Thermo Fisher Scientific ABI PRISM® Genotyper® Owner's manual

ABI PRISM® Genotyper® 2.0

Software

Applications Tutorials

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

ABI PRISM and the ABI PRISM design, Applied Biosystems, GeneScan, Genotyper, INHERIT and Sequence Navigator are

registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

ABI, AmpFlSTR, AutoAssembler and BaseSprinter are trademarks of Applera Corporation or its subsidiaries in the U.S. and

certain other countries.

AmpErase, AmpliTaq, EnviroAmp, GeneAmp and TaqMan are registered trademarks, and AmpliTaq Gold is a trademark of

Roche Molecular Systems, Inc.

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

P/N 904649B

Contents

iii

Software License and Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Genotyper® Software Tutorials . . . . . . . . . . . . . . . . 1-1

Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

How This Tutorial is Organized. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Parts of the Tutorial. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

What You Will Learn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

ABI PRISM® Software Genotyping Application Kits . . . . . . . . . . . . 1-3

Use Genotyper® Software Features . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Guidelines for Running Genotyping Applications . . . . . . . . . . . . . . . . . . . . 1-4

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Preparing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Creating Sample Sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Choosing Data- Collection Parameters. . . . . . . . . . . . . . . . . . . . . . . . 1-4

Choosing Size- Calling Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

iv

Guidelines for Using Genotyper Software . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

Planning out Results Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

Recording Genotyper® Software Steps . . . . . . . . . . . . . . . . . . . . . . . 1-5

Running Macros . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

Importing GeneScan Software Files . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

Viewing Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

Filtering Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

2 Microsatellite Analysis Part 1 . . . . . . . . . . . . . . . . . .2-1

Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

Overview of Part 1 of the Microsatellite Tutorial . . . . . . . . . . . . . . . . . . . . . 2-2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Genotyper Software Features You Will Use. . . . . . . . . . . . . . . . . . . . 2-2

Contents of the Fluorescent Genotyping Demonstration Kit . . . . . . . . . . . . 2-3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

How Fluorescent Genotyping Data Was Preprocessed. . . . . . . . . . . . . . . . . 2-4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

GeneScan Software Analysis Parameters. . . . . . . . . . . . . . . . . . . . . . 2-4

Source of DNA Template Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Pedigree Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Individual IDs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Abbreviations of Individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Create a Genotyper Software Document . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Create a New Genotyper Software Document . . . . . . . . . . . . . . . . . . 2-6

v

Define Categories for Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

What Is a Category? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Define Categories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

View the Category List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

Sort Categories by Dye Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9

Sort Categories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9

Import GeneScan® Software Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10

Import GeneScan Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10

Viewing GeneScan Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11

Label Allele Peak Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12

Selecting Dye/lanes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12

Label Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12

Filter Peak Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

Filter Peak Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

About the Filter Labels Command . . . . . . . . . . . . . . . . . . . . . . . . . . 2-14

Set Up an Allele Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15

Column Format of Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15

Set Up the Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16

The Concept of Overflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18

Append Rows of Filtered Peak Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-19

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-19

View Table Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-19

Edit Column Headings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-20

Sort the Table by Column Heading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21

Sort the Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21

Export the Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-22

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-22

Export the Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-22

vi

Make a Macro of Tutorial Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23

View the Steps in the Macro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23

Edit Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23

Create the Macro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-24

Run the Macro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-25

If the Macro Does Not Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-25

Make a Template for the Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26

Create a Template. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26

Use the Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-27

3 Microsatellite Analysis Part 2 . . . . . . . . . . . . . . . . . .3-1

Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

Overview of Part 2 of the Microsatellite Tutorial . . . . . . . . . . . . . . . . . . . . . 3-2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Genotyper Software Features You Will Use . . . . . . . . . . . . . . . . . . . 3-2

Import GeneScan Software files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Import GeneScan Software files . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Label Allele Peaks with Size Labels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Label Allele Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Filter Peak Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

