Thermo Fisher Scientific AmpFlSTR NGM PCR Amplification Kit User Guide
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AmpF ℓ STR
™
NGM
™
PCR
Amplification
Kit
USER GUIDE for use with:
200 reaction kit (Part no. 4415020)
1
,
000 reaction kit (Part no. 4415021)
Catalog Number 4415020 , 4415021
Publication Number 4425511
Revision H
For Forensic or Paternity Use Only.
Manufacturer: Thermo Fisher Scientific | 7 Kingsland Grange | Warrington, Cheshire WA1 4SR | United Kingdom
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. 4425511
Revision
H
G
F
E
D
C
B
A
Date
24 August 2018
February 2015
March 2012
July 2011
July 2011
September 20009
November 2009
September 2009
Description
Updated branding and trademarks, no technical changes.
Add information for the ProFlex ™ PCR System.
Change copyright information.
Added information for GeneAmp ™ 9700 and Veriti ™ Thermal Cycler emulation mode in PCR chapter.
Information on addition of SNP-Specific Primers at D2S441, D22S1045,
Amelogenin.
Patent upgrade.
Update copyright information.
Add Chapter 5 Experiments and Results
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.FTA is a trademark of
Whatman Limited. Windows and Windows Vista are trademarks of Microsoft Corporation. Adobe, Acrobat and Reader are trademarks of Adobe
Systems Incorporated.
©2018 Thermo Fisher Scientific Inc. All rights reserved.
Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Chapter 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Data Collection and GeneMapper
ID or ID-X Software . . . . . . . . . . . . . . . . . . . . . 16
Chapter 2 PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Amplification using bloodstained FTA
cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Chapter 3 Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide 3
Contents
Avant
and 3130/3130
xl
instruments . . . . . . . . . . . . 27
Avant and 3130/3130 xl instruments for electrophoresis . . . . . . . 27
Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . 27
Prepare samples for electrophoresis on the 3100/3100Avant
or
3130/3130 xl instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Section 3.2 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . 29
Prepare samples for electrophoresis on the 3500/3500xL instrument . . . . . . . . . . . . . . 29
Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . 31
Chapter 4 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
ID
Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
™
ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
™
ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Analyze and edit sample files with GeneMapper
ID Software . . . . . . . . . . . . . . . . . . . . 43
ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
™
ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4 AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Contents
™
ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Analyze and edit sample files with GeneMapper
ID-X Software . . . . . . . . . . . . . . . . . . 57
Chapter 5 Experiments and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide 5
Contents
New Primers added to the NGM ™
Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Comparison of primer sequences with other AmpF
l STR
™
Kits . . . . . . . . . . . . . . . . 91
Inclusion of three SNP-specific primers to address mutations at the Amelogenin,
Appendix A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Appendix B Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Appendix C PCR Work Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
6 AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Contents
Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . 117
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide 7
Contents
8 AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Purpose
About This Guide
The Applied Biosystems
™
AmpF l STR
™
NGM
™
PCR Amplification Kit User Guide provides information about our instruments, chemistries, and software associated with the AmpF l STR
™
NGM
™
PCR Amplification Kit.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
9
About This Guide
Purpose
10
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
1
Overview
■
■
■
■
Product overview
Purpose
The AmpF l STR
™
NGM ™ PCR Amplification Kit is a short tandem repeat (STR) multiplex assay that amplifies 14 tetranucleotide repeat loci and one trinucleotide repeat locus, D22S1045. The kit simultaneously coamplifies the 10 loci contained in the
AmpF l STR
™
SGM Plus
™
Kit (D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11,
D18S51, D19S433, TH01, and FGA) together with 2 highly polymorphic STR loci
(D1S1656 and D12S391), 3 “mini” STR loci (D10S1248, D22S1045 and D2S441), and the gender determination locus Amelogenin. The NGM ™ Kit delivers a 16-locus multiplex with a greater power of discrimination, better sensitivity, and improved robustness than earlier generation AmpF l STR
™
kits. The kit uses modified PCR cycling conditions for enhanced sensitivity, a new buffer formulation to improve performance with inhibited samples, more loci concentrated in the low molecularweight region of the profile to improve performance on degraded samples, and an improved process for synthesis and purification of the amplification primers to deliver a much cleaner electrophoretic background.
Product description
The NGM ™ Kit contains all the necessary reagents for the amplification of human genomic DNA.
The reagents are designed for use with the following Applied Biosystems instruments:
™
• ABI P RISM
™
3100/3100Avant Genetic Analyzer
• Applied Biosystems
™
3130/3130 xl Genetic Analyzer
• Applied Biosystems
™
3500/3500xL Genetic Analyzer
• Applied Biosystems
™
310 Genetic Analyzer
• GeneAmp
™
PCR System 9700 with the Silver 96-Well Block
• GeneAmp
™
PCR System 9700 with the Gold-plated Silver 96-Well Block
• Veriti
™
96-Well Thermal Cycler
• ProFlex
™
PCR System
11
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
1
Chapter 1 Overview
Product overview
About the primers
The NGM ™ Kit employs the latest improvements in primer synthesis and purification techniques to minimize the presence of dye-labeled artifacts. These improvements result in a much cleaner electropherogram background that enhances the assay's signal-to-noise ratio and simplifies the interpretation of results.
Loci amplified by the kit
The following table shows the loci amplified, their chromosomal locations, and the corresponding fluorescent marker dyes. The AmpF l STR
™
NGM
™
Allelic Ladder is used to genotype the analyzed samples. The alleles contained in the allelic ladder and the genotype of the AmpF l STR
™
Control DNA 007 are also listed in the table.
Table
1
NGM ™ Kit loci and alleles
Locus designation
D10S1248 vWA
D16S539
D2S1338
Amelogenin
D8S1179
D21S11
D18S51
D22S1045
D19S433
TH01
FGA
D2S441
D3S1358
D1S1656
D12S391
Chromosome location
10q26.3
12p13.31
16q24.1
2q35
X: p22.1-22.3
Y: p11.2
8q24.13
21q11.2-q21
18q21.33
22q12.3
19q12
11p15.5
4q28
2p14
3p21.31
1q42.2
12p13.2
Alleles included in AmpF l STR
™
NGM ™ Allelic Ladder
Dye label
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18
11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24
6-FAM ™
6-FAM
™
5, 8, 9, 10, 11, 12,13, 14, 15
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28
6-FAM ™
6-FAM ™
X, Y VIC
™
8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 VIC
™
VIC
™
24, 24.2, 25, 26, 27, 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, 37, 38
7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27
VIC
™
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15,
15.2, 16, 16.2, 17, 17.2
NED ™
NED
™
4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3
NED ™
NED ™ 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26.2, 27,
28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2,
44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2
9, 10, 11, 11.3, 12, 13, 14, 15, 16
12, 13, 14, 15, 16, 17, 18, 19
9, 10, 11, 12, 13, 14, 14.3, 15, 15.3, 16, 16.3,
17, 17.3, 18.3, 19.3, 20.3
PET
™
PET
™
PET
™
14, 15, 16, 17, 18, 19, 19.3, 20, 21, 22, 23, 24,
25, 26, 27
PET
™
Control DNA
007
12, 15
14, 16
9, 10
20, 23
X, Y
12, 13
28, 31
12, 15
11, 16
14, 15
7, 9.3
24, 26
14, 15
15, 16
13, 16
18, 19
12
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 1 Overview
Product overview
1
Allelic ladder profile
Figure 1 shows the allelic ladder for the NGM
™
Kit. See “Allelic ladder requirements” on page 25 for information on ensuring accurate genotyping.
Figure 1 GeneMapper
™
ID-X Software plot of the AmpF l STR
™
NGM ™ Allelic Ladder
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
13
1
Chapter 1 Overview
Product overview
Control DNA 007 profile
Figure 2 shows amplification of Control DNA 007 using the NGM
™ Kit.
Figure 2 1 ng of Control DNA 007 amplified with the NGM ™ Kit and analyzed on the Applied Biosystems
™
3130 xl
Genetic Analyzer
14
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Workflow overview
Perform
PCR
AutoMate Express
™
System + PrepFiler Express
™
Kit
Chapter 1 Overview
Workflow overview
1
Quantifiler
™
Duo DNA Quantification Kit Quantifiler
™
HP and Trio DNA Quantification Kits
AmpF l STR
™
NGM ™ PCR Amplification Kit
GeneAmp
™
PCR System 9700 Cycler ProFlex
™
PCR System Veriti
™
96-Well Thermal Cycler
Perform electrophoresis
Analyze data
310 Genetic
Analyzer
3100/3100Avant
Genetic Analyzer
3130/3130 xl
Genetic Analyzer
3500/3500xL
Genetic Analyzer
GeneMapper
™
ID-X or GeneMapper
™
ID Software
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
15
1
Chapter 1 Overview
Instrument and software overview
Instrument and software overview
This section provides information about the Data Collection Software versions required to run the NGM ™ Kit on specific instruments.
Data Collection and GeneMapper
™
ID
or
ID-X
Software
Instrument and software compatibility
The Data Collection Software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time. As the instrument measures sample fluorescence with its detection system, the Data
Collection Software collects the data and stores it. The Data Collection Software stores information about each sample in a sample file (.fsa files for 31xx instruments and .hid files for 3500 instruments), which is then analyzed by the GeneMapper
™
ID or ID-X Software.
Table
2
Software specific to each instrument
Instrument
Data Collection
Software
Analysis software
3500/3500xL 3500 Series Data
Collection Software v1.0
3.0
3130/3130 xl †
3100/3100Avant 1.1 (3100)
1.0 (3100Avant )
310
2.0
3.1
GeneMapper
Software v1.2 or higher
•
•
™
ID-X
GeneMapper
™
ID Software v3.2.1
and
GeneMapper
™
ID-X
Software v1.0.1 or higher
3.0
† We conducted validation studies for the NGM ™ Kit using this configuration.
About multicomponent analysis
Our fluorescent multi-color dye technology allows the analysis of multiple loci, including loci that have alleles with overlapping size ranges. Alleles for overlapping loci are distinguished by labeling locus-specific primers with different colored dyes.
Multicomponent analysis is the process that separates the 5 different fluorescent dye colors into distinct spectral components. The four dyes used in the NGM ™ Kit to label samples are 6-FAM ™ , VIC
™
, NED ™ , and PET
™ ™
, is used to label the GeneScan ™ 500 LIZ
™
dyes. The fifth dye, LIZ
Size Standard or GeneScan ™ 600 LIZ
™
Size Standard v2.0.
How multicomponent analysis works
Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength. During data collection on the Applied Biosystems
™
and ABI P RISM
™ instruments, the fluorescence signals are separated by diffraction grating according to their wavelengths and projected onto a charge-coupled device (CCD) camera in a predictably spaced pattern. The 6-FAM
™ is displayed as blue, followed by the VIC
dye emits at the shortest wavelength and it
™
dye (green), NED
™
dye (yellow), PET
™ dye (red), and LIZ
™
dye (orange).
16
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 1 Overview
Materials and equipment
1
Although each of these dyes emits its maximum fluorescence at a different
wavelength, there is some overlap in the emission spectra between the dyes (Figure 3).
The goal of multicomponent analysis is to correct for spectral overlap.
Figur e 3 Emission spectra of the five dyes used in the NGM ™ Kit
Dyes
6-FAM VIC NED PET LIZ
100
80
60
40
20
0
500 550 600
Wavelength (nm)
650 700
Materials and equipment
Kit contents and storage
The NGM ™ Kit contains materials sufficient to perform 200 (Part no. 4415020) or 1000
(Part no. 4415021) amplifications at a 25 µL reaction volume.
IMPORTANT!
The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standards from light when not in use. Keep freeze-thaw cycles to a minimum.
Table
3
Kit contents and storage
Component
AmpF l STR
Primer Set
AmpF l STR
Master Mix
AmpF l STR
™
NGM ™
Allelic Ladder
AmpF l STR
™
™
™
NGM
NGM
Control DNA 007
™
™
Description
Contains forward and reverse primers to amplify human DNA targets.
Contains enzyme, salts, dNTPs, carrier protein, and 0.05% sodium azide.
Contains amplified alleles.
See Table 1 on page 12 for a list
of alleles included in the allelic ladder.
Contains 0.10 ng/µL human male
007 DNA in 0.02% sodium azide and buffer † .
200
1 tube, 1.0 mL
2 tubes, 1.0 mL each
✕ Volume
1 tube, 50.0 µL
1 tube, 0.3 mL
1000 ✕ Volume
1 bottle, 5.0 mL
1 tube, 0.3 mL
Storage
-15 to -25°C on receipt, 2 to 8 °C after initial use
1 bottle, 10.0 mL -15 to -25°C on receipt, 2 to 8 °C after initial use
1 tube, 75.0 µL -15 to -25°C on receipt, 2 to 8°C after initial use
2 to 8°C
profile.
† The AmpF l STR
™
Control DNA 007 is included at a concentration appropriate to its intended use as an amplification control (to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype). The AmpF l STR
™
Control DNA 007 is not designed to be used as a DNA quantitation control, and laboratories may expect to see variation from the labelled concentration when quantitating aliquots of the AmpF l STR
™
Control DNA 007.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
17
1
Chapter 1 Overview
Materials and equipment
Standards for samples
For the NGM ™ Kit, the panel of standards needed for PCR amplification, PCR product sizing, and genotyping are:
• Control DNA 007 – A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF l STR
™
NGM
™
Allelic
Ladder.
• GeneScan
™
500 LIZ
™
Size Standard (GS 500) or GeneScan
™
600 LIZ
™
Size
Standard v2.0 (GS 600 v2.0) – Used for obtaining sizing results. These standards, which have been evaluated as internal size standards, yield precise sizing results for NGM
™
Kit PCR products. Order the GeneScan
™
500 LIZ
™
Size Standard
(Part no.
4322682) or the GeneScan
™
600 LIZ
™
(Part no.
4408399) separately.
Size Standard v2.0
• AmpF l STR
™
NGM ™ Allelic Ladder – Allelic ladder for accurate characterization of the alleles amplified by the NGM ™ Kit. The AmpF l STR
™
NGM contains most of the alleles reported for the 15
autosomal loci. Refer to Table 1 on page 12 for a list of the alleles included in the AmpF
l STR
™
NGM
™
™
Allelic Ladder
Allelic Ladder.
18
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
2
PCR Amplification
■
■
■
■
■
Amplification using bloodstained FTA
cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Required user-supplied materials and reagents
In addition to the NGM
™
Kit reagents, the use of low-TE buffer
(10 mM Tris, 0.1 mM EDTA, pH 8.0) is recommended. You can prepare the buffer as described in the procedure below or order it from Teknova (Cat. no. T0223).
To prepare low-TE buffer:
1.
Mix together:
• 10 mL of 1 M Tris-HCl, pH 8.0
• 0.2 mL of 0.5 M EDTA, pH 8.0
• 990 mL glass-distilled or deionized water
Note:
Adjust the volumes based on your specific needs.
2.
Aliquot and autoclave the solutions.
3.
Store at room temperature.
DNA quantification
Importance of quantification
Quantifying the amount of DNA in a sample before amplification allows you to determine whether or not sufficient DNA is present to permit amplification and to calculate the optimum amount of DNA to add to the reaction. The optimum amount of
DNA for the NGM
™
Kit is 1.0 ng in a maximum input volume of
10 µL amplified for 29 cycles.
19
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
2
Chapter 2 PCR Amplification
DNA quantification
If too much DNA is added to the PCR reaction, then the increased amount of PCR product that is generated can result in:
• Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument (“off-scale” data). Off-scale data are problematic because:
– Quantification (peak height and area) for off-scale peaks is not accurate. For example, an allele peak that is off-scale can cause the corresponding stutter peak to appear higher in relative intensity, thus increasing the calculated percent stutter.
– Multicomponent analysis of off-scale data is not accurate, and it results in poor spectral separation (“pull-up”).
• Incomplete A-nucleotide addition.
When the total number of allele copies added to the PCR is extremely low, allelic dropout can occur, resulting in a partial profile.
Methods of quantifying DNA
We provide several kits for quantifying DNA in samples. See the references cited in the following table for details about these kits.
Product
Quantifiler
™
Human DNA
Quantification Kit
(Part no.4343895) and
Quantifiler
™
Y Human Male
DNA Quantification Kit
(Part no. 4343906)
For more information, see
Quantifiler
™
Human DNA
Quantification Kits User’s Manual
(Part no. 4344790)
Quantifiler
Quantifiler
™
™
Duo DNA
Quantification Kit
(Part no. 4387746)
For more information, see
Duo DNA
Quantification Kit User's Manual
(Part no. 4391294)
Description
Properties:
The Quantifiler
™
Human and Quantifiler
™
Y Human Male Kits are highly specific for human DNA, and they detect total human or male DNA, respectively. The kits detect single-stranded and degraded DNA.
How they work:
The Quantifiler
™
DNA Quantification Kits consist of target-specific and internal control 5' nuclease assays.
The Quantifiler
™
Human and Quantifiler
™
Y Human Male Kits contain different targetspecific assays (human DNA or human male DNA, respectively) that each consist of two locus-specific PCR primers and one TaqMan
™
MGB probe labeled with FAM
™
dye for detecting the amplified sequence. The kits each contain a separate internal PCR control
(IPC) assay that consists of an IPC template DNA (a synthetic sequence not found in nature), two primers for amplifying the IPC template DNA, and one TaqMan
™
MGB probe labeled with VIC
™
dye for detecting the amplified IPC DNA.
