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

ABI P

RISM

® Genotyper ® 2.0

Software

Applications Tutorials

© Copyright 2001, Applied Biosystems. All rights reserved.

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

ABI P

RISM

and the ABI P

RISM

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, AmpF lSTR, 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

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

iii

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


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

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

v

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


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

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


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


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

vii

viii

Contents of the AmpF lSTR

™

Blue Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

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

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

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

How AmpF lSTR

™

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

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

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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

ix

x

Software License and Warranty

Applied

Biosystems

Software License and Limited

Product Warranty

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continued on next page

xi

xii

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xiii

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

“How This Tutorial is Organized”

“What You Will Learn”

“Guidelines for Running Genotyping Applications”

“Guidelines for Using Genotyper Software”

See page

1-2

1-3

1-4

1-5

1

Genotyper

®

Software Tutorials 1-1

How This Tutorial is Organized

Introduction

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

Tutorial

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

P

RISM

® DNA Sequencing Instruments and the GeneScan ® analysis software.

Refer to the particular ABI P

RISM

® 377 DNA Sequencer Protocols for detailed protocols for setting up the PCR reactions, and for running the amplified fluorescent products on the ABI P

RISM

® 377 instrument.

1-2 Genotyper

®

Software Tutorials

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 P

RISM

®

Software

Genotyping

Application Kits

You will use the following application kits in these tutorials:

♦

Fluorescent Genotyping Demonstration Kit

♦

ABI P

RISM

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

Genotyper

®


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.

1-4 Genotyper

®


Guidelines for Using Genotyper Software

Introduction

Ensure that you set up and follow the procedures in this tutorial step-bystep, 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.


Genotyper

®


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.

1-6 Genotyper

®


Microsatellite Analysis

Part 1

2


Introduction

This tutorial shows you how to use Genotyper software to analyze patterns of inheritance using family marker data available in the

Fluorescent Genotyping Demonstration Kit.

In This Chapter


Topic

“Overview of Part 1 of the Microsatellite Tutorial”

“Contents of the Fluorescent Genotyping Demonstration Kit”

“How Fluorescent Genotyping Data Was Preprocessed”

“Source of DNA Template Samples”

“Create a Genotyper Software Document”

“Define Categories for Markers”

“Sort Categories by Dye Colors”

“Import GeneScan ® Software Files”

“Label Allele Peak Data”

“Filter Peak Labels”

“Set Up an Allele Table”

“Append Rows of Filtered Peak Data”

“Sort the Table by Column Heading”

“Export the Table”

“Make a Macro of Tutorial Procedures”

“Make a Template for the Tutorial”

2-10

2-12

2-13

2-15

2-19

2-21

2-22

2-23

2-26

See page

2-2

2-3

2-4

2-5

2-6

2-7

2-9

2

Microsatellite Analysis Part 1 2-1

Overview of Part 1 of the Microsatellite Tutorial

Introduction

In Part 1, you will use Genotyper 2.0 software to process Fluorescent

Genotyping Demonstration Kit GeneScan files, and create an allelic size table to discriminate between heterozyote and homozygote individuals.

Goals

The goal of Part 1 of the microsatellite tutorial is to learn how to use

Genotyper features and commands to

♦

Process GeneScan software Sample files that have been generated from the Linkage Demonstration Kit

♦

Create a Genotyper software table that contains two columns (one for each allele) for each marker supplied in the kit

If a particular individual is a heterozygote for a marker, then there will be two peaks represented with their sizes in bp. If an individual is a homozygote, there will be only one allelic peak represented.

Genotyper

Software

Features You Will

Use

You will learn the following features of Genotyper software in this tutorial:

♦

Creating a new document

♦

Creating categories

♦

Sorting categories

♦

Labeling and filtering peaks

♦

Setting up and exporting tables

♦

Creating and running macros

♦

Saving a document as a template

2-2 Microsatellite Analysis Part 1

Contents of the Fluorescent Genotyping Demonstration Kit

Introduction

You will use data from the Fluorescent Genotyping Demonstration Kit for this Microsatellite Analysis tutorial.

Panels

The Fluorescent Genotyping Demonstration Kit contains six dinucleotide repeat markers from ABI P

RISM

Fluorescent Genotyping

Panels that are fluorescently labeled with FAM

™

, HEX, and TET amidites. The data from these markers were obtained by typing them on

14 individuals from the CEPH K1347 family.

Markers

The Fluorescent Genotyping Demonstration Kit markers used for this tutorial are shown in Table 2-1.

Table 2-1

Demonstration Kit Markers

Marker Name

D12S83

D7S517

D1S220

D3S1266

D2S391

D13S171

Dye Label

FAM

FAM

HEX

HEX

TET

TET

Allele Size Range

(bp)

98-113

Heterozygosity

0.81

235-261 0.83

226-260 0.82

281-303 0.81

139-153 0.79

171-197 0.73


How Fluorescent Genotyping Data Was Preprocessed

Introduction

Fluorescently labeled PCR products were electrophoresed on a ABI

P

RISM

377 DNA Sequencer under the following conditions:

♦

Gel Type – 4%, 36-cm WTR gel

♦

Run Module – GS 36C-2400 Run module for two hours

♦

Analysis Software – GeneScan Analysis 2.0.2 software

GeneScan

Software

Analysis

Parameters

Table 2-2 shows the parameters set in GeneScan software for sizing sample fragments.

Table 2-2

GeneScan Software Analysis Parameters

Peak

Detection

Threshold

50

Peak Detection

Minimum Half-width Sizing Method

3 Local Southern

Std. Peak Det.

Threshold

50


Source of DNA Template Samples

Introduction

This tutorial provides a pedigree for the samples being used. The pedigree information is part of the sample file name in GeneScan.

Family

The DNA template samples used in this kit are from the CEPH K1347 family, which includes two parents, two sets of grandparents, and eight siblings.

Pedigree

Structure

Figure 2-1 shows the pedigree structure for the CEPH K1347 family.

