Thermo Fisher Scientific GeneMapper® Software Quick start guide

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GeneMapper® Software Version 4.0

AFLP® System Analysis

Getting Started Guide

Front Cover

Getting Started

Setting Up

the Analysis

Analyzing and

Examining

the Data

Exporting and

Printing the

Analyzed Data

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GeneMapper® Software Version 4.0

AFLP® System Analysis

Getting Started Guide

Title Page

Getting Started

Setting Up

the Analysis

Analyzing and

Examining

the Data

Exporting and

Printing the

Analyzed Data

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iv GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

Copyright

For Research Use Only. Not for use in diagnostic procedures.

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that

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,

system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation,

either expressly, or by estoppel.

GeneMapper Software has not undergone specific developmental validation for human identification applications. Human

identification laboratories analyzing single-source or parentage samples which choose to use GeneMapper Software for data

analysis should perform their own developmental validation studies.

The AFLP process is covered by patents owned by Keygene N.V.

TRADEMARKS:

ABI PRISM, Applied Biosystems, GeneMapper, and SNaPshot are registered trademarks, and the AB Design, Applera, FAM,

GeneScan, ROX, and SNPlex are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries.

AFLP is a registered trademark of Keygene N.V.

rights reserved.

Windows is a registered trademarks of Microsoft Corporation.

Oracle is a registered trademark of Oracle Corporation.

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.

Part Number 4363079 Rev. B

06/2005

Contents

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GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide v

Preface vii

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . viii

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

Chapter 1 Getting Started 1

About Supported AFLP Reagent Chemistries . . . . . . . . . . . . . . . . . 2

About the Example Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

AFLP System Analysis Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

GeneMapper Software Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Starting the Software and Logging In . . . . . . . . . . . . . . . . . . . . . . . 8

Using This Guide With Your Own Sample Files . . . . . . . . . . . . . . . . 9

Using This Guide With the Version 3.7 Software . . . . . . . . . . . . . . . 9

Alternatives to the Procedures in This Guide . . . . . . . . . . . . . . . . . 10

Chapter 2 Setting Up the Analysis 11

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Creating a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Adding Sample Data to the Project . . . . . . . . . . . . . . . . . . . . . . . . 14

Creating an Analysis Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Configuring the Allele Tab Settings . . . . . . . . . . . . . . . . . . . . . 16

Configuring the Peak Detector Tab Settings . . . . . . . . . . . . . . 23

Configuring the Peak Quality Tab Settings . . . . . . . . . . . . . . . 25

Configuring the Quality Flags Tab Settings . . . . . . . . . . . . . . . 26

Applying the Analysis Parameters . . . . . . . . . . . . . . . . . . . . . . . . . 28

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Contents

vi GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

Chapter 3 Analyzing and Examining the Data 31

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Analyzing the Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Examining the Off-Scale Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Examining the Size Quality Data . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Sizing Solution #1: Adjusting the Analysis Method . . . . . . . . 40

Sizing Solution #2: Manually Correcting Miscalled Peaks . . . 42

Sizing Solution #3: Modifying the Size Standard Definition . . 45

Examining the Analyzed Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Reviewing the Analyzed Data in the Genotypes Table . . . . . . 50

Displaying the Peak Data in the Samples Plot . . . . . . . . . . . . 52

Reviewing the Size Standard Concordance . . . . . . . . . . . . . . 56

Visualizing Polymorphic Peaks . . . . . . . . . . . . . . . . . . . . . . . . 58

Editing the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Modifying the Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Modifying a Bin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Modifying Genotype Calls . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Saving the Generated Panel and Bin Set . . . . . . . . . . . . . . . . . . . 67

Completing the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Chapter 4 Exporting and Printing the Analyzed Data 75

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Exporting Results and Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Exporting Samples and Genotypes Tabs . . . . . . . . . . . . . . . . . . . 78

Exporting Plots and Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Exporting Data for Use in a Spreadsheet . . . . . . . . . . . . . . . . . . . 79

Exporting Projects and Reference Data . . . . . . . . . . . . . . . . . . . . 81

Printing Project Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Index 83

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GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide vii

Preface

How to Use This Guide

Purpose of This

Guide

This guide explains how to analyze the example amplified fragment

length polymorphisms (AFLP®) data provided with the

GeneMapper® Software. It is designed to quickly teach you how to

size, genotype, and visualize the band patterns of amplified fragment

length polymorphisms using the software. It also provides exercises

that illustrate basic troubleshooting techniques and methods for

exporting analyzed data for further analysis or presentation.

