)

Analyzing Sample Files 4-25

Analyzing a Sample File

Introduction The GeneScan Analysis Software analyzes raw data stored in sample
files according to parameters and standards that you select. You can
use the analyzed data to detect peaks associated with DNA fragments
and identify those peaks with an established size standard.

Procedure To analyze a sample file:

Step Action
1 Select Analyze “Sample File Name” from the Sample menu
(Ctrl+Y).
The Analyze Sample file dialog box appears.

2 From the Analyze Sample file dialog box, select one of the following
options:

Choose... To select...
Dyes to Analyze check boxes any number of dyes to analyze.
Analyze Parameters pop-up from the default parameters or
menu any parameter files in the
folder location specified in the
application preferences.
Size Standard pop-up menu from the default standard, or
any standard files in the folder
location that you specify in the
application preferences.
Standard Dye pop-up menu the inlane standard dye.

3 After analysis, evaluate the results.

For more information on evaluating the results, refer to Chapter 8,
“Evaluating Analysis Results.”

4-26 Analyzing Sample Files

Installing a New Use the following procedure to install a new matrix file for the Sample
Matrix File file that you want to analyze.
For information on attaching the new matrix to an ABI 373 or
ABI PRISM 377 gel file, refer to “Gel Processor User’s Manual.”

To install a new matrix file:

Step Action
1 Choose Install New Matrix from the Sample menu.
A directory dialog box appears.
The Folder Preferences settings determine where the GeneScan
Analysis Software looks for the matrix file.
For more information, refer to “Defining Folder Locations” on
page 3-16.
2 Select the new matrix file in the dialog box and click Open.
A message appears when the matrix is successfully assigned.
3 Re-Analyze the Sample file.
Applying a new matrix file clears previous analysis information, so
you must re-analyze the file.

Analyzing Sample Files 4-27

Working with Analysis
Parameters
Overview
5 5
In This Chapter Topics in this chapter include the following:

Topics See Page

About the Analysis Parameters 5-2
Sizecaller Algorithm Flowchart 5-4
Setting Analysis Parameters 5-5
Using Analysis Parameter Files 5-13

Working with Analysis Parameters 5-1

About the Analysis Parameters

What They Are Analysis parameter files are used by 3100 and 3700 Data Collection
software to inform the software of the parameters to use to
automatically analyze fragment data. Use the analysis parameters
provided, or follow the procedure in “Creating GeneScan Analysis
Modules” on page A-1 to create new analysis parameter files. The
auto-analysis feature is part of the 3100 and 3700 Data Collection
software.
These parameters are also used for analysis using the GeneScan ®
Analysis Software.

Why They Are Analysis parameter files are required because the auto-analysis feature
Necessary in 3100 and 3700 software has no user interface, so the parameters for
analysis cannot be directly configured in the analysis software. The
GeneScan Analysis Software creates the analysis parameters, and the
software saves the parameters in a folder that can be read by the Data
Collection system software that performs auto-analysis.

The analysis parameter file can also be used for manual analysis of
fragment data. It is then used by auto-analysis while it is processing run
data.

When to Specify a Before performing a run, specify the analysis parameter to use for
Parameter analysis of each sample. Do this when preparing the plate record for the
plate that contains the samples on 3100 and 3700 instruments and
when preparing the run sheet on 310 and 377 instruments.
Note If there is no analysis parameter specified for a particular sample in the
plate record, the sample will not be analyzed by auto-analysis. Use the
GeneScan Analysis Software to analyze the data.

5-2 Working with Analysis Parameters

 Analysis The following analysis parameter files are provided:
Parameter Files
Provided Analysis parameter file Suggested parameters for using...
GS120Analysis.gsp GeneScan 120 Size Standard
GS350Analysis.gsp GeneScan 350 Size Standard
GS400Cubic Analysis.gsp GeneScan 400 HD Size Standard
GS400HDAnalysis.gsp GeneScan 400 HD Size Standard
GS400Ord2Analysis.gsp GeneScan 400 HD Size Standard
GS500Analysis.gsp GeneScan 500 Size Standard

When Analysis Analysis parameter files are opened and read by the Data Extractor
Parameters Are program as each sample is extracted.
Used Note If you make a change to an existing analysis parameter file (without
changing its file name), this change will affect any sample extracted after the
change is saved.

Working with Analysis Parameters 5-3

Sizecaller Algorithm Flowchart

Flowchart The following flowchart shows how the sizecaller algorithm works:

Input
Electropherogram

Sizecalling
needed?

Limit Analysis
Range yes

Match Size
Standard
Baseline

Make Sizing Curve

Detect Peaks
no
Call Sizes

Analyzed
Data Ready
Clear
Extrapolation

Fragment
Select by Size Sizes
Ready

Output Results

5-4 Working with Analysis Parameters

Setting Analysis Parameters

Default Settings The following table describes the default settings for the analysis
parameters. Some of these parameters will be different depending on
the analysis setting you select. This is an example of one of the
settings.

Parameter Default setting

Analysis Range Full Range
Data Processing Smooth Options–the Light button is selected
Peak Detection Select Analysis Parameters from the Settings menu.The
Analysis Parameters dialog box opens.

Parameter Default setting

Peak Amplitude
Thresholds Dye Setting
Blue 50
Green 50
Yellow 50
Red 50
Orange 50

Min. Peak Half Width 2 points

Degree of Polynomial 3
differentiation
Peak Window Size 19 points
Slope Threshold for 0.0
Peak Start
Slope Threshold for 0.0
Peak End

Full Range Full Range

Sizecalling Local Southern Method
Method
Baselining Baseline Window Size 251 points
Auto Analysis Size Standard <None>

Working with Analysis Parameters 5-5

 Displaying Select Analysis Parameters from the Settings menu. The Analysis
Analysis Parameters dialog box opens.
Parameters Note To display the orange Peak Amplitude Threshold, use the scroll bar
under the values and scroll to the right.

5-6 Working with Analysis Parameters

 Analysis There are seven analysis parameters:
Parameters
Analysis parameter See Page
Analysis Range Options 5-7
Data Processing Options 5-7
Peak Detection Options 5-8
Sizecall Range Options 5-10
Sizecalling Method Options 5-10
Baselining Option 5-12
Auto-Analysis Only Option 5-12

Analysis Range The following are the Analysis Range options:

Options
Item Description
Full Range button Use to analyze all the data collected on the genetic
analysis instrument for each sample.
This Range Enter Start and Stop data point numbers in the entry
(Data Points) fields in order to specify only a limited range to be
button analyzed for each sample.
This affects what is displayed in the results display.
Normally, set the analysis range to start after the
primer peak.

Data Processing The Smooth Options only affect the appearance of the analyzed
Options electropherograms.
Note Since the tabulated peaks are calculated from unsmoothed data, they
might not be consistent with the smoothed display. For example, the reported
peak heights might not be the same as those visible from the smoothed
electropherogram. It is recommended that you select the None button.

Working with Analysis Parameters 5-7

 Peak Detection About the Peak Detection Options
Options The Peak Detection options locate peaks at the positive-to-negative
zero crossings of the first derivative of the baselined electropherogram.
The peak detector computes the first derivative at a data point i by fitting
a polynomial to a window centered on i.

Peak Detection Parameter Options Described

The following table describes the options:

Item Description For example
Peak Amplitude The sizecaller reports If you leave the default
Thresholds to the user only those value of 50, peaks with
peaks whose heights amplitude above 50 are
are at least the Peak analyzed and appear in
Amplitude Threshold the tabular data.
for that dye.
Lower amplitude peaks
Set the dye amplitude still appear in the
threshold at a level that electropherogram, but
allows the software to are not analyzed and
detect peaks, but do not appear in the
eliminate noise. tabular data.
For each dye, the
GeneScan Analysis
Software detects peaks
above the threshold
entered in the entry
field.
Minimum Peak Half Defines what If this number is large,
Width constitutes a peak. the software ignores
noise spikes.
Use to specify the
smallest full width at If the peaks in the data
half maximum for peak are narrow, set the
detection. value to a low number.
The range is 2–99. Experiment with this
value to determine the
A typical number might
best number for the
be 3 for microsatellites,
data.
or 10 for SSCPs.

5-8 Working with Analysis Parameters

The following table describes the options: (continued)

Item Description For example

Polynomial Degree Sets the degree of the These parameters
polynomial. control the sensitivity of
this process. Sensitivity
Min. Max. increases with the
setting setting polynomial degree and
2 5 decreases with the
window size.
Peak Window Size Sets the width of the
Use polynomials of
window.
degree 2 or 3 for
well-isolated peaks,
Min. setting Max. setting such as those from a
1 above the Degree of Number of scans size standard, and a
Polynomial between peaks. degree 4 for finer
differentiation setting. control.
For degree 4, the Peak
Window Size should be
1 to 2 times the full
width at half maximum
of the peaks that you
wish to detect.
These parameters
cannot be set for each
color independently.
Slope Threshold for Determines where a For example, a peak
Peak Start peak starts and stops. ends when the first
derivative again
Slope Threshold for
exceeds the Slope
Peak End
Threshold for Peak
End.
Slope Threshold for
peak start must be
non-negative and
Slope Threshold for
peak end must be
nonpositive.
Values other than 0 will
move the extent of the
peak toward its center.

Working with Analysis Parameters 5-9

 The following table describes the options: (continued)

Item Description For example

Baseline Window Size Controls the slope of To achieve symmetry,
the baseliner. you should set ß to an
odd value, though even
The sizecaller
values are acceptable.
determines a baseline
value for each scan i. Small ß will cause the
baseline to creep up
Basically, the sizecaller
into peaks.
sets the baseline to the
lowest Large ß will create a
electropherogram baseline that does not
value that it sees in a touch all peaks; that is,
window of size ß the peaks will not be
centered on scan i. baseline resolved after
the sizecaller subtracts
the baseline from the
electropherogram.

Sizecall Range About the Sizecall Range Options

Options Use the Sizecall Range parameter options to specify the range of size
fragment (in base pairs) to be included in the peak tabular data.

Sizecall Range Parameter Options Described

Item Description
Full Range button Select this choice to report all peaks within the range
of the matched size standard.
This Range Select this choice to limit the reported peaks by
(Base Pairs) button fragment size.

Sizecalling Method About Sizecalling Method Options

Options Click a button to select the desired sizecalling method. The GeneScan
Analysis Software uses these methods to determine the molecular
length of an unknown fragment.

5-10 Working with Analysis Parameters

Sizecalling Method Parameter Options Described

Item Description
2nd Order Least Squares and 3rd Both Least Squares Methods use
Order Least Squares regression analysis to build a best-fit
sizecalling curve.
For information on the Sizecalling
Methods, refer to page B-1.
Cubic Spline Interpolation Forces the sizing curve through all
the known points of the selected
GeneScan size standard.
For information on the Cubic Spline
Interpolation Method, refer to
page B-4.
Local Southern Method Determines the sizes of fragments
by using the reciprocal relationship
between fragment length and
mobility.
For information on the Local
Southern Method, refer to page B-5.
Global Southern Method Similar to the Least Squares Method
in that it compensates for standard
fragments that may run
anomalously.
For information on the Global
Southern Method, refer to page B-7.

Working with Analysis Parameters 5-11

 Baselining Option About the Baselining Option
The Baselining option controls the scope of the baseliner. Use this
option to set the size Beta of the Baseline Window.The sizecaller
computes a baseline for the electropherogram of each dye
independently.

How the Baselining Option Works

A baseline comprises a value at each data point i. Basically, the
baseline value at each data point i, is the lowest electropherogram
value in a window whose width Beta is set using the Baselining option,
and centered at each data point i.

More accurately, the baseline computed in this manner is intermediate.

The real baseline value at each data point i, is the highest intermediate
value, again in a window whose width Beta is set using the Baselining
options and centered at each data point i. The sizecaller baselines an
electropherogram by subtracting the baseline from the raw
electropherogram.

If the Baseline Window Is Too Small or Too Large

The following table describes what happens if the baseline window is
either too small or too large:

Using... Causes...
a small baseline window size the baseline to creep into the peaks,
resulting in shorter peaks in the
analyzed data.
a large baseline window size the baseline to ride too low, resulting
in elevated and possibly not
baseline-resolved peaks.

Auto-Analysis When performing auto-analysis, select the Size Standard to use from
Only Option this pop-up menu.

5-12 Working with Analysis Parameters

Using Analysis Parameter Files

In This Section This section contains the following topics:

Topic See Page

Assigning the Same Analysis Parameters to All Files 5-13
Assigning Different Parameters to Single Samples 5-15
Displaying Default Parameters 5-16
Creating Custom Analysis Parameter Files 5-16
Changing an Existing Analysis Parameters File 5-17
Deleting Custom Analysis Parameters 5-17

Assigning the To analyze samples using the same analysis parameters:

Same Analysis
Step Action
Parameters to All
Files 1 If the Analysis Control window is not displayed, then select Analysis
Control (Ctrl+1) from the Windows menu.
2 Click the arrow in the Parameters column heading and choose
parameters from the pop-up menu.
Your menu choice applies to all fields in the column.

Note Selecting this pop-up menu automatically selects ALL

samples. You cannot assign parameters to only a subset of
samples.

Pop-up menu

Working with Analysis Parameters 5-13

 To analyze samples using the same analysis parameters: (continued)

Step Action
3 The pop-up menu contains the following options:

Item Description
Analysis Parameters Applies the parameters that are stored
as preferences in the software.
Collection Setting Applies the analysis parameters file
specified in the Data Collection
software, which is embedded in the
sample file.
Custom parameters These are files that have been
that are listed at the predefined and they are located in the
bottom of the menu Params folder.
The path is:
D:\AppliedBio\Shared\Analysis\
Sizecaller\Params

5-14 Working with Analysis Parameters

 Assigning To apply separate analysis parameters to selected samples:
Different
Step Action
Parameters to
Single Samples 1 If the Analysis Control window is not displayed, then select Analysis
Control (Ctrl+1) from the Windows menu.
2 Click the arrow in the Parameters column for the sample parameter
settings that you want to change.
A pop-up menu opens.

Pop-up menu

3 The pop-up menu contains the following options:

Choose... To...
Analysis Parameters apply the parameters that are stored
as preferences in the software.
Define New display the Analysis Control dialog
box.
For information on completing the
fields, see “About the Analysis
Parameters” on page 5-2.

4 Repeat step 2 and step 3 for each sample.

Note You can also use the Cut, Copy, and Paste commands from
the Edit menu.

Working with Analysis Parameters 5-15

Displaying Default There are two ways to display the default parameters:
Parameters
You can... Then...
select Analysis Parameters from the the Analysis Parameters dialog box
Settings menu. opens with the default parameters.
double-click Analysis Parameters For information on defining the
from any entry in the Parameters Analysis Parameters, refer to
column in the Analysis Control page 5-2.
window.

Creating Custom To create a custom analysis parameter file:

Analysis
Step Action
Parameter Files
1 Select New from the File menu.
Note You can also select the Define New option from the pop-up
menu in the Parameters column of the Analysis Control window.

The Create New dialog box opens.

2 Click the Analysis Parameters icon.

The Analysis Parameters dialog box opens.
3 Change the parameters as necessary.
For more information on the analysis parameters, refer to page 5-2.
4 Choose Save (Ctrl+S) from the File menu.
A dialog box opens.
5 Enter a descriptive name and click Save.
The file now opens in the pop-up menu for analysis parameters in
the GeneScan Analysis Control window.
You can also select the file in the Data Collection software for
automatic analysis.

5-16 Working with Analysis Parameters

 Changing an To change an existing analysis parameter file:
Existing Analysis
Step Action
Parameters File
1 Select Open (Ctrl+O) from the File menu.
Note You can also double-click the file name in the Parameters
column of the Analysis Control window.

The Open Existing dialog box opens.

2 Click the Analysis Parameters icon.

An Open directory dialog box opens.

If... Then...
the Params folder does not navigate to the folder.
appear
The path is:
D:\AppliedBio\Shared\Analysis
\Sizecaller\Params

3 Select a file that you want to change and click Open.

4 Make the changes and close the window by clicking OK.

Deleting Custom To delete a custom analysis parameters file:

Analysis
Step Action
Parameters
1 Click the Start button, and then point to Programs.
2 Click Windows NT® Explorer and find the file in the folder you
specified as the location for the analysis parameters files.

Note The custom analysis parameters file is in the folder that is

normally called the GS Parameters Folder. This folder is inside the
ABI PRISM GeneScan folder.
3 Drag the custom analysis parameters file to the Recycle Bin.

Working with Analysis Parameters 5-17

Making a Matrix File6
Overview
6
This chapter describes the processes of making a matrix file, loading
and running samples and evaluating the file. You have the option of
selecting four or five dyes depending on the application when creating a
new matrix for data collection.
Note This chapter applies only to the 310 and 377 instruments.

In This Chapter Topics in this chapter include the following:

Topics See page

About Matrix Files 6-2
Process of Creating a New Matrix File 6-7
Loading and Running Dye Standards for the ABI PRISM 310 6-9
Loading and Running Dye Standards for the ABI PRISM 377 6-12
Generating Matrix Sample Files for the ABI PRISM 377 6-15
Instrument
Choosing a Scan Range for the Matrix Calculation 6-17
Generating a New Matrix File 6-20
Saving and Naming the Matrix File 6-22
Assigning the Matrix File to Sample Files 6-23
Evaluating the Matrix File 6-25
Causes for Bad Matrix Files 6-26

Making a Matrix File 6-1

About Matrix Files

Introduction There are three dye-labeling chemistries currently available to prepare

nucleic acid samples to use the GeneScan® Analysis Software on ABI
PRISM® instruments:
♦ Fluorescent NHS-Ester
♦ Fluorescent dNTP
♦ Fluorescent Phosphoramidite

Each chemistry has a set of dye labels that fluoresce at different

wavelengths when excited by a laser.

During data collection on the... The wavelengths are separated...

310 or 377, 377XL, or 96-lane by a spectrograph into a known
upgrade instrument spectral pattern across a detection
system with the sequencer.

Matrix File Matrix files are mathematical matrices that correct for spectral overlap
Definition of fluorescent emission spectra data collected from ABI PRISM®
instruments.

A matrix file allows you to account for spectral overlap when analyzing
Sample files.

Multicomponent This process of eliminating the bleed-through caused by spectral

Definition overlaps is called multicomponenting.
Applying a matrix file to raw data allows you to generate
multicomponented data.