Properties:
The Quantifiler
™
Duo Kit is highly specific for human DNA and combines the detection of both total human and male DNA in one PCR reaction.The kit detects single-stranded and degraded DNA.
How it works:
The Quantifiler
™
Duo DNA Quantification Kit consists of target-specific and internal control 5' nuclease assays.
The Quantifiler
™
Duo Kit combines two human-specific assays in one PCR reaction
(for total human DNA and human male DNA). The two human DNA specific assays each consist of two PCR primers and a TaqMan
™
probe. The TaqMan
™ human DNA and human male DNA assays are labeled with VIC
™
probes for the
and FAM ™ dyes, respectively. In addition, the kit contains an internal PCR control (IPC) assay similar in principle to that used in the other Quantifiler Kits, but labeled with NED ™ dye.
20
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 2 PCR Amplification
Prepare the amplification kit reactions
2
Product
Quantifiler
™
HP DNA
Quantification Kit (Cat. no.
4482911)
Quantifiler
™
Trio DNA
Quantification Kit (Cat. no.
4482910)
For more information, see
Quantifiler HP and Trio DNA
Quantification Kits User Guide
(Pub no. 4485354)
Description
Properties:
The Quantifiler
™
HP Kit is designed to quantify the total amount of amplifiable human
DNA in a sample. The Quantifiler
™
Trio Kit is designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample.
How they work:
The Quantifiler
™
HP and Trio DNA Quantification Kits use multiple-copy target loci for improved detection sensitivity. The human-specific target loci (Small Autosomal, Large
Autosomal, and Y-chromosome targets) each consist of multiple copies dispersed on various autosomal chromosomes (Small Autosomal and Large Autosomal).
To maximize the consistency of quantification results, genomic targets were selected with conserved primer- and probe-binding sites within individual genomes and also with minimal copy number variability between different individuals and population groups. As a result, the detection sensitivity of the Quantifiler
™
HP and Trio assays is improved over
Quantifiler
™
Duo, Human, and Y Human Male DNA Quantification Kit assays. The primary quantification targets (Small Autosomal and Y) consist of relatively short amplicons (75 to 80 bases) to improve the detection of degraded DNA samples. In addition, the Quantifiler
™
HP and Trio Kits each contain a Large Autosomal target with a longer amplicon (>200 bases) to aid in determining if a DNA sample is degraded.
Prepare the amplification kit reactions
1.
Calculate the volume of each component needed to prepare the reactions, using the table below.
DNA sample
AmpF l STR
™
NGM ™ Master Mix
AmpF l STR
™
NGM ™ Primer Set
Volume per reaction
10.0 µL
5.0 µL
Note:
Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers.
2.
Prepare reagents. Thaw the AmpF l STR
™
NGM ™ Master Mix and the AmpF l STR
™
NGM ™ Primer Set, then vortex the tubes for 3 seconds and centrifuge them briefly before opening.
IMPORTANT!
Thawing is required only during first use of the kit. After first use, reagents are stored at 2–8°C and, therefore, do not require subsequent thawing.
Do not refreeze the reagents.
3.
4.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Vortex the reaction mix for 3 seconds, then centrifuge briefly.
5.
Dispense 15 µL of reaction mix into each reaction well of a MicroAmp
™
Optical
96-
Well Reaction Plate or each MicroAmp
™
tube.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
21
2
Chapter 2 PCR Amplification
Perform PCR
6.
Prepare the DNA samples:
DNA sample To prepare...
Negative control Add 10 µL of low-TE buffer (10mM Tris, 0.1mM EDTA, pH 8.0).
Test sample
Positive control
Dilute a portion of the test DNA sample with low-TE buffer so that 1.0 ng of total DNA is in a final volume of 10 µL. Add 10 µL of the diluted sample to the reaction mix.
Add 10 µL of 007 control DNA (0.1 ng/µL) to provide 1.0 ng of total DNA in the positive control reaction.
The final reaction volume (sample or control plus reaction mix) should be 25 µL.
7.
Seal the MicroAmp
™
Optical 96-Well Reaction Plate with MicroAmp
Adhesive Film or MicroAmp
™
™
Clear
Optical Adhesive Film, or cap the tubes.
8.
Centrifuge the tubes or plate at 3000 rpm for about 20 seconds in a tabletop centrifuge (with plate holders if using 96-well plates) to remove bubbles.
9.
Amplify the samples in a GeneAmp
™
PCR System 9700 with the Silver 96-well block, or a GeneAmp
™
PCR System 9700 with the Gold-plated Silver 96-well block, or a Veriti
™
96-well Thermal Cycler, or a ProFlex ™ PCR System.
IMPORTANT!
The NGM
™
Kit is not validated for use with the GeneAmp
™
PCR System 9700 with the Aluminium 96-well block. Use of this thermal cycling platform may adversely affect the performance of the NGM
™
Kit.
Perform PCR
1.
Program the thermal cycling conditions.
• When using the GeneAmp
™
PCR System 9700 with either 96-well silver or gold-plated silver block, select the 9600 Emulation Mode .
• When using the Veriti
™
96-Well Thermal Cycler, refer to the following document for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode: User Bulletin: Veriti
™
96-Well Thermal Cycler
AmpF l STR
™
Kit Validation (Part no. 4440754).
• When using the ProFlex ™ PCR System, refer to the ProFlex ™ PCR System Kit
Validation User Bulletin (Pub. no. 100031595) for more information.
Final hold Initial incubation step
HOLD
95
°
C
11 min
Cycle (29/30 cycles)
Denature Anneal
94
°
C
20 sec
CYCLE
59
°
C
3 min
Final extension
HOLD
60
°
C
10 min
HOLD
4
°
C
∞
22
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 2 PCR Amplification
Amplification using bloodstained FTA
™
cards
2
IMPORTANT!
The NGM ™ Kit is validated for use at both 29 and 30 cycles. The optimum conditions for the NGM ™ Kit are 29 cycles of amplification with a 1 ng input DNA concentration. If using the NGM ™ Kit at 30 cycles, reduce the input
DNA concentration to 500 pg. Perform internal validation studies to evaluate kit performance at each cycle number intended for operational use.
2.
Load the plate or tubes into the thermal cycler and close the heated cover.
IMPORTANT!
If using the 9700 thermal cycler with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, be sure to place a MicroAmp
™
compression pad (Part no. 4312639) on top of the plate to prevent evaporation during thermal cycling.
3.
Start the run.
4.
On completion of the run, store the amplified DNA and protect from light.
If you are storing the DNA...
< 2 weeks
> 2 weeks
Then place at...
2 to 8°C
–15 to –25°C
IMPORTANT!
Store the amplified products so that they are protected from light.
Amplification using bloodstained FTA
™
cards
FTA
™
cards can be useful for the collection, storage, and processing of biological samples. A small punch disc of the card containing the sample can be placed directly into an amplification tube, purified, and amplified, without transferring the disc. Our studies indicate that a 1.2-mm bloodstained disc contains approximately 5–20 ng
DNA. An appropriate cycle number for this high quantity of DNA is 24 cycles, determined by our validation studies. Perform internal validation studies to evaluate kit performance at each cycle number intended for operational use.
In the example shown in Figure 4, a 1.2-mm disc of a bloodstained FTA
™
card was purified using three washes with FTA
™
Purification Reagent and two washes with
1 ✕ low-TE buffer. The punch was then amplified directly in the MicroAmp
™
tube for
24 cycles.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
23
2
Chapter 2 PCR Amplification
Amplification using bloodstained FTA
™
cards
Figure 4 NGM ™ Kit results from a 1.2-mm FTA
™
bloodstain disc (24-cycle amplification), analyzed on the Applied Biosystems
™
3130 xl Genetic Analyzer
24
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
3
Electrophoresis
■
■
■
Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis . . 27
Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
■
Set up the 3500/3500xL instrument for electrophoresis . . . . . . . . . . . . . . . . . . . . . 29
■
Prepare samples for electrophoresis on the 3500/3500xL instrument. . . . . . . . . . 29
■
■
Prepare samples for electrophoresis on the 310 instrument . . . . . . . . . . . . . . . . . 31
Allelic ladder requirements
To accurately genotype samples, you must run an allelic ladder sample along with the unknown samples.
Instrument
Number of allelic ladders to run
One injection equals
Number of samples per allelic ladder(s)
3100Avant or 3130 1 per 4 injections 4 samples
3100 or 3130 xl 1 per injection 16 samples
15 samples + 1 allelic ladder
15 samples + 1 allelic ladder
3500
3500xL
310
1 per 3 injections
1 per injection
1 per 10 samples
8 samples
24 samples
1 sample
23 samples + 1 allelic ladder
23 samples + 1 allelic ladder
9 samples + 1 allelic ladder
IMPORTANT!
Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single- and multiple-capillary runs
(with larger size variations seen between samples injected in multiple-capillary runs).
We recommend the above frequency of allelic ladder injections, which should account for normal variation in run speed. However, during internal validation studies, verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment.
25
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
3
Chapter 3 Electrophoresis
Allelic ladder requirements
It is critical to genotype using an allelic ladder run under the same conditions as the samples, because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions.
26
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Sectio n 3.1
3100/3100-Avant and 3130/3130xl instruments
Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis
3
Section 3.1
3100/3100Avant and 3130/3130 xl instruments
Set up the 3100/3100-
Avant
and 3130/3130
xl
instruments for electrophoresis
Reagents and parts
Appendix B, “Ordering Information” on page 107 lists the required materials not
supplied with the NGM ™ 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. Keep freeze-thaw cycles to a minimum.
Electrophoresis software setup and reference documents
Genetic
Analyzer
Data
Collection
Software
The following table lists data collection software and the run modules that can be used to analyze NGM ™ Kit PCR products. For details on the procedures, refer to the documents listed in the table.
Operating
System
Run modules and conditions References
Applied
Biosystems
™
3130/3130 xl
ABI P RISM
™
3100
ABI P RISM
™
3100Avant
3.0
†
2.0
1.1 Windows
™
NT
1.0
Windows
™
XP
Windows
™
2000
Windows
™
NT
• HIDFragmentAnalysis36_POP4_1
Injection conditions:
– 3130 = 3 kV/5 sec
– 3130 xl = 3 kV/10 sec
• Dye Set G5
• HIDFragmentAnalysis36_POP4_1
Injection condition: 3kV/10 sec
• Dye Set G5
• GeneScan36vb_DyeSetG5Module
Injection condition: 3kV/10 sec
• GS600v2.0Analysis.gsp
• GeneScan36Avb_DyeSetG5Module
Injection condition: 3 kV/5sec
• GS600v2.0Analysis.gsp
Applied Biosystems
™
3130/3130xl
Genetic Analyzers Using Data
Collection Software v3.0, Protocols for Processing AmpF l STR
™
PCR
Amplification Kit PCR Products User
Bulletin (Part no.
4363787)
ABI P RISM
™
3100/3100-Avant
Genetic Analyzers Using Data
Collection Software v2.0, Protocols for Processing AmpF l STR
™
PCR
Amplification Kit PCR Products User
Bulletin (Part no.
4350218)
ABI P RISM
™
3100/3100-Avant Genetic
Analyzers Protocols for Processing
AmpF l STR
™
PCR Amplification Kit
PCR Products User Bulletin
(Part no.
4332345)
ABI P RISM
™
3100/3100-Avant Genetic
Analyzers Protocols for Processing
AmpF l STR
™
PCR Amplification Kit PCR
Products User Bulletin
(Part no.
4332345)
† We conducted validation studies for the <Short form of the primary product name> using this configuration.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
27
3
Chapter 3 Electrophoresis
Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments
Prepare samples for electrophoresis on the 3100/3100-
Avant
or
3130/3130
xl
instruments
Prepare the samples for electrophoresis on the 3100/3100Avant or 3130/3130 xl immediately before loading.
1.
Calculate the volume of Hi-Di ™ Formamide and size standard needed to prepare the samples:
Reagent
GS 500 LIZ
GS 600 LIZ
™
™
Size Standard or
Size Standard v2.0
Hi-Di ™ Formamide
Volume per reaction
0.5 µL
9.5 µL
Note:
Include additional samples in your calculations 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.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
3.
Vortex the tube, then centrifuge briefly.
4.
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 Allelic Ladder
Note:
For blank wells, add 11 µL of Hi-Di ™ Formamide.
5.
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.
6.
Heat the reaction plate in a thermal cycler for 3 minutes at 95°C.
7.
Immediately place the plate on ice for 3 minutes.
8.
Prepare the plate assembly on the autosampler.
9.
Start the electrophoresis run.
28
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chap ter 3 Electrophoresis
Set up the 3500/3500xL instrument for electrophoresis
Section 3.2
3500/3500xL instruments
3
Set up the 3500/3500xL instrument for electrophoresis
Reagents and parts
Appendix B, “Ordering Information” on page 107 lists the required materials not
supplied with the NGM ™ 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. Keep freeze-thaw cycles to a minimum.
Electrophoresis software setup and reference documents
Genetic
Analyzer
Data
Collection
Software
The following table lists Data Collection Software and the run modules that can be used to analyze NGM ™ Kit PCR products. For details on the procedures, refer to the documents listed in the table.
Operating
System
Run modules and conditions References
Applied
Biosystems
™
3500
Applied
Biosystems
™
3500xL
3500 Data
Collection
Software v1.0
Windows
™
XP or
Windows
Vista
™
• HID36_POP4
Injection conditions: 1.2kV/15 sec
• Dye Set G5
• HID36_POP4
Injection conditions: 1.2kV/24 sec
• Dye Set G5
Applied Biosystems
™
3500/
3500xL Genetic Analyzer User
Guide (Part no. 4401661)
3500 and 3500xL Genetic
Analyzers Quick Reference Card
(Part no. 4401662)
Prepare samples for electrophoresis on the 3500/3500xL instrument
Prepare the samples for capillary electrophoresis on the 3500/3500xL instrument immediately before loading.
1.
Calculate the volume of Hi-Di
™
Formamide and Size Standard needed to prepare the samples, using the table below.
Reagent
GeneScan ™ 600 LIZ
™
Size Standard v2.0
Hi-Di ™ Formamide
Volume per reaction
0.5 µL
9.5 µL
Note:
Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
29
3
Chapter 3 Electrophoresis
Prepare samples for electrophoresis on the 3500/3500xL instrument
IMPORTANT!
The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your results and experiments.
2.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
3.
Vortex the tube, then centrifuge briefly.
4.
Into each well of a MicroAmp
™
Optical 96-Well Reaction Plate, or each MicroAmp
™
optical strip tube, add: a.
10 µL of the formamide: size standard mixture b.
1 µL of PCR product or allelic ladder
Note:
For blank wells, add 11 µL of Hi-Di ™ Formamide.
5.
Seal the reaction plate or strip tubes with the appropriate septa, then centrifuge to ensure that the contents of each well are collected at the bottom.
6.
Heat the reaction plate or strip tubes in a thermal cycler for 3 minutes at 95°C.
7.
Immediately put the plate or strip tubes on ice for 3 minutes.
8.
Prepare the plate assembly, then put it onto the autosampler.
9.
Ensure that a plate record is completed and link the plate record to the plate.
10.
Start the electrophoresis run.
30
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chap ter 3 Electrophoresis
Set up the 310 instrument for electrophoresis
3
Section 3.3
310 instruments
Set up the 310 instrument for electrophoresis
Reagents and parts
Appendix B, “Ordering Information” on page 107 lists the required materials not
supplied with the NGM ™ 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. Keep freeze-thaw cycles to a minimum.
Electrophoresis software setup and reference documents
Data
Collection
Software
Operating
System
The following table lists Data Collection Software and the run modules that can be used to analyze NGM ™ Kit PCR products. For details on the procedures, refer to the documents listed in the table.
Run modules and conditions References
3.1
† or
3.0
Windows XP or
Windows
NT
™
and
Windows
2000
• GS STR POP4 (1mL) G5 v2.md5
Injection condition:
15 kV/5 sec
ABI P RISM
™
310 Genetic Analyzer User’s Manual
(Windows) (Part no.
4317588)
ABI P
AmpF
RISM
™ l STR
™
310 Protocols for Processing
PCR Amplification Kit Products with Microsoft Windows NT Operating System:
User Bulletin (Part no.
4341742)
† We conducted concordance studies for the NGM ™ Kit using this configuration.
Prepare samples for electrophoresis on the 310 instrument
Prepare the samples for capillary electrophoresis on the 310 instrument immediately before loading.
1.
Calculate the volume of Hi-Di ™ Formamide and Size Standard needed to prepare the samples, using the table below.
Reagent
GeneScan ™ 500 LIZ
™
GeneScan
™
600 LIZ
™
Size Standard or
Size Standard v2.0
Hi-Di
™
Formamide
Volume per reaction
0.75 µL
24.25 µL
Note:
Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
31
3
Chapter 3 Electrophoresis
Prepare samples for electrophoresis on the 310 instrument
IMPORTANT!
The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your results and experiments.
2.
3.
Vortex the tube, then centrifuge briefly.
4.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Into each 0.2-mL or 0.5-mL sample tube, add: a.
25 µL of the formamide: size standard mixture b.
1.5 µL of PCR product or allelic ladder
Note:
For blank wells, add 25 µL of Hi-Di ™ Formamide.
5.
