Thermo Fisher Scientific VeriFiler Plus PCR Amplification Kit User Guide

VeriFiler™ Plus PCR Amplification Kit
USER GUIDE
Catalog Number A35495
Publication Number MAN0017493
Revision C.0
For Research, Forensic, or Paternity Use Only. Not for use in
diagnostic procedures.
Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0017493
Revision
Date
Description
C.0
10 August 2020
Add validation experiments for the updated formulation. Add information
about the D10S2148 marker.
B.0
17 October 2018
A.0
24 May 2018
Add new chapter: Chapter 6, “Experiments and results”. Minor edits.
New document.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Kit overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Internal quality control system for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Validated DNA amounts and PCR cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
About the primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Changes to the primers in the updated formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Primer comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Dyes used in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Loci amplified by the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Standards and controls that are required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Allelic ladder profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
DNA Control 007 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Instruments and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
■
CHAPTER 2 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Importance of quantification before STR analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of DNA quantity on results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods of quantifying DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
19
20
21
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
(Optional) Prepare low-TE buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Thaw reagents (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Direct amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
VeriFiler™ Plus PCR Amplification Kit User Guide
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Contents
■
CHAPTER 3 Perform electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . .
Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Create a 3500 QC protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27
27
28
29
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) . . . . . .
Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Obtain and activate 6-dye license . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31
31
31
33
Prepare samples for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
■
™
CHAPTER 4 Analyze data with GeneMapper ID‑X Software . . . . . . . . . . . . . . . . 35
™
Overview of the GeneMapper ID‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
™
Set up the GeneMapper ID‑X Software for analysis (before first use of the kit) . . . . . . . .
™
Workflow: Set up GeneMapper ID‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Check panel, bin, and stutter file versions on your computer . . . . . . . . . . . . . . . . . . . .
(If needed) Download newer versions of panel, bin, and stutter files . . . . . . . . . . . . . .
Import panels, bins, and marker stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(Optional) Define custom table or plot settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
37
37
37
38
38
42
Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Enter Analysis Method settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
About the GS600_LIZ_(60–460) size standard definition file . . . . . . . . . . . . . . . . . . . . . 51
Create a size standard definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Analyze and edit sample files with GeneMapper ID-X Software . . . . . . . . . . . . . . . . . . . . . . 53
Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
™
For more information on using the GeneMapper ID‑X Software . . . . . . . . . . . . . . . . . . . . . 54
■
CHAPTER 5 Assess the PCR reaction with the Internal Quality
Control System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Overview of the Internal Quality Control system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Evaluate the PCR reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Balanced profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ski slope profile with decreased IQCL peak height . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ski slope profile with IQC peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
No sample peaks with IQC peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
55
57
58
58
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VeriFiler™ Plus PCR Amplification Kit User Guide
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CHAPTER 6 Experiments and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Repeated validation experiments using the updated formulation . . . . . . . . . . . . . . . . . 61
Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 2.2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 3.9.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 3.9.6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal cycling temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
61
61
61
62
62
62
63
Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 3.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Accuracy observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precision and size window description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precision and size window observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
64
64
65
67
67
Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Causes of extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extra peaks: Stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extra peaks: Addition of 3' A nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extra peaks: Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
83
83
83
89
90
Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 3.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Loci in this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nature of polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
92
92
92
92
93
93
93
Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
SWGDAM Guideline 3.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Nonhuman studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
SWGDAM guideline 3.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Sensitivity observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 3.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Degraded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mixture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 3.8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mixture study overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mixture study observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VeriFiler™ Plus PCR Amplification Kit User Guide
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98
98
99
102
102
102
103
5
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Resolution of genotypes in mixed samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Limit of detection of the minor component . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SWGDAM guideline 3.7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Population data overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Loci in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Population samples used in these studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concordance studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Probability of Identity definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Probability of paternity exclusion observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
107
107
107
107
108
109
111
112
129
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APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
■
APPENDIX B Materials required but not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Sample preparation required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Thermal cycler required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
™
ProFlex PCR System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
™
Veriti Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
™
GeneAmp PCR System 9700 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
133
133
134
134
Genetic analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
3500 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
3130 Series Genetic Analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Analysis software required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
™
GeneMapper ID‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Miscellaneous required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Plates and tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Laboratory supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
■
APPENDIX C PCR work areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
■
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6
VeriFiler™ Plus PCR Amplification Kit User Guide
Contents
■
Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
VeriFiler™ Plus PCR Amplification Kit User Guide
7
1
Product information
■
■
■
■
■
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Instruments and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
Kit overview
The Applied Biosystems™ VeriFiler™ Plus PCR Amplification Kit is a 6-dye, short
tandem repeat (STR) multiplex assay for the amplification of specific loci in human
genomic DNA.
The kit amplifies:
• 23 autosomal STR loci: D3S1358, vWA, D16S539, CSF1PO, D6S1043, D8S1179,
D21S11, D18S51, D5S818, D2S441, D19S433, FGA, D10S1248, D22S1045,
D1S1656, D13S317, D7S820, Penta E, Penta D, TH01, D12S391, D2S1338, and
TPOX
• Two internal quality control markers (IQCS and IQCL)
• One insertion/deletion polymorphic marker on the Y chromosome (Y indel)
• Amelogenin (sex determining marker)
Internal quality control system for PCR
The VeriFiler™ Plus PCR Amplification Kit is an STR kit that includes an internal
quality control (IQC) system for PCR. The IQC system has two synthetic targets,
one low molecular weight and one high molecular weight, that are amplified with the
sample. The behavior of the IQC target peaks can be used to evaluate the success of
the PCR and indicate sample quality.
Validated DNA amounts and PCR cycles
The kit is validated for use with 500 pg of DNA for 29 PCR cycles. The DNA input
volume is 17.5 μL, for a total reaction volume of 25 µL.
8
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 1 Product information
Product description
1
About the primers
The VeriFiler™ Plus PCR Amplification Kit primers are manufactured to maximize the
assay signal‑to‑noise ratio and simplify the interpretation of results.
Table 1 compares the VeriFiler™ Plus kit primers to the VeriFiler™ Express PCR
Amplification Kit, GlobalFiler™ PCR Amplification Kit, GlobalFiler™ Express PCR
Amplification Kit, and NGM Detect™ PCR Amplification Kit. The VeriFiler™ Plus kit
primers do not contain any mobility modifiers.
Table 2 lists the dyes that are used in the kit.
Table 3 provides the following information about the loci amplified by the kit:
• Locus designation
• Chromosome location
• Alleles included in the VeriFiler™ Plus Allelic Ladder
• Dye label
• AmpFℓSTR™ DNA Control 007 genotype
Changes to the primers in the updated formulation
Artifacts, caused by primers amplifying non-human DNA, were reported by a small
number of laboratories with the original formulation of the VeriFiler™ Plus kit. After
internal investigations, we determined that a few primers caused artifact peaks
when DNA from certain bacterial species was present. Accordingly, we extended
primers for the D6S1043, D16S539, Penta E, D18S51, and CSF1PO markers by 1–2
nucleotides and leveraged the design of the IQCL marker from the NGM Detect™
kit and the GlobalFiler™ IQC kit. The primer change for the IQCL marker causes
the marker to size ~15 bp greater in the updated formulation than in the original
formulation. We did not make any other changes to the VeriFiler™ Plus kit formulation,
protocols, or workflow.
The updated formulation of the VeriFiler™ Plus kit was revalidated with internal
and external testing. Other than mitigating the artifact issue, the performance of
the updated formulation is fully comparable to that of the original formulation. In
Chapter 6, “Experiments and results”, all data from the revalidation studies are
labeled with "updated formulation".
VeriFiler™ Plus PCR Amplification Kit User Guide
9
1
Chapter 1 Product information
Product description
Primer comparison
Use Table 1 to compare the VeriFiler™ Plus kit to the four kits listed in the table. Do
not use Table 1 to make comparisons between the other kits; for example, do not
compare the VeriFiler™ Express kit to the GlobalFiler™ kit.
Note: A concordance population study was performed and genotypes were
compared between the same samples amplified with the VeriFiler™ Plus kit and
the VeriFiler™ Express kit and Huaxia™ Platinum™ kit. For more information, see
“Concordance studies” on page 109.
Table 1 VeriFiler™ Plus kit primer comparison
VeriFiler™ Plus kit compared to...
10
Marker
VeriFiler™ Express kit and
Huaxia™ Platinum™ kit
GlobalFiler™ kit and
GlobalFiler™ Express kit
NGM Detect™ kit
IQCS
Marker not included
Marker not included
Primers are the same
IQCL
Marker not included
Marker not included
Primers are the same
D2S441
1 primer is the same
1 primer is the same
Primers are different
D10S1248
Primers are different
Primers are different
Primers are different
D22S1045[1]
Primers are the same
Primers are the same
Primers are different
D3S1358[1]
Primers are different
Primers are different
Primers are different
D8S1179[1]
Primers are the same
Primers are the same
Primers are different
D19S433[1]
1 primer is the same
1 primer is the same
Primers are different
D5S818
Primers are different
Primers are different
Marker not included
vWA[1]
Primers are the same
Primers are the same
Primers are different
D1S1656
Primers are different
Primers are different
Primers are different
TH01
1 primer is the same
1 primer is the same
Primers are different
D21S11
Primers are the same
Primers are the same
Primers are different
D13S317
1 primer is the same
1 primer is the same
Marker not included
D12S391
Primers are different
Primers are different
Primers are different
D16S539[1]
1 primer is different
1 primer is different
Primers are different
FGA[1]
Primers are the same
Primers are the same
Primers are different
D18S51
1 primer is the same
1 primer is the same
Primers are different
D7S820
Primers are different
Primers are different
Marker not included
D2S1338
Primers are the same
Primers are the same
Primers are different
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 1 Product information
Product description
1
Table 1 VeriFiler Plus kit primer comparison (continued)
VeriFiler™ Plus kit compared to...
[1]
Marker
VeriFiler™ Express kit and
Huaxia™ Platinum™ kit
GlobalFiler™ kit and
GlobalFiler™ Express kit
NGM Detect™ kit
CSF1PO
1 primer is the same
1 primer is the same
Marker not included
TPOX
1 primer is the same
1 primer is the same
Marker not included
D6S1043[1]
Primers are different
Marker not included
Marker not included
Penta D[1]
Primers are different
Marker not included
Marker not included
Penta E
1 primer is the same
Marker not included
Marker not included
Y indel
Primers are the same
Primers are the same
Primers are the same
AMEL[1]
Primers are the same
Primers are the same
Primers are the same
The VeriFiler™ Plus kit has one or more additional SNP-specific primers
Dyes used in the kit
Table 2 Dyes used in the VeriFiler™ Plus PCR Amplification Kit
Dye
Color
Label
6‑FAM™
Blue
Samples, allelic ladders, and controls
VIC™
Green
TED™
Yellow
TAZ™
Red
SID™
Purple
LIZ™
Orange
GeneScan™ 600 LIZ™ Size Standard v2.0
Loci amplified by the kit
Table 3 VeriFiler™ Plus kit loci and alleles
Locus
designation
Chromosome
location
IQCS
—
1, 2
D3S1358
3p21.31
9–20
15, 16
vWA
12p13.31
11–24
14, 16
D16S539
16q24.1
5, 8–15
9, 10
CSF1PO
5q33.3-34
6–15
11, 12
Alleles included in Allelic Ladder
VeriFiler™ Plus PCR Amplification Kit User Guide
Dye label
DNA Control
007 genotype
6-FAM™
2
11
1
Chapter 1 Product information
Product description
Table 3 VeriFiler Plus kit loci and alleles (continued)
Locus
designation
Chromosome
location
D6S1043
6q15
7–25
IQCL
—
1, 2
Y indel
Yq11.221
1, 2
Amelogenin
12
Alleles included in Allelic Ladder
Dye label
DNA Control
007 genotype
6-FAM™
12, 14
2
VIC™
X p22.1– 22.3, X, Y
Y: p11.2
D8S1179
8q24.13
D21S11
21q11.2– q21
D18S51
2
X, Y
5–19
12, 13
24, 24.2, 25–28, 28.2, 29, 29.2, 30, 30.2, 31,
31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–
38
28, 31
18q21.33
7, 9, 10, 10.2, 11–13, 13.2, 14, 14.2, 15–27
12, 15
D5S818
5q21-31
7–18
D2S441
2p14
8–11, 11.3, 12–17
D19S433
19q12
5–12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2,
17, 17.2, 18.2, 19.2
14, 15
FGA
4q28
13–26, 26.2, 27–30, 30.2, 31.2, 32.2, 33.2, 42.2,
43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2,
51.2
24, 26
D10S1248
10q26.3
8–19
12, 15
D22S1045
22q12.3
8–19
D1S1656
1q42.2
9–14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18, 18.3,
19.3, 20.3
D13S317
13q22-31
5–16
11
D7S820
7q11.21-22
6–15
7, 12
Penta E
15q26.2
5–26
7, 12
Penta D
21q22.3
2.2, 3.2, 5–17
TH01
11p15.5
4–9, 9.3, 10–12, 13.3
7, 9.3
D12S391
12p13.2
14–19, 19.3, 20–27
18, 19
D2S1338
2q35-37.1
11–28
20, 23
TPOX
2p23-2per
5–15
8
11
TED™
TAZ™
14, 15
11, 16
13, 16
SID™
11, 12
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 1 Product information
Product description
1
Standards and controls that are required
For the VeriFiler™ Plus kit, the panel of standards needed for PCR amplification, PCR
product sizing, and genotyping are:
• DNA Control 007—A positive control for evaluating the efficiency of the
amplification step and STR genotyping using the VeriFiler™ Plus Allelic Ladder.
DNA Control 007 is present in the kit. See “DNA Control 007” on page 15.
• GeneScan™ 600 LIZ™ Size Standard v2.0—Used for obtaining sizing results.
This standard, which has been evaluated as an internal size standard, yields
precise sizing results for PCR products. Order the GeneScan™ 600 LIZ™ Size
Standard v2.0 (Cat. No. 4408399) separately.
• VeriFiler™ Plus Allelic Ladder—Developed for accurate characterization of the
alleles amplified by the kit. The Allelic Ladder is present in the kit and allows
automatic genotyping of most of the reported alleles for the loci in the kit. See
Figure 1.
Allelic ladder profile
The allelic ladder profile appears on the next page.
VeriFiler™ Plus PCR Amplification Kit User Guide
13
1
14
Product description
The IQCS and IQCL markers are visible as the smallest and largest markers in the FAM™ dye (blue) channel. Each IQC marker has two alleles in the allelic ladder (alleles 1
and 2). However, the IQC markers are not normally analyzed and genotyped because they are primarily intended to be qualitative indicators.
Chapter 1 Product information
VeriFiler™ Plus PCR Amplification Kit User Guide
Figure 1 GeneMapper™ ID‑X Software plot of the VeriFiler™ Plus Allelic Ladder (updated formulation)
Chapter 1 Product information
Product description
1
DNA Control 007
™
Figure 2 DNA Control 007 (500 pg) amplified with the VeriFiler Plus kit and analyzed on an 3500xL Genetic
Analyzer (Y-axis scale 0–18,000 RFU) (updated formulation)
The smallest and largest peaks in the FAM™ dye (blue) channel are the IQCS and IQCL markers, respectively.
VeriFiler™ Plus PCR Amplification Kit User Guide
15
1
Chapter 1 Product information
Contents and storage
Contents and storage
The VeriFiler™ Plus kit (Cat. No. A35495) contains sufficient reagents to perform
200 amplifications with a total reaction volume of 25 μL.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive.
Protect the primer set, amplified DNA, allelic ladder, and size standard from light
when not in use.
IMPORTANT! Do not refreeze kit components after thawing.
Table 4
VeriFiler™ Plus PCR Amplification Kit (Cat. No. A35495; 200 reactions)
Contents
VeriFiler™ Plus Master
Mix
Description
Amount
Contains enzyme, salts,
dNTPs, bovine serum
albumin, and 0.05% sodium
azide in buffer and salt.
2 × 0.50 mL
VeriFiler™ Plus Primer Set Contains forward and
reverse primers to amplify
DNA targets.
Storage
−25°C to −15°C on receipt.
2°C to 8°C after first use, for up to
6 months or up to the expiration
date stated on the kit (whichever
comes first).
2 × 0.25 mL
−25°C to −15°C on receipt.
2°C to 8°C after first use, for up to
6 months or up to the expiration
date stated on the kit (whichever
comes first).
Store protected from light.
VeriFiler™ Plus Allelic
Ladder
Contains amplified alleles.
1 × 0.065 mL
See Figure 1 for information.
−25°C to −15°C on receipt.
2°C to 8°C after first use, up to the
expiration date stated on the kit.
Store protected from light.
DNA Control 007
Contains 0.1 ng/µL human
male genomic DNA from cell
line in 0.05% sodium azide
and buffer[1]
1 × 0.3 mL
−25°C to −15°C on receipt.
2°C to 8°C after first use, up to the
expiration date stated on the kit.
See “DNA Control 007” on
page 15 for information.
[1]
DNA Control 007 is included at a concentration that is appropriate for use as an amplification control (that is, to provide confirmation of the
capability of the kit reagents to generate a profile of expected genotype). It is not designed for use as a DNA quantification control. If you
quantify aliquots of DNA Control 007, the concentration may differ from the labeled concentration.
Required materials not supplied
See Appendix B, “Materials required but not supplied”.
16
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 1 Product information
Instruments and software compatibility
1
Instruments and software compatibility
Type of instrument or
software
Thermal cyclers
Validated models or versions
• ProFlex™ 96‑well PCR System (Cat. No. 4484075)
• ProFlex™ 2 × 96‑well PCR System (Cat. No. 4484076)
• ProFlex™ 3 × 32‑Well PCR System (Cat. No. 4484073)
• Veriti™ 96‑Well Thermal Cycler (Cat. No. 4479071)
• GeneAmp™ PCR System 9700, 96-Well Silver (Cat. No. N8050001)
• GeneAmp™ PCR System 9700, 96-Well Gold-Plated (Cat. No. 4314878)
Genetic analyzers[1]
• 3500/3500xL Genetic Analyzer with any of the following:
– 3500 Series Data Collection Software 1 (Windows™ Vista operating system) and
HID Updater 3500 Data Collection Software v2 (Cat. No. 4480670)
– 3500 Series Data Collection Software 2 (Windows™ 7 operating system) and HID
Updater 3500 Data Collection Software v2 (Cat. No. 4480670)
– 3500 Series Data Collection Software 3.1 Upgrade (Windows™ 7 operating
system) (Cat. No. A26287)
– 3500 Series Data Collection Software 3.1 (Windows™ 7 operating system)
(Cat. No. 4475183)
– 3500 Series Data Collection Software 4, v4.0.1 software patch (Windows™ 10
operating system) (Cat. No. A40059 or A40376)
• 3130/3130xl Genetic Analyzer with:
– 3130 Data Collection Software v4 (Cat. No. 4475105) or 3130xl Data Collection
Software‑v4 (Cat. No. 4475126 ), Windows™ 7 operating system
– 3130/3730 Data Collection v4 6-Dye Module v1 (Cat. No. 4478404)
Analysis software
Any of the following GeneMapper™ ID‑X Software versions[2]:
• GeneMapper™ ID‑X Software v1.4 or v1.5; compatible with the Windows™ 7
operating system
• GeneMapper™ ID‑X Software v1.6 or later; compatible with the Windows™ 7
operating system or Windows™ 10 operating system
[1]
[2]
We conducted validation studies using the 3130xl, 3500, and 3500xL configurations.
GeneMapper™ ID‑X Software v1.2–v1.3 can be used to analyze VeriFiler™ Plus PCR Amplification Kit data. However, some genotype quality
assessment features of the VeriFiler™ Plus kit are not included in earlier versions of the software. See the Chapter 4, “Analyze data with
GeneMapper™ID‑X Software” for more details.
IMPORTANT! The VeriFiler™ Plus kit should NOT be used with the following
systems:
·
·
·
·
ProFlex™ 2 × Flat PCR System (Cat. No. 4484078)
ProFlex™ 2 × 384‑well PCR System (Cat. No. 4484077)
Veriti™ Fast 96‑Well Thermal Cycler (Cat. No. 4375305)
GeneAmp™ PCR System 9700 with the aluminium 96-well block (Cat. No.
4314879)
VeriFiler™ Plus PCR Amplification Kit User Guide
17
1
Chapter 1 Product information
Workflow
Workflow
Extract DNA, see:
www.thermofisher.com/hid-sampleprep
▼
Quantify DNA
“DNA quantification” on page 19
▼
Perform PCR
“Prepare the amplification kit reactions” on page 23
▼
“Perform PCR” on page 24
▼
Perform electrophoresis
“Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)” on page 27 or
“Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)” on page 31
▼
“Prepare samples for electrophoresis” on page 33
▼
Analyze data
“Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit)” on page 37
▼
“Create an analysis method” on page 42
▼
“Create a size standard definition file if needed” on page 51
▼
“Analyze and edit sample files with GeneMapper ID-X Software” on page 53
▼
“Examine or edit a project” on page 54
▼
Chapter 5, “Assess the PCR reaction with the Internal Quality Control System”
18
VeriFiler™ Plus PCR Amplification Kit User Guide
2
Perform PCR
■
■
■
■
■
DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Direct amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
DNA quantification
Importance of quantification before STR analysis
DNA quantification can be used to determine:
• If the sample contains sufficient human DNA and/or human male DNA to
proceed with short tandem repeat (STR) amplification.
• The amount of sample to use in STR analysis applications.
• The relative quantities of human male and female DNA in a sample (using the
Quantifiler™ Trio DNA Quantification Kit). This guides selection of the applicable
STR chemistry.
• The DNA quality can be evaluated for:
– Inhibition level (using any Quantifiler™ kit)
– DNA degradation level (using the Quantifiler™ Trio DNA Quantification Kit or
Quantifiler™ HP DNA Quantification Kit)
Quality metrics are useful for determining the likelihood of recovery of STR loci
with larger amplicon sizes.
• If the sample contains highly degraded DNA. Such samples may require an
alternative approach to STR analysis by capillary electrophoresis. The Thermo
Fisher Scientific Precision ID NGS System and Panels are optimized for
degraded samples. The Precision ID Identity Panel provides discrimination of
individuals similar to STR genotype match probabilities. Also, the Precision ID
Ancestry Panel infers biogeographical ancestry for investigative leads. See
“Related documentation” on page 142 for a list of documents that are related
to the Precision ID NGS System and Panels.
• If PCR inhibitors are present in a sample. Such samples may require additional
purification before proceeding to STR analysis.
VeriFiler™ Plus PCR Amplification Kit User Guide
19
2
Chapter 2 Perform PCR
DNA quantification
Effect of DNA quantity on results
If too much DNA is added to the PCR reaction, the increased amount of PCR product
that is generated can result in:
• Fluorescence intensity that exceeds the linear dynamic range for detection by
the capillary electrophoresis instrument ("off-scale" data).
Off-scale data are a problem because:
– Quantification (peak height and area) for off-scale peaks is not accurate.
For example, an allele peak that is off-scale can cause a corresponding
stutter peak to appear higher in relative intensity, therefore increasing the
calculated percent stutter.
– Multicomponent analysis of off-scale data are not accurate. This inaccuracy
results in poor spectral separation ("pull-up").
• False signals of inhibition by the IQC system, although none is present.
• A reduction in the IQCL peak height.
• Incomplete +A nucleotide addition.
To address these problems, rerun the amplification reaction using less DNA.
If too little DNA is added to the PCR reaction, the total number of allele copies added
to the PCR is extremely low. Unbalanced amplification of the alleles may occur
because of stochastic fluctuation.
20
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 2 Perform PCR
DNA quantification
2
Methods of quantifying DNA
For information on recent innovations in quantification chemistry, go to
thermofisher.com.
Kit and user guide
Quantifiler™ HP DNA
Quantification Kit
(Cat. No. 4482911)
For more information,
see Quantifiler™ HP and
Quantifiler™ Trio DNA
Quantification Kits User
Guide (Pub. No. 4485354)
Quantifiler™ Trio DNA
Quantification Kit
(Cat. No. 4482910)
For more information,
see Quantifiler™ HP and
Quantifiler™ Trio DNA
Quantification Kits User
Guide (Pub. No. 4485354)
Detects
How it works
• Total human DNA
(two targets—one
small amplicon and
one larger amplicon)
• Uses 5′ nuclease assays with multiple-copy target
loci, for improved detection sensitivity:[1]
– The human-specific target loci are multiple
copy, and dispersed on various autosomal
chromosomes.