Figure 2-1

Pedigree structure for CEPH K1347

Individual IDs

The number under each family member in Figure 2-1 refers to the individual ID in this family.

Abbreviations of

Individuals

Some of the abbreviations used in the sample file names in this data set are as follows:

PGM – Paternal grandmother

PGF – Paternal grandfather

MGM – Maternal grandmother

MGF – Maternal grandfather


Create a Genotyper Software Document

Introduction

To perform genotyping tasks on the Fluorescent Genotyping

Demonstration Kit marker data, you must import the related GeneScan software files into a Genotyper software document.

Create a New

Genotyper

Software

Document

The following steps are used to create a new document.

If...

Then...

Genotyper software is already open Select New from the File menu.

Genotyper software is closed Open Genotyper software by double-clicking the applications icon.


Define Categories for Markers

What Is a

Category?

Because each GeneScan software sample file can contain data from many markers in different size ranges (and up to three different colors) you will need to define the boundaries for each marker. The boundaries are based on the expected allele size range for that marker, and are used to define a category.

Define Categories

The categories you will be defining are from the Fluorescent

Genotyping Demonstration kit (see Table 2-1 on page 2-3)

.

Follow these steps to define categories for markers.

Step Action

1

Choose Clear Category list from the Analysis menu.

2

Note

This removes the default “Everything” category in the

Category window.

Choose Add Category... from the Category menu (or press

Command-L). The following window appears:

3

Type in the name of the first marker in the Fluorescent Genotyping

Demonstration Kit as the Category name (see Table 2-1 on page 2-3).


Follow these steps to define categories for markers.

(continued)

Step Action

4

In the pop-up menu next to the Category name, select the same color as the dye used to fluorescently label the marker. For example, choose blue for FAM-labeled markers.

5

6

Note

Use orange for the HEX markers and green for the TET markers.

Click the All Peaks radio button.

Type in the expected allele size range for the marker name you entered in Step 3.

7

8

9

Note

For marker allele size ranges, see Table 2-1 on page 2-3.

In the dye color checkboxes, select the dye color of the marker.

Click OK.

Repeat each of these steps for all six markers shown in Table 2-1 on page 2-3.

View the

Category

List

Choose Show Categories from the View menu or click on the

Categories window icon on the right edge of the main window. You should now have a Category list that looks like Figure 2-2.


Figure 2-2

Category list for linkage markers

Sort Categories by Dye Colors

\

Introduction

The default sort order of the Category list is by the name of the category. You will use the Category Sorting command to sort the categories by dye color.

Sort

Categories

Follow these steps to sort categories by dye color.

Step Action

1

Choose Category Sorting from the Views menu. The following window appears:

2

3

4

Set the sort order to Dye color in the first pop-up menu.

Click OK.

Close Categories window.


Import GeneScan

®

Software Files

Introduction

Once categories are defined, you must import the appropriate

GeneScan software files containing data for these categories.

Import

GeneScan

Software Files

Follow these steps to import GeneScan software files.

Step Action

1

Choose Import GeneScan software file(s) from the File menu and navigate to the “Microsatellite Sample Files” folder. The Import dialog box should appear as follows:


2

3

Uncheck the “Import raw data” checkbox.

Note

It is not always necessary to import the raw data for each sample file. Unchecking the “Import raw data” box will make importing 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 speed up the file-importing process and will be less taxing on computer memory.

Click Import All to import all 14 sample files from the folder.


Viewing

GeneScan

Software Files

Once you’ve imported GeneScan software files, you can view dye/lanes from the imported files in the Dye/lanes list.

From the Main window, click the Dye/lanes window icon to display the

Dye/lanes window. Or, choose Show Dye/lanes window from the Views menu.

The window should now list all of the imported sample files with all three colors. Each sample file is broken down into three dye/lanes, one for each color in that file. Four dye/lanes are displayed for each sample file if you include the red dye in the Import command.

Now close the Dye/lane window.


Label Allele Peak Data

Introduction

Next, you must label all the allele peaks for each marker with the respective size in base pairs.

Selecting Dye/lanes

Follow these steps to select the appropriate dye/lanes.

Step Action

1

Make the Dye/lanes pane active by clicking it, or by tabbing until a vertical bar appears next to the Dye/lane pane.

2

Choose Select All from the Edit menu, or click on the B, G, Y buttons while holding down the Shift key to select all the dye/lanes in these three colors.

Label Peaks

Follow these steps to label peaks.

Step Action

1

Select Label peaks from the Analysis menu. The following dialog box appears:


2

Check the “Size in bp” box and click OK.

Each peak that has been sized in GeneScan software will be labeled with the size in bp.

Filter Peak Labels

Introduction

Since you are interested in placing labels on only those peaks that correspond to the allelic peaks, you must filter out labels from the remainder of the unwanted peaks.

Note

You may wish the view the Plot window to see how the labels are filtered. Choose Show Plot window from the Views menu or click the Plot window icon on the right edge of the Main window.


Follow these steps to filter peak labels.

Step Action

1

Select Filter labels from the Analysis menu. The following dialog box appears:

2

Click OK.

Note

The last three of the four boxes for the filtering parameters have already been pre-selected as default.



About the Filter

Labels Command

The default parameters work well for most dinucleotide repeat markers.


This operation will remove labels from all the unwanted peaks and leave size labels on only the allele peaks of interest for each category. You are now ready to build a table containing the cleaned-up data.


Set Up an Allele Table

Introduction

Now you will create a table containing the following information: file name, sample info, category name, two columns for each allele designation per sample, and an overflow column to warn you if there are too many allele peaks (more than two) detected.

Column Format of

Table

When you have completed the setup for the table, the column format for the table should look like Figure 2-3.

Figure 2-3

Example of allele table column format



Set Up the Table

Follow these steps to set up the table in Figure 2-3.

Step Action

1

Choose Setup Table from the Table menu. The following window appears:

2

Uncheck all checkboxes by clicking the Uncheck all button.