6-2 Making a Matrix File

 Why Is a Matrix A matrix file is necessary because the four or five dyes used to label the
File Necessary fragments fluoresce at different wavelengths and may have spectral
overlaps, as shown below:

When to Create a Create a matrix file for each dye set used from that particular instrument
Matrix File before analyzing fragment data.
You may have to create new matrix files for different gel compositions or
unusual run conditions.

Making a Matrix File 6-3

Sample Files Using The figures below show examples of data analyzed with and without a
Matrix File matrix file.
You can see that peak data from a Sample file analyzed without a
matrix file displays the expected peak, along with extra peaks in other
dye colors, or bleed-through from other dye colors.

Sample file analyzed without a matrix file

Sample file analyzed with a matrix file

Installing a Matrix You can install a matrix file into a gel file or into a Sample file.
File
Normally the matrix from a matrix file is installed within a gel file or a
Sample file automatically upon generation during or after a run.
Additionally a matrix can be manually installed into a 377/310 Sample
file from within the GeneScan Analysis Software.

When to Assign a Before you can successfully analyze Sample files using the GeneScan
Matrix File Analysis Software, you must make a new matrix file or assign an
existing one to a set of Sample files.

Limitations to You can only assign a matrix file to Sample files generated on the same
Matrix Files instrument, under the same electrophoresis, gel matrix and buffer
conditions, and using the same dye set.
Note If you are using a fifth dye, then you need to create a new matrix file for
that dye.

6-4 Making a Matrix File

When to Create a Create a new matrix file in the following conditions:
New Matrix File ♦ For each dye set:
– NHS-Esters
– Phosphoramidite set
– Fluorescent dNTPs
♦ Whenever you change the dye set you use to label sample
fragments, for example, if you are using the fifth dye.
♦ When you use gel materials or buffers with pH values that differ
greatly from the pH value of the gel material or buffer on which the
existing matrix files were generated.
♦ When you use dyes other than those provided by Applied
Biosystems.
♦ When you run the same gel on a different instrument.
♦ When you see multiple unexpected peaks of different colors under
an expected peak.
♦ When you recalibrate your CCD camera (310 and 377 instruments)
and the change is greater than 3 pixels from the original pixel
position.
♦ When you replace the CCD camera (ABI PRISM 310 and
ABI PRISM 377).

Making a Matrix File 6-5

 Considerations The following table lists some of the considerations before making a
Before Making a matrix file:
Matrix File
Consideration Comment
How much dye matrix standard to With the ABI PRISM 377, loading
load? more than 3 µL, produces too much
signal.
Any amount that results in a signal
over 4,000 FUs is too strong.
Which lanes to load with the dye For gel electrophoresis, load the
matrix standards? matrix standards with an empty lane
between each sample to avoid
contamination of the individual dyes
by residual material leaking adjacent
samples.
What exact gel data will be used for After generating a gel image, for
matrix creation? ABI PRISM 377 instrument, check
that the tracking of the gel file is
adequate.

Where to Store Store matrix files intended for use by Data Collection software in:
Matrix Files
D:\AppliedBio\Shared\Analysis\Sizecaller\Matrix\

If Data Collection and Analysis are installed on different computers, the

location is the same. Remember to copy the matrix file from the
analysis computer to the Data Collection computer.

6-6 Making a Matrix File

Process of Creating a New Matrix File

Process Diagram The following diagram shows the procedure for making a new matrix
file:

1 2
Prepare Dye Load Dye
Matrix Matrix
Standards Standards

9 3
Evaluate the Generate
Matrix File Sample Files

Creating a New Matrix

8 4
Assign the View
Matrix File to Raw Data
Select Sample
Files

7 5
Save and Choose
Name the a Scan Range
Matrix File
6
Generate a
New Matrix
File

For sample preparation and loading information, refer to the appropriate

instrument user manual

Steps to Create a The following table lists the steps to create a new matrix file:
New Matrix
Step Process See Page
1 Loading and Running Dye Standards for the 6-9
ABI PRISM 310
2 Loading and Running Dye Standards for the 6-12
ABI PRISM 377
3 Choosing a Scan Range for the Matrix Calculation 6-17
4 Generating a New Matrix File 6-20

Making a Matrix File 6-7

 The following table lists the steps to create a new matrix file: (continued)

Step Process See Page

5 Saving and Naming the Matrix File 6-22
6 Assigning the Matrix File to Sample Files 6-23
7 Evaluating the Matrix File 6-25
8 Causes for Bad Matrix Files 6-26

6-8 Making a Matrix File

Loading and Running Dye Standards for the ABI PRISM 310

Introduction This section describes how to do the following tasks before running
matrix standards on the ABI PRISM 310:

Topic See Page

Creating a GeneScan Sample Sheet 6-9
Creating a GeneScan Injection List 6-10
Starting the Data Collection Software 6-11

Note When loading the matrix standards on an instrument, note which colors
you load in which autosampler positions.

Creating a To create a GeneScan Sample Sheet:

GeneScan Sample
Step Action
Sheet
1 Open the ABI PRISM 310 Data Collection software.
2 Choose New from the File menu.
3 Click the GeneScan Sample Sheet 48 or 96 tube icon.
The GeneScan Sample Sheet opens.

4 Enter the appropriate colors (Blue, Green, Yellow, Red, or Orange)

in the positions for each sample name.
5 Enter any additional information about the sample in the Comments
column.

Making a Matrix File 6-9

 To create a GeneScan Sample Sheet: (continued)

Step Action
6 Use the Save As command and save the Sample Sheet to the
Sample Sheet folder.

Creating a The Sample Sheet is imported into the Injection List, which defines the
GeneScan sample names and the initial injection order.
Injection List To create a GeneScan Injection List:

Step Action
1 Choose New from the File menu.
2 Click the GeneScan Injection List icon.
The GeneScan Injection List opens.

3 Select a Sample Sheet from the Sample Sheet pop-up menu.

4 Select the appropriate module in the Module pop-up menu for lines
1 through 4, for example, A1, A3, A5, and A7.
5 Unselect Auto-Anlz (analysis).
6 Unselect Auto-Print.

6-10 Making a Matrix File

Starting the Data To start the Data Collection software:
Collection
Step Action
Software
1 Click the Run button.
The following windows open:

Window Description
Raw Data window Shows the real-time chromatogram of the
run.
Log Window Shows the real-time written record of run
events.

2 Choose Status from the Window menu.

The current run is in italics in the Injection List.
You can monitor activities such as electrophoresis current, laser
power, running time, and gel temperature.

If the Run was If the run was cancelled and you are using Data Collection Software
Cancelled Version 1.0.4 or later (Macintosh or Windows version), the sample file is
saved if you skip to the next sample or cancel a run.

Run Time Run time is approximately 30 minutes for the GS STR POP-4 module,
so the total run time will be about 120 minutes.

Assigning Matrix For information on “Assigning the Matrix File to Sample Files,” refer to
File page 6-23.

Making a Matrix File 6-11

Loading and Running Dye Standards for the ABI PRISM 377

Introduction This section describes how to do the following tasks using the 377 and
the 377 with XL Upgrade instruments:

Topic See page

Creating a GeneScan Sample Sheet 6-12
Loading Matrix Standards 6-13
Running the Matrix Standards 6-14

Note When loading the matrix standards on an instrument, note which colors
you load in which lanes for gel-based systems.

Creating a The GeneScan Sample Sheet assigns sample and dye information to
GeneScan Sample the appropriate lane.
Sheet To create a GeneScan Sample Sheet:

Step Action
1 Open the ABI PRISM 377 Data Collection software.
Define or verify the data collection preferences.
2 Choose New from the File menu.
3 Click the GeneScan Sample Sheet icon.
The GeneScan Sample Sheet opens.

6-12 Making a Matrix File

 To create a GeneScan Sample Sheet: (continued)

Step Action
4 Enter the individual colors in the appropriate lanes where the matrix
standards are loaded.

Note It is important to fill out the Sample Sheet completely.

5 Enter any additional information about the sample in the Comments
column.
6 Use the Save As command and save the Sample Sheet to the
Sample Sheet folder.

Loading Matrix To load matrix standards:

Standards
Step Action
1 For denaturing gels load:
♦ 0.5–2 µL of matrix standard per lane.
♦ 8 lanes with different colors, leaving an empty lane between
each lane of matrix standard.
2 Complete the information in the data collection Run sheet, making
sure to choose the appropriate PreRun and Run modules.
Take the following action:

Choose modules
For this matrix... that use... Module file
Dye Primer matrix Virtual Filter A GS 36A...
Fluorescent Virtual Filter C GS36C...
Amidite

3 Electrophorese samples according to conditions specified in your

instrument manual.

Making a Matrix File 6-13

 Running the Run the matrix standards under the precise conditions you want to
Matrix Standards generate a matrix file.
To run the matrix standards:
Step Action
1 Complete the information in the data collection Run sheet.
Refer to the figure below to select the appropriate PreRun and Run
Modules.

2 Take the following action:

Choose modules
For this matrix... that use... Module file
Dye Primer or Virtual Filter A GS 36A...
dNTP matrix
GS Amidite matrix Virtual Filter C GS36C...

3 Start the electrophoresis run according to the conditions specified

in your instruction manual.
4 Go to “Generating Matrix Sample Files for the ABI PRISM 377
Instrument” on page 6-15.

6-14 Making a Matrix File

Generating Matrix Sample Files for the ABI PRISM 377 Instrument

Who Should Use Follow these guidelines to use this step:

This Step
Who should use this step Who should not use this step
This step is only necessary if you The ABI PRISM 310 Genetic
are using an: Analyzer automatically processes
collection data and generates
♦ ABI PRISM 377 instrument
Sample files when the run
♦ XL-upgraded instrument completes.
♦ 96-lane upgrade If you ran your Dye Matrix Standards
on the ABI PRISM 310, go to
“Choosing a Scan Range for the
Matrix Calculation” on page 6-17.

Gel Handling on The 377 Data Collection for Windows generates gel files. There is a new
Windows program on Windows called Gel Processor. This program handles all
gel file-related tasks, including tracking and sample file generation. For
more information on the Gel Processor, refer to the Gel Processor
user’s manual.

Verify Tracking Before generating Sample files, verify that the lanes were set and
tracked correctly. Once you have successfully completed your run of
Dye Matrix Standards, open the gel file in the Gel Processor and
visually verify the positions of the tracker lines.

Generating To generate Sample files from within Gel Processor:

Sample Files
Step Action
1 Click the appropriate Lane Indicators at the top of the Gel window to
make sure the tracker line is optimally aligned over each band in all
lanes.
If any of the lanes are not properly tracked, use the tracker line
editing tools to align the tracker lines in each lane. Refer to the Gel
Processor User’s Manual.
2 If you change any of the lane assignments in any way, save the
changes.

Making a Matrix File 6-15

 To generate Sample files from within Gel Processor: (continued)

Step Action
3 From the Gel menu, choose Track & Extract Lanes.

Note If the gel is already tracked, choose Extract Lanes from the
Gel menu.

The project Analysis Control window opens containing each of the

Sample files.

6-16 Making a Matrix File

Choosing a Scan Range for the Matrix Calculation

Introduction Depending on how well your Matrix Standards run, it may be necessary
for you to choose a specific range of data points to be considered for
your matrix calculation.

In order to choose appropriate values for the Scan range, you must first
view the Sample file raw data from each of the matrix standard files, so
you can decide where to choose the start and stop points for the scan
range.

Viewing the Raw When you have multiple Sample files, raw data can be accessed more
Data easily through a project’s Analysis Control window. Raw data can
provide useful information about the Sample files you have created.
Note You can view Sample files without opening a project. However, this
procedure is easier if you use a project to organize the Sample files.

To view raw data:

Step Action
1 Use the following steps to create a project for the Dye Matrix
Standards:
a. Choose New from the File menu.The Create New dialog box
opens.
b. Click the Project icon. An untitled Analysis Control window
opens.
c. Choose Add Sample Files from the Project menu.
d. Find and open your matrix run folder.
e. Select the five Sample files representing the blue, green, yellow,
red, and orange dye-labeled runs, and click Add.
f. Click Done after the Sample files are transferred.
For more information, refer to “Creating a New Project” on
page 2-9.
2 From the Analysis Control window, select the matrix standard
Sample files by clicking on the first Sample file, holding down the
mouse button, and releasing on the last Sample file.

Making a Matrix File 6-17

 To view raw data: (continued)

Step Action
3 From the Project menu, choose Raw Data (Ctrl+R).
Electropherograms displaying raw data from the matrix standard
Sample files appear.

Note For the ABI PRISM 377 instrument, you can also view raw
data from the gel display by selecting one of the lanes containing
Dye Matrix Standard and looking at the Slice View to the left of the
gel image.

For more information about viewing the gel image, refer to the Gel
Processor User’s Manual.

What to Look For In the raw data display of the Sample files verify the following:
in the Raw Data ♦ Data peaks are present in all four of the matrix standards.
Display
♦ There are no anomalies.
♦ The baseline is stable.
♦ Peaks should be on-scale—no more than 4000 relative fluorescent
units—and the peaks of the dye of interest should have a value of at
least 200.

If peak data does not show these characteristics, refer to “Causes for
Bad Matrix Files” on page 6-26, for possible interpretations of your peak
data.

Choosing a Scan To choose a scan range:

Range
Step Action
1 Move the cursor well away from the primer peak, in a region at the
beginning of the run and in a flat part of the baseline, and record
the scan numbers.

Note When choosing the start point, do not include primer peaks
in the scan range (refer to “Eliminating Primer Peaks”). Also, the
region for both the start and stop points should be flat points at the
baseline.
2 Record the data point values for both the start and stop points of
the scan range.
You will need to enter these values in the next step when
generating the new matrix file (refer to page 6-20).

6-18 Making a Matrix File

 To choose a scan range: (continued)

Step Action
3 Close the raw data boxes and the project by clicking in the upper
left-hand corner of the window.

Note Holding down the Alt key while clicking in the upper
left-hand corner of the window will close the windows
simultaneously.

Eliminating Both the primer peaks and the data peaks are displayed when viewing
Primer Peaks the raw data of your matrix standards. Any time you run dye-labeled
samples on a gel (377 instrument), or capillary (310 instrument), you
always have excess dye-labeled primer in the reaction. The primer peak
displays as the first peak, usually off-scale because it is in molar
excess.

Eliminate the primer peak when making a matrix, by choosing the start
point after the primer peak in a flat area with a stable baseline.

Making a Matrix File 6-19

Generating a New Matrix File

Procedure To generate a new matrix file:

Step Action
1 Choose New from the File menu.
The Create New dialog box opens.
2 Click the Matrix icon.
The Make New Matrix dialog box opens.

3 Choose the number of dyes from the Number of Dyes pop-up menu.
If 5 dyes are selected, a button is added to the bottom of the list.
4 The B, G, Y, R, and O buttons represent dye colors.
a. Click a button to display a pop-up menu.
b. Use the pop-up menu to access a Sample file to link to each of
the dye-labeled primers.
c. Choose the Sample file that represents the dye color for that
button.
5 Enter the start point that you determined when choosing a scan
range in the Start at field.
Refer to “Choosing a Scan Range” on page 6-18.

6-20 Making a Matrix File

 To generate a new matrix file: (continued)

Step Action
6 Enter the total number of data points to include to calculate the
matrix in the Points field.

Note You must have at least five peaks to make a matrix.

In most cases, leave the default value, unless you must exclude a
portion of your data because of artifacts or bleed-through.
7 Click OK.
This generates a new matrix file.

Matrix File The following is an example of the Matrix Values window that opens
Example showing the values used to calculate the overlap correction.

For each dye, the value where the dye fluorescence is read by the
appropriate filter is 1.000. The adjacent colors show the amount of
overlap for which the system must compensate. The adjacent values, in
most cases, should be less than 1.000, but equal to or greater than
0.0000.

Making a Matrix File 6-21

Saving and Naming the Matrix File

Introduction The matrix file is instrument-specific. You cannot apply a matrix file you
made on the ABI PRISM 377 to data you collected on an ABI PRISM 310,
nor can you apply a matrix file made on an ABI PRISM 377 to a Sample
or gel file made on another ABI PRISM 377. In other words, you cannot
apply matrix files created on one instrument to other instruments of the
same model.

Naming When naming a matrix file, consider including the following information
Considerations in the name:
Item For example
Instrument type ABI PRISM 377, or ABI PRISM 310.
Filter set used D, C, F, G5 and E5
Gel conditions native or denaturing.

Saving the Matrix To save the matrix file:

File
Step Action
1 Select Save from the File menu.
A dialog box opens.
2 Enter a descriptive name for the new matrix file and click Save.

Where to Store the Store matrix files that are intended for use by data collection to assign
Matrix File to collection runs in:
D:\AppliedBio\Shared\Analysis\Sizecaller\Matrix\

When Data Be sure to copy matrix files generated on the analysis computer (the
Collection and computer running GeneScan analysis) to the Data Collection computer,
Analysis Are if different. This will ensure that the correct matrix is stored in the
Performed on GeneScan sample file. The proper matrix is required for accurate
Different analysis of 310 and 377 sample files.
Computers

6-22 Making a Matrix File

Assigning the Matrix File to Sample Files

Introduction After generating the new matrix file, assign it to all the Sample files that
you want to analyze.
IMPORTANT After assigning your matrix file to Sample files, refer to
“Evaluating the Matrix File” on page 6-25.

Procedure To assign a matrix file to Sample files and review them:

Step Action
Assigning a matrix file to Sample files:
1 From the project that contains your matrix standard Sample files,
open the Analysis Control window.

2 Select the matrix standard Sample files.

3 Select Assign New Matrix from the Project menu.
4 Select the matrix file you just created.
5 Select numbers 1, 2, 3, and 4 on the left side of the window to
highlight the colors for each row.
6 Select Set Analysis Parameters from the Settings menu.
7 Enter the appropriate range for the Analysis Range and click OK to
return to the Analysis Control window.
8 Click Analyze.

Making a Matrix File 6-23

 To assign a matrix file to Sample files and review them: (continued)

Step Action
Reviewing the results:
1 Choose Results Control from the Windows menu.
The Results Control window opens as shown below.

Dye/Samples # of Panels
pull-down menu

2 Select 4 from the # of Panels pull-down menu (see above).

3 Click 1 under Dye/Samples (see above).
4 Click 1 on the Sample Files side of the Results Control window.
5 For the rest of the matrices, click Analyze and repeat steps 1 and 2.