Seal the tubes with the appropriate septa, then briefly centrifuge to ensure that the contents of each tube are mixed and collected at the bottom.
6.
Heat the tubes in a thermal cycler for 3 minutes at 95°C.
7.
Immediately place the tubes on ice for 3 minutes.
8.
Place the sample tray on the autosampler.
9.
Ensure that an injection list is prepared.
10.
Start the electrophoresis run.
32
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
4
Data Analysis
ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
■
■
■
ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . 34
Analyze and edit sample files with GeneMapper
ID Software. . . . . . . . . . . . . . 43
■
■
ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
■
■
■
ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . 46
Analyze and edit sample files with GeneMapper
ID-X Software. . . . . . . . . . . . 57
■
■
Section 4.1
GeneMapper ™ ID Software
Overview of GeneMapper
™
ID
Software
GeneMapper
™
ID Software is an automated genotyping software for forensic casework, databasing, and paternity data analysis.
After electrophoresis, the Data Collection Software stores information for each sample in an .fsa file. Using GeneMapper
™
ID Software v3.2.1 software, you can then analyze and interpret the data from the .fsa files.
Instruments
Refer to “Instrument and software overview” on page 16 for a list of compatible
instruments.
33
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
4
Chapter 4 Data Analysis
Set up GeneMapper
™
ID S of tw are for data analysis
Before you start
When using GeneMapper
™
ID Software v3.2.1 to perform human identification
(HID) analysis with AmpF l STR
™
kits, be aware that:
• HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis.
For multiple ladder samples, the GeneMapper
™
ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within 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 ladder(s) within 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. Failure to apply this setting for ladder samples results in failed analysis.
• 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 AmpF l STR
™
Allelic Ladders do exist. Off-ladder (OL) alleles may contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the ±0.5-nt 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’s protocol.
Set up GeneMapper
™
ID
Software for data analysis
File names
The file names shown in this section may differ from the file names you see when you download or import files. If you need help determining the correct files to use, contact your local Life Technologies Human Identification representative, or go to www.appliedbiosystems.com
.
Overview
To analyze sample (.fsa) files using GeneMapper
™
ID Software v3.2.1 for the first time:
1.
Import panels and bins into the Panel Manager, as explained in “Import panels and bins” on page 35.
2.
Create an analysis method, as explained in “Create an analysis method” on page 38.
3.
Create a size standard, as explained in “Create a size standard” on page 42.
4.
Define custom views of analysis tables.
Refer to the GeneMapper
™
ID Software Versions 3.1 and 3.2 Human Identification
Analysis Tutorial (Part no.
4335523) for more information.
5.
Define custom views of plots.
Refer to the GeneMapper
™
ID Software Versions 3.1 and 3.2 Human Identification
Analysis Tutorial (Part no.
4335523) for more information.
34
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
S ection 4.1
GeneMapper
™
ID Software
Set up GeneMapper
™
ID Software for data analysis
4
Import panels and bins
To import the NGM ™ Kit panel and bin set from www.appliedbiosystems.com
into the GeneMapper
™
ID Software v3.2.1 database:
1.
Download and open the file containing panels and bins: a.
From the Support menu of www.appliedbiosystems.com
, select
Support Software Downloads, Patches & Updates GeneMapper
™
ID
Software v 3.2 Updates & Patches , and download the file NGM Analysis
Files GMID .
b.
Unzip the file.
2.
Start the GeneMapper
™
ID Software, then log in with the appropriate user name and password.
IMPORTANT!
For logon instructions, refer to the GeneMapper
™
ID Software
Version 3.1 Human Identification Analysis User Guide (Part no.
4338775).
3.
Select Tools Panel Manager .
4.
Find, then open the folder containing the panels, bins, and marker stutter: a.
Select Panel Manager in the navigation pane.
b.
Select File Import Panels to open the Import
Panels dialog box.
c.
Navigate to, then open the NGM Analysis Files
GMID
folder that you unzipped in step 1 above.
5.
Select NGM_panel_v2.txt
, then click Import .
Note:
Importing this file creates a new folder in the navigation pane of the Panel
Manager, AmpFLSTR_NGM_v2. This folder contains the panel and associated markers.
6.
Import NGM_bins_v2.txt:
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
35
36
4
Chapter 4 Data Analysis
Set up GeneMapper
™
ID S of tw are for data analysis a.
Select the AmpFLSTR_NGM_v2 folder in the navigation pane.
b.
Select File Import Bin Set to open the Import Bin Set dialog box.
c.
Navigate to, then open the NGM Analysis Files GMID folder.
d.
Select NGM_bins_v2.txt
, then click Import .
Note:
Importing this file associates the bin set with the panels in the
NGM_panel_v2 folder.
7.
View the imported panels in the navigation pane: a.
Double-click the AmpFLSTR_NGM_v2 folder to view the NGM_panel_v2 folder.
b.
Double-click the NGM_panel_v2 folder to display the panel information in the right pane.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
S ection 4.1
GeneMapper
™
ID Software
Set up GeneMapper
™
ID Software for data analysis
4
8.
Select D10S1248 to display the Bin view for the marker in the right pane.
9.
Click Apply , then OK to add the NGM ™ Kit panel and bin set to the
GeneMapper
™
ID Software database.
IMPORTANT!
If you close the Panel Manager without clicking OK, the panels and bins are not imported into the GeneMapper
™
ID Software database.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
37
4
Cha pter 4 Data Analysis
Set up GeneMapper
™
ID S of tw are for data analysis
Create an analysis method
Use the following procedure to create an HID analysis method for the
AmpF l STR
™
NGM ™ Kit.
1.
Select Tools GeneMapper Manager to open the GeneMapper Manager.
General tab settings
2.
Select the Analysis Methods tab, then click New to open the New Analysis
Method dialog box.
3.
Select HID and click OK to open the Analysis Method Editor with the General tab selected.
Enter the settings shown in the figures on the following pages.
4.
After you enter settings in all tabs, click Save .
38
In the Name field, either type the name as shown for consistency with files supplied with other AmpF l STR
™
kits, or enter a name of your choosing. The
Description and Instrument fields are optional.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Allele tab settings
S ection 4.1
GeneMapper
™
ID Software
Set up GeneMapper
™
ID Software for data analysis
4
• In the Bin Set field, select the NGM_bins_v2 bin set imported previously and configure the stutter distance parameters as shown.
• GeneMapper
™
ID Software v3.2.1 allows you to specify four types of marker repeat motifs: tri, tetra, penta, and hexa. You can enter parameter values for each type of repeat in the appropriate column.
• The “Use marker-specific stutter ratio if available” check box is selected by default. Consequently, the software applies the stutter ratio filters supplied in the
NGM_panel_v2 file. GeneMapper
™
ID Software v3.2.1 specifies locus-specific filter ratios for minus stutters, but not for plus stutters, in the panel file. However, validation studies with the NGM ™ Kit show that the trinucleotide repeat
D22S1045 locus produces a relatively large amount of plus stutter compared to tetranucleotide repeat loci. The relatively large amount of stutter may cause the stutter peak to be labeled during routine analysis.
• The plus stutter at the D22S1045 locus can be filtered by assigning a global plus stutter filter for trinucleotide repeat loci in the Analysis Parameter file. Because
D22S1045 is the only trinucleotide repeat locus in the NGM ™ Kit, this stutter filter setting is applied only to plus stutter peaks at the D22S1045 locus. The settings shown above resulted in little or no labeling of D22S1045 plus stutter peaks during our validation studies. Perform internal validation studies to determine the settings to use in your laboratory.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
39
4
Cha pter 4 Data Analysis
Set up GeneMapper
™
ID S of tw are for data analysis
Peak Detector tab settings
Perform internal validation studies to determine settings
40
IMPORTANT!
Perform the appropriate internal validation studies to determine the peak amplitude thresholds for interpretation of NGM ™ Kit data.
Fields include:
• Peak amplitude thresholds – The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks. Although
GeneMapper
™
ID Software displays peaks that fall below the specified amplitude in electropherograms, the software does not label or determine the genotype of these peaks.
• Size calling method – The NGM ™ Kit has been validated using the 3
Least Squares sizing method in combination with the GeneScan
Standard. If using the GeneScan ™ 600 LIZ
™
™ rd Order
500 LIZ
™
Size
Size Standard v2.0, select the Local
Southern Method. Select alternative sizing methods only after you perform the appropriate internal validation studies.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Peak Quality tab settings
S ection 4.1
GeneMapper
™
ID Software
Set up GeneMapper
™
ID Software for data analysis
4
Perform internal validation studies to determine settings
IMPORTANT!
Perform the appropriate internal validation studies to determine the heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of NGM
™
Kit data.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
41
4
Cha pter 4 Data Analysis
Set up GeneMapper
™
ID S of tw are for data analysis
Quality Flags tab settings
Create a size standard
IMPORTANT!
The values shown are the software defaults and are the values we used during developmental validation. Perform the appropriate internal validation studies to determine the appropriate values to use in your laboratory.
The size standards for the NGM ™ Kit uses the following size standard peaks in their definitions:
500 LIZ
™
Size Standard
75, 100, 139, 150, 160, 200, 300, 350, 400, and
450
GeneScan ™ GeneScan ™ 600 LIZ
™
Size Standard v2.0
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
Note:
The 250-nt and the 340-nt peak are not included in the size standard definition.
These peaks can be used as an indicator of precision within a run.
Use the following procedure to create the size standard for the NGM ™ Kit.
42
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™
NGM ™ PCR Amplification Kit User Guide
S ection 4.1
GeneMapper
™
ID Software
Analyze and edit sample files with GeneMapper
™
ID Software
4
1.
Select Tools GeneMapper Manager to open the GeneMapper Manager.
2.
Select the Size Standards tab, then click New .
3.
Enter a name as shown below or enter a name of your choosing. In the Size
Standard Dye field, select Orange . In the Size Standard Table, enter the sizes
specified in on page 42. The example below is for the GeneScan
™
500 LIZ
™
Size
Standard.
Analyze and edit sample files with GeneMapper
™
ID
Software
1.
In the Project window, select File Add Samples to Project , then navigate to the disk or directory containing the sample files.
2.
Apply analysis settings to the samples in the project. The names of the settings shown are the names suggested in the sections above.
Parameter Settings
Sample Type Select the sample type.
Analysis Method NGM_AnalysisMethod_v2 (or the name of the analysis method you created)
Panel
Size Standard
NGM_panel_v2
CE_G5_NGM_GS500 (or the name of the size standard you created)
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™
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4
Chapter 4 Data Analysis
Examine and edit a project
For more information about how the Size Caller works, refer to the ABI Prism
™
GeneScan
™
Analysis Software for the Windows NT
™
Operating System Overview of the Analysis Parameters and Size Caller User Bulletin (Part no. 4335617). For additional information about size standards, refer to the GeneMapper
™
ID
Software Version 3.1 Human Identification Analysis User Guide (Part no. 4338775).
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 both:
– A completion bar extending to the right with the percentage completed indicated
– With text messages on the left
• The table displays the row of the sample currently being analyzed in green
(or red if analysis failed for the sample).
• The Genotypes tab becomes available after analysis.
Examine and edit a project
You can display electropherogram plots from the Samples and Genotypes tabs of the
Project window to examine the data. These procedures start with the Samples tab of the Project window (assuming the analysis is complete).
For more information
For details about GeneMapper
™
ID Software features, allele filters, peak detection algorithms, and project editing, refer to:
• GeneMapper
™
ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial
(Part no.
4335523)
• GeneMapper
™ no.
4338775)
ID Software Version 3.1 Human Identification Analysis User Guide (Part
• Installation Procedures and New Features for GeneMapper
™
Version v3.2 User Bulletin (Part no.
4352543)
ID Software Software
44
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™
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Chapter 4 GeneMapper
™
ID-X Software
Overview of GeneMapper
™
ID-X Software
Section 4.2
GeneMapper ™ ID-X Software
4
Overview of GeneMapper
™
ID-X
Software
GeneMapper
™
ID-X Software is an automated genotyping software for forensic casework, databasing, and paternity data analysis.
After electrophoresis, the data collection software stores information for each sample in a .fsa file. Using GeneMapper
™
ID-X Software v1.0.1 or higher you can then analyze and interpret the data from the .fsa or .hid files.
Note:
.hid files can only be analyzed using GeneMapper
™
ID-X Software v1.2 or higher
Instruments
Refer to “Instrument and software overview” on page 16 for a list of compatible
instruments.
Before you start
When using GeneMapper
™
ID-X Software v1.0.1 or higher to perform human identification (HID) analysis with AmpF l STR
™
kits, be aware that:
• HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis.
For multiple ladder samples, the GeneMapper
™
ID-X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within 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 ladder(s) within 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. Failure to apply this setting for ladder samples results in failed analysis.
• 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 AmpF l STR
™
Allelic Ladders do exist. Off-ladder (OL) alleles may contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the ±0.5-nt 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.
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™
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45
4
Cha pter 4 GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X S of tw are for data analysis
Set up GeneMapper
™
ID-X
Software for data analysis
File names
The file names shown in this section may differ from the file names you see when you download or import files. If you need help determining the correct files to use, contact your local Life Technologies Human Identification representative, or go to www.appliedbiosystems.com
.
Overview
To analyze sample (.fsa) files using GeneMapper
™
ID-X Software v1.0.1 or higher for the first time:
1.
Import panels, bins, and marker stutter into the Panel Manager, as explained in
“Import panels, bins, and marker stutter” below.
2.
Create an analysis method, as explained in “Create an analysis method” on page
3.
Create a size standard, as explained in “Create a size standard” on page 55.
4.
Define custom views of analysis tables.
Refer to the GeneMapper
™
ID-X Software Version 1.0 Getting Started Guide
(Part no.
4375574) for more information.
5.
Define custom views of plots.
Refer to the GeneMapper
™
ID-X Software Version 1.0 Getting Started Guide
(Part no.
4375574) for more information.
Import panels, bins, and marker stutter
To import the NGM ™ Kit panels, bin sets, and marker stutter from the Applied
Biosystems web site into the GeneMapper
™
ID-X Software database:
1.
Download and open the file containing panels, bins, and marker stutter: a.
From the Support menu of www.appliedbiosystems.com
, select
Support Software Downloads, Patches & Updates GeneMapper
™
ID-X
Software Updates & Patches , and download the file NGM Analysis Files
GMIDX .
b.
Unzip the file.
2.
Start the GeneMapper
™
ID-X Software, then log in with the appropriate user name and password.
IMPORTANT!
For logon instructions, refer to the GeneMapper
™
ID-X
Software Version 1.0 Getting Started Guide (Part no.
4375574).
3.
Select Tools Panel Manager .
4.
Find, then open the folder containing the panels, bins, and marker stutter: a.
Select Panel Manager in the navigation pane.
b.
Select File Import Panels to open the Import
Panels dialog box.
c.
Navigate to, then open the NGM Analysis Files
GMIDX
folder that you unzipped in step 1 of this
section.
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™
NGM ™ PCR Amplification Kit User Guide
Secti on 4.2
GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X Software for data analysis
4
5.
Select NGM_panel_v2X , then click Import .
Note:
Importing this file creates a new folder in the navigation pane of the Panel
Manager “AmpFLSTR_NGM_v2X”. This folder contains the panel and associated markers.
6.
Import NGM_bins_v2X.txt: a.
Select the AmpFLSTR_NGM_v2X folder in the navigation pane.
b.
Select File Import Bin Set to open the Import Bin Set dialog box.
c.
Navigate to, then open the NGM Analysis Files GMIDX folder.
d.
Select NGM_bins_v2X.txt
, then click Import .
Note:
Importing this file associates the bin set with the panels in the
NGM_panel_v2X folder.
7.
View the imported panels in the navigation pane:
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™
NGM ™ PCR Amplification Kit User Guide
47
4
Cha pter 4 GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X S of tw are for data analysis a.
Double-click the AmpFLSTR_NGM_v2X folder.
b.
Double-click the NGM_panel_v2X folder to display the panel information in the right pane and the markers below it.
8.
Select D10S1248 to display the Bin view for the marker in the right pane.
48
9.
Import NGM_stutter_v2X.txt:
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Secti on 4.2
GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X Software for data analysis
4 a.
Select the AmpFLSTR_NGM_v2 folder in the navigation panel.
b.
Select File Import Marker Stutter to open the Import Marker Stutter dialog box.
c.
d.
Navigate to, then open the NGM Analysis Files GMIDX folder.
Select NGM_stutter_v2X.txt
, then click Import .
Note:
Importing this file associates the marker stutter ratio with the bin set in the NGM_bins_v2X folder.
10.
View the imported marker stutters in the navigation pane: a.
Select the NGM_panel_v2X folder to display its list of markers in the right pane.
b.
Double-click the it.
NGM_panel_v2X folder to display its list of markers below c.
Double-click D22S1045 to display the Stutter Ratio & Distance view for the marker in the right pane.
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™
NGM ™ PCR Amplification Kit User Guide
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4
Cha pter 4 GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X S of tw are for data analysis
Because D22S1045 has a trinucleotide repeat unit, it produces a higher level of plus stutter than tetranucleotide markers, and so requires the use of a plus stutter filter. The settings for the D22S1045 plus stutter filter can be seen in the table in the right pane. Other markers may not require a plus stutter filter, in which case the settings for plus stutter are left blank.
11.
Click Apply , then OK to add the NGM
™
Kit panels, bin sets, 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 will not be imported into the GeneMapper
™
ID-X Software database.