Step Action

3

Select the following checkboxes:

♦

Name of Genescan software file

♦

Lane and dye

♦

Name of Category (use default options)

♦

Labels (use default options)

♦

Text if >N labels (use default options)

The Set Up Table window should now look like this:

4

Note

You can change the order of the selected fields by unchecking and then checking the checkboxes.

Click OK.



The Concept of

Overflow

If there are any anomalies (such as three peaks labeled for a particular individual/marker), a text entry, “Overflow,” will be displayed in the

Overflow column, indicating that there is a problem with the data from that particular individual/marker.

Note

“Text if > N labels” is the option in the Set up Table window to create an

Overflow column.


Append Rows of Filtered Peak Data

Introduction

Next, you must append to the table the “cleaned up” data.

View Table Data

Follow these steps to view table data.

Step Action

1

Select Append to Table from the Table menu.

2

Select Show Table from the Views menu, or click the checkered table icon on the right edge of the Main window.

The final table should now appear as follows.



Edit Column

Headings

The column headings for the size labels can be edited with different text.

Follow these steps to edit column headings.

Step Action

1

2

Click the column heading you want to edit (Peak 1, for example).

Select Edit Cell ( a - E) from the Edit menu. The following dialog box appears.

3

4

Type in a new name in the Edit Cell dialog box.

For example, Peak 1 and Peak 2 column headings can be edited to

Allele 1 and Allele 2.

Click OK.


Sort the Table by Column Heading

Introduction

The contents of the table can be alphanumerically sorted to group the data by any of the column headings.

Sort the Table

Follow these steps to change sort order.

Step Action

1

Select Sort Table from the Table menu. The following dialog box appears:

2

3

Choose Category in the first pop-up and File Name in the second pop-up.

Note

It is easier to view the table if you sort the contents by category and then by file name.

Click OK.


Export the Table

Introduction

The table can now be exported as a separate tab-and-carriage-returndelimited text file.


Follow these steps to export the table as a text file.

Step Action

1

Select Export to File from the Table menu.

2

In the dialog box, assign a name to the table.

3

4

Click Save.

The exported table file will have the Microsoft Excel icon and can be directly opened in Microsoft Excel by double-clicking the file icon.

Choose Clear Table from the Analysis menu.


Make a Macro of Tutorial Procedures

Introduction

Most of the steps (commands that you have used so far) have been recorded in the Step List window in the bottom-right corner of the Main window.

View the Steps in the Macro

If you choose the Show Step Window command from the Views menu, appear similar to Figure 2-4.

Figure 2-4

Show Step window

Edit Steps

You can now edit and save this list as a Macro. There are some steps in this list that you do not need to use the next time you want to process more data for the same project.

Follow this procedure to edit steps.

Step Action

1

While pressing the Command key ( a), click and select the following steps in the Step window:

2

♦

Clear all categories

♦

Show the Categories window

♦

Other Show... commands (optional)

Clear these two steps by selecting Clear from the Edit menu or by pressing the Clear key on the keyboard.

Note

The standard Macintosh Cut ( a-X), Copy (a-C), and

Paste ( a-V), commands also work in this window.


Follow this procedure to edit steps.

Step Action

3

Select and cut the “Clear Table” step using the Cut command from the Edit menu.

4

Select the “Remove labels from peaks” step and choose the Paste command from the Edit menu.

This will insert the step “Clear Table” after the “Remove labels” step and before the “Set up Table” step.

You should also clear from the step list any other commands that you don’t want to be included in the macro. The step list should now look like Figure 2-5.

Figure 2-5

Edited Step window

Create the Macro

Follow these steps to create a macro.

Step Action

1

Select Save Step Log from the Macro menu. The following dialog box appears:


2

3

Enter a unique name for the macro.

Click the checkbox and type in a number for the command key shortcut.

Step Action

4

Click OK.

Note

This will clear the Step window.

Run the Macro

Follow these steps to test the macro.

Step Action

1

2

3

Select Clear Dye/Lane List from the Analysis menu.

Import the same GeneScan software sample files you used in the preceding tasks.

Run the macro by pressing the Command key short-cut ( a - N, where N is the number you entered when saving the macro).

or

Run the macro by double-clicking the macro name in the Macro list window located in the lower-left corner of the Main window.

Note

To run a macro by double-clicking, you must choose Set

Preferences in the Edit menu and check the box “double-clicking runs macros & steps.”

Genotyper will automatically process the sample files and generate a table containing the allele sizes.

If the Macro Does

Not Work

If the macro doesn’t generate the table, go back through the preceding steps for generating a macro, and check the steps in the Step window against procedures in this chapter.


Make a Template for the Tutorial

Introduction

Once you have set up the required categories and created a macro, you can create a template (stationery pad) out of the Genotyper software file. You can then use this file to perform large-scale data processing automatically without having to set up the categories or write the macros again.

Create a

Template

Follow these steps to create a template.

3

4

5

6

Step Action

1

Select Clear Dye/Lane List and Clear Table from the Analysis menu.

2

You should now have a document that contains only the categories and the macro.

Select Current Step Log from the pane in the lower left corner of the

Main window and choose Clear Step Log from the Macro menu.

Save and close this document.

Open the folder that contains the newly saved document.

Click once to highlight the document file name.

Choose Get Info ( a – I) from the File menu. The Information window appears.


7

Check the Stationery pad box.

Step Action

8

Close the Get Info window.

The document is now a stationery pad, and will always retain the original categories and macro information.

Note

Stationery pads are permanent documents whose contents cannot be changed. When you open a stationery pad a new, untitled copy of the document is opened, containing the categories and macros.

Use the Template

Follow this step to use the template.

Step Action

1

Double-click the template icon.

or

Choose Open from the File menu and navigate to the folder containing the template. Select the template and click the Open button.

A new, untitled document will be opened that contains the categories and macros into which you can now import GeneScan files.


Microsatellite Analysis

Part 2

3


Introduction

This is the second part of the Microsatellite tutorial. This chapter shows you how to use features of Genotyper software to analyze patterns of inheritance using family marker data available in the Fluorescent

Genotyping Demonstration Kit.

