ABI PRISM® Genotyper® 2.0
Software
Applications Tutorials
© Copyright 2001, Applied Biosystems. All rights reserved.
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
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
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
iv
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
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
vi
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
vii
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
viii
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
ix
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xiii
Genotyper® Software
Tutorials
1
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
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 The tutorial is organized into three genotyping applications
Tutorial ♦ 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.
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 PRISM® You will use the following application kits in these tutorials:
Software ♦ Fluorescent Genotyping Demonstration Kit
Genotyping
Application Kits ♦ ABI PRISM linkage mapping panels
♦
AmpFlSTR Blue kit
Use In these tutorials, you will use the following Genotyper features:
Genotyper® ♦ Importing GeneScan software files
Software Features
♦
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® Software Tutorials 1-3
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 Adjust the pooling conditions to dilute the amplified products that
Samples consistently yield off-scale data. Optimal results can be obtained with
peak heights of approximately 1000 fluorescent units.
Creating Enter sample information in the GeneScan Sample Sheet for each
Sample Sheets 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- Follow these guidelines when choosing data collection parameters:
Collection ♦ Ensure that gel run parameters are consistent from run-to-run (for
Parameters
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- To ensure consistency in size calling, use the same size-calling method
Calling Methods 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® Software Tutorials
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 You can simplify the use of Genotyper software by thinking of how you
Results Tables 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 Most of the steps (commands) used in Genotyper software will be
Genotyper® recorded in the Step List (the bottom-right box in the Main window).
Software Steps
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
Genotyper® Software Tutorials 1-5
Importing It is not always necessary to import the raw data for each sample file.
GeneScan Unchecking the “Import raw data” box will make importing a large
Software Files 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 You can view full-screen windows that show the part of a Genotyper
Documents 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® Software Tutorials
2
Microsatellite Analysis
Part 1
2
Chapter Overview
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 This chapter contains the following topics.
Topic
See page
“Overview of Part 1 of the Microsatellite Tutorial”
2-2
“Contents of the Fluorescent Genotyping Demonstration Kit”
2-3
“How Fluorescent Genotyping Data Was Preprocessed”
2-4
“Source of DNA Template Samples”
2-5
“Create a Genotyper Software Document”
2-6
“Define Categories for Markers”
2-7
“Sort Categories by Dye Colors”
2-9
“Import GeneScan® Software Files”
2-10
“Label Allele Peak Data”
2-12
“Filter Peak Labels”
2-13
“Set Up an Allele Table”
2-15
“Append Rows of Filtered Peak Data”
2-19
“Sort the Table by Column Heading”
2-21
“Export the Table”
2-22
“Make a Macro of Tutorial Procedures”
2-23
“Make a Template for the Tutorial”
2-26
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 You will learn the following features of Genotyper software in this
Software tutorial:
Features You Will ♦ Creating a new document
Use
2-2 Microsatellite Analysis Part 1
♦
Creating categories
♦
Sorting categories
♦
Labeling and filtering peaks
♦
Setting up and exporting tables
♦
Creating and running macros
♦
Saving a document as a template
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 PRISM 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
Dye Label
Allele Size Range
(bp)
Heterozygosity
D12S83
FAM
98-113
0.81
D7S517
FAM
235-261
0.83
D1S220
HEX
226-260
0.82
D3S1266
HEX
281-303
0.81
D2S391
TET
139-153
0.79
D13S171
TET
171-197
0.73
Marker Name
Microsatellite Analysis Part 1 2-3
How Fluorescent Genotyping Data Was Preprocessed
Introduction Fluorescently labeled PCR products were electrophoresed on a ABI
PRISM 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 Table 2-2 shows the parameters set in GeneScan software for sizing
Software sample fragments.
Analysis Table 2-2 GeneScan Software Analysis Parameters
Parameters
2-4 Microsatellite Analysis Part 1
Peak
Detection
Threshold
Peak Detection
Minimum Half-width
Sizing Method
Std. Peak Det.