Create an analysis method
Use the following procedure to create an analysis method for the NGM
™
Kit.
IMPORTANT!
Analysis methods are version-specific, so you must create an analysis method for each version of the software. For example, an analysis method created for GeneMapper
™ of GeneMapper
™
ID-X Software version 1.2 is not compatible with earlier versions
ID-X Software, or with GeneMapper
™
ID Software version 3.2.1.
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™
NGM ™ PCR Amplification Kit User Guide
Secti on 4.2
GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X Software for data analysis
4
1.
Select Tools GeneMapper
™
GeneMapper
™
ID-X Manager to open the
ID-X Manager.
General tab settings
2.
Select the Analysis Methods tab, then click New to open the Analysis Method
Editor with the General tab selected.
3.
Enter the settings shown in the figures on the following pages.
Note:
you finish entering settings in all of the tabs.
4.
After you enter the settings on all tabs, click Save .
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™
NGM ™ PCR Amplification Kit User Guide
51
4
Cha pter 4 GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X S of tw are for data analysis
In the Name field, either type the name as shown for consistency with files supplied with other AmpF l STR
™
kits or enter a name of your choosing. In the Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. The Description and Instrument fields are optional.
Allele tab settings
52
• In the Bin Set field, select the NGM_bins_v2X bin set imported previously and configure the stutter distance parameters as shown.
• GeneMapper
™
ID-X Software allows you to specify 4 types of marker repeat motifs: tri, tetra, penta, and hexa. You can enter parameter values for each type of repeat in the appropriate column.
• The “Use marker-specific stutter ratio if applicable” check box is selected by default. When this box is checked, the software applies the stutter ratio filters in the NGM_stutter_v2X.txt file.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Peak Detector tab settings
Secti on 4.2
GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X Software for data analysis
4
Perform internal validation studies to determine settings
IMPORTANT!
Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of NGM
™
Kit data.
Fields include:
• Peak amplitude thresholds – The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks. 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.
• Size calling method – The NGM ™ Kit has been validated using the 3 rd Order
Least Squares sizing method with the GeneScan ™ 500 LIZ
™
Size Standard. If you use GeneScan ™ 600 LIZ
™
Size Standard v2.0, select the Local Southern Method.
Select alternative sizing methods only after you perform the appropriate internal validation studies.
• Normalization (v1.2 or higher) – For use with 3500 data. Perform internal validation studies to determine whether to use the Normalization feature for analysis of NGM ™ Kit data.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
53
4
Cha pter 4 GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X S of tw are for data analysis
Peak Quality tab settings
Perform internal validation studies to determine 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
NGM ™ Kit data.
54
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™
NGM ™ PCR Amplification Kit User Guide
SQ & GQ tab settings
Secti on 4.2
GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X Software for data analysis
4
Create a size standard
IMPORTANT!
The values shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use.
The size standards for the NGM ™ Kit uses the following size standard peaks in their definitions:
GeneScan ™ 500 LIZ
™
Size Standard
75, 100, 139, 150, 160, 200, 300, 350, 400, and
450
GeneScan ™ 600 LIZ
™
Size Standard v2.0
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
Note:
The 250-nt and the 340-nt peaks are not included in the size standard definition.
These peaks can be used as an indicator of precision within a run.
Use the following procedure to create the size standard for the NGM ™ Kit.
1.
Select Tools GeneMapper
™
ID-X Manager to open the GeneMapper
™
ID-X
Manager.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
55
4
Cha pter 4 GeneMapper
™
ID-X Software
Set up GeneMapper
™
ID-X S of tw are for data analysis
2.
Select the Size Standards tab, then click New .
3.
Complete the Name field as shown below or with a name of your choosing. In the
Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. In the Size Standard Dye field, select
Orange
. In the Size Standard Table, enter the sizes specified on page 55. The
example below is for the GeneScan ™ 500 LIZ
™
Size Standard.
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™
NGM ™ PCR Amplification Kit User Guide
Secti on 4.2
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 File Add Samples to Project , then navigate to the disk or directory containing the sample files.
2.
Apply analysis settings to the samples in the project. The names of the settings shown are the names suggested in the sections above. If you named the settings differently, select the names you specified.
Parameter
Sample Type
Analysis Method
Panel
Size Standard
Settings
Select the sample type.
NGM_AnalysisMethod_v2X (or the name of the analysis method you created)
NGM_panel_v2X
CE_G5_NGM_GS500 (or the name of the size standard you created)
For more information about how the Size Caller works, or about size standards, refer to the GeneMapper
™
ID-X Software v1.2 Reference Guide (Part no. 4426481A).
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 extending to the right with the percentage completed indicated.
• 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 upon completion of the analysis.
The figure below shows the analysis summary window after analysis.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
57
4
Chapter 4 GeneMapper
™
ID-X Software
Examine and edit a project
Examine and edit a project
You can display electropherogram plots from the Samples and Genotypes tabs of the
Project window to examine the data. These procedures start with the Analysis
Summary tab of the Project window (assuming the analysis is complete).
For more information
For more information about any of these tasks, refer to:
• For quick set-up instructions, refer to the GeneMapper
Getting Started Guide (Part no.
4375574).
™
ID-X Software Version 1.0
• For details about GeneMapper
™
ID-X Software features, allele filters, peak detection algorithms, and project editing, refer to:
– GeneMapper
™
ID-X Software Version 1.0 Getting Started Guide
(Part no.
4375574)
– GeneMapper
™
ID-X Software Version 1.0 Quick Reference Guide
(Part no.
4375670)
– GeneMapper
™
ID-X Software Version 1.0 Reference Guide (Part no.
4375671)
– GeneMapper
™
ID-X Software Version 1.1 (Mixture Analysis Tool) Getting Started
Guide (Part no.
4396773)
– GeneMapper
™
ID-X Software Version 1.1 (Mixture Analysis Tool) Quick
Reference Guide (Part no.
4402094)
– GeneMapper no.
4426482)
™
ID-X Software Version 1.2 Quick Reference Guide (Part
– GeneMapper
™
ID-X Software Version 1.2 Reference Guide (Part no. 4426481)
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™
NGM ™ PCR Amplification Kit User Guide
5
Experiments and Results
■
■
■
■
■
■
■
■
■
■
■
■
■
■
Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Overview
Importance of validation
Experiment conditions
Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure’s efficiency, reliability, and performance characteristics. By challenging the procedure with samples commonly encountered in forensic and parentage laboratories, the validation process uncovers attributes and limitations that are critical for sound data interpretation in casework (Sparkes, Kimpton, Watson et al.,
1996; Sparkes, Kimpton, Gilbard et al., 1996; Wallin et al., 1998).
We performed experiments to evaluate the performance of the NGM
™
Kit. The experiments were performed according to the revised guidelines from the Scientific
Working Group on DNA Analysis Methods (SWGDAM, July 10, 2003). Based on these guidelines, we conducted experiments that comply with guidelines 1.0 and 2.0 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).
59
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™
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5
Chapter 5 Experiments and Results
Developmental validation
This chapter discusses many of the experiments performed by us and provides examples of results obtained. We chose 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.
IMPORTANT!
Perform internal validation studies before using the NGM ™ Kit.
Developmental validation
SWGDAM guideline
1.2.1
“Developmental validation is the demonstration of the accuracy, precision, and reproducibility of a procedure by the manufacturer, technical organization, academic institution, government laboratory, or other party.” (SWGDAM, July 2003)
SWGDAM guideline
2.10.1
“The reaction conditions needed to provide the required degree of specificity and robustness must be determined. These include thermocycling parameters, the concentration of primers, magnesium chloride, DNA polymerase, and other critical reagents.” (SWGDAM, July 2003)
PCR components
We examined the concentration of each component of the NGM
™
Kit and established that the concentration of each component was within the range where data indicated that the amplification met the required performance criteria for specificity, sensitivity, and reproducibility. For example, 1.0 ng of Control DNA 007 was amplified in the presence of varying concentrations of magnesium chloride, and the results were analyzed on an Applied Biosystems
™
3130 xl Genetic Analyzer. Results are shown in
Figure 5. The performance of the multiplex is most robust within ±20% of the optimal
magnesium chloride concentration.
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AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Thermal cycler parameters
Chapter 5 Experiments and Results
Developmental validation
5
Figure 5 1.0
ng of control DNA 007 amplified for 29 cycles with the NGM ™ Kit in the presence of varying concentrations of magnesium chloride and analyzed on an
Applied Biosystems
™
3130 xl Genetic Analyzer
+ 30%
+ 20%
+ 10%
Optimal
–10%
–20%
– 30%
Thermal cycling parameters were optimized using a Design of Experiments (DOE) approach that attempts to identify the combination of temperatures and hold times that produce the best assay performance. Optimal assay performance was determined through evaluation of assay sensitivity, peak-height balance and resistance to PCR inhibitors.
For example, annealing temperatures of 55, 57, 59, 61, and 63°C were tested using a
Silver 96-Well GeneAmp
™
PCR System 9700 (Figure 6). The PCR products were
analyzed using an Applied Biosystems
™
3130 xl Genetic Analyzer.
Of the tested annealing temperatures, 55 to 61°C produced robust profiles. At 63°C the yield of the majority of loci was significantly reduced. 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 strongly recommended.
AmpF l STR
™
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61
5
Chapter 5 Experiments and Results
Developmental validation
Figur e 6 Electropherograms obtained from amplification of 1.0
ng of control DNA 007 for 29 cycles at annealing temperatures of 55, 57, 59, 61, and 63°C, analyzed on an
Applied Biosystems
™
3130 xl Genetic Analyzer, (Y-axis scale 0 to 3,000 RFU)
55 °C
57 °C
59 °C
61 °C
63 °C
PCR cycle number
NGM ™ Kit reactions were amplified for 27, 28, 29, 30, and 31 cycles on the Silver 96-
Well GeneAmp
™
PCR System 9700 using 1.0
ng of each of three DNA samples. 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 30 and 31 cycles (Figure 7).
The NGM ™ Kit was optimized for use with both 29 or 30 cycles and validated on the
Applied Biosystems
™
3130 xl Genetic Analyzer. The optimal DNA input amount per
PCR was found to be 1.0 ng and 0.5 ng, respectively, for 29- and 30-cycle amplifications. None of the cycle numbers tested produced nonspecific peaks.
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™
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Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
5
Figure 7 Representative NGM ™ Kit profiles obtained from amplification of 1.0
ng DNA template using 27, 28, 29, 30, and 31 cycles, analyzed on an Applied Biosystems
™
3130 xl
Genetic Analyzer, (Y-axis scale 0 to 8,000 RFU)
27 cycles
28 cycles
29 cycles
30 cycles
31 cycles
Accuracy, precision, and reproducibility
SWGDAM guideline
2.9
“The extent to which a given set of measurements of the same sample agree with their mean and the extent to which these measurements match the actual values being measured should be determined.” (SWGDAM, July 2003)
Accuracy
Laser-induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology (Holt et al., 2000; and Wallin et al., 2002). However, accuracy and reproducibility of NGM
™
Kit profiles have been determined from
various sample types. Figure 8 shows the size differences that are typically observed
between sample alleles and allelic ladder alleles on the Applied Biosystems
™
3130 xl
Genetic Analyzer with POP-4
™
polymer. The x-axis in Figure 8 represents the
nominal nucleotide sizes for the AmpF l STR
™
NGM
™
Allelic Ladder. The dashed 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.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
63
5
Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
Figure 8 Allele Size vs. Allelic Ladder Sizing for 42 samples analyzed on an Applied Biosystems
™
3130 xl Genetic
Analyzer.
Size and ladder sizing for the NGM ™ Kit were calculated using the GeneScan ™ 500 LIZ
™
Size Standard using the 3rd
Order Least Squares Method
Precision and size windows
Sizing precision enables the determination of accurate and reliable genotypes. Sizing precision was measured on an Applied Biosystems
™
3130 xl Genetic Analyzer. The recommended method for genotyping is to employ a ±0.5-nt “window” around the size obtained for each allele in the AmpF l STR
™
NGM ™ 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:
• An “off-ladder” allele, that is, an allele of a size that is not represented in the
AmpF l STR
™
NGM ™ Allelic Ladder or
• An allele that does correspond to an allelic ladder allele, 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 or in several lanes of one gel.
Table 7 on page 86 shows typical precision results obtained from five runs
(16 capillaries/run) of the AmpF l STR
™
NGM ™ Allelic Ladder on an
Applied Biosystems
™
3130 xl Genetic Analyzer (36-cm capillary and POP-4 ™ polymer), using the GeneScan ™ 500 LIZ
™
Size Standard. The results were obtained within a set of injections on a single capillary array.
64
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
5
Sample alleles may 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 having the smallest standard deviations in
sizing. Figure 8 on page 64 illustrates the tight clustering of allele sizes obtained on the
Applied Biosystems
™
3130 xl Genetic Analyzer, 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).
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 with an allele in the allelic ladder. Repeat analysis, when necessary, provides an added level of confidence in the final allele assignment.
GeneMapper
™
ID Software and GeneMapper
™
ID-X Software automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling 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 a number of 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.
We strongly recommend that the allele sizes be compared to the sizes obtained for known alleles in the AmpF l STR
™
NGM ™ Allelic Ladder from the same run and then
be converted to genotypes (as described in “Before you start” on page
45). See Table 4 for the results of five runs of the AmpF l STR
on an Applied Biosystems
™
3130 xl Genetic Analyzer. For more information on precision and genotyping, see Lazaruk et al., 1998 and Mansfield et al.
, 1998.
In Table 4, the mean sizes for all the alleles in each run (16 capillaries) were calculated.
The mean range shown in the table represents the lowest- and highest-mean size values obtained across all five runs. Similarly, the standard deviation for the allele sizing was calculated for all the alleles in each run. The standard deviation range
shown in Table 4 represents the lowest and highest standard deviation values obtained
across all five runs.
Table 4 Precision results of five runs (16 capillaries/run) of the AmpF l STR
™
NGM
™
Allelic
Ladder
Mean Standard Dev.
Allele
Amelogenin
X
Y
D10S1248
11
12
13
14
8
9
10
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
100.54–100.68
106.21–106.34
76.64–76.67
80.85–80.9
85.03–85.09
89.19–89.25
93.31–93.37
97.4–97.48
101.41–101.48
0.052–0.064
0.045–0.061
0.031–0.045
0.029–0.04
0.024–0.045
0.032–0.043
0.027–0.046
0.035–0.049
0.029–0.053
65
66
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Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
Allele (continued)
15
16
17
18
D12S391
19.3
20
21
22
14
15
16
17
18
19
23
24
25
26
27
D16S539
13
14
15
5
8
9
10
11
12
D18S51
7
9
10
10.2
11
12
13
Mean
105.32–105.4
109.24–109.31
113.18–113.24
117.14–117.21
229.97–230.12
233.9–234.05
237.87–238.01
241.75–241.91
245.68–245.83
249.59–249.73
252.59–252.75
253.54–253.68
257.45–257.58
261.3–261.47
265.27–265.43
269.29–269.44
273.28–273.42
277.22–277.38
281.31–281.45
Standard Dev.
0.03–0.05
0.035–0.046
0.032–0.048
0.025–0.044
0.042–0.056
0.035–0.057
0.036–0.063
0.046–0.051
0.042–0.056
0.038–0.062
0.034–0.054
0.034–0.058
0.04–0.052
0.037–0.05
0.039–0.063
0.039–0.05
0.034–0.059
0.045–0.053
0.049–0.066
228.68–228.78
240.7–240.83
244.72–244.83
248.73–248.84
252.73–252.87
256.78–256.89
260.8–260.93
264.82–264.95
268.86–268.99
0.04–0.05
0.04–0.052
0.05–0.055
0.048–0.059
0.046–0.063
0.04–0.055
0.046–0.061
0.037–0.056
0.04–0.054
262.24–262.34
270.36–270.49
274.43–274.56
276.45–276.56
278.5–278.62
282.56–282.71
286.64–286.78
0.04–0.068
0.046–0.062
0.04–0.063
0.05–0.065
0.048–0.069
0.059–0.075
0.038–0.073
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Allele (continued)
23
24
25
26
27
19
20
21
22
13.2
14
14.2
15
16
17
18
D19S433
13.2
14
14.2
15
9
10
11
12
12.2
13
15.2
16
16.2
17
17.2
D1S1656
9
10
11
12
13
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
5
127.1–127.2
130.98–131.06
134.88–134.96
138.83–138.9
140.96–141.03
143.04–143.13
145.21–145.28
147.28–147.37
149.44–149.52
151.48–151.57
153.54–153.63
155.53–155.64
157.58–157.68
159.55–159.67
161.54–161.66
175.77–175.96
179.67–179.85
183.53–183.71
187.39–187.57
191.24–191.4
Mean
288.67–288.79
290.76–290.88
292.75–292.9
294.83–294.98
298.95–299.08
303.1–303.23
307.24–307.39
311.39–311.52
315.53–315.65
319.66–319.8
323.86–323.99
327.92–328.04
332.03–332.16
336.16–336.27
340.27–340.39
344.39–344.5
0.027–0.05
0.033–0.05
0.032–0.052
0.023–0.045
0.027–0.043
0.03–0.049
0.029–0.041
0.026–0.051
0.03–0.047
0.028–0.04
0.036–0.047
0.032–0.048
0.039–0.051
0.04–0.056
0.039–0.047
0.046–0.09
0.047–0.08
0.054–0.08
0.056–0.078
0.04–0.069
Standard Dev.