6-24 Making a Matrix File

Evaluating the Matrix File

Introduction After creating a new matrix file and assigning it to select Sample files,
the next step is evaluating the quality of the matrix file. The quality of the
matrix file has a direct impact on the quality of the results data.

Procedure To evaluate the matrix file:

Step Action
1 Analyze the Sample files used to make the matrix.
2 Display Results data for all the Dye Matrix Standard Sample files on
one screen, showing only electropherogram data.
3 For each displayed Sample file:

You should see... If not...

that the only visible peaks you probably have a bad matrix
represent the color of the Dye file.
Matrix Standard run in that
For instructions on how to
lane, or for that injection
identify and correct problems
(ABI PRISM 310).
with bad matrix files see,
all other lines should be “Causes for Bad Matrix Files”
relatively flat. on page 6-26.
This indicates that the matrix
properly compensated for the
spectral overlap.
For example, for the blue
matrix standard Sample file,
you should only see blue.
sharp, well-defined, singularly
colored peak data.

Making a Matrix File 6-25

Causes for Bad Matrix Files

If an Error There are two possible causes for the error messages shown in the
Message Appears following table:
For this cause... Take this action...
designated the wrong files. reassign the matrix files.
Refer to “Assigning the Matrix File to
Sample Files” on page 6-23.
signal is too weak to make a matrix. rerun the matrix standards.

Two Causes of Bad The following table lists two common causes of bad matrix
Matrix Files files:
Problem Cause What to do
Artifact peaks of Loading too much dye Complete another run
different colors under when running matrix and recreate the
the true peaks. standards, resulting in matrix.
dye bleed-through.
Refer to the figure
below.

Noisy baseline. If the matrix subtracts Complete another

too much of a matrix run and make
particular color from sure you do not have
the sample, then the any off-scale data.
baseline may become
too elevated, resulting
in false peaks.
Refer to the figure
below.

6-26 Making a Matrix File

The following table lists two common causes of bad matrix
files: (continued)
Problem Cause What to do

Making a Matrix File 6-27

Working with Size
Standards
Overview
7 7
In This Chapter Topics in this chapter include the following:

Topics See Page

About Size Standards 7-2
Defining the Size Standard 7-3
Using Size Standards 7-9

Working with Size Standards 7-1

About Size Standards

What Are Size Size standards are specific DNA fragments of known sizes. After
Standards defining the peaks of a size standard, the GeneScan ® Analysis
Software matches this definition to the internal size standard included
with the run. The software assigns the defined size values to the
appropriate peaks of the internal size standard, and uses this
information with the selected sizecalling method to size all unknown
fragments.

Advantages of Running an internal size standard results in determining accurate and

Using Size precise molecular length. This is because the internal size standard and
Standard the unknown fragments undergo exactly the same electrophoretic
forces. The GeneScan Analysis Software can then compensate for
band-shift artifacts caused by variations in the gel and in the sample
from lane to lane or injection to injection.

Size Standards Applied Biosystems provides several fluorescently labeled size

Provided standards, which are described in Appendix C.
You can also label and use other fragments if they better suit the
fragment sizes with which you are working.

When to Define Normally, a size standard is defined using the GeneScan Analysis
Size Standards Software after running the size standard with samples on the
instrument. The software detects peaks for a selected dye color in a
selected sample file and allows you to define the peak sizes. You can
save the defined size standard in a file and use it to automatically
analyze other samples run with the same size standard and under the
same conditions.
For a list of the size standards that are supplied with the GeneScan
Analysis Software, refer to Appendix C, “GeneScan Size Standards.”

If Split Peaks Split peaks might appear in size standards in which both DNA strands
Appear are labeled. For some peaks, the two strands migrate at different rates
when running under denaturing conditions, and they appear as two
peaks approximately half the height of normal, non-split, peaks. One
peak of the two runs is true to size. Assign a size to that peak for the
size standard definition, and assign zero to the other peak.

7-2 Working with Size Standards

Defining the Size Standard

Two Ways to There are two ways to define a new size standard:
Define the Size
Standard Topic See Page
Using the New Command 7-3
Using the Analysis Control Window 7-7

Using the New To use the New command to define a new size standard:
Command
Step Action
1 Select New from the File menu.
The Create New dialog box opens.

2 Click the Size Standard icon.

The following dialog box opens:

Working with Size Standards 7-3

 To use the New command to define a new size standard: (continued)

Step Action
3 Select the sample file that contains the dye standard you want to
use as a template for the new size standard and click Open.
The Select Dye and Analysis Parameters dialog box opens.

4 From the Dye pop-up menu, select the code that represents the dye
label of the size standard in the selected sample File.
5 From the Analysis Parameters pop-up menu, select the analysis
parameters to use.
The pop-up menu contains the following options:
Item Description
Analysis Parameters Applies the parameters that
are stored as preferences in
the software.
custom parameters that are These are files that you
listed at the bottom of the defined and they are located in
menu the Params folder.
The path is:
D:\AppliedBio\Shared
\Analysis\Sizecaller\Params.

7-4 Working with Size Standards

To use the New command to define a new size standard: (continued)

Step Action
6 Click OK.
A window opens (see below) showing the electropherogram and a
table of peaks for the dye color and sample selected.
You should be able to recognize the peak pattern of the size
standard in the electropherogram.

Note You can only change the peak size value in the right column
of the table. You cannot change or rearrange the peak numbers.

If... Then...
too many peaks appear in the you may need to adjust the
electropherogram or the analysis parameters.
baseline is too high
See “Using Analysis
Parameter Files” on page 5-13.

The software assigns a number to each peak found in the

electropherogram in order, from left to right.

Working with Size Standards 7-5

 To use the New command to define a new size standard: (continued)

Step Action
7 Specify the peaks of the size standards and their sizes as follows:

Step Action
a. Click the peak you want to define either in the
electropherogram or in the table.
Use the Zoom In (Ctrl+ Plus sign) and Zoom Out
(Ctrl+ Minus sign) commands from the View menu to
zoom the electropherogram for easier viewing.
– If you click a peak in the electropherogram, the
corresponding row in the table is highlighted.
– If you click a peak number in the table, the
corresponding peak in the electropherogram is
highlighted.
b. Type the value for the selected peak in the
corresponding Size field in the table.
Refer to Appendix C, “GeneScan Size Standards,” for
values and peaks patterns.

Note Leave a zero in the Size field to ignore a peak for

the size standard definition.
c. Press Enter to automatically move to the next size
standard peak.

8 When you finish defining the peaks, save the size standard by
selecting Save As from the File menu.

Note You can also click the Close button.

9 Enter a descriptive name for the size standard and click Save.
Note Run conditions are not stored in the Size Standard file. Use
a name that clearly defines the size standard for future use.

This file is automatically saved in the SizeStandards folder.

The path is:
D:\AppliedBio\Shared\Analysis\Sizecaller\SizeStandards

7-6 Working with Size Standards

Using the Analysis To define a new size standard:
Control Window
Step Action
1 Open an existing project or create a new project.
For information on... See Page
Opening an Existing Project 2-8
Creating a New Project 2-9

The Analysis Control window should open. If it does not, select

Analysis Control (Ctrl+1) from the Windows menu.
2 In the Analysis Control window, find the row that contains the
sample for which you want to define the size standard.
3 In that row, Ctrl+click the dye color cell that represents your size
standard.
A diamond symbol (") appears in the cell, identifying it as the size
standard.
4 Click the arrow in the Parameters field of the same row, and select
an option from the pop-up menu that opens.
The pop-up menu contains the following options:
Item Description
Analysis Parameters Applies the parameters that are
stored as preferences in the
software.
filename Applies the settings specified in
the data collection run file.
Custom parameters that are These are files that you defined
listed at the bottom of the and they are located in the
menu Params folder.
The path is:
D:\AppliedBio\Shared\Analysis\
Sizecaller\Params

5 In the same row, click the arrow in the Size Standard field, and
select Define New from the pop-up menu.
A window opens showing the electropherogram and a table of
peaks for the dye color and sample you selected.
You should be able to recognize the peak pattern of the size
standard in the electropherogram.

Working with Size Standards 7-7

 To define a new size standard: (continued)

Step Action
6 Follow step 7 to step 9 on page 7-6.
The name of the size standard appears in the Size Standard pop-up
menu in the Analysis Control window.

7-8 Working with Size Standards

Using Size Standards

In This Section This section contains the following topics:

Topic See Page

Changing the Number of Peaks Detected 7-9
Editing the Size Standard Definition 7-9
Using the Open Command to Edit an Existing Size Standard 7-10
Using the Analysis Control Window to Edit an Existing Size 7-11
Standard
Deleting an Existing Size Standard 7-12
Analyzing Samples Using the Same Size Standard 7-13
Selecting Separate Size Standards for Samples 7-14

Verifying Size For information, see “Verifying Size Calculations” on page 9-33.
Calculations

Changing the Use the Analysis Parameters dialog box to change the number of peaks
Number of Peaks detected in the Define New Standard window.
Detected For information, refer to “Peak Detection Options” on page 5-8.

Editing the Size The following procedure describes how to edit the size standard
Standard definition (Collection Setting standard) that is embedded in the sample
Definition file. Double-clicking the size standard definition will not open the file.
To edit the size standard definition:
Step Action
1 Choose Open from the File menu.
The Open Existing dialog box opens.
The Collection Setting does not change.
2 Click the Size Standard icon.
An Open dialog box opens.

Working with Size Standards 7-9

 To edit the size standard definition: (continued)

Step Action
3 Navigate to the SizeStandards folder, select the file that you want to
edit, and click Open.
The path is:
D:\AppliedBio\Shared\Analysis\Sizecaller\SizeStandards
4 Make any changes to the file and click the Close button.
5 Choose the edited file from the Size Standard pop-up menu to apply
the changes.

Editing an Existing The two ways to edit a previously defined size standard are by:
Size Standard ♦ Using the Open Command to Edit an Existing Size Standard
♦ Using the Analysis Control Window to Edit an Existing Size
Standard

Using the Open Command to Edit an Existing Size Standard

Step Action
1 Choose Open (Ctrl+O) from the File menu.
The Open Existing dialog box opens.

2 Click the Size Standard icon.

An Open dialog box opens.
3 Navigate to the SizeStandards folder, select the size standard file
that you want to modify, and click Open.
The path is:
D:\AppliedBio\Shared\Analysis\Sizecaller\SizeStandards
4 Edit the peak size values by following steps 7 to 9 on page 7-6.
5 Select Save (Ctrl+S) from the File menu to update an existing file, or
Save As to specify a new name.

7-10 Working with Size Standards

Using the Analysis Control Window to Edit an Existing Size Standard

Step Action
1 If the Analysis Control window is not open, select Analysis Control
(Ctrl+1) from the Windows menu.
2 Click the arrow in the Size Standard column for the sample that you
want to change.

3 Select a size standard from the pop-up menu.

4 Edit the peak size values by following steps 7 to 9 on page 7-6
5 Select Save (Ctrl+S), or Save As from the File menu to save
changes and specify a new file name.

Working with Size Standards 7-11

 Deleting an The following procedure describes how to delete a user-defined size
Existing Size standard from the Params Folder so that it no longer appears in the
Standard Size Standard pop-up menus. The size standard is permanently
removed, and you must redefine it to use it again.

To delete an existing size standard:

Step Action
1 Click the Start button, and then point to Programs.
2 Click Windows NT® Explorer to find and open the SizeStandards
folder.
The path is:
D:\AppliedBio\Shared\Analysis\Sizecaller\SizeStandards
3 Select the size standard file that you want to delete.
4 Drag the file to the Recycle Bin.
Note You can also drag the size standard to another folder for
storage.

7-12 Working with Size Standards

Analyzing Samples To select the same size standard to analyze all samples:
Using the Same
Step Action
Size Standard
1 If the Analysis Control window is not displayed, select Analysis
Control (Ctrl+1) from the Windows menu.
2 Click the arrow in the Size Standard column heading and choose a
size standard file from the pop-up menu.
Your menu choice applies to all fields in the column.

Note Alternatively, you can choose a value from the pop-up

menu, click the header to select the entire column, and select Fill
Down (Ctrl+D) from the Edit menu.
Pop-up menu

3 The pop-up menu contains the following options:

Item Description
None Apply no standard definition.
Collection Setting Apply the size standard specified in
the Data Collection software, which is
embedded in the sample file.
For information on editing this file, see
“Editing the Size Standard Definition”
on page 7-9.
Custom standards that These are files that you defined and
are listed at the bottom they are located in the SizeStandards
of the menu folder.
The path is:
D:\AppliedBio\Shared\Analysis\Sizeca
ller\SizeStandards

Working with Size Standards 7-13

Selecting Separate To apply separate size standards to selected samples, click the arrow in
Size Standards for the Size Standard column for the sample that you want to change (refer
Samples to the figure below) to open the pop-up menu.
For information on using the pop-up menu, see step 3 above.

Pop-up menu

7-14 Working with Size Standards

Evaluating Analysis
Results
Overview
8 8
In This Chapter Topics in this chapter include the following:

Topic See Page

Process of Evaluating Analysis Results 8-2
Ways to Display Analysis Results 8-3
About the Results Display Window 8-4
Using the Results Control Window 8-7
Changing How the Results Are Displayed and Printed 8-13
About the Sample Results View 8-15
Updating the Results 8-16
Saving and Renaming the Results Control Format 8-17

Evaluating Analysis Results 8-1

Process of Evaluating Analysis Results

Evaluating The following table describes the steps to evaluate the analysis results:
Analysis Results
Step Action For information, see...
1 Displaying analysis results “Ways to Display Analysis
Results” on page 8-3
2 Using electropherogram and “About Electropherogram and
tabular data displays Tabular Data Displays” on
page 9-2
3 Viewing electropherograms “Displaying Electropherogram
Data” on page 9-9
4 Verifying analysis results “Process of Verifying Results”
on page 9-31
5 Displaying other sample file ♦ “Sample Info View” on
data page 4-11
♦ “Size Curve View” on
page 4-20
♦ “Raw Data View” on
page 4-22
♦ “EPT Data View” on
page 4-24
6 Using the analysis log “Using the Analysis Log” on
page 9-36
7 Remembering and renaming “Saving and Renaming the
the results display Results Control Format” on
page 8-17

8-2 Evaluating Analysis Results

Ways to Display Analysis Results

Two Ways to The following table describes two ways to display analysis results:
Display Analysis
Results You can use... Description See Page
About the This window is created from the Results 8-4
Results Control window of a project.
Display
Use to group and view multiple sample files
Window
as electropherogram and tabular data.
About the This view displays one sample file at a time, 8-15
Sample like the Results Display window, but is more
Results View convenient to view analysis results from a
single sample file.
It also allows quick access to supporting
information views.

Evaluating Analysis Results 8-3

About the Results Display Window

Introduction The Results Display window is created from the Results Control window
of a project. It allows you to group and view multiple sample files as
electropherogram or tabular data. You can use the window to show up
to eight panels, with multiple dye/samples per panel.

Displaying the When you select Results Control (Ctrl+2) from the Windows menu, a
Window window opens like the example shown below. The callouts are
described in the table that follows this example.

1 2 3 4 5

7
8

9
10

13 12 11

8-4 Evaluating Analysis Results

 Results Control The following table describes the callouts in the previous figure of the
Window Callouts Results Control window:
Described
Callout Description
1 Click one of the dye color fields (B, G, Y, R, O) to select dye/sample
information.
2 Click to display electropherograms for the selected samples.
3 Click to show which sample is selected for this panel.
4 Choose the number of electropherogram panels available for
display from the pop-up menu.
5 Click to display tabular data for the selected samples.
6 Identifies the sample by row number and dye code.
Sample information is displayed as specified in the project
options.
7 Plot color indicator

If you... Then...
double-click the plot the Choose a Plot Color dialog box
color indicator opens.

For more information, refer to

“Defining Individual Plot Colors” on
page 9-26.
Ctrl+double-click the the plot color indicator returns to
dye color indicator the default color.
For more information, refer to
“Setting Dye Indicator
Preferences” on page 3-14.

Note If you change the plot color, a vertical line appears beside
the indicator, showing that it has been modified.

Evaluating Analysis Results 8-5

 The following table describes the callouts in the previous figure of the
Results Control window: (continued)
Callout Description
8 Dye color indicator

If you... Then...
double-click the dye color the Choose a Dye Scale
indicator dialog box opens.

For more information, see

“Changing the Dye Scale in
Electropherograms” on
page 9-28.
Ctrl+double-click the dye the dye color indicator
color indicator returns to the default scale.
For more information, see
“Changing the Dye Scale
Preferences” on page 9-30.

Note If you change the scale, a vertical line appears beside the
indicator, showing that it has been modified.
9 Quick Tile buttons.
See “Creating Tiled Electropherogram Displays” on page 8-10.
10 Display button.
See “Displaying the Results” on page 8-12.
11 Clear Panel button.
See “Removing Samples” on page 8-12.
12 Print button.
See “Printing the Results” on page 8-12.
13 Clear All button.
See “Removing Samples” on page 8-12.

8-6 Evaluating Analysis Results

Using the Results Control Window

Selecting Display Choose the results display format by clicking the electropherogram or
Format tabular icons, or both. You must select one of the icons to display or
print data.

Click this icon... To display... And the...

electropherogram data panel information below
the icon is enabled. Refer
to the figure below.

tabular data Tabular data appears in a

single table, so
electropherogram panel
configuration is not
relevant.

Selecting In electropherograms, the data appears in panels. You can overlay up to

Electropherogram 16 samples within up to eight panels. Select the number of available
Panels panels (up to eight) from the pop-up menu labeled # of Panels.

Pop-up menu

Note Set the number of panels when using the Quick Tile option. Using this
option causes the GeneScan ® Analysis Software to automatically change
panels as you select samples to display (see “Creating Tiled Electropherogram
Displays” on page 8-10).

Evaluating Analysis Results 8-7

 Selecting Samples Use the dye color field on the left side of the Results Control window to
to Display select the dye to display.
You can select any or all dye colors, including the standard, for each
sample file.

To select samples to display:

Step Action
1 Click the Electropherogram icon.

The panel below the button is enabled.

2 Click a panel number in the list to the left of the Dye/Samples list.
If fewer than eight panels are available and you want to use more,
choose a larger number (up to eight) from the # of Panels pop-up
menu.

