Section 3 New Features and Procedures: Yfiler Kit. Applied Biosystems GeneMapper ID
GeneMapper ID is a software for analyzing genetic profiles from the AmpFlSTR® Yfiler™ PCR Amplification Kit. It allows you to quickly identify peaks visually and perform a size precision test. The software can also export data in the Export Combined Table format, which allows you to track all samples in an electrophoresis run.
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Section 3 New Features and Procedures: Yfiler Kit
Section 3 New Features and Procedures:
Yfiler Kit
This section covers:
New Features and Procedures Summary. . . . . . . . . . . . . . . . . . . . . 25
Allele Calling Parameters for New Marker Repeat Types . . . . . . . 27
−2-bp Filtering Workaround. . . . . . . . . . 27
Creating HID Analysis Methods for the Yfiler Kit. . . . . . . . . . . . . 30
Creating a Table Setting and Uploading Exported Haplotype(s) . . 37
New Features and Procedures Summary
Two features have been added in GeneMapper ID Software v3.2 to facilitate analysis of the AmpF lSTR ®
Yfiler
™
PCR Amplification
Kit:
• Allele calling parameters for new marker repeat types
• Plus stutter filtering
Additionally, Applied Biosystems has developed several procedures for using GeneMapper ID Software v3.2 with the Yfiler kit:
• Workaround for the DYS19 locus (
−2-bp filtering)
• Creating HID analysis methods for the Yfiler kit
• Exporting haplotypes from GeneMapper ID Software v3.2
provides a brief summary of these new features and procedures. For more detailed information, see the pages referenced in the table.
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Table 2 New features and procedures summary
Feature/Procedure Description
New feature: Allele calling parameters for new marker repeat types
In GeneMapper ID Software v3.1, allele calling parameters were only available for markers with tetranucleotide repeat motifs. In GeneMapper ID Software v3.2, however, allele calling parameters are available for four marker repeat types: tri-, tetra-, penta-, and hexanucleotide.
The four allele calling parameters appear in the Allele tab of the
Analysis Method Editor (as shown in
). You may enter all related analysis values directly into the Tri, Tetra,
Penta, or Hexa column fields, which allows you to see all values at once.
New feature: Plus stutter filtering (for the DYS392 locus)
To aid in interpreting genotype profiles, two new fields have been added to the Allele tab of the Analysis Method Editor specifically to filter out the DYS392 plus stutter:
• Plus Stutter Ratio
• Plus Stutter Distance
Procedure: Workaround for the DYS19 locus
( −2 bp filtering)
Procedure: Creating HID analysis methods for the
Yfiler kit
Laboratories may choose to implement a workaround for the
DYS19 locus by using the Minus A Ratio and Minus A Distance fields in the Allele tab of the Analysis Method Editor. These fields can be used to filter out the −2-bp stutter that is observed at the DYS19 locus.
Use the Analysis Method Editor in GeneMapper ID Software v3.2 to set analysis parameter values for analyzing the Yfiler kit data.
Procedure: Creating a table setting and uploading exported haplotype(s) for searching profiles with the Yfiler ™
Haplotype Database
Using the Table Setting Editor, you can create a table setting in
GeneMapper ID Software v3.2 to export haplotypes specifically for searching the Yfiler Haplotype Database for profile match estimation.
See
Page
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Allele Calling Parameters for New Marker Repeat Types
Allele Calling Parameters for New Marker Repeat
Types
Peak Detection
Analysis
Parameters
New Marker
Repeat Types
The peak detection analysis parameters allow you to specify the minimum peak height to be detected for analysis. This, in turn, controls the number of peaks analyzed. Peaks falling below the parameters specified are displayed in the electropherogram, but are not labeled and will not be genotyped.
In the Allele tab of the Analysis Method Editor, a number of parameters can be set to control allele calling, including:
• Bin set
• Marker-specific stutter ratio
• Amelogenin cutoff
• Marker repeat type
In GeneMapper ID Software v3.1, allele calling parameters were only available for markers with tetranucleotide repeat motifs. In
GeneMapper ID Software v3.2, however, allele calling parameters are available for four marker repeat types: tri-, tetra-, penta-, and hexanucleotide.
The four allele calling parameters appear in the Allele tab of the
Analysis Method Editor (as shown in
). You may enter all related analysis values directly into the Tri, Tetra, Penta, or
Hexa column fields, which allows you to see all values at once.
Plus Stutter Filtering and
−2-bp Filtering
Workaround
The following have been reported:
• Plus stutter at the DYS392 locus
•
−2-bp stutter at the DYS19 locus
Further descriptions of these loci are provided on page 28 . Allele
calling parameters that can be used as workarounds are provided on
.
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GeneMapper ® ID Software
Plus Stutter
(DYS392)
The DYS392 trinucleotide repeat locus displays the typical
−3-bp stutter but also a larger +3-bp stutter. Sequencing analysis of this
+3-bp stutter revealed that the product contains an additional repeat unit relative to the true allele peak.