0.057–0.073
0.046–0.069
0.061–0.075
0.055–0.082
0.056–0.077
0.053–0.088
0.06–0.079
0.066–0.086
0.056–0.087
0.062–0.077
0.063–0.082
0.061–0.076
0.058–0.079
0.056–0.085
0.072–0.079
0.057–0.077
67
68
5
Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
Allele (continued)
14
14.3
15
15.3
16
16.3
17
17.3
18.3
19.3
20.3
D21S11
31
31.2
32
32.2
29
29.2
30
30.2
24
24.2
25
26
27
28
28.2
35
35.2
36
37
38
33
33.2
34
34.2
D22S1045
8
Mean
195.13–195.29
198.01–198.16
198.87–199.02
201.86–202.01
202.72–202.88
205.76–205.91
206.63–206.79
209.66–209.82
213.56–213.73
217.48–217.64
221.4–221.56
185.02–185.14
187.02–187.12
188.98–189.08
192.92–193.01
196.87–196.96
200.75–200.83
202.71–202.81
204.69–204.78
206.73–206.82
208.71–208.8
210.68–210.78
212.7–212.79
214.67–214.76
216.69–216.78
218.65–218.75
220.67–220.76
222.62–222.72
224.74–224.85
226.65–226.74
228.7–228.8
230.64–230.73
232.62–232.74
236.68–236.78
240.61–240.71
79.99–80.08
0.034–0.058
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Standard Dev.
0.04–0.062
0.04–0.061
0.045–0.07
0.046–0.062
0.034–0.06
0.05–0.06
0.044–0.063
0.048–0.064
0.047–0.061
0.044–0.073
0.042–0.064
0.042–0.069
0.042–0.066
0.045–0.066
0.037–0.056
0.036–0.052
0.032–0.052
0.036–0.053
0.035–0.045
0.039–0.049
0.04–0.048
0.039–0.049
0.034–0.05
0.037–0.049
0.04–0.046
0.04–0.048
0.031–0.056
0.036–0.059
0.039–0.048
0.042–0.055
0.042–0.057
0.041–0.052
0.042–0.046
0.037–0.052
0.04–0.052
Allele (continued)
12
13
14
15
9
10
11
16
17
18
19
D2S1338
26
27
28
22
23
24
25
18
19
20
21
15
16
17
D2S441
13
14
15
16
9
10
11
12
D3S1358
12
13
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
5
289.59–289.7
293.68–293.78
297.73–297.82
301.81–301.9
305.9–306
310.03–310.11
314.12–314.22
318.22–318.31
322.3–322.39
326.38–326.46
330.46–330.55
334.54–334.61
338.63–338.71
342.95–343.05
80.29–80.38
84.41–84.5
88.51–88.62
91.66–91.76
92.58–92.69
96.49–96.62
100.51–100.63
104.4–104.53
Mean
83.08–83.18
86.14–86.25
89.2–89.33
92.26–92.37
95.29–95.43
98.33–98.46
101.3–101.42
104.2–104.32
107.13–107.24
110.06–110.17
113.02–113.13
134.88–134.99
138.98–139.12
0.038–0.054
0.036–0.053
0.042–0.055
0.046–0.064
0.043–0.06
0.047–0.057
0.042–0.062
0.034–0.06
0.044–0.062
0.044–0.058
0.038–0.06
0.047–0.056
0.046–0.057
0.037–0.061
0.028–0.054
0.033–0.057
0.03–0.062
0.046–0.067
0.045–0.062
0.036–0.069
0.045–0.067
0.038–0.056
Standard Dev.
0.037–0.058
0.041–0.063
0.043–0.065
0.047–0.067
0.045–0.069
0.044–0.07
0.054–0.066
0.043–0.064
0.054–0.068
0.052–0.069
0.046–0.059
0.043–0.064
0.042–0.064
69
70
5
Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
Allele (continued)
17
18
19
14
15
16
D8S1179
12
13
14
15
8
9
10
11
16
17
18
19
FGA
27
28
29
30
24
25
26
26.2
20
21
22
23
17
18
19
30.2
31.2
32.2
Mean
143.23–143.38
147.4–147.55
151.78–151.91
155.99–156.16
160.04–160.21
163.88–164.06
123.54–123.64
127.61–127.71
131.7–131.81
135.86–135.95
140.1–140.2
144.67–144.77
149.13–149.23
153.45–153.57
157.7–157.85
161.86–162
165.94–166.07
169.99–170.15
Standard Dev.
0.035–0.051
0.047–0.057
0.042–0.063
0.051–0.074
0.063–0.075
0.064–0.079
0.04–0.068
0.039–0.071
0.045–0.064
0.045–0.07
0.046–0.056
0.043–0.058
0.046–0.07
0.053–0.069
0.054–0.073
0.056–0.086
0.054–0.085
0.054–0.096
233.22–233.34
237.01–237.13
240.8–240.93
244.61–244.74
248.43–248.55
252.24–252.35
256.09–256.19
259.9–260.01
263.74–263.86
267.59–267.7
269.46–269.57
271.5–271.62
275.35–275.47
279.2–279.31
282.97–283.07
285.24–285.36
289.12–289.23
292.98–293.09
0.032–0.044
0.036–0.047
0.04–0.053
0.04–0.049
0.038–0.045
0.034–0.057
0.028–0.047
0.041–0.051
0.035–0.049
0.035–0.055
0.029–0.045
0.037–0.054
0.036–0.046
0.03–0.051
0.036–0.053
0.035–0.053
0.028–0.065
0.042–0.054
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
TH01
Allele (continued)
33.2
42.2
43.2
44.2
45.2
46.2
47.2
48.2
50.2
51.2
8
9
9.3
10
4
5
6
7
11
13.3
vWA
11
12
13
14
15
16
17
18
19
20
21
22
23
24
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 5 Experiments and Results
Accuracy, precision, and reproducibility
5
Mean
296.88–296.97
332.47–332.54
336.37–336.45
340.44–340.51
344.38–344.45
348.02–348.09
351.82–351.85
355.71–355.78
363.15–363.2
366.92–366.96
181.53–181.67
185.44–185.58
189.32–189.46
193.2–193.33
197.05–197.17
200.93–201.03
203.93–204.03
204.85–204.95
208.78–208.9
219.64–219.75
153.74–153.84
157.91–158.03
162.04–162.18
166.28–166.42
170.22–170.34
0.043–0.087
178.23–178.36
182.17–182.29
186.13–186.25
190.06–190.2
193.95–194.08
197.85–197.97
201.69–201.79
206.01–206.12
Standard Dev.
0.044–0.051
0.03–0.062
0.031–0.055
0.034–0.055
0.033–0.055
0.036–0.044
0.036–0.05
0.031–0.052
0.032–0.045
0.034–0.052
0.044–0.065
0.039–0.063
0.037–0.066
0.028–0.058
0.032–0.053
0.036–0.047
0.037–0.052
0.031–0.051
0.036–0.048
0.038–0.047
0.041–0.065
0.038–0.067
0.039–0.069
0.054–0.078
0.047–0.081
0.043–0.087
0.039–0.074
0.044–0.074
0.044–0.075
0.039–0.067
0.039–0.058
0.04–0.057
0.032–0.051
0.032–0.063
71
5
Chapter 5 Experiments and Results
Extra peaks in the electropherogram
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 DAB Standard 8.1.2.2).
Stutter products
Stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller (or less frequently, one repeat larger) than the major STR product (Butler, 2005; Mulero et al., 2006). Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele (Walsh et al., 1996).
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. Peak heights were measured for amplified samples (n = 1023) at the loci used in the NGM ™ Kit. All data were generated on the Applied Biosystems
™
3130 xl Genetic
Analyzer.
Due to the enhanced buffer system developed for the NGM
™ chemistries (for example, the SGM Plus
™
Kit, plus stutter peaks may be encountered at other loci more frequently than with older AmpF l STR
™
Kit). Laboratories should evaluate the occurrence of plus stutter peaks and develop appropriate interpretation guidelines as part of an internal validation study.
Some conclusions from these measurements and observations are:
• For each NGM
™
Kit locus, the percent stutter generally increases with allele length,
as shown in Figure 9 to Figure 13 on page 73 to 75.
• Smaller alleles display a lower level of stutter relative to the longer alleles within each locus.
• Each allele within a locus displays a percent stutter that is consistent.
• Stutter filter sets in GeneMapper
™
ID Software and GeneMapper
™
ID-X Software, calculated as the mean stutter for the locus plus three standard deviations
(n = 1023), are shown in Table 5 on page 75. Peaks in the stutter position that are
above the stutter filter percentage specified in the software are not filtered. Peaks in the stutter position that have not been filtered and remain labeled can be further
evaluated. For evaluation of mixed samples, see Figure 24 on page 90.
• The measurement of percent stutter for allele peaks that are off-scale may be unusually high due to artificial truncation of the main allele peak.
72
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™
NGM ™ PCR Amplification Kit User Guide
Chapter 5 Experiments and Results
Extra peaks in the electropherogram
5
Figur e 9 Stutter percentages for D10S1248, D12S391 and D16S539 loci (Blue and red colors indicate loci labeled with FAM ™ and PET ™ dyes respectively)
Figure 10 Stutter percentages for the D18S51, D19S433 and D1S1656 loci (Green, black, and red colors indicate loci labeled with VIC
™
, NED
™
, and PET
™
dyes, respectively)
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
73
5
Chapter 5 Experiments and Results
Extra peaks in the electropherogram
Figure 11 Stutter percentages for the D21S11, D22S1045 and D2S1338 loci (Green, black/ gray, and blue colors indicate loci labeled with VIC
™
, NED ™ and FAM ™ dyes, respectively.
Black and gray data points associated with the D22S1045 locus indicate minus- and plusstutter, respectively)
Figur e 12 Stutter percentages for the FGA and TH01 loci (Black data points indicate loci labeled with NED ™ dye)
74
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Chapter 5 Experiments and Results
Extra peaks in the electropherogram
5
Figure 13 Stutter percentages for the D2S441, D3S1358, D8S1179, and vWA loci (Red, green, and blue colors indicate loci labeled with and PET ™ , VIC
™
, and FAM ™ dyes, respectively)
Table
5
Marker-specific stutter filter percentages for NGM
™
Kit loci
Locus % Stutter
D10S1248 vWA
D16S539
D2S1338
D8S1179
D21S11
D18S51
D22S1045 (-3nt)
D22S1045 (+3 nt)
D19S433
12.39
11.83
10.12
12.83
10.31
10.87
14.08
18.05
7.63
11.20
TH01
FGA
D2S441
D3S1358
4.27
12.10
9.45
13.07
D1S1656 (-4nt)
D1S1656 (-2nt) †
14.46
4.70
D12S391 15.27
† The - 2nt stutter filter is not included in GeneMapper
™
ID Software v3.2.1NGM_panel_v2 due to functional limitations of the software.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
75
5
Chapter 5 Experiments and Results
Extra peaks in the electropherogram
Addition of 3´ A nucleotide
IMPORTANT!
The values shown are the values we determined during developmental validation studies. We recommend that laboratories perform internal validation studies to determine the appropriate values to use.
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 non-template 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.
The efficiency of +A addition is related to the particular sequence of the DNA at the 3´ end of the PCR product. The NGM ™ Kit includes two main design features that promote maximum +A addition:
• The primer sequences have been optimized to encourage +A addition.
• The new, highly robust PCR chemistry allows complete +A addition with a short final incubation at 60 °C for 10 minutes.
This final extension step gives the DNA polymerase additional time to complete +A
addition to all double-stranded PCR products. See Figure 14 on page 76 for examples
of incomplete and normal +A addition. Final extension incubation for longer than the recommended 10 minutes may 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, and is therefore to be avoided.
Figure 14 Omitting the final extension step results in shoulders on main allele peaks due to incomplete A nucleotide addition. Data are from an Applied Biosystems
™
3130 xl Genetic
Analyzer using the NGM
™
Kit
0 min. final extension
0 min. final extension
10 min. final extension
10 min. final extension
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™
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Artifacts
Chapter 5 Experiments and Results
Extra peaks in the electropherogram
5
Due to improved PCR buffer chemistry, the lack of +A addition is generally less an issue with the NGM ™ Kit than with earlier generation kits. However, “shouldering” of allele peaks may still be observed if the amount of input DNA is greater than that recommended by the NGM ™ Kit protocol. Amplification of excess input DNA may also result in offscale data.
Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible while anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise).
Due to improvements in PCR primer manufacturing processes, the incidence of artifacts has been greatly reduced in the NGM
™
Kit. NGM
™
Kit electropherograms are essentially free of reproducible dye artifacts within the Kit's read region of 68-407 nt.
Figure 15 on page 78 shows the very low baseline level fluorescence of a typical
negative control PCR using the NGM
™
Kit.
Most STR loci produce minus-stutter peaks as a by-product of PCR amplification. A process of “slippage” has been proposed as a molecular mechanism for stutter, where the Taq DNA polymerase enzyme “slips” on the template DNA during replication and produces a minority PCR product that is shorter than the template strand, usually by one repeat unit. The stutter process may also occur in the opposite direction to produce amplicon DNA that is usually one repeat unit longer than the template strand, termed plus-stutter. While 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. The D22S1045 locus in the NGM ™
Kit is a trinucleotide repeat locus, and shows an elevated level of plus-stutter. For
example, Figure 17 on page 79 is an electropherogram of the D22S1045 locus showing
plus stutter. GeneMapper
™
ID Software and GeneMapper
™
ID-X Software analysis parameter files supplied for use with the NGM ™ Kit contain a plus-stutter filter for the
D22S1045 locus to prevent these peaks from being called in normal profiles.
Plus stutter may also be visible at tetranucleotide repeat loci in next generation NGM
™
Kits due to improvements in buffer formulation over previous kits. Laboratories should evaluate the occurrence of plus stutter across the profile as part of internal validation studies.
Figure 16 on page 78 shows an example of a non-standard (minus 2-nt) stutter that
may be observed in certain STR loci such as D1S1656 with more complex nucleotide sequences that include regions of dinucleotide TG repeats. Genotyping may result in the detection of these artifacts as off-ladder (OL) alleles.
It is important to consider possible noise and artifacts when interpreting data from the
NGM
™
Kit on the Applied Biosystems
3100/3100Avant , and ABI P
™
3500/3500xL and 3130/3130 xl , ABI P RISM
™
RISM
™
310 Genetic Analyzers. Note that a high degree of
magnification is used in the sample electropherograms shown in Figure 15 to Figure 17
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5
Chapter 5 Experiments and Results
Extra peaks in the electropherogram
Figure 15 Examples of fluorescence background in data produced on an Applied Biosystems
™
3130 xl Genetic Analyzer,
(Y-axis scale 0–100 RFU)
Figure 16 Example of a –2 nt reproducible artifact at the D1S1656 locus. Data produced on an Applied Biosystems
™
3130 xl Genetic Analyzer
78
-2 nt stutter
Main allele peak
Normal -4 nt stutter
AmpF l STR
™
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Chapter 5 Experiments and Results
Characterization of loci
5
Figure 17 NGM ™ Kit electropherogram showing plus stutter associated with the D22S1045 STR locus. Data produced on an Applied Biosystems
™
3130 xl Genetic Analyzer
Main allele peak
Minus stutter
Plus stutter
Characterization of loci
SWGDAM guideline
2.1
“The basic characteristics of a genetic marker must be determined and documented.”
(SWGDAM, July 2003)
This section describes basic characteristics of the 15 loci and the sex-determining marker, Amelogenin, which are amplified with the NGM
™
Kit. Most of these loci have been extensively characterized by other laboratories.
Nature of the polymorphisms
The primers for the Amelogenin locus flank a 6-nucleotide deletion within intron 1 of the X homolog. Amplification results in 104-nt and 110-nt products from the X and Y chromosomes, respectively. (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.) With the sole exception of D22S1045, a trinucleotide STR, the remaining NGM ™ Kit loci are tetranucleotide short tandem repeat (STR) loci. The length differences among alleles of a particular locus result from differences in the number of repeat units.
All the alleles in the AmpF l STR
™
NGM ™ Allelic Ladder, including microvariants, have been subjected to sequencing at Life Technologies. 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
NGM ™ Kit loci, there is consensus on the repeat patterns and structure of the STRs.
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™
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5
Chapter 5 Experiments and Results
Characterization of loci
Inheritance
Mapping
Genetic linkage
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).
The NGM ™ Kit loci 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).
Two sets of STR loci in the NGM ™ Kit are located on the same chromosomes. vWA and
D12S391 are located approximately 6.3 million bp apart on the p arm of chromosome
12, while D2S1338 and D2S441 are located approximately 150 million bp apart on opposite arms of chromosome 2. Linkage disequilibrium analysis was conducted on
analyzed using the Linkage Disequilibrium module of GenePop software version
4.0.10 (Raymond and Rousset, 1995; Rousset, 2008). See Table 6 for results.
The relatively high probability values indicate that there is no statistically significant linkage disequilibrium found between the pairs of loci located on the same chromosome.
An independent analysis of the same set of population data by Budowle et al . (2010) concluded that there was no evidence of LD between or within any of the NGM ™ Kit, and that the multiplication rule could therefore be used for all markers contained in the 15 STR markers contained in the kit for the purpose of calculating the rarity of a
DNA profile. However, they cautioned that, while there was no evidence of LD at the population level, the independence of vWA and D12S391 could not be assumed for the purpose of kinship analysis, due to the proximity of the loci on Chromosome 12.
