Advertisement
Advertisement
GlobalFiler
™
Express PCR Amplification Kit
USER GUIDE
Catalog Numbers
4476609 and 4474665
Publication Number 4477672
Revision G
For Research, Forensic, or Paternity Use
Only. Not for use in diagnostic procedures.
Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition .
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history : Pub. No. 4477672
Revision
G
Date
13 October 2020
F
C
B
E
D
A
09 June 2020
21 December 2016
06 October 2016
May 2014
April 2014
October 2012
Description
Add the SeqStudio ™ Genetic Analyzer. Consolidate sample preparation for electrophoresis; it is the same for all instruments.
In kit overview, change amplification time from ~80 to ~45 minutes. Update copyright page to latest template. On cover, update regulatory statement and remove licensing statement link.
Revised the Peak Detector tab settings for GeneMapper ™ ID ‑ X Software analysis.
Updated 3730 Peak Detector settings in Chapter 4. Add references to 3500 Series
Data Collection 3 and GeneMapper ID-X v1.5.
Non-technical changes: Reorganized Chapter 1 and Chapter 5.
Added data to Chapter 5 about the evaluation of Hardy-Weinberg equilibrium.
Added Master Mix Additive instructions. Updated the HID Updater 3500 DC v2.0
instructions, including sizing method information. Added 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. Windows and Windows Vista are trademarks of Microsoft Corporation. EasiCollect , Whatman, and FTA are trademarks of Whatman Limited.
NUCLEIC-CARD, FLOQ,Swabs, and Copan are trademarks of Copan Flock Technologies, and used by Thermo Fisher Scientific under their permission. Bode Buccal DNA Collector is a trademark of Bode Technology Group, Inc. Harris Micro-Punch is a trademark of Harris, Joel S. TA Shunderson Communications. VWR Scientific is a trademark of VWR International, Inc. Robbins Scientific is a trademark of Molecular Bioproducts, Inc. Agilent is a trademark of Agilent Technologies, Inc. Adobe, Acrobat, and Reader are trademarks of Adobe Systems Incorporated.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1
Product information .................................................. 8
■
CHAPTER 2
Perform PCR ....................................................... 18
Thaw reagents and prepare Master Mix (before first use of the kit) . . . . . . . . . . . . . . . 19
GlobalFiler ™ Express PCR Amplification Kit User Guide 3
Contents
4
■
CHAPTER 3
Perform electrophoresis ............................................ 29
™
instruments for electrophoresis (before first use of the kit) . . . . . . 30
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . . 32
Set up the 3130/3130 xl instruments for electrophoresis (before first use of the kit) . . . . . . 36
Set up the 3730/3730 xl instruments for electrophoresis (before first use of the kit) . . . . . . 38
■
CHAPTER 4
™
ID ‑ X Software ................ 42
™
ID ‑ X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
™
ID ‑ X Software for analysis (before first use of the kit) . . . . . . . . 44
™
ID ‑ X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
(If needed) Download newer versions of panel, bin, and stutter files . . . . . . . . . . . . . . 45
™
polymer on a 3730 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Analyze and edit sample files with GeneMapper
™
ID ‑ X Software . . . . . . . . . . . . . . . . . . . . . 60
GlobalFiler ™ Express PCR Amplification Kit User Guide
Contents
For more information on using the GeneMapper
™
ID ‑ X Software . . . . . . . . . . . . . . . . . . . . . 61
■
CHAPTER 5
Experiments and results ........................................... 62
GlobalFiler ™ Express PCR Amplification Kit User Guide 5
6
Contents
™
™
sample collectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
■
APPENDIX A
Troubleshooting .................................................. 135
■
APPENDIX B
Materials required but not supplied ............................ 138
™
™
™
™
■
APPENDIX C
Plate layouts ...................................................... 145
GlobalFiler ™ Express PCR Amplification Kit User Guide
Contents
■
APPENDIX D
PCR work areas .................................................. 146
■
APPENDIX E
Safety .............................................................. 148
■
Documentation and support ...................................................... 151
References
Index ..................................................................................... 158
GlobalFiler ™ Express PCR Amplification Kit User Guide 7
8
1
Product information
■
■
■
■
■
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix in this document.
Product description
Kit overview
The Applied Biosystems ™ GlobalFiler ™ Express PCR Amplification Kit is a 6-dye, short tandem repeat
(STR) multiplex assay for the amplification of human genomic DNA.
The kit amplifies:
• 21 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51,
D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248,
D1S1656, D12S391, D2S1338)
• 1 Y-STR (DYS391)
• 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel)
• Amelogenin (sex determining marker)
The GlobalFiler ™ Express PCR Amplification Kit combines the 13 original CODIS loci with 7 from the expanded European Standard Set of Loci (ESSL) and the highly discriminating SE33 locus. The kit delivers a 24-locus multiplex with the highest discrimination power of any Thermo Fisher Scientific
Human Identification Kit, along with high sensitivity and tolerance to inhibitors. The concentration of 10 mini-STR loci that are entirely below 220 bp maximizes performance on degraded samples. The highly optimized buffer formulation contains an enzyme that allows completion of amplification in ~45 minutes.
The GlobalFiler ™ Express PCR Amplification Kit uses the same improved process for synthesis and purification of the amplification primers developed for other next-generation Thermo Fisher Scientific
STR chemistries. The improved amplification primers deliver clean electrophoretic backgrounds that assist interpretation.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 1 Product information
Product description
1
Single-source sample types supported
The GlobalFiler ™ Express PCR Amplification Kit is optimized to allow direct amplification from the following types of single-source samples without the need for sample purification:
• Blood and buccal samples on treated paper substrates.
• Blood and buccal samples collected on untreated paper substrates and treated with Prep ‑ n ‑ Go ™
Buffer.
• Buccal samples collected on swab substrates and treated with Prep ‑ n ‑ Go ™ Buffer.
Substrate examples
• Treated paper: NUCLEIC-CARD ™ system or Whatman FTA ™ cards
• Untreated paper: Bode Buccal DNA Collector ™ or 903 paper
• Swab: FLOQSwabs ™ or cotton swabs
Note: Our testing does not include blood samples on swab substrates. This sample type is not typically used for the collection of reference samples.
About the primers
The GlobalFiler ™ Express PCR Amplification Kit primers are manufactured using the same synthesis and purification improvements as the primers in the NGM SElect ™ and the Identifiler ™ Plus kits. These improvements enhance the assay signal ‑ to ‑ noise ratio and simplify the interpretation of results.
The primers used in the kit are:
• For all loci except AMEL—The same primer sequences as the NGM SElect ™ kit and the Identifiler
Plus kit including SNP-specific primers for the vWA, D16S539, AMEL, D2S441, D22S1045, and
™
D8S1179 loci.
• For AMEL—The same primer sequences as the NGM SElect ™
Identifiler ™ Plus kit).
kit (which are different from the
The GlobalFiler ™ Express PCR Amplification Kit also includes the following primer additions and modifications:
• Addition of DYS391 and a novel Y indel.
• The TPOX reverse primer has been redesigned to relocate the amplicon into the higher size range of the multiplex and optimize marker spacing.
• Addition of 8 new SNP-specific primers for the D3S1358, vWA, D18S51, D19S433, TH01, FGA,
D5S818, and SE33 loci. The second degenerate primer was added to the vWA locus to address two different SNPs in the primer binding site.
Non-nucleotide linkers are used in primer synthesis for the following loci: D19S433, vWA, CSF1PO,
D2S441, TH01, FGA, and D12S391. For these primers, non-nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis (Butler 2005, Grossman et al ., 1994).
Non-nucleotide linkers enable reproducible positioning of the alleles to facilitate interlocus spacing. The combination of a 6-dye fluorescent system and the use of non-nucleotide linkers allows simultaneous amplification and efficient separation of all 24 markers during automated DNA fragment analysis.