Filter Peak Labels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

About the Filter Labels Command. . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

Define Category Groups for Marker Alleles. . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Create Category Groups for Markers. . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Why Create an Unknown Category . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8

vii

Make New Categories for Alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Setting Up the Statistics Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Selecting Peaks for Individual Marker . . . . . . . . . . . . . . . . . . . . . . . 3-10

Creating New Category Members for Each Marker . . . . . . . . . . . . . 3-11

Finishing the Remainder of the Categories. . . . . . . . . . . . . . . . . . . . 3-14

Building an Allele Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

Select Dye/lanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

Label Allele Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

Filter Unwanted Labels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

About the Filter Labels Command . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

Set Up the Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

Column Format of Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19

The Concept of Overflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19

Append Rows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20

Edit Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21

View Plot Data for Overflows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21

Remove Labels from Overflow Alleles . . . . . . . . . . . . . . . . . . . . . . 3-21

Manually Call Alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21

Change Size Labels to Allele Names . . . . . . . . . . . . . . . . . . . . . . . . 3-22

Export the Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-23

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-23

Export the Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-23

4 Human Identification Tutorial . . . . . . . . . . . . . . . . 4-1

Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

Overview of The Human Identification Tutorial . . . . . . . . . . . . . . . . . . . . . . 4-2

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Genotyper Software Features You Will Use . . . . . . . . . . . . . . . . . . . . 4-2

viii

Contents of the AmpFlSTR™ Blue Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Using Allelic Ladders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Loci Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

How AmpFlSTR™ Blue Data Was Preprocessed. . . . . . . . . . . . . . . . . . . . . 4-4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Data-Collection Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

GeneScan Software Analysis Parameters . . . . . . . . . . . . . . . . . . . . . 4-4

Create a Genotyper Software Document . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5

Create a New Genotyper Software Document . . . . . . . . . . . . . . . . . . 4-5

Import GeneScan Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Import GeneScan Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Define Category Groups for Allele Markers. . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

View Allelic Ladders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Define Category Boundaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Add Category Group For Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Create Allele Categories for Marker Groups . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Mark Category Groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Label Peaks for Each Allele. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Filter Unwanted Peak Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

Make Categories from Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11

Example of Category List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12

Create Category Groups for Other Markers. . . . . . . . . . . . . . . . . . . 4-12

Edit Categories for FGA Alleles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

How to Edit FGA Categories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Example of Edited FGA Categories. . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Label Alleles with Names and Numbers . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Label Allele Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Example of Labeled Allelic Ladder. . . . . . . . . . . . . . . . . . . . . . . . . 4-14

ix

Set Up an Allele Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15

Column Format of Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15

Set Up the Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

Append Rows to Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-17

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-17

How to Append Rows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-17

Editing Table Cell Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-18

Removing Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-18

Adding Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-18

Updating the Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-18

Addendum to Human Identification Tutorial . . . . . . . . . . . . . . . . . . . . . . . 4-19

Sample File Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-19

x

xi

Software License and Warranty

Applied

Biosystems

Software License

and Limited

Product Warranty

PURCHASER, CAREFULLY READ THE FOLLOWING TERMS AND

CONDITIONS (THE “AGREEMENT”), WHICH APPLY TO THE

SOFTWARE ENCLOSED (THE “SOFTWARE”). YOUR OPENING OF

THIS PACKAGE INDICATES YOUR ACCEPTANCE OF THESE

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PROMPTLY RETURN THE COMPLETE PACKAGE AND YOUR

MONEY WILL BE RETURNED. THE LAW PROVIDES FOR CIVIL AND

CRIMINAL PENALTIES FOR ANYONE WHO VIOLATES THE LAWS

OF COPYRIGHT.

Copyright The SOFTWARE, including its structure, organization, code, user

interface, and associated documentation, is a proprietary product of

Applied Biosystems and is protected by international laws of copyright.

Title to the SOFTWARE, and to any and all portion(s) of the

SOFTWARE shall at all times remain with Applied Biosystems.

