GeneMapper® Software Version 4.

Loss of Heterozygosity Getting Started

(LOH) Analysis
Getting Started Guide
Setting Up
the Loss of
Heterozygosity
Analysis

Analyzing and
Examining
Results

Sorting Data and

Evaluating Loss of
Heterozygosity

Printing and
Exporting Results
GeneMapper® Software Version 4.1

Loss of Heterozygosity Getting Started

(LOH) Analysis
Getting Started Guide
Setting Up
the Loss of
Heterozygosity
Analysis

Analyzing and
Examining
Results

Sorting Data and

Evaluating Loss of
Heterozygosity

Printing and
Exporting Results
For Research Use Only. Not for use in diagnostic procedures.
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may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall
Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use
of this document.
Notice to Purchaser: License Disclaimer.
Purchase of this software product alone does not imply any license under any process, instrument or other apparatus,
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GeneMapper Software has not undergone specific developmental validation for human identification applications. Human
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TRADEMARKS:
Applied Biosystems, AB (Design), ABI PRISM, GeneMapper, LIZ and SNaPshot are registered trademarks and GeneScan and
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This product includes software developed by the Apache Software Foundation (http://www.apache.org/). Copyright © 1999-2000
The Apache Software Foundation. All rights reserved.
This product includes software developed by the ExoLab Project (http://www. exolab.org/). Copyright 2000 © Intalio Inc. All
rights reserved.
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AFLP is a registered trademark of Keygene N.V.
Oracle is a registered trademark of Oracle Corporation.
All other trademarks are the sole property of their respective owners.
© Copyright 2009, Applied Biosystems. All rights reserved.

Part Number 4403621 Rev. A

04/2009

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Contents

Preface v
How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . vi
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

Chapter 1 Getting Started 1

About LOH Microsatellite Analyses . . . . . . . . . . . . . . . . . . . . . . . . . 2
About the Example Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
LOH Microsatellite Analysis Workflow . . . . . . . . . . . . . . . . . . . . . . . 6
GeneMapper® Software Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Starting the Software and Logging In . . . . . . . . . . . . . . . . . . . . . . . 7
Using This Guide With Your Own Sample Files . . . . . . . . . . . . . . . . 8
Alternatives to the Procedures in This Guide . . . . . . . . . . . . . . . . . . 8

Chapter 2 Setting Up the Loss of Heterozygosity

Analysis 9
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Creating a Kit, Panel, and Markers . . . . . . . . . . . . . . . . . . . . . . . . 11
Creating a New Project and Adding Sample Files . . . . . . . . . . . . . 15
Setting Analysis Parameters and Table Settings for
the Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Performing the Initial Analysis on the Project . . . . . . . . . . . . . . . . . 26
Creating a Bin Set and Generating Bins
Using the Auto Bin Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

GeneMapper® Software Version 4.1 Microsatellite Analysis Getting Started Guide iii
Contents

Chapter 3 Analyzing and Examining Results 45

Editing the Analysis Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Analyzing the Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Examining the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Chapter 4 Sorting Data and Evaluating Loss of

Heterozygosity 53
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Sorting the LOH Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Generating a Report to Calculate and Evaluate LOH . . . . . . . . . . 58
Editing or Creating a Custom LOH Report Setting . . . . . . . . . . . . 59

Chapter 5 Printing and Exporting Results 65

Printing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Exporting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Index 69

iv GeneMapper® Software Version 4.1 Microsatellite Analysis Getting Started Guide

 Preface

How to Use This Guide

Purpose of This The GeneMapper® Software Version 4.1 LOH Analysis Getting
Guide Started Guide provides brief, step-by-step instructions for sizing,
genotyping, and evaluating LOH microsatellite data generated using
any of the compatible Applied Biosystems electrophoresis
instruments and Data Collection Software. It describes how to
troubleshoot, print and export data, and create reports. It is designed
to help you quickly learn to use basic functions of the GeneMapper
Software.

Audience This guide is intended for novice GeneMapper Software users.

Assumptions This guide assumes that:

• You have installed GeneMapper Software version 4.1 as
described in the GeneMapper® Software Version 4.1
Installation and Administration Guide (PN 4403614).
• You have a working knowledge of the Microsoft® Windows®
operating system.

Text Conventions This guide uses the following conventions:

• Bold indicates user action. For example:
Type 0, then press Enter for each of the remaining fields.
• Italic text indicates new or important words and is also used for
emphasis. For example:
Before analyzing, always prepare fresh matrix.
• A symbol separates successive commands you select from a
drop-down or shortcut menu. For example:
Select FileOpenSpot Set.
Right-click the sample row, then select View FilterView All
Runs.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide v

Preface
How to Obtain More Information

User Attention Two user attention words appear in Applied Biosystems user
Words documentation. Each word implies a particular level of observation
or action as described below:

Note: Provides information that may be of interest or help but is not

critical to the use of the product.

IMPORTANT! Provides information that is necessary for proper

instrument operation, accurate chemistry kit use, or safe use of a
chemical.

Examples of the user attention words appear below:

Note: The size of the column affects the run time.

Note: The Calibrate function is also available in the Control

Console.

IMPORTANT! To verify your client connection to the database, you

need a valid Oracle user ID and password.

IMPORTANT! You must create a separate Sample Entry Spreadsheet

for each 96-well plate.

Safety Alert Safety alert words also appear in user documentation. For more
Words information, see the GeneMapper® Software Version 4.1 Installation
and Administration Guide (PN 4403614).

How to Obtain More Information

Safety For safety information, see the GeneMapper® Software Version 4.1
Information Installation and Administration Guide (PN 4403614).

Software For all warranty and licensing information, see the GeneMapper®
Warranty and Software Version 4.1 Installation and Administration Guide
License (PN 4403614).

vi GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Preface
How to Obtain More Information

Related The following related documents are shipped with the software:
Documentation • GeneMapper® Software Version 4.1 Installation and
Administration Guide (PN 4403614) – Provides procedures for
installing, securing, and maintaining version 4.1 of the
GeneMapper Software.
• GeneMapper® Software Version 4.1 Getting Started Guides for
microsatellite analysis (PN 4403672), loss of hetereozygosity
(LOH) analysis (PN 4403621), AFLP® system analysis
(PN 4403620), SNaPshot® kit analysis (PN 4403618), and
SNPlex™ system analysis (PN 4403617) – Five guides that
explain how to analyze the application-specific example data
provided with the GeneMapper Software. The guides provide
brief, step-by-step procedures for the analysis of microsatellite,
LOH, AFLP® system, SNaPshot® kit, and SNPlex™ system data
generated by compatible Applied Biosystems electrophoresis
instruments and Data Collection Software. The guides are
designed to help you quickly learn to use basic functions of the
GeneMapper Software.
• GeneMapper® Software Version 4.1 Online Help – Describes
the GeneMapper Software and provides procedures for common
tasks. Access online help by pressing F1, selecting Help
Contents and Index, or clicking in the toolbar of the
GeneMapper window.
• GeneMapper® Software Version 4.1 Quick Reference Guide
(PN 4403615) – Provides workflows for specific analysis types
and lists instruments, software, and analysis applications
compatible with the GeneMapper Software.
• GeneMapper® Software Version 4.1 Reference and
Troubleshooting Guide (PN 4403673) – Provides reference
information such as theory of operation and includes
troubleshooting information.
Portable document format (PDF) versions of this guide and the other
documents listed above are available on the GeneMapper Software
Version 4.1 Documentation CD.

Note: For additional documentation, see “How to Obtain Support”

on page viii.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide vii
Preface
How to Obtain Support

Obtaining The GeneMapper Software features an online help system that

Information from describes how to use each feature of the user interface. Access online
Online Help help by pressing F1, selecting Help Contents and Index, or
clicking in the toolbar of the GeneMapper window.