• Degraded DNA
• Total human DNA
(two targets—one
small amplicon and
one larger amplicon)
• Human male DNA
• Degraded DNA
– The primary quantification targets have
relatively short amplicons (75–80 bases), to
improve the detection of degraded DNA
samples.
• Uses features that maximize the consistency of
quantification:
– Genomic targets have conserved primer- and
probe-binding sites.
– Minimal copy number variation between
different individuals and population groups.
• Contains a Large Autosomal target with a longer
amplicon (>200 bases) to help determine if a DNA
sample is degraded.
• Contains an Internal PCR control (IPC) 5′ nuclease
assay that amplifies an integrated synthetic DNA
sequence. The performance of this assay can be
used to assess whether real-time PCR of the sample
has been impacted by inhibition.
[1]
The detection sensitivity of the Quantifiler™ HP Kit and the Quantifiler™ Trio Kit is improved over the Quantifiler™ Duo Kit.
VeriFiler™ Plus PCR Amplification Kit User Guide
21
2
Chapter 2 Perform PCR
Before you begin
Before you begin
(Optional) Prepare low-TE buffer
We recommend Invitrogen™ Nuclease-Free Water (Cat. No. AM9937) for sample
preparation. However, you can also use low-TE buffer. Prepare the low-TE buffer
as described in this procedure or use Invitrogen™ TE Buffer (Cat. No. 12090015).
IMPORTANT! Ensure that the low-TE buffer has final concentrations of
10 mM Tris‑HCl and 0.1 mM EDTA, pH 8.0. Do not use regular TE buffer because
it has a higher EDTA concentration that can chelate Mg2+ ions, therefore reducing
PCR amplification efficiency and robustness.
1. Mix:
• 10 mL of 1 M Tris-HCl, pH 8.0
• 0.2 mL of 0.5 M EDTA, pH 8.0
• 990 mL of nuclease-free water
Note: Adjust the volumes proportionally for specific needs. The final
concentration should be 10 mM Tris‑HCl and 0.1 mM EDTA, pH 8.0.
2. Aliquot, then autoclave the solutions.
3. Store the aliquots at room temperature.
Thaw reagents (before first use of the kit)
Thaw the Master Mix and Primer Set.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive.
Protect the primer set, amplified DNA, allelic ladder, and size standard from light
when not in use.
IMPORTANT! Thawing is required only before first use of the kit. After first use, the
reagents are stored at 2–8°C. Do not refreeze the reagents.
22
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Prepare the amplification kit reactions
2
Prepare the amplification kit reactions
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive.
Protect the primer set, amplified DNA, allelic ladder, and size standard from light
when not in use.
IMPORTANT! Use adhesive film for plate sealing to provide a consistent seal across
all wells and prevent evaporation. Caps may not provide a consistent seal across all
plate wells.
1. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes,
remove droplets from the caps by centrifuging the tubes briefly.
2. Pipet the required volumes of components into an appropriately sized clear
(non-colored) polypropylene tube:
Component
Volume per reaction
Master Mix
5.0 µL
Primer Set
2.5 µL
Note: Include volume for additional reactions to provide excess volume for the
loss that occurs during reagent transfers.
3. Vortex the reaction mixture for 3 seconds, then centrifuge briefly.
4. Dispense 7.5 µL of reaction mixture into each reaction well of a MicroAmp™
Optical 96-Well Reaction Plate or each MicroAmp™ tube.
5. Adjust the sample input amount and volume as needed:
Note: We recommend a DNA input amount of 500 pg.
• If the total sample input amount is >500 pg of DNA, dilute with nucleasefree water or low‑TE buffer to achieve a 500‑pg input in a 17.5-μL volume.
• If the total sample input volume is <17.5 μL, bring to volume with nucleasefree water or low‑TE buffer to achieve a 17.5-μL input volume.
6. Prepare the samples as shown in the following table, then add the samples to
the appropriate well or tube (the final reaction volume is 25 µL).
Sample
Add
Negative control
17.5 μL of nuclease-free water or low‑TE buffer
Test sample
VeriFiler™ Plus PCR Amplification Kit User Guide
17.5 μL of DNA[1]
23
2
Chapter 2 Perform PCR
Perform PCR
(continued)
Sample
Add
Positive control
Combine, then add to the reaction well or tube:
• 5 μL of DNA Control 007 (0.1 ng/μL)
• 12.5 μL of nuclease-free water or low‑TE
buffer
[1]
Prepared in step 5 .
7. Mix until the solution is homogenous.
8. Seal the MicroAmp™ Optical 96-Well Reaction Plate with MicroAmp™ Clear
Adhesive Film or MicroAmp™ Optical Adhesive Film.
9. Centrifuge the tubes or plate at 3,000 rpm for approximately 30 seconds in a
tabletop centrifuge (with plate holders, if using 96-well plates).
Perform PCR
IMPORTANT! This protocol is for casework only. For a direct amplification protocol,
see the technical note Direct Amplification of Reference Samples Using the VeriFiler™
Plus PCR Amplification Kit.
IMPORTANT! This kit is optimized for use with the thermal cyclers listed in
“Instruments and software compatibility” on page 17.
1. Program the thermal cycling conditions.
IMPORTANT! If you are using the:
™
· ProFlex PCR System, select 9700 Simulation Mode.
™
· GeneAmp PCR System 9700, select the Max ramping mode.
™
· Veriti Thermal Cycler, set up the method using the Convert a Method tool
and select 9700 Max Mode.
Do not use 9600 emulation mode.
Initial
incubation
First stage (2 cycles)
Denature
HOLD
[1]
Anneal/extend
Second stage (27 cycles)
Denature
Anneal/extend
CYCLE (29 cycles)
Final
extension
Final hold
HOLD
HOLD
95°C
96°C
62°C
96°C
59°C
60°C
4°C
1 minute
10 seconds
90 seconds
10 seconds
90 seconds
5 minutes
Up to
24 hours[1]
The infinity (∞) setting allows an unlimited hold time.
24
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Direct amplification
2
2. Load the plate into the thermal cycler, close the heated cover, then start the run.
IMPORTANT! If you are using a GeneAmp™ PCR System 9700 and adhesive
clear film instead of caps to seal the plate wells, place a MicroAmp™ Optical
Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent
evaporation during thermal cycling. The Veriti™ Thermal Cycler and the ProFlex™
PCR System do not require a compression pad.
3. When the run is complete, store the amplified DNA.
If you are storing the DNA...
Then place at...
<2 weeks
2°C to 8°C
>2 weeks
–25°C to –15°C
IMPORTANT! Protect the amplified DNA from light.
Direct amplification
The VeriFiler™ Plus PCR Amplification Kit is designed and optimized for casework
sample processing (including a pre-amplification extraction and cleanup step) with
VeriFiler™ Express PCR Amplification Kit as the partner kit. The VeriFiler™ Express kit
is designed and optimized to perform direct amplification of reference samples.
Some laboratories have expressed a preference for a single PCR amplification kit
capable of processing both casework and reference samples. To support those
laboratories, we have optimized a direct amplification PCR protocol. We performed
studies to demonstrate that the VeriFiler™ Plus kit can be used for direct amplification
of reference samples. Specifically, we evaluated the performance of the kit using a
direct amplification of blood and buccal samples collected on FTA™ cards, NUCLEICCARD™ Devices, Bode Buccal DNA Collectors, blood stain cards made out of filter
paper, and two types of swabs (Puritan™ Cotton Swabs and 4N6FLOQSwabs™).
Note: The direct amplification uses a different PCR protocol. Perform validation
studies before using this kit for direct amplification of single-source samples, or use
the VeriFiler™ Express kit. At PCR cycle numbers <29, the IQC peaks may not be
detected.
For more information on direct amplification studies, go to thermofisher.com,
then search for the technical note Direct Amplification of Reference Samples—
GlobalFiler™ PCR Amplification Kit, or contact your local Human Identification
representative.
VeriFiler™ Plus PCR Amplification Kit User Guide
25
3
Perform electrophoresis
■
■
■
■
■
Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Set up the 3500/3500xL instruments for electrophoresis (before first
use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Set up the 3130/3130xl instruments for electrophoresis (before first use
of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Prepare samples for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Allelic ladder requirements for electrophoresis
To accurately genotype samples, you must run the VeriFiler™ Plus Allelic Ladder with
the samples.
Instrument
3500
3500xL
3130
3130xl
Number of
allelic ladders to
run
One
injection
equals
Number of samples per allelic
ladder(s)
1 per 3
injections
8 samples
23 samples + 1 allelic ladder
1 per injection
24 samples
23 samples + 1 allelic ladder
1 per 4
injections
4 samples
15 samples + 1 allelic ladder
1 per injection
16 samples
15 samples + 1 allelic ladder
IMPORTANT! Variation in laboratory temperature can cause changes in fragment
migration speed and sizing variation between runs. Follow the guidelines in the
preceding table, which should account for normal variation in run speed. Perform
internal validation studies to verify the required allelic ladder injection frequency, to
ensure accurate genotyping of all samples in your laboratory environment.
It is critical to genotype using an allelic ladder run under the same conditions as the
samples. Size values obtained for the same sample can differ between instrument
platforms, because of different polymer matrices and electrophoretic conditions.
26
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Materials required for electrophoresis
3
Materials required for electrophoresis
Appendix B, “Materials required but not supplied” lists the required materials that are
not supplied with this kit.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive.
Protect the primer set, amplified DNA, allelic ladder, and size standard from light
when not in use.
Set up the 3500/3500xL instruments for electrophoresis (before
first use of the kit)
Electrophoresis software setup
The following table lists the data collection software and the run modules that you
can use to analyze PCR products that are generated with this kit. For details on the
procedures, see the documents that are listed in “Documentation and support” on
page 142.
Genetic
Analyzer[1]
Operating
system
Data Collection
Software
Additional
software
3500
Windows™ Vista
3500 Data Collection
Software v1
HID Updater
3500 DC v2
(Cat. No.
4480670)
3500xL
Instrument protocols, run
modules, and conditions
Set up the following conditions:
• Run module: HID36_POP4
(HID36_POP4xl for 3500xL)
• Injection conditions[2]:
1.2 kV/15 sec (24 sec for
3500xL)
• Run conditions: 13 kV/1,500
sec (13 kV/1,500 sec for
3500xL)
• Dye Set J6-T
3500
3500xL
Windows™ 7
3500 Data Collection
Software v2
VeriFiler™ Plus PCR Amplification Kit User Guide
HID Updater
3500 DC v2
(Cat. No.
4480670)
Same as 3500 Data Collection
Software v1 listed above
27
3
Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
(continued)
Genetic
Analyzer[1]
Operating
system
Data Collection
Software
Additional
software
3500
Windows™ 7
3500 Data Collection
Software v3
None
Same as 3500 Data Collection
Software v1 listed above
Windows™ 10
3500 Data Collection
Software v4, v4.0.1
software patch
None
Same as 3500 Data Collection
Software v1 listed above
3500xL
3500
3500xL
[1]
[2]
Instrument protocols, run
modules, and conditions
We conducted validation studies using the 3130xl, 3500, and 3500xL configurations.
Our studies indicate that the injection conditions that are documented generate profiles from 0.5 ng of input DNA with heterozygous peak
height averages between 4,000– 10,000 RFU (3500 or 3500xL) with no instances of allelic dropout and minimal occurrence of off-scale allele
peaks. However, individual CE instrument signal intensities can vary, therefore changes to injection parameters may need to be explored and
validated to deliver the best results on your specific system. Large deviations from the recommended injection parameters could affect the
performance of the size standard and/or allelic ladder, therefore internal validation studies are recommended.
Create a 3500 QC protocol
The VeriFiler™ Plus kit has been validated with data that was analyzed using both the
3rd Order Least Squares method (80–460 base pairs) and the Local Southern method
(60–460 base pairs).
1. In the Library tab, open the QC Protocol window.
2. Create a new QC protocol :
a. Name the new QC protocol according to your laboratory naming
convention.
b. Set the following parameters:
Setting
Parameter
28
Size Standard
GS600_LIZ_(60-460)
Size Range
Partial
Sizing Start Size
60 bp
Sizing Stop Size
460 bp
Size Calling Method
Local Southern or 3rd Order Least
Squares method
After checking the "Use Baselining"
box: Baseline Window Pts.
33
Peak Window Size
11
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
3
c. Click Save.
3. Add the QC protocol to the HID assay.
Perform spectral calibration
Perform a spectral calibration using the DS-37 (Dye set J6‑T, 6‑dye) Matrix Standard
Kit (J6-T Dye Set) (Cat. No. A31234).
Note: If it does not already exist, you must create a J6-T Dye Set in the
Data Collection Software before running a spectral calibration. For instructions
on creating a new dye set, see the "Create a New Dye Set" section of the
VeriFiler™ Plus PCR Amplification Kit User Guide
29
3
Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v2 User
Guide (Cat. No. 4476988). Use the J6 template to set up the J6-T dye set.
The following figure is an example of a passing 6-dye spectral calibration.
30
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)
3
Set up the 3130/3130xl instruments for electrophoresis (before first
use of the kit)
Electrophoresis software setup
The following table lists the Data Collection Software and the run modules that you
can use to analyze PCR products generated by this kit. For details on the procedures,
see the documents listed in “Documentation and support” on page 142.
Data
Collection
Software
Genetic
analyzer[1]
Operating
system
3130
Windows™ 7
Data
Collection
Software v4
Additional software
3130/3730 DC v4
6‑Dye Module v1
Run modules and conditions
Set up the following conditions:
• HIDFragmentAnalysis36_POP4_1
• Injection conditions[2]: 3 kV/4 sec
• Run conditions: 13 kV/1,600 sec
• Dye Set J6-T
3130xl
Set up the following conditions:
• HIDFragmentAnalysis36_POP4_1
• Injection conditions[2]: 3 kV/6 sec
• Run conditions: 13 kV/1,600 sec
• Dye Set J6-T
[1]
[2]
We conducted validation studies using the 3130xl, 3500, and 3500xL configurations.
Our studies indicate that the injection conditions that are documented generate profiles from 0.5 ng of input DNA with heterozygous peak
height averages between 2,000– 4,000 RFU (3130 or 3130xl) with no instances of allelic dropout and minimal occurrence of off-scale allele
peaks. However, individual CE instrument signal intensities can vary, therefore changes to injection parameters may need to be explored and
validated to deliver the best results on your specific system. Large deviations from the recommended injection parameters could affect the
performance of the size standard and/or allelic ladder, therefore internal validation studies are recommended.
Obtain and activate 6-dye license
1. Confirm that you are running Data Collection Software v4 (Help4About).
2. Obtain a 3130 DC v4 6-Dye Module v1 License key. Contact your local Human
Identification representative for information.
3. Ensure that all network cards in the computer are enabled.
IMPORTANT! You can run the 3130 Series Data Collection Software v4 using
only the network cards that are enabled when you activate the software license.
For example, if you activate the software when your wireless network card is
disabled, you will not be able to run the software when the wireless network
card is enabled.
VeriFiler™ Plus PCR Amplification Kit User Guide
31
3
Chapter 3 Perform electrophoresis
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)
4. Select Tools4License Manager to display the Software Activation dialog box.
5. Request the software license file by performing steps 1a, 1b, and 1c as listed on
the activation screen. The license file will be emailed to you.
6. Obtain the software license file from your email.
7. Make a copy of the software license file and keep it in a safe location.
8. Copy the software license file to the desktop of the Data Collection Software v4
computer.
9. If the Software Activation dialog box has closed, select Tools4License
Manager.
10. Click Browse, then navigate to the software license file saved on your computer.
11. Click Install and Validate License.
A message is displayed when the license is installed and validated.
12. Click Close.
32
VeriFiler™ Plus PCR Amplification Kit User Guide
nd/or line-length, as needed.
xt, this rectangle, and unused
ws, or other SVG elements
this SVG to the repository.
1
Chapter 3 Perform electrophoresis
Prepare samples for electrophoresis
3
Perform
spectral calibration
1
1
Perform a spectral calibration using the DS-37 (Dye set J6‑T, 6‑dye) Matrix Standard
Kit (J6-T Dye Set) (Cat. No. A31234).
The following figure is an example of a passing 6-dye spectral calibration.
Prepare samples for electrophoresis
This procedure applies to the 3130/3130xl Genetic Analyzer and
3500/3500xL Genetic Analyzer.
Prepare the samples for electrophoresis immediately before loading.
1. Pipet the required volumes of components into an appropriately sized
polypropylene tube:
Reagent
Volume per reaction
™
GeneScan 600 LIZ Size Standard v2.0
0.4 μL
Hi‑Di™ Formamide
9.6 μL
™
Note: Include volume for additional samples to provide excess volume for the
loss that occurs during reagent transfers.
IMPORTANT! The volume of size standard indicated in the table is a suggested
amount. Determine the appropriate amount of size standard based on your
experiments and results.
2. Vortex the tube, then briefly centrifuge.
3. Into each well of a MicroAmp™ Optical 96-Well Reaction Plate, add:
• 10 µL of the formamide/size standard mixture
• 1 µL of PCR product or VeriFiler™ Plus Allelic Ladder
VeriFiler™ Plus PCR Amplification Kit User Guide
33
3
Chapter 3 Perform electrophoresis
Prepare samples for electrophoresis
Note: For blank wells, add 10 µL of Hi‑Di™ Formamide.
4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge
the plate to ensure that the contents of each well are mixed and collected at the
bottom.
5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.
6. Immediately place the plate on ice for 3 minutes.
7. Place the sample tray on the autosampler, then start the electrophoresis run.
34
VeriFiler™ Plus PCR Amplification Kit User Guide
4
Analyze data with GeneMapper™
ID‑X Software
■
■
■
■
■
■
■
■
■
Overview of the GeneMapper™ ID‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Set up the GeneMapper™ ID‑X Software for analysis (before first use of
the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Analyze and edit sample files with GeneMapper ID-X Software . . . . . . . . . . . . 53
Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
For more information on using the GeneMapper™ ID‑X Software . . . . . . . . . . 54
Overview of the GeneMapper™ ID‑X Software
GeneMapper™ ID‑X Software is an automated genotyping software application for
forensic casework, databasing, and paternity data analysis.
GeneMapper™ ID‑X Software v1.4 or later analyzes 4-dye, 5-dye, and 6-dye data
and is required to analyze data that is generated using the VeriFiler™ Plus kit. After
electrophoresis, the data collection software stores information for each sample in
a .fsa or .hid file. The GeneMapper™ ID‑X Software v1.4 or later allows you to analyze
and interpret the data from the .fsa or .hid files.
Earlier versions of GeneMapper™ ID‑X Software (v1.2 and above) are capable of
analyzing data that is generated using the VeriFiler™ Plus kit. However, some quality
assessment tools are not fully functional on versions earlier than v1.4. Details on
VeriFiler™ Plus PCR Amplification Kit User Guide
35
4
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Allelic ladder requirements for data analysis
the data assessment functionality added to specific versions of GeneMapper™ ID‑X
Software are found in the release notes for each version. To summarize:
• In versions earlier than v1.4, samples show a red flag for the CGQ if the Y‑indel
peak is missing, for example, with a female DNA sample. This occurs because
the absence of the Y‑indel peak triggers the Allele Number Rule flag.
Allelic ladder requirements for data analysis
• HID analysis requires at least one allelic ladder sample per run folder. Perform
the appropriate internal validation studies before you use multiple allelic ladder
samples in an analysis.
For multiple allelic ladder samples, the GeneMapper™ ID‑X Software calculates
allelic bin offsets by using an average of all allelic ladders that use the same
panel in a run folder.
• Allelic ladder samples in an individual run folder are considered to be from a
single run. When the software imports multiple run folders into a project, only the
ladders in their respective run folders are used for calculating allelic bin offsets
and subsequent genotyping.
• Allelic ladder samples must be labeled as "Allelic Ladder" in the Sample Type
column in a project. Analysis will fail if the Allelic Ladder Sample Type is not
specified.
• Injections containing the allelic ladder must be analyzed with the same analysis
method and parameter values that are used for samples, to ensure proper allele
calling.
• Alleles that are not in the allelic ladders do exist. Off-ladder (OL) alleles can
contain full and/or partial repeat units. An off-ladder allele is an allele that occurs
outside the bin window of any known allelic ladder allele or virtual bin.
Note: If a sample allele peak is called as an off-ladder allele, verify the sample
result according to your laboratory protocol.
File names and versions used in this section
The file names and version numbers of panel, bin, and stutter files that are shown in
this section may differ from the file names that you see when you download or import
files.
If you need help to determine the correct files to use, contact your local Human
Identification representative, or go to thermofisher.com/support.
36
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit)
4
Set up the GeneMapper™ ID‑X Software for analysis (before first
use of the kit)
Workflow: Set up GeneMapper™ ID‑X Software
Before you use GeneMapper™ ID‑X Software to analyze data for the first time, you
must do the following:
“Check panel, bin, and stutter file versions on your computer” on page 37
▼
“(If needed) Download newer versions of panel, bin, and stutter files” on page 38
▼
“Import panels, bins, and marker stutter” on page 38
▼
“(Optional) Define custom table or plot settings” on page 42
Check panel, bin, and stutter file versions on your computer
Common Callouts and Arrows
1.
1. Start the GeneMapper™ ID‑X Software , then log in with the appropriate user
Copy-paste
a callout
arrow to use in this SVG.
name
andorpassword.
Note: If you need more advanced callouts or arrows
2. Select Tools4Panel Manager.
use the TechComm_Inkscape_Callout&Arrow_Libary.
3. Check the version of files that are currently available in the Panel Manager:
a. Select Panel Manager in the
navigation pane.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding
SVG to the
the repository.
b. this
Expand
Panel Manager
1
folder and any subfolders to
identify the analysis file version
1 already installed
1
that is
for your kit choice.
1
4. Check the version of files available for import into the Panel Manager:
a. Select Panel Manager, then select File4Import Panels to open the
Import Panels dialog box.
b. Navigate to, then open the Panels folder, then check the version of panel,
bin, and stutter files installed.
5. Check for newer versions of the files as described in the next procedure.
VeriFiler™ Plus PCR Amplification Kit User Guide
37
4
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit)
(If needed) Download newer versions of panel, bin, and stutter files
1. Go to www.thermofisher.com/GMIDXsoftware.
2. If the file versions listed are newer than the versions on your computer,
download the file VeriFiler Plus Analysis Files.
Note: When downloading new versions of analysis files, see the associated
Read Me file for details of changes between software file versions. Perform the
appropriate internal validation studies before using new file versions for analysis.
3. Unzip the file.
Import panels, bins, and marker stutter
Common Callouts and Arrows
To import the latest panel, bin set, and marker stutter from the website into the
GeneMapper ID‑X Software database:
1. Copy-paste a callout or™arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
Start the GeneMapper™ ID‑X Software,
use the 1.
TechComm_Inkscape_Callout&Arrow_Libary.
then log in with the appropriate user
name and password.
2. Edit number and/or line-length, as needed.
2. Select Tools4Panel Manager.
3. Delete this text, this rectangle, and unused
Find,
openelements
the folder containing
callouts,3.
arrows,
or then
other SVG
before adding
thispanels,
SVG to the
repository.
the
bins,
and marker stutter:
1
1
1
a. Select
Panel Manager,
then select File4Import Panels to open the
Import Panels dialog box. 1
b. Navigate to, then open the VeriFiler Plus Analysis Files folder that you
unzipped in the previous procedure.
4. Select VeriFiler_Plus_Panel.txt, then click Import.
Note: Importing this file creates a new folder in the Panel Manager navigation
pane, VeriFiler_Plus_Panel. This folder contains the panel and associated
markers.
38
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit)
outs and Arrows
4
5. Import the bins file:
a. In the Panel Manager navigation pane, click the VeriFiler_Plus folder to
display the VeriFiler_Plus_Panel.
out or arrow to use in this SVG.
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b. Select File4Import Bin Set to open the Import Bin Set dialog box.
c. Navigate to, then open the VeriFiler Plus Analysis Files folder.
t number and/or line-length, as needed.
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louts, arrows, or other SVG elements
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VeriFiler™ Plus PCR Amplification Kit User Guide
39
4
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit)
d. Select VeriFiler_Plus_Bins.txt, then click Import.
Note: Importing this file associates the bin set with the panels in the
VeriFiler_Plus_Panel folder.
Callouts and Arrows
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u need more advanced callouts or arrows
chComm_Inkscape_Callout&Arrow_Libary.
6. (Optional) View information about panels and bins: In the Panel Manager
navigation pane, click + next to the VeriFiler_Plus folder, then click + next to
the VeriFiler_Plus_Panel folder to reveal marker-level information.