License 1. You may use the SOFTWARE on a single computer (or on a single

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transfer the SOFTWARE to another single computer (or network, if a

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previous computer or network. You may never have operational

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terms and conditions of this Agreement. If you transfer the SOFTWARE

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or destroy those not transferred. Any such transfer terminates your

license.

continued on next page

xii

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SOFTWARE, or the associated documentation, in whole or in part,

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continued on next page

xiii

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concerning the SOFTWARE.

Genotyper® Software Tutorials 1-1

Genotyper® Software

Tutorials 1

Chapter Overview

Introduction This manual is designed to familiarize you with using Genotyper®

software. The manual consists of three tutorials that introduce you to

two common genotyping applications:

♦Microsatellite (Parts 1 and 2) – Analyze inheritance patterns and

prepare the allele labels for export to linkage applications.

♦Human Identification Tutorial – Create allelic tables for unknown

forensic samples.

In This Chapter This chapter contains the following topics.

Topic See page

“How This Tutorial is Organized”1-2

“What You Will Learn”1-3

“Guidelines for Running Genotyping Applications”1-4

“Guidelines for Using Genotyper Software”1-5

1

1-2 Genotyper® Software Tut o ria l s

How This Tutorial is Organized

Introduction This manual consists of three different tutorials. It is designed as a step-

by-step guide learning several features of Genotyper 2.0 software. The

instructions and illustrations assume that you have followed the tutorial

step-by-step.

Parts of the

Tutor i al

The tutorial is organized into three genotyping applications

♦Microsatellite analysis (part 1)

♦Microsatellite analysis (part 2)

♦Human Identification

Assumptions This tutorial assumes that you are familiar with the operations of the ABI

PRISM® DNA Sequencing Instruments and the GeneScan® analysis

software.

Refer to the particular ABI PRISM® 377 DNA Sequencer Protocols for

detailed protocols for setting up the PCR reactions, and for running the

amplified fluorescent products on the ABI PRISM® 377 instrument.

Genotyper® Software Tutorials 1-3

What You Will Learn

Introduction Each of the tutorials provides a step by step way to learn the features

and uses of Genotyper software. Each chapter emphasizes different

features in the software.

Goals After completing all of the tutorials, you should be able to

♦Run two different kinds of genotyping applications

♦Use features of Genotyper software

♦Prepare allele information for use in linkage applications

ABI PRISM®

Software

Genotyping

Application Kits

You will use the following application kits in these tutorials:

♦Fluorescent Genotyping Demonstration Kit

♦ABI PRISM linkage mapping panels

♦AmpFlSTR Blue kit

Use

Genotyper®

Software Features

In these tutorials, you will use the following Genotyper features:

♦Importing GeneScan software files

♦Defining categories and markers

♦Editing categories

♦Labeling allele peaks

♦Filtering unwanted allele peaks

♦Constructing and filling tables

♦Constructing and running macros

♦Exporting Genotyper software data

1-4 Genotyper® Software Tut o ria l s

Guidelines for Running Genotyping Applications

Introduction In order for you to use this manual and Genotyper software, it is

important to follow basic guidelines when preparing samples and

analyzing them using the GeneScan analysis software.

Preparing

Samples

Adjust the pooling conditions to dilute the amplified products that

consistently yield off-scale data. Optimal results can be obtained with

peak heights of approximately 1000 fluorescent units.

Creating

Sample Sheets

Enter sample information in the GeneScan Sample Sheet for each

sample so that sample tracking will be easier in the database. It is quite

possible that several samples of blood may be taken from the same

individual, or that several DNA preps may have been carried out from

the single blood sample for an individual. In order to keep track of this

information, it is important to enter the sample information (tube

number, DNA number, sample number, and so on) in the Sample

Sheet.