Send Us Your Applied Biosystems welcomes your comments and suggestions for
Comments improving its user documents. You can e-mail your comments to:
techpubs@appliedbiosystems.com

How to Obtain Support

For the latest services and support information for all locations, go to
http://www.appliedbiosystems.com, then click the link for
Support.
At the Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates
of analysis, and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Support page provides access to worldwide telephone
and fax numbers to contact Applied Biosystems Technical Support
and Sales facilities.

viii GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide
 Chapter 1

Getting Started

Chapter 1
This chapter includes:
■ About LOH Microsatellite Analyses . . . . . . . . . . . . . . . . . . . 2
Getting Started
■ About the Example Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
■ LOH Microsatellite Analysis Workflow . . . . . . . . . . . . . . . . 6
■ GeneMapper® Software Terms . . . . . . . . . . . . . . . . . . . . . . . 7
■ Starting the Software and Logging In . . . . . . . . . . . . . . . . . . 7
Chapter 2 ■ Using This Guide With Your Own Sample Files . . . . . . . . . . 8
Setting Up the
Loss of Heterozygosity ■ Alternatives to the Procedures in This Guide. . . . . . . . . . . . . 8
Analysis

Chapter 3
Analyzing and
Examing Results

Chapter 4
Sorting Data and
Evaluating Loss of
Heterozygosity

Chapter 5
Printing and
Exporting Results

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 1

 Chapter 1 Getting Started
About LOH Microsatellite Analyses

About LOH Microsatellite Analyses

Microsatellite Microsatellite markers, also known as short tandem repeats (STRs),
Markers are polymorphic DNA loci consisting of a repeated nucleotide
sequence. The repeat sequence can be from 2 to 7 base pairs long.
The number of repeat units varies in a population, thereby creating
multiple alleles for a microsatellite locus.

Microsatellite In a typical microsatellite analysis, microsatellite loci are amplified

Analysis by PCR using fluorescently labeled forward and unlabeled reverse
primers. The PCR amplicons are separated by size using
electrophoresis; then the dye labeled products are identified by
fluorescence detection. You can then use the GeneMapper Software
to size and genotype the alleles.

What is LOH? In the two-hit model used to describe inactivation of tumor

suppressor genes (TSGs), the first mutation or “hit” results in a
heterozygous state for the TSG with one wild-type and one mutant
allele. If this is followed by a second “hit,” in which all or part of the
chromosome that contains the wild-type allele of the TSG is deleted,
the chances of tumorigenesis increase. This phenomenon is known as
Loss of Heterozygosity or LOH. While this is a rare event, it occurs
more frequently in familial forms of cancer, in which a mutation of
one of the TSG alleles is inherited.

LOH An LOH microsatellite analysis is the screening of tumor samples for

Microsatellite LOH using microsatellite markers. Because LOH can be caused by
Analysis deletion of genomic DNA regions containing the wild-type copy of a
TSG, researchers can use microsatellite markers to screen samples for
LOH.
A typical LOH assay compares amplified microsatellite markers
from the suspected cancerous tissue to the same markers from the
healthy tissue, both from the same individual. The healthy tissue
should show two alleles for a given heterozygous microsatellite
marker. If a given tumor sample has undergone LOH, it will be
evident by a decrease in peak height of one of the two alleles (relative
to the healthy allele peak heights) (Figure 1-1). The reason the tumor
sample shows a decrease in peak height (instead of an absence of the
peak) is that during isolation, the tumor sample is contaminated with
healthy cells, thus introducing some wild-type DNA into the tumor
specimen.

2 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 1 Getting Started
About LOH Microsatellite Analyses

Allele 1
Allele 1

Allele 2

Allele 2

Healthy Sample Diseased Sample

Figure 1-1 Diseased sample shows loss in peak height of Allele 2

After performing a microsatellite analysis, you can use the Report

Manager in the GeneMapper® Software to calculate and compare
peak height ratios of microsatellite alleles between healthy and
diseased tissues, as well as identify and flag LOH candidates based
on set peak-height ratio thresholds. You can alter these thresholds
based on the observed level of wild-type DNA contamination in the
tumor samples.

Custom Primers Applied Biosystems provides custom primers for PCR amplification
of LOH microsatellite markers. For more information, visit the
Applied Biosystems Web site at www.appliedbiosystems.com.

Compatible For information about Applied Biosystems electrophoresis

Instruments instruments that are compatible with LOH microsatellite analyses,
see the GeneMapper® Software Version 4.1 Quick Reference Guide
(PN 4403615).

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 3

 Chapter 1 Getting Started
About the Example Data

About the Example Data

Sample File The naming convention for the sample files used in this guide allows
Naming you to take advantage of the LOH Default report setting provided
Conventions with the GeneMapper® Software. Specifically, healthy and tumor
samples from the same individual start with the same letter or
number, so they list consecutively when sorted by sample file and
marker.
Example: 1_Healthy.fsa, 1_Tumor.fsa, 2_Healthy.fsa,
2_Tumor.fsa, and so on
When analyzing your own LOH data, if you want to use the LOH
Default report setting, your sample files must be named as described
above.
If your LOH data sample files are not named as described above, you
can still use the sorting and LOH reporting features in the
GeneMapper® Software, but you will have to edit the report setting
as described in “Editing or Creating a Custom LOH Report Setting”
on page 59. Below is an example naming convention in which
healthy and tumor samples from the same individual do not list
consecutively after being sorted by sample file and marker.
Example: Healthy_1.fsa, Healthy_2.fsa, …,Tumor_2.fsa,
Tumor_1.fsa, and so on

Sample File To perform the exercise described in this getting started guide, use
Location the four sample files (.fsa) located on your computer hard drive at:
<drive>:\AppliedBiosystems\GeneMapper\Example
Data\LOH

Note: The above location will vary depending on the installation of

the GeneMapper® Software. The default installation is the D drive.

Instrument and Sample files were generated by running PCR-amplified and

Size Standard fluorescently tagged LOH samples on an ABI PRISM® 3100 Genetic
Analyzer using the GeneScan™ 500 LIZ® Size Standard.

4 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 1 Getting Started
About the Example Data

Marker The sample files contain the following six markers.

Information
Marker Dye Label Allele Size Range (bp)

R5 Blue 120 – 186

R26 Blue 193 – 295
R14 Blue 300 – 470
R21 Yellow 100 – 160
R24 Yellow 170 – 250
R2 Green 157 – 204

You will use this marker information when creating a panel and
markers in Chapter 2, “Setting Up the Loss of Heterozygosity
Analysis.”

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 5

 Chapter 1 Getting Started
LOH Microsatellite Analysis Workflow

LOH Microsatellite Analysis Workflow

The following flowchart summarizes the steps for performing an
LOH analysis using the GeneMapper® Software:

Set Up the Loss of Heterozygosity Analysis

(Chapter 2)

1. Create a kit, panel, and markers for the project.

2. Create a new project and add sample files.
3. Set the analysis parameters and table settings
for the project.
4. Perform an initial analysis.
5. Create a bin set and generate bins (using
Auto Bin).

Analyze and Examine Results

(Chapter 3)

1. Edit the analysis method to specify a bin set.

2. Analyze the samples in the project.
3. Examine the results.

Sort Data and

Evaluate Loss of Heterozygosity
(Chapter 4)

1. Sort the LOH data in the Genotypes tab by

sample file and marker.
2. Generate a report to calculate and evaluate LOH.
3. (Optional) Edit or create a custom LOH report
setting.

Print and Export Results (Optional)

(Chapter 5)

• Print results.
• Export results.

6 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 1 Getting Started
GeneMapper® Software Terms

GeneMapper® Software Terms

Term Definition

analysis A collection of user-defined settings (including an

parameters analysis method, size standard, and panel) that
determine the sizing and genotyping algorithms used
by the GeneMapper® Software to analyze all sample
files in a project.
bin A fragment size (bp) and dye color that define an allele
within a marker. You create a bin for each possible
allele associated with a marker.
bin set A collection of bins (allele definitions), typically specific
to a set of experimental conditions.
marker A microsatellite marker is defined by a name, fragment
size range (bp), dye color, and repeat length.
panel A group of markers. In the GeneMapper Software, you
associate a panel with a bin set to provide bin
definitions for the markers.
kit A group of panels.

Starting the Software and Logging In

To start the GeneMapper® Software and log in:
1. Select StartAll ProgramsApplied Biosystems
GeneMapperGeneMapper 4.1.
2. In the Login to GeneMapper dialog box:
a. Type the User Name and Password assigned by your
system administrator.
b. Click OK.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 7

 Chapter 1 Getting Started
Using This Guide With Your Own Sample Files

Using This Guide With Your Own Sample Files

In addition to using this guide to analyze the example data provided
with the software, you can use this guide to lead you through the
general LOH microsatellite analysis workflow when analyzing your
own sample files. For information on advanced software features, see
the GeneMapper® Software Online Help.

Alternatives to the Procedures in This Guide

Overview This guide presents one of several possible solutions for analyzing
microsatellite LOH data using the GeneMapper® Software. Once you
have completed the exercises in this document, you will most likely
want to tailor the process to fit the requirements of your laboratory.
This section provides you with a summary of several alternatives and
where to go for further information.