In This Chapter


Topic

“Overview of Part 2 of the Microsatellite Tutorial”

“Import GeneScan Software files”

“Label Allele Peaks with Size Labels”

“Filter Peak Labels”

“Define Category Groups for Marker Alleles”

“Make New Categories for Alleles”

“Building an Allele Table”

“Edit Table”

“Export the Table”

See page

3-2

3-3

3-4

3-6

3-7

3-9

3-15

3-21

3-23

3


Overview of Part 2 of the Microsatellite Tutorial

Introduction

Most linkage analysis applications accept alleles from genotyping data as numeric integers. For example, alleles must be called as

1,2,3,4...and so on, or can be numbered based on the size of the alleles

(for example, alleles that fall in the range of 99.82 to 100.16 can be called 100).

Using the template you created in Chapter 2 “Microsatellite Analysis

Tutorial Part 1”, you will use Genotyper 2.0 software to create a table that contains allele numbers instead of sizes in base pairs. This kind of table can then be exported as a text file to be used in linkage-analysis applications.

Goals

The goal of the Part 2 of the microsatellite tutorial is to learn how to use

Genotyper software features and commands to

♦

Use the template you created in Part 1 of the Microsatellite tutorial, and import GeneScan files from the Fluorescent Genotyping

Demonstration Kit

♦

Create a Genotyper software table that contains allele numbers instead of sizes in base pairs using the Histogram window

♦

Export the table in a format that can be used by third-party analysis applications

Genotyper

Software


Use

In this tutorial, you will use the following Genotyper software features:

♦

Importing GeneScan software data

♦

Labeling and filtering peaks

♦

Defining Category groups for markers using the Histogram window

♦

Creating categories from labels

♦

Creating and filling tables

♦

Editing tables

♦

Changing size labels to allele names

♦

Exporting tables as files


Import GeneScan Software files

Introduction

You will be importing GeneScan software files into the template you created in Chapter 2, “Microsatellite Analysis Part 1.” If you don’t have that template open already, open it now.

Import

GeneScan

Software files


Step Action

1

Choose Import Genescan software file(s) from the File menu and navigate to the “Microsatellite Sample Files” folder. The Import dialog box should appear as follows:

2

3

4

Uncheck the Red dye color checkbox.

You will not be using the red dye information in this tutorial.

Uncheck the “Import raw data” checkbox.

This will improve performance of the file-importing process.

Click Import All to import all 14 sample files from the folder.

The Dye/lanes pane should now display all of the sample files.


Label Allele Peaks with Size Labels

Introduction

Next, you must label all of the allele peaks for each marker with the respective size in base pairs.

Label Allele Peaks

This procedure will assign labels to all allele peaks in each marker.

Follow these steps to label allele peaks.

Step Action

1

Make the Dye/lane pane active by clicking it or by tabbing until a vertical bar appears next to the active list.

2

Choose the Select All command from the Edit menu.

or

Click on the B, G, and Y buttons while pressing the Shift key to select all the dye/lanes.

Note

If you prefer to view more of the dye/lanes at one time, choose the Show Dye/lanes command from the Views menu, or click the Dye/lanes icon located on the right side of the Main window to open the Dye/lane window.

3

Choose Label peaks from the Analysis menu. The following dialog box appears:



(continued)

Step Action

4

Check the “Size in bp” box (default) and click OK.

You can view the labels by selecting individual dye/lanes. Each and every peak that has been sized in GeneScan software (including stutters, +A and background peaks) will be labeled with the size in bp.

Note

You can view multiple plots by selecting Show Plot window from the Views menu (or clicking the Plot window icon in the Main window).



Introduction

Since you are interested in placing labels on only those peaks that correspond to the allelic peaks, you must filter out labels from the remainder of the unwanted peaks.


Follow these steps to filter peak labels.

Step Action

1

Select Filter Labels from the Analysis menu. The following dialog box appears:

2

Click OK.

Note

The last three of the four boxes for the filtering parameters have already been preselected as default.


Labels Command



This operation will remove labels from all the unwanted peaks and leave size labels on only the allele peaks of interest for each category.


Define Category Groups for Marker Alleles

Introduction

You now must redefine each of the categories from Chapter 2,

“Microsatellite Analysis Part 1.”

In Chapter 2 (“Microsatellite Analysis Part 1”), each category referred to a marker. You will now create several category members for each marker and group them under the marker name.

Create Category

Groups for

Markers

Each of these new members for a particular group (marker) will represent a single allele.

Follow these steps to create category groups for markers.

Step Action

1

Choose the Show Categories window command from the Views menu, or click on the Categories window icon on the right side of the Main window to display the six categories (markers).

2

3

4

Click once to select the first category (D12S83).

Choose the Clear command from the Edit menu, or press the Clear key on the keyboard.

The corresponding information (name, size range and so on) for that category will still be displayed in the upper part of the window.

Highlight and delete the name of this category (D12S83) and type in a new name (for example, “unknown”).


Follow these steps to create category groups for markers.

(continued)

Step Action

5

Click the Group checkbox and type in the name of the marker

(D12S83) as the group name.

6

7

8

Note

You can use the Cut and Paste commands from the Edit menu in Steps 4 and 5.

Click the Add button.

This will create a single category member called “Unknown” under the Group D12S83.

Repeat Steps 2 through 6 for the other categories.

Verify that the window appears as follows:

9

10

Sort categories by Color and Size by choosing Category Sorting from the Views menu.

Close Categories window.

Why Create an

Unknown

Category

The “Unknown” category will find and label any peaks that do not belong to any of the allelic bins, but are still within the size range for that marker. These alleles will be labeled as an “unknown allele.”


Make New Categories for Alleles

Introduction

Now you will make new categories representing each allele for the selected marker. To do this, you will use the Statistics and Histogram options to bin the alleles for each marker.

You will use the range of peaks with labels in the Plot window to bin the peaks together for each allele, using the information from the Histogram display. The histograms are linked to the Statistics window, and are displayed according to the options that you set up for the statistics. The source of the data for the histograms in this exercise will be the peaks selected in the Plot window. Keep the bin size to 0.1 bp so that you can bin the alleles more precisely.