GlobalFiler ™ Express PCR Amplification Kit User Guide 9
1 Chapter 1 Product information
Product description
Dyes used in the kit
Dye
6 ‑ FAM ™
VIC ™
NED ™
TAZ ™
SID ™
LIZ ™
Color
Blue
Green
Yellow
Red
Purple
Orange
Label
Samples, allelic ladders, and controls
GeneScan ™ 600 LIZ ™ Size Standard v2.0
Loci amplified by the kit
Table 1 GlobalFiler ™ Express PCR Amplification Kit loci and alleles
Locus designation
D3S1358 vWA
D16S539
CSF1PO
TPOX
Y indel
Chromosome location
3p21.31
12p13.31
Alleles included in Allelic Ladder
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24
16q24.1
5q33.3-34
2p23-2per
Yq11.221
5, 8, 9, 10, 11, 12,13, 14, 15
6, 7, 8, 9, 10, 11, 12, 13, 14, 15
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
1, 2
Amelogenin X: p22.1-22.3
Y: p11.2
X, Y
D8S1179
D21S11
D18S51
DYS391
D2S441
D19S433
TH01
8q24.13
21q11.2-q21 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,
18q21.33
Yq11.21
2p14
19q12
11p15.5
5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
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
7, 8, 9, 10, 11, 12, 13
8, 9, 10, 11, 11.3, 12, 13, 14, 15, 16, 17
6, 7, 8, 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15,
15.2, 16, 16.2, 17, 17.2, 18.2, 19.2
4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3
Dye label
6-FAM ™
VIC ™
NED ™
DNA Control
007
15, 16
14, 16
9, 10
11, 12
8, 8
2
X, Y
12, 13
28, 31
12, 15
11
14, 15
14, 15
7, 9.3
10 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 1 Product information
Product description
1
Table 1 GlobalFiler Express PCR Amplification Kit loci and alleles (continued)
Locus designation
Chromosome location
Alleles included in Allelic Ladder
FGA
D22S1045
D5S818
D13S317
D7S820
SE33
D10S1248
D1S1656
D12S391
D2S1338
4q28 13, 14, 15, 16, 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
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 22q12.3
5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18
13q22-31 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
6q14 4.2, 6.3, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 20.2, 21, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2,
27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35,
35.2, 36, 37
10q26.3
1q42.2
12p13.2
2q35
8, 9, 10, 11, 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
14, 15, 16, 17, 18, 19, 19.3, 20, 21, 22, 23, 24,
25, 26, 27
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28
Dye label
NED ™
TAZ ™
SID ™
DNA Control
007
24, 26
11, 16
11, 11
11, 11
7, 12
17, 25.2
12, 15
13, 16
18, 19
20, 23
Standards and controls that are required
For the GlobalFiler ™ Express PCR Amplification Kit, the panel of standards needed for PCR amplification, PCR product sizing, and genotyping are:
• DNA Control 007 —A positive control for evaluating the efficiency of the amplification step and STR genotyping using the GlobalFiler ™ Express Allelic Ladder. DNA Control 007 is present in the kit. See
“DNA Control 007 profile” on page 13
.
• GeneScan ™ 600 LIZ ™ has been evaluated as an internal size standard, yields precise sizing results for PCR products.
Order the GeneScan ™
Size Standard v2.0
—Used for obtaining sizing results. This standard, which
600 LIZ ™ Size Standard v2.0 (Cat. No. 4408399) separately.
• GlobalFiler ™ Express Allelic Ladder —Developed for accurate characterization of the alleles amplified by the kit. The Allelic Ladder is present in the kit and allows automatic genotyping of
most of the reported alleles for the loci in the kit. See “Allelic ladder profile” on page 12
.
GlobalFiler ™ Express PCR Amplification Kit User Guide 11
1 Chapter 1 Product information
Product description
Allelic ladder profile
Figure 1 GeneMapper ™ ID ‑ X Software plot of the GlobalFiler ™ Express Allelic Ladder
12 GlobalFiler ™ Express PCR Amplification Kit User Guide
DNA Control 007 profile
Chapter 1 Product information
Product description
1
Figure 2 DNA Control 007 (1 ng) amplified with the GlobalFiler ™ an Applied Biosystems ™
Express PCR Amplification Kit and analyzed on
3500xL Genetic Analyzer (Y-axis scale 0 to 8,000 RFU).
GlobalFiler ™ Express PCR Amplification Kit User Guide 13
1 Chapter 1 Product information
Contents and storage
Contents and storage
The GlobalFiler ™ Express PCR Amplification Kit contains sufficient quantities of the following reagents to perform 200 (Cat. No. 4476609) or 1,000 (Cat. No. 4474665) amplifications at 15 μL/amplification.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
IMPORTANT! Do not refreeze kit components after thawing.
Contents
GlobalFiler ™ Express
Master Mix
Master Mix Additive
GlobalFiler ™ Express
Primer Set
GlobalFiler ™ Express
Allelic Ladder
Description
Contains enzyme, salts, dNTPs, bovine serum albumin, enzyme, and
0.05% sodium azide in buffer and salt.
Reagent for one-time addition to the
GlobalFiler ™ Express
Master Mix following first thaw.
Contains forward and reverse primers to amplify human DNA targets.
200 reactions
(Cat. No.
4476609)
1 × 1.13 mL
1 × 0.1 mL
1,000 reactions
(Cat. No.
4474665)
1 × 5.64 mL
1 × 0.45 mL
Storage
−25°C to −15°C on receipt.
2°C to 8°C after first use, for up to 6 months or up to the expiration date stated on the kit (whichever comes first).
−25°C to −15°C on receipt.
Discard the tube after adding to the master mix.
1 × 1.2 mL 1 × 6 mL
Contains amplified alleles.
See “Allelic ladder profile” on page 12 for
information.
1 × 0.065 mL 1 × 0.15 mL
−25°C to −15°C on receipt.
2°C to 8°C after first use, for up to 6 months or up to the expiration date stated on the kit (whichever comes first).
Store protected from light.
−25°C to −15°C on receipt.
2°C to 8°C after first use, up to the expiration date stated on the kit.
Store protected from light.
14 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 1 Product information
Required materials not supplied
1
(continued)
Contents Description
200 reactions
(Cat. No.
4476609)
1,000 reactions
(Cat. No.
4474665)
Storage
DNA Control 007 Contains 2 ng/µL human male genomic DNA from cell line in 0.05% sodium azide and buffer [1]
See “DNA Control 007 profile” on page 13 for
information.
1 × 0.05 mL 1 × 0.1 mL −25°C to −15°C on receipt.
2°C to 8°C after first use, up to the expiration date stated on the kit.
[1] DNA Control 007 is included at a concentration that is appropriate for use as an amplification control (that is, to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype). It is not designed for use as a DNA quantification control. If you quantify aliquots of Control 007, the concentration may differ from the labeled concentration.
Required materials not supplied
See Appendix B, “Materials required but not supplied”.
Instrument and software compatibility
Instrument type
Thermal cyclers
Validated models
• ProFlex ™ 96 ‑ well PCR System (Cat. No. 4484075 )
• ProFlex ™ 2 × 96 ‑ well PCR System (Cat. No. 4484076)
• Veriti ™ 96 ‑ Well Thermal Cycler (Cat. No. 4479071)
• GeneAmp ™ PCR System 9700, 96-Well Silver (Cat. No. N8050001)
• GeneAmp ™ PCR System 9700, 96-Well Gold-Plated (Cat. No. 4314878)
IMPORTANT! GlobalFiler ™ Express PCR Amplification Kit is NOT validated for use with:
· Veriti ™ Fast 96 ‑ Well Thermal Cycler (Cat. No. 4375305)
· GeneAmp ™ PCR System 9700, 96-Well Aluminum (Cat. No. 4314879)
GlobalFiler ™ Express PCR Amplification Kit User Guide 15
1 Chapter 1 Product information
Instrument and software compatibility
(continued)
Instrument type
Genetic analyzers [1]
Validated models
3500/3500xL Genetic Analyzer
• 3500 Series Data Collection Software 1 (Windows ™ Vista operating system) and HID Updater
3500 Data Collection Software v2 (Cat. No. 4480670)
• 3500 Series Data Collection Software 2 (Windows ™ 7 operating system) and HID Updater
3500 Data Collection Software v2 (Cat. No. 4480670)
• 3500 Series Data Collection Software 3 (Windows ™ 7 operating system)
• 3500 Series Data Collection Software 3.1 (Windows ™ 7 operating system)
• 3500 Series Data Collection Software 4 (Windows ™ 10 operating system)
• 3500 Series HID Data Collection Software v4.0.1 (Windows ™ 10 operating system)
3130/3130 xl Genetic Analyzer
• 3130 Series Data Collection Software 4 (Windows ™ 7 operating system)
• 3130/3730 Data Collection 4 6-Dye Module v1
3730/3730xl DNA Analyzer
• 3730 Series Data Collection Software 4 (Windows ™ 7 operating system)
• 3730 Series Data Collection Software 4 6-Dye Module v1
• 3730xl Data Collection Software 5 (Windows ™ 10 operating system)
Note: For information on using the 3730xl DNA Analyzer, see the 3730xl Data
Collection Software 5 for HID User Bulletin: New Features and Developmental Validation
(Pub. No. MAN0019461)
SeqStudio ™ Genetic Analyzer
• SeqStudio ™ Data Collection Software v1.2
• SeqStudio ™ Data Collection Software v1.2.1
Analysis software
GeneMapper ™ ID ‑ X Software v1.4 or later
Windows ™ XP, Windows ™ 7, or Windows ™ 10 operating system
[1] We conducted validation studies using the 3130 xl , 3500, 3500xL, and 3730xl instruments. For validation information on the 3730xl instrument, see the 3730xl Data Collection Software 5 for HID User Bulletin: New Features and Developmental Validation (Pub. No. MAN0019461).