Using The GeneMapper Software includes an Autoanalysis feature that can

Autoanalysis to eliminate most of the tasks leading up to the analysis of a
Set Up Projects microsatellite LOH project. Much of Chapter 2, “Setting Up the Loss
of Heterozygosity Analysis,” explains how to manually create, add
samples to, and analyze projects for use in microsatellite LOH
projects. When configured for Autoanalysis, the GeneMapper
Software can automatically accomplish these tasks by coordinating
with the Data Collection Software. For a more detailed explanation
of how to use the Autoanalysis feature to set up microsatellite LOH
projects, see the GeneMapper® Software Version 4.1 Installation and
Administration Guide (PN 4403614).

Using the The GeneMapper Software features a command line interface that
Command Line can perform most of the major functions of the software. The
Interface to Set command line interface can be a useful tool when analyzing
Up Projects microsatellite LOH projects because it automate many of the tasks
explained in Chapter 2, “Setting Up the Loss of Heterozygosity
Analysis.” For a complete description of the command line interface
and how it can be used to automate the functions of the GeneMapper
Software, see the GeneMapper® Software Version 4.1 Installation
and Administration Guide (PN 4403614).

8 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2

Setting Up the Loss of

Heterozygosity Analysis

Chapter 1 This chapter includes:

Getting Started
■ Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
■ Creating a Kit, Panel, and Markers . . . . . . . . . . . . . . . . . . . . 11
■ Creating a New Project and Adding Sample Files. . . . . . . . 15
■ Setting Analysis Parameters and Table Settings for
the Project. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Chapter 2 ■ Performing the Initial Analysis on the Project . . . . . . . . . . . 26
Setting Up the
Loss of Heterozygosity ■ Creating a Bin Set and Generating Bins
Analysis Using the Auto Bin Feature . . . . . . . . . . . . . . . . . . . . . . . . . 35

Chapter 3
Analyzing and
Examing Results

Chapter 4
Sorting Data and
Evaluating Loss of
Heterozygosity

Chapter 5
Printing and
Exporting Results

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 9

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Overview

Overview
In This Chapter In this chapter you will learn how to:
• Create a kit, panel, and markers
• Create a new project and add sample files
• Set analysis parameters and display settings for the project
• Perform the initial analysis on the project
• Create a bin set and generate bins using the Auto Bin feature

For More This chapter contains basic procedures. It does not describe all
Information features and parameters in the GeneMapper Software. For more
detailed information on topics presented in this chapter, see the
following topics in the GeneMapper® Software Online Help:
• Creating a New Kit
• Creating a Custom Panel
• Creating Markers
• Creating a Project
• Adding Samples
• Applying Analysis Settings
• Starting Analysis
• Creating a New Bin Set
• Using the Auto Bin Function
Online help is available from the Help menu, by clicking , or by
pressing F1.

10 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Kit, Panel, and Markers

Creating a Kit, Panel, and Markers

Overview You create the following hierarchical objects in the Panel Manager:
• Kit – A group of panels
• Panel – A group of markers
• Marker – A fragment size range (bp), dye color, and repeat
length

Note: In this guide, you will learn how to create panels and markers.
However, you can also import panels (text files) that contain marker
information. For example, panel files are available in the
GeneMapper® Software for some of the LMS kits available from
Applied Biosystems. For more information on importing panels, see
the GeneMapper® Software Online Help.

Creating a Kit, To create a kit, panel, and markers:

Panel, and
1. Open the Panel Manager by clicking (ToolsPanel
Markers
Manager).
2. Select Panel Manager at the top of the Navigation Pane (left
side), then click (FileNew Kit).

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 11

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Kit, Panel, and Markers

3. In the New Kit dialog box, type LOH Kit for the Kit Name,
select Microsatellite for the Kit Type, then click OK.

LOH Kit appears in the Navigation Pane (left side).

4. Select the LOH Kit in the Navigation Pane, then click

(FileNew Panel).

12 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Kit, Panel, and Markers

5. In the right pane of the Panel Manager, select New Panel, type
LOH Panel for the Panel Name, then press Enter.

LOH Panel appears under LOH Kit in the Navigation Pane (left
side).

6. Select the LOH Panel in the Navigation Pane, then click

(FileNew Marker).
7. In the right pane of the Panel Manager, type the following
marker information:

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 13

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Kit, Panel, and Markers

8. Repeat steps 6 through 7 to create the following markers:

Dye Minimum Maximum Marker
Marker
Color Size (bp) Size (bp) Repeat

R26 Blue 193 295 2

R14 Blue 300 470 2
R21 Yellow 100 160 2
R24 Yellow 170 250 2
R2 Green 157 204 2

9. Click OK to apply your changes and close the Panel Manager.

Next Steps Create a new project and add sample files (.fsa) to it as described on
page 15.

14 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a New Project and Adding Sample Files

Creating a New Project and Adding Sample Files

Overview You create a project and add samples to the project in the
GeneMapper window.

Creating a To create a new project and add sample files:

New Project and
1. Click (FileNew Project).
Adding
Sample Files

2. In the New Project dialog box, select Microsatellite, then click

OK.
3. From the GeneMapper window, click (FileAdd Samples
to Project).
4. In the Add Samples to Project dialog box, in the Files tab,
navigate to:
<drive>:\AppliedBiosystems\GeneMapper\Example
Data

Note: The above location will vary depending on the

installation of the GeneMapper® Software. The default
installation is the D drive.

5. Select the LOH folder, click Add to List, then click Add.
Note: For this guide you added all sample files in the LOH
folder. However, you can add a subset of files from a folder by
expanding the folder in the left pane, pressing and holding Ctrl,
then selecting individual files before clicking Add To List.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 15

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a New Project and Adding Sample Files

The four sample files from the LOH Data folder appear in the
Samples tab, along with information entered in the Data
Collection Software on the compatible Applied Biosystems
electrophoresis instrument.

Next Steps Set analysis parameters and display settings for the project as
described on page 17.

16 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

Setting Analysis Parameters and Table Settings for

the Project
Overview You set analysis parameters and display settings for the project in the
GeneMapper window.
Analysis parameters include:
• Analysis method (including bin set)
• Panel (set of markers)
• Size standard
You set analysis parameters that determine the peak detection, sizing,
and genotyping algorithms used by the GeneMapper® Software to
analyze all sample files in a project.
Display settings include Table Settings and Plot Settings.

Setting Analysis To set analysis parameters for the project:

Parameters
1. Select the Samples tab in the GeneMapper window.
2. Click the first row in the Analysis Method column, then select
New Analysis Method from the drop-down list.

Note: You can also create a new analysis method from the
Analysis Method tab in the GeneMapper Manager.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 17

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

3. In the New Analysis Method dialog box, select Microsatellite

for Analysis Type, then click OK.

4. In the Analysis Method Editor dialog box, select and edit the
five tabs:
• General – This tab includes reference information about
the method. Type LOH Analysis Method for the Name.
Optionally, type a description and the instrument on which
the data was generated.

18 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

• Allele – This tab includes settings that determine allele

calling. Select None for the Bin Set. Leave the default
values for all other settings.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

• Peak Detector – This tab includes settings that determine

peak detection and sizing of peaks. Select Basic for the
Peak Detection Algorithm. Leave the default values for all
other settings.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

• Peak Quality – This tab includes settings that determine

when specific PQVs are left green (Pass) or flagged
yellow (Check).
Type 2.0. for the Max peak width (basepairs).
Leave the default values for all other settings.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

• Quality Flags – This tab includes:

– Settings that determine the importance of individual
flagged Process Quality Values (PQVs) to the overall
Genotype Quality (GQ). You can weight each PQV from
0 to 1, with 0 being of no importance and 1 meaning
very important.
– Threshold settings that determine when the SQ and GQ
are flagged as Pass , Check , or Low Quality .
The SQ and GQ are given initial scores of 1. The value
of any flagged PQVs are then subtracted from 1 to give
the final SQ and GQ scores.
– Assume Linearity Range, where the size calling
algorithm assumes the fragment migration is linear for a
given size range when calculating the Sizing Quality
(SQ).
Leave the default values for all settings.

For details on analysis method parameters, see the

GeneMapper® Software Online Help.

22 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

5. Click OK to save the method and close the Analysis Method

Editor dialog box.
6. Select the first row in the Panel column. From the Select a Panel
dialog box, expand the LOH Kit, then double-click LOH
Panel. (This is the Panel you created on page 11.)