Setting Up the

Statistics Options

Follow these steps to set up the statistics options.

Step Action

1

Select Set Statistics Options from the Analysis menu. The following window appears:

2

3

Set the Bin Size to 0.1 (default is 0.5).

Click OK.



Selecting Peaks for

Individual Marker

Follow these steps to select peaks for individual markers.

Step Action

1

Click the Blue lane button next to the Dye/lane pane to select blue lanes only.

2

Using the cursor, draw a rectangular box that includes all allelic peaks for the first marker/category (D12S83) in the plot displayed in the Main window, or the Plot window, as shown here:



Creating New

Category

Members for Each

Marker

You can now use the Histogram window to select and create new category members.

Follow these steps to use the Histogram window to create new category members.

Step Action

1

Choose Show Histogram Window from the Views menu. The following window appears:

2

3

Verify that the correct category name (in this case D12S83) is displayed in the Category pop-up menu.

If not, choose the correct category from the pop-up menu.

Choose Zoom Out from the Zoom submenu of the Views menu.



(continued)

Step Action

4

Using the cross-hair cursor, draw a box around the first group of peaks. The distance between the left and right edges of the bin should be approximately 1 bp, as show here:


5

The status bar at the bottom of the histogram displays the value

(size in bp) of the range selected by the drawn box in increments of

0.1 bp (set in the Statistics Option window) as you drag the cursor across the peaks. The count in the status bar displays the number of peaks found within that bin.

While pressing the Shift key, choose Add Category from the

Category menu.

Note

If you choose Add Category without holding down the Shift key, the category size will appears as a range (for example, 100.39 to 101.39). If you hold down the Shift key, the size will appear as a center value and tolerance (for example, 100.89 ± 0.5). The center value will be calculated as the weighted average of the selected bins.


(continued)

Step Action

6

Type in the name or number of the first allele (1) in the following window:

7

8

Note

You can also name the alleles using the midpoint of the bins. For example, an allele falling in the bin 99.8 to 100.8 could be called “100.” Or, you can use an alternate naming system of your choice.

Click OK.

This will create a new, exclusive category member called “1” which is defined as a member of the group D12S83 with the highest peak in the size range of 100.89 ± 0.5 bp with blue dye.

Repeat Steps 3 through 6 for the remainder of the grouped peaks

(bins) from the same Histogram window. There are a total of five bins (alleles) for this marker and the allele names will be

1 through 5.



Finishing the

Remainder of the

Categories

Repeat “Selecting Peaks for Individual Marker,” and “Creating New

Category Members for Each Marker,” for the remainder of the markers.

The final Categories window should look like Figure 3-1.

Figure 3-1

Finished Category window

Each category group will have several members, including an

“Unknown” category member. If a labeled peak does not fall into any of the allelic bins, but is still within the size range of a marker, that particular peak will be labeled “Unknown.”


Building an Allele Table

Introduction

You will now build a table containing the number alleles for each marker.

This table will contain information about the samples, markers

(categories), and the corresponding numbered alleles for each individual.

Select Dye/lanes

Follow these steps to select dye/lanes.

Step Action

1

Choose Clear all labels from the Analysis menu.

2

3

This will remove any labels that may be left behind from previous steps.

Select the dye/lane pane.

Click each individual color button (B, G, Y) on the left side of the

Dye/lane pane with the Shift key held down.

or

Choose Select All from the Edit menu.





Step Action

1

Choose Label peaks from the Analysis menu. The following dialog box appears:

2

3

4

Check the Category name box only.

Uncheck all other boxes.

Click OK.

At this point, all allelic peaks that fall within the bin for each of the alleles as defined in the Categories window will be labeled with the respective allele number. All other peaks (stutters, +A peaks, background and so on) that do not fall in any of the allelic bins, but are still within the size range for that marker, will be labeled as

“unknown”.



Filter Unwanted

Labels

This operation will remove labels from most of the unwanted peaks and leave allele numbers on the allelic peaks.

Follow these steps to filter unwanted labels.

Step Action

1

Choose Filter labels from the Analysis menu. The following dialog box appears:

2

Click OK to accept the default settings.


Labels Command


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.

This operation will remove labels from all the unwanted peaks, and leave size labels on only the allele peaks of interest for each category.




You are now ready to build a table containing the cleaned-up data.

Follow these steps to set up a table.

Step Action

1

Choose Set up Table from the Table menu. The following window appears:

2

3

Check the column title boxes exactly as shown here.

Click the Options button next to Labels. The following dialog box appears:


Follow these steps to set up a table.

(continued)

Step Action

4

Click OK to accept the defaults.

The defaults of the label options create a table that includes a maximum of two peaks (alleles) per category (marker) with both alleles arranged next to each other in the table.

Note

The overflow column will be flagged if there are more than two alleles per category.


Table


Figure 3-2

Table column format

The Concept of

Overflow

If there are any anomalies (such as three peaks labeled for a particular individual/marker), a text entry, “Overflow,” will be displayed in the

Overflow column, indicating that there is a problem with the data from that particular individual/marker.

Note

“Text if > N labels” is the option in the Set up Table window to create an

Overflow column.



Append Rows

Follow these steps to append rows to your new table.

Step Action

1

2

Choose Append to Table command from the Table menu.

Choose Sort Table from the Table menu. The following dialog box appears:

3

4

5

Choose Category in the first pop-up menu.

Choose File Name in the second pop-up menu.

Click OK. The window should appear as follows:


Edit Table

Introduction

You should now edit the table to correct for overflows (more than two called alleles) by removing labels from the incorrectly labeled alleles.

View Plot Data for

Overflows

If there are any overflows indicated for any of the individuals, click the overflow cell. The corresponding plot area will be displayed in the Plot pane.

Remove Labels from Overflow

Alleles

Make appropriate edits as follows: if a background peak (stutter, +A, and so on) has not been filtered out leading to >2 alleles being called, then those labels can be removed.

Follow these steps to remove labels from a peak.