Note: Select the Y Marker checkbox to avoid flagging female samples.
r and/or line-length, as needed.
The panel view is shown below.
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VeriFiler™ Plus PCR Amplification Kit User Guide
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1
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit)
4
7. Import the stutter file:
a. Select the VeriFiler_Plus_Panel folder in the navigation pane.
b. Select File4Import Marker Stutter to open the Import Marker Stutter
dialog box.
c. Navigate to, then open the VeriFiler Plus Analysis Files folder.
d. Select VeriFiler_Plus_Stutter_v1.txt, then click Import.
Note: Importing this file associates the marker stutter ratio with the bin
set in the VeriFiler_Plus folder and overwrites any existing stutter ratios
associated with the panels and bins in that folder.
mon Callouts and Arrows
paste a callout or arrow to use in this SVG.
If you need more advanced callouts or arrows
he TechComm_Inkscape_Callout&Arrow_Libary.
The list of markers is displayed.
b. Double-click a marker to display the Stutter Ratio & Distance view for the
marker in the right pane.
umber and/or line-length, as needed.
e this text, this rectangle, and unused
ts, arrows, or other SVG elements
e adding this SVG to the repository.
1
8. View the imported marker stutters in the navigation pane:
a. Click + next to the VeriFiler_Plus and VeriFiler_Plus_Panel folders.
1
1
mmon Callouts and Arrows
py-paste a callout or arrow to use in this SVG.
te: If you need more advanced callouts or arrows
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VeriFiler™ Plus PCR Amplification Kit User Guide
it number and/or line-length, as needed.
lete this text, this rectangle, and unused
41
4
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
9. Click Apply, then click OK to add the panel, bin set, and marker stutter to the
GeneMapper™ ID‑X Software database.
IMPORTANT! If you close the Panel Manager without clicking Apply, the
panels, bin sets, and marker stutter are not imported into the GeneMapper™ ID‑X
Software database.
(Optional) Define custom table or plot settings
Default views for table and plot settings are provided with the software. For
information on defining custom views, see GeneMapper™ ID‑X Software Getting
Started Guide— Basic Features.
Create an analysis method
Create an analysis method
IMPORTANT! Analysis methods are version-specific, so you must create an
analysis method for each version of the software. For example, an analysis method
that is created in GeneMapper™ ID‑X Software version 1.2 is not compatible
with analysis methods that are created in earlier versions of software, or with
GeneMapper™ Software v3.2.1.
1. Select Tools4GeneMapper® ID-X Manager to open the GeneMapper ID-X
Manager.
n Callouts and Arrows
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1
2. Click the Analysis Methods tab, then click New to open the Analysis Method
Editor with the General tab selected.
1
1
3. Enter the settings shown in the figures on the following pages.
Note: The Analysis Method Editor closes when you save your settings. To
complete this step quickly, do not save the analysis method until you finish
entering settings in all of the tabs.
4. After you enter the settings on all tabs, click Save.
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
4
Enter Analysis Method settings
Enter General tab settings
1. Enter a Name and select the Security Group appropriate for your software
configuration.
allouts and Arrows
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nd/or line-length, as needed.
t, this rectangle, and unused
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this SVG to the repository.
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1
1
2. (Optional) Enter a Description and Instrument.
VeriFiler™ Plus PCR Amplification Kit User Guide
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Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
Enter Allele tab settings
IMPORTANT! Perform appropriate internal validation studies to determine the
appropriate settings to use.
1. Select the VeriFiler_Plus_Bins_v1 bin set.
outs and Arrows
ut or arrow to use in this SVG.
more advanced callouts or arrows
m_Inkscape_Callout&Arrow_Libary.
r line-length, as needed.
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1
Figure 3 Settings used in developmental validation of the kit
2. (Optional) To apply the stutter ratios contained in the
VeriFiler_Plus_Stutter_v1.txt, select the Use marker-specific stutter ratio
and distance if available checkbox (selected by default).
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
4
3. If you use global stutter filters instead of marker‑specific stutters, enter values
for Tri-, Tetra-, and Penta‑nucleotide repeats.
Note: Using global stutter filters instead of marker-specific stutters is not
typical.
Note: There are no hexa-nucleotide repeat markers in the VeriFiler™ Plus kit.
4. Enter the appropriate filter settings.
Enter Peak Detector tab settings
Enter the appropriate values:
Field
Ranges
Values to enter or select
Additional information
Enter the values shown in Figure 4.
—
™
Note: The read region for the VeriFiler Plus
kit is 75–426 bp.
Peak Detection
Enter the appropriate settings.
IMPORTANT! Perform appropriate internal
validation studies to determine the
appropriate peak amplitude thresholds for
interpretation of data.
The software uses the Peak Amplitude
Thresholds to specify the minimum
peak height. Although GeneMapper™ ID‑X
Software displays peaks that fall below the
specified amplitude in electropherograms,
the software does not label or determine the
genotype of these peaks.
Note: As shown in Figure 4, there
are additional Peak Detection parameters,
including Min. Peak Half Width, Polynomial
Degree, Peak Window Size, and Slope
Threshold.
Smoothing and
Baseline
Enter the values shown in Figure 4.
VeriFiler™ Plus PCR Amplification Kit User Guide
—
45
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Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
(continued)
Field
Values to enter or select
Additional information
Size Calling Method
Select Local Southern Method or 3rd Order
Least Squares, or another method that
has been validated during your internal
validation studies.
The VeriFiler™ Plus kit has been validated
using both the 3rd Order Least Squares
sizing method (80–460 bp) and the Local
Southern sizing method (60–460 bp).
Both sizing methods produced 100%
concordance and generated acceptable
data. Select alternative sizing methods only
after performing the appropriate internal
validation studies.
Normalization
(Optional) Select the Normalization
checkbox.
A Normalization checkbox is available on
this tab in GeneMapper™ ID‑X Software
for use with data that are run on the
3500/3500xL Genetic Analyzer.
IMPORTANT! Both the 3rd Order Least Squares Sizing algorithm and the Local
Southern Sizing algorithm have been validated for analysis of VeriFiler™ Plus kit data
on 3130- and 3500‑series instruments.
Note: The read region for the VeriFiler™ Plus kit is 75–426 bp.
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
4
nd Arrows
ow to use in this SVG.
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Figure 4 Settings used in developmental validation of the kit
IMPORTANT! TBD indicates values to be determined in your laboratory.
Laboratories must perform the appropriate internal studies to determine the peak
amplitude thresholds for interpreting the VeriFiler™ Plus kit data.
VeriFiler™ Plus PCR Amplification Kit User Guide
47
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
4
Create an analysis method
Enter Peak Quality tab settings
IMPORTANT! Perform the appropriate internal validation studies to determine the
heterozygous and homozygous minimum peak height thresholds, maximum peak
height threshold, and the minimum peak height ratio threshold for interpretation of
data.
Enter the following values:
Arrows
o use in this SVG.
ed callouts or arrows
Callout&Arrow_Libary.
as needed.
and unused
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epository.
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
4
IMPORTANT! TBD indicates values to be determined in your laboratory.
Laboratories must perform the appropriate internal studies to determine the peak
amplitude thresholds for interpreting the VeriFiler™ Plus kit data.
VeriFiler™ Plus PCR Amplification Kit User Guide
49
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Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create an analysis method
Enter SQ and GQ tab settings
IMPORTANT! The values that are shown are the software defaults and are the
values that we used during developmental validation. Perform appropriate internal
validation studies to determine the appropriate values to use.
Enter the following values:
uts and Arrows
ut or arrow to use in this SVG.
more advanced callouts or arrows
m_Inkscape_Callout&Arrow_Libary.
line-length, as needed.
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1
Note: Set the ACC GQ Weighting according to the values you determine during
internal validation studies of the ACC PQV. For example, set the ACC GQ Weighting
to 0.3 or greater to flag samples in which the Amelogenin result is anything other than
X, X or X, Y, or does not agree with the results for the Y indel marker.
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create a size standard definition file if needed
4
Create a size standard definition file if needed
If you cannot use the default settings that are provided, create a new size standard
definition file.
About the GS600_LIZ_(60–460) size standard definition file
The GS600_LIZ_(60– 460) size standard definition that is provided with GeneMapper™
ID‑X Software and used with the Local Southern size calling method contains the
following peaks: 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260,
280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440, and 460.
This size standard definition has been validated for use with this kit on the genetic
analyzers listed in “Instruments and software compatibility” on page 17. If you need
to create your own size standard definition, see “Create a size standard definition file”
on page 51.
Create a size standard definition file
1. Select Tools4GeneMapper ID-X Manager to open the GeneMapper ID-X
Manager.
2. Click the Size Standards tab, then click New.
mon Callouts and Arrows
y-paste a callout or arrow to use in this SVG.
e: If you need more advanced callouts or arrows
the TechComm_Inkscape_Callout&Arrow_Libary.
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re adding this SVG to the repository.
1
3. Specify settings in the Size Standard Editor:
a. Enter a name as shown in the following figure or enter a new name.
1
1
VeriFiler™ Plus PCR Amplification Kit User Guide
51
4
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Create a size standard definition file if needed
b. In the Security Group field, select the Security Group appropriate for your
software configuration.
c. In the Size Standard Dye field, select Orange.
d. In the Size Standard Table, enter the peak sizes that correspond to your
size standard.
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Analyze and edit sample files with GeneMapper ID-X Software
4
Analyze and edit sample files with GeneMapper ID-X Software
1. In the Project window, select Edit4Add Samples to Project, then navigate to
the disk or directory that contains the sample files.
2. Apply analysis settings to the samples in the project.
Parameter
Settings
Sample Type
Select the sample type.
Analysis Method
Select VeriFiler Plus Analysis Method (or the name of the
analysis method you created).
Panel
Select VeriFiler_Plus_Panel.
Size Standard
Use a size range of 60– 460 bp for Local Southern size-calling
method or a size range of 80– 460 bp for 3rd Order Least
Squares size-calling method. [1]
[1]
The VeriFiler™ Plus kit was originally validated for use with the GeneScan™ 600 LIZ™ Size Standard
v2.0. If you use a different size standard, perform the appropriate internal validation studies to support
the use of this size standard with the VeriFiler™ Plus kit.
3. Click Analyze, enter a name for the project (in the Save Project dialog box),
then click OK to start analysis.
• The status bar displays the progress of analysis as a completion bar.
• The table displays the row of the sample currently being analyzed in green
(or red if analysis failed for the sample).
• The Analysis Summary tab is displayed, and the Genotypes tab is
available when the analysis is complete.
allouts and Arrows
callout or arrow to use in this SVG.
eed more advanced callouts or arrows
omm_Inkscape_Callout&Arrow_Libary.
nd/or line-length, as needed.
VeriFiler™ Plus PCR Amplification Kit User Guide
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s, or other SVG elements
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Chapter 4 Analyze data with GeneMapper™ ID‑X Software
Examine or edit a project
Examine or edit a project
Display electropherogram plots from the Samples and Genotypes tabs of the Project
window to examine the data.
For more information on using the GeneMapper™ ID‑X Software
See “Related documentation” on page 142 for a list of available documents.
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VeriFiler™ Plus PCR Amplification Kit User Guide
5
Assess the PCR reaction with the
Internal Quality Control System
■
■
Overview of the Internal Quality Control system . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Evaluate the PCR reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Overview of the Internal Quality Control system
The Internal Quality Control (IQC) System is a tool that can help you evaluate the PCR
reaction and, along with the STR marker data, infer possible sample degradation or
inhibition.
The primers for the two IQC markers, IQCS and IQCL, amplify synthetic DNA targets
that are included in the primer mix. IQCS is a low molecular weight amplicon, with
mobility of 70 nt. IQCL is a higher molecular weight amplicon, with mobility of 451 nt.
Note: The IQC markers enable qualitative insight into the sample amplification. By
default, the IQC markers are not genotyped during analysis in the GeneMapper™
ID‑X Software. However, if needed, the VeriFiler™ Plus Allelic Ladder contains two
peaks each for the IQCS and IQCL markers (designated 1 and 2) to detect the IQC
sequences. Sample reactions contain one peak each for IQCS and IQCL, which
corresponds to the larger peak (peak 2) in each IQC marker.
The IQC System allows you to:
• Confirm the success or failure of the PCR reaction, by looking for the presence
or absence of the IQCS and IQCL primer peaks on the electropherogram.
• Determine if PCR inhibitors might be present in the PCR reaction, or if the PCR
reaction conditions are not optimal, by evaluating the peak heights of IQCS and
IQCL.
Evaluate the PCR reaction
To evaluate the PCR performance of the samples, review the peak heights of
the IQCS and IQCL. Under ideal PCR conditions, the peak heights of the IQCS
and IQCL should be >2,000 RFU using standard injection protocols on the
3500/3500xL Genetic Analyzer (see Table 5). Under suboptimal PCR conditions
(for example, moderate inhibition), the height of the IQCL is substantially reduced.
Under extremely high inhibition, even the small IQC peak heights are substantially
reduced. Note that when high inputs of DNA are amplified (greater than 2 ng) some
suppression of the IQCL peak may also be seen.
VeriFiler™ Plus PCR Amplification Kit User Guide
55
5
Chapter 5 Assess the PCR reaction with the Internal Quality Control System
Evaluate the PCR reaction
See Table 5 for outcome scenarios.
Note: The electropherograms that are shown in Figure 5 through Figure 10 are from
runs on a 3500xL Genetic Analyzer using standard injection protocols.
Table 5
IQC peak interpretation
Sample DNA
profile
IQC peaks
IQC interpretation
Typically indicates...
Recommended action for re‑amplification[1]
Balanced
Both IQC peaks
are >2,000 RFU
PCR performance within the
optimal range—no sample
degradation or inhibition (see
Figure 5)
Re-amplification should not be required. On
a case-by-case basis, you can evaluate the
profile and determine if re‑amplification is
required.
Balanced
The IQCS peak
height is >
2,000 RFU,
while the IQCL
peak height
is significantly
decreased
Mild inhibition (see Figure 6)
Evaluate the profile and determine if additional
input would recover alleles without saturating
the system.
Ski-slope
The IQCS peak
height is near
2,000 RFU or
greater, while the
IQCL peak is not
present
High inhibition (see Figure 7)
Perform a dilution or dilutions of the sample
to minimize the inhibitor while still allowing
for enough DNA to achieve amplification (for
example, 10–20% dilution).
Ski-slope
The IQCS
peak height
is significantly
decreased, while
the IQCL peak is
not present
Very high inhibition
(see Figure 8)
Perform a dilution or dilutions of the sample
to minimize the inhibitor while still allowing
for enough DNA to achieve amplification (for
example, 20–60% dilution).
Ski-slope
Both IQC peaks
are >2,000 RFU
Degraded sample DNA
(see Figure 9)
Evaluate the profile and determine if additional
input would recover alleles without saturating
the system.
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Chapter 5 Assess the PCR reaction with the Internal Quality Control System
Evaluate the PCR reaction
Table 5
5
IQC peak interpretation (continued)
Sample DNA
profile
IQC peaks
IQC interpretation
Typically indicates...
Recommended action for re‑amplification[1]
No peaks
Both IQC peaks
are >2,000 RFU
No DNA or very little sample
DNA (see Figure 10)
Check the quantification and normalization
calculations. If you have sufficient DNA to
generate a profile, increase the amount of DNA
in the amplification.
No peaks
No peaks
PCR failure or ultra-high
inhibition
Re-amplify the sample at the appropriate
target to determine if PCR failure occurred. If
DNA is still not detected, perform a dilution
or dilutions of the sample to minimize the
inhibitor while still allowing for enough DNA
to achieve amplification (for example, 20–60%
dilution).
[1]
The decision to re-amplify the sample should be based on individual laboratory protocols that identify how much information is sufficient for
reporting.
Balanced profile
In this example of a balanced profile, the IQC peaks and the DNA profile peaks are
balanced, which indicates that PCR has occurred optimally.
Figure 5 FAM™ dye channel electropherogram that shows IQCS and IQCL peaks with 0.5 ng of DNA (scaled
to 22,000 RFU) (updated formulation)
The red circle highlights the IQCS peak and the green circle highlights the IQCL peak.
VeriFiler™ Plus PCR Amplification Kit User Guide
57
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Chapter 5 Assess the PCR reaction with the Internal Quality Control System
Evaluate the PCR reaction
Ski slope profile with decreased IQCL peak height
Figure 6 shows a significantly lower IQCL peak height. This indicates that the PCR
reaction has been compromised by inhibition. Figure 7 shows the complete absence
of an IQCL peak, indicating a high level of inhibition.
Figure 6 Combined dyes electropherogram for the VeriFiler™ Plus kit in the presence of 150 ng/µL of humic acid
(scaled to 22,000 RFU) (updated formulation)
The red circle highlights the IQCS peak (a PCR inhibitor) and the green circle highlights the IQCL peak. While overall peak heights
are reduced relative to the uninhibited control sample, only a subtle ski-slope effect is observed.
ws
ary.
Figure 7 Combined dyes electropherogram for the VeriFiler™ Plus kit in the presence of 350 ng/µL of humic acid
(scaled to 5,000 RFU) (updated formulation)
The red circle highlights the IQCS peak; the IQCL peak is absent (green box). The arrow indicates the ski slope peak pattern
observed in the DNA profile.
Ski slope profile with IQC peaks
The presence of both IQCS and IQCL peaks >2,000 RFU indicates that PCR has
occurred optimally.
1
Figure 8 Combined dyes electropherogram for the VeriFiler™ Plus PCR Amplification Kit in the presence of
450 ng/µL of humic acid (updated formulation)
The red circle highlights the IQCS peak; the IQCL peak is absent (green circle). The arrow indicates the ski slope peak pattern
observed in the DNA profile. The Y‑axis is scaled to 250 RFU to make extremely low peaks visible.
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Chapter 5 Assess the PCR reaction with the Internal Quality Control System
Evaluate the PCR reaction
5
Figure 9 Combined dyes electropherogram from degraded DNA (scaled to 10,000 RFU) (updated formulation)
The red circle highlights the IQCS peak and the green circle highlights the IQCL peak. The arrow indicates the ski slope pattern
observed in the DNA profile.
ws
ary.
No sample peaks with IQC peaks
There are no DNA profile peaks in Figure 10. However, the presence of both the IQCS
and IQCL peaks indicates that normal amplification has occurred in the PCR.
1
Figure 10 Combined dyes electropherogram that shows IQCS and IQCL peaks with 0 ng of DNA (scaled to
16,000 RFU) (updated formulation)
The red circle highlights the IQCS peak and the green circle highlights the IQCL peak.
VeriFiler™ Plus PCR Amplification Kit User Guide
59
6
Experiments and results
■
■
■
■
■
■
■
■
■
■
■
■
Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Mixture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Importance of validation
Validation of a DNA typing procedure for human identification applications is
an evaluation of the efficiency, reliability, and performance characteristics of
the procedure. By challenging the procedure with samples that are commonly
encountered in forensic and parentage laboratories, the validation process uncovers
attributes and limitations that are critical for sound data interpretation (Sparkes,
Kimpton, Watson, 1996; Sparkes, Kimpton, Gilbard, 1996; Wallin, 1998).
Experiment conditions
We conducted developmental validation experiments according to the updated and
revised guidelines from the Scientific Working Group on DNA Analysis Methods
(SWGDAM, December 2016). Based on these guidelines, we conducted experiments
that comply with guidelines 2.0 and 3.0 and its associated subsections. This DNA
methodology is not novel. (Moretti et al., 2001; Frank et al., 2001; Wallin et al., 2002;
and Holt et al., 2000).
We used conditions that produced optimum PCR product yield and that met
reproducible performance standards. It is our opinion that while these experiments
are not exhaustive, they are appropriate for a manufacturer of STR kits intended for
forensic and/or parentage testing use.
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 6 Experiments and results
Laboratory requirements for internal validation
6
Repeated validation experiments using the updated formulation
Minor modifications were made to the VeriFiler™ Plus PCR Amplification Kit primer
mix to reduce the incidence of non-specific PCR artifacts that were observed in
certain forensic samples with high microbial DNA load. Accordingly, we used the
updated formulation to repeat the validation experiments that might be impacted by
the primer mix changes, as follows:
• Developmental validation—“PCR components” on page 62
• Extra peaks—“Dye artifact observation” on page 91
• Species Specificity—“Nonhuman studies” on page 95
• Sensitivity—“Sensitivity observation” on page 97
• Stability—“Degraded DNA” on page 98
• Stability—“Effect of inhibitors” on page 99
• Mixture Studies—“Limit of detection of the minor component” on page 104
Because most of the kit components (for example, the master mix) remain
unchanged, we did not repeat the remaining validation experiments.
Laboratory requirements for internal validation
Each laboratory using this kit must perform internal validation studies. Performance
of this kit is supported when used according to the following developmentally
validated parameters. Modifications to the protocol should be accompanied by
appropriate validation studies performed by the laboratory.
Developmental validation
Developmental validation studies were performed using the ProFlex™ 96‑well PCR
System, Veriti™ 96‑Well Thermal Cycler, and the GeneAmp™ PCR System 9700 96Well thermal cycler according to the protocol described in the Perform PCR chapter.
SWGDAM guideline 2.2.1
“Developmental validation is the acquisition of test data and determination of
conditions and limitations of a new or novel DNA methodology for use on forensic,
database, known or casework reference samples.” (SWGDAM, December 2016)
SWGDAM guideline 3.9.2
“The reaction conditions needed to provide the required degree of specificity
and robustness should be determined. These include, but are not limited to,
thermal cycling parameters, the concentration of primers, magnesium chloride, DNA
polymerase, and other critical reagents.” (SWGDAM, December 2016)
VeriFiler™ Plus PCR Amplification Kit User Guide
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Chapter 6 Experiments and results
Developmental validation
SWGDAM guideline 3.9.6
“Criteria for detection of amplified product should be determined based on the
platform and/or method. ” (SWGDAM, December 2016)
PCR components
We examined the concentration of each component of the kit. The concentration
of each component was in the range where data indicated that the amplification
met the required performance criteria for specificity, sensitivity, and reproducibility.
For example, 0.5 ng of DNA Control 007 was amplified in the presence of varying
concentrations of magnesium sulfate, and the results were analyzed on a 3500xL
Genetic Analyzer (Figure 11). The performance of the multiplex is most robust within
±20% of the optimal magnesium sulfate concentration.
d Arrows
w to use in this SVG.
anced callouts or arrows
e_Callout&Arrow_Libary.
™
Figure 11 DNA Control 007 (0.5 ng) amplified with the VeriFiler Plus kit in the
presence of varying concentrations of magnesium sulfate and analyzed on a 3500xL
Genetic Analyzer (Y-axis scale 0–25,000 RFU) (updated formulation)
th, as needed.
e, and unused
G elements
e repository.
Thermal cycling temperatures
1
1
Thermal cycling parameters were optimized using a Design of Experiments (DOE)
approach that seeks to identify the combination of temperatures and hold times that
produce the best assay performance. Optimal assay performance was determined
through evaluation of several factors, such as assay sensitivity, peak-height balance,
and resistance to PCR inhibitors.
For example, annealing/extension temperatures of 57, 58, 59, 60, and 61°C were
tested using a ProFlex™ PCR System (Figure 12). The PCR products were analyzed
using a 3500xL Genetic Analyzer.
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 6 Experiments and results
Developmental validation
6
Robust profiles were obtained between 57–61°C. The optimal combination of
specificity, sensitivity, and resistance to PCR inhibition was observed at 59°C.
Thermal cycler temperature is critical to assay performance; therefore routine,
regularly scheduled thermal cycler calibration is recommended.
and Arrows
arrow to use in this SVG.
advanced callouts or arrows
scape_Callout&Arrow_Libary.
™
Figure 12 VeriFiler Plus kit electropherograms obtained from amplification of 0.5 ng
of DNA Control 007 at annealing temperatures of 57, 58, 59, 60, and 61°C, analyzed on
a 3500xL Genetic Analyzer (Y-axis scale 0–25,000 RFU)
ength, as needed.
angle, and unused
r SVG elements
o the repository.
PCR cycle number
1
1
Reactions were amplified for 27, 28, 29, 30, and 31 cycles on the ProFlex™ PCR
System using 0.5 ng of DNA Control 007. As expected, the amount of PCR product
increased with the number of cycles. A full profile was generated for all numbers of
thermal cycles (27–31) and off-scale data were collected for several allele peaks at
31 cycles (Figure 13).
Additional experiments were performed not only to optimize for peak heights, peak
balance, sensitivity, and so on, but also to minimize the formation of non-target PCR
artifacts. The optimized end result is a "touchdown" PCR protocol in which the first
two cycles are performed at a slightly higher, more stringent annealing temperature
(62°C), followed by 27 cycles with an annealing temperature of 59°C.