8-8 Evaluating Analysis Results

To select samples to display: (continued)

Step Action
3 Click the dye color fields to select or unselect the corresponding
samples.
Take the following action:

To... Click the...

select the entire column header of the column.
unselect the entire column Clear All button, or click the
header if it is already selected.
select all colors for a index number to the left of the
sample file color columns.

The samples corresponding to the selected dyes appear in the

Dye/Samples list to the right of the Sample File list, as shown in the
figure below.

Dye color Plot color of

of sample electropherogram

Row number and dye code

followed by dye/sample information

Evaluating Analysis Results 8-9

 To select samples to display: (continued)

Step Action
4 When you change to a new panel, the dye colors of the samples
you selected in other panels appear dark gray to indicate that they
have been selected.
You can select them again in the current panel.

Creating Tiled Procedure

Electropherogram
To create tiled electropherogram displays:
Displays
Step Action
1 Choose the number of panels you want to display from # of Panels
pop-up menu.
2 Click the On button under Quick Tile.
3 Select samples by clicking color fields.
For information on:
Topic See Page
Selecting Samples to Display 8-8
Setting the tiled electropherogram preferences 8-13

Each time a sample is selected, the program automatically changes

to the next panel, so each selection is placed after the one
containing your previous selection.
After you select a sample for the last panel, the panel displays the
first panel again.

Example
The following table describes two examples of how to use the Quick Tile
feature:
If you... Then...
have four samples and choose four click the column heading for the blue
panels for display dye to select all four blue
dye-labeled samples.
The blue dye for each sample file
appears in a separate panel.

8-10 Evaluating Analysis Results

 The following table describes two examples of how to use the Quick Tile
feature: (continued)
If you... Then...
click the row index number for the each dye for that sample file
first sample file to select all dye appears in a separate panel.
colors for one sample file

Unselecting To unselect the samples that you have selected for display:
Samples
Step Action
1 If you specified the Electropherogram display, click the panel
number to the left of the Dye/Samples list to display the panel
containing the samples you want to unselect.

Dye color fields Panel numbers

2 On the left side of the Results Control window, unselect the dye
color fields corresponding to the samples you want to remove.
The dye/sample identifiers are removed from the Dye/Samples list
as you unselect samples.

Evaluating Analysis Results 8-11

Removing Samples To remove samples:
Step Action
1 Select the panel number on the buttons to the left of the
Dye/Samples list.
2 You can take the following action:

To remove… Click…
all the samples you selected to Clear Panel.
display in a panel
the samples you have selected Clear All.
to display in all panels

Displaying the To display the results on the screen, you can either:
Results ♦ Click the Display button.
♦ Press the Return or Enter key.

Printing the To print the results:

Results
Step Action
1 You can either:
♦ Click the Print button, or
♦ Select Print (Ctrl+ P) from the File menu.
2 Click OK in the dialog box that opens.

8-12 Evaluating Analysis Results

Changing How the Results Are Displayed and Printed

Procedure You can set certain display preferences that remain in effect each time
you display or print results data.

To change how results are displayed and printed:

Step Action
1 Select Preferences from the Settings menu and Results Display
from the submenu.
The following dialog box opens:

There are three Results Display preference categories:

♦ Default Display Attributes
♦ Stacked Electropherogram Panels
♦ Printing Preferences

Evaluating Analysis Results 8-13

 To change how results are displayed and printed: (continued)

Step Action
2 Set the Default Display Attributes to control the display attributes of
new results displays, as follows:

You can select the... For more information...

Align By Size check box “Showing Data by Fragment
Size” on page 9-20.
Show Peak Positions check box “Displaying Peak Positions” on
page 9-14.
Show Legends check box “Using Legends to Change the
Display” on page 9-15.
Show Offscale Region check “Showing Off-Scale Data” on
box page 9-18.
Standard “Defining Custom Colors” on
page 9-25.
or
Custom Plot Colors buttons
Opaque “Highlighting Peaks” on
page 9-15.
or
Transparent Peak Highlighting
buttons

3 Set the Stacked Electropherogram Panels, as follows:

Choose... To set...
Use Common Vertical Scale all panels in a display so they
check box have the same vertical scale.
The common scale is based on
the electropherogram with the
largest vertical scale.
Panel Height Resize Limits minimum and maximum values
for electropherogram panel
height in the results display.
Use to limit how much the
electropherogram panels
stretch or shrink to fit the size
of the window.

8-14 Evaluating Analysis Results

 To change how results are displayed and printed: (continued)

Step Action
4 Set the Printing Preferences, as follows:

If... Then...
the height of the panels that select the As Shown on Screen
appears on screen is button.
acceptable
you want to print the click the Fixed at button and
electropherogram at a enter a value in the field.
specified height
you want to force a page break select the Page Break before
after the electropherograms Tabular Data check box.
have been printed

5 Click OK.

About the Sample Results View

About the View The Sample Results view is displayed within a Sample File window. You
can access the window through a sample file or through a project’s
Analysis Control or Results Control window.

If... Then...
you are opening a sample file as a the Sample Results view is the
stand-alone file default display within the Sample File
window.
For more information on... See Page
the Sample Results view 4-9

Evaluating Analysis Results 8-15

Updating the Results

Re-analyzing the The Results Control and the Sample Results windows are dynamic.
Data
If you re-analyze your data with either window active, then the software
updates this window.

8-16 Evaluating Analysis Results

Saving and Renaming the Results Control Format

Introduction You can use the Results Control window to view multiple sample files in
electropherogram and tabular format. The GeneScan Analysis Software
allows you to save formats for future use. You can then redisplay or print
these formats without having to redefine them again.

Important The following are important considerations for saving a Results Control
Considerations format:
♦ You must save the project for the display to be available when you
open the project again.
♦ Remembering a display preserves the combination of
windows/panels/data and customized color settings.
It does not preserve any zooming you have performed.

Saving the Display To save a display format for future viewing:

Format
Step Action
1 With the Results Control window set for the display, either:
♦ Click the Display button, or
♦ Press the Enter key.
2 With the display on the screen, select Remember Display from the
Project menu.

The Remember Display dialog box opens.

3 Enter a name for the display and click OK.

Evaluating Analysis Results 8-17

 Working with a Use the Previous Displays dialog box to display, print, remove or
Previously Saved rename a saved display.
Display To work with a previously saved displays:

Step Action
1 Select Previous Displays from the Project menu.
The following is an example of the Previous Displays dialog box:

2 Select a display or multiple displays and take the following action:

To... Click an item in the list and click...
display the saved Display.
formats
print the saved formats Print.

The standard print dialog box opens.

remove the saved Remove.
formats
An alert opens.
rename a currently Rename.
saved format
The Rename dialog box opens.
Note You can only
See “Renaming the Current Results
rename one display at
Display” on page 8-19.
a time.

8-18 Evaluating Analysis Results

 Renaming the To rename the Results display that is currently on the screen and to
Current Results save the display under a different name:
Display
Step Action
1 Ensure that the display is the active window.
2 Select Rename Display from the Project menu.
The following is an example of the Rename Display dialog box:

3 Enter a new name for the display and click OK.

The new name opens in the Previous Displays dialog box.

Evaluating Analysis Results 8-19

Evaluating
Electropherograms 9
Overview
9
In This Chapter Topics in this chapter include the following:

Topic See Page

About Electropherogram and Tabular Data Displays 9-2
Displaying Electropherogram and Tabular Data 9-4
Displaying Electropherogram Data 9-9
Working with Electropherogram Data 9-12
Defining Custom Colors in Electropherograms 9-24
Changing the Dye Scale in Electropherograms 9-28
Process of Verifying Results 9-31
Verifying Size Calculations 9-33
Using the Analysis Log 9-36
Verifying Peak Detection 9-38

Evaluating Electropherograms 9-1

About Electropherogram and Tabular Data Displays

Introduction After analyzing the data, you can display the results for each sample in
electropherogram and tabular data. You can also customize the
electropherogram and tabular data display.
Note Altering the appearance of the electropherograms and the tabular data
displays does not change the analyzed data contained in the sample file on
which they are based.

How the Window When electropherogram and tabular data are displayed together, the
Is Divided window is divided into upper and lower windows.
Window Contains
Upper window electropherogram data
Lower window tabular data
For more information on... See
customizing the window’s “Adjusting Window Size” on
appearance by adjusting the relative page 9-7.
size of each window

What Tabular If you analyze samples and perform sizecalling, the tabular data
Data Contains contains the estimated sizes (in base pairs) of all detected fragments.
Use this information for detailed data analysis and further calculations.
The peaks matched to the defined size standard are identified by dots
next to the Dye/Sample Peak field.

Sample peaks that are larger (in base pairs) than the largest defined
peak in the selected standard are not sized. The corresponding size
fields are blank.
Note Tabular data displays only peaks that are detected based on the Dye
Amplitude Thresholds and Minimum Peak Half Width setting of the analysis
parameters.

How In the Results Display window, the GeneScan ® Analysis Software sizes
Electropherogram all electropherogram panels to fit within the electropherogram portion of
Panels Are Sized the window by using the largest size that fits them into the visible area.
You can scroll to see the portion of the display that is not visible.

9-2 Evaluating Electropherograms

 For More For more information, see the following topics:
Information
Topic See Page
Displaying Electropherogram and Tabular Data 9-4
Displaying Electropherogram Data 9-9
Working with Electropherogram Data 9-12

Evaluating Electropherograms 9-3

Displaying Electropherogram and Tabular Data

Procedure To display electropherogram and tabular data:

Step Action
1 Select Results Control (Ctrl+2) from the Windows menu to open the
Results Control window.
2 Click the Electropherogram button and the Tabular
button .

3 If applicable, select the number of electropherogram panels from

the # of Panels list.
4 Click the dye color fields to select or unselect the corresponding
samples.

Dye color fields

Take the following action:

To... Click the...
select the entire column header.
unselect the entire column Clear All button.
select all colors for a sample index number to the left of the
file color columns.

The samples corresponding to the selected dyes appear in the

Dye/Samples list to the right of the Sample File list.
5 Click Display.
The electropherogram and tabular data are displayed in the Results
Display window.

See also “Working with Electropherogram Data” on page 9-12.

9-4 Evaluating Electropherograms

 Example of The following is an example of tabular data with a corresponding
Tabular Data and electropherogram:
Electropherogram

Table Describing The following table describes the columns in the “Example of Tabular
Columns Data and Electropherogram” above:
Column heading Identifies...
Dye/Sample Peak ♦ Sample index number
♦ Dye color
♦ Peak number
Minutes The time, in minutes, from the start of the run to the
time the fragment was detected
Size The differences in fragment mobility
This value is calculated automatically only if you:
♦ Run the size standard in the same lane or injection
as the sample, and
♦ Perform sizecalling
Peak Height Signal size (RFU)

Evaluating Electropherograms 9-5

 The following table describes the columns in the “Example of Tabular
Data and Electropherogram” above: (continued)
Column heading Identifies...
Peak Area Area of the detected peak
Data point Data point of the fragment at its maximum peak
height

Why Some Peaks Peaks may be visible in the electropherogram and not listed in the
May Be Visible tabular data because:
Only in an
Electropherogram Reason For more information, see...
The software detects the peaks “Peak Detection Options” on
based on the Peak Amplitude page 5-8
Thresholds and Min Peak Half
Width.
Electropherograms display the “Sizecall Range Options” on
peaks that fall within the range page 5-10
specified by the Sizecall Range
parameters that are defined in the
Analysis Parameters dialog box.

Why Some Peak If negative values appear in peak areas in the electropherogram, it is
Areas May Have a because a portion of the peak is below the baseline. The GeneScan
Negative Number Analysis Software display does not show the part of the
electropherogram that is below the baseline.

Highlighting To highlight information for one peak in the electropherogram and

Information tabular data:
Click... Then...
the peak in the electropherogram the peak fills with color and the
corresponding row in the tabular
data window is highlighted.
the Dye/Sample Peak number in the highlights the corresponding peak in
tabular data window the electropherogram.

9-6 Evaluating Electropherograms

 Changing the To change the highlight transparency:
Highlight Color
Step Action
1 Select Peak Highlighting from the View menu.
2 Then select either Opaque or Transparent from the submenu.

Note For more information on highlighting peaks, see page 9-15.

Adjusting Window To adjust the relative size of the electropherogram and tabular windows:
Size
Step Action
1 Move the cursor to the window divider (the double line between the
two windows).
2 When the cursor changes to a bidirectional arrow ( ), click the
window divider line and drag it up or down.

Hiding Selected To hide selected rows of data:

Rows of Data
Step Action
1 Take the following action:

If you want to... Then...

select a row either:
♦ Click the first field in the row,
or
♦ Click the corresponding
peak in the
electropherogram.
select several rows that are not Ctrl+click the rows.
next to each other

2 Select Hide Selected Rows (Ctrl+H) from the View menu.

Evaluating Electropherograms 9-7

Limiting the Rows To limit the display to the selected rows of data:
to Display
Step Action
1 Select the rows you want to display.
2 Select Show ONLY Selected Rows (Ctrl+G) from the View menu.
Note Select Show All Rows (Ctrl+G) from the View menu to
display all of the tabular data after limiting the display.

9-8 Evaluating Electropherograms

Displaying Electropherogram Data

Definition Each electropherogram provides a profile of the selected dye samples it

represents. The y-axis represents the relative fluorescence of the
detected fragments as they occurred over time. The x-axis represents
time and can be displayed by data points or base pairs.

Base Pairs Versus The tick marks on the x-axis can represent size in base pairs instead of
Data Points data points. This option is only available for runs that include an internal
size standard with the sample (see “Showing Data by Fragment Size”
on page 9-20).

Procedure to To display electropherogram data:

Display Data
Step Action
1 Select Results Control (Ctrl+2) from the Windows menu to open the
Results Control window.
2 Click the Electropherogram button.
3 If applicable, select the number of electropherogram panels from
the # of Panels pop-up menu.
4 Click the dye color fields to select or unselect the corresponding
samples.

Dye color fields

Take the following action:

To... Click the...

select the entire column header.
unselect the entire column Clear All button.
select all colors for a sample index number to the left of the
file color columns.

The samples corresponding to the selected dyes appear in the

Dye/Samples list to the right of the Sample File list.
5 Click Display.
The electropherogram is displayed in the Results Display window
as shown in the example below.

Evaluating Electropherograms 9-9

Electropherogram The following is an example of an electropherogram:
Example
2

6 5 4 3

Electropherogram The following table describes the callouts in the figure above:
Callouts Described
Call
out Description See...
1 Cross hairs “Displaying X- and
Y-Axis Positions” on
page 9-13
2 Magnifying glass “Zooming In and Out”
on page 9-17
Use to zoom in a specific area or hold
down while pressing the Alt key to zoom
out to a smaller scale.
Or, click and drag a marque around an
area to zoom in to that area.
3 Legend “Using Legends to
Change the Display”
Text from the sample file that appear
on page 9-15
beneath electropherogram panels in the
Results Display window.

9-10 Evaluating Electropherograms

The following table describes the callouts in the figure above: (continued)

Call
out Description See...
4 Dye color indicator “Dye color indicator”
on page 8-6
5 Plot color indicator “Plot color indicator”
on page 8-5
6 Scroll bar “Scrolling the Display”
on page 9-16
Use the scroll bar to scroll horizontally.

Evaluating Electropherograms 9-11

Working with Electropherogram Data

In This Section This section describes how to perform the following tasks:

Task See Page

Displaying X- and Y-Axis Positions 9-13
Moving the Electropherogram 9-13
Changing the Dye Color 9-14
Displaying Peak Positions 9-14
Highlighting Peaks 9-15
Using Legends to Change the Display 9-15
Scrolling the Display 9-16
Zooming In and Out 9-17
Showing Off-Scale Data 9-18
Electropherogram Displaying Off-Scale Data 9-19
Electropherogram Displaying the Flat-Topped Effect 9-19
Showing Data by Fragment Size 9-20
Changing the Horizontal Scale 9-21
Changing the Vertical Scale 9-22
Assigning Standard or Custom Colors 9-23

9-12 Evaluating Electropherograms

Displaying X- and The following table describes how to display the x- and y-axis positions:
Y-Axis Positions
To... Then...
display the x- and y-axis positions. click the cross hairs and select an
area in the Electropherogram.
The x- and y-axis values appear in
the box in the lower left corner of the
electropherogram.
If tabular data is also displayed, the
row in the table is highlighted.

Moving the Move the associated electropherogram to the front by clicking one of
Electropherogram the following in the legend:
♦ Dye scale indicator
♦ Plot color indicator, or
♦ Text

Dye scale Plot color Text

indicator indicator

Evaluating Electropherograms 9-13

 Changing the Dye The following table describes how to change the dye color and how to
Color return to the default dye color:
To... Then...
change the dye color double-click the dye color indicator.
return to the default dye color Ctrl+double-click the dye color
indicator.
If... Then...
you change the dye color or scale a vertical line appears beside the
indicator, showing that it has been
modified.

Displaying Peak Use Show Peak Positions from the View menu to examine how the
Positions GeneScan Analysis Software defines peaks by displaying markers that
identify the beginning, center, and end of each peak.

9-14 Evaluating Electropherograms

Highlighting Peaks Use the Peak Highlighting command to highlight a selected peak with
the dye/sample’s plot color.

To highlight selected peaks:

Step Action
1 Select Peak Highlighting from the Views menus and either Opaque
or Transparent from the submenu.

Use this option... To...

Opaque fill the peak with a solid color that can
obscure peaks behind the selected peak.
Transparent use a slightly diffused plot color that allows
you to view overlapping peaks.

2 Click a detected peak in an electropherogram. The peak is

highlighted.

Using Legends to The following table shows how to use legends to change how
Change the electropherograms are displayed:
Display
If you want to... Then...
show or hide legends select Show Legends from the View
menu.
open sample file windows double-click the corresponding
legend text.
reorganize overlaid a. Display the electropherograms
electropherograms with legends.
b. Click either the dye scale
indicator, plot color indicator, or
the text for the sample you want
to move to the front.

Evaluating Electropherograms 9-15

 Scrolling the The following table describes ways to scroll the display:
Display
Use the Description
scroll bar
If you want to... Then...
shift the electropherogram click in the gray region of
to the right or the left the scroll bar to the right or
left of the scroll box.
scroll across the click an arrow at the end of
electropherogram the scroll bar.
control the amount of drag the scroll bar to the
scroll right or the left.

scroller a. Hold the mouse cursor over either the vertical or

symbols horizontal scale of the electropherogram.
Either a vertical scroller symbol ( ) or a horizontal
scroller symbol ( ) appears.
b. Hold down the mouse button and move the mouse in
the direction of the information you want to view.