Table
6
GenePop LD Result (p value for pairwise analysis of loci)
Locus
Chromosome
Map
Position †
Chromosome
Nuclear
Coordinates
(million bp)
African-
American
(N = 350)
Caucasian
(N = 350)
Hispanic
(N = 293) vWA p13.31
D12S391 p13.2
D2S441 p14
D2S1338 q35
5.9
12.2
68
218
0.86
0.29
0.27
0.11
0.32
0.19
† STR locus mapping data was obtained from the NCBI Map Viewer http://www.ncbi.nlm.nih.gov/projects/ mapview/map_search.cgi?taxid=9606 or the UCSC Genome Browser ( http://genome.ucsc.edu/ ). GenePop
LD analysis probability results (p values) greater than 0.05 were considered to indicate that linkage disequilibrium between the loci within the population tested was not statistically significant.
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™
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Chapter 5 Experiments and Results
Species specificity
5
Species specificity
SWGDAM Guideline
2.2
“For techniques designed to type human DNA, the potential to detect DNA from forensically relevant nonhuman species should be evaluated.” (SWGDAM, July 2003)
The NGM ™ Kit provides the required specificity for detecting human alleles.
Nonhuman studies
Nonhuman DNA may be present in forensic casework samples. The following species were tested (in the specified amounts) using standard PCR and capillary electrophoresis conditions for the NGM ™ Kit.
• Primates: gorilla, chimpanzee, and macaque (1.0 ng each)
• Non-primates: mouse, dog, sheep, pig, rabbit, cat, horse, hamster, rat, chicken, and cow (5.0 ng each)
• Microorganisms: Candida albicans, Staphylococcus aureus, Escherichia coli,
Neisseria gonorrhoeae, Bacillus subtilis, and Lactobacillus rhamnosus (equivalent to 105 copies)
Results were assessed for the presence of any amplified peaks that would indicate cross reactivity of the NGM ™ Kit with any of these non-human species.
Figure 18 on page 82 shows example electropherogram results from the species
specificity tests. The chimpanzee and gorilla DNA samples produced partial profiles within the 70 – 283 nucleotide region (gorilla data not shown). Macaque DNA produced a strong Amelogenin-X peak and two small out-of-marker-range peaks in
PET (data not shown).
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™
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5
Chapter 5 Experiments and Results
Sensitivity
Figur e 18 Representative electropherograms from a species specificity study including positive and non-template controls
(NTC)
Chimpanzee
Dog
Cat
Horse
Rat
Microbes
NTC
The microorganisms, cow, sheep, pig, dog, cat, chicken, hamster, mouse, rabbit, and rat did not yield detectable products. Of the non-primates, only horse DNA produced a
96-bp fragment near the amelogenin locus in the VIC
™
dye.
Sensitivity
SWGDAM guideline
2.3
“When appropriate, the range of DNA quantities able to produce reliable typing results should be determined.” (SWGDAM, July 2003)
Importance of quantification
The recommended amount of input DNA for the NGM ™ quantification using either the Quantifiler
™
Kit is 1.0 ng, based on
Human or Quantifiler
™
Duo
Quantification Kit and individual laboratories should determine the optimum input
DNA amount according to the quantification method in use in the laboratory. If the sample contains degraded or inhibited DNA, amplification of a higher concentration
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™
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Sensitivity
5
of DNA may be beneficial. In Figure 19 on page 84, the control DNA 007 was serially
diluted from 2.0 ng to 0.062 ng. Full profiles (32 PCR products) were consistently obtained at 0.125 ng, but occasional partial profiles missing 1 – 3 alleles were observed at 0.062 ng.
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 instrument (“off-scale” data).
• Off-scale data. Off-scale data is 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 the corresponding stutter peak to appear higher in relative intensity, thus increasing the calculated percent stutter.
– Multicomponent analysis of off-scale data is not accurate. This inaccuracy results in poor spectral separation (“pull-up”).
• Incomplete +A nucleotide addition.
To address these issues, reamplify the sample using less DNA.
When the total number of allele copies added to the PCR is extremely low, unbalanced amplification of the alleles may occur because of stochastic fluctuation.
Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA.
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™
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5
Chapter 5 Experiments and Results
Stability
Figure 19 Electropherograms for 29-cycle amplifications using 2 ng, 1 ng, 0.50
ng, 0.25
ng, 0.125
ng, and 0.062
ng of control DNA 007. Electrophoresis was performed on an Applied Biosystems
™
3130 xl Genetic Analyzer. Note that the y-axis scale is magnified for the smaller input amounts of DNA
2 ng
1 ng
0.50 ng
0.25 ng
0.125 ng
0.062 ng
Stability
SWGDAM guideline
2.4
“The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults has been extensively documented. 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 using known samples to determine the effects of such factors.” (SWGDAM, July 2003)
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.
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Stability
5
Degraded DNA was prepared to examine the potential for differential amplification of loci. High-molecular-weight Raji DNA was sonicated and incubated with increasing doses of DNase I (0 to 6 Units) for 20 minutes (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 1.0 ng of degraded DNA using the NGM ™ 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.
Figure 20 Amplification of Raji DNA samples sonicated and incubated with increasing doses of DNase I. PCR amplification was done for 29 cycles, with electrophoresis being performed on an Applied Biosystems
™
3130 xl . Panels 1,
2, 3, and 4 correspond to 0, 4, 5, and 6 units of DNase I. Note that the y-axis scale is magnified for more degraded samples, which generate lower peak heights
0 U DNase I
4 U
5 U
6 U
The larger loci contained in the NGM ™ Kit, which fail to amplify in significantly degraded samples may be recovered by using the AmpF l STR
™
MiniFler ™ Kit. The amplicon size for the larger loci has been reduced to facilitate performance on degraded samples. For more information please refer to the AmpF l STR
™
MiniFler ™
PCR Amplification Kit User's Guide (Part no. 4374618).
Effect of inhibitors
— hematin
Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains (DeFranchis et al., 1988; Akane et al., 1994). It is believed that the inhibitor is co-extracted and co-purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity.
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™
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5
Chapter 5 Experiments and Results
Stability
To examine the effects of hematin on the performance of the NGM ™ Kit, 1.0 ng of control DNA 007 was amplified in the presence of increasing concentrations of
hematin for 29 cycles of amplification (Figure 21 on page 86).The concentrations of
hematin used were 0 μ M, 50 μ M, 100 μ M, 150 μ M, and 200 μ
Figure 21 Electropherograms for the AmpF l STR
NGM ™
™
NGM
™
and SGM Plus
Kit in the presence of hematin compared with previous AmpF l STR
™
™
Kits show the improved performance of the
kits. In order from top to bottom, the panels show profiles for: NGM ™ Kit uninhibited control, NGM ™ Kit with 200 µM hematin, SGM Plus
™
Kit uninhibited control and SGM
Plus
™
Kit with 200 µM hematin. The SGM Plus
™
and NGM ™ Kits were amplified for 28 and 29 cycles, respectively
NGM ™ Kit
Uninhibited Control
NGM ™ Kit
200 μ M hematin
SGM Plus
™
Kit
Uninhibited Control
SGM Plus
™
Kit
200 μ M hematin
Table
7
NGM ™ Kit performance in simulated hematin inhibition (n = 3)
Hematin Concentration
0 µM
50 µM
100 µM
150 µM
200 µM
Number of Alleles Detected
32, 32, 32
32, 32, 32
32, 32, 32
32, 32, 32
32, 32, 32
†
† Only those peaks >50 RFU were counted. A complete profile with control DNA 007 yields 32 peaks using the NGM ™ Kit on an Applied Biosystems
™
3130 xl instrument.
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™
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Stability
5
Effect of inhibitors
— humic acid
Traces of humic acid may inhibit the PCR amplification of DNA evidence collected from soil. Amplification of 1 ng of control DNA 007 in the presence of increasing amounts of humic acid was performed using the NGM ™ Kit for 29 cycles of
amplification (see Figure 22). The concentrations of humic acid tested were 0, 20, 40,
60, and 80 ng/ μ
Figure 22 Electropherograms for the AmpF l STR
™
NGM
™
and SGM Plus
™
Kits show improved performance of the NGM
™
Kit in the presence of humic acid compared to previous AmpF l STR
™
kits. In order from top to bottom, the panels show profiles for: NGM ™ Kit uninhibited control, NGM ™ Kit with 80 ng/µL humic acid, SGM Plus
™
Kit uninhibited control, and
SGM Plus
™
Kit with 80 ng/µL humic acid. The SGM Plus
™
and NGM ™ Kits were amplified for 28 and 29 cycles, respectively
NGM ™ Kit
Uninhibited Control
NGM ™ Kit
80 ng/ μ L humic acid
SGM Plus
™
Kit
Uninhibited Control
SGM Plus
™
Kit
80 ng/ μ L humic acid
Table
8
NGM ™ Kit performance in simulated model of humic acid inhibition (n = 3)
Humic acid concentration
0 ng/ μ L
20 ng/ μ L
40 ng/
μ
L
60 ng/
μ
L
80 ng/ μ L
Number of alleles detected
32, 32, 32
32, 32, 32
32, 32, 32
32, 32, 32
32, 32, 32
†
† Only those peaks >50 RFU were counted. A complete profile with control DNA 007 yields 32 peaks using the NGM ™ Kit on an Applied Biosystems
™
3130 xl instrument.
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™
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5
Chapter 5 Experiments and Results
Mixture studies
Mixture studies
SWGDAM guideline
2.8
“The ability to obtain reliable results from mixed source samples should be determined.” (SWGDAM, July 2003)
Evidence samples may contain DNA from more than one individual. The possibility of multiple contributors should be considered when interpreting the results. We recommend that individual laboratories determine a minimum peak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures.
Mixture studies
Evidence samples that contain body fluids and/or tissues originating from more than one individual are an integral component of forensic casework. Therefore, it is essential to ensure that the DNA typing system is able to detect DNA mixtures. Mixed samples can be distinguished from single-source samples by:
• The presence of more than two alleles at a locus
• 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. Mean, median, minimum, and maximum peak height ratios observed for alleles in the NGM ™ Kit loci
in unmixed population database samples are shown in Figure 23 below.
Figur e 23 Heterozygote ratios for 1 ng of input DNA amplified for 29 cycles with the NGM ™ Kit.
The distribution of intra-locus peak height ratios are expressed as plus and minus percent, by locus. Green 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. Red diamonds are outlier data points more than 1.5
IQR from the median
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™
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Mixture studies
5
If an unusually low peak height ratio is observed for one locus, and there are no other indications that the sample is a mixture, the sample may be reamplified and reanalyzed 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
Resolution of genotypes in mixed samples
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 component(s) at a single locus.
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 sample(s).
Limit of detection of the minor component
Mixtures of two DNA samples were examined at various ratios (0:1, 1:1, 3:1, 7:1, 15:1,
1:0). The total amount of genomic input DNA mixed at each ratio was 1.0 ng. The samples were amplified in a GeneAmp
™
PCR System 9700, then electrophoresed and detected using an Applied Biosystems
™
3130 xl Genetic Analyzer.
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™
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5
Chapter 5 Experiments and Results
Mixture studies
The results of the mixed DNA samples are shown in Figure 24 on page 90 where
samples A and B 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 3:1 (0.750:0.250 ng) and 7:1 (0.875:0.125 ng).
Generally, 15:1 ratios resulted in partial profiles for the minor component. The profiles
of these samples are described in Table 9 on page 90.
Figur e 24 Amplification of DNA mixtures at various ratios. Panels show electropherograms for (top to bottom): Major contributor only, 1:1 mixture (maj:min), 3:1 mixture, 7:1 mixture, 10:1 mixture and 15:1 mixture. The experiment was performed with both 29- and 30 cycle amplification; electropherograms shown are from the 29-cycle amplification
90
Table
9
Genotypes of mixed DNA samples
Locus
D10S1248 vWA
D16S539
D2S1338
AMEL
DS1179
D21S11
Sample A Genotype
13, 14
14, 17
10, 11
17, 23
X, Y
11,14
29, 35
Sample B Genotype
12, 15
17, 19
9, 12
17, 20
X
10,11
31.2, 32.2
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™
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Chapter 5 Experiments and Results
Population data
5
Locus
D18S51
D22S10
D19S433
TH01
FGA
D2S441
D3S1358
D1S1656
D12S391
Sample A Genotype
15, 16
45, 15
11, 17.2
7, 8
19, 25
11, 12
14, 17
12, 18.3
18, 18.3
Sample B Genotype
13, 14
15, 16
13
6, 9.3
22, 23
11, 14
15, 16
11, 15
18, 22
Population data
SWGDAM guideline
2.7
“The distribution of genetic markers in populations should be determined in relevant population groups.” (SWGDAM, July 2003)
Overview
The NGM
™
Kit contains loci for which extensive population data are available. For additional information on 11 loci shared between the kits, see the population data and additional studies section of the AmpF l STR
™
PCR Amplification Kit
User’s Manual (Part no.
44309589).
SGM Plus
™
Population samples used in these studies
The NGM ™ Kit was used to generate the population data provided in this section.
Whole blood samples, provided by the Insterstate Blood Bank (Memphis, Tennessee), were collected in the United States (with no geographical preference) from randomly selected individuals of known ethnicities. Ethnicities of sample donors were:
• African-American – 344 samples
• Caucasian – 346 samples
• Hispanic – 390 samples
DNA was extracted using an ABI P RISM
™
6100 Nucleic Acid PrepStation.
In addition to the alleles that were observed and recorded in our databases, other alleles have been published or reported to us by other laboratories (see the STRBase at www.cstl.nist.gov/div831/strbase ).
New Primers added to the NGM ™ Kit
Comparison of primer sequences with other
AmpF
l
STR
™
Kits
Both the NGM
™
and NGM SElect
™
Kits contain an additional unlabeled primer for the
D8S1179 locus to allow detection of a rare population-specific SNP-containing allele affecting one of the primer binding regions for that locus. The additional D8 primer was first introduced in the Identifiler
™
kit (released 2001) and has been included in all other AmpF l STR
™
kits containing the D8S1179 locus since that tine. This primer is not included in AmpF l STR
™
kits released before 2001, including the SGM Plus
™
kit.
Laboratories may therefore see occasional non-concordance at the D8S1179 locus when comparing results from different AmpF l STR
™
kits.
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™
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5
Chapter 5 Experiments and Results
New Primers added to the NGM ™ Kit
Both the NGM ™ and NGM SElect ™ Kits also contain slight modifications to the
Amelogenin primers to reduce inter-species cross-reactivity compared to previous
AmpF l STR
™
kits.
Inclusion of three
SNP-specific primers to address mutations at the
Amelogenin,
D2S441, and
D22S1045 loci
After the initial release of the NGM ™ Kit in early 2010, more recent population study results showed the existence of certain mutations that affected primer binding sites at three of the NGM ™ Kit loci: amelogenin, D2S441, and D22S1045 (Carolyn Hill and
John Butler, National Institute of Standards and Technology, personal communication).
The mutations, when present, caused the drop-out of affected alleles. While the mutations are relatively rare and restricted primarily to specific population groups, the decision was made to include one additional PCR primer for each of the affected loci to allow the mutant alleles to be detected by the NGM ™ Kit. The inclusion of the additional primers also required a minor re-optimization of the NGM ™ Kit Master Mix to support the expanded primer mix.
Subsequent validation experiments showed that other aspects of kit performance remained unaffected by the additional primers and the minor re-optimization of the
Master Mix. Current versions of both the AmpF l STR
™
NGM
™
and NGM SElect
™
Kits contain identical sets of primers for the 16 loci they have in common; the only difference between the kits is the presence of primers for the SE33 locus in the NGM
SElect
™
Kit.
The first commercially available batches of the NGM ™ Kit to include the additional primers and benefit from the minor Master Mix re-optimisation were 200 Reaction Kit
Lot 1105011 (Part no. 4415020) and 1000 Reaction Kit Lot 1106009 (Part no. 4415021).
These lots were released in late summer 2011.
Note:
The inclusion of the three additional primers in the NGM SElect ™ Kit and the associated buffer optimisation studies were conducted as part of the original development of the NGM SElect ™ Kit.
Figure 25 shows in the new version of the NGM
™
Kit electropherograms with examples where the additional primers for amelogenin, D2S441 and, D22S1045 allow mutant alleles at these loci to be detected, whereas they had not been detected by the original version of the NGM
™
Kit.
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Mutation rate
5
Figur e 25 New PCR primers added to D22S1045, amelogenin and D2S441 loci. The electropherograms below show the effects of adding additional PCR primers to amplify known mutant alleles that would otherwise not be detected. Panels A and B show the D22S1045 locus for individual CV133 without (A) and with (B) the extra primer; the additional primer allowed the variant of allele 15 to be detected. Panels C and D show the amelogenin locus for individual
CV423 without (C) and with (D) the extra primer; the additional primer allowed the variant of the
X allele to be detected. Panels E and F show the D2S441 locus for individual CV1054 without (E) and with (F) the extra primer; when amplified, the variant allele was always seen to type as a
9.1 microvariant
Mutation rate
Estimating germline mutations
Estimation of spontaneous or induced germ-line mutation at genetic loci can be achieved by comparing the genotypes of offspring to those of their parents. From such comparisons the number of observed mutations are counted directly.