Click to expand
LOH Kit
Double-click

7. Select the first row in the Size Standard column, then select
GS500(-35, -250, -340)LIZ from the drop-down list.
Note: In the GeneMapper software, the following size standards
are available for use with samples run with the GeneScan™ 500
LIZ® Size Standard:
• GS500(-250)LIZ – excludes the 250-bp peak
• GS500(-35,-250,-340)LIZ – excludes the 35-, 250-,
and 340-bp peaks
• GS500LIZ – includes all peaks present in the actual
GeneScan™ 500 LIZ® Size Standard
Depending on your instrument, polymer type, and primer, it may
be appropriate to choose one of the other size standards that
omits peaks. Specifically, the 35-bp peak can be eclipsed by the
neighboring primer peak, or the 250- and 340-bp peaks can
migrate abnormally on the capillary electrophoresis instrument.
Additionally, you can create your own custom size standards.
For information on creating custom size standards, see the
GeneMapper® Software Online Help.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

8. Fill down your selections to all sample rows in the Samples tab:
a. Click-drag across the Analysis Method, Panel, and Size
Standard column headers to highlight all rows in all three
columns.

b. Select EditFill Down (or press Ctrl-D).

Selecting Table At the top of the GeneMapper window, select Microsatellite Default
Setting from the Table Settings drop-down list.

Table Settings control the information displayed in the Samples tab

and Genotypes tab after analysis. Microsatellite Default is one of the
default Table Settings provided with the GeneMapper Software.
You can also edit and create custom Table Settings in the
GeneMapper Manager. For more information, see the GeneMapper®
Software Online Help.

24 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Setting Analysis Parameters and Table Settings for the Project

Saving the To save the project:

Project
1. Click (FileSave Project).
2. In the Save Project dialog box, type LOH Project, then click
OK.

LOH Project appears in the title bar of the GeneMapper window.

Next Steps Perform the initial analysis on the project as described on page 26.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

Performing the Initial Analysis on the Project

Overview Now that you have added sample files to and set analysis parameters
for the project, perform an initial analysis to size the data so you have
sample files available as reference data to create bins (allele
definitions).

Note: Because you selected a Panel for the sample files in the
project, the GeneMapper® Software will not only size the data but
also try to genotype the data. However, because you did not specify a
bin set in the analysis method, the Genotype Quality (GQ) will fail.

To perform the initial analysis:

• Analyze the project
• Review the SQ and contributing PQVs
• Examine the size standard
• View sample information (including raw data)
• Viewing samples plots

Analyzing the Click (AnalysisAnalyze).

Project
The GeneMapper Software analyzes each sample in the project,
displaying its progress in the Status Bar (lower left) of the
GeneMapper window.

26 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

Reviewing the To review the Size Quality (SQ) and contributing PQVs:
SQ and PQVs
1. Make sure “Analysis Completed” appears in the Status Bar
(lower left) of the GeneMapper window.
2. Review the SQ by scrolling to the right in the Samples tab.

Click-drag scroll bar to right to view SQ column

If you followed the procedures and used the example data

indicated in this guide, the SQ for each sample is (Pass).
Most of the Process Quality Values (PQVs) that contribute to the
SQ (SFNF, MNF, SNF, and OS) should also be .

Investigating IMPORTANT! When analyzing your own data, you may find the
Yellow and SQ to be (Check) or (Low Quality) and associated PQVs
Red SQs (SFNF, MNF, SNF, and OS) to be , indicating issues with the
size standard, data, or analysis parameters. To investigate and
correct these issues, see “Examining the Size Standard” on
page 28.

Note: Click to sort the samples by SQ score. Samples with

a SQ will be listed at the top of the Samples tab.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

Examining the To examine the size standard:

Size Standard
1. Select all samples in the Samples tab by selecting EditSelect
All.
2. Open the Size Match Editor by clicking (AnalysisSize
Match Editor).

Size Quality (SQ) Score

Figure 2-1 Size Match Editor – Size Matches tab

3. Click the Size Matches tab to view the following for the
selected sample:
• Size Quality (SQ) score
• Size standard peaks
• Size standard peak labels
4. Note the Sizing Quality score (Figure 2-1) for the sample. This
score reflects how well the data from the size standard match the
size standard you selected in the software. This score determines
whether the SQ displays (Pass), (Check) or (Low
Quality).
If you followed the instructions in this guide, the Sizing Quality
is > 0.75 and the SQ displays (Pass).
However, when analyzing you own data you may notice the
Sizing Quality is less and the SQ displays (Check) or
(Low Quality). For troubleshooting help, see Table 2-1 on
page 30.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

5. Determine if all peaks in the size standard are present and

labeled correctly.
If you followed instructions in this guide, all peaks are present
and labeled correctly as shown in Figure 2-1.
However, when analyzing you own data you may find some size
standards peaks to be incorrectly labeled or missing. For
troubleshooting help, see Table 2-1 on page 30.
6. Click the Size Calling Curve tab to view the size standard curve
for the selected sample. You will see red data points
representing the fragments from the size standard and a black
best-fit curve.

7. Select another sample from the left pane of the Size Match
Editor, then repeat steps 3 through 6.
8. Click OK to close the Size Match Editor.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

Table 2-1 Troubleshooting the size standard

Problem Action

Sizing Quality score is low and Override the Sizing Quality by

the SQ displays (Check) or clicking Override SQ at the top of the
(Low Quality), but all size Size Matches tab (Figure 2-1).
standard peaks are present and Overriding changes the Sizing Quality
labeled correctly. score to 1.0, indicating the user
verified the size standard.
Some size standard peaks are Edit, delete, and add size labels in
not labeled correctly. the Size Matches tab, then click
Apply to reanalyze the data with the
updated sizing information. For more
information, see the GeneMapper®
Software Online Help.
Some size standard peaks are Create a custom size standard in the
not present. software. For more information, see
the GeneMapper® Software Online
Help.

For additional help in troubleshooting sizing problems, see the

GeneMapper® Software Reference and Troubleshooting Guide.

Viewing Sample To view information and raw data associated with individual sample
Information files, select a sample file in the Navigation Pane (left), then select the
Info or Raw Data tabs.

Select
sample file
to view
sample
information
and raw data

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

Viewing To view the plots of the samples:

Sample Plots
1. Select ViewSamples to display the Samples tab.
2. Select a sample (row) in the Samples tab. To select multiple
samples, press and hold Shift or Ctrl. To select all samples,
select EditSelect All.
3. Click (AnalysisDisplay Plots).
The Samples Plot window displays an electropherogram for
each selected sample.

Select
Microsatellite Default
for the Plot Setting

Zoom by
click-dragging
on top x-axis

Select the
number of plots
to display

4. Select Microsatellite Default for the Plot Setting.

Note: Plot Settings control the information displayed in the
Samples Plot window after analysis. Microsatellite Default is
one of the default Plot Settings provided with the GeneMapper
Software. You can also edit and create custom Plot Settings in
the GeneMapper Manager. For more information, see the
GeneMapper® Software Online Help.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

5. Zoom on the x- and y-axes in the Samples Plot:

To … Then …

Zoom on a specific Place the cursor on the top x-axis, then

region of the x-axis click-drag the right or left to zoom all
plots. Press and hold Shift while
click-dragging to zoom only the selected plot.
or
Right-click the top x-axis, select Zoom To,
type range, then click OK.
Zoom on a specific Place the cursor on the left y-axis, then
region of the y-axis click-drag the up or down.
or
Right-click the left y-axis, select Zoom To,
type maximum, optionally, select Apply to all
electropherograms, then click OK.
Unzoom Double-click the x-axis or y-axis.
or
Right-click the x-axis or y-axis, then select
Full View.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Performing the Initial Analysis on the Project

Examining Data Other tasks you can perform in the Samples Plot window include:
in the Samples • Adjust the scale of the x-axes (basepairs or data points)
Plot Window • Adjust the scale of the y-axes (scale to individual maximum,
global maximum, or a specific value)
• Show and hide specific dye color peaks
• Display a status line for individual peaks
• Display a Sizing Table, which displays a row of sizing
information for each detected peak
• Display a Genotypes Table, which displays a row of genotyping
information for each detected peak
• Select peaks, which highlights a corresponding row of data in
the Sizing Table
See Figure 2-2 on page 34 for an illustration of some of the above
features.
For more information on using the above features, press F1, then
select the desired topic from the GeneMapper® Software Online
Help.
When done viewing the Samples Plot, click to close the window.