9-16 Evaluating Electropherograms

Zooming In and About Zooming In and Out
Out By default, the GeneScan Analysis Software scales each
electropherogram horizontally to show all peaks detected during the
run. While this provides a good overview of the run, some peaks may
be quite compressed.

Improving Visibility
To improve visibility, you can change the horizontal scale of the
electropherograms by zooming.

Zooming affects:
♦ Only the horizontal scale, and zooms the middle portion of the
window
♦ All displayed electropherogram panels

How to Change the View Scale

If you want to... Then...

see views with greater detail ♦ Select Zoom In (Ctrl++) from the
View menu, or
♦ Click the magnifying glass in the
upper-left corner of the window,
and drag around a specific area
to zoom in on the
electropherogram.
see a smaller scale view of the data ♦ Select Zoom Out (Ctrl+ -) from
after zooming in the View menu, or
♦ Click the magnifying glass cursor,
hold down the Alt key, and click
the electropherogram.
The data appears in successively
smaller scale views.
quickly scale the data so that the Select Zoom Out (Full Range) from
entire length fits within the window, the View menu.
again

Evaluating Electropherograms 9-17

Showing Off-Scale This section contains the following information:
Data
Topic See Page
Procedure 9-18
About Flat-Topped Peaks 9-18
Electropherogram Displaying Off-Scale Data 9-19
Electropherogram Displaying the Flat-Topped Effect 9-19

Procedure

To show off-scale data:

Step Action
1 Select Preferences from the Settings menu and Results Display
from the submenu.
The Results Display Preferences dialog box opens.
2 Select the Show Offscale Regions check box to highlight with a red
bar regions in the electropherogram that contain off-scale data (see
“Electropherogram Displaying Off-Scale Data” on page 9-19).

Note Select the Zoom In (Ctrl++) command from the View menu
to more clearly show the areas of off-scale data.

If... Then...
the sample was sized the Analysis Log lists the
numbers of off-scale regions in
the analysis range for each
sample file.

Note You can toggle this command for individual

electropherograms by selecting Hide/Show Offscale Regions
(Ctrl+`) from the View menu.

About Flat-Topped Peaks

An additional feature is that peaks that contain off-scale data points are
drawn in the electropherograms as “flat topped;” that is, the top section
of the peak is flat rather than pointed (see “Electropherogram
Displaying the Flat-Topped Effect” on page 9-19).

9-18 Evaluating Electropherograms

 This feature can be seen when the data is analyzed with no or light
smoothing; the flat-topped peaks may not be apparent with heavy
smoothing.

Select Analysis Parameters from the Settings Menu (see “Data

Processing Options” on page 5-7).

Electropherogram The following is an example of an electropherogram displaying off-scale

Displaying data:
Off-Scale Data

Off-scale data

Electropherogram The off-scale peaks in the electropherogram in the expanded view

Displaying the below illustrate the flat-topped effect.
Flat-Topped Effect

Evaluating Electropherograms 9-19

 Showing Data by The following table explains how to show data by fragment size:
Fragment Size
If... Then...
you analyze your samples with an use the Align by Size (Ctrl+T)
internal size standard command from the View menu to
align the horizontal scale of the
electropherograms by fragment size
instead of by data point.

Note You can display data by size

only if you analyzed and performed
sizecalling of your samples using a
size standard.
For example, if Then...
you run two identical samples in the Align by Size command adjusts
different runs for run-to-run variations by aligning
peaks by size value. This eliminates
any apparent differences that were
caused by run discrepancies.

Note You can display overlaid

samples in the same dye in different
colors. See “Defining Custom Colors
in Electropherograms” on page 9-24.

How to Switch Between Size and Data Point Display

If you want to... Then...

show data by size select Align by Size (Ctrl+ T) from the View menu.
When the data is aligned by size, the menu command
changes to Align by Data Point.
Select the command again, to show the data aligned
by data point.
set the default peak select Preferences from the Settings menu, and select
alignment Results Display from the submenu.

You can use the Results Display Preferences dialog

box to set certain preferences that remain in effect
each time you display or print results data.
For more information, see “Changing How the Results
Are Displayed and Printed” on page 8-13.

9-20 Evaluating Electropherograms

 Changing the Changing the Horizontal Scale for All Electropherograms
Horizontal Scale
To change the scale of the horizontal axis for all electropherograms:
1 Display the electropherogram panels you want to change.
2 Select Horizontal Scale from the View menu.
The Horizontal Scale Parameters dialog box opens.
You can also move the cursor over the horizontal axis of a displayed
electropherogram and double-click.

3 Enter the increments represented by the tick marks for the

horizontal axis in the Tick Spacing box.
4 Enter a range in the entry fields labeled Display from and Display to.
5 Click OK.

Changing the Horizontal Scale for Individual Electropherograms

To change the horizontal scale for individual electropherograms:
Step Action
1 Display the electropherograms.
2 Move the cursor over the horizontal axis of the panel that you want
to change and double-click.
The Horizontal Scale Parameters dialog box opens.
3 Enter the increments represented by the tick marks for the
horizontal axis in the Tick Spacing box.
4 Enter a range in the entry fields labeled Display from and Display to.
5 Click OK.
The horizontal scale changes.

Evaluating Electropherograms 9-21

 Changing the Changing the Vertical Scale for All Electropherograms
Vertical Scale
To change the vertical scale for all electropherograms:
Step Action
1 Display the electropherogram panel that you want to change.
2 Select Vertical Scale from the View menu.
The Vertical Scale dialog box opens.

3 Enter the increments represented by the tick marks for the vertical
axis in the Tick Spacing box.
4 Enter a range in the entry fields labeled Display from and Display to.
5 Click OK.

9-22 Evaluating Electropherograms

 Changing the Vertical Scale for Individual Electropherograms

To change the vertical scale for individual electropherograms:

Step Action
1 Display the electropherograms.
2 Move the cursor over the vertical axis of the panel that you want to
change, and double-click.
The following dialog box opens:

3 Enter tick mark increments and a range.

4 Ensure that the Apply to all Electropherogram panels check box is
not selected.
Select the check box only to apply the changes to all displayed
electropherogram panels.
5 Click OK.
The vertical scale changes only for the electropherogram panel that
you selected.

Assigning The following table describes how to assign standard or custom colors
Standard or and where to look for more information:
Custom Colors
To... Then...
assign standard or custom colors select the Plot Colors command from
the View menu and either Standard
or Custom from the submenu.
For information on defining custom
colors, see “Defining Custom Colors
in Electropherograms” on page 9-24.

Evaluating Electropherograms 9-23

Defining Custom Colors in Electropherograms

Introduction The GeneScan Analysis Software assigns a plot color to each

dye/sample added to an electropherogram. Normally, it is the color
associated with the individual dye/sample by the Dye Indicators
Preferences.
Note To change the default dye colors in the Analysis Control window and the
Results displays, see “Setting Dye Indicator Preferences” on page 3-14.

Note Custom plot colors are not available in the Sample Results view.

Why Change Change the colors in the electropherogram to:

Colors in the ♦ Differentiate between different samples labeled with the same color
Electropherogram dye
♦ Improve contrast between different dye colors
♦ Show data in a special color for a presentation
♦ Optimize plot colors for a particular printer

Saving the Display The following table describes the options to save the display format
Format after customizing the display colors:
If you... And then...
specify saving the display format of open it at a later time, the custom
a Results Display window after colors still appear.
customizing the display
For more information, see “Saving
and Renaming the Results Control
Format” on page 8-17.
do not save the display format after display the same results again. The
manually customizing the colors electropherograms are redrawn
using default colors.

9-24 Evaluating Electropherograms

Defining Custom To define custom colors for all electropherograms:
Colors
Step Action
1 Select Project Options from the Settings menus and Choose
Custom Plot Colors from the submenu.

The Custom Plot Colors dialog box opens.

2 Select new colors from the pop-up menus beside the 16 plot
numbers.

Note The plot numbers indicate the order of the samples in the
electropherogram legend.
3 Select Other from the pop-up menu to specify a color that does not
appear in the pop-up menu.
The following is an example of a color picker that opens:

Evaluating Electropherograms 9-25

 To define custom colors for all electropherograms: (continued)

Step Action
4 Position the cross hairs pointer on the color you want, and click.
The color appears in the Custom Color box.
5 To save the custom color, click Add to Custom Colors.
6 Click OK.
The Color Picker window closes.
7 In the Custom Plot Colors dialog box, select the check box labeled
Save As Defaults to save the customized colors.
8 Click OK.

Defining To define the individual plot colors:

Individual Plot
Step Action
Colors
1 Display the electropherograms with legends.
2 Double-click the plot color indicator next to the sample you want to
change.

Plot color
indicator

The Choose a Plot Color dialog box opens:

Pop-up menu

3 Select a new color from the pop-up menu.

If you select Other, then a color picker opens.

9-26 Evaluating Electropherograms

To define the individual plot colors: (continued)

Step Action
4 Click OK.
The color of the electropherogram for the individual sample
changes, and a vertical line appears beside the plot color indicator
to signify that it has been modified.

Note You can change the plot color in the same way from the
Results Control window. When you do so, the dye/sample is plotted
with the set color each time you open the applicable Results Display
window.

Note Press Ctrl and double-click the plot color indicator to reset it
to the original color.

Evaluating Electropherograms 9-27

Changing the Dye Scale in Electropherograms

What the Dye The dye scale defines how dyes in an electropherogram appear relative
Scale Defines to each other. You can compensate for peaks with different intensities
by redefining the dye scale.
Note Changing the dye scale affects only the display, not the underlying data.

Increasing the Dye The following table describes one way to increase the dye scale:
Scale Example
If... Then... Action...
you loaded a smaller the peaks for the green To make it easier to
amount of green sample might appear view both samples on
sample in relation to half as tall as those of the same scale,
the red sample the red sample. increase the dye scale
value of the green
sample to make the
peaks appear similar.

9-28 Evaluating Electropherograms

Changing the Dye To change the dye scale of an individual electropherogram:
Scale of an
Step Action
Electropherogram
1 Display the electropherograms with legends.
2 Double-click the dye color indicator next to the sample you want to
change.

Plot color
indicator

The Choose a Dye Scale dialog box opens.

3 Enter a new scale in the dialog box and click OK.

The dye scale for the individual sample changes, and a vertical line
appears beside the dye color indicator to signify that it is modified.

Note You can change the dye color in the same way from the
Results Control window. The dye is scaled each time you open the
Results Display window.

Evaluating Electropherograms 9-29

 Changing the Dye To change the dye scale preferences:
Scale Preferences
Step Action
1 Select Preferences from the Settings menu and select Results Dye
Scales from the submenu.

The Preferences dialog box opens with the Results Dye Scales
pop-up menu displayed.

2 Enter a positive number between 0.1 and 100 for each sample
relative to any other sample, and click OK.

Note Dye scale values do not automatically revert to default

values. Change them back to the defaults before examining results
of another run.

9-30 Evaluating Electropherograms

Process of Verifying Results

Introduction You can use the electropherogram and tabular displays to verify the
results of analysis by checking the GeneScan Analysis Software
calculated sizes and peaks.
Note The sizecalling of the standard and of sample fragments varies
according to the sizecalling method you defined in the Analysis Parameters and
the accuracy of the defined standard.

Steps to Verify Size To verify size calculations:

Calculation
Step Action See
1 Compare how well multiple size “Verifying Size Calculations” on
standard electropherograms page 9-33.
line up within a Results Display
window when aligned by size.
2 View the sizing curve calculated “Example of Size Curve View”
by the GeneScan Analysis on page 4-21.
Software.
3 Determine how well the defined “Sample Info View” on
size standard matches the size page 4-11.
standard run with your sample.
Use the Peak Total information in
the Sample Info view of the
Sample File window.
4 View the Analysis Log, which “Using the Analysis Log” on
provides messages for each page 9-36.
analyzed sample file.
If there is a problem or a
questionable condition during
sizecalling, a warning message
is displayed in the Analysis Log.
5 Use the Raw Data view to “Example of Raw Data View” on
display information about the page 4-23.
raw data for a sample.
Analyzed sample files contain
raw and analyzed data.

Evaluating Electropherograms 9-31

 To verify size calculations: (continued)

Step Action See

6 Use EPT Data to troubleshoot “EPT Data View Example” on
problems caused by poor run page 4-25.
conditions, such as:
♦ EP voltage
♦ EP current
♦ Laser power
♦ Run temperature versus time
EPT data can be displayed for
each sample file.

9-32 Evaluating Electropherograms

Verifying Size Calculations

Introduction This section describes the following topics:

♦ Verifying for the GeneScan-350 Standard
♦ Evaluating for Multiple Size Standards

Verifying for the About the Standard

GeneScan-350 In the GeneScan-350 standard, the size of the first peak should be
Standard approximately 50 bp, the second 75 bp, and so on, assuming that
processing started after the 35-bp fragment passed the scan region. If
peaks appear to be correctly measured for your run, measurement of
the sample fragments that ran with the standard should also be correct.
Note For a complete list of fragment sizes, refer to Appendix C, “GeneScan
Size Standards.”

Procedure

To verify the size calculation for the GeneScan-350 standard:

Step Action
1 In the Results Control window, select the Electropherogram button
and the Tabular button.
2 Click the Clear All button to clear the panels.
3 Click the dye color for the size standard you want to view.

Dye color fields

4 Click Display.
Note You can also open the Sample File window for the sample
file of interest to verify sizecalling.

Evaluating Electropherograms 9-33

 To verify the size calculation for the GeneScan-350 standard: (continued)

Step Action
5 Click each peak in the electropherogram and check the tabular data
to ensure it is the correct size (see figure below).

Note The peaks matched to the defined size standard are

identified by dots next to the Dye/Sample Peak field.
Indicates peak matched to defined size standard

6 If you find that the sizes were not calculated correctly, you can:
♦ Redefine the size standard, or
♦ Change the analysis parameters and re-analyze the affected
samples (see “Sizecaller Algorithm Flowchart” on page 5-4)

9-34 Evaluating Electropherograms

Evaluating for To evaluate size calculations for multiple size standards:
Multiple Size
Step Action
Standards
1 In the Results Control window, select the Electropherogram button.
2 Click the Clear All button to clear the panels.
3 Click the On button to turn on the Quick Tile option.
4 Click the dye colors for the size standards you want to view.

Dye color fields

When the Quick Tile option is on, the GeneScan Analysis Software
inserts each in a separate panel.

Note Click the header of the appropriate dye/sample column to

display all standards in the project that are the same color dye.
5 Click Display.
The standards appear in tiled electropherogram displays.
For information on setting the tiled electropherogram displays, see
“Creating Tiled Electropherogram Displays” on page 8-10.
6 Select Align by Size (Ctrl+T) from the View menu if the
electropherograms are not already aligned by size.
The size standards should line up when aligned by size.

Note You can set preferences so that all new displays show data
aligned by size by selecting Preferences from the Settings menu
and Results Display from the submenu. Click the Align By Size
check box.

For more information, see “Saving and Renaming the Results

Control Format” on page 8-17.

Evaluating Electropherograms 9-35

Using the Analysis Log

What Is the The Analysis Log maintains a running record of analysis performed by
Analysis Log the GeneScan Analysis Software. If a problem occurs during analysis of
a sample file, the Analysis Log automatically opens in the foreground as
an alert.

Displaying the Select Analysis Log (Ctrl+0) from the Windows menu.
Analysis Log
The following is an example of the Analysis Log:

9-36 Evaluating Electropherograms

What to Evaluate Evaluate the following:

What to evaluate What not to evaluate

Potential problems that the The GeneScan Analysis Software
GeneScan Analysis Software might will not alert you to any consecutive
have had during sizecalling. peaks at the end of the definition.
Analysis Log will alert you if more This is to avoid logging warnings
than two defined size standard when your sample was not run long
peaks were not matched. enough to include all the defined
size standard peaks.
This prevents you from having to
create a new standard for shorter
runs.
The Analysis Log will, however, alert
you if less than 50% of the defined
size standard peaks were not
matched, regardless of the peak
locations in the definition.

Removing To remove information from the Analysis Log:

Information from
Step Action
the Analysis Log
1 Select the information you want to remove.

Note Choose Select All (Ctrl+A) from the Edit menu to select all
the information.
2 Select Clear from the Edit menu.

Closing the You can either:

Analysis Log ♦ Click the Close button in the upper-left corner, or
♦ Select Close (Ctrl+W) from the File menu

Evaluating Electropherograms 9-37

Verifying Peak Detection

Introduction Use the Show Peak Positions command from the View menu, while the
electropherogram and associated tabular data are displayed, to verify
results by examining how the GeneScan Analysis Software defined the
total area that comprises each peak and the center of the peak.

Verifying Peak To verify peak detection:

Detection
Step Action
1 Select Show Peak Positions from the View menu.
Markers appear that identify the beginning, center, and end of each
peak.

Note For a better view, select Zoom In from the View menu, or use
the Zoom tool (see “Zooming In and Out” on page 9-17).

2 Examine the display to ensure that each peak’s center, beginning,

and end points are correct.

9-38 Evaluating Electropherograms

To verify peak detection: (continued)

Step Action
3 Select Hide Peak Positions from the View menu to suppress the
display of the peak markers.

Note You can also use the Sample Info view to display information
about the peaks detected and matched.

For more information, see “Description of Information” on

page 4-14.

Evaluating Electropherograms 9-39

Saving, Archiving, and
Copying Files
Overview
10 10
In This Section Topics in this chapter include the following:

Topics See Page

Why Save GeneScan Files 10-2
Saving GeneScan Files 10-3
Archiving Sample Files 10-4
Transferring Data to Other Applications 10-5

Saving, Archiving, and Copying Files 10-1

Why Save GeneScan Files

Reasons for Saving The following table explains why you save projects, sample files, and
Files Results Displays. For information on archiving files, see “Archiving
Sample Files” on page 10-4.

Save Because See

GeneScan ® Analysis It protects the links to “Saving Projects” on
Software projects sample files and their page 10-3.
preferences.
Projects contain links
to sample files and
preferences regarding
display and analysis.
Sample files It protects the links to “Saving Sample Files”
projects and their on page 10-3.
preferences.
Sample files also
contain raw data and
critical information
about the run, settings,
and analysis control.
Results Displays It saves the Results “Saving Results
Display settings in Displays” on
projects when you page 10-3.
have a display format
that suits your needs.