Audience This guide is intended for trained laboratory personnel. Applied

Biosystems is not liable for damage or injury that results from use of

this guide by unauthorized or untrained parties.

Assumptions This guide assumes that:

• You have installed GeneMapper® Software Version 4.0 as

described in the

GeneMapper

®

Software Version 4.0

Installation

and Administration Guide (PN 4363080).

• 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 right arrow bracket () separates successive commands you

select from a drop-down or shortcut menu. For example:

Select FileOpenSpot Set.

Right-click the sample row, then select View FilterView All.

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Preface

How to Obtain More Information

viii GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

User Attention

Words

Two user attention words appear in Applied Biosystems user

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.

How to Obtain More Information

Safety

Information

See the

GeneMapper

®

Software Version 4.0

Installation and

Administration Guide

(PN 4363080) for safety information.

Software

Warranty and

License

See the GeneMapper® Software Version 4.0 Installation and

Administration Guide (PN 4363080) for warranty and licensing

information.

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Preface

How to Obtain More Information

GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide ix

The following related documents are shipped with the software:

•

GeneMapper

®

Software Version 4.0 Installation and

Administration Guide

– Provides procedures for installing,

securing, and maintaining version 4.0 of the GeneMapper Software.

•GeneMapper® Software Version 4.0 Getting Started Guides –

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 4.0 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 4.0 Quick Reference Guide

–

Provides workflows for specific analysis types and lists

instruments, software, and analysis applications compatible with

the GeneMapper Software.

•

GeneMapper

®

Software Version 4.0 Reference and

Troubleshooting Guide

– 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 4.0 Documentation CD.

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

on page x.

Send Us Your

Comments

Applied Biosystems welcomes your comments and suggestions for

improving its user documents. You can e-mail your comments to:

[email protected]

Obtaining

Information from

the Online Help

The GeneMapper® Software features an online help system that

describes how to use each feature of the user interface. To access the

online help, click in any window or dialog box (HelpContents

and Index if available) for more information.

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Preface

How to Obtain Support

xGeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

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.

Chapter 1

Getting Started

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Chapter 1

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GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide 1

Getting Started

This chapter covers:

■About Supported AFLP Reagent Chemistries . . . . . . . . . . . . 2

■About the Example Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■AFLP System Analysis Workflow . . . . . . . . . . . . . . . . . . . . . 7

■GeneMapper Software Terms. . . . . . . . . . . . . . . . . . . . . . . . . 8

■Starting the Software and Logging In . . . . . . . . . . . . . . . . . . 8

■Using This Guide With Your Own Sample Files . . . . . . . . . . 9

■Using This Guide With the Version 3.7 Software . . . . . . . . . 9

■Alternatives to the Procedures in This Guide. . . . . . . . . . . . 10

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Chapter 1 Getting Started

About Supported AFLP Reagent Chemistries

2GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

About Supported AFLP Reagent Chemistries

About the

AFLP System

Amplified fragment length polymorphism (AFLP®) is a mapping

technique used to visualize polymorphisms in genomic DNA. The

AFLP system combines the well-known restriction fragment length

polymorphism (RFLP) technique and the polymerase chain reaction

(PCR) to generate a large number of amplified restriction fragments

from prepared, genomic DNA. When separated by electrophoresis,

the samples yield unique band patterns that, when visualized by

southern blot or fluorescence-based fragment analysis, can be used

for high-resolution genotyping, polymorphism detection, or

cladistics.‡

Applications Supported by the Workflows in This Guide

The flexibility and robustness of the AFLP system provides a broad

number of applications for the technology. This guide contains a

general analysis workflow that can be used to support two of the

most common: sample/strain identification and backcross analysis

for mapping. The procedures focus on sample identification analysis,

however many of them are applicable to the alternative applications.

Compatible

AFLP Assays

The GeneMapper Software can analyze samples that have been:

• Prepared using an AFLP chemistry that incorporates the

Applied Biosystems fluorescent dye-labeling and detection

technology

• Run on a compatible Applied Biosystems electrophoresis

instrument

Applied Biosystems has adapted the AFLP technique for use with its

fluorescent dye-labeling and detection technology. In the modified

system, a 5′ dye-labeled primer has been substituted for one of the

selective primers used in the final amplification step. The following

section describes the Applied Biosystems chemistry.