Next Steps Create a bin set and generate bins using the Auto Bin feature and
reference data as described on page 35.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 33

34 Click to
show and hide
dye colors

Performing the Initial Analysis on the Project

Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Click to display
Sizing Table
below
GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

Place cursor
over peak to
display status
line and data in
Status Bar
Click, Ctrl-click, (lower left)
or Shift-click
peaks to select them
and highlight the
corresponding
rows of data in the
Sizing Table below

Hint: Press Ctrl-G

to clear the Sizing
Table, then click
individual peaks
Sizing Table to add their
associated
information to the
Sizing Table.

Status Bar

Figure 2-2 Examining and comparing data from different sample files in the Samples Plot
 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

Creating a Bin Set and Generating Bins

Using the Auto Bin Feature
Overview Use the Panel Manager to create bin sets and generate bins (allele
definitions).
Before you create a bin set, you must select a kit. You can then
associate the bin set with any panels in that kit.
Before you generate bins, you must select a panel and a bin set. Only
sample files in projects analyzed with that panel are available to add
as reference data to the selected panel to generate the bins. The bins
will be associated with markers in the selected panel and stored in the
selected bin set.

Note: In this guide, you will learn how to create bins using reference
data and the Auto Bin feature. However, you can also import bin sets
(text files) that contain bin information. Or you can create bins
manually. For more information on importing bin sets or creating
bins manually, see the GeneMapper® Software Online Help.

Creating a To create a bin set:

Bin Set
1. Open the Panel Manager by clicking (ToolsPanel
Manager).
2. In the Navigation Pane (left), select the LOH Kit you created on
page 11.
3. Click (BinsNew Bin Set).
4. In the New Bin Set dialog box, type LOH Bin Set for the Bin
Set Name, then click OK.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

The LOH Bin Set is added to the Bin Set drop-down list at the
top of the Panel Manager. The LOH Bin Set can now be
associated with the LOH Panel (or any other panels added to the
LOH Kit).

Adding Note: You can add all or only a subset of the sample files in a project
Reference Data as reference data. Because the tumor sample files would contain the
to a Panel and same alleles as their healthy counterparts, it is not necessary to add
Bin Set all the samples files. In this guide, you will use only the healthy
sample files as the reference data for use in creating a panel and bin
set.

To add reference data to the LOH Panel and LOH Bin Set:
1. Make sure the LOH Bin Set is selected in the Bin Set
drop-down list.

2. In the Navigation Pane (left), expand the LOH Kit, then select
the LOH Panel you created on page 11.

All the markers you created display on the right pane of the
Panel Manager (Figure 2-3 on page 38).

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

3. Click (BinsAdd Reference Data).

The Add Microsatellite Reference Data dialog box opens
displaying the LOH Project you created in the lower left pane.
Note: The lower left pane always displays all projects that have
been analyzed using the selected panel.

4. In the Add Microsatellite Reference Data dialog box:

a. Expand LOH Project.
b. Expand the LOH folder.
c. Press and hold Ctrl to select both healthy sample files.
d. Click Add to List.
e. Click Add.

Click to expand
LOH Project
and LOH Data folder

Select 2 healthy
sample files

Click Add To List,

then click Add

The selected sample files are added as reference samples to the

LOH Panel and appear in the lower half of the Navigation Pane
in the Panel Manager (Figure 2-3 on page 38).

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

Figure 2-3 Panel Manager displaying markers and reference

sample files for selected panel

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

Generating Bins To generate bins using the Auto Bin feature:

using Auto Bin 1. Make sure the LOH Panel is selected in the Navigation Pane and
LOH Bin Set is selected in the Bin Set drop-down list, then
click (BinsAuto Bin).
2. In the Auto Bin dialog box, select:
• Leave Minimum quality value set to 0.1
• Rounded basepair for Allele Naming Scheme
• Auto Bin (clear existing bins) for Auto Bin Options

3. Click OK.
4. When “Autobinning completed” displays, click OK.

Reviewing the To review the markers and bins generated from the reference data:
Markers and Bins 1. Expand the LOH Panel by clicking , then select a marker in
the upper half of the Navigation Pane.
A plot (Figure 2-4) displays:
• Marker (pink line)
• Bins (grey columns) for that marker
• Reference alleles (red cross hatches) for each bin
2. With a marker selected in the upper half of the Navigation Pane,
select a reference sample in the lower half of the Navigation Pane.
The plot updates (Figure 2-5) to show the electropherogram
peaks for the selected sample.
Note: Applied Biosystems recommends selecting each marker
to confirm that bins were created for it. If no bins are present,
investigate why. See the GeneMapper® Software Reference and
Troubleshooting Guide.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 39

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

Bins Reference
(bp range) alleles (bp)

Select
marker

Marker
(bp range)

Figure 2-4 Selecting a marker

Select
marker

Select
reference
sample

Figure 2-5 Selecting a marker and reference sample

40 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

Accepting the Click OK to accept the new bin set and close the Panel Manager.
Bin Set

Adding, Editing, To complete the experiment in this guide, you do not need to add,
and Deleting Bins edit, or delete any bins or markers. However, you may wish to test
and Markers these functions by opening the Panel Manager, then selecting the
(Optional) LOH Kit and LOH Panel.

IMPORTANT! If you edit or delete any bins or markers, make sure

you click Cancel at the bottom of the Panel Manager. Clicking OK or
Apply can adversely affect the results of the analysis.

Adding a Bin to a Marker

1. Select the marker in the upper Navigation Pane, then click

(BinsAdd Bin).
2. Click in the plot at the location where you want to add the bin.
3. In the Add Bin dialog box, type a Name, Location, and Offsets
for the bin, then click OK.

Editing a Bin

1. Click the bin (grey vertical bar) to select it.

2. Click (BinsEdit Bin) or right-click the bin, then select
Edit Bin.
3. In the Edit Bin dialog box, edit the Name, Location, and
Offsets for the bin, then click OK.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

Editing a Bin Graphically

1. Click the bin (grey vertical bar) to select it (Figure 2-6).

2. Click-drag the blue center line that defines the bin location.
3. Click-drag the left of right handles that define the bin offsets
(range).
Note: To correct any undesired change, select EditUndo.

Click-drag
blue center line to
edit bin location

Click-drag handle
to edit bin range

Figure 2-6 Editing a bin graphically

Deleting a Bin
To delete a bin from a marker:
• Select the bin, then click (BinsDelete Bin)
or
• Select the bin, right-click the bin, then select Delete Bin.

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 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

Editing a Marker

1. Select the marker in the upper Navigation Pane (Figure 2-7 on

page 43).
2. Click-drag the left or right handles that define the marker range.
Note: To correct any undesired change, select EditUndo.

Click-drag
handle to edit
marker range

Figure 2-7 Editing a marker

Deleting a Marker From a Panel

1. Select the marker in the upper Navigation Pane.

2. Click (EditClear Marker).

Next Steps Analyze and examine the data in the LOH project as described in
Chapter 3.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 43

 Chapter 2 Setting Up the Loss of Heterozygosity Analysis
Creating a Bin Set and Generating Bins Using the Auto Bin Feature

44 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 3

Analyzing and
Examining Results

Chapter 1 This chapter includes:

Getting Started
■ Editing the Analysis Method . . . . . . . . . . . . . . . . . . . . . . . . 46
■ Analyzing the Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
■ Examining the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Chapter 2
Setting Up the
Loss of Heterozygosity
Analysis

Chapter 3
Analyzing and
Examining Results

Chapter 4
Sorting Data and
Evaluating Loss of
Heterozygosity

Chapter 5
Printing and
Exporting Results

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 45

 Chapter 3 Analyzing and Examining Results
Editing the Analysis Method

Editing the Analysis Method

Overview When you created the LOH Analysis Method on page 17, for the
initial analysis, you did not select a bin set in the method. Now that
you have created a bin set, select that bin set in the analysis method
before analyzing the data. The bin set will allow the GeneMapper®
Software to make allele calls when you analyze the sample files in
the project.

Editing the To edit the LOH Analysis Method:

Analysis Method
1. Open the GeneMapper Manager by clicking
(ToolsGeneMapper Manager).
2. Select the Analysis Methods tab.

3. Select the LOH Analysis Method, then click Open.

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 Chapter 3 Analyzing and Examining Results
Editing the Analysis Method

4. In the Analysis Method Editor, select the Allele tab, select LOH
Bin Set for the Bin Set, then click OK.

5. Click Done to close the GeneMapper Manager.

Note: You can also access the Analysis Method Editor by
double-clicking any row in the Analysis Method column in the
Samples tab of the GeneMapper window (see page 17).