Threshold
50
3
Local Southern
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 Figure 2-1 shows the pedigree structure for the CEPH K1347 family.
Structure
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 Some of the abbreviations used in the sample file names in this data set
Individuals are as follows:
PGM – Paternal grandmother
PGF – Paternal grandfather
MGM – Maternal grandmother
MGF – Maternal grandfather
Microsatellite Analysis Part 1 2-5
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 The following steps are used to create a new document.
Genotyper
Then...
Software If...
Document Genotyper software is already open Select New from the File menu.
Genotyper software is closed
2-6 Microsatellite Analysis Part 1
Open Genotyper software by
double-clicking the applications icon.
Define Categories for Markers
What Is a Because each GeneScan software sample file can contain data from
Category? 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
1
Action
Choose Clear Category list from the Analysis menu.
This removes the default “Everything” category in the
Note
Category window.
2
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).
Microsatellite Analysis Part 1 2-7
Follow these steps to define categories for markers. (continued)
Step
4
Action
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.
Use orange for the HEX markers and green for the TET
Note
markers.
5
Click the All Peaks radio button.
6
Type in the expected allele size range for the marker name you
entered in Step 3.
Note
For marker allele size ranges, see Table 2-1 on page 2-3.
7
In the dye color checkboxes, select the dye color of the marker.
8
Click OK.
9
Repeat each of these steps for all six markers shown in Table 2-1 on
page 2-3.
View the Choose Show Categories from the View menu or click on the
Category Categories window icon on the right edge of the main window. You
List should now have a Category list that looks like Figure 2-2.
Figure 2-2
2-8 Microsatellite Analysis Part 1
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 Follow these steps to sort categories by dye color.
Categories
Step
Action
1
Choose Category Sorting from the Views menu. The following
window appears:
2
Set the sort order to Dye color in the first pop-up menu.
3
Click OK.
4
Close Categories window.
Microsatellite Analysis Part 1 2-9
Import GeneScan® Software Files
Introduction Once categories are defined, you must import the appropriate
GeneScan software files containing data for these categories.
Import Follow these steps to import GeneScan software files.
GeneScan
Step Action
Software Files
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
Uncheck the “Import raw data” checkbox.
It is not always necessary to import the raw data for each
Note
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.
3
Click Import All to import all 14 sample files from the folder.
continued on next page
2-10 Microsatellite Analysis Part 1
Viewing Once you’ve imported GeneScan software files, you can view dye/lanes
GeneScan from the imported files in the Dye/lanes list.
Software Files
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.
Microsatellite Analysis Part 1 2-11
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.
2-12 Microsatellite Analysis Part 1
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.
You may wish the view the Plot window to see how the labels are
Note
filtered. Choose Show Plot window from the Views menu or click the Plot
window icon on the right edge of the Main window.
Filter Peak Labels 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.
The last three of the four boxes for the filtering parameters
Note
have already been pre-selected as default.
continued on next page
Microsatellite Analysis Part 1 2-13
About the Filter The default parameters work well for most dinucleotide repeat markers.
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.
2-14 Microsatellite Analysis Part 1
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 When you have completed the setup for the table, the column format for
Table the table should look like Figure 2-3.
Figure 2-3
Example of allele table column format
continued on next page
Microsatellite Analysis Part 1 2-15
Set Up the Table Follow these steps to set up the table in Figure 2-3.
Step
2-16 Microsatellite Analysis Part 1
Action
1
Choose Setup Table from the Table menu. The following window
appears:
2
Uncheck all checkboxes by clicking the Uncheck all button.
Step
3
Action
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:
You can change the order of the selected fields by
Note
unchecking and then checking the checkboxes.
4
Click OK.
continued on next page
Microsatellite Analysis Part 1 2-17
The Concept of If there are any anomalies (such as three peaks labeled for a particular
Overflow 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.
“Text if > N labels” is the option in the Set up Table window to create an
Note
Overflow column.