‡ Savelkoul, P. H. M., Aarts, H. J. M., de Haas, J., Dijkshoorn, L., Duim, B.,

Otsen, M., Rademaker, J. L. W., Schouls, L., and Lenstra, J. A., J Clin

Microbio, 1999, 37(10), 3083–3091.

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Chapter 1 Getting Started

About Supported AFLP Reagent Chemistries

GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide 3

Template Preparation and Adaptor Ligation

Isolated, genomic DNA is exposed to two restriction endonucleases

(EcoRI and MseI in the example) to generate restriction fragments.

The samples are then ligated to double-stranded, oligonucleotide

adaptors that contain sequences complimentary to the ends of the

digested fragments (see Figure 1-1).

Figure 1-1 Example of template preparation and adaptor ligation

Preselective Amplification (Optional)

Following ligation, the fragments are amplified using a set of forward

and reverse primers that target the combined sequences of the

adaptors and restriction sites. The primary products of this

“preselective” PCR are amplicons generated from the restriction

fragments that were ligated to adaptors at both ends (see Figure 1-2).

Figure 1-2 Preselective amplification

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Chapter 1 Getting Started

About Supported AFLP Reagent Chemistries

4GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

Selective Amplification

Following preselective amplification, the fragments are amplified

again using several “selective” primers, including a fluorescent, 5′

dye-labeled primer. Figure 1-3 illustrates the selective amplification

of fragments that have been digested by the EcoRI and MseI

restriction endonuclease. The primary products of the PCR are

amplicons generated from EcoRI/MseI-ended fragments. In this way,

the combination of selective primers act to further simplify the band

pattern for the sample.

Note: When run, the compatible Applied Biosystems electrophoresis

instrument detects only the products of the EcoRI-ended fragments.

The MseI-MseI fragments will not be visualized because they are not

fluorescently labeled.

Figure 1-3 Selective amplification with fluorescent, 5′ dye-

labeled primer

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Chapter 1 Getting Started

About Supported AFLP Reagent Chemistries

GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide 5

Electrophoresis and Data Collection

After a size standard is added to the amplified samples, they are

loaded onto a compatible Applied Biosystems electrophoresis

instrument for electrophoretic separation and fluorescence detection.

During the electrophoresis, the instrument monitors the passage of

the fluorescent, 5′ dye-labeled fragments through the polymer by

detecting fluctuations in emitted light when the fragments migrate

past a fixed laser beam. When finished, the instrument assembles the

spectral data for each sample (see Figure 1-4) and stores it as a

sample file, or saves it to the application database.

Figure 1-4 Signal data (electropherograms) of the example files

provided for this guide (see “About the Example Data” on page 6)

Compatible

Instruments

See the GeneMapper® Software Version 4.0 Quick Reference Guide

(PN 4362816) for a list of compatible

Applied Biosystems

electrophoresis

instruments and chemistries.

Supported AFLP

Chemistry Kits

The GeneMapper Software can analyze data generated using several

AFLP fragment analysis chemistry kits. For a complete list of AFLP

chemistries available from Applied Biosystems, visit the

Applied Biosystems web site (www.appliedbiosystems.com).

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Chapter 1 Getting Started

About the Example Data

6GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

About the Example Data

Location and

Function of the

Sample Files

This guide explains how to analyze

AFLP

sample data by guiding

you through the analysis of an example data set from an

AFLP

experiment. The example sample files referred to in the exercises of

this guide install automatically with the GeneMapper Software and

can be found at the following location:

<drive>:\AppliedBiosystems\GeneMapper\

Example Data\AFLP Data

Note: The location shown above may vary depending on the

installation of the software.

About the

Experiment

The example AFLP sample files provided with the GeneMapper

Software were generated as part of a sample identification study. The

samples were prepared using a the Applied Biosystems AFLP® Plant

Mapping Kit that included 5′ FAM™ dye-labeled selective primer.

The samples were run on an Applied Biosystems 3100 Genetic

Analyzer using the GeneScan™-500 (ROX™) size standard available

from Applied Biosystems.

Example Panel

and Bin Set

The panel and bin set used to analyze the example AFLP data set are

generated automatically by the GeneMapper Software. See

“Automatic Panel Generation” on page 19 for more information.