Next Steps Analyze the project as described on page 48.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 47

 Chapter 3 Analyzing and Examining Results
Analyzing the Project

Analyzing the Project

Overview Now that the analysis method specifies a bin set, when you analyze
your samples files, the GeneMapper® Software will size and
genotype the data.
Note the following in the Samples tab of the GeneMapper window:
• The icon displays in the Status column, indicating that the
samples are ready to be analyzed and have not been analyzed
with the current analysis parameters selected in the Samples tab.
This icon displays because you modified the LOH Analysis
Method.
• The icon displays in the REF column, indicating these
samples were used as reference data for creating a bin set.

Analyzing Click (AnalysisAnalyze).

The GeneMapper Software analyzes each sample in the project,
displaying its progress in the Status Bar (lower left) of the
GeneMapper window.

Next Steps Examine the results as described below.

Examining the Results

Overview To examine the sizing and genotyping results:
• Review the SQ, associated PQVs, size standard, sample
information, and samples plots (described on pages 27 through
33)
• Review the GQ and contributing PQVs (page 49)
• Review the allele calls for each sample (page 50)
• View genotype plots (page 50)
• Examine data in Genotypes Plot window (page 52)
• View project alleles (page 52)

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 Chapter 3 Analyzing and Examining Results
Examining the Results

Reviewing the To review the Genotype Quality (GQ) of the data, select the
GQ and PQVs Genotypes tab and scroll to the right.

Select Genotypes tab GQ column

Click-drag scroll bar to right to view GQ column

If you followed the procedures and used the example data indicated
in this guide, the GQ for most samples should be (Pass). The
Process Quality Values (PQVs) that contribute the GQ (AN, BD,
BIN, LPH, OBA, OS, PHR, SHP, SP, SPA, SPU, and XTLK) should
also be , except for a few samples with PHR (Peak Height
Ratio). However, yellow PHR is expected for LOH data.

Hint: When analyzing LOH data, to reduce the likelihood of PHR

being flagged, you may want to edit the Analysis Method to reduce
the Minimum Peak Height Ratio in the Heterozygote Balance section
of the Peak Quality tab (see page 21).

Investigating IMPORTANT! When analyzing your own data, you may find the GQ
Yellow and to be (Check) or (Low Quality) and the contributing PQVs
Red GQs (AN, BD, BIN, LPH, OBA, OS, PHR, SHP, SP, SPA, SPU, and
XTLK) to be , indicating issues with the data, marker or bin
definitions, or analysis parameters. To investigate and correct these
issues, see the GeneMapper® Software Reference and
Troubleshooting Guide.

Note: Click to sort the samples by GQ score. Samples with a

red GQ will be listed at the top of the Genotypes tab.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 49

 Chapter 3 Analyzing and Examining Results
Examining the Results

Reviewing the To review the allele calls for each marker in each sample, select the
Allele Calls Genotypes tab, then view the Allele 1 and Allele 2 columns.
View allele calls for
each marker

Viewing To view the genotypes plots of the samples:

Genotype Plots
1. Select a sample and marker (row) in the Genotypes tab. To
select multiple markers, press and hold Shift or Ctrl. To select
all markers, select EditSelect All.
2. Click (AnalysisDisplay Plots).
The Genotypes Plot window displays an electropherogram for
each selected marker.

Select
Microsatellite Default
for the Plot Setting

Select the
number of plots
to display

Zoom by
click-dragging
on top x-axis

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 Chapter 3 Analyzing and Examining Results
Examining the Results

3. Select Microsatellite Default for the Plot Setting.

Note: Plot Settings control the information displayed in the
Genotypes Plot window after analysis. Microsatellite Default is
one of the default Plot Settings provided with the GeneMapper
Software. You can also edit and create custom Plot Settings in
the GeneMapper Manager. For more information, see the
GeneMapper® Software Online Help.

4. Zoom on the x- and y-axes in the Genotypes Plot:

To … Then …

Zoom on a specific Place the cursor on the top x-axis, then

region of the x-axis click-drag the right or left to zoom that
plot.
or
Right-click the top x-axis, select Zoom To,
type range, then click OK.
Zoom on a specific Place the cursor on the left y-axis, then
region of the y-axis click-drag the up or down.
or
Right-click the left y-axis, select Zoom To,
type maximum, optionally, select Apply to all
electropherograms, then click OK.
Unzoom Double-click the x-axis or y-axis.
or
Right-click the x-axis or y-axis, then select
Full View.

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 Chapter 3 Analyzing and Examining Results
Examining the Results

Examining Data Other tasks you can perform in the Genotypes Plot window include:
in the Genotypes • Adjust the scale of the x-axes (basepairs or data points)
Plot Window • Adjust the scale of the y-axes (scale to individual maximum,
global maximum, or a specific value)
• Show and hide specific dye color peaks
• Display a status line for individual peaks
• Add, rename, and delete allele calls
• Edit and delete markers and bins
For more information on using the above features, press F1, then
select the desired topic from the GeneMapper® Software Online
Help.
When done viewing the Genotypes Plot, click to close the
window.

Viewing All To view all the alleles detected in the sample data for each marker:
Project Alleles
1. Open the Panel Manager by clicking (ToolsPanel
Manager).
2. In Navigation Pane, expand the LOH Kit, expand the LOH
Panel, then select a marker in the Navigation Pane (left).
3. Select BinsShow Project Alleles.
The project alleles (alleles detected in the sample data) appear as
blue asterisks in each bin. The y-axis position of each
indicates the GQ score for that marker and sample.

52 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 4

Sorting Data and Evaluating

Loss of Heterozygosity

Chapter 1 This chapter includes:

Getting Started
■ Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
■ Sorting the LOH Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
■ Generating a Report to Calculate and Evaluate LOH . . . . . 58
■ Editing or Creating a Custom LOH Report Setting . . . . . . . 59

Chapter 2
Setting Up the
Loss of Heterozygosity
Analysis

Chapter 3
Analyzing and
Examing Results

Chapter 4
Sorting Data and
Evaluating Loss of
Heterozygosity

Chapter 5
Printing and
Exporting Results

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 53

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Overview

Overview
An LOH analysis investigates and compares healthy tissue and
suspected diseased tissue from the same individual. Both the healthy
and diseased samples should show two alleles for a given
heterozygous microsatellite marker. If LOH is occurring in the
diseased tissue, it will be evident by a decrease in peak height of one
of the two alleles (relative to the healthy allele peak heights). The
reason the tumor sample shows a decrease in peak height (instead of
an absence of the peak) is that during isolation, the tumor sample is
contaminated with healthy cells, thus introducing some wild-type
DNA into the tumor specimen.
The Report Manager in the GeneMapper Software includes an LOH
Default report setting that calculates and compares peak height ratios
of microsatellite alleles between healthy and diseased tissues
(Figure 4-1). It then evaluates and flags any samples that show
potential LOH based on set peak-height ratio thresholds, so these
samples can be further investigated. You can alter these thresholds
based on the observed level of wild-type DNA contamination in the
tumor samples.

Allele 1
Allele 1

Allele 2

Allele 2

Healthy Sample Diseased Sample

Figure 4-1 Diseased sample shows loss in peak height of Allele 2

54 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Overview

The calculations and LOH criteria used by the LOH Default report
setting are summarized below:

1. Calculates the Allele Ratio between 2 allele peaks

for each marker for each sample using the following equation:

Peak Height of Allele 1

Allele Ratio = ---------------------------------------------------
Peak Height of Allele 2

2. Calculates Allelic Imbalance between Allele Ratios for

the healthy sample and diseased sample for each marker using
the following equation:

Allele Ratio of Healthy Sample

Allelic Imbalance = -------------------------------------------------------------------------
Allele Ratio of Diseased Sample

3. Evaluates the Allelic Imbalance for each marker and sample pair
then flags those with scores above or below set thresholds:

If Allelic Imbalance > 1.35, then flag as LOH Candidate

OR
If Allelic Imbalance < 0.67, then flag as LOH Candidate

Figure 4-2 Calculations and thresholds used by the LOH Default

report setting

IMPORTANT! You can edit the LOH Default report setting or create
your own report setting as appropriate for your data (see page 59).

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 55

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Sorting the LOH Data

Sorting the LOH Data

Overview Before you can apply the LOH Default report setting to calculate and
evaluate potential LOH, it is critical to sort the data so that healthy
samples and diseased samples for each marker are listed
consecutively.