2-18 Microsatellite Analysis Part 1
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.
continued on next page
Microsatellite Analysis Part 1 2-19
Edit Column The column headings for the size labels can be edited with different
Headings text.
Follow these steps to edit column headings.
Step
Action
1
Click the column heading you want to edit (Peak 1, for example).
2
Select Edit Cell (
box appears.
3
Type in a new name in the Edit Cell dialog box.
a - E) from the Edit menu. The following dialog
For example, Peak 1 and Peak 2 column headings can be edited to
Allele 1 and Allele 2.
4
2-20 Microsatellite Analysis Part 1
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
Choose Category in the first pop-up and File Name in the second
pop-up.
It is easier to view the table if you sort the contents by
Note
category and then by file name.
3
Click OK.
Microsatellite Analysis Part 1 2-21
Export the Table
Introduction The table can now be exported as a separate tab-and-carriage-returndelimited text file.
Export the Table 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
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.
4
2-22 Microsatellite Analysis Part 1
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 If you choose the Show Step Window command from the Views menu,
the Macro 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
1
2
Action
a
While pressing the Command key ( ), click and select the following
steps in the Step window:
♦
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.
a
a
The standard Macintosh Cut ( -X), Copy ( -C), and
Note
Paste ( -V), commands also work in this window.
a
Microsatellite Analysis Part 1 2-23
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
2-24 Microsatellite Analysis Part 1
Action
1
Select Save Step Log from the Macro menu. The following dialog
box appears:
2
Enter a unique name for the macro.
3
Click the checkbox and type in a number for the command key
shortcut.
Step
4
Action
Click OK.
Note
This will clear the Step window.
Run the Macro Follow these steps to test the macro.
Step
Action
1
Select Clear Dye/Lane List from the Analysis menu.
2
Import the same GeneScan software sample files you used in the
preceding tasks.
3
Run the macro by pressing the Command key short-cut ( - N,
where N is the number you entered when saving the macro).
a
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.
To run a macro by double-clicking, you must choose Set
Note
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 If the macro doesn’t generate the table, go back through the preceding
Not Work steps for generating a macro, and check the steps in the Step window
against procedures in this chapter.
Microsatellite Analysis Part 1 2-25
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 Follow these steps to create a template.
Template
Step
1
Action
Select Clear Dye/Lane List and Clear Table from the Analysis
menu.
You should now have a document that contains only the categories
and the macro.
2-26 Microsatellite Analysis Part 1
2
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.
3
Save and close this document.
4
Open the folder that contains the newly saved document.
5
Click once to highlight the document file name.
6
Choose Get Info (
window appears.
7
Check the Stationery pad box.
a – I) from the File menu. The Information
Step
8
Action
Close the Get Info window.
The document is now a stationery pad, and will always retain the
original categories and macro information.
Stationery pads are permanent documents whose
Note
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
1
Action
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 1 2-27
Microsatellite Analysis
Part 2
3
3
Chapter Overview
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 This chapter contains the following topics.
Topic
“Overview of Part 2 of the Microsatellite Tutorial”
See page
3-2
“Import GeneScan Software files”
3-3
“Label Allele Peaks with Size Labels”
3-4
“Filter Peak Labels”
3-6
“Define Category Groups for Marker Alleles”
3-7
“Make New Categories for Alleles”
3-9
“Building an Allele Table”
3-15
“Edit Table”
3-21
“Export the Table”
3-23
Microsatellite Analysis Part 2 3-1
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 In this tutorial, you will use the following Genotyper software features:
Software ♦ Importing GeneScan software data
Features You Will
Use ♦ Labeling and filtering peaks
3-2 Microsatellite Analysis Part 2
♦
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
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
Uncheck the Red dye color checkbox.
You will not be using the red dye information in this tutorial.
3
Uncheck the “Import raw data” checkbox.
This will improve performance of the file-importing process.
4
Click Import All to import all 14 sample files from the folder.
The Dye/lanes pane should now display all of the sample files.
Microsatellite Analysis Part 2 3-3
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.