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Chapter 1 Getting Started

AFLP System Analysis Workflow

GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide 7

AFLP System Analysis Workflow

Analysis

Workflow

Figure 1-5 summarizes the process for studying band patterns from

many AFLP

samples simultaneously.

Figure 1-5 Analyzing AFLP system data

Chapter 2

Set Up the AFLP Analysis

1. Create a panel for the project.

2. Create a new project and add samples to it.

3. Set the analysis parameters in the Samples tab.

4. Set the table settings.

5. Perform an initial analysis.

6. Create a new bin set and generate bins.

Chapter 4

Print or Export the Results (Optional)

If necessary:

• Print or export the desired views, plots, and tables

for use in reports or presentations.

• Export desired views, plots, and tables for further

analysis by third-party software.

Chapter 3

Analyze and Examine the Data

1. Analyze the project.

2. Examine the results.

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Chapter 1 Getting Started

GeneMapper Software Terms

8GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

GeneMapper Software Terms

Starting the Software and Logging In

Starting the

GeneMapper

Software

1. In the desktop, double-click GeneMapper v4.0 (Start

All ProgramsApplied BiosystemsGeneMapper

GeneMapper v4.0).

2. In the Login to GeneMapper dialog box, type the User Name

and Password assigned by your system administrator.

3. Click OK.

Table 1-1 Common terms used in this guide

Term Defini t i o n

analysis

parameters

A collection of user-defined settings (including an analysis

method) that determine the sizing and genotyping

algorithms used by the GeneMapper Software to analyze all

sample files in a project

bin

A fragment size and dye color that define an allele. You

typically create a bin for each possible allele associated with

a marker

bin set

A set of bins (allele definitions), typically specific to a set of

experimental conditions

kit

A group of panels

marker

A known segment of DNA that has two or more allelic forms.

A marker exists at a known chromosomal loci and can be a

gene or a non-gene. A marker is defined by a name,

fragment size range (bp), dye color, and repeat length.

panel

A group of markers, typically specific to a set of

experimental conditions

RFU

Relative Fluorescent Units

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Chapter 1 Getting Started

Using This Guide With Your Own Sample Files

GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide 9

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 AFLP analysis workflow when analyzing your own sample

files. For information on advanced software features, see the

GeneMapper® Software Online Help.

Using This Guide With the Version 3.7 Software

The workflows and procedures presented in this guide are also valid

for Version 3.7 of the GeneMapper Software.

IMPORTANT! GeneMapper Software Version 3.7 does not contain

the correct example data used in the exercises of this guide.

Therefore, if you have Version 3.7 of the GeneMapper Software, do

not use this guide to analyze the example data that it references;

instead, use it to lead you through the AFLP analysis workflow when

analyzing your own sample files.

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Chapter 1 Getting Started

Alternatives to the Procedures in This Guide

10 GeneMapper® Software Version 4.0 AFLP® System Analysis Getting Started Guide

Alternatives to the Procedures in This Guide

Overview This guide presents one of several possible solutions for analyzing

AFLP data using the GeneMapper Software. Once you have

completed the exercises in this guide, this section provides you with a

summary of several alternatives and other resources for customizing

the process to fit the requirements of your laboratory.

Using

Autoanalysis to

Set Up Projects

The GeneMapper Software includes an Autoanalysis feature that can

eliminate most of the tasks leading up to the analysis of an AFLP

project. Much of Chapter 2, “Setting Up the Analysis,” explains how

to manually create, add samples to, and analyze projects. When

configured for Autoanalysis, the GeneMapper Software can

accomplish these tasks automatically by coordinating with the Data

Collection Software. For a more detailed explanation of how to use

the Autoanalysis feature to set up AFLP projects, see the

GeneMapper® Software Version 4.0 Installation and Administration

Guide (PN 4363080).

Using the

Command Line

Interface to Set

Up Projects

The GeneMapper Software features a command line interface that can

perform most of the major functions of the software. The command

line interface can be a useful tool when analyzing AFLP projects

because it automates many of the tasks explained in Chapter 2,

“Setting Up the 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 4.0

Installation and Administration Guide

(PN 4363080).

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Thermo Fisher Scientific GeneMapper® Software Quick start guide

Thermo Fisher Scientific GeneMapper® Software Quick start guide

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