In previous studies, genotypes of ten STR loci that were amplified by the AmpF l STR
™
SGM Plus
™
PCR Amplification Kit were determined for a total of 146 parentoffspring allelic transfers (meioses) at the Forensic Science Service, Birmingham,
England. One length-based STR mutation was observed at the D18S11 locus; mutations were not detected at any of the other nine STR loci. The D18S11 mutation was represented by an increase of one 4-nt repeat unit, allele 17 was inherited as allele
18 (single-step mutation). The maternal/paternal source of this mutation could not be distinguished.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
93
5
Chapter 5 Experiments and Results
Probability of identity
Additional mutation studies
Additional studies (Edwards et al., 1991; Edwards et al., 1992; Weber and Wong, 1993;
Hammond et al., 1994; Brinkmann et al., 1995; Chakraborty et al., 1996; Chakraborty et al., 1997; Brinkmann et al., 1998; Momhinweg et al., 1998; Szibor et al., 1998) of direct mutation rate counts produced:
• Larger sample sizes for some of the NGM ™ Kit loci.
• Methods for modifications of these mutation rates (to infer mutation rates indirectly for those loci where the rates are not large enough to be measured directly and/or to account for those events undetectable as Mendelian errors).
Probability of identity
Table
10
Allele frequencies (%) by population group for NGM
™
Kit STR loci. (The † symbol indicates alleles not detected or, where values appear in parentheses, alleles not detected in significant quantities.)
Allele
African American
(N = 344)
Caucasian
(N = 346)
Hispanic
(N = 390)
16
17
18
19
12
13
14
15
D10S1248
6
7
8
9
10
11
20
D12S391
13
14
15
15.1
16
16.1
17
17.1
†
3.63
13.95
22.67
28.2
18.6
9.88
2.18
7.12
5.09
15.7
3.47
29.05
29.77
19.65
13.44
3.76
4.19
3.47
10.55
4.23
25.51
36.03
22.95
8.08
2.56
3.97
5.13
7.31
94
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Allele
(continued)
16
D18S51
7
8
12.2
13
14
15
9
9.2
10
9
10
11
12
5
6
7
8
22
23
24
25
26
27
28
D16S539
20
20.3
21
21.3
17.3
18
18.3
19
19.1
19.3
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
African American
(N = 344)
25.73
1.16
14.1
11.92
6.98
5.09
3.34
0.87
3.49
22.24
11.63
29.07
19.33
12.94
1.31
Chapter 5 Experiments and Results
Probability of identity
5
1.45
12.43
4.05
32.23
30.78
17.2
1.73
1.16
Caucasian
(N = 346)
2.02
16.18
2.17
12.28
9.83
13.73
10.69
8.09
3.61
2.02
1.92
10.38
15.77
31.92
24.49
13.97
1.15
0.64
Hispanic
(N = 390)
1.15
20
2.05
18.59
1.15
17.31
8.72
6.92
3.72
1.79
1.28
95
96
5
Chapter 5 Experiments and Results
Probability of identity
Allele
(continued)
23.2
24
25
26
27
D19S433
9
9.2
21.2
22
22.2
23
19.2
20
20.2
21
10
10.2
11
17.2
18
18.2
19
15.2
16
16.2
17
13.2
14
14.2
15
10.2
11
11.2
12
12.2
13
African American
(N = 344)
6.25
4.07
5.81
17.3
1.02
9.16
4.22
2.03
18.31
15.7
14.1
1.16
9.74
Caucasian
(N = 346)
0.87
14.74
11.85
17.49
15.32
11.85
10.98
8.53
4.34
1.3
1.01
17.05
8.46
3.46
1.79
2.31
0.64
Hispanic
(N = 390)
1.15
10.77
11.54
15.77
12.31
12.82
1.54
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Allele
(continued)
17.1
17.3
18
18.3
16
16.1
16.3
17
14
14.3
15
15.3
10
11
12
13
19
19.3
20.3
16
16.2
17
17.2
18
18.2
D1S1656
9
14
14.2
15
15.2
11.2
12
12.1
12.2
13
13.2
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
African American
(N = 344)
10.76
3.78
28.63
5.23
18.31
5.81
6.25
4.22
1.45
3.34
7.27
2.62
5.96
1.89
1.31
5.52
7.99
11.19
24.27
1.02
17.44
1.89
10.32
Chapter 5 Experiments and Results
Probability of identity
5
15.26
0.77
4.36
0.64
16.03
2.95
15.26
5.26
6.92
3.85
9.49
7.18
11.03
Hispanic
(N = 390)
8.46
1.41
18.72
7.05
30.38
4.49
12.69
7.31
4.1
2.31
4.91
4.91
12.72
5.92
1.73
5.92
16.04
6.65
6.36
15.17
8.53
9.97
Caucasian
(N = 346)
7.23
27.46
1.59
34.68
2.17
16.18
3.47
5.92
97
98
5
Chapter 5 Experiments and Results
Probability of identity
Allele
(continued)
37
37.2
38
38.2
35
35.2
36
36.2
33
33.2
34
34.2
31
31.2
32
32.2
39
D22S1045
5
29.2
29.3
30
30.2
27.2
28
28.2
29
D21S11
23.2
24
24.2
25
25.2
26
26.2
27
27.1
African American
(N = 344)
15.7
20.93
1.6
8.72
4.94
1.31
6.98
0.87
3.2
3.49
0.73
5.52
25.29
Caucasian
(N = 346)
16.91
6.79
8.67
2.31
9.54
23.55
23.41
2.75
2.02
2.75
Hispanic
(N = 390)
-- (0.26)
1.41
11.03
21.15
27.95
1.67
5
11.28
1.28
12.44
-- (0.13)
5.26
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Allele
(continued)
28
29
D2S441
8
24
25
26
27
9
10
11
20
21
22
23
16
17
18
19
16
17
18
20
D2S1338
13
14
15
12
13
14
15
8
9
6
7
10
11
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
African American
(N = 344)
0.73
4.07
14.53
5.96
8.58
23.55
19.91
20.35
2.03
5.23
10.03
4.8
15.99
10.03
12.79
12.65
9.3
8.58
6.98
2.47
0.73
8.87
35.03
Chapter 5 Experiments and Results
Probability of identity
5
Hispanic
(N = 390)
0.64
7.82
1.03
1.03
2.18
42.56
35.64
7.95
1.03
3.59
17.69
6.54
17.82
13.72
3.59
6.28
14.87
8.85
5.38
1.41
31.15
31.67
4.19
18.79
8.38
14.31
15.46
2.75
1.73
10.12
9.97
11.85
1.73
19.8
33.82
Caucasian
(N = 346)
13.58
1.01
3.47
36.56
36.27
7.51
99
5
Chapter 5 Experiments and Results
Probability of identity
100
Allele
(continued)
D8S1179
7
8
9
10
11
12
13
18
18.2
19
20
16
16.2
17
17.2
14
15
16
D3S1358
9
11
12
13
14
15
15.2
14.3
15
16
17
11.3
12
12.3
13
13.3
14
African American
(N = 344)
3.34
20.06
3.63
26.89
1.89
3.34
5.81
11.05
18.31
36.05
17.73
5.96
9.16
28.34
32.85
22.09
5.81
15.17
27.31
23.99
19.8
11.85
1.45
Caucasian
(N = 346)
5.06
4.05
3.18
28.32
4.48
2.02
1.3
10.84
6.65
15.03
33.53
18.64
8.67
2.89
0.64
9.49
4.87
12.44
33.33
23.46
11.54
3.33
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
9.49
34.49
26.54
17.95
10.13
0.77
Hispanic
(N = 390)
4.36
3.97
1.92
22.56
3.33
Allele
(continued)
23.3
24
24.2
25
22
22.2
23
23.2
20
20.2
21
21.2
18
18.2
19
19.2
FGA
16
16.1
16.2
17
17.2
17
18
19
20
27.2
28
--
29
25.2
26
26.2
27
29.2
30
30.2
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
African American
(N = 344)
1.02
0.87
0.73
6.83
6.69
12.06
18.17
16.86
18.75
9.3
3.92
2.62
1.16
Chapter 5 Experiments and Results
Probability of identity
5
Caucasian
(N = 346)
14.31
6.79
1.88
1.16
5.35
15.61
18.35
18.93
0.87
14.6
Hispanic
(N = 390)
0.64
8.72
13.59
14.1
0.64
12.69
15.9
14.1
6.79
2.95
0.9
0.64
7.82
101
5
Chapter 5 Experiments and Results
Probability of identity
Allele
(continued)
10
10.3
11
12
8
8.3
9
9.3
13
13.3
6.1
6.3
7
7.3
4
5
5.3
6
50.2
51.2
TH01
3
46.2
47.2
48.2
49.2
42.2
43.2
44.2
45.2
31
31.2
32
32.2
33.2
34.2
African American
(N = 344)
37.06
21.22
15.84
8.43
1.45
15.41
102
17.77
11.42
17.05
31.07
0.87
21.68
Caucasian
(N = 346)
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
31.92
8.46
12.56
17.69
1.41
27.95
Hispanic
(N = 390)
Chapter 5 Experiments and Results
Probability of identity
5
Allele
(continued)
African American
(N = 344)
Caucasian
(N = 346)
Hispanic
(N = 390)
20
21
22
23
17.3
18
18.2
19
24
25 vWA
10
11
12
13
14
15
15.2
16
17
0.73
7.41
20.93
27.47
19.48
13.81
6.98
2.03
8.38
12.14
22.69
27.31
18.06
9.97
1.3
6.41
9.87
30
27.18
18.46
6.67
0.77
† A minimum allele frequency (0.7% for the African-American database, 0.7% for the U.S. Caucasian database, 0.9% for the U.S. Hispanic database, and 1.3% for the Native American database) is suggested by the National Research Council in forensic calculations.
Table 11 shows the allele frequencies at NGM
™ Kit loci by population group. The P
I value is the probability that two individuals selected at random will have an identical
NGM ™ Kit genotype (Sensabaugh, 1982). The P in this section are then 6.74 ✕ 10 -20 ng/
μ
L–22
I
values for the populations described
(African-American), 2.76
✕ 10 –19 (U.S.
Caucasian) and 4.00 ✕ 10 –19 (U.S. Hispanic).
Table
11
Probability of identity (P
I
) values for the NGM ™ Kit STR loci
Locus
African-American
(N = 344)
U.S.-Caucasian
(N = 346)
U.S.-Hispanic
(N = 390)
D10S1248 vWA
D16S539
D2S1338
D8S1179
D21S11
0.069
0.062
0.072
0.023
0.076
0.045
0.094
0.065
0.104
0.032
0.063
0.052
0.111
0.091
0.082
0.032
0.068
0.050
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
103
5
Chapter 5 Experiments and Results
Probability of paternity exclusion
Locus
D18S51
D22S1045
D19S433
TH01
FGA
D2S441
D3S1358
D1S1656
D12S391
Combined
African-American
(N = 344)
0.031
0.056
0.040
0.094
0.033
0.101
0.100
0.034
0.039
6.74 x 10 –20
U.S.-Caucasian
(N = 346)
0.031
0.133
0.085
0.080
0.039
0.098
0.075
0.022
0.023
2.76 x 10 –19
U.S.-Hispanic
(N = 390)
0.028
0.161
0.048
0.091
0.028
0.107
0.095
0.025
0.032
4.00 x 10 –19
Probability of paternity exclusion
Table 12 Probability of Paternity Exclusion values for the NGM
™
Kit
™
STR loci
Locus
D10S1248 vWA
D16S539
D2S1338
D8S1179
D21S11
D18S51
D22S1045
D19S433
TH01
FGA
D2S441
D3S1358
D1S1656
D12S391
Combined
African-American
(N = 344)
0.727
0.786
0.698
0.636
0.515
0.727
0.522
0.476
0.659
0.603
0.556
0.798
0.587
0.745
0.745
0.999999931
Caucasian
(N = 346)
0.568
0.644
0.579
0.752
0.616
0.729
0.758
0.455
0.507
0.502
0.666
0.497
0.522
0.811
0.799
0.999999835
Hispanic
(N = 390)
0.692
0.718
0.397
0.647
0.539
0.708
0.477
0.482
0.469
0.638
0.557
0.738
0.557
0.723
0.662
0.999999376
The P
E
value is the probability, averaged over all possible mother-child pairs, that a random alleged father will be excluded from paternity after DNA typing of the NGM ™
Kit STR loci (Chakraborty, Stivers, and Zhong, 1996).
104
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
A
Troubleshooting
Observation
Faint or no signal from both, the 007
AmpF l STR
™
Control
DNA and the DNA test samples at all loci
Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis.
Possible causes Recommended actions
Incorrect volume or absence of either
AmpF l STR
™
AmpF l STR
™
NGM ™
NGM
™
Master Mix or
Primer Set
No activation of enzyme
Repeat amplification using correct reagent volumes.
Repeat amplification, making sure to hold reactions initially at 95°C for 11 min.
Master Mix not vortexed thoroughly before aliquoting
Vortex Master Mix thoroughly.
AmpF l STR
™
NGM ™ Primer Set exposed to too much light
Store Primer Set protected from light.
GeneAmp
™
PCR System malfunction Refer to the thermal cycler user’s manual and check instrument calibration.
Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters.
Tubes/plate not seated tightly in the thermal cycler during amplification
Wrong PCR reaction tubes or plate
Push reaction tubes/plate firmly into contact with block after first cycle. Repeat test.
Use Applied Biosystems
™
MicroAmp Reaction
Tubes with Caps or the MicroAmp Optical 96-
Well Reaction Plate for the GeneAmp
™
PCR
System 9700 or Veriti
™
96-well Thermal Cycler.
MicroAmp ™ Base used with tray/ retainer set and tubes in
GeneAmp
™
PCR System 9700
Insufficient PCR product electrokinetically injected
Remove MicroAmp Base from tray/retainer set and repeat test.
Refer to Chapter 3, “Electrophoresis” on page 25,
for instructions on recommended actions on the
ABI P RISM
™
3100/ 3100Avant or
Applied Biosystems
™
3130/3130 xl , 3500/ 3500xL, and the ABI P RISM
™
310 instruments.
Degraded formamide Check the storage of formamide; do not thaw and refreeze multiple times. Try Hi-Di ™ Formamide.
105
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
A
Appendix A
Troubleshooting
Observation
Positive signal from
AmpF l STR
™
Control
DNA 007 but partial or no signal from DNA test samples
More than two alleles present at a locus
Poor peak height balance
Possible causes Recommended actions
Quantity of test DNA sample is below assay sensitivity
Test sample contains high concentration of PCR inhibitor (for example, heme compounds, certain dyes
Test sample DNA is severely degraded
Quantify DNA and add either 1.0 ng or 500 pg of
DNA depending upon cycle number being used.
Repeat test.
Quantify DNA and add minimum necessary volume. Repeat test.
Wash the sample in a Centricon
™
-100 centrifugal filter unit. Repeat test.
If possible, evaluate the quality of DNA sample by running an agarose gel. If DNA is degraded, reamplify with an increased amount of DNA or use the AmpF l STR
™
MiniFiler ™ Kit.
Redilute DNA using low-TE Buffer (with 0.1 mM
EDTA).
Dilution of test sample DNA in water or wrong buffer (for example, TE formula with incorrect EDTA concentration)
Presence of exogenous DNA
Amplification of stutter product
Mixed sample
Incomplete 3´ A base addition
(n-1 nt position)
Signal exceeds dynamic range of instrument (off-scale data)
Use appropriate techniques to avoid introducing foreign DNA during laboratory handling.
Interpret according to laboratory procedures.
Note:
Additional information will be provided on completion of validation.
Addition of excess DNA to the reaction will contribute to the occurrence of incomplete 3' base addition. Quantify DNA and add 1.0 ng of DNA to the reaction. Repeat test. Also be sure to include the final extension step of 60°C for 10 min in the
PCR.
Ensure cycle number is optimized according to
instructions on page 22. Repeat PCR amplification
using fewer PCR cycles or use your laboratory’s
SOP to analyze off-scale data.
Poor spectral separation (bad matrix) Follow the steps for creating a spectral file.
Confirm that Filter Set G5 modules are installed and used for analysis.
Too much DNA in reaction
Incomplete denaturation of double stranded DNA
Use recommended amount of template DNA:
1.0 ng at 29 cycles; 500 pg at 30 cycles.
Use the recommended amount of Hi-Di
™
Formamide and perform heat denaturation
according to instructions in Chapter 3,
Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters.
GeneAmp
™
PCR System 9700 with Aluminum 96-Well block or third-party thermal cyclers
Use Applied Biosystems
™
GeneAmp
™
PCR
System 9700 with silver, gold-plated silver blocks or Veriti
™
96-well Thermal Cycler only.
106
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
B
Ordering Information
Materials and equipment not included
The tables below list optional equipment and materials not supplied with the NGM
™
Kit. Unless otherwise noted, many of the items are available from major laboratory suppliers (MLS).