16 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 1 Product information
Workflow
1
Workflow
Perform PCR on treated or untreated paper substrates
“Prepare the amplification kit reactions: treated paper substrate” on page 21 or
“Prepare the amplification kit reactions: untreated paper substrate” on page 23
▼
Obtain punch with Harris Manual Punch or BSD Semi-
Automated Dried Sample Punch Instrument
▼
Untreated paper only: Process with Prep ‑ n ‑ Go ™ Buffer
Perform PCR on swab substrates
“Prepare the reactions: swab substrate”
▼
▼
Process with the GlobalFiler ™
Amplification Kit
Express PCR
▼
Lyse in Prep ‑ n ‑ Go ™ Buffer
▼
Process with the GlobalFiler ™
Amplification Kit
Express PCR
▼
Amplify with a recommended thermal cycler
▼
Perform electrophoresis
“Set up the SeqStudio ™ instruments for electrophoresis (before first use of the kit)” on page 30
or
“Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)” on page 32
or
“Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)” on page 36 or
“Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit)” on page 38
▼
“Prepare samples for electrophoresis” on page 41
▼
Analyze data
“Set up the GeneMapper ™ ID ‑ X Software for analysis (before first use of the kit)” on page 44
“Create an analysis method” on page 49
“Create a size standard definition file if needed” on page 57
“Analyze and edit sample files with GeneMapper ™ ID ‑ X Software” on page 60
“Examine or edit a project” on page 61
GlobalFiler ™ Express PCR Amplification Kit User Guide 17
2
Perform PCR
■
■
■
■
■
■
Optimize PCR cycle number (before first use of the kit)
Before using the GlobalFiler ™ Express PCR Amplification Kit for the first time, perform a single initial sensitivity experiment to determine the appropriate cycle number to use during internal validation studies and operational use of the kit. This experiment accounts for instrument ‑ to ‑ instrument and sample ‑ to ‑ sample variations. If you are processing multiple sample type and substrate combinations
(for example, buccal samples on treated paper and buccal samples on swabs), perform separate sensitivity experiments for each sample type and substrate to be used for testing.
Procedural guidelines when optimizing PCR cycle number
• (Recommended) Use 26 samples so that you can complete electrophoresis using a single 96 ‑ well plate. This minimizes the impact of run ‑ to ‑ run variation on the results. Examples of PCR and
electrophoresis plate layouts are provided on page 145.
• To maximize result quality, prepare and amplify Plate 1, then repeat for Plates 2 and 3. Do not prepare all 3 plates before amplification.
• To minimize the effect of instrument ‑ to ‑ instrument variation, use the same thermal cycler to amplify all 3 plates.
Select samples and prepare plates
1.
Select 26 of each sample+substrate type. Ensure that the selected samples represent a "typical" range of samples analyzed in your laboratory.
2.
Prepare the samples and the reactions as described in the appropriate protocols later in this chapter. Prepare sufficient PCR reagents to complete amplification of three replicate plates.
3.
Create the first of 3 identical PCR plates (see page 145 for a suggested plate layout).
18 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Before you begin
2
4.
Amplify each plate using a different cycle number to determine the optimum conditions for use in your laboratory.
Suggested cycle numbers for different sample type and substrate combinations are listed in the following table.
Sample type
Blood
Buccal
Treated paper
25, 26, 27 cycles
26, 27, 28 cycles
Substrate
Untreated paper
25, 26, 27 cycles
26, 27, 28 cycles
Swab
N/A
25, 26, 27 cycles
Determine optimum PCR conditions
1.
Run the PCR products on the appropriate CE platform using the recommended protocol that is
described in Chapter 3, “Perform electrophoresis”.
2.
Based on the results of the sensitivity study, select the appropriate PCR cycle number for future experiments.
Our studies indicate the optimum PCR cycle number should generate profiles with the following heterozygote peak heights, with no instances of allelic dropout and minimal occurrence of off ‑ scale allele peaks:
Instrument
3500 Series
3130 Series
3730 Series
SeqStudio ™ Genetic Analyzer
Heterozygous peak height
3,000–12,000 RFU
1,000–3,000 RFU
3,000–12,000 RFU
3,000–12,000 RFU
When amplifying single ‑ source, unpurified samples, you will see greater sample ‑ to ‑ sample variation in peak height than you see with purified samples. Careful optimization of the cycle number helps to minimize this variation.
Before you begin
Thaw reagents and prepare Master Mix (before first use of the kit)
1.
Thaw the Master Mix, Master Mix Additive, and Primer Set, then vortex for 3 seconds.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to 8°C and, therefore, do not require subsequent thawing. Do not refreeze the reagents.
GlobalFiler ™ Express PCR Amplification Kit User Guide 19
2 Chapter 2 Perform PCR
Treated paper substrates: prepare the amplification kit reactions
2.
Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly and tapping the bottles on the bench.
3.
Add the following volumes of Master Mix Additive to the Master Mix:
Kit
200 reactions
1,000 reactions
Master Mix Additive volume
80 µL
390 µL
4.
Gently invert the Master Mix tube 10 times, then centrifuge the tube briefly or tap the bottle on the bench.
5.
Mark the cap of the Master Mix with a (+) to indicate that the Master Mix Additive has been added.
6.
Discard the Master Mix Additive tube.
Treated paper substrates: prepare the amplification kit reactions
Sample preparation guidelines: treated paper substrate
• Do not add water to the wells on the reaction plate before adding the punches. If you observe static issues with the paper discs, you can prepare and dispense the 15-µL reaction mix into the wells of the reaction plate before adding the punches.
Alternatively, dispense 3 µL of low-TE Buffer into each sample and negative amplification control well (NOT the positive amplification control wells) before adding the punches.
• Make the punch as close as possible to the center of the sample to ensure optimum peak intensity.
Increasing the size of the punch may cause inhibition during PCR amplification.
• For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate.
• For automated punching: See the User Guide of your automated or semiautomated disc punch instrument for proper guidance.
Prepare low-TE buffer
For optimal results, we recommend using low-TE buffer for sample preparation. Prepare it as described in this procedure or buy it from Teknova (Cat. No. T0223).
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 accordingly for specific needs.
20 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Treated paper substrates: prepare the amplification kit reactions
2
2.
Aliquot, then autoclave the solutions.
3.
Store the aliquots at room temperature.
Prepare the amplification kit reactions: treated paper substrate
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.
Mix (before first use of the kit)” on page 19
before proceeding.
1.
Add samples to the MicroAmp ™ Optical 96 ‑ well Reaction Plate:
To these wells of the plate ...
Negative control
Test samples
Positive control
IMPORTANT! Do not add a blank disc to the positive control well.
1.2 mm blank disc
1.2 mm sample disc
For 25 and 26 cycles
For 27 cycles
For 28 cycles
Add...
3 μL of Control DNA 007
2 μL of Control DNA 007
1 μL of Control DNA 007
Note: The volumes of positive control are suggested amounts and can be adjusted if peak heights are too high or too low for your optimized cycle number.
2.
Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
3.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix
Primer Set
Low-TE buffer
Volume per reaction
6.0 μL
6.0 μL
3.0 μL
Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! This kit is optimized for a 15-μL PCR volume to overcome the PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.