VeriFiler™ Plus PCR Amplification Kit User Guide
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
d Arrows
to use in this SVG.
nced callouts or arrows
_Callout&Arrow_Libary.
h, as needed.
, and unused
elements
repository.
1
™
Figure 13 Representative VeriFiler Plus kit profiles obtained from amplification of
0.5 ng of DNA Control 007 using 27, 28, 29, 30, and 31 cycles, analyzed on a 3500xL
Genetic Analyzer (Y-axis scale 0–35,000 RFU)
Accuracy, precision, and reproducibility
SWGDAM guideline 3.5
1
64
“Precision and accuracy of the assay should be demonstrated: Precision
characterizes the degree of mutual agreement among a series of individual
measurements, values and/or results. Precision depends only on the distribution of
random errors and does not relate to the true value or specified value. The measure
of precision is usually expressed in terms of imprecision and computed as a standard
deviation of the test results. Accuracy is the degree of conformity of a measured
quantity to its actual (true) value. Accuracy of a measuring instrument is the ability
of a measuring instrument to give responses close to a true value.” (SWGDAM,
December 2016)
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
6
Accuracy observation
The accuracy and reproducibility of STR profiles have been determined from various
sample types. Important factors include: Sizing accuracy and precision, and the
consistency in observed sizing between sample STR alleles and corresponding allelic
ladder alleles.
Figure 14, Figure 15, and Figure 16 show the size differences that were observed
between sample alleles and allelic ladder alleles on the 3130xl, 3500, and 3500xL
Genetic Analyzers with POP-4™ Polymer. The X‑axis in the following figures
represents the nominal nucleotide sizes for the VeriFiler™ Plus Allelic Ladder. The
horizontal lines parallel to the X-axis represent the ±0.25-nt windows. The Y‑axis
represents the deviation of each sample allele size from the corresponding Allelic
Ladder allele size. All sample alleles are within ±0.5 nt from a corresponding allele in
the Allelic Ladder, irrespective of the capillary electrophoresis platforms.
Note: The IQCS and IQCL markers were omitted from this study because they are
not used for genotyping.
and Arrows
row to use in this SVG.
dvanced callouts or arrows
ape_Callout&Arrow_Libary.
Figure 14 Allele size vs. Allelic Ladder sizing for 84 samples analyzed on a 3130xl
™
Genetic Analyzer. Size and ladder sizing for the VeriFiler Plus kit were calculated
™
™
using the GeneScan 600 LIZ Size Standard v2.0.
ngth, as needed.
ngle, and unused
SVG elements
the repository.
1
1
VeriFiler™ Plus PCR Amplification Kit User Guide
65
Chapter 6 Experiments and results
6
Accuracy, precision, and reproducibility
Arrows
o use in this SVG.
ed callouts or arrows
Callout&Arrow_Libary.
Figure 15 Allele size vs. Allelic Ladder sizing for 84 samples analyzed on a 3500
™
Genetic Analyzer. Size and ladder sizing for the VeriFiler Plus kit were calculated
™
™
using the GeneScan 600 LIZ Size Standard v2.0.
as needed.
and unused
elements
epository.
1
1
d Arrows
to use in this SVG.
nced callouts or arrows
_Callout&Arrow_Libary.
Figure 16 Allele size vs. Allelic Ladder sizing for 84 samples analyzed on a 3500xL
™
Genetic Analyzer. Size and ladder sizing for the VeriFiler Plus kit were calculated
™
™
using the GeneScan 600 LIZ Size Standard v2.0.
h, as needed.
, and unused
G elements
repository.
1
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
6
Precision and size window description
Sizing precision enables the determination of accurate and reliable genotypes. The
recommended method for genotyping is to use a ±0.5-nt “window” around the size
obtained for each allele in the allelic ladder. A ±0.5-nt window allows for the detection
and correct assignment of alleles. Any sample allele that sizes outside the specified
window could be either:
• An “off-ladder” allele, that is, an allele of a size that is not represented in the
allelic ladder.
• An allele that does correspond to an allele in the allelic ladder, but whose size is
just outside a window because of measurement error.
The measurement error inherent in any sizing method can be defined by the degree
of precision in sizing an allele multiple times. Precision is measured by calculating
the standard deviation in the size values obtained for an allele that is run in several
injections on a capillary instrument.
Precision and size window observation
Table 7 lists typical precision results obtained from multiple runs of the VeriFiler™ Plus
Allelic Ladder using the GeneScan™ 600 LIZ™ Size Standard v2.0. The results were
obtained within a set of injections on a single capillary array. The number of repeated
injections for each genetic analyzer is shown in Table 6.
Table 6 Number of repeated injections for each genetic analyzer
Genetic analyzer
Capillaries
Repeated
injections
Sizing method
3130xl
16/injection
5
Local Southern, 60–460 bp
3500
8/injection
12
Local Southern, 60–460 bp
3500xL
24/injection
4
Local Southern, 60–460 bp
The mean sizes and the standard deviation for the allele sizing were calculated for all
the alleles in each run (Table 7). The mean range and the standard deviation range
show the lowest and highest values obtained across multiple runs.
Sample alleles can occasionally size outside of the ±0.5‑nt window for a respective
Allelic Ladder allele because of measurement error. The frequency of such an
occurrence is lowest in detection systems with the smallest standard deviations in
sizing. The figures in “Accuracy observation” on page 65 illustrate the tight clustering
of allele sizes obtained on the Applied Biosystems™ genetic analyzers, where the
standard deviation in sizing is typically less than 0.15 nt. The instance of a sample
allele sizing outside the ±0.5‑nt window because of measurement error is relatively
rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith,
1995).
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
For sample alleles that do not size within a ±0.5‑nt window, the PCR product must
be rerun to distinguish between a true off–ladder allele versus measurement error of
a sample allele that corresponds to an allele in the Allelic Ladder. Repeat analysis,
when necessary, provides an added level of confidence in the final allele assignment.
The GeneMapper™ ID‑X Software automatically flags sample alleles that do not size
within the prescribed window around an allelic ladder allele by labeling the allele as
OL (off‑ladder).
Maximum sizing precision is obtained within the same set of capillary injections.
Cross–platform sizing differences occur due to several factors including type and
concentration of polymer, run temperature, and electrophoresis conditions. Variations
in sizing can also occur between runs on the same instrument and between runs on
different instruments of the same platform type because of these factors.
IMPORTANT! To minimize the variation in sizing between runs and to ensure
accurate genotyping, follow the guidelines in “Allelic ladder requirements for data
analysis” on page 36 and use allelic ladders obtained from the same run as samples
to analyze the samples.
For more information on precision and genotyping, see (Lazaruk et al., 1998;
Mansfield et al., 1998).
Note: The IQCS and IQCL markers were omitted from this study because they are
not used for genotyping.
Table 7 Precision results of multiple runs of the VeriFiler™ Plus Allelic Ladder
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
9
89.28–89.33
0.013–0.046
89.48–89.52
0.006–0.039
89.37–89.44
0.010–0.045
10
93.45–93.52
0.012–0.039
93.67–93.70
0.005–0.046
93.55–93.63
0.013–0.039
11
97.62–97.69
0.012–0.042
97.83–97.89
0.006–0.042
97.75–97.79
0.008–0.054
12
101.78–101.86
0.018–0.040
102.00–102.06
0.010–0.040
101.91–101.96
0.010–0.043
13
105.95–105.99
0.014–0.041
106.13–106.20
0.010–0.040
106.04–106.11
0.008–0.034
14
110.07–110.13
0.014–0.043
110.28–110.34
0.013–0.038
110.19–110.26
0.010–0.040
15
114.21–114.26
0.017–0.039
114.41–114.46
0.010–0.039
114.33–114.38
0.005–0.033
16
118.33–118.38
0.016–0.042
118.53–118.58
0.005–0.036
118.45–118.51
0.008–0.043
17
122.45–122.51
0.015–0.050
122.66–122.70
0.006–0.038
122.57–122.63
0.013–0.050
18
126.64–126.70
0.010–0.041
126.85–126.90
0.010–0.044
126.76–126.83
0.005–0.041
19
130.73–130.78
0.016–0.037
130.92–130.97
0.013–0.044
130.84–130.89
0.008–0.044
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Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
20
134.72–134.80
0.008–0.036
134.93–134.98
0.013–0.043
134.83–134.91
0.006–0.044
11
151.38–151.45
0.015–0.052
151.76–151.80
0.010–0.045
151.65–151.76
0.019–0.048
12
155.39–155.45
0.021–0.048
155.78–155.83
0.005–0.044
155.69–155.77
0.010–0.049
13
159.46–159.52
0.018–0.049
159.85–159.90
0.010–0.037
159.76–159.83
0.008–0.045
14
163.68–163.74
0.024–0.055
164.07–164.12
0.010–0.041
163.97–164.05
0.010–0.053
15
167.62–167.69
0.026–0.047
168.01–168.06
0.010–0.045
167.93–168.00
0.010–0.050
16
171.66–171.73
0.021–0.049
172.05–172.11
0.010–0.050
171.98–172.06
0.010–0.059
17
175.71–175.77
0.017–0.053
176.10–176.15
0.005–0.046
176.05–176.10
0.013–0.047
18
179.72–179.77
0.014–0.054
180.10–180.18
0.005–0.034
180.03–180.11
0.013–0.060
19
183.76–183.83
0.029–0.054
184.16–184.22
0.008–0.046
184.12–184.17
0.000–0.057
20
187.79–187.87
0.028–0.054
188.20–188.26
0.005–0.056
188.12–188.21
0.015–0.057
21
191.80–191.86
0.023–0.048
192.20–192.26
0.005–0.040
192.13–192.21
0.013–0.067
22
195.83–195.88
0.021–0.055
196.23–196.29
0.006–0.051
196.14–196.24
0.013–0.051
23
199.77–199.84
0.027–0.048
200.20–200.25
0.010–0.046
200.12–200.19
0.010–0.062
24
204.14–204.20
0.027–0.058
204.54–204.60
0.013–0.049
204.48–204.53
0.013–0.057
5
226.98–227.05
0.029–0.055
227.54–227.60
0.008–0.043
227.44–227.50
0.029–0.077
8
239.10–239.18
0.037–0.067
239.67–239.73
0.013–0.043
239.59–239.64
0.034–0.079
9
243.17–243.24
0.029–0.070
243.73–243.79
0.000–0.046
243.60–243.70
0.025–0.090
10
247.21–247.27
0.041–0.078
247.77–247.83
0.013–0.050
247.67–247.75
0.017–0.068
11
251.26–251.33
0.025–0.066
251.84–251.87
0.010–0.045
251.72–251.80
0.026–0.081
12
255.30–255.36
0.043–0.061
255.88–255.93
0.015–0.045
255.76–255.83
0.029–0.075
13
259.33–259.39
0.040–0.074
259.92–259.98
0.008–0.053
259.81–259.88
0.017–0.088
14
263.39–263.45
0.038–0.077
263.96–264.02
0.008–0.057
263.86–263.94
0.021–0.077
15
267.42–267.48
0.037–0.077
268.01–268.07
0.005–0.056
267.89–267.99
0.025–0.094
vWA
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
6
281.95–282.03
0.035–0.077
282.56–282.63
0.010–0.057
282.49–282.58
0.029–0.107
7
285.99–286.07
0.042–0.076
286.58–286.65
0.016–0.045
286.53–286.61
0.024–0.099
8
290.02–290.10
0.047–0.068
290.61–290.69
0.016–0.047
290.57–290.65
0.021–0.090
9
294.04–294.13
0.042–0.071
294.65–294.72
0.008–0.042
294.60–294.69
0.027–0.104
10
298.07–298.15
0.039–0.069
298.68–298.75
0.010–0.050
298.65–298.73
0.028–0.087
11
302.10–302.18
0.033–0.074
302.72–302.79
0.006–0.059
302.66–302.75
0.040–0.081
12
306.12–306.20
0.032–0.075
306.77–306.83
0.013–0.061
306.72–306.79
0.029–0.093
13
310.15–310.23
0.042–0.069
310.78–310.85
0.013–0.054
310.76–310.83
0.024–0.095
14
314.17–314.27
0.045–0.081
314.83–314.89
0.013–0.051
314.78–314.87
0.033–0.091
15
318.19–318.28
0.021–0.072
318.85–318.93
0.010–0.065
318.84–318.91
0.033–0.085
7
334.73–334.82
0.029–0.074
335.38–335.44
0.006–0.055
335.32–335.38
0.024–0.074
8
338.78–338.87
0.028–0.067
339.43–339.48
0.008–0.055
339.37–339.45
0.028–0.097
9
342.72–342.83
0.023–0.079
343.42–343.48
0.008–0.059
343.32–343.42
0.029–0.087
10
346.78–346.87
0.027–0.071
347.46–347.52
0.008–0.054
347.40–347.48
0.021–0.083
11
350.84–350.93
0.035–0.072
351.52–351.57
0.000–0.049
351.46–351.54
0.025–0.079
12
354.84–354.94
0.047–0.069
355.53–355.61
0.006–0.062
355.47–355.56
0.029–0.076
13
358.88–358.97
0.049–0.085
359.57–359.62
0.008–0.059
359.47–359.57
0.013–0.081
14
362.92–363.01
0.041–0.083
363.61–363.66
0.014–0.060
363.53–363.61
0.013–0.076
15
366.91–367.02
0.037–0.069
367.63–367.69
0.013–0.067
367.55–367.63
0.021–0.085
16
370.90–370.99
0.042–0.065
371.60–371.65
0.010–0.057
371.51–371.60
0.037–0.083
17
374.91–374.99
0.039–0.062
375.63–375.69
0.013–0.060
375.55–375.63
0.019–0.096
18
378.89–378.98
0.026–0.066
379.63–379.70
0.010–0.061
379.55–379.63
0.008–0.089
19
382.92–382.99
0.030–0.088
383.67–383.73
0.008–0.051
383.58–383.67
0.018–0.074
20
386.92–386.99
0.028–0.075
387.69–387.76
0.010–0.061
387.60–387.69
0.026–0.098
21
390.90–390.99
0.029–0.082
391.71–391.76
0.006–0.050
391.62–391.69
0.034–0.091
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Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
22
394.88–394.95
0.021–0.070
395.70–395.76
0.015–0.066
395.57–395.68
0.024–0.081
23
398.85–398.93
0.024–0.082
399.68–399.74
0.008–0.051
399.57–399.68
0.029–0.081
24
402.82–402.91
0.034–0.088
403.70–403.74
0.010–0.057
403.60–403.67
0.027–0.098
25
406.80–406.89
0.035–0.082
407.68–407.75
0.013–0.053
407.57–407.68
0.030–0.085
1
81.20–81.26
0.019–0.047
81.43–81.47
0.005–0.051
81.33–81.38
0.008–0.036
2
86.50–86.56
0.023–0.041
86.73–86.78
0.008–0.045
86.62–86.70
0.010–0.042
X
102.45–102.50
0.014–0.044
102.53–102.57
0.008–0.034
102.44–102.50
0.013–0.041
Y
108.41–108.46
0.016–0.047
108.52–108.60
0.013–0.032
108.47–108.52
0.008–0.042
5
114.53–114.58
0.019–0.035
114.64–114.68
0.008–0.038
114.57–114.62
0.005–0.054
6
118.63–118.69
0.014–0.052
118.74–118.79
0.005–0.039
118.67–118.74
0.006–0.044
7
122.74–122.79
0.009–0.040
122.84–122.89
0.006–0.046
122.75–122.83
0.010–0.047
8
126.84–126.89
0.009–0.039
126.94–126.99
0.010–0.047
126.88–126.93
0.005–0.045
9
130.93–131.00
0.008–0.045
131.03–131.08
0.010–0.040
130.97–131.05
0.010–0.042
10
135.02–135.08
0.014–0.041
135.12–135.16
0.010–0.047
135.04–135.11
0.005–0.061
11
139.11–139.16
0.012–0.047
139.19–139.24
0.008–0.058
139.13–139.18
0.000–0.048
12
143.20–143.27
0.015–0.039
143.29–143.34
0.013–0.040
143.23–143.29
0.013–0.047
13
147.39–147.44
0.012–0.035
147.47–147.51
0.010–0.039
147.40–147.47
0.008–0.052
14
151.47–151.53
0.016–0.046
151.52–151.58
0.006–0.039
151.48–151.54
0.010–0.054
15
155.54–155.59
0.010–0.037
155.58–155.64
0.005–0.039
155.54–155.60
0.010–0.048
16
159.61–159.65
0.016–0.032
159.65–159.71
0.010–0.048
159.58–159.65
0.010–0.046
17
163.66–163.72
0.010–0.032
163.70–163.75
0.010–0.041
163.66–163.71
0.006–0.059
18
167.73–167.78
0.015–0.035
167.76–167.82
0.013–0.047
167.72–167.76
0.010–0.045
19
171.79–171.86
0.008–0.027
171.82–171.86
0.010–0.044
171.77–171.83
0.010–0.051
Y Indel
AMEL
D8S1179
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6
Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
24
182.85–182.91
0.033–0.079
183.23–183.28
0.006–0.049
183.17–183.25
0.005–0.070
24
184.88–184.94
0.028–0.061
185.26–185.31
0.008–0.056
185.23–185.28
0.013–0.071
25
186.88–186.95
0.037–0.068
187.28–187.32
0.005–0.056
187.23–187.28
0.019–0.062
26
190.94–190.98
0.034–0.076
191.32–191.37
0.008–0.044
191.27–191.33
0.017–0.072
27
194.98–195.04
0.042–0.065
195.38–195.42
0.008–0.047
195.31–195.40
0.014–0.077
28
198.99–199.05
0.035–0.077
199.37–199.43
0.010–0.044
199.34–199.40
0.013–0.067
28
200.99–201.03
0.039–0.061
201.38–201.43
0.010–0.045
201.34–201.39
0.008–0.074
29
203.00–203.05
0.045–0.077
203.39–203.44
0.008–0.052
203.37–203.42
0.005–0.067
29
205.06–205.10
0.042–0.079
205.44–205.50
0.015–0.047
205.40–205.47
0.021–0.078
30
207.05–207.10
0.039–0.074
207.45–207.51
0.013–0.045
207.39–207.47
0.017–0.082
30
209.05–209.11
0.042–0.072
209.45–209.49
0.010–0.043
209.39–209.47
0.021–0.067
31
211.08–211.14
0.032–0.067
211.49–211.55
0.008–0.045
211.43–211.51
0.010–0.072
31
213.08–213.15
0.039–0.073
213.49–213.52
0.010–0.043
213.44–213.50
0.014–0.081
32
215.12–215.17
0.042–0.074
215.52–215.57
0.005–0.040
215.47–215.54
0.010–0.069
32
217.10–217.16
0.050–0.069
217.52–217.56
0.005–0.048
217.44–217.54
0.010–0.069
33
219.14–219.22
0.048–0.075
219.55–219.61
0.006–0.039
219.52–219.59
0.017–0.080
33
221.10–221.16
0.044–0.071
221.50–221.57
0.010–0.050
221.48–221.54
0.020–0.085
34
223.23–223.29
0.035–0.078
223.65–223.69
0.013–0.040
223.59–223.66
0.022–0.075
34
225.17–225.23
0.043–0.074
225.58–225.64
0.010–0.042
225.54–225.61
0.021–0.090
35
227.26–227.30
0.030–0.080
227.66–227.71
0.005–0.046
227.62–227.68
0.024–0.077
35
229.20–229.25
0.042–0.073
229.61–229.67
0.005–0.048
229.57–229.64
0.022–0.076
36
231.20–231.26
0.037–0.082
231.63–231.69
0.010–0.051
231.58–231.65
0.017–0.075
37
235.28–235.34
0.042–0.076
235.71–235.75
0.006–0.051
235.65–235.73
0.030–0.070
38
239.25–239.32
0.040–0.082
239.67–239.75
0.008–0.045
239.62–239.71
0.028–0.072
261.02–261.08
0.037–0.075
261.35–261.42
0.005–0.044
261.32–261.38
0.037–0.064
D21S11
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
6
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
9
269.17–269.23
0.028–0.068
269.49–269.56
0.010–0.051
269.44–269.51
0.010–0.068
10
273.24–273.30
0.034–0.062
273.56–273.62
0.013–0.045
273.52–273.59
0.013–0.071
10
275.24–275.30
0.034–0.079
275.56–275.61
0.010–0.041
275.51–275.60
0.024–0.060
11
277.31–277.37
0.025–0.063
277.63–277.69
0.010–0.046
277.58–277.65
0.015–0.062
12
281.38–281.45
0.031–0.073
281.70–281.75
0.008–0.056
281.64–281.72
0.015–0.078
13
285.46–285.53
0.037–0.069
285.77–285.81
0.010–0.051
285.69–285.79
0.016–0.063
13
287.48–287.54
0.027–0.075
287.77–287.82
0.017–0.051
287.71–287.79
0.010–0.062
14
289.53–289.60
0.034–0.058
289.82–289.89
0.013–0.044
289.80–289.87
0.013–0.068
14
291.56–291.61
0.032–0.069
291.82–291.89
0.013–0.051
291.79–291.85
0.005–0.070
15
293.62–293.68
0.020–0.057
293.91–293.96
0.008–0.046
293.85–293.93
0.010–0.064
16
297.70–297.77
0.035–0.064
297.97–298.03
0.010–0.059
297.92–298.00
0.010–0.059
17
301.76–301.83
0.029–0.071
302.03–302.09
0.017–0.051
302.01–302.08
0.019–0.062
18
305.85–305.90
0.025–0.078
306.11–306.18
0.010–0.055
306.08–306.14
0.021–0.053
19
309.94–309.98
0.029–0.061
310.16–310.22
0.016–0.051
310.15–310.21
0.008–0.067
20
314.00–314.06
0.021–0.054
314.25–314.29
0.013–0.045
314.21–314.28
0.010–0.059
21
318.10–318.13
0.021–0.057
318.31–318.37
0.010–0.043
318.26–318.37
0.008–0.054
22
322.22–322.28
0.015–0.055
322.44–322.50
0.005–0.053
322.42–322.49
0.010–0.062
23
326.22–326.28
0.025–0.064
326.43–326.51
0.013–0.057
326.42–326.49
0.015–0.076
24
330.27–330.34
0.022–0.067
330.51–330.56
0.010–0.041
330.49–330.57
0.013–0.073
25
334.34–334.41
0.018–0.062
334.56–334.63
0.008–0.056
334.56–334.63
0.010–0.059
26
338.44–338.49
0.022–0.055
338.63–338.69
0.005–0.050
338.63–338.72
0.010–0.055
27
342.48–342.56
0.024–0.068
342.71–342.76
0.006–0.052
342.72–342.79
0.014–0.059
7
370.14–370.21
0.037–0.079
370.66–370.71
0.015–0.068
370.60–370.70
0.006–0.077
8
374.15–374.21
0.029–0.059
374.68–374.75
0.010–0.064
374.65–374.73
0.013–0.079
9
378.14–378.20
0.019–0.063
378.71–378.76
0.010–0.074
378.67–378.75
0.018–0.076
10
382.14–382.20
0.016–0.068
382.71–382.79
0.010–0.065
382.69–382.77
0.013–0.076
D5S818
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6
Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
11
386.13–386.20
0.024–0.072
386.74–386.80
0.008–0.057
386.71–386.78
0.024–0.090
12
390.12–390.19
0.037–0.072
390.75–390.82
0.013–0.061
390.73–390.80
0.021–0.082
13
394.10–394.18
0.038–0.102
394.76–394.83
0.015–0.067
394.72–394.82
0.032–0.087
14
398.10–398.16
0.032–0.071
398.77–398.82
0.010–0.068
398.74–398.82
0.031–0.091
15
402.08–402.14
0.026–0.073
402.77–402.84