If you prefer to view more of the dye/lanes at one time,
Note
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
3-4 Microsatellite Analysis Part 2
Choose Label peaks from the Analysis menu. The following dialog
box appears:
Follow these steps to label allele peaks. (continued)
Step
4
Action
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.
You can view multiple plots by selecting Show Plot window
Note
from the Views menu (or clicking the Plot window icon in the Main
window).
Microsatellite Analysis Part 2 3-5
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.
Filter Peak Labels
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.
The last three of the four boxes for the filtering parameters
Note
have already been preselected as default.
About the Filter The default parameters work well for most dinucleotide repeat markers.
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.
3-6 Microsatellite Analysis Part 2
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 Each of these new members for a particular group (marker) will
Groups for represent a single allele.
Markers
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
Click once to select the first category (D12S83).
3
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.
4
Highlight and delete the name of this category (D12S83) and type
in a new name (for example, “unknown”).
Microsatellite Analysis Part 2 3-7
Follow these steps to create category groups for markers. (continued)
Step
5
Action
Click the Group checkbox and type in the name of the marker
(D12S83) as the group name.
You can use the Cut and Paste commands from the Edit
Note
menu in Steps 4 and 5.
6
Click the Add button.
This will create a single category member called “Unknown” under
the Group D12S83.
7
Repeat Steps 2 through 6 for the other categories.
8
Verify that the window appears as follows:
9
Sort categories by Color and Size by choosing Category Sorting
from the Views menu.
10
Close Categories window.
Why Create an The “Unknown” category will find and label any peaks that do not
Unknown belong to any of the allelic bins, but are still within the size range for that
Category marker. These alleles will be labeled as an “unknown allele.”
3-8 Microsatellite Analysis Part 2
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 Follow these steps to set up the statistics options.
Statistics Options
Step
Action
1
Select Set Statistics Options from the Analysis menu. The following
window appears:
2
Set the Bin Size to 0.1 (default is 0.5).
3
Click OK.
continued on next page
Microsatellite Analysis Part 2 3-9
Selecting Peaks for Follow these steps to select peaks for individual markers.
Individual Marker
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:
continued on next page
3-10 Microsatellite Analysis Part 2
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
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.
3
Choose Zoom Out from the Zoom submenu of the Views menu.
Microsatellite Analysis Part 2 3-11
Follow these steps to use the Histogram window to create new category
members. (continued)
Step
4
Action
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:
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.
5
While pressing the Shift key, choose Add Category from the
Category menu.
If you choose Add Category without holding down the Shift
Note
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.
3-12 Microsatellite Analysis Part 2
Follow these steps to use the Histogram window to create new category
members. (continued)
Step
6
Action
Type in the name or number of the first allele (1) in the following
window:
You can also name the alleles using the midpoint of the
Note
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.
7
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.
8
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.
continued on next page
Microsatellite Analysis Part 2 3-13
Finishing the Repeat “Selecting Peaks for Individual Marker,” and “Creating New
Remainder of the Category Members for Each Marker,” for the remainder of the markers.
Categories
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.”
3-14 Microsatellite Analysis Part 2
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
1
Action
Choose Clear all labels from the Analysis menu.
This will remove any labels that may be left behind from previous
steps.
2
Select the dye/lane pane.
3
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.
continued on next page
Microsatellite Analysis Part 2 3-15
Label Allele Peaks Follow these steps to label allele peaks.
Step
Action
1
Choose Label peaks from the Analysis menu. The following dialog
box appears:
2
Check the Category name box only.
3
Uncheck all other boxes.
4
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”.
continued on next page
3-16 Microsatellite Analysis Part 2
Filter Unwanted This operation will remove labels from most of the unwanted peaks and
Labels 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.
About the Filter The default parameters work well for most dinucleotide repeat markers.
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.
continued on next page
Microsatellite Analysis Part 2 3-17
Set Up the Table You are now ready to build a table containing the cleaned-up data.
Follow these steps to set up a table.
