Thermo Fisher Scientific ABI PRISM® GeneMapper™ Software Owner's manual

Brand: Thermo Fisher Scientific

Model: ABI PRISM® GeneMapper™ Software

Category: Software manuals

Type: Owner's manual

ABI PRISM® GeneMapper™

Software Version 3.0

SNP Genotyping

Tutorial

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ABI PRISM® GeneMapper™

Software Version 3.0

SNP Genotyping

Tutorial

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September 25, 2002 1:20 pm, 7x9_Title.fm

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

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 PE Corporation (NY),

either expressly, or by estoppel.

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.

ABI PRISM and its design, Applied Biosystems, GeneScan, Genotyper, LIZ, and SNaPshot are registered trademarks of Applera

Corporation or its subsidiaries in the U.S. and certain other countries.

AB (Design), ABI, Applera, GeneMapper, GeneScan, and Primer Focus are trademarks of Applera Corporation or its subsidiaries

in the U.S. and certain other countries.

Macintosh is a registered trademark of Apple Computer, Inc.

All other trademarks are the sole property of their respective owners.

Part Number 4335524 Rev. B

09/2002

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SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial iv

Contents

Chapter 1 Introduction

About the SNP Genotyping Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Data Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

New Features in GeneMapper Software Version 3.0 . . . . . . . . . . . . . . 1-5

Process Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7

Chapter 2 Working With Kits, Bin Sets, and Panels

Process Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Importing and Sizing Reference Data . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

Setting Up Kits, Bin Sets, and Panels . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

Creating Markers and Bins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

Chapter 3 Data Analysis

Process Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Creating an Analysis Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Performing Analysis on SNP Genotyping Data . . . . . . . . . . . . . . . . . . 3-10

Examining the Analyzed Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13

Performing Tasks in the Plot Windows . . . . . . . . . . . . . . . . . . . . . . . . 3-18

Chapter 4 Auto Panel Feature for Primer Focus

Process Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Importing and Sizing Reference Data . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Setting Up Kits and Bin Sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Using the Auto Panel Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

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v SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

Appendix A Peak Detection Algorithms

Index

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SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial 1-1

Introduction 1

In This Chapter This chapter contains the following topics:

About the SNaPshot Tutorial. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-2

Data Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-4

New Features in GeneMapper Software Version 3.0. . . . . . . . . . . .1-5

Process Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-7

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Chapter 1 Introduction

1-2 SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

About the SNP Genotyping Tutorial

Overview This tutorial is intended to help you get quickly up to speed in

performing analyses of SNaPshot® data with ABI PRISM®

GeneMapper™ Software Version 3.0.

Note: This tutorial contains basic information only. For more

advanced information, please refer to the ABI PRISM®

GeneMapper™ Genotyping Software User’s Manual (PN 4335526).

Goals After completing this tutorial, you should be able to:

• Set up a GeneMapper software project.

• Import sample files for analysis.

• Define new SNP markers and bin sets using the Auto Panel

feature. This feature is only for reference data generated by the

SNaPshot® Primer Focus™ kit.

How to Use This

Tutorial \When you are following the procedures in this tutorial, it is important

that you:

• Perform every step in the order listed.

• Do not introduce any extraneous samples or bins.

The procedures are written using menu selections, for example:

Select Tools > GeneMapper Manager.

You may also use the icons located on the toolbar.

Terms Used in

This Tutorial The following terms are used in this tutorial:

Table 1-1 Terms and Definitions

Term Definition

Analysis Method A collection of parameters set by the user to

determine the bin set and analysis algorithms. See

page 3-2.

Bin A fragment size or basepair range and dye color

that define an allele.

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About the SNP Genotyping Tutorial

SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial 1-3

For More

Information For more information about the GeneMapper software, refer to the

documents listed below.

Bin set A set of bins (that is, allele definitions) for one

source or set of experimental conditions, usually

an instrument. Bin sets are available inside a kit.

Marker A known SNP location.

Panel A set of markers.

Project settings Parameters set by the user to prepare a project for

analysis. See page 3-12.

Table 1-1 Terms and Definitions (continued)

Term Definition

Document Part Number

ABI PRISM® GeneMapper™ Genotyping Software

User’s Manual 4335526

Microsatellite Analysis with ABI PRISM®

GeneMapper™ Software Version 3.0 Tutorial 4335525

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Chapter 1 Introduction

1-4 SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

Data Provided

Overview GeneMapper Software Version 3.0 comes with the data listed below.

Algorithms and

Quality Values To support SNaPshot multiplex analysis, the following algorithms

and quality values are provided:

•Double Peak: Two peaks with the same color in the same bin

•Narrow Bin: Peak of the same color as a bin within 0.5 basepairs

of the bin

•SNaPshot Cutoff Value: Examines the ratio (Y/X) between two

peaks (X and Y) within a marker; if the ratio is less than the cutoff

value, the second peak will not be called

Default Settings The following SNaPshot default settings are listed below.