4.
Vortex the reaction mix for 3 seconds, then centrifuge briefly.
5.
Dispense 15 µL of the reaction mix into each reaction well of a MicroAmp ™
Reaction Plate.
Optical 96-Well
GlobalFiler ™ Express PCR Amplification Kit User Guide 21
2 Chapter 2 Perform PCR
Untreated paper substrates: prepare the amplification kit reactions
6.
Seal the plate with MicroAmp ™ Clear Adhesive Film (Cat. No. 4306311 ) or MicroAmp ™
Adhesive Film (Cat. No. 4311971).
Optical
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across all wells.
IMPORTANT! If you are using the GeneAmp ™ PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp ™ Optical
Film Compression Pad (Cat. No. 4312639 ) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.
7.
Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
8.
Amplify the samples as described in Chapter 2, “Perform PCR”.
IMPORTANT! This kit is not validated for use with the GeneAmp ™ PCR System 9700 with the aluminum 96-well block. Use of this thermal cycling platform may adversely affect performance of this kit.
Untreated paper substrates: prepare the amplification kit reactions
Sample preparation guidelines: untreated paper substrate
• Make a 1.2 mm punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification.
• If you are using a Bode Buccal DNA Collector ™ , make a 1.2 mm punch as close as possible to the tip of the DNA collector to ensure optimum peak intensity.
A larger punch may cause inhibition during PCR amplification.
1
• For manual punching: Place the tip of a 1.2 mm Harris
Micro-Punch on the card, hold the barrel of the Harris
Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate.
• For automated punching: See the User Guide of your automated or semiautomated disc punch instrument for proper guidance.
1 Location of punch with a Bode Buccal
DNA Collector ™
22 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Untreated paper substrates: prepare the amplification kit reactions
2
Prepare the amplification kit reactions: untreated paper substrate
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.
Mix (before first use of the kit)” on page 19
before proceeding.
1.
Add Prep ‑ n ‑ Go ™ Buffer (Cat. No. 4467079) to the MicroAmp ™ Optical 96-Well Reaction Plate:
To these wells ...
Negative control
Test samples
Positive control
Add...
3 μL of Prep ‑ n ‑ Go ™ Buffer
3 μL of Prep ‑ n ‑ Go ™ Buffer
For 25 and 26 cycles
For 27 cycles
For 28 cycles
0 μL of Prep ‑ n ‑ Go ™ Buffer
1 μL of Prep ‑ n ‑ Go ™ Buffer
2 μL of Prep ‑ n ‑ Go ™ Buffer
2.
Add samples to the reaction plate:
To these wells ...
Negative control
Test samples
Positive control
IMPORTANT! Do not add a blank disc to the positive control well.
1.2 mm blank disc
1.2 mm sample disc
For 25 and 26 cycles
For 27 cycles
For 28 cycles
Add...
3 μL of Control DNA 007
2 μL of Control DNA 007
1 μL of Control DNA 007
Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number.
3.
Centrifuge the plate to ensure that the punches are immersed in the Prep ‑ n ‑ Go ™ Buffer.
4.
Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
5.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix
Primer Set
Volume per reaction
6.0 μL
6.0 μL
GlobalFiler ™ Express PCR Amplification Kit User Guide 23
2 Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! This kit is optimized for a 15-μL PCR volume to overcome the PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.
6.
Vortex the reaction mix for 3 seconds, then centrifuge briefly.
7.
Dispense 12 µL of the reaction mix into each reaction well of a MicroAmp ™
Reaction Plate.
Optical 96-Well
The final volume in each well is 15 µL (reaction mix plus Prep ‑ n ‑ Go ™ control).
Buffer and sample or positive
8.
Seal the plate with MicroAmp ™ Clear Adhesive Film (Cat. No.
Adhesive Film (Cat. No. 4311971 ).
4306311 ) or MicroAmp ™ Optical
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across all wells.
IMPORTANT! If you are using the GeneAmp ™ PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp ™ Optical
Film Compression Pad (Cat. No. 4312639 ) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.
9.
Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
10.
Amplify the samples as described in Chapter 2, “Perform PCR”.
IMPORTANT! This kit is not validated for use with the GeneAmp ™ PCR System 9700 with the aluminum 96-well block. Use of this thermal cycling platform may adversely affect performance of this kit.
Swab substrates: prepare the amplification kit reactions
Sample preparation guidelines: swab substrate
• Detach each buccal swab head from the swab shaft before lysis.
• If you are using the heated lysis protocol, perform lysis in either of the following formats:
– 1.5-mL tubes with a heat block (VWR ™ Scientific Select dry heat block or similar)
– PrepFiler ™ 96-Well Processing Plates (Cat. No. A47010)
– Robbins Scientific ™ Model 400 Hybridization Incubator or similar
24 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
2
– Agilent ™ Benchtop Rack for 200 μL Tubes/V Bottom Plates (metal) or similar (Cat. No. 410094)
IMPORTANT! Do not use a plastic plate adaptor.
• For optimum performance, lyse the entire swab. If you need to preserve the sample, use half of the lysate prepared from the entire swab.
Prepare the sample lysate: room temperature
This protocol may improve the performance for challenging or aged samples.
1.
Add 400 µL Prep ‑ n ‑ Go ™ Buffer (Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a
PrepFiler ™ 96-Well Processing Plate (Cat. No. A47010).
2.
Into each tube or well, put the entire head of each swab, then let stand for 20 minutes at room temperature (20°C to 25°C) to lyse the sample.
3.
After 20 minutes, transfer the sample lysate out of the sample plate into tubes or plates for storage, then discard the deep ‑ well plate containing the swab heads.
Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate.
4.
Go to “Prepare the reactions: swab substrate” on page 26
or “Store the sample lysate” on page 27
.
Prepare the sample lysate: heat protocol
This protocol may improve the performance for challenging or aged samples.
1.
Preheat the heat block to 90°C or the oven with metal plate adaptor to 99°C.
2.
Add 400 µL Prep ‑ n ‑ Go ™ Buffer (for buccal swabs, Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a PrepFiler ™ 96-Well Processing Plate (Cat. No. A47010).
3.
Into each tube or well, put the entire head of each swab. If you are using tubes, cap the tubes. Let the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample.
4.
After 20 minutes, remove the tubes or the deep ‑ well plate from the heat block or oven.
5.
Let the lysate stand at room temperature for at least 15 minutes to cool the lysate (for accurate pipetting).
6.
Transfer the sample lysate out of the 1.5-mL tubes or sample plate into tubes or plates for storage.
Discard the 1.5-mL tubes or deep ‑ well plate containing the swab heads.
Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate.
7.
Go to “Prepare the reactions: swab substrate” on page 26
or “Store the sample lysate” on page 27
.
GlobalFiler ™ Express PCR Amplification Kit User Guide 25
2 Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
Prepare the reactions: swab substrate
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.
Mix (before first use of the kit)” on page 19
before proceeding.
1.
Add Prep ‑ n ‑ Go ™ Buffer (Cat. No. 4471406) to the control wells in the MicroAmp ™ Optical 96-Well
Reaction Plate:
To these wells ...
Negative control
Positive control
Add...
3 μL of Prep ‑ n ‑ Go ™ Buffer
For 25 and 26 cycles 0 μL of Prep ‑ n ‑ Go ™ Buffer
For 27 cycles
For 28 cycles
1 μL of Prep ‑ n ‑ Go
2 μL of Prep ‑ n ‑ Go
™
™
Buffer
Buffer
2.
Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
3.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix
Primer Set
Volume per reaction
6.0 μL
6.0 μL
Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! This kit is optimized for a 15-μL PCR volume to overcome the PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.
4.
Vortex the reaction mix for 3 seconds, then centrifuge briefly.
5.
Dispense 12 μL of the reaction mix into each reaction well of a MicroAmp ™
Reaction Plate.
Optical 96-Well
The final volume in each well is 15 μL (reaction mix plus Prep ‑ n ‑ Go ™ positive control).
Buffer or sample lysate or
26 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
2
6.
Add samples to the reaction plate:
To these well(s) of a MicroAmp ™ Optical 96-
Well Reaction Plate...
Test samples
Positive control
3 μL of sample lysate
For 25 and 26 cycles
Add...