Based on developmental validation studies conducted by
Applied Biosystems, the highest percent stutter observed for any
DYS392 allele was 7.9%.
−2-bp DYS19
Stutter
It has been reported that the DYS19 tetranucleotide repeat locus displays the typical
−4-bp stutter but also a smaller −2-bp stutter.
Based on developmental validation studies conducted by
Applied Biosystems, the highest percent stutter observed for any
DYS19 locus was 10.21%.
Allele Calling
Parameters
The Allele tab of the Analysis Method Editor is used to set allele calling parameters for data processing.
Select the Use marker-specific stutter ratio if available checkbox.
When this checkbox is selected, the software uses the stutter ratios defined in the panel. These values are provided in the panels supplied by Applied Biosystems.
To filter out the reproducible DYS19 tetranucleotide stutter, the
Minus A Ratio and Minus A Distance fields can be used.
Additionally, two new fields specifically developed to filter out the
DYS392 trinucleotide repeat plus stutter have been added to the
Analysis Method Editor: Plus Stutter Ratio and Plus Stutter Distance.
Figure 6 on page 29 shows allele calling parameters that can be used
with GeneMapper ID Software v3.2.
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User Bulletin
Plus Stutter Filtering and
−
2-bp Filtering Workaround
Figure 6 Allele calling parameters
Note:
The circled values shown in Figure 6
above are based on developmental validation studies conducted at Applied Biosystems and should only be used as reference values. Each laboratory should perform their own internal validation studies to determine the appropriate values to enter. The DYS19 workaround is not locusspecific. All peaks for the tetranucleotide loci falling within the range specified in the Minus A Ratio and Minus A Distance fields will be filtered.
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GeneMapper ® ID Software
Creating HID Analysis Methods for the Yfiler Kit
Use the Analysis Method Editor to set analysis parameter values for analyzing the Yfiler kit data.
Note: The size distribution for the Yfiler kit is ~100 to 330 bp. As a result, the 450-bp size standard peak does not need to be included in the size standard definition for analysis.
Workflow
1. Import the Yfiler kit panels and bin sets.
See the GeneMapper ™ ID Software Version 3.1 Human
Identification Analysis Tutorial (PN 4335523) for procedures on importing panels and bin sets.
2. Create a new HID_Advanced analysis method for use with the
AmpFlSTR Yfiler kit (below).
3. Create a new HID_Classic analysis method for use with the
AmpFlSTR Yfiler kit (
Create the
HID_Advanced
Analysis Method
To create the HID_Advanced analysis method:
1.
Select Tools > GeneMapper Manager to open the
GeneMapper Manager.
2.
Select the Analysis Methods tab, then click New to open the
New Analysis Method dialog box.
30
3.
Select HID, then click OK to open the Analysis Method
Editor with the General tab selected.
4.
Select the analysis method settings shown in
.
IMPORTANT!
You must select your settings on all the tabs before you click OK to save the analysis method and return to GeneMapper Manager!
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Creating HID Analysis Methods for the Yfiler Kit
Analysis Method
Settings
IMPORTANT!
The values shown in
Table 3e on page 35 are based on developmental validation studies
conducted at Applied Biosystems and should only be used as reference values. Each laboratory should perform their own internal validation studies to determine the appropriate values to enter.
Table 3a Analysis method settings: General tab
Settings Tab
General
Enter a name of your choosing here. See the
Notes below.
Notes for the General tab:
1. Enter a name of your choosing. For example, enter:
– AmpFlSTR_Yfiler_AdvancedMode, if you are creating an HID_Advanced analysis method, or
– AmpFlSTR_Yfiler_ClassicMode, if you are creating an HID_Classic analysis method
2. Select your instrument from the drop-down list.
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GeneMapper ® ID Software
Table 3b Analysis method settings: Allele tab
Allele
Tab Settings
Notes for the Allele tab:
1. Be sure to select the Use marker-specific stutter ratio if available checkbox. When this checkbox is selected:
– The software uses the stutter ratios defined in the panel.
– The stutter ratios include all loci except for the DYS392 +3 stutter and the DYS19 −2 stutter. The stutter ratios are defined in the Analysis Method Editor under the Allele tab, if applicable.
2. See
“Plus Stutter Filtering and −2-bp Filtering Workaround” on page 27
for a discussion of the
Tetra column values in the Minus A Ratio/Distance fields and Tri column values in the Plus Stutter
Ratio/Distance fields.
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Creating HID Analysis Methods for the Yfiler Kit
Table 3c Analysis method settings: Peak Detector tab
Settings Tab
Peak Detector
Select
Advanced or
Classic here, depending on the analysis method you are creating. See the
Notes below.