Equipment
Applied Biosystems
™
3500/3500xL Genetic Analyzer for Human Identification
ABI P
RISM
™
3100/3100Avant Genetic Analyzer
Applied Biosystems
™
3130/3130 xl Genetic Analyzer
Applied Biosystems
™
310 Genetic Analyzer
GeneAmp
™
PCR System 9700 with the Silver 96-Well Block
GeneAmp
™
PCR System 9700 with the Gold-plated Silver 96-Well Block
Silver 96-Well Sample Block
Gold-plated Silver 96-Well Sample Block
Veriti
™
96-well Thermal Cycler
ProFlex ™ 96-Well PCR System
Tabletop centrifuge with 96-Well Plate Adapters (optional)
Part number
Contact your local
Life Technologies sales representative
N8050001
4314878
N8050251
4314443
4375786
4484075
MLS
Item
3500/3500xL Analyzer materials
Anode buffer container (ABC)
Cathode buffer container (CBC)
POP-4 ™ polymer (960 samples) for 3500/3500xL Genetic Analyzers
POP-4 ™ polymer (384 samples) for 3500/3500xL Genetic Analyzers
Conditioning reagent
8-Capillary array, 36 cm for 3500 Genetic Analyzers
24-Capillary array, 36 cm for 3500xL Genetic Analyzers
96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers
8-Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers
8-Strip Septa for 3500/3500xL Genetic Analyzers
96-Well Septa for 3500/3500xL Genetic Analyzers
Septa Cathode Buffer Container, 3500 series
GeneScan ™ 600 LIZ
™
Size Standard v2.0
Part number
4393927
4408256
4393710
4393715
4393718
4404683
4404687
4410228
4410231
4410701
4412614
4410715
4408399
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
107
B
Appendix B
Ordering Information
Materials and equipment not included
Note:
For a complete list of parts and accessories for the 3500/3500xL instrument, refer to the
Applied Biosystems
™
3500/3500xL Genetic Analyzer User Guide (Part no. 4401661)
AmpF l STR
™
NGM ™ PCR Amplification Kit (200x/1000x)
3100/3100-
Avant
Analyzer materials
96-Well Plate Septa
Item
Reservoir Septa
3100/3100Avant Genetic Analyzer Capillary Array, 36-cm
POP-4
™
Polymer for 3100/3100Avant Genetic Analyzers
3100/3100Avant Genetic Analyzer Autosampler Plate Kit, 96-well
GeneScan
™
500 LIZ
™
Size Standard Or
GeneScan ™ 600 LIZ
™
Size Standard v2.0
Part number
4415020/4415021
4315933
4315932
4333464
4316355
4316471
4322682
Or
4408399
Running Buffer, 10 ✕
DS-33 Matrix Standard Kit (Dye Set G5)
MicroAmp
™
Optical 96-Well Reaction Plate
250-
μ
L Glass Syringe (array-fill syringe)
402824
4345833
N8010560
4304470
5.0-mL Glass Syringe (polymer-reserve syringe) 628-3731
Note:
For a complete list of parts and accessories for the 3100 instrument, refer to Appendix B of the ABI
P RISM
™
3100 Genetic Analyzer and 3100-Avant Genetic Analyzer User Reference Guide (Part no.
4335393).
3130/3130 xl Analyzer materials
96-Well Plate Septa 4315933
Reservoir Septa
3100/3130 xl Genetic Analyzer Capillary Array, 36-cm
POP-4 ™ Polymer for 3130/3130 xl Genetic Analyzers
4315932
4315931
4352755
4316471 3130/3130 xl Genetic Analyzer Autosampler Plate Kit, 96-well
GeneScan
™
500 LIZ
™
Size Standard Or
GeneScan
™
600 LIZ
™
Size Standard v2.0
4322682
Or
4408399
Running Buffer, 10
✕
DS-33 Matrix Standard Kit (Dye Set G5)
MicroAmp
™
Optical 96-Well Reaction Plate
402824
4345833
N8010560
For a complete list of parts and accessories for the 3130 xl instrument, refer to Appendix A of the Applied Biosystems
3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (Part no. 4352716).
310 DNA Analyzer materials
310 DNA Analyzer Capillary Array, 47-cm
0.5 mL Sample Tray
96-Well Tray Adaptor (for 9700 thermal cycler trays)
402839
5572
4305051
108
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Appendix B
Ordering Information
Materials and equipment not included
B
Item Part number
GeneScan
GeneScan
™
™
500 LIZ
600 LIZ
™
™
Size Standard Or
Size Standard v2.0
4322682
Or
4408399
Running Buffer, 10 ✕
Genetic Analyzer Septa Retainer Clips for 96-Tube Sample Tray
4335643
402866
Genetic Analysis Sample Tubes (0.5-mL) 401957
Septa for 0.5-mL Sample Tubes
DS-33 Matrix Standard Set (6-FAM ™ , VIC
™
, NED ™ , PET
™
, and LIZ
™
dyes) for
ABI PRISM
™
310/377 systems
MicroAmp
™
8-Tube Strip, 0.2-mL
MicroAmp
™
96-Well Base (holds 0.2-mL reaction tubes)
MicroAmp
™
96-Well Full Plate Cover
MicroAmp
™
96-Well Tray/Retainer Set
401956
4318159
N8010580
N8010531
N8010550
403081
POP-4
™
Polymer for the 310 Genetic Analyzer 402838
For a complete list of parts and accessories for the 310 instrument, refer to Appendix B of the ABI PRISM
™
310
Genetic Analyzer User Guide (Part no. 4317588).
PCR Amplification
MicroAmp
™
96-Well Tray
MicroAmp
™
Reaction Tube with Cap, 0.2-mL
MicroAmp
™
8-Tube Strip, 0.2-mL
MicroAmp
™
8-Cap Strip
MicroAmp
™
96-Well Tray/Retainer Set
MicroAmp
™
96-Well Base
MicroAmp
™
Clear Adhesive Film
MicroAmp
™
Optical Adhesive Film
MicroAmp
™
Optical 96-Well Reaction Plate
Other user-supplied materials
Hi-Di
™
Formamide, 25-mL
Aerosol resistant pipette tips
N8010541
N8010540
N8010580
N8010535
403081
N8010531
4306311
4311971
N8010560
4311320
MLS
Microcentrifuge tubes
Pipettors
Tape, labeling
Tube, 50-mL Falcon
MLS
MLS
MLS
MLS
Tube decapper, autoclavable
Deionized water, PCR grade
Tris-HCL, pH 8.0
EDTA, 0.5 M
Vortex
MLS
MLS
MLS
MLS
MLS
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
109
B
Appendix B
Ordering Information
Materials and equipment not included
110
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
C
PCR Work Areas
■
■
■
Work area setup and lab design
Many resources are available for the appropriate design of a PCR laboratory. If you are using the AmpF l STR
™
NGM ™ PCR Amplification Kit for:
• Forensic DNA testing, refer to “Forensic Laboratories: Handbook for Facility
Planning, Design, Construction and Moving,” National Institute of Justice, 1998
( www.nij.org/publications )
• Parentage DNA testing, refer to the “Guidance for Standards for Parentage
Relationship Testing Laboratories,” American Association of Blood Banks, 7th edition, 2004
The sensitivity of the NGM
™
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).
Also take care while handling and processing samples 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
IMPORTANT!
These items should never leave the PCR Setup Work Area.
• Calculator
• Gloves, disposable
• Marker pen, permanent
• Microcentrifuge
• Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate clean tube (for
Master Mix preparation)
• Microcentrifuge tube rack
• Pipette tips, sterile, disposable hydrophobic filter-plugged
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
111
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.
You can use the following systems:
• GeneAmp
™
PCR System 9700 with the Silver 96-Well Block
• GeneAmp
™
PCR System 9700 with the Gold-plated Silver 96-Well
Block
IMPORTANT!
The NGM ™ Kit is not validated for use with the GeneAmp
™
PCR System 9700 with the Aluminium 96-Well Block. Use of this thermal cycling platform may adversely affect performance of the NGM ™ Kit.
• Veriti
™
96-well Thermal Cycler
112
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
D
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, etc). To obtain SDSs, see the “Documentation and
Support” section in this document.
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
113
D
Appendix D
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, and 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 adequate ventilation (for example, fume hood).
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's 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 necessary) 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.
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.
114
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Appendix D
Safety
Biological hazard safety
D
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. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices).
Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially infectious materials.
Read and follow the applicable guidelines and/or regulatory requirements in the following:
In the U.S.:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories found at: www.cdc.gov/biosafety
• Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030), found at: www.access.gpo.gov/nara/cfr/waisidx_01/
29cfr1910a_01.html
• Your company’s/institution’s Biosafety Program protocols for working with/ handling potentially infectious materials.
• Additional information about biohazard guidelines is available at: www.cdc.gov
In the EU:
Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization
(WHO) Laboratory Biosafety Manual, third edition, found at: www.who.int/ csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
115
D
Appendix D
Safety
Biological hazard safety
116
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
Documentation and Support
Related documentation
The following related documents are shipped with the system:
Document
ABI P RISM
™
3100/3100-Avant Data Collection v2.0 User Guide
ABI P RISM
™
3100/3100-Avant Genetic Analyzers Using Data
Collection Software v2.0 User Bulletin
ABI P RISM
™
3100 Genetic Analyzer User Manual (Data Collection v1.1)
ABI P RISM
™
AmpF l STR
3100/3100-Avant Genetic Analyzers Protocols for Processing
™
PCR Amplification Kit PCR Products User Bulletin
AmpF l STR
™
NGM ™ PCR Amplification Kit - PCR Setup Quick Reference Card
AmpF l STR
™
NGM ™ PCR Amplification Kit - CE Quick Reference Card
Veriti
™
96-Well Thermal Cycler AmpF l STR
™
Kit Validation User Bulletin
ProFlex ™ PCR System Kit Validation User Bulletin
Applied Biosystems
™
3130/3130xl Genetic Analyzers Using Data
Collection Software v3.0 User Bulletin
Applied Biosystems
™
3130/3130xl Genetic Analyzers Getting Started Guide
Applied Biosystems
™
3130/3130xl Genetic Analyzers
Maintenance, Troubleshooting, and Reference Guide
Applied Biosystems
™
3130/3130xl Genetic Analyzers Quick Reference Card
Applied Biosystems
™
3130/3130xl Genetic Analyzers AB Navigator
Software Administrator Guide
Applied Biosystems
™
3130/3130xl DNA Analyzers User Guide
Applied Biosystems
™
3730/3730xl Genetic Analyzer Getting Started Guide
Quantifiler
™
Kits: Quantifiler
™
Human DNA Quantification Kit and
Quantifiler
™
Y Human Male DNA Quantification Kit User’s Manual
Identifiler
™
™ Forensic DNA Extraction Kit User Guide
GeneMapper
™
ID Software Version 3.1 Human Identification Analysis User
Guide
GeneMapper
™
ID Software Versions 3.1 and 3.2 Human
Identification Analysis Tutorial
Installation Procedures and New Features for GeneMapper
™
ID
Software v3.2 User Bulletin
GeneMapper
™
ID-X Software Version 1.0 Getting Started Guide
Part number
4347102
4350218
4315834
4332345
4442401
4442693
4440754
100031595
4363787
4352715
4352716
4362825
4359472
4331468
4359476
4344790
4390932
4338775
4335523
4352543
4375574
AmpF l STR
™
NGM ™ PCR Amplification Kit User Guide
117
Documentation and Support
Obtain support
Document Part number
GeneMapper
™
ID-X Software Version 1.0 Quick Reference Guide
GeneMapper
™
ID-X Software Version 1.0 Reference Guide
GeneMapper
™
ID-X Software Version 1.1 (Mixture Analysis) Getting
Started Guide
GeneMapper
™
ID-X Software Version 1.1 (Mixture Analysis) Quick
Reference Guide
GeneMapper
™
ID-X Software Version 1.2 Quick Reference Guide
GeneMapper
™
ID-X Software Version 1.2 Reference Guide
4375670
4375671
4396773
4402094
4426482
4426481
Note:
To open the user documentation, use the Adobe
™
Reader
™
software available from www.adobe.com
Note:
For additional documentation, see “Obtain support” on page 118.
Obtain support
For HID 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: www.lifetechnologies.com
At the website, you can:
• Access worldwide telephone and fax numbers to contact Technical Support and
Sales facilities
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents
• Obtain information about customer training
• Download software updates and patches
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 found on Life Technologies' website 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.
118
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™
NGM ™ PCR Amplification Kit User Guide
Bibliography
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J. Forensic Sci.
39:362–372.
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Relationship Testing Laboratories . 7th ed. Bethesda, Md: American Association of Blood
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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.
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108: 31-35.
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.
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108:
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Brinkman, B., Moller, A. and Wiegand, P. 1995. Structure of new mutations in 2 STR systems. Intl. J. Legal Med.
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Butler, J.M. 2005. Forensic DNA Typing . Burlington, MA:Elsevier Academic Press.
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Drabek, J., Chung, D.T., Butler, J.M., McCord, B.R. 2004. Concordance study between
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Edwards, A., Civitello, A., Hammond, H., and Caskey, C. 1991. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet .
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Frank, W., Llewellyn, B., Fish, P., et al.
2001. Validation of the AmpF l STR
™
Profiler
Plus ™ PCR Amplification Kit for use in forensic casework. J. Forensic Sci.
46:642–646.
Gamero, J. J., Romero, J. L., Gonzalez, J. L., Arufe, M. I., Cuesta, M. I., Corte-Real, F.,
Carvalho, M., Anjos, M. J., Vieira, D. N., and Vide, M. C. 6-5-2000. A study on ten short tandem repeat systems: African immigrant and Spanish population data. Forensic
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Glock, B., Dauber, E.M., Schwartz, D.W., Mayr W.R. 1997. Additional variability at the
D12S391 STR locus in an Austrian population sample: sequencing data and allele distribution. Forensic Sci. Int.
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™
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13 short tandem repeat loci for use in personal identification applications. Am J. Hum.
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(STR) systems based on sizing precision in a capillary electrophoresis instrument.
Electrophoresis 19:86–93.
Li, H. Schmidt, L., Wei, M-H., Hustad, T. Leman, M.I., Zbar, B. and Tory, K. 1993. Three tetranucleotide polymorphisms for loci:D3S1352; D3S1358; D3S1359. Hum. Mol. Genet.
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Magnuson, V.L., Ally, D.S., Nylund, S.J., Karanjawala, Z.E., Rayman, J.B., Knapp, J.I.,
Lowe, A.L., Ghosh, S., Collins, F.S. 1996. Substrate nucleotide-determined nontemplated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. Biotechniques 21:700–709.
Mansfield, E.S., Robertson, J.M., Vainer, M., Isenberg, A.R., Frazier, R.R., Ferguson, K.,
Chow, S., Harris, D.W., Barker, D.L., Gill, P.D., Budowle, B., McCord, B.R. 1998.
Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis. Electrophoresis 19:101–107.
Mills, K.A., Even, D., and Murrau, J.C. 1992. Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus (FGA). Hum. Mol. Genet . 1:779.
Möller, A. and Brinkmann, B. 1994. Locus ACTBP2 (SE33): Sequencing data reveal considerable polymorphism. Int. J. Leg. Med. 106: 262-267
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Puers C, Hammond HA, Jin L, Caskey CT, Schumm JW., Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus
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Raymond M. & Rousset F., 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism.
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Bibliography
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Index
Symbols
+A nucleotide addition
Numerics
3100/3100-Avant instruments 27
A
accuracy and reproducibility 63
alleles
off-ladder 64 alleles,off-ladder 64
allelic ladder
requirements for accurate genotyping 25
amplification
using bloodstained FTA cards 23
annealing temperatures, validation of 61
B
C
characterization of loci, validation 79
control DNA
AmpF l STR
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NGM ™ PCR Amplification Kit User Guide
D data
accuracy, precision, reproducibility 63
DNA
mixture studies figure 89 mixtures, limit of detection 89
negative-control reaction 22 positive-control reaction 22
sample preparation 22 test sample 22
E electrophoresis
,
preparing samples on the 310 instrument 31
preparing samples on the 3500/3500xL instrument 29 reagents and parts 29
,
set up of 3100/3100-Avant instruments 27
equipment, not included in kit 107
F
FTA cards
amplification 23 bloodstained 23
123
Index
G
GeneMapper
™
ID Software data analysis
overview 16
ID-X Software
GeneScan size standard
H
Hi-Di formamide, volume per reaction 28
I
import
instrumentation
3100/3100-Avant genetic analyzer 16
3130/3130xl genetic analyzer 16
3500/3500xL genetic analyzer 29
K kit
thermal cyclers for use with 112
kit performance, comparisons
124
L
LIZ size standard
loci
M
master mix, volume per reaction 21
materials and equipment
mixed samples, resolution of genotypes 89
N
negative control, sample preparation 22
O
P
paternity exclusion, probability of 104
PCR
thermal cycling conditions, programming 22
positive control, sample preparation 22
primers
probability of identity, definition 103
project examination and editing 58
Q
AmpF l STR
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NGM ™ PCR Amplification Kit User Guide
R
run module, electrophoresis 27
S safety
size deviation, sample alleles and ladder alleles 63
software
software, instrument compatibility 16
split peaks, +A nucleotide addition 76
stutter percentages, marker-specific 75
T
thermal cyclers
thermal cycling
U
V validation
accuracy, precision, reproducibility 63
AmpF l STR
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NGM ™ PCR Amplification Kit User Guide
probability of paternity exclusion 104
size deviation, sample and ladder alleles 63
W
work area
PCR setup 111 setup and lab design 111
Index
125
Index
126 AmpF l STR
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NGM ™ PCR Amplification Kit User Guide
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