0.012–0.058
402.73–402.81
0.017–0.095
16
406.06–406.12
0.032–0.110
406.78–406.85
0.013–0.075
406.73–406.81
0.017–0.085
17
410.04–410.10
0.044–0.114
410.78–410.85
0.008–0.079
410.72–410.82
0.021–0.100
18
414.00–414.08
0.043–0.103
414.77–414.85
0.017–0.057
414.73–414.80
0.037–0.106
8
83.24–83.27
0.008–0.034
83.25–83.30
0.005–0.050
83.19–83.25
0.005–0.036
9
87.34–87.37
0.012–0.031
87.37–87.42
0.006–0.038
87.31–87.40
0.008–0.036
10
91.40–91.42
0.010–0.034
91.45–91.50
0.005–0.041
91.40–91.45
0.008–0.035
11
95.52–95.56
0.014–0.046
95.60–95.65
0.006–0.046
95.56–95.61
0.013–0.038
11
98.49–98.53
0.015–0.032
98.57–98.63
0.008–0.039
98.54–98.58
0.008–0.048
12
99.59–99.64
0.015–0.037
99.69–99.73
0.008–0.041
99.65–99.70
0.008–0.035
13
103.52–103.55
0.018–0.036
103.62–103.68
0.013–0.036
103.58–103.64
0.013–0.045
14
107.59–107.63
0.015–0.049
107.71–107.76
0.014–0.038
107.67–107.71
0.008–0.046
15
111.64–111.69
0.015–0.045
111.78–111.82
0.014–0.045
111.75–111.80
0.006–0.039
16
115.73–115.78
0.010–0.033
115.85–115.90
0.010–0.034
115.81–115.89
0.010–0.044
17
119.88–119.93
0.014–0.033
120.02–120.07
0.013–0.045
120.01–120.05
0.013–0.039
5
141.04–141.09
0.016–0.043
141.42–141.46
0.006–0.050
141.37–141.44
0.021–0.059
6
145.02–145.07
0.019–0.059
145.41–145.45
0.008–0.038
145.39–145.45
0.013–0.071
7
148.99–149.05
0.015–0.048
149.39–149.44
0.014–0.051
149.38–149.44
0.010–0.063
8
152.95–153.02
0.014–0.056
153.38–153.42
0.010–0.046
153.33–153.42
0.018–0.069
9
156.91–156.98
0.026–0.055
157.35–157.41
0.008–0.042
157.35–157.40
0.014–0.073
10
160.86–160.94
0.029–0.051
161.33–161.38
0.010–0.041
161.32–161.38
0.013–0.065
D2S441
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
6
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
11
164.82–164.89
0.017–0.048
165.30–165.36
0.010–0.052
165.28–165.36
0.017–0.070
12
168.78–168.85
0.029–0.054
169.26–169.32
0.008–0.052
169.24–169.33
0.024–0.084
12
170.89–170.97
0.032–0.059
171.38–171.44
0.006–0.046
171.37–171.45
0.005–0.076
13
172.74–172.81
0.018–0.060
173.24–173.29
0.005–0.048
173.22–173.30
0.027–0.080
13
174.72–174.80
0.025–0.062
175.22–175.28
0.010–0.045
175.21–175.30
0.027–0.079
14
176.68–176.76
0.030–0.065
177.20–177.26
0.008–0.054
177.16–177.27
0.028–0.082
14
178.65–178.74
0.026–0.069
179.17–179.25
0.005–0.037
179.16–179.27
0.018–0.088
15
180.62–180.70
0.029–0.070
181.14–181.21
0.005–0.039
181.11–181.23
0.024–0.098
15
182.63–182.69
0.028–0.064
183.15–183.22
0.006–0.046
183.15–183.22
0.028–0.088
16
184.56–184.66
0.034–0.060
185.10–185.17
0.006–0.046
185.07–185.19
0.031–0.077
16
186.57–186.66
0.028–0.063
187.11–187.17
0.005–0.043
187.13–187.20
0.029–0.083
17
188.49–188.59
0.028–0.070
189.06–189.13
0.005–0.053
189.05–189.15
0.032–0.087
17
190.53–190.62
0.031–0.064
191.09–191.15
0.005–0.062
191.07–191.16
0.030–0.096
18
194.56–194.64
0.032–0.060
195.12–195.19
0.008–0.032
195.12–195.20
0.037–0.077
19
198.49–198.58
0.031–0.062
199.08–199.14
0.010–0.051
199.08–199.17
0.028–0.105
13
207.89–207.92
0.012–0.039
207.88–207.93
0.008–0.052
207.83–207.89
0.010–0.042
14
211.94–211.97
0.013–0.030
211.93–211.97
0.010–0.051
211.88–211.94
0.013–0.055
15
215.99–216.02
0.011–0.035
215.96–216.02
0.008–0.051
215.93–215.97
0.005–0.049
16
220.05–220.09
0.016–0.032
220.01–220.07
0.008–0.047
219.97–220.04
0.010–0.046
17
224.11–224.15
0.015–0.039
224.05–224.11
0.010–0.043
224.01–224.08
0.010–0.040
18
228.16–228.20
0.013–0.031
228.11–228.17
0.006–0.053
228.07–228.12
0.005–0.049
19
232.22–232.27
0.013–0.033
232.15–232.20
0.010–0.042
232.12–232.17
0.010–0.048
20
236.27–236.33
0.010–0.041
236.20–236.25
0.010–0.046
236.17–236.22
0.005–0.043
21
240.33–240.38
0.008–0.052
240.25–240.30
0.010–0.045
240.21–240.27
0.006–0.039
22
244.39–244.44
0.012–0.034
244.30–244.35
0.000–0.045
244.25–244.33
0.012–0.041
23
248.46–248.50
0.012–0.044
248.34–248.40
0.013–0.038
248.31–248.36
0.008–0.050
FGA
VeriFiler™ Plus PCR Amplification Kit User Guide
75
6
Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
24
252.51–252.56
0.014–0.040
252.39–252.45
0.010–0.046
252.38–252.43
0.010–0.044
25
256.58–256.62
0.016–0.033
256.46–256.51
0.014–0.044
256.41–256.49
0.010–0.042
26
260.63–260.68
0.014–0.046
260.48–260.54
0.005–0.052
260.46–260.52
0.010–0.050
26
262.66–262.71
0.014–0.063
262.53–262.59
0.010–0.050
262.49–262.55
0.005–0.041
27
264.66–264.70
0.012–0.040
264.52–264.56
0.010–0.041
264.48–264.53
0.010–0.048
28
268.72–268.76
0.010–0.046
268.58–268.61
0.010–0.048
268.53–268.59
0.008–0.045
29
272.78–272.83
0.017–0.063
272.63–272.69
0.013–0.038
272.61–272.66
0.005–0.049
30
276.86–276.92
0.011–0.048
276.71–276.78
0.008–0.045
276.68–276.73
0.006–0.045
30
278.69–278.75
0.012–0.044
278.55–278.60
0.010–0.053
278.51–278.58
0.006–0.048
31
282.76–282.81
0.012–0.034
282.59–282.67
0.008–0.045
282.55–282.63
0.008–0.048
32
286.83–286.88
0.018–0.038
286.68–286.73
0.010–0.042
286.62–286.69
0.013–0.051
33
290.89–290.94
0.014–0.036
290.71–290.77
0.013–0.041
290.67–290.75
0.008–0.049
42
327.62–327.67
0.014–0.050
327.39–327.43
0.010–0.053
327.38–327.46
0.008–0.057
43
331.67–331.73
0.006–0.061
331.44–331.49
0.010–0.049
331.42–331.51
0.015–0.063
44
335.72–335.79
0.015–0.046
335.50–335.57
0.010–0.046
335.53–335.59
0.013–0.062
45
339.80–339.86
0.019–0.048
339.57–339.63
0.000–0.050
339.59–339.65
0.010–0.068
46
343.77–343.84
0.013–0.054
343.52–343.59
0.005–0.057
343.54–343.61
0.005–0.077
47
347.84–347.90
0.013–0.047
347.57–347.64
0.013–0.041
347.59–347.67
0.013–0.067
48
351.92–351.98
0.012–0.043
351.66–351.72
0.010–0.048
351.67–351.75
0.006–0.076
49
355.98–356.04
0.014–0.044
355.71–355.77
0.000–0.045
355.74–355.80
0.012–0.060
50
360.03–360.07
0.018–0.054
359.73–359.80
0.013–0.050
359.75–359.82
0.017–0.079
51
364.08–364.14
0.018–0.078
363.80–363.85
0.014–0.056
363.82–363.88
0.017–0.073
8
371.77–371.82
0.023–0.063
372.17–372.23
0.010–0.057
372.12–372.21
0.010–0.071
9
375.73–375.80
0.023–0.061
376.16–376.23
0.010–0.059
376.13–376.20
0.017–0.069
10
379.73–379.79
0.031–0.058
380.16–380.23
0.010–0.047
380.14–380.19
0.016–0.056
11
383.68–383.76
0.024–0.060
384.12–384.19
0.008–0.054
384.09–384.16
0.010–0.068
D10S1248
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VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
6
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
12
387.70–387.76
0.020–0.056
388.15–388.22
0.012–0.054
388.13–388.18
0.021–0.058
13
391.69–391.75
0.019–0.163
392.14–392.20
0.013–0.062
392.11–392.17
0.010–0.072
14
395.65–395.72
0.028–0.088
396.10–396.17
0.013–0.054
396.07–396.15
0.008–0.076
15
399.64–399.72
0.028–0.128
400.11–400.17
0.013–0.054
400.07–400.14
0.017–0.074
16
403.61–403.67
0.024–0.062
404.10–404.19
0.013–0.058
404.07–404.14
0.017–0.077
17
407.63–407.68
0.025–0.081
408.13–408.18
0.005–0.057
408.08–408.14
0.017–0.085
18
411.56–411.60
0.019–0.115
412.04–412.12
0.008–0.052
412.00–412.09
0.014–0.067
19
415.49–415.57
0.021–0.102
416.02–416.10
0.015–0.056
416.00–416.05
0.017–0.080
8
88.43–88.47
0.012–0.037
88.46–88.53
0.005–0.039
88.40–88.45
0.005–0.052
9
91.42–91.46
0.013–0.037
91.46–91.54
0.005–0.042
91.41–91.46
0.010–0.042
10
94.42–94.46
0.010–0.030
94.50–94.54
0.005–0.042
94.43–94.47
0.012–0.039
11
97.43–97.46
0.013–0.038
97.49–97.56
0.008–0.045
97.43–97.49
0.005–0.054
12
100.42–100.45
0.010–0.042
100.51–100.56
0.006–0.043
100.45–100.50
0.005–0.036
13
103.41–103.46
0.012–0.038
103.50–103.57
0.008–0.040
103.44–103.49
0.013–0.041
14
106.40–106.44
0.008–0.039
106.51–106.55
0.015–0.045
106.43–106.48
0.014–0.041
15
109.39–109.44
0.013–0.044
109.51–109.57
0.013–0.039
109.44–109.50
0.010–0.043
16
112.38–112.42
0.017–0.038
112.50–112.54
0.010–0.039
112.43–112.49
0.000–0.037
17
115.37–115.40
0.008–0.028
115.49–115.56
0.010–0.044
115.41–115.49
0.013–0.040
18
118.35–118.41
0.012–0.032
118.50–118.55
0.013–0.053
118.41–118.48
0.008–0.041
19
121.34–121.40
0.010–0.040
121.48–121.53
0.005–0.042
121.41–121.46
0.006–0.042
9
146.83–146.86
0.011–0.039
146.87–146.93
0.010–0.043
146.86–146.91
0.005–0.041
10
150.72–150.76
0.015–0.034
150.77–150.83
0.010–0.039
150.78–150.81
0.017–0.043
11
154.72–154.77
0.010–0.038
154.78–154.83
0.008–0.044
154.77–154.84
0.014–0.050
12
158.81–158.85
0.014–0.031
158.86–158.92
0.010–0.042
158.88–158.91
0.013–0.048
13
162.74–162.76
0.017–0.034
162.80–162.85
0.014–0.037
162.80–162.85
0.010–0.043
D22S1045
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
14
166.81–166.85
0.012–0.038
166.89–166.93
0.013–0.048
166.87–166.94
0.010–0.051
14
169.77–169.80
0.010–0.034
169.85–169.89
0.008–0.043
169.85–169.89
0.010–0.047
15
170.79–170.83
0.010–0.047
170.87–170.92
0.006–0.036
170.86–170.93
0.000–0.051
15
173.75–173.79
0.014–0.035
173.84–173.90
0.005–0.042
173.83–173.89
0.006–0.043
16
174.80–174.85
0.014–0.035
174.88–174.93
0.000–0.036
174.88–174.94
0.008–0.053
16
177.75–177.80
0.016–0.040
177.83–177.89
0.008–0.044
177.84–177.89
0.010–0.048
17
178.88–178.92
0.012–0.064
178.95–179.00
0.008–0.040
178.95–179.00
0.013–0.053
17
181.75–181.79
0.009–0.043
181.84–181.89
0.005–0.039
181.82–181.89
0.014–0.044
18
182.78–182.82
0.010–0.034
182.87–182.92
0.005–0.050
182.85–182.92
0.005–0.051
18
185.75–185.78
0.012–0.042
185.84–185.89
0.005–0.041
185.85–185.90
0.010–0.040
19
189.77–189.80
0.017–0.036
189.88–189.93
0.006–0.043
189.87–189.93
0.006–0.052
20
193.82–193.86
0.014–0.035
193.93–193.98
0.005–0.033
193.92–193.98
0.008–0.059
5
201.31–201.35
0.020–0.036
201.46–201.52
0.010–0.047
201.45–201.50
0.013–0.053
6
205.33–205.37
0.015–0.031
205.47–205.52
0.015–0.038
205.46–205.51
0.005–0.045
7
209.35–209.39
0.011–0.039
209.49–209.54
0.010–0.050
209.48–209.53
0.017–0.056
8
213.37–213.42
0.015–0.033
213.49–213.55
0.005–0.039
213.50–213.55
0.014–0.049
9
217.42–217.46
0.014–0.036
217.53–217.58
0.005–0.039
217.50–217.56
0.005–0.048
10
221.43–221.48
0.019–0.036
221.55–221.60
0.010–0.054
221.53–221.60
0.006–0.045
11
225.48–225.50
0.018–0.038
225.58–225.63
0.010–0.043
225.57–225.62
0.017–0.048
12
229.49–229.54
0.014–0.039
229.60–229.66
0.008–0.049
229.59–229.64
0.010–0.043
13
233.54–233.58
0.012–0.044
233.62–233.68
0.008–0.043
233.63–233.66
0.015–0.051
14
237.55–237.61
0.012–0.034
237.66–237.72
0.005–0.044
237.64–237.69
0.006–0.054
15
241.59–241.64
0.005–0.041
241.69–241.74
0.006–0.048
241.67–241.72
0.010–0.053
16
245.64–245.70
0.015–0.043
245.72–245.78
0.008–0.042
245.71–245.76
0.015–0.066
259.89–259.94
0.010–0.048
260.03–260.07
0.008–0.049
259.99–260.05
0.008–0.046
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
6
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
7
263.90–263.96
0.008–0.045
264.06–264.11
0.013–0.042
264.00–264.07
0.014–0.049
8
267.92–267.97
0.018–0.044
268.08–268.13
0.010–0.048
268.05–268.09
0.010–0.056
9
271.95–271.99
0.009–0.045
272.10–272.15
0.017–0.045
272.07–272.12
0.010–0.050
10
275.96–276.01
0.017–0.042
276.13–276.19
0.010–0.047
276.10–276.16
0.010–0.061
11
279.97–280.02
0.012–0.057
280.16–280.22
0.010–0.047
280.10–280.19
0.014–0.052
12
284.00–284.04
0.017–0.042
284.20–284.25
0.012–0.039
284.14–284.21
0.012–0.056
13
288.02–288.08
0.012–0.040
288.21–288.28
0.014–0.042
288.18–288.24
0.008–0.061
14
292.05–292.09
0.013–0.045
292.24–292.30
0.013–0.048
292.21–292.27
0.010–0.062
15
296.07–296.11
0.017–0.040
296.26–296.33
0.010–0.052
296.24–296.28
0.010–0.053
5
310.56–310.66
0.075–0.110
311.23–311.28
0.010–0.059
311.20–311.26
0.044–0.096
6
315.69–315.78
0.068–0.113
316.34–316.39
0.014–0.055
316.30–316.38
0.014–0.097
7
320.72–320.82
0.073–0.121
321.41–321.47
0.005–0.051
321.39–321.46
0.028–0.099
8
325.78–325.88
0.060–0.108
326.44–326.50
0.010–0.057
326.42–326.48
0.029–0.096
9
330.82–330.91
0.060–0.109
331.48–331.54
0.008–0.052
331.46–331.54
0.025–0.090
10
335.88–335.97
0.071–0.103
336.53–336.59
0.010–0.058
336.53–336.60
0.040–0.097
11
340.87–341.01
0.064–0.117
341.58–341.66
0.005–0.056
341.59–341.65
0.040–0.094
12
345.96–346.05
0.078–0.118
346.63–346.69
0.013–0.070
346.64–346.71
0.036–0.081
13
351.02–351.12
0.065–0.120
351.69–351.75
0.005–0.055
351.70–351.78
0.017–0.091
14
356.06–356.18
0.075–0.123
356.74–356.80
0.018–0.066
356.74–356.83
0.030–0.090
15
361.13–361.23
0.093–0.137
361.80–361.86
0.013–0.055
361.78–361.87
0.033–0.090
16
366.21–366.29
0.073–0.122
366.83–366.91
0.013–0.067
366.82–366.91
0.019–0.082
17
371.24–371.35
0.069–0.117
371.90–371.98
0.013–0.070
371.87–371.96
0.021–0.080
18
376.29–376.36
0.066–0.116
376.96–377.02
0.019–0.068
376.93–377.00
0.030–0.083
19
381.31–381.41
0.070–0.104
382.00–382.07
0.014–0.059
381.98–382.03
0.012–0.083
20
386.35–386.43
0.069–0.108
387.04–387.09
0.010–0.070
387.01–387.10
0.031–0.109
21
391.38–391.46
0.068–0.106
392.07–392.15
0.008–0.056
392.05–392.14
0.005–0.087
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
22
396.39–396.48
0.064–0.108
397.11–397.17
0.012–0.067
397.07–397.16
0.017–0.095
23
401.41–401.51
0.071–0.111
402.15–402.21
0.010–0.054
402.11–402.21
0.030–0.101
24
406.40–406.52
0.077–0.120
407.17–407.23
0.005–0.052
407.14–407.22
0.013–0.092
25
411.44–411.54
0.081–0.109
412.20–412.25
0.008–0.051
412.16–412.24
0.029–0.083
26
416.45–416.52
0.076–0.113
417.22–417.27
0.017–0.067
417.21–417.28
0.026–0.095
2
78.64–78.68
0.008–0.055
78.99–79.05
0.010–0.048
78.87–78.95
0.010–0.061
3
83.57–83.62
0.013–0.057
83.98–84.05
0.005–0.049
83.86–83.94
0.017–0.063
5
91.30–91.35
0.023–0.053
91.78–91.86
0.010–0.037
91.65–91.74
0.018–0.062
6
96.21–96.27
0.023–0.067
96.71–96.79
0.010–0.041
96.62–96.69
0.022–0.064
7
101.32–101.37
0.023–0.060
101.85–101.92
0.008–0.035
101.72–101.81
0.013–0.059
8
105.99–106.05
0.028–0.060
106.60–106.65
0.012–0.044
106.47–106.57
0.022–0.077
9
110.88–110.96
0.028–0.063
111.52–111.57
0.010–0.033
111.38–111.48
0.025–0.071
10
115.91–115.96
0.025–0.060
116.55–116.60
0.005–0.041
116.44–116.51
0.021–0.073
11
120.62–120.69
0.038–0.075
121.30–121.38
0.008–0.041
121.17–121.28
0.029–0.076
12
125.49–125.57
0.035–0.073
126.21–126.27
0.017–0.042
126.07–126.19
0.030–0.077
13
130.45–130.53
0.031–0.078
131.21–131.28
0.010–0.038
131.06–131.18
0.031–0.081
14
135.19–135.27
0.033–0.077
135.95–136.04
0.008–0.049
135.82–135.94
0.029–0.094
15
140.05–140.13
0.038–0.073
140.85–140.93
0.010–0.043
140.73–140.85
0.039–0.092
16
145.02–145.10
0.046–0.077
145.85–145.90
0.008–0.042
145.70–145.81
0.029–0.098
17
149.75–149.84
0.048–0.081
150.61–150.67
0.010–0.061
150.44–150.59
0.043–0.102
4
169.86–169.92
0.029–0.060
170.35–170.40
0.005–0.045
170.30–170.38
0.017–0.069
5
173.87–173.94
0.018–0.055
174.34–174.42
0.000–0.047
174.30–174.39
0.013–0.068
6
177.86–177.93
0.023–0.060
178.36–178.41
0.008–0.048
178.31–178.38
0.017–0.076
7
181.86–181.94
0.030–0.053
182.36–182.40
0.005–0.036
182.32–182.40
0.021–0.075
8
185.86–185.94
0.022–0.060
186.35–186.43
0.005–0.043
186.33–186.40
0.016–0.078
Penta D
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
6
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
9
189.87–189.95
0.035–0.059
190.39–190.43
0.008–0.051
190.34–190.41
0.024–0.076
9
192.94–193.01
0.028–0.069
193.43–193.49
0.010–0.050
193.41–193.48
0.019–0.080
10
193.88–193.95
0.031–0.062
194.37–194.44
0.005–0.051
194.35–194.42
0.015–0.074
11
197.87–197.96
0.027–0.060
198.40–198.47
0.006–0.046
198.37–198.44
0.021–0.078
12
201.89–201.97
0.029–0.070
202.39–202.48
0.013–0.050
202.37–202.44
0.034–0.071
13
208.95–209.03
0.036–0.065
209.45–209.52
0.010–0.048
209.43–209.52
0.025–0.081
14
218.79–218.84
0.014–0.034
218.81–218.88
0.006–0.034
218.81–218.87
0.010–0.047
15
222.81–222.84
0.016–0.041
222.81–222.87
0.010–0.046
222.82–222.87
0.012–0.042
16
226.76–226.80
0.010–0.033
226.76–226.83
0.012–0.050
226.77–226.82
0.015–0.056
17
230.84–230.89
0.013–0.036
230.84–230.90
0.008–0.054
230.85–230.90
0.008–0.064
18
234.88–234.92
0.015–0.036
234.87–234.92
0.005–0.058
234.87–234.91
0.010–0.043
19
238.94–238.99
0.015–0.037
238.94–238.99
0.010–0.047
238.92–238.98
0.005–0.048
19
241.95–242.00
0.010–0.076
241.93–241.98
0.005–0.043
241.93–241.97
0.010–0.055
20
242.89–242.95
0.012–0.043
242.91–242.95
0.010–0.052
242.89–242.95
0.008–0.044
21
247.01–247.06
0.010–0.043
246.99–247.04
0.010–0.041
246.97–247.04
0.005–0.046
22
251.12–251.17
0.012–0.038
251.09–251.15
0.014–0.041
251.08–251.13
0.013–0.050
23
255.12–255.18
0.011–0.033
255.10–255.15
0.010–0.040
255.09–255.15
0.013–0.042
24
259.13–259.19
0.018–0.034
259.11–259.16
0.010–0.042
259.09–259.15
0.010–0.033
25
263.16–263.22
0.017–0.035
263.13–263.19
0.013–0.053
263.12–263.18
0.008–0.046
26
267.23–267.27
0.015–0.048
267.19–267.23
0.006–0.038
267.16–267.22
0.005–0.043
27
271.22–271.27
0.012–0.041
271.18–271.24
0.013–0.046
271.16–271.23
0.010–0.045
11
280.86–280.95
0.042–0.072
281.45–281.48
0.008–0.058
281.40–281.48
0.040–0.085
12
284.85–284.94
0.031–0.069
285.45–285.50
0.017–0.043
285.41–285.48
0.041–0.093
13
288.87–288.94
0.031–0.075
289.42–289.51
0.013–0.057
289.41–289.48
0.033–0.089
14
292.86–292.96
0.024–0.065
293.43–293.48
0.010–0.062
293.40–293.50
0.021–0.088
D12S391
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Chapter 6 Experiments and results
Accuracy, precision, and reproducibility
Table 7 Precision results of multiple runs of the VeriFiler Plus Allelic Ladder (continued)
3130xl
Marker/allele
3500
3500xL
Mean
Standard
deviation
Mean
Standard
deviation
Mean
Standard
deviation
15
296.82–296.91
0.025–0.061
297.35–297.39
0.006–0.048
297.31–297.39
0.021–0.089
16
300.87–300.94
0.034–0.067
301.39–301.45
0.015–0.053
301.37–301.44
0.029–0.086
17
304.87–304.94
0.033–0.063
305.40–305.46
0.010–0.054
305.39–305.46
0.021–0.088
18
308.91–308.98
0.029–0.070
309.41–309.48
0.014–0.053
309.41–309.47
0.029–0.082
19
312.91–312.98
0.034–0.058
313.44–313.50
0.013–0.053
313.41–313.48
0.039–0.091
20
316.92–317.00
0.020–0.058
317.45–317.51
0.013–0.060
317.43–317.49
0.021–0.088
21
320.97–321.05
0.021–0.063
321.48–321.54
0.005–0.051
321.47–321.54
0.027–0.084
22
324.97–325.06
0.033–0.070
325.48–325.53
0.010–0.053
325.46–325.53
0.015–0.070
23
328.98–329.06
0.024–0.060
329.49–329.55
0.013–0.073
329.48–329.58
0.015–0.088
24
333.00–333.08
0.024–0.061
333.53–333.58
0.010–0.054
333.51–333.61
0.015–0.075
25
337.01–337.10
0.028–0.063
337.53–337.58
0.010–0.066
337.54–337.63
0.015–0.080
26
341.06–341.13
0.026–0.063
341.54–341.60
0.005–0.059
341.56–341.64
0.015–0.078
27
345.14–345.22
0.028–0.067
345.61–345.68
0.008–0.039
345.61–345.69
0.006–0.079
28
349.38–349.46
0.021–0.062
349.77–349.81
0.006–0.055
349.80–349.86
0.010–0.074
5
364.37–364.50
0.073–0.107
365.42–365.42
0.013–0.013
365.34–365.44
0.043–0.119
6
368.42–368.52
0.061–0.110
369.45–369.45
0.010–0.010
369.37–369.50
0.040–0.112
7
372.46–372.59
0.054–0.102
373.51–373.51
0.008–0.008
373.45–373.54
0.040–0.133
8
376.44–376.57
0.053–0.087
377.49–377.49
0.010–0.010
377.44–377.54
0.044–0.110
9
380.42–380.54
0.043–0.090
381.52–381.52
0.014–0.014
381.43–381.55
0.050–0.113
10
384.44–384.56
0.037–0.093
385.53–385.53
0.005–0.005
385.47–385.58
0.046–0.142
11
388.43–388.55
0.054–0.092
389.57–389.57
0.010–0.010
389.51–389.58
0.055–0.128
12
392.41–392.53
0.046–0.098
393.59–393.59
0.015–0.015
393.50–393.60
0.065–0.121
13
396.42–396.53
0.042–0.095
397.59–397.59
0.010–0.010
397.52–397.63
0.066–0.124
14
400.41–400.53
0.050–0.102
401.61–401.61
0.013–0.013
401.50–401.66
0.061–0.127
15
404.39–404.51
0.057–0.098
405.63–405.63
0.008–0.008
405.51–405.64
0.047–0.141
TPOX
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Extra peaks in the electropherogram
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Extra peaks in the electropherogram
Causes of extra peaks
Peaks other than the target alleles may be detected on the electropherogram.