Step Action

1

In the Plot window, place the cross-hair cursor on the peak in question.

2

3

The vertical peak locator line will be aligned with the midpoint of the peak.

Click once to remove the label.

Choose Update Table command from the Table menu to update the table with the new edits.

Manually Call

Alleles

If an allele was not called for some reason, you can manually call that allele (provided that allele is still falls in the bin for one of the alleles).

Follow these steps to manually call an allele.

Step Action

1

Locate the peak of interest in the Plot window.

2

3

Place the cross-hair cursor on the peak until the vertical peak locator line is in the middle of the peak.

Click once to add the size in bp label to that peak (“Size in bp” is the default for the Click options under Analysis).

4



Change Size

Labels to Allele

Names

Follow these steps to change the size label to the corresponding allele name.

Step Action

1

1

Select the Dye/lane pane by tabbing or clicking inside the pane.

Click each individual color button (B, G, Y) on the left side of the

Dye/lane pane while pressing the Shift key.

or

1

2

3

4


Choose Change label from the Analysis menu.

Check the “Categories name” box.

Click OK.




Introduction

Once all the edits are finished, and you are satisfied with the contents and format of the table you can export the table as a tab-and-carriagereturn-delimited text file.


Follow these steps to export a table.

Step Action

1

Select Export to File from the Table menu.

2

Assign a unique file name and click Save.

The exported file is compatible with Microsoft Excel and will display the

Microsoft Excel icon. If you have Microsoft Excel installed on your computer, then you can directly open the exported file by doubleclicking the file name.


Human Identification

Tutorial

4


Introduction

This tutorial takes you through a series of steps in Genotyper software that are required to process data obtained from GeneScan software using the AmpF l

STR Blue™ Kit, and create a table containing the allelic information for the unknown forensic samples.

In This Chapter


Topic

“Overview of The Human Identification Tutorial”

“Contents of the AmpFlSTR Blue™ Kit”

“How AmpFlSTR Blue™ Data Was Preprocessed”

“Create a Genotyper ® Software Document”

“Import GeneScan ® Software Files”

“Define Category Groups for Allele Markers”

“Create Allele Categories for Marker Groups”

“Edit Categories for FGA Alleles”

“Label Alleles with Names and Numbers”

“Set Up an Allele Table”

“Append Rows to Table”

“Addendum to Human Identification Tutorial”

See page

4-2

4-3

4-4

4-5

4-6

4-7

4-9

4-13

4-14

4-15

4-17

4-19

4

Human Identification Tutorial 4-1

Overview of The Human Identification Tutorial

Introduction

In this tutorial, you will use Genotyper 2.0 software to process

AmpF l

STR Blue™ GeneScan software files, and create an allelic-size table to discriminate between heterozyote and homozygote individuals.

Goals

The goal of the Human Identification tutorial is to learn how to use

Genotyper software features and commands to

♦

Process GeneScan Sample files and work with two sets of allelic ladders that have been generated from the AmpF l

STR Blue Kit

♦

Create category groups for each marker supplied, and categories for each allele within a marker group

♦

Create a Genotyper Table containing allelic information that will identify 32 unknown forensic samples

Genotyper

Software


Use

In this tutorial, you will use the following Genotyper software features.

♦

Creating a new document

♦

Importing GeneScan software data

♦

Defining category groups

♦

Creating and editing allele categories

♦

Labeling alleles with names and numbers

♦

Creating tables

4-2 Human Identification Tutorial

Contents of the AmpF

lSTR Blue™ Kit

Introduction

A PCR-based DNA typing kit for fluorescent analysis of Short Tandem

Repeat (STR) markers is available from Applied Biosystems. The

AmpF l

STR Blue™ Kit includes reagents and protocols for the coamplification and detection of three STR loci – D3S1358, vWA and

FGA.

All three loci are tetranucleotide-repeat containing markers.

One primer from each locus is labeled with FAM that is detected as blue on the ABI P

RISM

Instruments.

Using Allelic

Ladders

The allele assignments to the unknown samples are made using a known set of allelic ladders for the three loci in the kit.

The example data in this tutorial consists of GeneScan software sample files for 32 unknown sample files and two sets of allelic ladders obtained using the ABI P

RISM

377 DNA Sequencer.

Loci Markers

The AmpF l

STR Blue Kit markers used for this tutorial are shown in

Table 4-1.

Table 4-1

AmpF l

STR Blue Kit markers

Marker Name

D3S1358 vWA

FGA

Chromosome

Location

3

12

4

Allele Size Range

(bp) a Number of Alleles

113-141 8

156-196 11

212-268 14 a. The size range and number of alleles refers to the allelic ladder only for each marker.


How AmpF

lSTR Blue™ Data Was Preprocessed

Introduction

The GeneScan software files you import to perform procedures in this tutorial are the result of data collection and GeneScan software analysis of AmpF l

STR Blue Kit reagents.

Data-Collection

Process

Fluorescently labeled products were electrophoresed on a 5 percent

Long Ranger gel, 36-cm WTR on the ABI P

RISM

377 DNA Sequencer using the GS36A-2400 Run module for 2.25 hours. The raw data was analyzed using GeneScan version 2.0.2 software and GS-350 ROX as an internal lane size standard.

GeneScan

Software

Analysis

Parameters

Table 4-2 shows the parameters used in the GeneScan analysis.

Table 4-2

GeneScan Software Analysis Parameters

Peak Detection

Threshold

50

Peak Detection

Minimum

Half-width

5

Sizing Method

Local Southern

Std. Peak Det.

Threshold

150


Create a Genotyper

®

Software Document

Introduction

To perform genotyping tasks on AmpF l

STR Blue™ kit marker data, you must import the related GeneScan files into a Genotyper Document.

Create a New

Genotyper

Software

Document

The following steps are used to create a new Genotyper software document.

Creating a new Genotyper software document.

If you have...

Not started Genotyper software

Already started Genotyper software

Then...

Launch the application by double-clicking it.

A new “Untitled” Genotyper software document appears

Choose the New command from the File menu.