10-2 Saving, Archiving, and Copying Files

Saving GeneScan Files

Introduction This section describes how to save projects, sample files, and results
displays.

Saving Projects Note You do not need to save a sample file after analysis. The analyzed data
is written directly to the sample file during analysis.

The following table describes the options to save a project:

If you choose... Then...

Save Project you can take the following action:
(Ctrl+S)
If you... Then...
previously saved the it is automatically saved
project using the same name.
had not saved the the Save this document
project as dialog box opens.

Select a location for the

file, enter a name, and
click Save.

Save Project As the Save this document as dialog box opens.

Select a location for the file, enter a name, and click
Save.

Saving Sample To save sample files, select Save (Ctrl+S) from the File menu. If you
Files select Close from the File menu or click the Close button when you have
not saved the changes, a dialog box opens with a message asking if
you want to save them.

Saving Results You can combine electropherograms and tabular data in many ways for
Displays display, and the GeneScan Analysis Software allows you to save
display combinations and formats for future viewing.

For more information on saving a display for future viewing, refer to

“Saving and Renaming the Results Control Format” on page 8-17.

Saving, Archiving, and Copying Files 10-3

Archiving Sample Files

When to Archive Archive sample files when you feel confident that the channel selections
Sample Files (tracking) used to generate them were correct.

Procedure A sample file is 60 KB to 150 KB in size, depending on the length of the

run.
Step Action
1 To archive sample files, drag the file icon or the run folder
containing the files to the floppy disk icon or to an alternative
storage device.
2 A 1.4 MB high-density disk holds about 12 files.

10-4 Saving, Archiving, and Copying Files

Transferring Data to Other Applications

Genotyper GeneScan Analysis Software files can be read by Genotyper ® software

Software for Windows NT ® platform.

Cutting and To cut and paste tabular data:

Pasting Tabular
Step Action
Data
1 Display the tabular data you want to copy.
2 Select the rows you want to copy by taking the following action:

If you want to select... Then...

all tabular data Choose Select All (Ctrl+A) from
the File menu.
several consecutive rows Shift-click the first and last row
in the group you want to select.
several rows that are not listed Ctrl+click the rows.
next to each other

3 Select Copy (Ctrl+C) from the Edit menu.

4 Optional: To view the contents of the clipboard before pasting, from
the Edit menu, select Show Clipboard.
5 Open the new application and click where you want to place the
information.
6 Select Paste (Ctrl+V) from the Edit menu.

Creating a Text To create a text file from tabular data:

File
Step Action
1 Display the tabular data.
2 Select Export Table from the File menu.
The Save As dialog box opens.
3 Choose a name and file location in the dialog box and click Save.

Saving, Archiving, and Copying Files 10-5

Printing Results
Overview
11 11
In This Chapter Topics in this chapter include the following:

Topics See Page

About Printing 11-1
Printing Run Results Automatically 11-2
Printing Selected Sample Files 11-3

About Printing

Ways You Can You can print the results of the run automatically at the end of the run or
Print interactively, as selected sample files or display combinations.

If You Get You might initially get unexpected results from autoprinting or the Print
Unexpected One command if you switch printers.
Results
Step Action
1 The first time you print after changing the printer configuration,
select Print Setup (Ctrl+J) from the File menu and click OK.
2 Select Print (Ctrl+P) from the File menu and use the Print dialog
box.

Printing Results 11-1

Printing Run Results Automatically

Introduction You can specify that the results are printed in the 310 and 377 Data
Collection software or in the GeneScan® Analysis Software.

From the Data When you choose automatic printing from the 310 and 377 Data
Collection Collection software, the GeneScan Analysis Software prints a separate
Software page for each designated Sample file, showing electropherograms and
tabular data as specified in the Auto-Analysis Defaults (see step 2 on
page 2-6).

Choose automatic printing in the 310 and 377 Data Collection software
as follows:

On this instrument... Choose...

ABI PRISM® 310 Auto-Print in the Injection list.
ABI PRISM ® 377 Auto-Print in the Run Sheet.

For information about setting up your run, refer to the instrument user
manual.

Procedure To print the results automatically as the samples are analyzed:

Step Action
1 In the Analysis Control window, select the check box labeled Print
Results.

2 Select the samples you want to analyze.

For more information, see “Analyzing a Sample File” on page 4-26.
3 Click the Print Setup button to specify the samples and the format.
For more information, see “Specifying the Format for Printed
Results” on page 3-8.
4 Click OK.
The results are printed after the results are analyzed.

11-2 Printing Results

Printing Selected Sample Files

Introduction Print selected sample files by using the Results Control window or by
choosing the sample file.

Setting Printing To set the printing options:

Options
Step Action
1 Select Preferences from the Settings menu and Results Display
from the submenu.
The Results Display Preferences dialog box opens.
2 Use the Printing Preferences section, change the electropherogram
height and page breaks.

Depending on how you set these options, the format that prints may
be different from what is on the screen.
For information on printing saved Results Control formats, see
“Working with a Previously Saved Display” on page 8-18.

Printing from the Print selected samples after analysis, regardless of whether you
Results Control choose automatic printing.
Window To print results for selected sample files after analysis:

Step Action
1 In the Results Control window, select the dye/samples and format
you want to print.
Use the same technique as you did to select the format and the
dye/samples to display the data.
For more information, see “Using the Results Control Window” on
page 8-7.

Printing Results 11-3

 To print results for selected sample files after analysis: (continued)

Step Action
2 You can take the following action:
You can either... Then...
Click the Print button. The Print dialog box opens.
Select Print (Ctrl+P) from the Make any changes to the
File menu. settings and click OK.
Select Print One from the File The sample files are printed.
menu.
Note The Print dialog box
does not appear.

Printing from the To print a sample file from the File menu:
File Menu
Step Action
1 Select Open from the File menu.
The Open Existing dialog box opens.
Note You can also double-click the sample file name in the folder
containing the files. If the GeneScan ® Analysis Software is not
running, the software starts and opens the sample file.
2 Click the Sample icon.
An Open dialog box opens.
3 In the dialog box, navigate to the folder and select the sample file
that you want to open.
4 Click Open.
The Sample File window opens.
For more information about the Sample File window, see page 4-8.
5 Select one of the five views of the Sample File window, and select
Print or Print One from the File menu.

Note If you select Print One, then the Print dialog box does not
appear.

11-4 Printing Results

Creating GeneScan
Analysis Modules A
Overview
A
In This Chapter Analysis modules provide the auto-analysis feature with the parameters
to use for the GeneScan ® Analysis Software. For more information
about analysis parameters, refer to Chapter 5, “Working with Analysis
Parameters.”

This appendix includes the following topics:

Topic See Page

About GeneScan Analysis Modules A-2
Creating GeneScan Analysis Modules A-5

Creating GeneScan Analysis Modules A-1

About GeneScan Analysis Modules

Introduction The GeneScan Analysis Software modules contain the following

analysis options:
♦ Analysis range to use
♦ Statistical method used to fit the standard curve to the size
standards data

Analysis Module GeneScan analysis modules have the file name format filename.gsp
Format and are stored in the Params folder.
The path is: D:\AppliedBio\Shared\Analysis\Sizecaller\Params

Files Referenced GeneScan analysis modules reference two types of companion files
by the Module that contain other analysis parameter information.

Default Settings The following table describes the default settings for the analysis
parameters:
File type Description
Sizecaller Size standards are specific DNA fragments of known
standard file sizes. After defining the peaks of a size standard, the
GeneScan ® Analysis Software matches this definition
to the internal size standard included with the run. The
software assigns the defined size values to the
appropriate peaks of the internal size standard, and
uses this information with the selected sizecalling
method to size all unknown fragments.
Size standard setting is used only during auto-analysis
by 3100 and 3700 Data Collection software.
For more information, refer to “What Are Size
Standards” on page 7-2.

A-2 Creating GeneScan Analysis Modules

The following table describes the default settings for the analysis
parameters: (continued)
File type Description
Sizecaller There are two sizecaller parameter files. The format is
parameter file filename.scp, and they are stored in the SizeStandards
folder at the following directory location:
D:\AppliedBio\Shared\Analysis\Sizecaller\Params

File Use this file...

ABISizecallerAu when the run data is being
toAnalysis.scp analyzed automatically (the
first time it is analyzed).
ABISizecallerGS if the data is re-analyzed.
Analysis.scp

Note Do not move or delete these files.

Creating GeneScan Analysis Modules A-3

 Relationship The relationship between an analysis module and the two companion
Between a Module files is illustrated below.
and the Files
Filename.scp

Filename.gsp

Filename.scp

Filename.szs Filename.szs

Written in ABI Format, a The sizecaller standard fie and

GeneScan Analysis sizecaller parameter file are
Software module contains referenced while the analysis
the name of the basecaller module is being read
settings file to be referenced. automatically.

Never Modify the Never modify the sizecaller standard file or the sizecaller parameter file.
Files If you modify one of these files, it will no longer work.

A-4 Creating GeneScan Analysis Modules

Creating GeneScan Analysis Modules

Summary of the Follow these steps to create an analysis module:

Procedure
Step Action
1 Review the size standards data and select the analysis parameters
that remove unwanted noise and peaks outside of the size range of
the standards.
See step 1 in the “Creating a Size Standard File” procedure below.
2 Create a size standard file (filename.szs) for the reviewed
standards.
See step 2 on page A-6 to step 10 on page A-9.
3 Review the sample file of a sample to be sized, selecting the
analysis parameters that optimize the appearance of the data.
See step 1 on page A-9 to step 4 on page A-11.
4 Save the analysis parameters as a new .gsp file, referencing the
size standard file just created.
See step 5 on page A-11 to step 8 on page A-12.

Creating GeneScan Analysis Modules A-5

 Creating a Size To create a size standard file:
Standard File
Step Action
Reviewing and Choosing a Size Standard as a Template
1 Review the size standard data and optimize the analysis
parameters.
2 Select New (Ctrl+N) from the File menu.
The Create New dialog box opens.

3 Click the Size Standard icon.

The Select Sample File browser box opens.

4 Navigate to the Completed folder at the following directory location:

D:\AppliedBio\DataExtractor\Completed

A-6 Creating GeneScan Analysis Modules

To create a size standard file: (continued)

Step Action
5 Select the GeneScan Analysis Software sample file, with the
extension .fsa, that you want to use as a template.

6 Click Open.
The Select Dye and Analysis Parameters dialog box opens.

7 From the Dye pop-up menu, select the dye that was used to label
the size standard DNA fragments.

Creating GeneScan Analysis Modules A-7

 To create a size standard file: (continued)

Step Action
8 From the Analysis Parameters pop-up menu, select Analysis
Parameters.

This references the current analysis parameter setting rather than a

specific analysis parameter file.
9 Click OK.
The following is an example of the dialog box that opens:

A-8 Creating GeneScan Analysis Modules

To create a size standard file: (continued)

Step Action
10 In the Size column, enter the known sizes of the standard’s peaks.

Saving the Size Standard

1 Select Save from the File menu.
The Save this document as dialog box opens.
2 Navigate to and open the Size Standards folder (see below).
The folder contains size standards (.szs) files.
The path is
D:\AppliedBio\Shared\Analysis\Sizecaller\SizeStandards

3 Enter a name in the File name text box for the size standards files,
and click Save.
The dialog box closes and the file is saved to the correct location for
auto-analysis to read.
4 Click the Close button ( ) in the newly created Filename.szs
dialog box.
The dialog box closes.

Creating GeneScan Analysis Modules A-9

 Creating an To create an analysis parameter file:
Analysis
Step Action
Parameter File
1 In the GeneScan Analysis Software, select New from the File menu.
The Create New dialog box opens.

2 Click the Analysis Parameters icon. An untitled Analysis Parameters

dialog box opens.

Note To display the orange Peak Amplitude Threshold use the

scroll bar under the values and scroll to the right.

Note The Size Standard option is only specific to the 3100 and
3700 instruments.

A-10 Creating GeneScan Analysis Modules

To create an analysis parameter file: (continued)

Step Action
3 Complete the dialog box using the definitions in the “Sizecaller
Algorithm Flowchart” on page 5-4.
4 In the AutoAnalysis Only group box, select from the pop-up menu
the size standard that you just created.

5 Select Save from the File menu.

The Save this document as dialog box opens.
6 Navigate to and open the Params folder.
The path is:
D:\AppliedBio\Shared\Analysis\Sizecaller\Params

Creating GeneScan Analysis Modules A-11

 To create an analysis parameter file: (continued)

Step Action
7 Enter a file name for the analysis parameter file in the File name text
box, and click Save.
This saves the file to the correct location for auto-analysis to read.
8 Click the Close button in the newly created Filename.gsp dialog
box.

A-12 Creating GeneScan Analysis Modules

Sizecalling Methods B

Overview
B
In This Appendix Topics in this appendix includes the following:

Topic See Page

Least Squares Method B-2
Cubic Spline Interpolation Method B-4
Local Southern Method B-5
Global Southern Method B-7

Sizecalling Methods B-1

Least Squares Method

About This Both Least Squares Methods (2nd Order and 3rd Order) use regression
Method analysis to build a best-fit sizecalling curve. This curve compensates for
any fragments that may run anomalously. As a result, this method
normally results in the least amount of error for all the fragments,
including the size standards and the samples.

Depending on whether you choose the 2nd or 3rd Order Least Squares
Method in the Analysis Parameters dialog box, the resulting size curve
is either a quadratic function or a cubic function. The software uses the
known standard fragments and the associated scan number positions
to produce a sizing curve based on multiple linear regression.

Least Squares The first figure below shows the 2nd Order Least Squares sizecalling
Sizecalling curve, and the second figure shows the 3rd Order Least Squares
Examples sizecalling curve.

Figure B-1 2nd Order Least Squares sizecalling curve

B-2 Sizecalling Methods

 Figure B-2 3rd Order Least Squares sizecalling curve

Advantages In nearly all instances in the “Least Squares Sizecalling Examples”

above, the mobility of an individual DNA fragment is coincident with the
best curve fit of the entire data set. Stated differently, the mobility of
most DNA fragments is strictly length dependent. This method
automatically compensates for fragments that run anomalously.

GeneScan ® Analysis Software calculates a best-fit least squares curve

for all samples, regardless of the sizecalling method you choose. The
curve is black in the Standard Sizing Curve window.

Sizecalling Methods B-3

Cubic Spline Interpolation Method

About This By definition, the Cubic Spline Method forces the sizing curve through
Method all the known points of the selected GeneScan size standard. Although
this produces exact results for the values of the standards themselves,
it does not compensate for standard fragments that may run
anomalously.

Possible Local Mobility of any DNA fragment can be affected by its sequence and by
Sizing Inaccuracy secondary and tertiary structure formation. If any internal size standard
fragment has anomalous mobility, the Cubic Spline Method may exhibit
local sizing inaccuracy.
For example: Assume that a standard fragment is close in molecular
length to an unknown sample fragment. Assume further that the
standard fragment runs anomalously. The Cubic Spline Method assigns
the official value to this standard fragment, even though it may be
slightly incorrect. The size of the unknown fragment is then likely to be
calculated incorrectly as well.
Note This method does not determine the amount of sizing accuracy error.

B-4 Sizecalling Methods

Local Southern Method

About This The Local Southern Method determines the sizes of fragments by using
Method the reciprocal relationship between fragment length and mobility, as
described by E. M. Southern (1979).

The Equation The following equation attempts to describe the reciprocal relationship
between the mobility, m, and the length, L0, of the standard fragments:
L = [c/(m – m0)] + L0

Sizecalling Methods B-5

 How This Method This method, which is similar to the Cubic Spline Method, uses the four
Works fragments closest in size to the unknown fragment to determine a
best-fit line value. Using this method, only the region of the size ladder
near the fragment of unknown length is analyzed.
Note Size estimates may be off if any of the standard fragments run
anomalously.

The following table summarizes how the Local Southern Method works:
Step Action
1 The fitting constants of the curve are calculated for each group of
three neighboring points on the standard.
A separate curve is created for each set of three points.
2 A curve is then created by using three standard points (two points
below and one point above the fragment), and a fragment size is
determined.
3 Another curve is created by looking at an additional set of three
points (one point below and two points above the fragment) and
another value is assigned.
4 The two size values are averaged to determine the unknown
fragment length.

B-6 Sizecalling Methods

Global Southern Method

About This This method is similar to the Least Squares Method in that it
Method compensates for standard fragments that may run anomalously. The
method creates a best-fit line through all the available points, and then
uses values found on that line to calculate the fragment values.

The Equations The following table describes how the equations work:

Equation Description
L = [c/(m – m0)] + L0 Attempts to describe the reciprocal
relationship between the mobility, m,
and the length, L0, of the standard
fragments.
∑i(Li - (c/(mi – m0) + L0))2 The fitting constants L0, m0, and c
are calculated by a least squares fit
to minimize the following quantity.

Sizecalling Methods B-7

 How This Method All points in the standard are weighted equally, and the curve is not
Works constrained to go through any specific point. The software can analyze
a large range of fragment sizes with this method.

DNA fragments that are... Are sized using...

not bracketed within the size a second-order least squares curve
standard curve extrapolation.
bracketed within the size standard the method that was chosen.
curve

For best results, use a standard that brackets all the fragments of
interest.

B-8 Sizecalling Methods

GeneScan Size
Standards
Overview
C C
About the Size The GeneScan Analysis Software comes with several ready-to-use size
Standards standard definition files that you can choose from to analyze fragments
run on the ABI PRISM ® 3700 DNA Analyzer. The size standards are
stored in the SizeStandards folder.

The path is:

D:\AppliedBio\Shared\Analysis\Sizecaller\SizeStandards

See also “About Size Standards” on page 7-2.

Size Standards The following table lists the ready-to-use size standards:
Included
Size Standard See Page
GS 120.szs C-2
GS 350 All.szs C-4
GS 400HD.szs C-9
GS 500 All.szs C-11

The table below lists size standards in which some of the fragments
have been set to 0.
Note You can easily remove any of these sizes by opening the definition and
setting any unwanted sizes to 0.