Notes for the Peak Detector tab:
From the Peak Detection Algorithm drop-down list, select:
• Advanced, if you are creating an HID_Advanced analysis method, or
• Classic, if you are creating an HID_Classic analysis method
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Table 3d Analysis method settings: Peak Quality tab
Settings Tab
Peak Quality
Notes for the Peak Quality tab:
The values shown in the Peak Quality tab have been chosen based on empirical data generated at
Applied Biosystems and should only be used as reference values. Each laboratory should perform their own internal validation studies to determine the appropriate values to enter.
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Creating HID Analysis Methods for the Yfiler Kit
Table 3e Analysis method settings: Quality Flags tab
Settings Tab
Quality Flags
Notes for the Quality Flags tab:
The weighted values in the Quality Flag Settings section were ranked on a scale from most important to least important. This ranking was performed by the Applied Biosystems HID Group.
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Create the
HID_Classic
Analysis Method
To create the HID_Classic analysis method:
1.
Select Tools > GeneMapper Manager to open the
GeneMapper Manager.
2.
Select the Analysis Methods tab, then click New to open the
New Analysis Method dialog box.
3.
Select HID, then click OK to open the Analysis Method
Editor.
4.
Select the analysis method settings shown in
.
IMPORTANT!
You must select your settings on all the tabs before you click OK to save the analysis method and return to GeneMapper Manager!
What Next?
After creating analysis methods for the Yfiler kit, continue with data analysis as follows:
1. Import sample files
2. Select analysis parameters
3. Analyze the project
For More
Information
For more information, refer to the following documents:
• GeneMapper ™ ID Software Version 3.1 Human Identification
Analysis User Guide (PN 4338775)
• GeneMapper ™ ID Software Version 3.1 Human Identification
Analysis Tutorial (PN 4335523)
• AmpF lSTR ® Yfiler
(PN 4358101)
™ PCR Amplification Kit User’s Manual
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Creating a Table Setting and Uploading Exported Haplotype(s)
Creating a Table Setting and Uploading Exported
Haplotype(s)
Using the Table Setting Editor, you can create a table setting in
GeneMapper ID Software v3.2 to export haplotypes specifically for searching the Yfiler ™ Haplotype Database for profile match estimation.
Database Online
Search Tool
The Yfiler Haplotype Database online search tool searches for haplotypes generated with the Yfiler kit. The online search tool allows you to:
• Estimate the frequency of a given Y-chromosome haplotype in specified populations. (Frequency calculations are based on the haplotype data generated from the 17 loci included in the Yfiler kit. This data was compiled from more than 3000 samples from a range of populations.)
• Estimate the frequency of haplotypes in a number of reference populations.
• Search complete or partial profiles generated with the Yfiler kit.
• Compare the discrimination capacity of the Yfiler kit relative to the recombination of Ystr loci.
Workflow
1. Use the Table Setting Editor to create a table setting in
GeneMapper ID Software v3.2 (below).
2. Upload the exported haplotype(s) into the Yfiler Haplotype
Database for profile match estimation (
).
Create a Table
Setting
Define custom Samples and Genotypes views for displaying the
Yfiler kit data.
To create a table setting:
1.
From the GeneMapper Manager, select the Table Settings tab.
2.
Click New to open the Table Setting Editor.
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GeneMapper ® ID Software
To create a table setting: (continued)
3.
Select the General tab and complete it as follows: a. Enter a Name of your choosing. b. If desired, enter a Description.
4.
Select the Samples tab and complete it as follows: a. In the Column Settings section, deselect all options. b. In the Font Settings section, leave the default values.
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Creating a Table Setting and Uploading Exported Haplotype(s)
To create a table setting: (continued)
5.
Select the Genotypes tab and complete it as follows: a. In the Column Settings section, select:
– Sample Name
– Marker
– Allele
Deselect all of the other options.
b. In the Font Settings section, leave the default values.
c. In the Allele Settings section:
– Enter 2 in the Number of Alleles field.
– Deselect the Keep Allele, Size... check box.
6.
Click OK to save the table settings, close the Table Setting
Editor, and return to the GeneMapper Manager.
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Upload the
Exported
Haplotype(s)
Upload the exported haplotype(s) into the Yfiler Haplotype
Database.
To upload the exported haplotype(s):
1.
If you are not already there, open the project in GeneMapper
ID Software v3.2.
2.
From the Table Settings drop-down list, select the table setting you created specifically to export haplotype(s) for searching the Yfiler Haplotype Database.
3.
For the remaining upload procedures, see:
• The Yfiler Haplotype Database: http://www.appliedbiosystems.com/yfilerdatabase/ or
• The AmpF l STR
®
Yfiler
™
PCR Amplification Kit User’s
Manual (PN 4358101)
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Public link updated
The public link to your chat has been updated.
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Key features
- Export Combined Table format
- Labeled peak assignments
- Allele calling parameters for different repeat types
- Plus stutter filtering
- HID analysis methods for the Yfiler kit
- Exporting haplotypes