Causes for the appearance of extra peaks include stutter products, incomplete 3´
A nucleotide addition (at the n-1 position), dye artifacts, and mixed DNA samples
(see DNA Advisory Board (DAB) Standard 8.1.2.2).
Extra peaks: Stutter
Stutter definition
Stutter is a well-characterized PCR artifact that refers to the appearance of a minor
peak one repeat unit smaller than the target STR allele product (minus stutter), or
less frequently, one repeat larger (plus stutter) (Butler, 2005; Mulero et al., 2006).
Sequence analysis of stutter products at tetranucleotide STR loci has revealed that
the minus stutter product is missing a single tetranucleotide core repeat unit relative
to the main allele (Walsh et al., 1996). Although plus-stutter is normally much less
significant than minus-stutter in STR loci with tetranucleotide repeats, the incidence
of plus-stutter may be more significant in trinucleotide repeat-containing loci.
Contact HID Support for more information on plus stutter.
The proportion of the stutter product relative to the main allele (percent stutter) is
measured by dividing the height of the stutter peak by the height of the main allele
peak.
Stutter observations
Peak heights were measured for amplified samples at the loci that are used in the
kit. All data were generated on the 3500xL Genetic Analyzer. Some conclusions from
these measurements and observations are:
• For each locus, the stutter percentage generally increases with allele length.
• Each allele within a locus displays a relatively consistent average stutter
percentage.
• Peaks in the stutter position that are above the stutter filter percentage specified
in the software are not filtered.
• The measurement of stutter percentage for allele peaks that are off-scale may be
unusually high due to artificial truncation of the main allele peak.
• Stutter can be elevated when minus stutter and plus stutter overlap. This is
typically observed when a given allele flanks another allele that is 2 repeat units
away (as seen with the FGA locus in control 007 DNA).
• The magnitude and/or variability of stutter may increase with low DNA input
amounts.
Figure 17 through Figure 21 show the stutter observed in the population study that
are one repeat unit away from the alleles recorded. All data were generated on the
3500xL Genetic Analyzer.
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6
Extra peaks in the electropherogram
The stutter filter settings that are derived from this data are listed in “Stutter
percentage filter settings provided with GeneMapper™ ID‑X Software” on page 88.
Plus-stutter was regularly observed and was more significant in trinucleotide repeatcontaining loci (see “Plus-stutter peaks at the D22S1045 locus” on page 87).
Arrows
Figure 17 Stutter percentages for the VeriFiler™ Plus kit FAM™ dye (blue) channel loci:
D3S1358, vWA, D16S539, CSF1PO, and D6S1043.
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Figure 18 Stutter percentages for the VeriFiler™ Plus kit VIC™ dye (green) channel loci:
D8S1179, D21S11, D18S51, and D5S818.
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6
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Figure 19 Stutter percentages for the VeriFiler™ Plus kit TED™ dye (yellow) loci:
D2S441, D19S433, FGA, and D10S1248.
Note: In the electropherogram plot, yellow is displayed as black.
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Figure 20 Stutter percentages for the VeriFiler™ Plus kit TAZ™ dye (red) loci:
D22S1045, D1S1656, D13S317, D7S820, and Penta E.
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Chapter 6 Experiments and results
Extra peaks in the electropherogram
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Figure 21 Stutter percentages for the VeriFiler™ Plus kit SID™ dye (purple) loci:
Penta D, TH01, D12S391, D2S1338, and TPOX.
th, as needed.
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Plus-stutter peaks at the D22S1045 locus
The D22S1045 STR locus in the VeriFiler™ Plus kit is a trinucleotide repeat locus, and
shows an elevated level of plus stutter (Figure 22). Other loci, such as FGA, may also
exhibit elevated plus stutter.
Figure 22 VeriFiler™ Plus kit electropherogram showing minus and plus stutter
associated with the D22S1045 STR locus. Data produced on a 3500xL Genetic
Analyzer.
STR loci such as D1S1656 (Figure 23) contain more complex nucleotide sequences
including regions of dinucleotide repeats which can yield additional stutter peaks. If
these stutter peaks exceed the peak amplitude threshold (typically 175 RFU), they
may be detected as additional alleles in the profile. The stutter file that is provided
with the GeneMapper™ ID‑X Software for analysis of VeriFiler™ Plus kit data contains
a minus 2−nt stutter filter for D1S1656, as well as filters for commonly observed plus
stutter, to prevent these peaks from being called in normal profiles.
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Figure 23 Example of non-standard stutter peaks in the D1S1656 locus. In addition to
the main allele peaks and standard (–4 nt) stutter peaks, –2 nt minus stutter peaks and
+4 nt plus stutter peaks can also be observed. Data produced on a 3500xL Genetic
Analyzer.
1
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Chapter 6 Experiments and results
Extra peaks in the electropherogram
Stutter percentage filter settings provided with GeneMapper™ ID‑X
Software
The settings in Table 8 were derived using the data that is shown earlier in this
section. The proportion of the stutter product relative to the main allele (stutter
percent) is measured by dividing the height of the stutter peak by the height of the
main allele peak.
Analysis showed that observed stutter data points were not normally distributed. As
such, at each locus a best-fit, non-parametric statistical model was applied to the
data and a threshold filter level that minimized the occurrence of non-filtered stutter
peaks while allowing maximum peak-detection sensitivity was determined.
IMPORTANT! The values that are shown in the table are the values that were
determined during developmental validation studies using specific data sets. To
determine the appropriate values to use for your applications, always perform internal
validation studies.
Table 8 Minus and plus stutter percentage filter settings provided with the
GeneMapper™ ID‑X Software
Note: Penta D and Penta E do not require plus stutter filters because their stutter levels are so low.
88
Locus [1]
Minus Stutter (%)
Plus Stutter (%)
D3S1358
12.61
3.74
vWA
11.86
4.46
D16S539
11.19
4.05
CSF1PO
11.00
5.42
D6S1043
12.05
4.58
D8S1179
11.54
4.85
D21S11
13.83
5.04
D18S51
13.73
5.96
D5S818
10.90
4.64
D2S441
9.71
3.71
D19S433
11.00
5.91
FGA
14.01
7.96
D10S1248
12.48
3.13
D22S1045
19.04
8.63
D1S1656
15.20
6.40
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Extra peaks in the electropherogram
6
Table 8 Minus and plus stutter percentage filter settings provided with the
GeneMapper ID‑X Software (continued)
[1]
Locus [1]
Minus Stutter (%)
Plus Stutter (%)
D13S317
11.00
6.25
D7S820
9.83
4.27
Penta E
7.99
—
Penta D
4.59
—
TH01
5.55
3.95
D12S391
15.61
6.10
D2S1338
16.69
8.00
TPOX
5.82
3.49
These percentages are used as stutter filters in VeriFiler_Plus_Stutter_v1.txt
Extra peaks: Addition of 3' A nucleotide
3′ A nucleotide addition definition
Many DNA polymerases can catalyze the addition of a single nucleotide
(predominantly adenosine) to the 3′ ends of double-stranded PCR products (Clark,
1988; Magnuson et al., 1996). This nontemplate addition results in a PCR product
that is one nucleotide longer than the actual target sequence. The PCR product with
the extra nucleotide is referred to as the “+A” form.
3′ A nucleotide addition observations
The efficiency of +A addition is related to the particular sequence of the DNA at the 3´
end of the PCR product.
The VeriFiler™ Plus kit includes two main design features that promote maximum +A
addition:
• The primer sequences have been optimized to encourage +A addition.
• The PCR chemistry allows complete +A addition with a final incubation at 60°C
for 5 minutes.
This final extension step gives the DNA polymerase additional time to complete
+A addition to all double-stranded PCR products.Figure 24 shows examples of
incomplete and normal +A addition. Final extension incubation for longer than the
recommended time can result in double +A addition, in which two nontemplate
adenosine residues are added to the PCR product. Double +A addition can cause
"shoulders" on the right side of main allele peaks.
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Extra peaks in the electropherogram
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Figure 24 The top panel shows normal vWA peaks that are fully adenylated, with no
visible shouldering and correct genotype calls. The bottom panel shows vWA peaks in
which adenylation was incomplete, resulting in minus-A shoulders that were called as
off-ladder (OL) peaks by the GeneMapper™ ID‑X Software.
If the amount of input DNA is greater than recommended concentration,
"shouldering" of allele peaks can be observed. Amplification of excess input DNA
can also result in off-scale data and lowered IQCL peak heights. In this situation, the
IQCL may also exhibit some shouldering due to incomplete +A nucleotide addition.
Other adverse conditions may also result in incomplete adenylation, for example:
PCR inhibition, expired PCR reagents, or not completing the final hold step during
thermal cycling.
Extra peaks: Artifacts
Artifact definition
Artifacts and anomalies are seen in all molecular biological systems. Artifacts are
typically reproducible. Anomalies are non-reproducible, intermittent occurrences that
are not observed consistently in a system (for example, spikes and baseline noise).
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Dye artifact observation
Because of improvements in PCR primer manufacturing processes, the incidence
of artifacts has been greatly reduced in the VeriFiler™ Plus kit. Internal population
studies show that kit electropherograms are free of reproducible dye artifacts in the
kit read region of 64–458 nt. Two exceptions are as follows:
• A low-level 113–117 nt dye artifact in the VIC™ dye channel that has been
detected.
• A low-level ~66 nt dye artifact in the TED™ dye channel. This artifact was
approximately 40–80 RFU in our studies. The peak height observed may vary
depending on the sensitivity of individual CE instruments.
Figure 25 shows the low baseline-level fluorescence that is observed in a typical
negative control PCR. However, it is important to consider noise and other
amplification-related artifacts when interpreting data.
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Figure 25 Examples of fluorescence background in data produced on a 3500xL
Genetic Analyzer (Y-axis scale 0–200 RFU) (updated formulation)
Note the two FAM™ dye-labeled IQC peaks (IQCS and IQCL) that should normally be present in
negative samples.
ength, as needed.
Some small PCR artifacts were occasionally observed in negative control reactions
prepared with the VeriFiler™ Plus kit reagents that were subjected to long-term
storage at elevated temperature as part of stability testing. The most prominent and
consistent artifacts that were sometimes observed were:
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• An artifact sizing at 120–130 bp in the FAM™ dye channel
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• An artifact sizing at 110–120 bp in the VIC™ dye channel
For examples of these dye artifacts, see Figure 26. Such artifacts may form
spontaneously upon long-term storage of PCR reagents, but their formation can be
minimized by following the recommended storage conditions.
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Chapter 6 Experiments and results
Characterization of loci
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Figure 26 Examples of FAM™ dye and VIC™ dye artifacts in negative (NTC) reactions
with VeriFiler™ Plus kit reagents that were subjected to long-term storage at elevated
temperature
In this example, both artifact peaks are below 100 RFU.
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Characterization of loci
SWGDAM guideline 3.1
“The basic characteristics of a genetic marker should be determined and
documented.” (SWGDAM, December 2016)
Loci in this kit
This section describes basic characteristics of the 23 autosomal STR loci, Y indel
locus, and sex-determining marker (Amelogenin), that are amplified with the VeriFiler™
Plus kit. Most of these loci have been extensively characterized by other laboratories.
Nature of polymorphisms
The primers for the Amelogenin locus flank a 6-nucleotide deletion in intron 1 of
the X homolog. Amplification generates 102‑nt and 108‑nt products from the X
and Y chromosomes, respectively. The primers for the Y indel flank a region in
the q arm of the Y chromosome (Yq11.221). Depending on the haplotype of the
sample, the amplification generates either a 81‑nt or a 87‑nt product. (Sizes are
the actual nucleotide size according to sequencing results, including 3´ A nucleotide
addition, and size may not correspond exactly to allele mobility observed on capillary
electrophoresis platforms.) Most of the STR loci present in the VeriFiler™ Plus kit
contain tetranucleotide repeat units. However, the kit also contains one locus with
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Chapter 6 Experiments and results
Characterization of loci
6
trinucleotide repeats (D22S1045) and two loci with pentanucleotide repeats (Penta D
and Penta E). The length differences among alleles of a particular locus are caused
by differences in the number of repeat units.
We have sequenced all the alleles in the VeriFiler™ Plus Allelic Ladder, including
microvariants. In addition, other groups in the scientific community have sequenced
alleles at some of these loci (Nakahori et al., 1991; Puers et al., 1993; Möller et al.,
1994; Barber et al., 1995; Möller and Brinkmann, 1995; Barber et al., 1996; Barber
and Parkin, 1996; Brinkmann et al., 1998; Momhinweg et al., 1998; Watson et al.,
1998). Among the various sources of sequence data on the loci, there is consensus
on the repeat patterns and structure of the STRs.
Inheritance
The Centre d'Etude du Polymorphisme Humain (CEPH) has collected DNA from
families of Utah Mormon, French Venezuelan, and Amish descent. These DNA
sets have been extensively studied all over the world and are routinely used to
characterize the mode of inheritance of various DNA loci. Each family set contains
three generations, generally including four grandparents, two parents, and several
offspring. Consequently, the CEPH family DNA sets are ideal for studying inheritance
patterns (Begovich et al., 1992).
Mapping
The loci present in the VeriFiler™ Plus kit have been mapped, and the chromosomal
locations have been published (Nakahori et al., 1991; Edwards et al., 1992; Kimpton
et al., 1992; Mills et al., 1992; Sharma and Litt, 1992; Li et al., 1993; Straub et al.,
1993; Barber and Parkin, 1996; and Lareu, et al., 1996).
Genetic linkage
As shown in Table 9, four sets of STR loci in the VeriFiler™ Plus kit are located on
the same chromosomes. Of these, the most closely spaced are vWA and D12S391,
which are located 6.3 million bp apart on the p arm of chromosome 12. Linkage
disequilibrium analysis was conducted on vWA and D12S391 genotype results from
1,034 individuals of three ethnic groups (350 African American, 349 Caucasian, and
335 Hispanic) using the Linkage Disequilibrium module of GenePop software version
4.0.10 (Raymond and Rousset, 1995; Rousset, 2008).
The results of this analysis (not shown) indicated that there is no statistically
significant linkage disequilibrium found between vWA and D12S391.
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Chapter 6 Experiments and results
Species specificity
However, an inheritance analysis of the CEPH pedigree families demonstrated a
degree of linkage between vWA and D12S391 that does not support the assumption
of independence for kinship analysis (Budowle, et al., 2010).
Table 9 Four sets of STR loci in the VeriFiler™ Plus kit that are located on the
same chromosomes
Locus
Chromosome location
Map
Mb units
D5S818
5q23.2
123.139
CSF
5q33.1
149.436
vWA
12p13.31
5.963
D12S391
12p13.2
12.341
D21S11
21q21.1
19.476
Penta D
21q22.3
43.88
TPOX
2p25.3
1.472
D2S441
2p14
68.213
D2S441
2p14
68.213
D2S1338
2q35
218.705
Distance apart (Mb)
26.297
6.378
24.404
66.741
150.492
Species specificity
SWGDAM Guideline 3.2
“The ability to detect genetic information from non-targeted species (e.g., detection
of microbial DNA in a human assay) should be determined. The detection of genetic
information from non-targeted species does not necessarily invalidate the use of the
assay, but may help define the limits of the assay.” (SWGDAM, December 2016)
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Species specificity
6
Nonhuman studies
The VeriFiler™ Plus kit provides the required specificity for detecting human alleles.
Species specificity testing was performed to confirm that there is minimal crossreactivity with nonhuman DNA that may be present in forensic casework samples,
and to document the rarely observed examples of cross-species detection.
The following species were tested (in the specified amounts) using standard PCR and
capillary electrophoresis conditions for the VeriFiler™ Plus kit kit:
• Primates: chimpanzee, orangutan, and macaque (0.5 ng each)
• Non-primates: mouse, dog, sheep, pig, rabbit, cat, horse, rat, and cow (5.0 ng
each)
• Microorganisms: Streptococcus salivarius, Neisseria gonorrhea, Bacillus
subtilis, Candida albicans, Lactobacillus delbrueckii, Escherichia coli, and
Staphylococcus aureus (pooled genomic DNAs, with approximately 100,000
copies of DNA from each species, per reaction)
Results were evaluated for the presence of any amplified peaks that would indicate
cross reactivity of the VeriFiler™ Plus kit with any of these non-human species.
Figure 27 shows example electropherogram results from the species specificity tests.
Primate species (chimpanzee, macaque) produced more extensive cross-reactivity
than non-primate species, and for a closely related species like chimpanzee, many
peaks fell into allele bins. A few of the more distantly related mammals (bovine, dog,
horse) produced peaks over the 175 RFU threshold, all of which genotyped as "OL"
because they fell outside of allele bins. None of the remaining species that were
tested gave peaks over the detection threshold.
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Chapter 6 Experiments and results
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Species specificity
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Figure 27 Representative electropherograms for some species tested in a species
specificity study. Data produced on a 3500xL Genetic Analyzer (updated formulation)
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Note: The individual panes are at different magnifications (zoom function) on the Y-axis.
Table 10 shows the most significant cross-reactive peaks that were observed among
non-human, non-primate, genomic DNAs (that is, peaks over a 175 RFU Peak
Amplitude Threshold on the 3500xL Genetic Analyzer), with the original and updated
formulations of the VeriFiler™ Plus kit. Peaks were observed for dog, horse, hamster,
bovine, and sheep. No peaks fell into human STR locus bins, and would therefore not
be confused with human STR alleles. This data shows that the likelihood of obtaining
an allelic profile consistent with that from a human sample, from non-primates or
microorganisms, is extremely low.
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Table 10 Observed cross-reactive peaks for non-human, non-primate animals for the original and updated
formulations of the VeriFiler™ Plus kit
Species
Dog
96
Dye channel
Size (bp)
VeriFiler™ Plus kit—Original
formulation
Average peak
height (RFU)
Genotype call
Blue
341
214
D6 OL
Red
307
692
Penta E OL
Horse
Green
98
630
Hamster
Green
130
243
VeriFiler™ Plus kit—Updated
formulation
Average peak
height (RFU)
Genotype call
—
—
716
Penta E OL
Out of Marker 828
Range (OMR)
D8 OL
—
OMR
—
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Sensitivity
6
Table 10 Observed cross-reactive peaks for non-human, non-primate animals for the original and updated
formulations of the VeriFiler Plus kit (continued)
Species
Dye channel
Size (bp)
VeriFiler™ Plus kit—Original
formulation
Average peak
height (RFU)
Genotype call
VeriFiler™ Plus kit—Updated
formulation
Average peak
height (RFU)
Genotype call
Bovine
Red
221
399
D13 OL
489
D13 OL
Sheep
Purple
211
345
TH01 OL
301
TH01 OL
Sensitivity
SWGDAM guideline 3.3
“The ability to obtain reliable results from a range of DNA quantities, to include the
upper and lower limits of the assay, should be evaluated.” (SWGDAM, December
2016)
Sensitivity observation
The recommended amount of input DNA for the VeriFiler™ Plus kit is 0.5 ng for
29 cycles of amplification based on real-time PCR quantification, such as with the
Quantifiler™ Trio DNA Quantification Kit or the Quantifiler™ HP DNA Quantification Kit.
To determine the optimum input DNA, perform studies according to the quantification
kit that you use.
If the sample contains degraded DNA, amplification of a higher amount of DNA may
be beneficial. In Figure 28, DNA Control 007 was serially diluted from 1.0–0.016 ng.
Full profiles (46 human alleles) were consistently obtained at 0.125 ng, but occasional
partial profiles resulted at lower concentrations.
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Stability
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Figure 28 Electropherograms for amplifications using 1,000, 500, 250, 125, 63, 31,
and 16 pg of DNA Control 007 (updated formulation)
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Electrophoresis was performed on a 3500xL Genetic Analyzer. Note that because the DNA input is
serially diluted by 2-fold, the Y-axis scale is also magnified by 2-fold for the smaller input amounts of
DNA.
Stability
SWGDAM guideline 3.4
“The ability to obtain results from DNA recovered from biological samples deposited
on various substrates and subjected to various environmental and chemical insults
should be evaluated. In most instances, assessment of the effects of these factors
on new forensic DNA procedures is not required. However, if substrates and/or
environmental and/or chemical insults could potentially affect the analytical process,
then the process should be evaluated to determine the effects of such factors.”
(SWGDAM, December 2016)
Degraded DNA
As the average size of degraded DNA approaches the size of the target sequence,
the amount of PCR product generated is reduced, because of the reduced number
of intact templates in the size range necessary for amplification. Degraded DNA was
prepared to examine the potential for differential amplification of loci. High-molecular-
98
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Stability
6
weight DNA was sonicated and incubated with increasing doses of DNase I (Bender
et al., 2004). The DNA was examined by agarose gel analysis to determine the
average size of the DNA fragments at each time point. Amplification of 0.5 ng of
degraded DNA using the VeriFiler™ Plus kit was performed. As the DNA became
progressively degraded, the loci failed to amplify robustly in order of decreasing size.
Preferential amplification was not observed.
s and Arrows
r arrow to use in this SVG.
e advanced callouts or arrows
kscape_Callout&Arrow_Libary.
Figure 29 Amplification of a single donor DNA sample sonicated and incubated with
increasing doses of DNase I (updated formulation)
e-length, as needed.
The panels show the control (undegraded), low-, medium-, and high-degraded samples. Note that
the Y-axis scale is magnified for more degraded samples, which generate lower peak heights.
Effect of inhibitors
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1
Because of the substrates and environments from which they are collected, forensic
DNA samples may be contaminated with substances that inhibit PCR amplification.
The PCR chemistry for the VeriFiler™ Plus kit was optimized to be as robust
as possible in the presence of such inhibitors. Model inhibitors tested during
developmental validation were: hematin, a representative compound often extracted
from blood stains (DeFranchis et al., 1988; Alkane et al., 1994); humic acid, a
simulant for soil-based inhibition; and tannic acid, a compound often co-extracted
from leather sample substrates. 0.5 ng of DNA Control 007 was amplified in the
presence of increasing concentrations of each model inhibitor. All samples were then
amplified using the standard 29-cycle "touchdown" thermal cycling program.