Primer Focus

Sample Files Six Primer Focus sample files are provided. Each of these is a single

primer that corresponds with the SNaPshot® Multiplex Control

Primers.

SNaPshot

Sample Files Five sample files of SNaPshot multiplex data are provided:

• Four sample files were generated using the SNaPshot Multiplex

Control Primers on different individuals.

• One sample file contains the GeneScan™-120 LIZ® size standard

only.

Type Name Parameters

Analysis

Method SNaPshot Default • Analysis Type = SNaPshot

•Bin Set = none

• Analysis Algorithm = basic

Plot Setting SNP Default N/A

Table Setting SNP Default

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New Features in GeneMapper Software Version 3.0

SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial 1-5

Size Standard

Definition The GeneScan™ –120 LIZ® (GS120LIZ) size standard definition is

provided.

Note: Custom size standard definitions can be created. For

procedures, refer to the ABI PRISM® GeneMapper™ Genotyping

Software User’s Manual (PN 4335526).

New Features in GeneMapper Software

Version 3.0

List of New

Features GeneMapper Software Version 3.0 is an enhancement of

GeneMapper Software Version 2.0. The new features in GeneMapper

Software Version 3.0 are listed below.

•ABIP

RISM® GeneScan® software functionality, including:

– EPT Data tab added to the Samples tab (accessed via the

Project window)

– Sizing Table tab added to the Plot Settings Editor (accessed via

the Plots window)

– Standard Curve tab added to the Size Match Editor

•ABIP

RISM® Genotyper® software functionality, including:

– Enhanced allele call labels in the Plots window

– Customized allele labeling options in the Plots window

– Marker and bin editing within the Plots window

• User interface consistency across products, including new or

revised menus and shortcut keys

For detailed information about these new features, refer to the

ABI PRISM® GeneMapper™ Genotyping Software User’s Manual

(PN 4335526).

How These

Features Affect

Data Analysis

When you perform SNaPshot data analysis with GeneMapper

Software Version 3.0 you will:

• Have increased editing flexibility

• Be able to save panels that may pre-exist (for Primer Focus data)

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Chapter 1 Introduction

1-6 SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

Auto Panel

Feature GeneMapper software contains an Auto Panel feature that takes

advantage of the reference data generated by the Primer Focus kit to

define new SNP markers and bin sets quickly. The software

determines the overlap potential of SNP markers and recommends

panels to achieve accurate SNaPshot multiplexing results.

Note: Auto Panelizing procedures are provided in Chapter 4.

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Process Flowchart

SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial 1-7

Process Flowchart

Analyzing

SNaPshot Data The flowchart below provides an overview of the tasks required to

analyze SNaPshot data with the GeneMapper software. For detailed

procedures, see the references below.

Rules for SNP

Kits, Panels, and

Bins

The following rules apply to SNP kits, panels, and bins:

• Bins of the same color may not overlap anywhere within a panel.

• A bin is one color. There are up to four bins or colors per marker,

one for each of the ddNTP bases (A, C, T, G).

• Markers in a panel may overlap as long as bins of the same color

do not overlap.

Set up a new project:

• Import and size reference data

• Manually create panels/bins or use the Auto

Panel feature with Primer Focus sample files

Define a size standard, if necessary

Examine the analyzed data:

• Adjust panels or bins

• Edit allele calls

Import data, convert Macintosh® files,

if necessary

Analyze data, apply project settings

Create an Analysis Method:

• Select the analysis type (SNaPshot)

• Select a bin set

• Select an analysis algorithm

See Chapter 2

and Chapter 4

See page 3-5

See User Manual

See page 3-10

See page 3-13

See page 2-3

and User Manual

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Chapter 1 Introduction

1-8 SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

• Bins in a marker are single continuous ranges; there is only one

bin of each color in a marker.

• Bins in a marker must be different colors and may overlap; for

almost all markers there will be some overlap of their constituent

bins.

• The marker size range is the minimum and maximum of its bin

size ranges. A margin may be necessary to accommodate alleles

not seen in the SNP Reference Data samples.

• Duplicate bin names are not allowed in a marker.

• Each kit can only have one bin set.

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SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial 2-1

Working With Kits, Bin Sets,

and Panels 2

In This Chapter This chapter contains the following topics:

Process Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2

Importing and Sizing Reference Data. . . . . . . . . . . . . . . . . . . . . . .2-3

Setting Up Kits, Bin Sets, and Panels . . . . . . . . . . . . . . . . . . . . . . .2-8

Creating Markers and Bins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13

Assumptions This chapter guides you through setting up a project in ABI PRISM®

GeneMapper™ Software Version 3.0 in order to analyze

SNaPshot®data.

It is assumed that you have already installed and logged on to the

GeneMapper software.

Note: This chapter provides instructions for manually creating kits,

bins sets and panels. To create these using the Auto Panel feature

with SNaPshot® Primer Focus™ kit sample files, please refer to

Chapter 4.