Sorting To sort the LOH data:

1. Select the Genotypes tab in the GeneMapper window.
2. Sort by sample files by pressing and holding the Shift key, then
clicking the Sample File column header.
All markers from the 1_Healthy sample file are listed first,
followed by all samples from the 1_Tumor sample file, and so
on.
Shift-click
Sample File
header

Figure 4-3 Sorting by sample file

3. Sort by Marker by pressing and holding the Shift key, then

clicking the Marker column header.
This sorts the results so that healthy samples and diseased
samples for the same marker are listed consecutively.

56 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Sorting the LOH Data

Shift-click
Marker
header

Figure 4-4 Sorting by sample file and marker

Next Steps Generate a report to calculate and evaluate the LOH as described on
page 58.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 57

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Generating a Report to Calculate and Evaluate LOH

Generating a Report to Calculate and Evaluate LOH

To generate a report that calculates and evaluates loss of
heterozygosity:
1. Make sure you sorted the data as described on page 56.
2. Select all the samples in the Genotypes tab by selecting
EditSelect All.
3. Select AnalysisReport Manager.
4. In the Report Manager dialog box, select LOH Default from the
Report Setting drop-down list.

Select
LOH
Default

The LOH Default report setting is applied to the LOH Project

data. The resulting report shows the Allele Ratio, Allelic
Imbalance, and LOH Assessment for each sample or pair of
samples.
5. Save, print, or export the generated report by selecting the
appropriate command from the File menu.

IMPORTANT! When generating a report for your own data, make

sure samples do not have a Low Quality SQ score in the Samples
tab. If they do, the samples and associated markers appear in the
Genotypes tab, but with no data to be used by the report setting.

58 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Editing or Creating a Custom LOH Report Setting

Editing or Creating a Custom LOH Report Setting

Overview The LOH Default report setting provided with the GeneMapper
Software assumes the following:
• Allelic Imbalance Row Calculation – When calculating the
Allelic Imbalance (ratio of peak-height ratios) between two
samples, the report setting compares every two rows in the
Genotypes tab. This calculation assumes your sample files are
named so that healthy samples and diseased samples for each
individual and marker are listed consecutively (in pairs) after
being sorted by sample file and marker.
• LOH Assessment Thresholds – The threshold values used to
flag samples as LOH candidates are > 1.35 and < 0.67.
You can edit the above settings in the LOH Default report setting as
appropriate for your samples and experiment.

Editing the Allelic Depending on how your sample files are named, healthy and tumor
Imbalance Row files from the same individual may or may not list consecutively after
Calculation being sorted by sample file and marker (Figure 4-5).

1_Healthy.fsa Healthy_1.fsa
1_Tumor.fsa Healthy_2.fsa
2_Healthy.fsa Healthy_3.fsa
2_Tumor.fsa .
3_Healthy.fsa .
3_Tumor.fsa .
. Healthy_n.fsa
. Tumor_1.fsa
. Tumor_2.fsa
n_Healthy.fsa Tumor_3.fsa
n_Tumor.fsa .
.
.
Tumor_n.fsa

Healthy and tumor files from same All healthy files list first, followed
individual list consecutively by all tumor files

Figure 4-5 How sample file naming convention affects the order
files list after being sorted by sample and marker

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 59

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Editing or Creating a Custom LOH Report Setting

To change the row calculation for the Allelic Imbalance in the LOH
Default report setting:
1. Open the GeneMapper Manager by clicking
(ToolsGeneMapper Manager).
2. Select the Report Settings tab (Figure 4-5).
3. Select the LOH Default report setting, then click Open.

Select
LOH Default
report setting

Click Open

Figure 4-6 GeneMapper Manager Report Settings tab

4. In the Report Settings Editor (Figure 4-5):

a. Select the Calculation tab.
b. Select Allelic Imbalance from the Selected Columns list
(right pane).
c. Click to move Allelic Imbalance to the Calculation tab.

60 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Editing or Creating a Custom LOH Report Setting

Select
Calculation
tab

Select
Allelic
Imbalance

Click

Figure 4-7 Report Settings Editor

5. Select Allelic Imbalance in the Calculation tab, then click Edit.

6. In the Edit Calculation dialog box, replace 2 with n + 1 in the
two fields shown in Figure 4-5.
Note: The value n is the sample set number, that is the number
of healthy samples or the number of tumor samples (see
Figure 4-5 on page 59). For example, if your sample set contains
5 healthy and 5 tumor samples, you would type 6.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 61

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Editing or Creating a Custom LOH Report Setting

Replace 2
with n + 1

Figure 4-8 Edit Calculation dialog box

7. Click OK to close the Edit Calculation dialog box.

8. In the Report Settings Editor (Figure 4-5), click to move
Allelic Imbalance from the Calculation tab to the Selected
Columns list.
9. In the Report Settings Editor (Figure 4-5):
a. Select the Analysis tab.
b. Select LOH Assessment from the Selected Columns list
(right pane).
c. Click to move LOH Assessment to the Analysis tab.
10. Select LOH Assessment in the Analysis tab, then click Edit.

62 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Editing or Creating a Custom LOH Report Setting

11. In the Analysis section of the Edit Analysis dialog box, select
For every group of, then type n + 1 in the two fields shown in
Figure 4-5.
Note: The value n is the sample set number, that is the number
of healthy samples or the number of tumor samples (see
Figure 4-5 on page 59). For example, if your sample set contains
5 healthy and 5 tumor samples, you would type 6.

Replace 2
with n + 1

Figure 4-9 Edit Analysis dialog box

12. Click OK to close the Edit Analysis dialog box.

13. In the Report Settings Editor (Figure 4-5 on page 61), click
to move LOH Assessment from the Analysis tab to the Selected
Columns list.
14. Click OK to close the Report Settings Editor. Click Done to
close the GeneMapper Manager.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 63

 Chapter 4 Sorting Data and Evaluating Loss of Heterozygosity
Editing or Creating a Custom LOH Report Setting

Editing the LOH Select LOH Assessment thresholds appropriate for the observed level
Assessment of wild-type DNA contamination in tumor samples. For more
Thresholds information, see the discussion on page 54.

To change the thresholds for the LOH Assessment in the LOH

Default report setting:
1. Open the GeneMapper Manager by clicking
(ToolsGeneMapper Manager).
2. Select the Report Settings tab (Figure 4-5 on page 60).
3. Select the LOH Default report setting, then click Open.
4. In the Report Settings Editor (Figure 4-5 on page 61):
a. Select the Analysis tab.
b. Select LOH Assessment from the Selected Columns list
(right pane).
c. Click to move LOH Assessment to the Analysis tab.
5. Select LOH Assessment in the Analysis tab, then click Edit.
6. In the Edit Analysis dialog box, in the Value column, replace the
lower and upper thresholds of 0.67 and 1.35 with thresholds
appropriate for your data.

Change these values

as appropriate for
your data

7. Click OK to close the Edit Analysis dialog box.

8. In the Report Settings Editor (Figure 4-5), click to move
LOH Assessment from the Analysis tab to the Selected Columns
list.
9. Click OK to close the Report Settings Editor. Click Done to
close the GeneMapper Manager.

64 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 5

Printing and Exporting Results

Chapter 1 This chapter includes:

Getting Started ■ Printing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
■ Exporting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Chapter 2
Setting Up the
Loss of Heterozygosity
Analysis

Chapter 3
Analyzing and
Examing Results

Chapter 4
Sorting Data and
Evaluating Loss of
Heterozygosity

Chapter 5
Printing and
Exporting Results

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 65

 Chapter 5 Printing and Exporting Results
Printing Results

Printing Results
You can print results from the following windows and tabs by
selecting FilePrint:

Access From GeneMapper Project

Window/Tab
Window by selecting

GeneMapper window – ViewSamples

Samples tab
GeneMapper window – ViewGenotypes
Genotypes tab
GeneMapper window – ViewSample Info
Info tab
GeneMapper window – ViewRaw Data
Raw Data tab
GeneMapper window – ViewEPT Data
EPT Data tab
Samples Plot window The Samples tab, then AnalysisDisplay
Plots
Genotypes Plot window The Genotypes tab, then
AnalysisDisplay Plots
Report Manager window AnalysisReport Manager

Note: You can also print reports. For information on creating report
settings and generating reports, see the GeneMapper® Software
Online Help.