Size Standard See Page

GS 350 377.szs C-7
GS 350-250.szs C-8
GS 500 377.szs C-13
GS 500-250.szs C-14

GeneScan Size Standards C-1

GeneScan 120 Size Standard

About This Size You can use the GeneScan-120 LIZ size standard to determine
Standard fragment lengths between 15 and 120 base-pairs.

Special Uses This size standard was designed to provide accurate sizing of short
DNA fragments. Therefore, it is particularly useful for SNP analysis. All
fragments have been checked for migration that is true to size under a
wide variety of run conditions. There are no anomalous fragments.

How It Is Prepared All aspects of the preparation of the GeneScan 120 LIZ size standard
are proprietary. Each fragment contains a single LIZ fluorophore.

Fragment Lengths The following table lists the lengths of the nine fragments that comprise
the GeneScan 120 LIZ size standard:

15
20
25
35
50
62
80
110
120

Denaturing The GeneScan 120 LIZ size standard is made of single-stranded DNA
Electropherogram fragments. The following figure shows the peak patterns of GeneScan
120 fragments run under denaturing conditions. Fragments were run
using the 3700 POP-5 polymer at 60 °C.

C-2 GeneScan Size Standards

Electropherogram The following is an electropherogram of GeneScan 120 LIZ:
of GeneScan 120
LIZ

GeneScan Size Standards C-3

GeneScan 350 All Size Standard

About This Size The GeneScan 350 All size standard contains sizes for all fragments in
Standard the GS 350 size standard. This size standard is useful for sizing
fragments between 35 and 350 base-pairs. The native fragments are
uniformly spaced to provide accurate sizecalling.

How It Is Prepared The GeneScan 350 All size standard is prepared by Pst 1 digestion of
plasmid DNA, followed by ligation of a TAMRA or ROX-labeled 22-mer
oligodeoxynucleotide to the cut ends. A subsequent enzymatic
digestion with BstU 1 yields DNA fragments containing a single TAMRA
or ROX dye (see “GeneScan 350 Molecular Lengths” below).

GeneScan 350 The following table lists the GeneScan 350 Denatured Fragment
Molecular Lengths Molecular Lengths (Nucleotides):
35 160
50 200
75 250
100 300
139 340
150 350

C-4 GeneScan Size Standards

 Running Under The following table describes running the GeneScan 350 All standard
Denaturing under denaturing conditions:
Conditions

Like the GeneScan 2500 and However, like the GeneScan

GeneScan 1000 standard... 250 standard... Consequently
the GeneScan 350 standard is the GeneScan 350 standard under denaturing conditions,
made of double-stranded DNA has only one labeled strand. even if the two strands migrate
fragments. at different rates, only the
labeled strand is detected.
Refer to “Electropherogram of
GeneScan 350” below.
Because of this, split peaks are
avoided that result when two
strands move through a
denaturing polymer at different
rates.

GeneScan Size Standards C-5

Electropherogram The following is an electropherogram of GeneScan 350 run under
of GeneScan 350 denaturing conditions:

Double-Stranded The following figure shows the sizes of double-stranded GeneScan 500
GeneScan 500 fragments. Use these values to size fragments run under native
Fragments conditions.
IMPORTANT An asterisk (*) for the 250 and 340 base-pair peaks denotes
peaks resulting from abnormal migration of double strands that did not
completely separate under denaturing conditions when analyzed on the 3100
and 3700 instruments. Do not use these peaks to size samples. The peaks
show smaller values than the actual size of the fragments.

C-6 GeneScan Size Standards

GeneScan 350 377 Size Standard

About This Size The GeneScan 350 377 size standard contains all GS 350 fragment
Standard sizes except the 35 and 50 base-pair sizes. The two smallest fragments
(35 and 50 base-pairs) are often lost in the primer peak on gel
instruments. The construction of this size standard differs from the GS
500 377.szs. This is because its electropherogram begins only after the
50 base-pair peak (depending on the run conditions), so the two
smallest sizes are not present and do not need to be set to 0 (as in the
GS 500 377 size standard).

This size standard can be used on any instrument.

GeneScan 350 377 The following table lists the GeneScan 350 denatured fragment
Molecular Lengths molecular lengths (nucleotides):
75 200
100 250
139 300
150 340
160 350

Electropherogram The following is an electropherogram of GeneScan 350 377 run under

of GeneScan denaturing conditions:
350 377

GeneScan Size Standards C-7

GeneScan 350-250 Size Standard

About This Size The GeneScan 350-250 size standard contains all GS 350 fragment
Standard sizes except the 250 base-pair size, which has been set to 0.

GeneScan 350-250 The following table lists the GeneScan Denatured Fragment Molecular
Molecular Lengths Lengths (Nucleotides):
35 160
50 200
75 0
100 300
139 340
150 350

Electropherogram The following is an electropherogram of GeneScan 350-250 run under

of GeneScan denaturing conditions:
350-250

C-8 GeneScan Size Standards

GeneScan-400HD Size Standard

About This Size Use the GeneScan-400HD (High Density) size standard to determine
Standard fragment lengths between 50 and 400 base-pairs.

Special Uses The high density of marker bands in this standard makes it particularly
useful for microsatellite analysis. All fragments have been checked for
migration that is true to size under a wide variety of run conditions on all
ABI PRISM ® instruments. There are no anomalous fragments
(compared with the 250-bp fragment in GeneScan 350 or 500 on the
3700 Analyzer).

GeneScan-400HD is the recommended size standard for use with the

ABI PRISM Linkage Mapping Sets.

How It Is Prepared All aspects of the preparation of the GeneScan-400HD size standard
are proprietary. Each fragment contains a single ROX fluorophore.

Fragment Lengths The following table lists the lengths of the 21 fragments that make up
the GeneScan-400HD size standard:

50 160 260 360

60 180 280 380
90 190 290 400
100 200 300
120 220 320
150 240 340

Denaturing Although the GeneScan-400HD size standard is made of

Electropherogram double-stranded DNA fragments, only one of the strands is labeled.
Consequently, even if the two strands migrate at different rates under
denaturing conditions you will not need to worry about peak splitting.
The following figure shows the peak patterns of GeneScan-400HD
fragments run under denaturing conditions. Fragments were run using
the 3700 POP-6™ polymer at 50 °C.

GeneScan Size Standards C-9

Electropherogram The following is an electropherogram of GeneScan-400HD:
of
GeneScan-400HD

C-10 GeneScan Size Standards

GeneScan 500 All Size Standard

About This Size The GeneScan 500 All size standard contains all fragments in the GS
Standard 500 size standard. This size standard is useful for sizing fragments
between 35 and 500 base-pairs. The native fragments are uniformly
spaced to provide accurate base calling.

How It Is Prepared The GeneScan 500 All size standard is prepared by Pst 1 digestion of
plasmid DNA, followed by ligation of a TAMRA or ROX-labeled 22-mer
oligodeoxynucleotide to the cut ends. A subsequent enzymatic
digestion with BstU 1 yields DNA fragments containing a single TAMRA
or ROX dye (see “GeneScan 350 Molecular Lengths” below).

GeneScan 500 The following table lists the GeneScan 500 denatured fragment
Molecular Lengths molecular lengths (nucleotides):
35 250
50 300
75 340
100 350
139 400
150 450
160 490
200 500

Running Under Like the GeneScan 2500 and GeneScan 1000 standard, the GeneScan
Denaturing 500 standard is made of double-stranded DNA fragments. However,
Conditions with the GeneScan 500, only one strand of the double-stranded DNA is
labeled, whereas the other two standards have labels on both strands.
Consequently, under denaturing conditions, even if the two strands
migrate at different rates, only the one labeled strand is detected.
Because of this, split peaks are avoided that result when two strands
move through a denaturing polymer at different rates.

Refer to “Electropherogram of GeneScan 500” below.

GeneScan Size Standards C-11

Electropherogram The following is an electropherogram of GeneScan 500 run under
of GeneScan 500 denaturing conditions:

Double-Stranded The following figure shows the sizes of double-stranded GeneScan 500
GeneScan 500 fragments. Use these values to size fragments run under native
Fragments conditions.

C-12 GeneScan Size Standards

GeneScan 500 377 Size Standard

About This Size The GeneScan 500 377 size standard includes all GS 500 fragment
Standard sizes except the 35 and 50 base-pairs, which have been set to 0. The
two smallest fragments (35 and 50 base-pairs) are often lost in the
primer peak on gel instruments.

This size standard can be used on any instrument.

GeneScan 500 377 The following table lists the GeneScan 500 377 denatured fragment
Molecular Lengths molecular lengths (nucleotides):
0 250
0 300
75 340
100 350
139 400
150 450
160 490
200 500

Electropherogram The following is an electropherogram of GeneScan 500 377 run under

of GeneScan 500 denaturing conditions:
377

GeneScan Size Standards C-13

GeneScan 500-250 Size Standard

About This Size The GeneScan 500-250 size standard has the 250 base-pair peak set
Standard to 0, since this peak does not migrate as it should on capillary
instruments.
Note The 340 base-pair peak is still present in this file and may not migrate
properly. You can check this for the run conditions by changing the 340
base-pair definition to 0, and then use the GeneScan Analysis Software to size
the size standard. If you are not happy with GeneScan’s size for the 340
base-pair fragment, then it is not migrating properly and you should not use it for
your definition.

GeneScan 500-250 The following table lists the GeneScan 500-250 denatured fragment
Molecular Lengths molecular lengths (nucleotides):
35 0
50 300
75 340
100 350
139 400
150 450
160 490
200 500

C-14 GeneScan Size Standards

Electropherogram The following is an electropherogram of GeneScan 500-250 run under
of GeneScan denaturing conditions:
500-250

GeneScan Size Standards C-15

Troubleshooting the
GeneScan Software D
Introduction
D
In This Appendix The tables in this section present information about problems you might
experience with your GeneScan ® Analysis Software runs and suggest
possible causes and corrections.

Topics in this appendix include the following:

Topics See Page

Troubleshooting Projects and Results D-2
Troubleshooting Gel Data D-4
Troubleshooting Genotyping Results D-7
GeneScan Analysis Software Error Messages D-8

Troubleshooting the GeneScan Software D-1

Troubleshooting Projects and Results

Table Description The following table describes the problem, probable cause, and
correction for troubleshooting projects and results:
Problem Probable Cause Correction
File name is dimmed in The file has been a. Move the file back to
Size Standard or moved from the folder its original location.
Parameters column in of its original location. b. Reset preferences
Analysis Control to specify the new
window. folder location.
c. Create or select a
new file.
Peaks appear on ♦ Peak Amplitude a. Adjust minimum
display but the Threshold set too peak height to
GeneScan Analysis high. include smallest
Software does not peaks desired and
♦ Minimum Peak Half
detect them (cannot re-analyze.
Width set too high.
select them in b. Reduce minimum
electropherogram ♦ Electrophoresis run peak half-width
display). too quickly resulting setting and
in poor resolution. re-analyze.
c. Repeat
electrophoresis at
reduced power.
For more information,
refer to “Peak
Detection Options” on
page 5-8.
At the position of one Off-scale data not a. Repeat
strong peak additional multicomponented electrophoresis;
colors appear correctly. load less sample.
underneath the peak. b. Regenerate sample
files.
Peaks appearing in a Bleed-through from Repeat
dye color that should other colors because of electrophoresis; load
not be present. off-scale data. less sample.

D-2 Troubleshooting the GeneScan Software

The following table describes the problem, probable cause, and
correction for troubleshooting projects and results: (continued)
Problem Probable Cause Correction
Peak centers seem to ♦ Resolution of the Repeat electrophoresis
be incorrect in gel might be at lower power.
electropherogram. inadequate (ABI
PRISM® 377).
♦ Signal-to-noise ratio
might be too low.
Software cannot ♦ Sample’s in-lane ♦ Re-analyze the
display the sizing curve size standard does sample file with a
for a sample. not match defined different size
size standard. standard or create a
new one.
♦ Sample file was not
sizecalled. In the Analysis Control
window:
a. Ctrl+click
dye/sample that
represents Size
Standard for the
sample file.
b. Select a size
standard definition
file and re-analyze.
Peaks disappear in the Included the primer Re-analyze sample file
electropherogram. peak in the analysis. without primer peak.

Troubleshooting the GeneScan Software D-3

Troubleshooting Gel Data

Table Description The following table lists the problem, probable cause, and correction for
troubleshooting of gel data:
Problem Probable Cause Correction
At the position of one ♦ Off-scale data not a. Repeat
strong peak, additional multicomponented electrophoresis;
colors appear correctly. load less sample.
underneath the peak.
♦ Poor / incorrect b. Attach a new gel
matrix. matrix, regenerate
the gel image.
♦ Gel Image not
multicompo- c. Regenerate the gel
nented. image with
multicomponenting
selected.
Peaks appearing in a Bleed-through from Repeat
dye color that should other colors because of electrophoresis; load
not be present. off-scale data. less sample.
TAMRA-labeled size Collected using: Repeat electrophoresis
standard appears with:
♦ Filter set A
yellow on the gel
(ABI 373), or ♦ Filter set B
display.
(ABI 373), or
♦ Virtual filter A
(ABI PRISM 377). ♦ Virtual filter C
(ABI PRISM 377).
TET-labeled products Collected using: Repeat electrophoresis
not seen on gel display. with:
♦ Filter set A
(ABI 373), or ♦ Filter set B
(ABI 373), or
♦ Virtual filter A
(ABI PRISM 377). ♦ Virtual filter C
(ABI PRISM 377).

D-4 Troubleshooting the GeneScan Software

The following table lists the problem, probable cause, and correction for
troubleshooting of gel data: (continued)
Problem Probable Cause Correction
Signal showing up in Leaking wells of gel. ♦ Consider using a
neighboring lanes. square-tooth comb
instead of a
shark-tooth comb.
♦ If using 96 lanes,
then rerun gel
using protocol in
the ABI PRISM 377
DNA Sequencer
96-Lane Upgrade
User’s Manual
(P/N 4305423).
Signal intensity very Move tracker lane
high and signal is position from center of
being detected in band to edge of the
neighboring lanes due band away from strong
to closeness of signal and extract as
spacing. usual.
Use 1- or 2-lane
averaging to extract
lanes.
HEX-labeled products Collected using: Repeat electrophoresis
appear green on gel with:
♦ Filter set A
display.
(ABI 373), or ♦ Filter set B
(ABI 373), or
♦ Virtual filter A
(ABI PRISM 377). ♦ Virtual filter C
(ABI PRISM 377).
Collection time was ♦ Gel Image a. Regenerate gel
sufficient, but only a Processing image with new
small portion of gel preferences did scan range.
displayed. not include b. Adjust to correct
enough scans to settings; repeat
display entire gel. electrophoresis.
♦ Electrophoresis
power too low.

Troubleshooting the GeneScan Software D-5

 The following table lists the problem, probable cause, and correction for
troubleshooting of gel data: (continued)
Problem Probable Cause Correction
Improper tracking Bad matrix. Attach new matrix.
results. Sample Sheet not filled Fill out Sample Sheet
out properly. properly.
Comb types set a. Fix and type in gel
improperly. preferences.
b. Retrack gel.
Peak height or red Rerun gel with more
signal too low. size standard.

D-6 Troubleshooting the GeneScan Software

Troubleshooting Genotyping Results

Table Description The following table describes the problem, probable cause, and
correction for troubleshooting the Genotyper ® software results:

Problem Probable Cause Correction

Allele peaks seen in ♦ Bleed-through from a. Repeat
correct molecular other colors because electrophoresis;
weight range, with of off-scale data. load/inject less
additional peaks sample.
♦ Primers not fully
seen outside this b. Check optimization.
optimized.
range.
With allele peaks of ♦ Background above a. Adjust minimum
high intensity, the minimum peak peak height
GeneScan Analysis height. threshold;
Software calls many re-analyze.
♦ Too much PCR
small peaks. b. Repeat
product loaded.
electrophoresis;
load/inject less
sample.
For more information,
refer to “Peak Detection
Options” on page 5-8.
A homozygous Truncated single peak Repeat electrophoresis;
individual shows a because of off-scale load/inject less sample.
dip at the top of an data can appear as two
allele peak which peaks.
may be called as two
separate peaks.
Warning message: The first allele peak for a. Adjust minimum
“Could not complete one or more loci in the peak height;
‘Run Macro’ allelic ladder is lower re-analyze.
command because than the preset b. Repeat
the labeled peak minimum peak height electrophoresis;
could not be found.” specification in the load/inject less
categories list. sample.
For more information,
refer to “Peak Detection
Options” on page 5-8.

Troubleshooting the GeneScan Software D-7

GeneScan Analysis Software Error Messages

Introduction This section includes two tables:

♦ Analysis Log Error Messages
♦ Error Messages When Defining Size Standards

Analysis Log The following table describes the error messages you might encounter
Error Messages in the GeneScan Analysis Software Log:
Analysis Log Error
Message Comment/Correction Refer To
The Analysis Range Make sure the Analysis “Setting Analysis
parameter does not Range in your analysis Parameters” on
include enough data parameters contains at page 5-5.
points. least 250 data points.
Check your analysis
parameters.
The Range of Data Specify a smaller “Setting Analysis
Points parameter to range in the analysis Parameters” on
analyze is too large. parameters. page 5-5.
Check your analysis
parameters.
The analysis Make sure the Analysis “Setting Analysis
Parameters could not Parameters file Parameters” on
be accessed. specified in the page 5-5.
Analysis Control
Check your Analysis
window is valid and
Parameters Setting.
accessible.

D-8 Troubleshooting the GeneScan Software

Error Messages The following table describes the error messages you might encounter
When Defining while defining size standards:
Size Standards
Error Message Comment/Correction Refer To
The affected sample If the sample file name “Finding Missing
file is not available. is dim in the Analysis Sample Files” on
Control window, the page 2-14.
Locate the sample file
GeneScan Analysis
and try again.
Software has not
located the sample file.
You can instruct the
program to search for
the sample file.
A Dye Standard is not Select the dye/sample
selected for the that represents the
affected sample file. standard by
Ctrl+clicking the
Select a Dye Standard
appropriate
and try again.
dye/sample field.
The affected sample Select either: “Using Analysis
file does not have a Parameter Files” on
♦ The default program
valid Analysis page 5-13.
parameters
Parameters Selection.
(<Analysis
Select new analysis Parameter >), or
parameters and try ♦ A valid analysis
again. parameters file in
the Analysis Control
window.
No peaks were found ♦ Make sure the Peak “Sizecaller Algorithm
within the Analysis AmplitudeThreshold Flowchart” on
Range. setting allows for page 5-4.
detection of the
Check your analysis
peaks in your
parameters.
sample.
♦ If peaks in your data
are narrow, make
sure the Minimum
Peak Half Width is a
small number.