For 27 cycles
For 28 cycles
3 μL of Control DNA
007
2 μL of Control DNA
007
1 μL of Control DNA
007
Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number.
7.
Seal the plate with MicroAmp ™ Clear Adhesive Film (Cat. No. 4306311 ) or MicroAmp ™
Adhesive Film (Cat. No. 4311971 ).
Optical
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across all wells.
IMPORTANT! If you are using the GeneAmp ™ PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp ™ Optical
Film Compression Pad (Cat. No. 4312639 ) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.
8.
Vortex the reaction mix at medium speed for 3 seconds.
9.
Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
10.
Amplify the samples as described in Chapter 2, “Perform PCR”.
IMPORTANT! This kit is not validated for use with the GeneAmp ™ PCR System 9700 with the aluminum 96-well block. Use of this thermal cycling platform may adversely affect performance of this kit.
Store the sample lysate
1.
Cap the sample lysate storage tubes or seal the sample lysate storage plate with MicroAmp ™
Adhesive Film.
Clear
2.
Store the sample lysate as needed:
If you are storing the sample lysate...
<2 weeks
>2 weeks
Then place at...
2°C to 8°C
–25°C to –15°C
GlobalFiler ™ Express PCR Amplification Kit User Guide 27
2 Chapter 2 Perform PCR
Perform PCR
Note: The effects of multiple freeze/thaw cycles on the lysate have not been fully evaluated.
Therefore, multiple freeze/thaw cycles are not recommended.
Perform PCR
IMPORTANT!
.
1.
Program the thermal cycling conditions.
IMPORTANT! If you are using the GeneAmp ™
If you are using the ProFlex ™ 96 ‑ simulation mode. If you are using the Veriti ™
PCR System 9700, select the Max ramping mode.
well PCR System, select the GeneAmp ™ PCR System 9700
Thermal Cycler, select the 100% ramping rate. Do not use 9600 emulation mode.
Initial incubation step
Optimum cycle number [1]
Denature Anneal/Extend
Final extension Final hold
HOLD CYCLE HOLD HOLD
95°C, 1 minute 94°C, 3 seconds 60°C, 30 seconds 60°C, 8 minutes 4°C, up to 24 hours [2]
[1]
[2]
See “Optimize PCR cycle number (before first use of the kit)” on page 18
.
The infinity (∞) setting allows an unlimited hold time.
2.
Load the plate into the thermal cycler, close the heated cover, then start the run.
IMPORTANT! If you are using adhesive clear film instead of caps to seal the plate wells, be sure to place a MicroAmp to prevent evaporation during thermal cycling. The Veriti
System, and ProFlex ™
™ Optical Film Compression Pad (Cat. No. 4312639 ) on top of the plate
2 × 96 ‑
™ Thermal Cycler, ProFlex well PCR System do not require a compression pad.
™ 96 ‑ well PCR
3.
When the run is complete, store the amplified DNA.
If you are storing the DNA...
<2 weeks
>2 weeks
Then place at...
2°C to 8°C
–25°C to –15°C
IMPORTANT! Protect the amplified DNA from light.
28 GlobalFiler ™ Express PCR Amplification Kit User Guide
3
Perform electrophoresis
■
■
■
■
■
■
■
Allelic ladder requirements for electrophoresis
To accurately genotype samples, you must run an allelic ladder with the samples.
Instrument
3500
3500 xl
3130
3130 xl
3730/3730 xl ,
48 ‑ capillary
SeqStudio ™
Number of allelic ladders to run
1 per 3 injections
1 per injection
1 per 4 injections
1 per injection
3 per injection
1 per 6 injections
One injection equals
8 samples
24 samples
4 samples
16 samples
48 samples
4 samples
Number of samples per allelic ladder(s)
23 samples + 1 allelic ladder
23 samples + 1 allelic ladder
15 samples + 1 allelic ladder
15 samples + 1 allelic ladder
15 samples + 1 allelic ladder
23 samples + 1 allelic ladder
IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between runs. Follow the guidelines in the preceding table, which should account for normal variation in run speed. Perform internal validation studies to verify the required allelic ladder injection frequency, to ensure accurate genotyping of all samples in your laboratory environment.
It is critical to genotype using an allelic ladder run under the same conditions as the samples. Size values obtained for the same sample can differ between instrument platforms, because of different polymer matrices and electrophoretic conditions.
29 GlobalFiler ™ Express PCR Amplification Kit User Guide
3 Chapter 3 Perform electrophoresis
Materials required for electrophoresis
Materials required for electrophoresis
this kit.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
Set up the SeqStudio
™
instruments for electrophoresis
(before first use of the kit)
Electrophoresis software setup
The following table lists the data collection software and the run modules that you can use to analyze
PCR products generated by this kit. For details on the procedures, see the documents listed in
“Documentation and support” on page 151 .
Genetic
Analyzer
SeqStudio ™
Data Collection
Software
SeqStudio ™
Data Collection
Software v1.2.1
Additional software
None
Run modules and conditions
• Run Module: HIDAnalysis
• Injection Conditions: 1.2 kV/10 sec
• Run Conditions: 11 kV/1,120 sec
• Dye Set J6
• Kit: GlobalFiler Express (to enable marker-to-marker pull-up reduction feature)
30 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Set up the SeqStudio ™ instruments for electrophoresis (before first use of the kit)
3
Perform spectral calibration
Perform a spectral calibration using the DS ‑ 36 Matrix Standard Kit (Dye set J6, 6 ‑ dye)
(Cat. No. 4425042).
You need to perform manual calibration for each dye set only once before first use. To determine if a dye set requires manual calibration, review the calibration history for the dye set.
The following figure is an example of a passing 6-dye spectral calibration.
GlobalFiler ™ Express PCR Amplification Kit User Guide 31
3 Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
Set up the 3500/3500xL instruments for electrophoresis
(before first use of the kit)
Electrophoresis software setup
The following table lists the data collection software and the run modules that you can use to analyze
PCR products generated by this kit. For details on the procedures, see the documents listed in
“Documentation and support” on page 151 .
Note: We conducted original validation studies for the kit using the 3130 xl , 3500, or 3500xL configurations. Subsequent validation studies for the kit were performed using the 3730 xl 48-capillary configuration and the SeqStudio ™ Genetic Analyzer.
Genetic analyzer
3500
3500xL
Operating system
Windows ™ 10
3500 Data
Collection
Software v4.0.1
3500
3500xL
3500
3500xL
3500
3500xL
Windows ™ 7
Windows ™ 7
Windows ™
Vista v3, v3.1
v2 v1
Additional software
Plate templates, assays, run modules, and conditions
None Assays: AB_J6_LS_POP4 (and _xl) and AB_J6OSR_LS_POP4 (and _xl), which contain instrument protocols
AB_HID36_POP4_J6_NT3200 (and _xl) and
AB_HID36_POP4_J6OSR_NT3200 (and _xl)
All assays use the following conditions:
• Run Module: HID36_POP4(xl)
• Injection Conditions: 1.2kV/15 sec
(24 sec for xl)
• Run Conditions: 13kV/1,550 sec
• Dye Set J6 or J6-OSR
None
HID Updater
3500 DC v2
(Cat. No. 4480670)
HID Updater
3500 DC v2
(Cat. No. 4480670)
Plate templates: 6dye_36_POP4 (and _xl)
Assays (DCv3.1 and earlier): GF+Norm_POP4
(and _xl) and GF_POP4 (and _xl), which contain instrument protocol HID36_POP4
(and_xl)_J6_NT3200.
All assays use the following conditions:
• Run module: HID36_POP4(xl)
• Injection conditions: 1.2 kV/15 sec
(24 sec for xl)
• Run conditions: 13 kV/1,550 sec
• Dye Set J6
32 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
3
Obtain and run the HID Updater (v1 and v2 software only)
Perform this procedure if you are using 3500 Series Software v1 or v2.
You can run 6-dye samples on 3500 Data Collection Software. Before running on either system for the first time, run the HID Updater 3500 DC v2 (Cat. No. 4480670). The HID Updater installs plate templates, assays, and instrument protocols that can be used to run GlobalFiler ™ Express PCR
Amplification Kit samples. For more information, refer to the release notes provided with the Updater.
Note: If you have a new instrument installed by a Thermo Fisher Scientific representative, the updater may have been run during installation.
1.
Shut down the 3500/3500xL Data Collection Software.
2.