A new “Untitled” Genotyper software document appears.


Import GeneScan

®

Software Files

Introduction

You must import the appropriate GeneScan software files containing data for the categories you define.

Import

GeneScan

Software Files


Step Action

1

Choose Import Genescan software file(s) from the File menu and navigate to the “HID Sample Files” folder. The import dialog box should appear as follows:

2

3

Because you will be working with only blue-colored dye/lanes, you can uncheck the other colors.

You may also uncheck the “Import raw data” box in order to speed up the file-importing process.

Click Import All to import all 34 Sample files from the folder.

The Dye/lanes window should now display all the Sample files in blue.


Define Category Groups for Allele Markers

Introduction

Each GeneScan software file contains data from three markers in different size ranges. You will define the boundaries for each marker based on the expected allele size range for that marker. These boundaries define a category group.

View Allelic

Ladders

The allelic ladder contains all the common alleles for each locus

(marker). The allelic ladder is run in at least one lane of each gel, or in at least one injection in a set of runs on a capillary. Genotypes are assigned to the samples by comparing the sizes obtained for the unknown sample alleles with the sizes obtained for the alleles in the allelic ladder. This method of genotyping normalizes potential run-to-run differences in sizing.

Follow these steps to view allelic ladders.

Step Action

1

Choose the Clear Category List command from the Analysis menu to remove the default “Everything” category in the Category list.

2

Click once to select and highlight the first sample file in the

Dye/lanes pane.

3

The first Sample file in this list contains the allelic ladder with alleles for all the markers that are displayed in the Plot pane.

From the Views menu choose the Zoom command and choose

Zoom in to better view the allelic ladder.

Define Category

Boundaries

Using the cursor in the Plot pane (which turns into a cross-hair cursor), draw a rectangular box for the first marker. Start a few bp before the leftmost peak, and end a few base pairs after the rightmost peak for that marker as shown in Figure 4-1.

Figure 4-1

Example of an allelic ladder for

AmpF l

STR

marker alleles



Add Category

Group For

Markers

You will now use the cursor in the Plot pane to mark the boundaries of a category group.

Follow these steps to define category groups for marker alleles.

Step Action

1

Choose Add Category from the Category menu (or press c-L).

The Add Category dialog box appears.


2

3

Fill out the Add Category dialog box as follows:

♦

Type in “New” as the category name. (You may also call this category “OL Allele?” where OL means “off ladder”).

♦

Check the Member of Group box and enter the name of the marker as the group name (D3S1358 in this example).

♦

Leave the following as defaults: All peaks, dye color blue only.

The size range in bp displayed is the outer limit of the category, as determined by the rectangular box drawn when defining category boundaries.

Click OK.

This creates a new category group called D3S138 with a category called New in the size range (106-147 bp).

Create Allele Categories for Marker Groups

Introduction

Since the allelic ladder for D3S1358 has eight alleles, you need to create additional categories, one for each allele, in the group D3S1358.

Each allele category will have a defined size with a

±

0.5 bp width so that all peaks in the unknown samples within that

±

0.5 bp range will be labeled with the same allele number.

Mark Category

Groups

Follow these steps to mark a category group.

Step Action

1

Ensure the group D3S1358 has been marked (that is, has a • mark).

2

If not, click and select the group name. Choose the Mark command from the Edit menu, or double-click the group name to mark it.

Note

Marking or unmarking a group will automatically mark/unmark all the categories belonging to that group.

Label Peaks for

Each Allele

Now, choose the Label peaks... command from the Analysis menu, and label all peaks with the size in bp.

Note

In addition to the alleles being labeled, some of the background noise peaks (if present) will also be labeled.



Filter Unwanted

Peak Labels

Follow these steps to remove unwanted labels.

Step Action

1

Choose Filter labels from the Analysis menu. The following dialog box appears:

2

3

4

Uncheck all the boxes except the second checkbox.

Set the filtering parameter to 25%.

Click OK to apply filter.



Make Categories from Labels

Follow these steps to make categories from labels.

Step Action

1

2

Choose Make from Labels from the Category menu.

Choose the options as follows:

3

Click OK.

This procedure creates a set of eight member categories (alleles) for the group “D3S1358” that are numbered sequentially from 12 through

19. The size in bp for each allele is the midpoint of the category with a

0.5 bp window on either side. All eight categories will be marked as exclusive (X) categories, meaning that any peak that falls within that window will belong to that particular allele.

Any peak(s) that falls outside of this window and does not belong to any of the other seven categories (alleles) will be called as “New” (or “OL

Alleles?”) alleles.



Example of

Category List

Figure 4-2 shows the resulting Category list after making allele categories for the D3S1358 category.

Figure 4-2

Category list for D3S1358 category group

Create Category

Groups for Other

Markers

Next, you will need to create groups for the remaining two markers.

Follow these steps to create category group for the other markers.

Step Action

1

Choose Clear all labels from the Analysis menu.

2

3

Note

Ensure the other groups are unmarked while creating new categories for each marker.

Define category groups for allele markers (see page 4-7).

Create allele categories for all marker groups (see page 4-9).

Note

vWA has 11 alleles numbered from 11 to 21, and FGA has 14 alleles numbered from 18 to 30.


Edit Categories for FGA Alleles

Introduction

Even though most of the alleles in these markers are 4 bp apart, there is a 2 bp variant allele in FGA. To account for this, you must do some manual editing of the categories/alleles for FGA.

How to Edit FGA

Categories

FGA allele 27 in this example is the 2 bp variant and must be renamed as allele 26.2.

Follow these steps to Edit FGA categories.

Step Action

1

Select the category/allele 27 in the group FGA and choose Edit category from the Analysis menu.

2

Change the name of the category to 26.2 and click Replace.

3

Change the names of the next five alleles to 27, 28, 29, 30 and 31 respectively, by using the Edit category command.

Example of Edited

FGA

Categories

The FGA group should now look like Figure 4-3.