Step
3-18 Microsatellite Analysis Part 2
Action
1
Choose Set up Table from the Table menu. The following window
appears:
2
Check the column title boxes exactly as shown here.
3
Click the Options button next to Labels. The following dialog box
appears:
Follow these steps to set up a table. (continued)
Step
4
Action
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.
The overflow column will be flagged if there are more than
Note
two alleles per category.
Column Format of When you have completed the setup for the table, the column format for
Table the table should look like Figure 3-2.
Figure 3-2
Table column format
The Concept of If there are any anomalies (such as three peaks labeled for a particular
Overflow 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.
“Text if > N labels” is the option in the Set up Table window to create an
Note
Overflow column.
continued on next page
Microsatellite Analysis Part 2 3-19
Append Rows Follow these steps to append rows to your new table.
Step
3-20 Microsatellite Analysis Part 2
Action
1
Choose Append to Table command from the Table menu.
2
Choose Sort Table from the Table menu. The following dialog box
appears:
3
Choose Category in the first pop-up menu.
4
Choose File Name in the second pop-up menu.
5
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 If there are any overflows indicated for any of the individuals, click the
Overflows overflow cell. The corresponding plot area will be displayed in the Plot
pane.
Remove Labels Make appropriate edits as follows: if a background peak (stutter, +A,
from Overflow and so on) has not been filtered out leading to >2 alleles being called,
Alleles then those labels can be removed.
Follow these steps to remove labels from a peak.
Step
1
Action
In the Plot window, place the cross-hair cursor on the peak in
question.
The vertical peak locator line will be aligned with the midpoint of the
peak.
2
Click once to remove the label.
3
Choose Update Table command from the Table menu to update the
table with the new edits.
Manually Call If an allele was not called for some reason, you can manually call that
Alleles 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
Place the cross-hair cursor on the peak until the vertical peak
locator line is in the middle of the peak.
3
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
Choose Update Table command from the Table menu to update the
table with the new edits.
Microsatellite Analysis Part 2 3-21
Change Size Follow these steps to change the size label to the corresponding allele
Labels to Allele name.
Names
Step
Action
1
Select the Dye/lane pane by tabbing or clicking inside the pane.
1
Click each individual color button (B, G, Y) on the left side of the
Dye/lane pane while pressing the Shift key.
or
Choose Select All from the Edit menu.
3-22 Microsatellite Analysis Part 2
1
Choose Change label from the Analysis menu.
2
Check the “Categories name” box.
3
Click OK.
4
Choose Update Table command from the Table menu to update the
table with the new edits.
Export the Table
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.
Export the Table 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.
Microsatellite Analysis Part 2 3-23
Human Identification
Tutorial
4
4
Chapter Overview
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 AmpFlSTR Blue™ Kit, and create a table containing the
allelic information for the unknown forensic samples.
In This Chapter This chapter contains the following topics.
Topic
See page
“Overview of The Human Identification Tutorial”
4-2
“Contents of the AmpFlSTR Blue™ Kit”
4-3
“How AmpFlSTR Blue™ Data Was Preprocessed”
4-4
“Create a Genotyper® Software Document”
4-5
“Import
GeneScan®
Software Files”
4-6
“Define Category Groups for Allele Markers”
4-7
“Create Allele Categories for Marker Groups”
4-9
“Edit Categories for FGA Alleles”
4-13
“Label Alleles with Names and Numbers”
4-14
“Set Up an Allele Table”
4-15
“Append Rows to Table”
4-17
“Addendum to Human Identification Tutorial”
4-19
Human Identification Tutorial 4-1
Overview of The Human Identification Tutorial
Introduction In this tutorial, you will use Genotyper 2.0 software to process
AmpFlSTR 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 AmpFlSTR 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 In this tutorial, you will use the following Genotyper software features.