Download the updater from www.thermofisher.com/us/en/home/technical-resources/ software-downloads/3500-Series-Genetic-Analyzers-for-Human-Identification.html
.
3.
Open the Read me file and review the software release notes.
4.
Click the updater .exe
file.
5.
Follow the on-screen prompts.
6.
Restart the computer.
Modify 3500 QC protocol
The GlobalFiler ™ Express PCR Amplification Kit has been validated with data that was analyzed using both the 3rd Order Least Squares method (80–460 base pairs) and Local Southern method (60–460 base pairs).
Before using the QC protocol to acquire data, modify it to:
• Change the Baseline Window and Peak Window Settings default settings to the settings shown in the following figure.
• Change the size calling default setting to to Local Southern, if needed.
1.
In the Library tab, open the QC Protocol window.
2.
Create a new QC protocol: a.
Name the new QC protocol according to your laboratory naming convention.
b.
Set the following parameters:
Parameter
Size Standard
Size Range
Sizing Start Size
Sizing Stop Size
GS600_LIZ_(60-460)
Setting
Partial
60 bp
460 bp
GlobalFiler ™ Express PCR Amplification Kit User Guide 33
3 Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
(continued)
Parameter
Size Calling Method
After checking the "Use Baselining" box:
Baseline Window Pts.
Peak Window Size c.
Click Save .
Setting
Local Southern Method or 3rd Order Least
Squares
33
13
34
3.
Add the QC protocol to the HID assay.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)
3
Perform spectral calibration
Perform a spectral calibration using the DS ‑ 36 Matrix Standard Kit (Dye set J6, 6 ‑ dye)
(Cat. No. 4425042).
The following figure is an example of a passing 6-dye spectral calibration.
To enable off-scale recovery (OSR) with 3500 Series Data Collection Software 4.0.1, select the J6-OSR dye set in the spectral calibration and use the J6 OSR assay to run samples.
GlobalFiler ™ Express PCR Amplification Kit User Guide 35
3 Chapter 3 Perform electrophoresis
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)
Set up the 3130/3130
xl
instruments for electrophoresis
(before first use of the kit)
Electrophoresis software setup
The following table lists the data collection software and the run modules that you can use to analyze
PCR products generated by this kit. For details on the procedures, see the documents listed in
“Documentation and support” on page 151 .
Note: We conducted original validation studies for the GlobalFiler ™ Express PCR Amplification Kit using the 3130xl, 3500, or 3500xL configurations. Subsequent validation studies for the kit were performed using the 3730 xl 48-capillary configuration and the SeqStudio ™ Genetic Analyzer.
Genetic
Analyzer
3130
3130 xl
Operating
System
Windows ™ 7
Data
Collection
Software
Data
Collection
Software v4 [1]
Additional software
3130/3730 DC v4
6 ‑ Dye Module v1
Run modules and conditions
• HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/5 sec
• Run conditions: 15 kV/1500 sec
• Dye Set J6
• HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/10 sec
• Run conditions: 15 kV/1500 sec
• Dye Set J6
[1] Requires activation of 6 ‑ dye license.
Obtain and activate 6-dye license
1.
Confirm that you are running Data Collection Software v4 ( Help 4 About ).
2.
Obtain a 3130 DC v4 6-Dye Module v1 License key. Contact your local Human Identification representative for information.
3.
Ensure that all network cards in the computer are enabled.
IMPORTANT! You can run the 3130 Series Data Collection Software v4 using only the network cards that are enabled when you activate the software license. For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled.
36 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)
3
4.
Select Tools 4 License Manager to display the Software Activation dialog box.
5.
Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you.
6.
Obtain the software license file from your email.
7.
Make a copy of the software license file and keep it in a safe location.
8.
Copy the software license file to the desktop of the Data Collection Software v4 computer.
9.
If the Software Activation dialog box has closed, select Tools 4 License Manager .
10.
Click Browse , then navigate to the software license file saved on your computer.
11.
Click Install and Validate License .
A message is displayed when the license is installed and validated.
12.
Click Close .
GlobalFiler ™ Express PCR Amplification Kit User Guide 37
3 Chapter 3 Perform electrophoresis
Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit)
Perform spectral calibration
Perform a spectral calibration using the DS ‑ 36 Matrix Standard Kit (Dye set J6, 6 ‑ dye)
(Cat. No. 4425042).
The following figure is an example of a passing 6-dye spectral calibration.
Set up the 3730/3730
xl
instruments for electrophoresis
(before first use of the kit)
Electrophoresis software setup
The following table lists the data collection software and the run modules that you can use to analyze
PCR products generated by this kit. For details on the procedures, see the documents listed in
“Documentation and support” on page 151 .
Genetic
Analyzer
3730 xl
Operating
System
Windows ™ 10
Data
Collection
Software
Data
Collection
Software v5
3730 Windows ™ 7 Data
Collection
Software v4 [1]
[1] Requires activation of 6 ‑ dye license.
Additional software
None
3130/3730 DC v4
6-Dye Module v1
Run modules and conditions
• GeneMapper36_POP7_1 Injection conditions: 2 kV/10 sec
• Run conditions: 15 kV/1,200 sec
• Dye Set J6
38 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit)
3
Obtain and activate the 6-dye license
1.
Confirm that you are running Data Collection Software v4 ( Help 4 About )
2.
Obtain a 3730 DC v4 6-Dye Module v1 License key. Contact Thermo Fisher Scientific for information.
3.
Ensure that all network cards in the computer are enabled.
IMPORTANT! You can run the 3730 Series Data Collection Software v4 using only the network cards enabled when you activate the software license. For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled.
4.
Select Tools 4 License Manager to display the Software Activation dialog box.
GlobalFiler ™ Express PCR Amplification Kit User Guide 39
3 Chapter 3 Perform electrophoresis
Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit)
5.
Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you.
6.
Obtain the software license file from your email.
7.
Make a copy of the software license file and keep in a safe location.
8.
Copy the software license file to the desktop of the Data Collection Software v4 computer.
9.
If the Software Activation dialog box has closed, select Tools 4 License Manager .
10.
Click Browse , then navigate to the software license file saved on your computer.
11.
Click Install and Validate License .
A message is displayed when the license is installed and validated.
12.
Click Close .
Perform spectral calibration
Perform a spectral calibration using the DS ‑ 36 Matrix Standard Kit (Dye set J6, 6 ‑ dye)
(Cat. No. 4425042).
The following figure is an example of a passing 6-dye spectral calibration.
40 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis
Prepare samples for electrophoresis
3
Prepare samples for electrophoresis
Prepare the samples for electrophoresis immediately before loading.
1.
Pipet the required volumes of components into an appropriately sized polypropylene tube:
Reagent
GeneScan ™ 600 LIZ ™ Size Standard v2.0
Hi ‑ Di ™ Formamide
Volume per reaction
0.5 μL
9.5 μL
Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers.
IMPORTANT! The volume of size standard indicated in the table is a suggested amount.
Determine the appropriate amount of size standard based on your experiments and results.
2.
Vortex the tube, then briefly centrifuge.
3.
Into each well of a MicroAmp ™ Optical 96-Well Reaction Plate, add:
• 10 µL of the formamide/size standard mixture
• 1 µL of PCR product or Allelic Ladder
Note: For blank wells, add 10 µL of Hi ‑ Di ™ Formamide.
4.
Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom.
5.
Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.
6.
Immediately place the plate on ice for 3 minutes.
7.
Place the sample tray on the autosampler, then start the electrophoresis run.
GlobalFiler ™ Express PCR Amplification Kit User Guide 41
42
4
Analyze data with GeneMapper
™
ID ‑ X
Software
■
■
■
■
■
■
■
■
■
Overview of GeneMapper
™ ID ‑ X
Software
GeneMapper ™ ID ‑ X Software is an automated genotyping software application for forensic casework, databasing, and paternity data analysis.
GeneMapper electrophoresis, the data collection software stores information for each sample in a .fsa or .hid file. The or .hid files.
™
GeneMapper ™
ID ‑ X Software v1.4 or later analyzes 4-dye, 5-dye, and 6-dye data and is required to correctly analyze data that is generated using the GlobalFiler
ID ‑ X
™ Express PCR Amplification Kit. After
Software v1.4 or later allows you to analyze and interpret the data from the .fsa
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Allelic ladder requirements for data analysis
4
Allelic ladder requirements for data analysis
• HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies before you use multiple allelic ladder samples in an analysis.