Figure 4-3

FGA category list


Label Alleles with Names and Numbers

\

Introduction

Now that all the alleles have been defined for each marker, you can label alleles from both the allelic ladder and the unknown samples with the allele names/numbers.



Step Action

1

Tab to the Category pane or open the Category window by clicking the Category window icon.

6

7

8

4

5

2

3


Choose Mark from the Edit menu to mark all category members in all Groups (they will now have a • mark).

Close the Categories window if it is still open.

Choose Clear all labels from the Analysis menu to clear any existing labels.

Click the “Blue” button next to the Dye/Lanes pane to select all bluecolored dye/lanes.

Choose Label Peaks from the Analysis menu, and click the

“Category name” box.

Click OK.

Choose Filter labels from the Analysis menu, and click OK to accept default settings.

Example of

Labeled Allelic

Ladder

Figure 4-4 is an example of the allelic ladder labeled with the allele numbers.


Figure 4-4

Allelic ladder labeled with allele numbers

Set Up an Allele Table

Introduction

Now you will create a table containing the following information: file name, sample name, category name, two columns for each allele designation per sample, and an overflow column to put “New” alleles in.

New alleles are alleles that do not fall within the 0.5bp width of any category within a marker group.


Table


Figure 4-5

Example of allele table column format




Follow these steps to set up the table in Figure 4-5.

Step Action

1

Choose Setup Table from the Table menu. The following window appears:


2

3

Select these checkboxes as they appear in the preceding figure:

♦

Name of Genescan software file

♦

Lane & Dye

♦

Name of category (use default options)

♦

Labels (use default options)

♦

Text if >N labels (use default options)

Click OK.

Append Rows to Table

Introduction

Next, you can append the allele numbers to the table rows. If you need to edit any of the data in the columns, you can do so with the Edit Cell command from the Edit menu.

How to Append

Rows

Follow these steps to append rows to the table you created.

Step Action

1

From the Main window, select Append to table from the Table menu.

2

In the Table window, the following table appears:



Editing Table Cell

Contents

Follow these steps to change any of the column headings.

Step Action

1

2

3

Select the cell you want to edit.

Select Edit cell from the Edit menu.

Type in a new name in the edit box and click OK.

Note

Editing most commonly involves either removing or adding labels to certain peaks.

Removing Labels

Follow these steps to remove labels.

Step Action

1

Select a particular cell. (This will display corresponding peaks in the

Plot pane.)

2

3

Place the cross-hair cursor on the peak in the Plot pane that you want to edit.

Click once to remove the label.

Adding Peaks

Follow these steps to add peaks.

Step Action

1

Place the cross hairs cursor in the middle peak so that the vertical line locator is in the middle of the peak.

2

Click once.

3

4

5

A “size in bp” label will be added to the peak.

Choose Change labels from the Analysis menu.

Check the “Category name” box.

Click OK.

Updating the Table

To update the table with the new edits, choose Update table from the

Table menu.


Addendum to Human Identification Tutorial

Sample File

Labeling

Some peaks in a couple of the sample files will not be labeled by

Genotyper software in this exercise because they were not sized by

GeneScan software due to inadequate analysis settings. Some of the analysis settings that could lead to problems are

♦

Peak Amplitude Threshold set too high

♦

Min. Peak Half Width set too wide

♦

Different Size Calling methods used for different samples

The affected sample files are

♦

08•8 – A peak at approximately 224 bp was not sized in GeneScan software.

♦

18•18 – Allele 17 of D3S1358 at approximately 134 bp was not sized in GeneScan.

Note

Inadequate GeneScan software analysis settings is the most common reason why alleles or peaks are not called in Genotyper software. Such sample files need to be reanalyzed in GeneScan software with the proper Analysis

Parameters and then processed in Genotyper software.


Index

Symbols

+A peaks 3-19

“OL Allele?” 4-8

“unknown allele” 3-8

“Unknown” category 3-8

A

ABI Prism linkage mapping panels 1-3

Add Category 3-14

Allele Table 2-14

Alleles bins 3-11 calling manually 3-23 changing size labels to names 3-24

FGA 4-13 making new categories 3-11 markers 4-7

Allelic Ladders viewing definition 4-7

Allelic size table 4-2

AmpFlSTR

Blue GeneScan files 4-2

AmpFlSTR

Blue kit 1-3 anomalous peaks 1-4 append 2-17

Append to Table 3-22

C

Categories defining 2-7 sorting 2-9

Category Boundaries defining 4-7 category members 3-7

CEPH K1347 family 2-3

Choosing Data Collection Parameters 1-4 column format 3-21

Creating Sample Sheets 1-4 cross-hair cursor 3-14

D

dinucleotide repeat markers 1-6

E

Edit Cell 2-18

Edit Steps 2-21 exclusive (X) categories 4-11

Export 2-20, 3-25

F

FGA alleles 4-13

Fluorescent Genotyping Demonstration Kit 1-3 markers 2-3

G

gel 1-4 gel run parameters 1-4

GeneScan analysis parameters 2-4 importing files 2-10

GeneScan analysis software 1-4

GeneScan Files importing 1-6

GeneScan Sample Sheet 1-4

Genotyper creating new 2-6

Get Info window 2-24

H

heterozygote 2-2

HID tutorial folder 4-6

Index-1

Histogram Window 3-13

Histogram window marking peaks 3-14 homozygote 2-2

Human Identification Tutorial 1-1, 4-2

I

Import raw data 3-3

L

Labels filtering 1-6

M

Macros creating 2-22 definition 1-5 running 1-5 testing 2-23 matrix 1-4

Microsatellite 1-1

N

noise peaks 1-6

O

Overflow 2-16

P

Peaks filtering 2-13 labeling 2-12

Plot window 4-7

S

Set up Table 3-20

Show Step Window 2-21

Size Calling Methods 1-4 size-standard peaks 1-4

Sort Table 2-19

Statistics Options 3-11

Step List 1-5 stutters 3-18

Index-2

T

Tables exporting 2-20 tables updating 4-18

Template creating 2-24

Templates using 2-25

Text if > N labels 2-16

Z

Zoom 4-7

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