66 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Chapter 5 Printing and Exporting Results
Exporting Results

Exporting Results
Exporting To export the results displayed in the Samples tab and Genotypes
Samples Tab and tab of the GeneMapper window:
Genotypes Tab 1. Prepare the content and format of the data to export:
a. Select the desired Table Setting from the drop-down list at
the top of the GeneMapper window. The Table Setting
controls which columns display and the sorting order for
the samples.
b. Optionally, sort the data to determine the order that the
samples appear. Select EditSort or Shift-click the
column header in the Samples tab or Genotypes tab. You
can also click (AnalysisLow Quality on Top) to
sort the samples by GQ score.
Note: For more information on editing or creating Table
Settings and sorting data, see the GeneMapper® Software Online
Help.

2. Select one of the following commands:

• FileExport Table – Exports information displayed in the
selected tab.
• FileExport Combined Table – Exports information
displayed in both tabs. (This command is available only
when the Samples tab is selected.)

Exporting Kits To export all kits in the Panel Manager:

1. Open the Panel Manager by clicking (ToolsPanel
Manager).
2. Select FileExport All Kits.

Exporting Panels To export all panels in a kit:

1. Open the Panel Manager by clicking (ToolsPanel
Manager).
2. Select the kit in the Navigation Pane (left).
3. Select FileExport Panels.

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 67

 Chapter 5 Printing and Exporting Results
Exporting Results

Exporting To export a bin set:

Bin Sets
1. Open the Panel Manager by clicking (ToolsPanel
Manager).
2. In the Navigation Pane, select the kit with which the bin set is
associated.
3. Select the bin set from the Bin Set drop-down list.
4. Select FileExport Bin Set.

Exporting To export projects, analysis methods, table settings, plot settings,

Projects, reports settings, and size standards:
Methods, 1. Open the GeneMapper Manager by clicking
Settings, and (ToolsGeneMapper Manager).
Size Standards
2. Select one of the following tabs:
• Projects
• Analysis Methods
• Table Settings
• Plot Settings
• Report Settings
• Size Standards
3. Select the object(s) you want to export. Press and hold Shift or
Ctrl to select multiple objects.
4. Click Export.

Exporting You can also export reports. For information on creating report
Reports settings and generating reports, see the GeneMapper® Software
Online Help.

68 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Index

A Auto Bin 39
adding
bins to markers 41 B
sample files to project 15 bin
size standard labels 30 adding to a marker 41
Advanced peak detection algorithm 20 creating manually 35
allele calls, reviewing 50 definition 7, 35
Allele Ratio 55 deleting 42
editing 41, 42
Allele tab of Analysis Method Editor 19, 47 generating using Auto Bin 39
alleles, project 52 reviewing 39
Allelic Imbalance 55, 59 bin set
analysis method accepting 41
creating 17 creating 35
editing 46 definition 7, 35
exporting 68 exporting 68
saving 23 importing 35
selecting bin set 19, 47 selecting in analysis method 19, 47
Analysis Method Editor bold text, when to use v
Allele tab 19, 47
General tab 18 C
Peak Detector tab 20
Peak Quality tab 21 conventions
Quality Flags tab 22 bold text v
analysis parameters for describing menu commands v
IMPORTANTS! vi
definition 7, 17
in this guide v
setting 17
italic text v
analysis. See LOH microsatellite analysis Notes vi
analyzing a project 26, 48 user attention words vi
Applied Biosystems creating
contacting viii analysis method 17
customer feedback on documentation viii bin set 35
Information Development bins 39
department viii kit 11
Technical Support viii markers 11
assumptions for using this guide v panel 11
project 15

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 69

Index

report setting 68 F
customer feedback, on Applied Biosystems files. See sample files
documents viii
fill down 24

D G
data
See example data General tab of Analysis Method Editor 18
See reference data generating
See sample files report for LOH 58
deleting reports 68
bins 42 genotype quality. See GQ
markers 43 Genotypes Plot window
size standard labels 30 displaying 50
disclaimer, license ii examining data in 52
documentation, related vii exporting 67
printing 66
zooming 51
E Genotypes tab 49
editing Genotypes Table 33
Allelic Imbalance 59 GQ
analysis method 46
investigating 49
bins 41, 42 reviewing 49
LOH Assessment Thresholds 64
markers 43
report setting 59 H
size standard labels 30 help, online, accessing vii, viii
example data hiding dye color peaks 33, 52
instrument used 4
marker information 5, 14
overview 4 I
size standard for 4 importing
exporting bin set 35
analysis methods 68 panels 11
bin sets 68 Information Development department,
Genotypes Plot window 67
contacting viii
kits 67
panels 67 instruments
plot settings 68 compatible with LOH microsatellite
projects 68 analyses 3
report settings 68 for example data 4
reports 58, 68 italic text, when to use v
results 67
Samples Plot window 67
size standards 68 K
table settings 68 kits

70 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Index

creating 11 overriding SQ 30
definition 7, 11
exporting 67
type 12 P
Panel Manager
L opening 11
panels
license disclaimer ii adding reference data 36
LOH creating 11
definition 2, 54 definition 7, 11
evaluating 53 exporting 67
LOH Assessment thresholds 55, 59, 64 importing 11
LOH Default report setting 55, 59 selecting for analysis 23
peak detection algorithm 20
LOH microsatellite analysis
compatible instruments 3 Peak Detector tab of Analysis Method
definition 2, 54 Editor 20
evaluating LOH 53 Peak Quality tab of Analysis Method
flowchart 6 Editor 21
sorting data 56 plot setting 31, 51, 68
Plot window
M See Genotypes Plot window
markers See Samples Plot window
adding bins to 41 plots. See sample plots
creating 11 primers, custom 3
definition 2, 7, 11
printing
deleting from a panel 43
editing 43 Genotypes Plot window 66
for example data 5, 14 reports 58, 66
plots, viewing 50 results 66
reviewing 39 Samples Plot window 66
viewing plots 50 project
menu commands, conventions for adding sample files to 15
describing v analyzing 26, 48
creating 15
method. See analysis method exporting 68
microsatellite analysis saving 25
definition 2 setting analysis parameters 17
markers 2 setting table settings 17
See also LOH microsatellite analysis project alleles
setting up 9 viewing 52
MSDSs, obtaining viii
Q
O Quality Flags tab of Analysis Method
online help, accessing vii, viii Editor 22

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 71

Index

R examining data in 33
exporting 67
reference data printing 66
adding to a panel 36 zooming 32
icon 48
Samples tab 17
Report Manager 58
saving
report setting analysis method 23
creating 68 project 25
editing 59 reports 58
LOH Default 55, 59 setting up analysis 9
Report Settings Editor 60
showing and hiding dye color peaks 33, 52
report settings, exporting 68
size calling curve 29
report variables
Size Match Editor 28
Allele Ratio 55
Allelic Imbalance 55, 59 size quality. See SQ
LOH Assessment 55, 59, 64 size standard
reports custom 30
exporting 58, 68 examining 28
generating 68 exporting 68
generating for LOH 58 for example data 4
printing 58, 66 labels, adding 30
saving 58 labels, deleting 30
results labels, editing 30
selecting for analysis 23
exporting 67
troubleshooting 30
printing 66
Sizing Table 33
software
S starting and logging in 7
sample files terms defined 7
adding to project 15 sorting
location 4 LOH samples 56
naming convention 4, 59 samples 27, 49
plots, viewing 31, 50
SQ
sorting 27, 49, 56
viewing information on 30 investigating 27
viewing plots 31, 50 overriding 30
zooming 32, 51 reviewing 27
score 28
sample plots
Status Bar 26
viewing 31, 50
zooming 32, 51
samples T
sorting 27, 49 table settings 17, 24, 68
sorting for LOH evaluation 56
zooming 32, 51 Technical Support, contacting viii
Samples Plot window text conventions v
thresholds. See LOH Assessment thresholds

72 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

 Index

training, information on viii

troubleshooting the size standard 30

U
unzooming 32, 51
user attention words, described vi

X
x-axis
scale 33, 52
zooming 32, 51

Y
y-axis
scale 33, 52
zooming 32, 51

Z
zooming
Genotypes Plot window 51
Samples Plot window 32
unzooming 32, 51
x-axis 32, 51
y-axis 32, 51

GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide 73

Index

74 GeneMapper® Software Version 4.1 LOH Analysis Getting Started Guide

Part Number 4403621 Rev. A 04/2009

Applied Biosystems Technical Resources and Support

850 Lincoln Centre Drive For the latest technical resources and
Foster City, CA 94404 USA support information for all locations,
Phone 650.638.5800 | Toll Free 800.345.5224 please refer to our Web site at
www.appliedbiosystems.com www.appliedbiosystems.com/support