For multiple allelic ladder samples, the GeneMapper ™ ID ‑ X Software calculates allelic bin offsets by using an average of all allelic ladders that use the same panel in a run folder.
• Allelic ladder samples in an individual run folder are considered to be from a single run. When the software imports multiple run folders into a project, only the ladders in their respective run folders are used for calculating allelic bin offsets and subsequent genotyping.
• Allelic ladder samples must be labeled as " Allelic Ladder " in the Sample Type column in a project.
Analysis will fail if the Allelic Ladder Sample Type is not specified.
• Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples, to ensure proper allele calling.
• Alleles that are not in the allelic ladders do exist. Off-ladder (OL) alleles can contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the bin window of any known allelic ladder allele or virtual bin.
Note: If a sample allele peak is called as an off-ladder allele, verify the sample result according to your laboratory protocol.
File names and versions used in this section
The file names and version numbers of panel, bin, and stutter files that are shown in this section may differ from the file names that you see when you download or import files.
If you need help to determine the correct files to use, contact your local Human Identification representative, or go to thermofisher.com/support .
GlobalFiler ™ Express PCR Amplification Kit User Guide 43
4 Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Set up the GeneMapper ™ ID ‑ X Software for analysis (before first use of the kit)
Set up the GeneMapper
™
first use of the kit)
ID ‑ X
Software for analysis (before
Workflow: Set up GeneMapper
™
ID
‑
X Software
Before you use GeneMapper ™ following:
ID ‑ X Software to analyze data for the first time, you must do the
“Check panel, bin, and stutter file versions on your computer” on page 44
▼
“(If needed) Download newer versions of panel, bin, and stutter files” on page 45
▼
“Import panels, bins, and marker stutter” on page 45
▼
“(Optional) Define custom table or plot settings” on page 48
Check panel, bin, and stutter file versions on your computer
1.
Start the GeneMapper ™ ID ‑ X Software , then log in with the appropriate user name and password.
2.
Select Tools 4 Panel Manager .
3.
Check the version of files that are currently available in the Panel Manager : a.
Select Panel Manager in the navigation pane.
b.
Expand the Panel Manager folder and any subfolders to identify the analysis file version that is already installed for your kit choice.
4.
Check the version of files available for import into the
Panel Manager : a.
Select Panel Manager , then select File 4 Import Panels to open the Import Panels dialog box.
b.
Navigate to, then open the Panels folder, then check the version of panel, bin, and stutter files installed.
5.
Check for newer versions of the files as described in the next procedure.
44 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Set up the GeneMapper ™ ID ‑ X Software for analysis (before first use of the kit)
4
(If needed) Download newer versions of panel, bin, and stutter files
1.
Go to www.thermofisher.com/GMIDXsoftware .
2.
If the file versions listed are newer than the versions on your computer, download the file
AmpFLSTR Analysis Files .
Note: When downloading new versions of analysis files, see the associated Read Me file for details of changes between software file versions. Perform the appropriate internal validation studies before using new file versions for analysis.
3.
Unzip the file.
Import panels, bins, and marker stutter
To import the latest panel, bin set, and marker stutter from the website into the GeneMapper ™
Software database:
ID ‑ X
1.
Start the GeneMapper ™ ID ‑ X Software, then log in with the appropriate user name and password.
2.
Select Tools 4 Panel Manager .
3.
Find, then open the folder containing the panels, bins, and marker stutter: a.
Select Panel Manager , then select File 4 Import Panels to open the Import Panels dialog box.
b.
Navigate to, then open the AmpFLSTR Analysis Files folder that you unzipped in the previous procedure.
4.
Select AmpFLSTR_Panels.txt, then click Import .
Note: Importing this file creates a new folder in the navigation pane of the Panel Manager ,
AmpFLSTR_Panels. This folder contains the panel and associated markers.
GlobalFiler ™ Express PCR Amplification Kit User Guide 45
4 Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Set up the GeneMapper ™ ID ‑ X Software for analysis (before first use of the kit)
5.
Import the bins file: a.
Select the AmpFLSTR_Panels folder in the navigation pane.
46 b.
Select File 4 Import Bin Set to open the Import Bin Set dialog box.
c.
Navigate to, then open the AmpFLSTR Analysis Files folder.
d.
Select AmpFLSTR_Bins.txt, then click Import .
Note: Importing this file associates the bin set with the panels in the AmpFLSTR_Panels folder.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Set up the GeneMapper ™ ID ‑ X Software for analysis (before first use of the kit)
4
6.
(Optional) View the imported panels and bins in the navigation pane: Double-click the
AmpFLSTR_Panels folder.
The panel information is displayed in the right pane and the markers are displayed below it.
7.
Import the stutter file: a.
Select the AmpFLSTR_Panels folder in the navigation panel.
b.
Select File 4 Import Marker Stutter to open the Import Marker Stutter dialog box.
c.
Navigate to, then open the AmpFLSTR Analysis Files folder.
GlobalFiler ™ Express PCR Amplification Kit User Guide 47
4 Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Set up the GeneMapper ™ ID ‑ X Software for analysis (before first use of the kit) d.
Select AmpFLSTR_Stutter.txt, then click Import .
Note: Importing this file associates the marker stutter ratio with the bin set in the
AmpFLSTR_Panels folder and overwrites any existing stutter ratios associated with the panels and bins in that folder.
8.
View the imported marker stutters in the navigation pane: a.
Double-click the AmpFLSTR_Panels folder to display the folder.
b.
Double-click the folder to display its list of markers below it.
c.
Double-click a marker to display the Stutter Ratio & Distance view for the marker in the right pane.
9.
Click Apply , then click OK to add the panel, bin set, and marker stutter to the GeneMapper ™ ID ‑ X
Software database.
IMPORTANT! If you close the Panel Manager without clicking Apply , the panels, bin sets, and marker stutter are not imported into the GeneMapper ™ ID ‑ X Software database.
(Optional) Define custom table or plot settings
Default views for table and plot settings are provided with the software. For information on defining custom views, see GeneMapper ™ ID ‑ X Software Getting Started Guide— Basic Features .
48 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Create an analysis method
4
1
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
Create an analysis method
Create an analysis method
IMPORTANT! Analysis methods are version-specific, so you must create an analysis method for each version of the software. For example, an analysis method that is created in GeneMapper ™ ID ‑ X Software version 1.2 is not compatible with analysis methods that are created in earlier versions of software, or with GeneMapper ™ Software v3.2.1.
1.
Select Tools 4 GeneMapper ® ID-X Manager to open the GeneMapper ID-X Manager .
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1
1
2.
Click 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: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs.
4.
After you enter the settings on all tabs, click Save .
GlobalFiler ™ Express PCR Amplification Kit User Guide 49
4 Chapter 4 Analyze data with GeneMapper ™
Create an analysis method
ID ‑ X Software
Enter Analysis Method settings
Enter General tab settings
1.
Enter a Name and select the Security Group appropriate for your software configuration.
50
2.
(Optional) Enter a Description and Instrument .
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Create an analysis method
4
Enter Allele tab settings
IMPORTANT! Perform appropriate internal validation studies to determine the appropriate settings to use.
1.
Select the AmpFlSTR_Bins_v3X bin set.
Figure 3 Settings used in developmental validation of the kit
2.
(Optional) To apply the stutter ratios contained in the AmpFLSTR_Stutter.txt
, select the Use marker-specific stutter ratio and distance if available checkbox (selected by default).
3.
If using GeneMapper ™ ID ‑ X Software v1.4 or later, enter values for the 4 Marker Repeat Types .
4.
Enter the appropriate filter settings.
GlobalFiler ™ Express PCR Amplification Kit User Guide 51
4 Chapter 4 Analyze data with GeneMapper ™
Create an analysis method
ID ‑ X Software
Enter Peak Detector tab settings
Figure 5 shows the settings for use on the 3730/3730xl DNA Analyzer.
Enter the appropriate values for each field:
Field
Ranges
Peak
Detection
Smoothing and Baseline
Values to enter or select
Enter the values shown in Figure 4, or adjust as
needed depending on the polymer and genetic analyzer that you are using.
Enter the appropriate settings.
IMPORTANT! Perform appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of data.