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Chapter 2 Working With Kits, Bin Sets, and Panels

2-2 SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

Process Flowchart

Setting Up a

Project The flowchart below provides an overview of the tasks required to set

up a GeneMapper project when you are manually creating kits, bin

sets, and panels.

Panel Manager Project Window

Create a new kit

Import reference data into a new project

Size the reference data:

• Select an Analysis Method with

SNaPshot as the analysis type

• Set the panel to None

• Select a size standard

Create a new panel

Add reference data to the kit

Create markers and bins

Add reference data to the panel

Create a new bin set

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Importing and Sizing Reference Data

SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial 2-3

Importing and Sizing Reference Data

Overview Predefined panel and bin information does not exist for SNaPshot

multiplex samples, since every user has a different set of primers,

primer lengths, and multiplexing levels. Therefore, the first tasks

required when creating panels and bins are to import reference data

into a new project and perform sizing analysis.

The steps required are:

• Converting the sample files, if necessary

• Importing reference data (that is, SNP sample files) into a

new project

• Performing sizing analysis on the reference data

Converting

Sample Files Note: Information on converting sample files is not necessary for

this tutorial. However, you may find it useful when you are working

with your own sample files.

If necessary, convert ABI PRISM® GeneScan® Analysis Software

sample files generated by the Apple Macintosh® software to the .fsa

format. The conversion is described in the ABI PRISM®

GeneMapper™ Genotyping Software User’s Manual (PN 4335526).

Importing

Reference Data

into a New

Project

To import reference data into a new project:

1. In the Project window, select

File >Add Samples to Project.

The Add Samples to Project window opens.

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Chapter 2 Working With Kits, Bin Sets, and Panels

2-4 SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

2. Navigate to the disk/directory containing the GeneMapper

software application.

3. Add sample files from the SNaPshot folder to this project, as

follows:

a. Open the following folders in this order:

GMdistribution > Tutorial Data > SNaPshot >

SNaPshot

b. Select the SNaPshot folder.

c. Click Add To List.

The SNaPshot folder moves to the Samples To Add list in the

Add Samples to Project window.

Note: If you move the wrong folder to the Samples To Add

list, select the folder and click Clear to remove it.

4. Clicking Add performs these functions:

a. Imports the sample files into a new project

b. Closes the Add Samples to Project window.

c. Reveals the Project window (previously hidden

behind the Add Samples to Project window) now

populated with the imported sample files (in the

Samples tab).

To import reference data into a new project: (continued)

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Importing and Sizing Reference Data

SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial 2-5

Performing Sizing

Analysis To perform sizing analysis on the reference data:

1. Go to the Samples tab view in the Project window.

2. Select an Analysis Method:

a. Select the top cell in the Analysis Method column.

b. Select SNaPshot Default from the drop-down list.

Note: When you are analyzing your own SNaPshot

reference data, make sure the Analysis Method you choose

has SNaPshot as the analysis type.

3. For this tutorial, the Panel column should already be set to

None. If it is not:

a. Select the top cell in the Panel column. The Select a Panel

window opens.

b. Double-click None.

The Select a Panel window closes and None is entered into

the first cell of the Panel column in the Samples tab.

Note: Setting the Panel column to None ensures that a

sizing-only analysis is performed.

4. Select a size standard:

a. Select the top cell in the Size Standard column.

b. Select GS120LIZ from the drop-down list.

Note: The GS120LIZ size standard has been predefined for

you. To learn how to create your own size standard, refer to

the ABI PRISM® GeneMapper™ Genotyping Software User’s

Manual (PN 4335526).

5. Apply the selections to the selected samples, as follows:

a. Click and drag the mouse across the three column

headings (Analysis Method, Panel, and Size Standard) to

select the entire columns.

b. Select Edit > Fill Down (or press Ctrl+D) to apply the

selection to the selected samples.

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Chapter 2 Working With Kits, Bin Sets, and Panels

2-6 SNP Genotyping with ABI PRISM GeneMapper Software Version 3.0 Tutorial

6. Select Analysis > Analyze.

The Save Project dialog box opens.

7. Type SNaPshot Tutorial and click OK.

The program initiates analysis, then saves each analyzed

sample to the project you have just named. While analysis is

proceeding, progress is displayed as follows:

• The progress indicator at the bottom of the Project

window shows progress in two ways:

– As a bar graph extending from the left

– As a percentage indicator

• The current sample undergoing analysis is indicated by

the sample row in the table displayed in green (or red if

analysis failed for the sample).

When the program has finished analyzing the samples, the

message “Analysis Completed” displays on the Status bar of

the Project window (lower left corner).

When samples are sized successfully:

• A green square is displayed in the SQ column.

•The Status column is cleared.

Note: Auto-saving takes place after every 10 sample files

are analyzed or before the “Analysis Completed”

message displays.

To perform sizing analysis on the reference data: (continued)

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Thermo Fisher Scientific ABI PRISM® GeneMapper™ Software Owner's manual

Brand: Thermo Fisher Scientific

Model: ABI PRISM® GeneMapper™ Software

Category: Software manuals

Type: Owner's manual

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