Figure 30 and Figure 31 show representative electropherograms from the PCR
inhibitor studies with humic acid, hematin, and tannic acid. Table 11 shows inhibitor
final concentrations in 25‑µL PCR reactions and average allele counts obtained in
replicate reactions with the VeriFiler™ Plus kit.
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Chapter 6 Experiments and results
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Stability
Arrows
to use in this SVG.
™
ced callouts or arrows
_Callout&Arrow_Libary.
, as needed.
and unused
elements
repository.
Figure 30 Electropherograms show the results of testing the VeriFiler Plus kit with
the humic acid PCR inhibitor (updated formulation)
The top panel is the experimental control with 0.5 ng (total) of DNA Control 007 without humic acid.
The remaining panels show 0.5 ng of DNA Control 007 with increasing levels of humic acid (150,
250, and 350 ng/µL). Note the behavior of the IQCS and IQCL peaks in response to increasing levels
of inhibition, with the IQCL peak decreasing in peak height and eventually disappearing as the humic
acid concentration increases.
Note: The individual panes are at different magnifications (zoom function) on the Y-axis.
1
1
100
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ne-length, as needed.
Each sample contained 0.5 ng (total) of human male DNA Control 007 with varying levels of hematin
or tannic acid. Note that the IQCL peak was significantly reduced or absent while the IQCS peak still
remained in the inhibitor-containing samples.
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Figure 31 Example electropherograms show the results of testing hematin and tannic
acid PCR inhibitors (updated formulation)
Note: The individual panes are at different magnifications (zoom function) on the Y-axis.
Table 11 VeriFiler™ Plus kit performance with PCR inhibitors (N = 4) (updated
formulation)
1
1
Sample
Inhibitor concentration
Mean allele count
0
46
Hematin low
400 µM
46
Hematin medium
500 µM
46
Hematin high
600 µM
8.75
Humic acid low
150 ng/µL
46
Humic acid medium
250 ng/µL
46
Humic acid high
350 ng/µL
42.5
Tannic acid low
250 ng/µL
46
Tannic acid medium
350 ng/µL
46
Tannic acid high
450 ng/µL
46
Control (no inhibitor)
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Chapter 6 Experiments and results
Mixture studies
Mixture studies
SWGDAM guideline 3.8
“The ability to obtain reliable results from mixed source samples should be
determined.” (SWGDAM, December 2016)
Mixture study overview
Evidence samples may contain DNA from more than one individual. The possibility of
multiple contributors should be considered when interpreting the results. Perform
studies to determine a minimum peak height threshold to avoid typing when
stochastic effects are likely to interfere with accurate interpretation of mixtures.
Evidence samples that contain body fluids and/or tissues originating from more than
one individual are an important category of forensic casework.
It is essential to ensure that the DNA typing system is able to detect DNA mixtures.
Typically, mixed samples can be distinguished from single-source samples by:
• The presence of more than two alleles at one or more loci
• The presence of a peak at a stutter position that is significantly greater in
percentage than typically observed in a single-source sample
• Significantly imbalanced alleles for a heterozygous genotype
The peak height ratio is defined as the height of the lower peak (in RFU) divided by
the height of the higher peak (in RFU), expressed as a percentage.
If an unusually low peak height ratio is observed for one locus, and there are no
other indications that the sample is a mixture, reamplify and reanalyze the sample to
determine if the imbalance is reproducible. Possible causes of imbalance at a locus
are:
• Degraded DNA
• Presence of inhibitors
• Extremely low amounts of input DNA
• A mutation in one of the primer binding sites
• Presence of an allele containing a rare sequence that does not amplify as
efficiently as the other allele
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Mixture studies
Mixture study observation
Median, minimum, and maximum peak height ratios observed for alleles in the
VeriFiler™ Plus kit loci in single-source human population database samples are
shown in Figure 32. The population samples that are used are listed in “Population
samples used in these studies” on page 108.
allouts and Arrows
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eed more advanced callouts or arrows
omm_Inkscape_Callout&Arrow_Libary.
Figure 32 Heterozygote ratios for ~0.5 ng of input DNA. Intra-locus peak height ratio
is calculated per individual profile by dividing the lower peak by the higher peak in
each heterozygote allele pair, per marker. Box plot boxes and data points are colored
according to the dye channel. Boxes show the middle 50% or interquartile range (IQR).
Box halves below and above median show the second and third quartile, respectively.
"Whiskers" indicate 1.5 IQR from the upper and lower margins of the IQR. Black dots
are outlier data points more than 1.5 IQR from the median.
nd/or line-length, as needed.
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Resolution
of genotypes in mixed samples
1
1
A sample containing DNA from two sources can comprise (at a single locus) any of
the seven genotype combinations (see below).
• Heterozygote + heterozygote, no overlapping alleles (four peaks)
• Heterozygote + heterozygote, one overlapping allele (three peaks)
• Heterozygote + heterozygote, two overlapping alleles (two peaks)
• Heterozygote + homozygote, no overlapping alleles (three peaks)
• Heterozygote + homozygote, overlapping allele (two peaks)
• Homozygote + homozygote, no overlapping alleles (two peaks)
• Homozygote + homozygote, overlapping allele (one peak)
Specific genotype combinations and input DNA ratios of the samples contained in a
mixture determine whether or not it is possible to resolve the genotypes of the major
and minor components at a single locus.
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Chapter 6 Experiments and results
Mixture studies
The ability to obtain and compare quantitative values for the different allele peak
heights on Applied Biosystems™ instruments provides additional valuable data to aid
in resolving mixed genotypes.
Ultimately, the likelihood that any sample is a mixture must be determined by the
analyst in the context of each particular case, including the information provided from
known reference samples.
Note: Peak detection is a complex process that involves the STR chemistry,
capillary electrophoresis conditions, and the data analysis software. Contact HID
Support for a Technical Note with additional information on detecting peaks in
electropherograms.
Limit of detection of the minor component
Mixtures of two DNA samples were examined at various ratios (0:1, 1:1, 1:3, 1:7,
1:15, 1:30, 30:1, 15:1, 7:1, 3:1 and 1:0). The total amount of genomic input DNA
mixed at each ratio was 0.5 ng. The samples were amplified in a ProFlex™ 96‑well
PCR System, then electrophoresed and detected using an 3500xL Genetic Analyzer.
The results of the mixed DNA samples are shown in Figure 33. The two human
genomic DNAs, male 007 and female 9947a, were mixed according to the ratios
indicated. The minor component allele calls at non-overlapping loci are highlighted.
Detection of full profiles for the minor contributor was possible at ratios of 7:1
(0.438 ng of 007 and 0.063 ng of 9947a) and 1:7 (0.063 ng of 007 and 0.438 ng
of 9947a), with 3/3 replicates of both samples giving full profiles of the respective
minor contributor. 15:1, 30:1, 1:30, and 1:15 ratios resulted in partial profiles for the
minor component (Table 13). The genotypes of each contributor DNA are shown in
Table 12.
Table 13 shows mixture sample compositions and mean STR allele counts from
VeriFiler™ Plus kit assays.
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6
and Arrows
row to use in this SVG.
Figure 33 Amplification of DNA mixtures at various ratios (updated formulation)
dvanced callouts or arrows
cape_Callout&Arrow_Libary.
Panels show electropherograms for 1:0 (male 007 DNA only), 1:7 mixture, 1:1 mixture, 7:1 mixture,
and 0:1 (female 9957a DNA only).
Table 12 Genotypes of mixture sample contributors (updated formulation)
ngth, as needed.
Asterisks denote alleles that are unique for each individual, and also apart from plus- or minusstutter positions of other alleles present in the individuals.
ngle, and unused
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the repository.
Marker
1
1
Male 007 genotype
Female 9947a genotype
Allele 1
Allele 2
Allele 1
Allele 2
D3S1358
15
16*
14
15
vWA
14*
16
17*
18*
D16S539
9*
10
11*
12*
CSF1PO
11
12
10
12
TPOX
8
—
8
—
Yindel
2*
—
—
—
AMEL
X
Y*
X
—
D8S1179
12
13
13
—
D21S11
28*
31*
30
—
D18S51
12*
15
15
19*
D2S441
14
15*
10*
14
D19S433
14
15
14
15
TH01
7
9.3
8*
9.3
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Chapter 6 Experiments and results
Mixture studies
Table 12 Genotypes of mixture sample contributors (updated
formulation) (continued)
Marker
Male 007 genotype
Female 9947a genotype
Allele 1
Allele 2
Allele 1
Allele 2
FGA
24
26*
23
24
D22S1045
11
16*
11
14*
D5S818
11
—
11
—
D13S317
11
—
11
—
D7S820
7*
12*
10*
11
D10S1248
12
15
13*
15
D1S1656
13*
16*
18.3*
—
D12S391
18
19
18
20*
D2S1338
20*
23
19
23
D6S1043
12
14*
12
18*
Penta E
7*
12
12
13*
Penta D
11
12
12
—
Table 13 Mixture sample compositions and mean STR allele counts from
VeriFiler™ Plus kit assays (updated formulation)
106
DNA amounts (ng/reaction)
Mean allele count[1]
Mixture
sample
Ratio (A:B)
Male 007
("A")
Female
9947a ("B")
Male 007
("A")
Female
9947a ("B")
A
1:0
0.500
0.000
18.00
0.00
B
1:1
0.250
0.250
18.00
14.00
C
1:3
0.125
0.375
18.00
14.00
D
1:7
0.063
0.438
18.00
14.00
E
1:15
0.031
0.469
15.33
14.00
F
1:30
0.016
0.484
12.67
14.00
G
30:1
0.484
0.016
18.00
7.33
H
15:1
0.469
0.031
18.00
10.67
I
7:1
0.438
0.063
18.00
14.00
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Population data
Table 13 Mixture sample compositions and mean STR allele counts from VeriFiler
Plus kit assays (updated formulation) (continued)
[1]
DNA amounts (ng/reaction)
Mean allele count[1]
Mixture
sample
Ratio (A:B)
Male 007
("A")
Female
9947a ("B")
Male 007
("A")
Female
9947a ("B")
J
3:1
0.375
0.125
18.00
13.67
K
0:1
0.000
0.500
0.00
14.00
Mixture sample compositions and STR allele counts from VeriFiler™ Plus kit assays. The average number of
unique, distinct alleles (that is, alleles neither present in the other contributor genotype nor located in a stutter
position) obtained in 3 replicate reactions per mixture sample. The full profiles of unique alleles in 007 and
9947a DNAs were 18 and 14, respectively.
Population data
SWGDAM guideline 3.7
“The distribution of genetic markers in populations should be determined in relevant
population groups.” (SWGDAM, December 2016)
Population data overview
To interpret the significance of a match between genetically typed samples, you must
know the population distribution of alleles at each locus in question. If the genotype
of the relevant evidence sample is:
• Different from the genotype of the reference sample for a suspect, then the
suspect is excluded as the donor of the biological evidence that was tested.
An exclusion is independent of the frequency of the two genotypes in the
population.
• The same as the genotype of the reference sample for a suspect, then the
suspect is included as a possible source of the evidence sample.
The probability that another, unrelated individual would also match the evidence
sample is estimated by the frequency of that genotype in the relevant populations.
Loci in the kit
The VeriFiler™ Plus PCR Amplification Kit was designed and optimized primarily for
the analysis of forensic casework samples. It was designed as a companion kit to
the VeriFiler™ Express PCR Amplification Kit (Cat. No. A32014) and the Huaxia™
Platinum™ PCR Amplification Kit (Cat. No. A31323), and contains the same set of 25
loci.
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Chapter 6 Experiments and results
Population data
Population samples used in these studies
The VeriFiler™ Plus kit has high genotypic concordance (>99.5%) to its databasing
counterparts, the VeriFiler™ Express kit and Huaxia™ Platinum™ kit (all three kits
contain the same 25 loci). An initial population study of individuals of different
ethnic populations within the United States and China was conducted with
2,475 individuals. This initial study was the basis for the allele frequency tables and
associated random match probability tables at the end of this section.
After the minor redesign of certain primers in the VeriFiler™ Plus kit multiplex to
reduce the incidence of artifact peaks in samples with high levels of bacterial DNA, a
new population study was performed to check:
• Concordance between the original and redesigned VeriFiler™ Plus kits
• Concordance with the GlobalFiler™ kit reference genotypes
As with the original population study, the study with the updated formulation included
in-house testing of domestic United States individuals as well as population samples
collected and tested by laboratories in China. See Table 14.
Table 14 Population samples (updated formulation)
Sample source
(population)
In-house testing
of domestic
United States
individuals
Chinese
laboratories
108
Total
sample
s
1,308
987
Sample type
No. of samples by ethnic
origin
• Extracted DNAs from
1,108 blood samples
• African-American—285
• 200 samples from
blood cards that
were tested in direct
amplification mode
• Caucasian—322
• Asian—316
• Hispanic—385
Blood cards that were
Individuals of Chinese origin
tested in direct amplification —987
mode
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Chapter 6 Experiments and results
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Population data
Concordance studies
Concordance between the original VeriFiler™ Plus kit and the redesigned kit was
100% for 2,288 samples tested in-house and externally. When genotype results were
compared between the VeriFiler™ Plus kit and the GlobalFiler™ kit, the following level
of concordance was observed:
Sample source
(population)
In-house testing of
domestic United
States individuals
Sample type
Level of discordance
Extracted DNAs from
1,108 blood samples
1,099/1,108 (discordances
observed in D10S1248 and
D3S1358)
200 samples from blood cards
that were tested in direct
amplification mode
200/200 (0 discordances
observed)
Chinese laboratories Blood cards that were tested in
direct amplification mode
986/987 (1 discordance
observed in D5S818)
Among the in-house extracted samples, several instances of discordance were
observed that were caused by a 1‑bp insertion in the flanking region of D10S1248.
Affected individuals genotyped as off-ladder (OL). The same individuals tested with
the GlobalFiler™ kitdid not give the OL genotypes. All individuals that contained
the 1‑bp insertion were of African descent, and subsequent database searches
confirmed that this indel is found almost exclusively in individuals of African ancestry.
D10S2148 in the VeriFiler™ Plus kit
The VeriFiler™ Express kit and Huaxia™ Platinum™ kit were designed for database
workflows that involve pristine single-source samples. The VeriFiler™ Plus kit was
designed for casework workflows that involve challenging samples. Although the
three kits are complementary, the VeriFiler™ Plus kit was modified to optimize
casework performance and discrimination power with degraded samples. In the
VeriFiler™ Plus kit, more discriminating markers (such as the Penta D marker)
were given priority for amplification and located in the smaller molecular weight
regions, while the D10S1248 marker, because of its lower discrimination power, was
positioned in the larger molecular weight region.
To move the D10S1248 marker from a smaller base pair size in the SID™ dye channel
to the larger base pair size in the TED™ dye channel, the flanking region that borders
the primer binding and STR sites was extended. By sequencing additional base
pairs in the flanking region of the D10S1248 marker, the opportunity to observe rare,
sample-specific mutations increases.
In the case of six African-American (out of 330) and one Hispanic (out of 393)
population samples, an insertion in the flanking region for the D10S1248 marker
caused a 1‑bp difference when comparing results from the VeriFiler™ Plus kit to
the VeriFiler™ Express kit, Huaxia™ Platinum™ kit, and GlobalFiler™ kit (Figure 34).
This was seen with alleles 13, 14, and 15 reported as 13.1, 14.1, and 15.1. The
discrepancy has not been observed in any other population samples tested (1,335
VeriFiler™ Plus PCR Amplification Kit User Guide
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6
Chapter 6 Experiments and results
Population data
total combined Caucasian and Asian samples). Subsequent sequencing of the
sample showed that it is consistent with African lineage. This D10S1248 insertion
is observed in the original and updated formulations of the VeriFiler™ Plus kit.
Figure 34 D10S1248 marker from an African American population sample amplified
™
with the VeriFiler Plus kit
ts and Arrows
or arrow to use in this SVG.
ore advanced callouts or arrows
Inkscape_Callout&Arrow_Libary.
Supporting data
ne-length, as needed.
Additional studies were performed at the National Institute of Standards and
Technology (NIST, C.R. Steffan). Those studies confirmed the above conclusions,
showing the 1‑bp difference in nine out of 355 African-American samples.
ectangle, and unused
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1
The off-ladder (OL) peak sizes at the 14.1 base pair position because of a single base pair insertion
in the flanking region between the primer and STR region. When amplified with the VeriFiler™
Express kit, Huaxia™ Platinum™ kit, and GlobalFiler™ kit, the genotype at this marker would be 14,
15.
For expanded frequency analysis of this insertion, we examined the sequencing data
for the specific insertion at the D10S1248 marker from the following organizations:
1
1
• Broad Institute (https://gnomad.broadinstitute.org/variant/10-131092326-TTA?dataset=gnomad_r2_1)
• 1,000 Genomes Project (https://www.internationalgenome.org/about)
• NCBI (https://www.ncbi.nlm.nih.gov/snp/rs567190580#frequency_tab)
Out of 5,044 total African-related samples, there were 80 observations. Out of
771 total Hispanic-related samples, the insertion was not observed.
110
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Chapter 6 Experiments and results
6
Population data
Next steps and considerations
The potential to observe discrepant genotypes among STR PCR kits that use
different primer sequences is a known phenomenon in the forensic community.
Depending on workflow, interpretation protocols, and database search parameters,
each laboratory may take a different approach to handling discrepancies across STR
PCR kits.
If your laboratory has an established protocol for handling these discrepancies, we
recommend that you follow that protocol for this occurrence. If your laboratory does
not have an established protocol, we recommend that you consider different analysis
and database search approaches, some of which include:
• Evaluate previously generated VeriFiler™ Plus kit files that contain
0.1 microvariant calls at the D10S1248 marker.
• Confirm the genotype through amplification with a secondary STR PCR kit, such
as the GlobalFiler™ kit.
• Exclude the D10S1248 marker from upload to a database if the 0.1 microvariant
is observed.
• Perform database searches that include one mismatch when a database is
composed of profiles from different STR PCR kits.
Probability of Identity definition
The PI value is the probability that two individuals selected at random will have an
identical genotype (Sensabaugh, 1982).
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Chapter 6 Experiments and results
Population data
Probability of identity
Figure 35 through Figure 39 show the autosomal STR allele frequencies at VeriFiler™ Plus kit loci by population
group.
Note: These tables show observed allele frequencies among different population groups. To ensure a
conservative approach when performing statistical calculations such as profile frequency estimates, minimum
allele frequencies should be used for very rare alleles that may be insufficiently sampled in a population. For
autosomal markers, minimum allele frequencies are typically calculated as 5/2N, where N is the number of
individuals sampled in a given population (Butler, 2010).
Table 15 shows the Random Match Probability (RMP) values of the VeriFiler™ Plus kit loci individually and
combined.
112
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Population data
Chapter 6 Experiments and results
Figure 35 Combined China populations (minimum allele frequency = 0.0010)
6
113
6
114
Population data
Chapter 6 Experiments and results
VeriFiler™ Plus PCR Amplification Kit User Guide
Combined China populations (continued)
VeriFiler™ Plus PCR Amplification Kit User Guide
Population data
Chapter 6 Experiments and results
Combined China populations (continued)
6
115
6
116
Population data
Chapter 6 Experiments and results
VeriFiler™ Plus PCR Amplification Kit User Guide
Figure 36 United States—African-American population (minimum allele frequency = 0.0082)
VeriFiler™ Plus PCR Amplification Kit User Guide
Population data
Chapter 6 Experiments and results
United States—African-American population (continued)
6
117
6
118
Population data
Chapter 6 Experiments and results
United States—African-American population (continued)
VeriFiler™ Plus PCR Amplification Kit User Guide
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Population data
Chapter 6 Experiments and results
Figure 37 United States—Asian-American population (minimum allele frequency = 0.0066)
6
119
6
120
Population data
Chapter 6 Experiments and results
VeriFiler™ Plus PCR Amplification Kit User Guide
United States—Asian-American population (continued)
VeriFiler™ Plus PCR Amplification Kit User Guide
United States—Asian-American population (continued)
Population data
Chapter 6 Experiments and results
6
121
6
122
Population data
Chapter 6 Experiments and results
VeriFiler™ Plus PCR Amplification Kit User Guide
Figure 38 United States—Caucasian population (minimum allele frequency = 0.0074)
VeriFiler™ Plus PCR Amplification Kit User Guide
Population data
Chapter 6 Experiments and results
United States—Caucasian population (continued)
6
123
6
124
Population data
Chapter 6 Experiments and results
United States—Caucasian population (continued)
VeriFiler™ Plus PCR Amplification Kit User Guide
VeriFiler™ Plus PCR Amplification Kit User Guide
Population data
Chapter 6 Experiments and results
Figure 39 United States—Hispanic population (minimum allele frequency = 0.0065)
6
125
6
126
Population data
Chapter 6 Experiments and results
VeriFiler™ Plus PCR Amplification Kit User Guide
United States—Hispanic population (continued)
VeriFiler™ Plus PCR Amplification Kit User Guide
United States—Hispanic population (continued)
Table 15 Random Match Probability (RMP) values for VeriFiler™ Plus kit markers
Caucasian
Asian-American
Hispanic
CSF1PO
0.1168
0.0792
0.1289
0.1221
0.1289
D10S1248
0.0938
0.0672
0.0931
0.1007
0.0931
D12S391
0.0427
0.0362
0.0198
0.0408
0.0198
D13S317
0.0643
0.1363
0.0735
0.0675
0.0735
D16S539
0.0799
0.0727
0.0981
0.0836
0.0981
D18S51
0.0343
0.0300
0.0289
0.0366
0.0289
D19S433
0.0526
0.0361
0.0895
0.0549
0.0895
D1S1656
0.0440
0.0315
0.0195
0.0490
0.0195
D21S11
0.0509
0.0419
0.0466
0.0508
0.0466
D22S1045
0.0883
0.0504
0.1367
0.0844
0.1367
D2S1338
0.0318
0.0198
0.0279
0.0321
0.0279
D2S441
0.0858
0.0996
0.0976
0.0897
0.0976
6
African-American
Population data
Combined China
Chapter 6 Experiments and results
127
Allele
6
128
Table 15 Random Match Probability (RMP) values for VeriFiler Plus kit markers (continued)
African-American
Caucasian
Asian-American
Hispanic
D3S1358
0.1262
0.1033
0.0757
0.1222
0.0757
D5S818
0.0870
0.0952
0.1290
0.0907
0.1290
D6S1043
0.0287
0.0248
0.0584
0.0313
0.0584
D7S820
0.0799
0.0747
0.0678
0.0905
0.0678
D8S1179
0.0449
0.0700
0.0604
0.0462
0.0604
FGA
0.0345
0.0301
0.0370
0.0393
0.0370
Penta D
0.0594
0.0237
0.0530
0.0577
0.0530
Penta E
0.0115
0.0188
0.0199
0.0121
0.0199
TH01
0.1490
0.0885
0.0811
0.1641
0.0811
TPOX
0.2129
0.0860
0.1785
0.2141
0.1785
vWA
0.0674
0.0602
0.0624
0.0641
0.0624
Cumulative
1.34 x 10-28
2.13 x 10-30
1.24 x 10-28
3.09 x 10- 28
1.24 x 10-28
Population data
Combined China
Chapter 6 Experiments and results
Allele
VeriFiler™ Plus PCR Amplification Kit User Guide
Chapter 6 Experiments and results
Population data
6
Probability of paternity exclusion observation
If your laboratory requires the probability of paternity exclusion calculation, contact
your Thermo Fisher Scientific HID representative.
VeriFiler™ Plus PCR Amplification Kit User Guide
129
A
Troubleshooting
Observation
Faint or no signal from both the
DNA Control 007 and the DNA
test samples at all loci, including
the IQC markers
130
Possible cause
Recommended action
The incorrect volume of Master
Mix or Primer Set was used.
Use the correct volume of Master Mix or Primer
Set.
The DNA Polymerase was not
activated.
Repeat the amplification with an initial hold at
95°C for 1 minute.
The Master Mix was not
vortexed thoroughly before
aliquoting.
Vortex the Master Mix thoroughly.
The Primer Set was exposed to
too much light.
Replace the Primer Set and store it protected
from light.
Evaporation.
Ensure that the plate is properly sealed with
film and that a compression pad was used
with the GeneAmp™ PCR System 9700. (Do not
use a compression pad with the other validated
thermal cyclers.)
The thermal cycler
malfunctioned.
See the thermal cycler user manual and check
the instrument calibration.
Incorrect thermal cycler
conditions were used.
Use correct thermal cycler conditions.