Troubleshooting the GeneScan Software D-9

GeneScan Analysis
Software Files E E
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.

Table E-1 GeneScan Analysis Software files

File Type Created by Location Where is it used
.fsa 3700 Data Extractor In a folder often with the title GeneScan software
3100 Data Extractor "Run Folder" can be used to display,
Gel Processor (377) view and edit any .fsa
310 Data Collection file.
.gel 377 Data Collection In a folder often with the title The gel file contains
"Run Folder" the raw data collected
during the instrument
run. The gel file is
tracked and data is
extracted into sample
files by Gel Processor.
filename.gsp Shipped with D:\AppliedBio\Shared\Analysis These files specify
GeneScan software. \Sizecaller\Params certain ranges and
Users can create methods used during
custom files. data analysis. Users
can create custom .gsp
files.
filename.szs Shipped with D:\AppliedBio\Shared\Analysis These files are used to
GeneScan software. \Sizecaller\SizeStandards identify peak sizes for
Users can create specified size
custom files. standards run under
certain conditions.
Users can define the
standards after running
them on the
instruments.

GeneScan Analysis Software Files E-1

Table E-1 GeneScan Analysis Software files (continued)

File Type Created by Location Where is it used

filename.prj GeneScan Software User can specify location. This Projects contain
file is often stored in a folder references to Sample
with the sample files in of the files. Sample files of a
corresponding project single project can be
from one or multiple
runs. Projects allow a
group of data to be
organized, displayed
and analyzed together.
filename.mxt GeneScan Software, Matrix files must be
using the Matrix created for each
making feature instrument. The matrix
standards are collected
on 310 and 377
instruments. These
files are used to create
.mxt files using
GeneScan Analysis
software.
Sample GeneScan Software D:\AppliedBio\GeneScan\Bin Contains a running
log.log record of analysis
performed by the
software
Analysis GeneScan Software D:\AppliedBio\GeneScan\Bin Contains a running
log.log record of analysis
performed by the
software

E-2 GeneScan Analysis Software Files

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Technical Support F-1

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F-2 Technical Support

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Technical Support F-3

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F-4 Technical Support

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Technical Support F-7

License and
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License and Warranty G-1

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License and Warranty G-3

Glossary
This glossary defines special terminology used in the GeneScan Analysis Software User’s
Manual. The terms are listed in alphabetical order. Many terms are defined in the text of the
manual, so if you do not find a term here, check the index to see if you can locate it in the
manual.

align-by-size curve Curve created by the GeneScan ® Analysis Software for aligning data by
size.
The software calculates a best-fit, least-squares curve for all samples.
This is a third-order curve when you use the Third Order Least Squares
sizecalling method; for all other sizecalling methods it is a second-order
curve.
This curve is black in the Standard Sizing Curve window, although when
the sizing curve and this curve match, they overlap so you see only the
sizing curve.
See also sizecalling curve and size standard spline interpolation curve.

Align By Size/Data Shows the horizontal scale of the electropherograms by fragment size or
Point by data point.
You can display data by size only if you ran an internal size standard with
your samples and sizecalled the data.

analysis parameters Options that specify certain ranges and methods used during analysis
using the GeneScan Analysis Software.
The software has default analysis parameters that are stored in the project
itself.
These parameters apply globally, unless you create your own parameters
files for use with specific protocols.

baselining Adjusting the baselines of detected dye colors to the same level for a
better comparison of relative signal intensity.

Glossary-1
 Sequence Collector Sequence Collector (formerly BioLIMS™) is a genetic information
management system that provides a relational database for storage and
retrieval of DNA sequence and fragment data.
In addition to the database itself, Sequence Collector contains a set of
software applications and tools for maintaining and interacting with the
database.
The Sequence Collector database resides on a UNIX workstation and
uses a Sybase ® or an Oracle ® database server.
The client applications run on Windows NT ®-based computers and/or on
UNIX workstations.

data point The 3700 Data Collection program samples data as it passes by the
detector.
Each “sampling” is stored as a data point.

dye color indicator Left color box in the Results Control window and the legend of the Results
Display.
In the Results Display, click this box to move the associated
electropherogram to the front.
In the Results Control and the Results Display windows, double-click this
box to change the dye scale, or Ctrl+double-click it to reset the dye scale
to the default.

dye/sample Individual sample labeled with a single dye within a sample file.
Sample files normally contain up to four dye/samples, depending on how
many labeled samples you included in each lane or injection of your 3700
Data Collection program run.

electropherogram Four-color picture of a sequence, showing peaks that represent the bases.
The term is used interchangeably with chromatogram in this manual.

grid Spreadsheet-like display used for entering data in tabular format.

The Analysis Control and Results Control windows display grids for
entering sample information.

Glossary-2
internal size standard Also called internal lane or injection size standard, DNA fragment of
known size that you include with your run. On the ABI PRISM 3700 DNA
Analyzer you include the size standard with each injection.
Running an internal lane standard results in particularly accurate and
precise molecular length determination because the internal lane standard
and the unknown fragments undergo exactly the same electrophoretic
forces.
The software can then compensate for band-shift artifacts caused by
variations in the run.

legend Informational text that appears beneath electropherogram panels in the

results displays.
You can show or hide legends, and use the color boxes displayed in them
to bring specified electropherograms to the front of the panel, or customize
the colors.

matrix File used to adjust for the spectral overlap between the fluorescent dyes
file/multicomponent used on the ABI PRISM® instruments. A mathematical matrix of the
matrix spectral overlaps is created and the inverse matrix is used to correct the
data during analysis. Matrix files are stored in the AppliedBio folder inside
the Macintosh® computer System folder, or in the matrix folder in the
GeneScan Analysis Software folder. The values of the matrix are stored in
the gel file (ABI 373 and ABI PRISM 377) and in the Sample files.
For more information, see Chapter 6, “Making a Matrix File.”

overlaid Displayed together so they overlap.

In the GeneScan Analysis Software Results Display window, all
electropherograms in a single panel are overlaid.
You can bring a specific one to the front by clicking the color box that
represents it in the legend.

plot color indicator Right color box in the Results Control window and the legend of the
Results Display.
In the Results Display window, click this box to move the associated
electropherogram to the front.
In the Results Control and the Results Display windows, double-click this
box to change the plot color, or Ctrl+double-click it to reset the plot color to
the default.

Glossary-3
 preferences Defaults you can set so that certain parameters are automatically applied
when you are working with a project.
The GeneScan Analysis Software remembers preferences and applies
them globally to all new projects.

project File containing links to a set of sample files that you want to analyze and
display together.
A project can contain sample files from multiple runs. Adding a sample file
to a project creates a reference to the file. It does not copy the file into the
project.

project options Formatting information you can set for the current project. Project options
are remembered by the project when you open it again.

sample files Computer files that contain raw and analyzed data.
Sample files are created directly by the 3700 DNA Analyzer. Sample files
contain data such as peak locations, sizecalling values, and a record of
analysis settings.

sizecalling curve Curve created by the GeneScan Analysis Software for sizecalling.
The software calculates this curve based on the sizecalling method you
specify for data analysis.
This curve is read in the Standard Sizing Curve window. When it matches
the align-by-size curve, the two overlap so you see only this curve.
See also “align-by-size curve” and “size standard spline interpolation
curve.”

size standard Specific DNA fragments of known sizes.

After you define the peaks of a size standard, the GeneScan Analysis
Software matches this definition to the internal lane or injection standard
that you include with your run.
The software assigns the defined size values to the appropriate peaks of
the internal lane or injection standard, and uses this information with the
selected sizecalling method to size all unknown fragments.

Glossary-4
size standard spline Curve created by the GeneScan Analysis Software for aligning data by
interpolation curve size.
The software creates this curve if you use the Local Southern or Cubic
Spline Interpolation sizecalling method and the size standard data does
not match the best-fit curve, which is normally used for aligning the data by
size.
This curve is blue in the Sizing Curve window. See also align-by-size curve
and sizecalling curve.

tiled Displayed so they do not overlap.

The GeneScan Analysis Software displays tiled electropherogram panels
in the Results Display.
If you display more than one electropherogram in each panel, all
electropherograms in the panel are overlaid.

Glossary-5
 Index
A analysis parameters (continued)
ABI 373 using (continued)
generating sample files the same parameters 5–13 to 5–14
procedure 6–15 to 6–16 analysis results
ABI Prism 310 changing how displayed and printed 8–13
automatic analysis diagram 2–2, 2–3 to 8–15
ABI PRISM 310 Results Display window
loading and running dye standards 6–9 to about 8–4 to 8–6
6–11 using 8–7 to 8–12
ABI PRISM 377 steps to evaluating
generating sample files 6–15 to 6–16 evaluating results 8–2
loading and running dye standards 6–12 to updating the results 8–16
6–14 using the Analysis Log 9–36 to 9–37
Analysis Control window ways to display 8–3
using to analyze project files 3–2 to 3–5 See Also Sample Results View
using to analyze sample file 3–6 to 3–15 analyzing
Analysis Log, using 9–36 to 9–37 project files 3–2 to 3–5
analysis modules sample files 3–6 to 3–15
about A–2 to A–4 AppliedBio folder, files installed in 7–6
creating archiving 10–4
analysis parameter file A–10 to A–12 Auto Analysis, setting analysis parameters 5–12
size standard file A–6 to A–9 automatic analysis, setting up 2–4 to 2–6
analysis parameter file A–10 to A–12
analysis parameters B
about 5–2 Baseline Window Size, setting analysis
setting 5–5 to 5–12 parameters 5–12
Analysis Range options 5–7
Auto Analysis Only option 5–12
Baselining options 5–12 C
Data Processing options 5–7 CD-ROM drive 1–10
default settings 5–5, A–2 colors
displaying analysis parameters 5–6 assigning to electropherograms 9–23
Peak Detection options 5–8, 5–9 to defining in electropherograms 9–24 to 9–27
5–10 copying data to other applications 10–5
Sizecall Range options 5–10 Cubic Spline Method B–4
Sizecalling Method options 5–10 to customer support. See technical support F–1
5–11
using 5–13 to 5–17 D
changing existing parameters 5–17 Data Collection software
creating custom parameters 5–16 automatically printing results from 11–2
deleting custom parameters 5–17 deleting, custom analysis parameters 5–17
different parameters 5–15 disk space recommended 1–10
displaying default parameters 5–16

Index-1
Documents on Demand F–6 G
dye color, changing in electropherograms 9–14 gel files
dye scale, changing 9–28 to 9–30 troubleshooting gel data D–4 to D–5
GeneScan
E about the size standards C–1
electropherograms analysis modules
about 9–2 to 9–3 about A–2 to A–4
changing dye scale 9–28 to 9–30 creating A–6 to A–12
defining custom colors 9–24 to 9–27 creating analysis parameter file A–10
displaying electropherogram data 9–9 to to A–12
9–11 See Also analysis parameters
displaying electropherograms and tabular archiving 10–4
data 9–4 to 9–8 copying, data to other applications 10–5
using to verify peak detection 9–38 registering 1–8
using to verify results 9–31 to 9–32 related manuals 1–7
verifying size calculations 9–33 to 9–35 saving
working with electropherogram data 9–12 projects 10–3
to 9–23 Results Displays 10–3
assigning standard colors 9–23 sample files 10–3
changing horizontal scale 9–21 why save 10–2
changing the dye color 9–14 size standard
changing vertical scale 9–22 to 9–23 GeneScan 350 All C–4 to C–7
displaying peak positions 9–14 GeneScan 400HD C–9
displaying x-and y-axis positions 9–13 GeneScan 500 All C–11 to C–12
highlighting peaks 9–15 GeneScan 500 to 250 C–14
making active window 9–13 software files, table of E-1 to E-2
scrolling the display 9–16 GeneScan 500 All size standard C–11 to C–12
showing data by fragment size 9–20 GeneScan-400 HD size standard C–9 to C–10
Genotyper software, troubleshooting software
about 9–20 results D–7
showing off-scale data 9–18
Global Southern Method B–7 to B–8
using legends to change the
display 9–15
zooming in and out 9–17 H
e-mail, address for technical support F–1 hard drive partitions 1–11
EPT Data View 4–24 to 4–25 hardware and software requirements to run
displaying the view 4–24 program 1–10
example 4–25 help. See technical support F–2
what it displays 4–24 horizontal scale, changing in
error messages D–8 to D–9 electropherograms 9–21

F I
files, software files, table of E-1 to E-2 Internet address
folders Documents on Demand F–6
AppliedBio folder, files installed in 7–6
defining locations 3–16 L
Least Squares sizecalling method B–2 to B–3

Index-2
legends, using to change display in peak positions, displaying in
electropherograms 9–15 electropherograms 9–14
Local Southern Method B–5 to B–6 peaks, highlighting in electropherograms 9–15
printing
M about printing 11–1
automatically printing run results 11–2
Macintosh, converting sample files to the
printer recommended 1–10
NT 4–3
sample files 11–3 to 11–4
macintosh, converting sample files to the
projects
Windows NT platform 4–4
analyzing project files 3–2 to 3–5
manual
creating
related manuals 1–7
finding missing sample files 2–14 to
matrix files
2–15
about 6–2 to 6–6
process diagrams, using the ABI Prism
assigning to sample files 6–23 to 6–24
310 2–2, 2–3
bad matrix files, causes 6–26 to 6–27
using to manage sample files 2–6 to
choosing a scan range 6–17 to 6–19
2–7
defined Glossary–3
working with 2–8 to 2–13
evaluating the matrix file 6–25
creating a new project 2–9 to 2–13
generating files (ABI 373 and ABI PRISM
defining folder locations 3–16
377) 6–15 to 6–16
importance of saving 10–2
installing new file
opening an existing project 2–8
sample file 4–27
removing samples from a project 2–13
loading and running dye standards
saving, procedure 10–3
ABI PRISM 310 6–9 to 6–11
troubleshooting D–2 to D–3
ABI PRISM 377 6–12 to 6–14
new matrix file, generating 6–20 to 6–21
process of creating new file 6–7 to 6–8 R
saving and naming 6–22 Raw Data View 4–22 to 4–23
monitor, recommended 1–10 displaying the view 4–22
multicomponent matrix defined Glossary–3 example 4–23
what it displays 4–22
O what to evaluate 4–23
registering the software 1–8
off-scale data
Registration Number 1–8
showing in electropherograms 9–18
requirements, hardware and software 1–10
Operating System requirement 1–10
Results Control window
saving and renaming 8–17 to 8–19
P important considerations 8–17
partitions, hard drive 1–11 renaming current display 8–19
PCR Single-stranded Conformation saving the format 8–17
Polymorphism (SSCP) using previously saved formats 8–18
Minimum Peak Half Width setting 5–8 working with previously saved
peak detection displays 8–18
setting analysis parameters 5–8, 5–9 to Results Display window
5–10 about 8–4 to 8–6
verifying 9–38 using 8–7 to 8–12

Index-3
Results Displays saving (continued)
importance of saving 10–2 Results Control format 8–17 to 8–19
saving, procedure 10–3 Results Displays 10–3
sample files 10–3
S why save 10–2
Show Clipboard command 10–5
Sample File window
Size Curve View 4–20 to 4–21
about 4–8
size standard file A–6 to A–9
EPT Data View 4–24 to 4–25
size standards
Raw Data View 4–22 to 4–23
about size standards 7–2
Sample Info View 4–11 to 4–19
about the size standards C–1
Sample Results View 4–9 to 4–11
defining, how to 7–3 to 7–8
Size Curve View 4–20 to 4–21
file, creating for analysis module A–6 to
sample files
A–9
about 4–2 to 4–3
GeneScan 350 377 C–7
analyzing 4–26 to 4–27
GeneScan 350 All C–4 to C–7
installing new matrix file 4–27
GeneScan 400HD C–9
procedure 4–26
GeneScan 500 377 C–13
using Analysis Control window 3–6 to
GeneScan 500 All C–11 to C–12
3–15
GeneScan 500 to 250 C–14
archiving 10–4
using 7–9 to 7–14
converting Macintosh files to the NT 4–3
Sizecall Range, setting analysis
converting Macintosh files to the Windows NT
parameters 5–10
platform 4–4
sizecalling methods
file size 1–10
Cubic Spine Method B–4
finding missing sample files 2–14 to 2–15
Global Southern Method B–7 to B–8
matrix file, assigning to sample files 6–23 to
Least Squares Method B–2 to B–3
6–24
Local Southern Method B–5 to B–6
opening, procedure 4–7
setting analysis parameters 5–10 to 5–11
printing 11–3 to 11–4
software
removing from a project 2–13
files, table of E-1, E-1 to E-2
Sample File window
software, registering 1–8
about 4–8
SSCP, Minimum Peak Half Width setting 5–8
EPT Data view 4–24 to 4–25
Raw Data View 4–22 to 4–23
Sample Info View 4–11 to 4–19 T
Sample Results View 4–9 to 4–11 tabular data displays
Size Curve View 4–20 to 4–21 about 9–2 to 9–3
saving, procedure 10–3 displaying electropherogram and tabular
unlocking sample files 2–12 data 9–4 to 9–8
using project to manage files 2–6 to 2–7 using to verify peak detection 9–38
why save 10–2 using to verify results 9–31 to 9–32
Sample Info View 4–11 to 4–19 technical support F–1 to F–7
description of view 4–11 to 4–19 e-mail address F–1
Sample Results View 4–9 to 4–11 Internet address F–6
saving regional sales offices F–4 to F–5
matrix file 6–22 telephone/fax (North America) F–2
projects 10–3

Index-4
troubleshooting
bad matrix files, causes 6–26 to 6–27
error messages D–8 to D–9
gel data D–4 to D–5
Genotyping software results D–7
projects and results D–2 to D–3

U
unlocking sample files 2–12

V
vertical scale, changing 9–22 to 9–23
viewing scale, changing for
electropherograms 9–17

W
WWW address
Applied Biosystems F–6
Documents on Demand F–6

X
x-axis positions, displaying in
electropherograms 9–13

Y
y-axis positions, displaying in
electropherograms 9–13

Z
zooming, electropherograms 9–17

Index-5
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Printed in the USA, 01/2001

Part Number 4308923 Rev. B