Choosing Data-

Collection

Parameters

Follow these guidelines when choosing data collection parameters:

♦Ensure that gel run parameters are consistent from run-to-run (for

example, gel percentage, and Run Module files).

♦Choose the right matrix for the right type of gel and run parameters.

Using a poor/incorrect matrix will result in off-scale data that is not

baselined properly, leading to anomalous peaks being sized.

Choosing Size-

Calling Methods

To ensure consistency in size calling, use the same size-calling method

for every run. Setting the minimum peak height for red too high may

result in size-standard peaks being ignored. In lanes where extraneous

bands are being called as size markers, use the “User defined” option

to set the value of these peaks to zero.

Genotyper® Software Tutorials 1-5

Guidelines for Using Genotyper Software

Introduction Ensure that you set up and follow the procedures in this tutorial step-by-

step, and do not introduce any extraneous samples or categories while

working through the tutorials. Adhering to the following guidelines will

make the tutorial more useful.

Planning out

Results Tables

You can simplify the use of Genotyper software by thinking of how you

can best present your results data in tabular format. The earlier in the

genotyping process that you can decide what kind of table you want to

create, the easier it will be for you to create tables that reveal the

significance of your results data.

Recording

Genotyper®

Software Steps

Most of the steps (commands) used in Genotyper software will be

recorded in the Step List (the bottom-right box in the Main window).

This Step List can later be edited and saved as a macro. Macros are

very useful tools for repetitive processing of similar types of data.

For example, you can set up the categories for all the markers in a

linkage project, process the data from the first gel, and make a macro

with the common steps that you must go through to process the initial

data. You can then create a template containing the categories and the

macro that were set up. You can then import the next set of data and

process it automatically using the macro and the template without going

through all the steps again manually.

In this tutorial, you will create a macro and a template.

Running Macros Macros can be run by either pressing the command key that you

selected when saving the macro, or by double-clicking the macro name.

To run a macro or perform a step by double-clicking, choose Set

Preferences in the Edit menu and check the box for “Double-clicking

runs macros & steps” under Other options.

continued on next page

1-6 Genotyper® Software Tut o ria l s

Importing

GeneScan

Software Files

It is not always necessary to import the raw data for each sample file.

Unchecking the “Import raw data” box will make importing a large

number of files much faster. Also, since you don’t need data from the

red size standard markers for this exercise, you can uncheck the red

box. This will also speed up the file-importing process and is less taxing

on computer memory.

Viewing

Documents

You can view full-screen windows that show the part of a Genotyper

software document with which you are working.

If you prefer to see more of the categories at one time, choose the

Show Categories Window command from the Views menu or click on

the Categories Window icon at the right side of the Main window.

If you prefer to view more dye/lanes at one time, choose the Show

Dye/lanes command from the Views menu. You can also open the

Dye/lanes window by clicking on the Dye/lanes icon at the right side of

the Main window.

If you want to view more of the plots, choose the Show Plot Window

command from the Views menu, or click on the Plot icon on the right

side of the main window. The Plot window shows all the dye/lanes as

separate plots for each color, with all the peaks labeled with the size

label. Move this window to one side of the computer screen to see the

effects of the next step.

Filtering Labels When you open this dialog box by choosing the Filter Labels command

from the Analysis menu, you will see that the last three of the four boxes

for the filtering parameters have already been preselected by default.

These selections work quite well for most dinucleotide repeat markers.

The first of the three checked boxes will remove labels from most of the

small (noise) peaks that have a peak height of 32 percent or less than

the true alleles. The second will remove labels from the +A peaks if their

peak height is less than that of the true allele associated with it, and if

the +A peak is within 1.6 bp from the true allele. The third will remove

labels from stutter peaks. The filtering operations listed in this dialog

box are performed one at a time, in the order they are listed.

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Thermo Fisher Scientific ABI PRISM® Genotyper® Owner's manual

Thermo Fisher Scientific ABI PRISM® Genotyper® Owner's manual

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