A MicroAmp™ base was used
with a tray/retainer set and tubes
in GeneAmp™ PCR System
9700.
Remove the MicroAmp™ base.
The tubes or plate were not
seated tightly in the thermal
cycler during amplification.
Push the tubes or plate firmly into the block after
first cycle.
The wrong PCR reaction tubes
or plate were used.
Use MicroAmp™ Reaction Tubes with Caps or
the MicroAmp™ Optical 96‑well Reaction Plate
for the GeneAmp™ PCR System 9700 or Veriti™
Thermal Cycler.
Insufficient PCR product was
electrokinetically injected.
Use correct genetic analyzer settings.
Degraded formamide was used.
Check the storage of formamide. Do not
thaw and refreeze multiple times. Try Hi‑Di™
Formamide.
VeriFiler™ Plus PCR Amplification Kit User Guide
Appendix A Troubleshooting
A
Population data
Observation
Possible cause
Recommended action
Positive signal from DNA Control
007 but partial or no signal from
DNA test samples when IQC
peaks are present and balanced
The quantity of test DNA sample
is below the assay sensitivity.
Quantify DNA and (when possible) add 500 pg
of DNA. For low concentration samples, add up
to 17.5 µL of the DNA sample to the reaction
mix.
The test sample DNA is severely
degraded.
Use the Quantifiler™ HP DNA Quantification Kit
or the Quantifiler™ Trio DNA Quantification Kit to
evaluate sample quality during the quantification
step. If DNA is degraded, reamplify with an
increased amount of DNA or consider using the
Precision ID GlobalFiler™ NGS STR Panel.
The test sample was diluted in
the wrong buffer (for example, a
TE buffer with an incorrect EDTA
concentration).
Redilute DNA using nuclease-free water or lowTE buffer (with 0.1 mM EDTA).
The test sample contains a high
concentration of PCR inhibitor
(for example, heme compounds,
certain dyes).
Quantify the DNA, then use the minimum
necessary volume of test sample DNA.
The test sample was diluted in
the wrong buffer (for example, a
TE buffer with an incorrect EDTA
concentration).
Redilute DNA using nuclease-free water or lowTE buffer (with 0.1 mM EDTA).
Positive signal from DNA
Control 007 and elevated signal
from DNA test samples when
IQC peaks are present and
unbalanced
The quantity of the test
sample DNA is in excess of
recommended input amount,
which can cause loss of balance
in IQC peaks.
Quantify DNA, then use 500 pg.
More than two alleles present at
a locus
Exogenous DNA is present in
the sample.
Use appropriate techniques to avoid introducing
foreign DNA during laboratory handling.
Stutter product (–1 repeat unit
position) was amplified.
Ensure that you apply the provided locusspecific stutter filters.
Stutter filters apply to sample
input amounts equal or greater
than 250 pg. Samples well
below the recommended input
amount may exhibit stutters
values exceeding the filters due
to the stochastic effects of the
PCR.
Increase the sample input above 250 pg, if
possible. (Note, the optimum sample input is
500 pg.)
The test sample contained
mixed DNA.
If a mixed profile is not expected, check that
laboratory protocols relating to cleanliness are
followed.
Positive signal from DNA Control
007 but partial or no signal
from DNA test samples when
IQC peaks are present and
unbalanced
VeriFiler™ Plus PCR Amplification Kit User Guide
Wash the sample in a Centricon™-100
centrifugal filter unit.
131
A
Appendix A Troubleshooting
Population data
Observation
More than two alleles present at
a locus
(continued)
Possible cause
Incomplete 3´ A base addition
(n−1 nt position) occured.
Recommended action
Include the final extension step of 60°C for
5 minutes in the PCR.
Remove amplified plate from storage (thaw if
necessary) and place on thermal cycler at 60°C
for 15 minutes.
Check the quantity of the original sample
DNA to ensure input is less than 750 pg per
reaction. Adjust input as necessary during reamplification.
If the total amount of DNA in the reaction
exceeds 1 ng, adjust the final extension time to
15 minutes to minimize incomplete 3′ A base
addition.
The signal exceeds the dynamic
range of the instrument and
is causing signal "pull-up" into
adjacent channels.
Check that you are using the recommended
number of PCR cycles. Repeat PCR
amplification using reduced input DNA amount,
or interpret the off-scale data according to your
laboratory procedure.
Check that you are using the recommended
injection conditions on the instrument.
Poor spectral separation
occurred.
Perform a spectral calibration.
Too much DNA was present in
the reaction.
Use the recommended amount of template
DNA: 500 pg for 29 PCR cycles.
The double-stranded DNA was
not completely denatured.
Use the recommended amount of Hi‑Di™
Formamide and heat the sample plate at 95°C
for 3 minutes.
Poor peak height balance
Incorrect thermal cycler
conditions were used.
Use correct thermal cycler conditions.
Some but not all loci visible on
electropherogram of DNA Test
Samples
The DNA quantity was too low,
leading to stochastic effects.
Load more DNA sample or concentrate the
DNA.
STR profiles contain many offscale alleles
DNA quantification was not
performed or not accurate.
Ensure that DNA quantification is accurate.
132
Confirm that Filter Set J6-T modules are
installed and used for analysis.
VeriFiler™ Plus PCR Amplification Kit User Guide
B
Materials required but not supplied
■
■
■
■
■
Sample preparation required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Thermal cycler required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Genetic analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Analysis software required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Miscellaneous required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Unless otherwise indicated, all materials are available through thermofisher.com.
"MLS" indicates that the material is available from fisherscientific.com or another
major laboratory supplier.
Sample preparation required materials
Item
GeneScan™ 600 LIZ™ Size Standard v2.0, 2 × 200 µL
Source
4408399
IMPORTANT! Do not use GeneScan™ 350 ROX™, GeneScan™ 500 ROX™,
or GeneScan™ 500 LIZ™ Size Standards with this kit.
Hi‑Di™ Formamide, 25‑mL
4311320
Invitrogen™ Nuclease-Free Water (not DEPC-Treated)
AM9937
(Optional) Invitrogen™ TE Buffer (low-TE buffer; 10 mM Tris‑HCl pH 8.0 and
0.1 mM EDTA)
12090015
or see “(Optional) Prepare low-TE
buffer” on page 22.
Thermal cycler required materials
ProFlex™ PCR System
Item
Source
ProFlex™ 96‑well PCR System
4484075
ProFlex™ 2 × 96‑well PCR System
4484076
ProFlex™ 3 × 32‑Well PCR System
4484073
VeriFiler™ Plus PCR Amplification Kit User Guide
133
B
Appendix B Materials required but not supplied
Genetic analyzer required materials
Veriti™ Thermal Cycler
Item
Veriti™ 96‑Well Thermal Cycler
Source
4479071
(Optional) Tabletop centrifuge with 96-Well Plate Adapters
MLS
GeneAmp™ PCR System 9700
Item
GeneAmp™ PCR System 9700, 96-Well Silver
GeneAmp™ PCR System 9700, 96-Well Gold-Plated
Silver 96-Well Sample Block
Source
N8050001
4314878
N8050251
Gold-Plated 96-Well Block
4314443
Genetic analyzer required materials
3500 Series Genetic Analyzer
Item
3500 Series Data Collection Software 4, v4.0.1 software patch
Source
A40059 or A40376[1]
3500 Series Data Collection Software 3.1 Upgrade (RUO)
A26287[1]
3500 Series Data Collection Software 3.1 (RUO)
4475183[1]
HID Updater 3500 Data Collection Software v2
4480670
Anode buffer container (ABC)
4393927
Cathode buffer container (CBC)
4408256
POP-4™ Polymer (960 samples) for 3500/3500xL Genetic Analyzers
4393710
POP-4™ Polymer (384 samples) for 3500/3500xL Genetic Analyzers
4393715
DS-37 Matrix Standard Kit (Dye Set J6-T)
A31234
Conditioning reagent
4393718
8-Capillary array, 36 cm for 3500 Genetic Analyzers
4404683
24-Capillary array, 36 cm for 3500xL Genetic Analyzers
4404687
96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers
4410228
8-Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers
4410231
134
VeriFiler™ Plus PCR Amplification Kit User Guide
Appendix B Materials required but not supplied
Analysis software required materials
B
(continued)
Item
Source
8-Strip Septa for 3500/3500xL Genetic Analyzers
4410701
96-Well Septa for 3500/3500xL Genetic Analyzers
4412614
Septa Cathode Buffer Container, 3500 series
4410715
[1]
Contact your Thermo Fisher Scientific HID representative.
3130 Series Genetic Analyzer required materials
Item
Source
3130 Data Collection Software v4
4475105
3130xl Data Collection Software‑v4
4475126
3130/3730 Data Collection Software‑v4 6‑Dye Module v1
4478404
96‑Well Plate Septa
4315933
Reservoir Septa
4315932
3130/3130xl Genetic Analyzer 16‑Capillary Array, 36 cm
4315931
POP-4™ Polymer for the 3130/3130xl Genetic Analyzer
4352755
Running Buffer, 10✕
402824
DS‑37 Matrix Standard Kit (Dye Set J6-T)
A31234
MicroAmp™ Optical 96-Well Reaction Plate
N8010560
Analysis software required materials
GeneMapper™ ID‑X Software
Item
Source
GeneMapper™ ID‑X Software v1.6 Full Installation
A39975
GeneMapper™ ID‑X Software v1.6 Client Installation
A39976
GeneMapper™ ID‑X Software v1.5 Full Installation
A27884
GeneMapper™ ID‑X Software v1.5 Client Installation
A27886
GeneMapper™ ID‑X Software v1.4 Full Installation
4479707
GeneMapper™ ID‑X Software v1.4 Client Installation
4479711
VeriFiler™ Plus PCR Amplification Kit User Guide
135
B
Appendix B Materials required but not supplied
Miscellaneous required materials
Miscellaneous required materials
Plates and tubes
Item
Source
MicroAmp™ 96-Well Tray
N8010541
MicroAmp™ Reaction Tube with Cap, 0.2 mL
N8010540
MicroAmp™ 8-Tube Strip, 0.2 mL
N8010580
MicroAmp™ Optical 8-Cap Strips
4323032
MicroAmp™ 96-Well Tray/Retainer Set
403081
MicroAmp™ 96-Well Base
N8010531
MicroAmp™ Clear Adhesive Film
4306311
MicroAmp™ Optical Adhesive Film
4311971
MicroAmp™ Optical 96-Well Reaction Plate
N8010560
Laboratory supplies
Item
Source
Various procedures
Aerosol resistant pipette tips
MLS[1]
Microcentrifuge tubes
MLS
Pipettors
MLS
Tape, labeling
MLS
Tube, 50-mL Falcon™
MLS
Tube decapper, autoclavable
MLS
Deionized water, PCR grade
MLS
Vortex
MLS
[1]
Major laboratory supplier
136
VeriFiler™ Plus PCR Amplification Kit User Guide
C
PCR work areas
■
■
■
Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Work area setup and lab design
Many resources are available for the appropriate design of a PCR laboratory. If you
are using this kit for:
• Forensic DNA testing, see "Forensic Laboratories: Handbook for Facility
Planning, Design, Construction, and Moving", National Institute of Justice, 1998
• Parentage DNA testing, see the "Guidance for Standards for Parentage
Relationship Testing Laboratories", American Association of Blood Banks, 7th
edition, 2004
The sensitivity of this kit (and other PCR-based tests) enables amplification of minute
quantities of DNA, necessitating precautions to avoid contamination of samples yet
to be amplified (Kwok and Higuchi, 1989).
Process samples carefully to prevent contamination by human DNA. Wear gloves at
all times and change them frequently. Close sample tubes when not in use. Limit
aerosol dispersal by handling sample tubes and reagents carefully.
Note: We do not intend these references for laboratory design to constitute all
precautions and care necessary for using PCR technology.
PCR setup work area materials
IMPORTANT! Do not remove these items from the PCR Setup Work Area.
• Calculator
• Gloves, disposable
• Marker pen, permanent
• Microcentrifuge
• Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate nuclease-free
tube (for master mix preparation)
• Microcentrifuge tube rack
• Pipette tips, sterile, disposable hydrophobic filter-plugged
VeriFiler™ Plus PCR Amplification Kit User Guide
137
C
Appendix C PCR work areas
Amplified DNA work area
• Pipettors
• Tube decapper, autoclavable
• Vortex
Amplified DNA work area
IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area.
Use only the validated thermal cyclers listed in “Instruments and software
compatibility” on page 17.
138
VeriFiler™ Plus PCR Amplification Kit User Guide
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in
the user documentation may result in personal injury or damage to the instrument
or device. Ensure that anyone using this product has received instructions in
general safety practices for laboratories and the safety information provided in this
document.
· Before using an instrument or device, read and understand the safety information
·
provided in the user documentation provided by the manufacturer of the
instrument or device.
Before handling chemicals, read and understand all applicable Safety Data Sheets
(SDSs) and use appropriate personal protective equipment (gloves, gowns, eye
protection, and so on). To obtain SDSs, see the “Documentation and Support”
section in this document.
VeriFiler™ Plus PCR Amplification Kit User Guide
139
Safety
Chemical safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure
laboratory personnel read and practice the general safety guidelines for chemical
usage, storage, and waste provided below. Consult the relevant SDS for specific
precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the chemical
·
·
·
manufacturer before you store, handle, or work with any chemicals or hazardous
materials. To obtain SDSs, see the "Documentation and Support" section in this
document.
Minimize contact with chemicals. Wear appropriate personal protective equipment
when handling chemicals (for example, safety glasses, gloves, or protective
clothing).
Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
only with sufficient ventilation (for example, fume hood).
Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer cleanup procedures as recommended in the SDS.
Handle chemical wastes in a fume hood.
·
· Ensure use of primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks from
the primary container. Both containers must be compatible with the waste material
and meet federal, state, and local requirements for container storage.)
After emptying a waste container, seal it with the cap provided.
·
· Characterize (by analysis if needed) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of
·
according to all local, state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require special handling,
and disposal limitations may apply.
WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the
instrument is potentially hazardous. Follow the guidelines noted in the preceding
General Chemical Handling warning.
WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and waste
bottles can crack and leak. Each 4-liter bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in the
upright position.
140
VeriFiler™ Plus PCR Amplification Kit User Guide
Safety
Biological hazard safety
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used on this
instrument, the surface may be considered a biohazard. Use appropriate
decontamination methods when working with biohazards.
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,
infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Conduct all work in properly equipped facilities with
the appropriate safety equipment (for example, physical containment devices). Safety
equipment can also include items for personal protection, such as gloves, coats,
gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles.
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially biohazardous materials.
Follow all applicable local, state/provincial, and/or national regulations. The following
references provide general guidelines when handling biological samples in laboratory
environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological and
·
Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112,
Revised December 2009; found at:
https://www.cdc.gov/labs/pdf/CDCBiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf
World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
VeriFiler™ Plus PCR Amplification Kit User Guide
141
Documentation and support
Related documentation
Document title
Pub. No.
STR kits
VeriFiler™ Plus PCR Amplification Kit—PCR Amplification and CE Quick Reference
MAN0017495
VeriFiler™ Plus PCR Amplification Kit—PCR Setup Quick Reference
MAN0017494
Quantification kits
Quantifiler™ HP and Quantifiler™ Trio DNA Quantification Kits User Guide
4485354
Thermal cyclers
ProFlex™ PCR System User Guide
MAN0007697
Veriti™ Thermal Cycler User Guide
4375799
GeneAmp™ PCR System 9700 Base Module User Manual
4303481
Direct Amplification of Reference Samples Using the VeriFiler™ Plus PCR Amplification Kit
—
3500 Series Genetic Analyzer
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v1 User Guide
4401661
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v2 User Guide
4476988
HID Updater 3500 Data Collection Software v2.0 User Bulletin
—
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software 3.1 User Guide
100025036
3500 Series Data Collection Software v3 User Bulletin: New Features and HID Validation
Summary
MAN0010812
3500 Series Data Collection Software v3.1 User Bulletin: New Features and HID Validation
Summary
MAN0014110
3500 Series Data Collection Software 4 User Bulletin: New Features and Developmental
Validation
100075298
3130xl Series Genetic Analyzer
3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide
4352716
3130/3130xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin
4363787
142
VeriFiler™ Plus PCR Amplification Kit User Guide
Documentation and support
Related documentation
(continued)
Document title
Pub. No.
3130/3130xl Genetic Analyzers Getting Started Guide
4352715
3130/3130xl Genetic Analyzers Quick Reference Card
4362825
3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide
4359472
GeneMapper™ ID‑X Software all versions
GeneMapper™ ID‑X Software Bin Overlap User Bulletin
100029546
GeneMapper™ ID‑X Software v1.0
GeneMapper™ ID‑X Software v1.0 Getting Started Guide— Basic Features
4375574
GeneMapper™ ID‑X Software v1.0 Quick Reference— Basic Features
4375670
GeneMapper™ ID‑X Software v1.0 Installation Guide
4476603
GeneMapper™ ID‑X Software v1.0 Administrator Guide
4376327
GeneMapper™ ID‑X Software v1.0 Reference Guide
4375671
GeneMapper™ ID‑X Software v1.1
GeneMapper™ ID‑X Software v1.1 Getting Started Guide— Mixture Analysis Tool
4396773
GeneMapper™ ID‑X Software v1.2
GeneMapper™ ID‑X Software v1.2 Verification Experiments and Installation Procedures User
Bulletin
4462639
GeneMapper™ ID‑X Software v1.2 Quick Reference— Mixture Analysis Tool
4426482
GeneMapper™ ID‑X Software v1.2 Reference Guide
4426481
GeneMapper™ ID‑X Software v1.3
GeneMapper™ ID‑X Software v1.3 Verification Experiments and Installation Procedures User
Bulletin
4470483
GeneMapper™ ID‑X Software v1.4
GeneMapper™ ID‑X Software v1.4 New Features and Installation Procedures User Bulletin
4477684
GeneMapper™ ID‑X Software v1.5
GeneMapper™ ID‑X Software v1.5 New Features and Verification User Bulletin
100031708
GeneMapper™ ID‑X Software v1.5 Getting Started Guide— Basic Features
100031701
GeneMapper™ ID‑X Software v1.5 Quick Reference— Basic Features
100031702
GeneMapper™ ID‑X Software v1.5 Getting Started Guide— Mixture Analysis Tool
100031704
GeneMapper™ ID‑X Software v1.5 Quick Reference— Mixture Analysis Tool
100031705
VeriFiler™ Plus PCR Amplification Kit User Guide
143
Documentation and support
Customer and technical support
(continued)
Document title
Pub. No.
GeneMapper™ ID‑X Software v1.5 Installation Guide
100031706
GeneMapper™ ID‑X Software v1.5 Administrator Guide
100031703
GeneMapper™ ID‑X Software v1.5 Reference Guide
100031707
GeneMapper™ ID‑X Software v1.6
GeneMapper™ ID‑X Software v1.6 New Features and Software Verification User Bulletin
100073905
Precision ID Ancestry Panel and Precision ID Identity Panel
Precision ID Panels with the Ion S5™ System Application Guide
MAN0015831
Customer and technical support
For support:
• In North America—Send an email to [email protected], or
call 888-821-4443 option 1.
• Outside North America—Contact your local support office.
For the latest services and support information for all locations, go to
thermofisher.com/support to obtain the following information.
• Worldwide contact telephone numbers
• Product support
• Order and web support
• Safety Data Sheets (SDSs; also known as MSDSs)
Additional product documentation, including user guides and Certificates of Analysis,
are available by contacting Customer Support.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as
set forth in the Life Technologies' General Terms and Conditions of Sale
at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you
have any questions, please contact Life Technologies at www.thermofisher.com/
support.
144
VeriFiler™ Plus PCR Amplification Kit User Guide
References
Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994.
Identification of the heme compound copurified with deoxyribonucleic acid (DNA)
from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.
J. Forensic Sci. 39:362–372.
Barber, M.D. and Parkin, B.H. 1996. Sequence analysis and allelic designation of the
two short tandem repeat loci D18S51 and D8S1179. Intl. J. Legal Med. 109:62–65
Barber, M.D., Piercy, R.C., Andersen, J.F. and Parkin, B.H. 1995. Structural variation
of novel alleles at the Hum vWA and Hum FES/FPS short tandem repeat loci. Int. J.
Leg. Med. 108:31–35.
Barber, M.D., McKeown, B.J. and Parkin, B.H. 1996. Structural variation in the alleles
of a short tandem repeat system at the human alpha fibrinogen locus. Int. J. Leg.
Med. 108:180–185.
Begovich A.B., McClure G.R., Suraj V.C., Helmuth R.C., Fildes N., Bugawan T.L.,
Erlich H.A., Klitz W. 1992. Polymorphism, recombination, and linkage disequilibrium
within the HLA class II region. J. Immunol. 148:249–258.
Bender, K., Farfan, M.J., Schneider, P.M. 2004. Preparation of degraded human DNA
under controlled conditions. Forensic Sci. Int. 139:134–140.
Brinkmann, B., Klintschar, M., Neuhuber, F., Huhne, J. and Rolf, B. 1998. Mutation
rate in human microsatellites: Influence of the structure and length of the tandem
repeat. Am. J. Hum. Genet. 62:1408–1415.
Budowle, B. et al. 2010. Population genetic analyses of the NGM STR loci. Int. J.
Legal Med. e-publication www.springerlink.com/content/p713q3w5440674u3/
Butler, J.M. 2005. Forensic DNA Typing. Burlington, MA:Elsevier Academic Press.
Butler, J.M. 2010. Fundamentals of Forensic DNA Typing. Burlington, MA:Elsevier
Academic Press.
Chakraborty, R., Stivers, D., and Zhong, Y. 1996. Estimation of mutation rates from
parentage exclusion data: applications to STR and VNTR loci. Mutat. Res. 354:41–48.
Clark J.M. 1988. Novel non-templated nucleotide addition reactions catalyzed by
procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16:9677–9686.
DeFranchis, R., Cross, N.C.P., Foulkes, N.S., and Cox, T.M. 1988. A potent inhibitor
of Taq DNA polymerase copurifies with human genomic DNA. Nucleic Acids Res.
16:10355. DNA Advisory Board, Federal Bureau of Investigation, U.S. Department of
Justice. 1998. Quality assurance standards for forensic DNA testing laboratories.
Edwards, A., Hammond, H.A., Lin, J., Caskey, C.T., and Chakraborty, R. 1992.
Genetic variation at five trimeric and tetrameric tandem repeat loci in four human
population groups. Genomics 12:241–253.
VeriFiler™ Plus PCR Amplification Kit User Guide
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148
VeriFiler™ Plus PCR Amplification Kit User Guide
Index
+A 89
+A nucleotide addition 89
3' A 89
3130 instrument 31
3500 instrument 27
3730/3730xl instrument, 6-dye license 31
6-dye
license activation for 3730/3730xl instrument 31
spectral calibration 29, 33
600 LIZ Size Standard v2.0 51
A
prepare samples 33
references 27, 31
run module 27, 31
setup of the 3130 instrument 31
setup of the 3130xl instrument 31
setup of the 3500 and 3500xL instruments 27
extra peaks 83
G
GeneScan 600 LIZ Size Standard v2.0 51
GeneScan size standard, about 13
I
accuracy and reproducibility 64
alleles, off-ladder 67
allelic ladder, requirements for electrophoresis 26
artifacts 90
import panels, bins, and marker stutter 38
instrument and software compatibility 17
B
limited product warranty 144
LIZ size standard
about 13
peak sizes 51
volume per reaction 33
LIZ Size Standard v2.0 51
bins, import 38
biohazard safety 141
C
characterization of loci, validation 92
control DNA
007 13
profile 15
D
developmental validation 61
direct amplification 25
DNA control profile 15
documentation, related 142
DS-37 matrix standard 29, 33
dye set for 6-dye samples 29, 33
E
L
M
marker stutter, import 38
materials not supplied 133
P
panels
check version 37
import 38
PCR
conditions 24
perform 24
setup 137
work areas 137
electrophoresis
data collection software 27, 31
VeriFiler™ Plus PCR Amplification Kit User Guide
149
Index
Q
quantification, DNA 19
R
required materials 133
run module for electrophoresis
3130 instrument 31
3130xl instrument 31
3500 and 3500xL instruments 27
S
safety, biohazard 141
sensitivity 97
size standard 51
spectral calibration 29, 33
stutter, peaks 87
150
stutter file, import 38
T
terms and conditions 144
thermal cyclers
for use with kit 17
programming 24
troubleshooting 130
V
validation, importance 60
W
warranty 144
work area, PCR setup 137
VeriFiler™ Plus PCR Amplification Kit User Guide
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