Software ♦ Creating a new document
Features You Will
Use ♦ Importing GeneScan software data
4-2 Human Identification Tutorial
♦
Defining category groups
♦
Creating and editing allele categories
♦
Labeling alleles with names and numbers
♦
Creating tables
Contents of the AmpFlSTR Blue™ Kit
Introduction A PCR-based DNA typing kit for fluorescent analysis of Short Tandem
Repeat (STR) markers is available from Applied Biosystems. The
AmpFlSTR 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 PRISM Instruments.
Using Allelic The allele assignments to the unknown samples are made using a
Ladders 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 PRISM 377 DNA Sequencer.
Loci Markers The AmpFlSTR Blue Kit markers used for this tutorial are shown in
Table 4-1.
Table 4-1
AmpFlSTR Blue Kit markers
Marker Name
Chromosome
Location
Allele Size Range
(bp)a
Number of Alleles
D3S1358
3
113-141
8
vWA
12
156-196
11
FGA
4
212-268
14
a. The size range and number of alleles refers to the allelic ladder only for each marker.
Human Identification Tutorial 4-3
How AmpFlSTR 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 AmpFlSTR Blue Kit reagents.
Data-Collection Fluorescently labeled products were electrophoresed on a 5 percent
Process Long Ranger gel, 36-cm WTR on the ABI PRISM 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 Table 4-2 shows the parameters used in the GeneScan analysis.
Software
Table 4-2 GeneScan Software Analysis Parameters
Analysis
Peak Detection
Parameters
4-4 Human Identification Tutorial
Peak Detection
Threshold
Minimum
Half-width
Sizing Method
Std. Peak Det.
Threshold
50
5
Local Southern
150
Create a Genotyper® Software Document
Introduction To perform genotyping tasks on AmpFlSTR Blue™ kit marker data, you
must import the related GeneScan files into a Genotyper Document.
Create a New The following steps are used to create a new Genotyper software
Genotyper document.
Software
Creating a new Genotyper software document.
Document
If you have...
Then...
Not started Genotyper
software
Launch the application by double-clicking it.
Already started Genotyper
software
Choose the New command from the File
menu.
A new “Untitled” Genotyper software
document appears
A new “Untitled” Genotyper software
document appears.
Human Identification Tutorial 4-5
Import GeneScan® Software Files
Introduction You must import the appropriate GeneScan software files containing
data for the categories you define.
Import Follow these steps to import GeneScan software files.
GeneScan
Step
Action
Software Files
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
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.
3
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.
4-6 Human Identification Tutorial
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 The allelic ladder contains all the common alleles for each locus
Ladders (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.
The first Sample file in this list contains the allelic ladder with alleles
for all the markers that are displayed in the Plot pane.
3
From the Views menu choose the Zoom command and choose
Zoom in to better view the allelic ladder.
Define Category Using the cursor in the Plot pane (which turns into a cross-hair cursor),
Boundaries 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 AmpFlSTR marker alleles
continued on next page
Human Identification Tutorial 4-7
Add Category You will now use the cursor in the Plot pane to mark the boundaries of a
Group For category group.
Markers
Follow these steps to define category groups for marker alleles.
Step
1
Action
Choose Add Category from the Category menu (or press c-L).
The Add Category dialog box appears.
2
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.
3
Click OK.
This creates a new category group called D3S138 with a category
called New in the size range (106-147 bp).
4-8 Human Identification Tutorial
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 Follow these steps to mark a category group.
Groups
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.
Marking or unmarking a group will automatically
Note
mark/unmark all the categories belonging to that group.
Label Peaks for Now, choose the Label peaks... command from the Analysis menu, and
Each Allele label all peaks with the size in bp.
In addition to the alleles being labeled, some of the background noise
Note
peaks (if present) will also be labeled.
continued on next page
Human Identification Tutorial 4-9
Filter Unwanted Follow these steps to remove unwanted labels.
Peak Labels
Step
Action
1
Choose Filter labels from the Analysis menu. The following dialog
box appears:
2
Uncheck all the boxes except the second checkbox.
3
Set the filtering parameter to 25%.
4
Click OK to apply filter.
continued on next page
4-10 Human Identification Tutorial
Make Categories Follow these steps to make categories from labels.
from Labels
Step
Action
1
Choose Make from Labels from the Category menu.
2
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.
continued on next page
Human Identification Tutorial 4-11
Example of Figure 4-2 shows the resulting Category list after making allele
Category List categories for the D3S1358 category.
Figure 4-2
Category list for D3S1358 category group
Create Category Next, you will need to create groups for the remaining two markers.
Groups for Other
Follow these steps to create category group for the other markers.
Markers
Step
1
Action
Choose Clear all labels from the Analysis menu.
Ensure the other groups are unmarked while creating new
Note
categories for each marker.
2
Define category groups for allele markers (see page 4-7).
3
Create allele categories for all marker groups (see page 4-9).
vWA has 11 alleles numbered from 11 to 21, and FGA has 14 alleles
Note
numbered from 18 to 30.
4-12 Human Identification Tutorial
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 FGA allele 27 in this example is the 2 bp variant and must be renamed
Categories 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 The FGA group should now look like Figure 4-3.
FGA
Categories
Figure 4-3
FGA category list
Human Identification Tutorial 4-13
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.
Label Allele Peaks Follow these steps to label allele peaks.
Step
Action
1
Tab to the Category pane or open the Category window by clicking
the Category window icon.
2
Choose Select All from the Edit menu.
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.
4
Choose Clear all labels from the Analysis menu to clear any
existing labels.
5
Click the “Blue” button next to the Dye/Lanes pane to select all bluecolored dye/lanes.
6
Choose Label Peaks from the Analysis menu, and click the
“Category name” box.
7
Click OK.
8
Choose Filter labels from the Analysis menu, and click OK to
accept default settings.
Example of Figure 4-4 is an example of the allelic ladder labeled with the allele
Labeled Allelic numbers.
Ladder
Figure 4-4
4-14 Human Identification Tutorial
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.
Column Format of When you have completed the setup for the table, the column format for
Table the table should look like Figure 4-5.
Figure 4-5
Example of allele table column format
continued on next page
Human Identification Tutorial 4-15
Set Up the Table Follow these steps to set up the table in Figure 4-5.
Step
Choose Setup Table from the Table menu. The following window
appears:
2
Select these checkboxes as they appear in the preceding figure:
3
4-16 Human Identification Tutorial
Action
1
♦
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 Follow these steps to append rows to the table you created.
Rows
Step
Action
1
From the Main window, select Append to table from the Table menu.
2
In the Table window, the following table appears:
continued on next page
Human Identification Tutorial 4-17
Editing Table Cell Follow these steps to change any of the column headings.
Contents
Step
Action
1
Select the cell you want to edit.
2
Select Edit cell from the Edit menu.
3
Type in a new name in the edit box and click OK.
Editing most commonly involves either removing or adding labels to
Note
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
Place the cross-hair cursor on the peak in the Plot pane that you
want to edit.
3
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.
A “size in bp” label will be added to the peak.
3
Choose Change labels from the Analysis menu.
4
Check the “Category name” box.
5
Click OK.
Updating the Table To update the table with the new edits, choose Update table from the
Table menu.
4-18 Human Identification Tutorial
Addendum to Human Identification Tutorial
Sample File Some peaks in a couple of the sample files will not be labeled by
Labeling 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.
Inadequate GeneScan software analysis settings is the most common
Note
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.
Human Identification Tutorial 4-19
Index
column format 3-21
Creating Sample Sheets 1-4
cross-hair cursor 3-14
Symbols
+A peaks 3-19
“OL Allele?” 4-8
“unknown allele” 3-8
“Unknown” category 3-8
D
dinucleotide repeat markers
1-6
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
E
Edit Cell 2-18
Edit Steps 2-21
exclusive (X) categories
Export 2-20, 3-25
4-11
F
FGA alleles 4-13
Fluorescent Genotyping Demonstration Kit
markers 2-3
1-3
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
1-4
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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