Additional information
—
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.
For more information, see the GeneMapper ™
ID ‑ X Software v1.4 New Features and Installation
Procedures User Bulletin (Pub. No. 4477684
Rev. B), "Known issues: 3730 DNA Analyzer allelic ladder failures".
Size Calling
Method
Enter the values shown in Figure 4, or adjust
as needed dependent on the polymer you are using.
3730/3730xl DNA Analyzer with POP-7 ™ polymer only: With the Smoothing setting of
None , the instances of spacing failures for the
D2S441 and D1S1656 markers in some allelic ladder samples are significantly reduced. With the default Smoothing setting of Light , failures of base-pair spacing quality assessment are observed.
Select 3rd Order Least Squares , or another method if validated by your internal validation.
IMPORTANT! Because of the small amplicon sizes that are generated by the this kit, the
3rd Order Least Squares sizing method is validated for use with this kit. Perform internal validation studies before using other sizing calling methods.
—
Normalization (Optional) Select the Normalization checkbox.
A Normalization checkbox is available on this tab in GeneMapper ™ ID ‑ X Software for use in conjunction with data run on the 3500 Series
Genetic Analyzers.
52 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Create an analysis method
4
Figure 4 Settings used in developmental validation of the kit
GlobalFiler ™ Express PCR Amplification Kit User Guide 53
4 Chapter 4 Analyze data with GeneMapper ™
Create an analysis method
ID ‑ X Software
54
Figure 5 Settings for use on the 3730/3730xl DNA Analyzer.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Create an analysis method
4
Enter Peak Quality tab settings
IMPORTANT! Perform the appropriate internal validation studies to determine the heterozygous and homozygous minimum peak height thresholds, maximum peak height threshold, and the minimum peak height ratio threshold for interpretation of data.
Enter the following values:
GlobalFiler ™ Express PCR Amplification Kit User Guide 55
4 Chapter 4 Analyze data with GeneMapper ™
Create an analysis method
ID ‑ X Software
Enter SQ and GQ tab settings
IMPORTANT! The values that are 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.
Enter the following values:
56
Note: Set the ACC GQ Weighting according to the values you determine during internal validation studies of the ACC PQV . For example, set the ACC GQ Weighting to 0.3 or higher to flag samples in which the Amelogenin result is anything other than X, X or X, Y, or does not agree with the results for the DYS391 or Y indel markers.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Create a size standard definition file if needed
4
Create a size standard definition file if needed
If you cannot use the default settings that are provided, create a new size standard definition file.
About the GS600_LIZ_ (60– 460) size standard definition file
The GS600_LIZ_(60– 460) size standard definition that is provided with GeneMapper ™ ID ‑ X Software and used with the Local Southern size calling method contains the following peaks: 60, 80, 100, 114,
120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420,
440, and 460.
This size standard definition has been validated for use with this kit on the genetic analyzers listed
in “Instrument and software compatibility” on page 15 . If you need to create your own size standard
definition, see “Create a size standard definition file” on page 58
.
If you use POP-7
™
polymer on a 3730 instrument
The 60 bp size-standard peak may occasionally be obscured by the primer peak. The issue can be addressed by either of the following steps:
• Re-inject samples in which the 60 base-pair peak is not recognized.
• Use the 80– 460 bp size-standard definition after performing appropriate validation studies (as a general rule, the 60 base-pair peak is not required for accurate fragment sizing with the 3rd Order
Least Squares sizing method).
For more information, see the GeneMapper ™ ID ‑ X Software v1.4 New Features and Installation
Procedures User Bulletin (Pub. No. 4477684 Rev. B), “Known issues: 3730 DNA Analyzer sizing failures”.
GlobalFiler ™ Express PCR Amplification Kit User Guide 57
4 Chapter 4 Analyze data with GeneMapper ™
Create a size standard definition file if needed
ID ‑ X Software
Create a size standard definition file
1.
Select Tools 4 GeneMapper ID-X Manager to open the GeneMapper ID-X Manager .
2.
Click the Size Standards tab, then click New .
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1 1
1
3.
Specify settings in the Size Standard Editor : a.
Enter a name as shown in the following figure or enter a new name.
b.
In the Security Group field, select the Security Group appropriate for your software configuration.
c.
In the Size Standard Dye field, select Orange .
58 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Create a size standard definition file if needed
4 d.
In the Size Standard Table , enter the peak sizes that correspond to your size standard.
GlobalFiler ™ Express PCR Amplification Kit User Guide 59
4 Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Analyze and edit sample files with GeneMapper ™ ID ‑ X Software
Analyze and edit sample files with GeneMapper
™
Software
ID ‑ X
1.
In the Project window, select Edit 4 Add Samples to Project , then navigate to the disk or directory that contains the sample files.
2.
Apply analysis settings to the samples in the project.
Parameter
Sample Type
Analysis Method
Settings
Select the sample type.
Select GlobalFilerExpress_AnalysisMethod (or the name of the analysis method you created).
Select GlobalFiler_Express .
Panel
Size Standard Use a size range of 60– 460 bp for Local Southern size calling method or a size range of 80– 460 bp for 3rd Order Least Squares size-calling method.
[1]
[1] The GlobalFiler ™ Express PCR Amplification Kit was originally validated with the GeneScan with the GlobalFiler ™ Express PCR Amplification Kit.
™ 600 LIZ ™ Size Standard v2.0. If you use a different size standard, perform the appropriate internal validation studies to support the use of this size standard
3.
Click Analyze , enter a name for the project (in the Save Project dialog box), then click OK to start analysis.
• The status bar displays the progress of analysis as a completion bar.
• The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample).
• The Analysis Summary tab is displayed, and the Genotypes tab is available when the analysis is complete.
60 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapper ™ ID ‑ X Software
Examine or edit a project
4
Examine or edit a project
Display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data.
For more information on using the GeneMapper
™
Software
ID ‑ X
See “Related documentation” on page 151 for a list of available documents.
GlobalFiler ™ Express PCR Amplification Kit User Guide 61
62
5
Experiments and results
■
■
■
■
■
■
■
■
■
■
■
Importance of validation
Validation of a DNA typing procedure for human identification applications is an evaluation of the efficiency, reliability, and performance characteristics of the procedure. By challenging the procedure with samples that are commonly encountered in forensic and parentage laboratories, the validation process uncovers attributes and limitations that are critical for sound data interpretation (Sparkes,
Kimpton, Watson, 1996; Sparkes, Kimpton, Gilbard, 1996; Wallin, 1998).
Experiment conditions
We conducted developmental validation experiments according to the updated and revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, December 2012). Based on these guidelines, we conducted experiments that comply with guidelines 2.0 and 3.0 and its associated subsections. This DNA methodology is not novel. (Moretti et al ., 2001; Frank et al.
, 2001; Wallin et al.
,
2002 ; and Holt et al ., 2000).
We used conditions that produced optimum PCR product yield and that met reproducible performance standards. It is our opinion that while these experiments are not exhaustive, they are appropriate for a manufacturer of STR kits intended for forensic and/or parentage testing use.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Laboratory requirements for internal validation
5
Laboratory requirements for internal validation
Each laboratory using this kit must perform internal validation studies. Performance of this kit is supported when used according to the following developmentally validated parameters. Modifications to the protocol should be accompanied by appropriate validation studies performed by the laboratory.
Developmental validation
Except where noted, all developmental validation studies were performed using the Veriti ™
Cycler according to the protocol described in the Perform PCR chapter.
Thermal
SWGDAM guideline 2.2.1
“Developmental validation is the acquisition of test data and determination of conditions and limitations of a new or novel DNA methodology for use on forensic, database, known or casework reference samples.” (SWGDAM, December 2012)
SWGDAM guideline 3.9.2
“The reaction conditions needed to provide the required degree of specificity and robustness should be determined. These include, but are not limited to, thermal cycling parameters, the concentration of primers, magnesium chloride, DNA polymerase, and other critical reagents.” (SWGDAM, December
2012)
PCR components
We examined the concentration of each component of the kit. We 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, blood and buccal samples on treated ‑ paper substrates or swab ‑ sample lysates were amplified in the presence of varying concentrations of magnesium chloride, and the results were analyzed on a 3500xL Genetic Analyzer
(Figure 6). The performance of the multiplex is most robust within ±20% of the optimal magnesium
chloride concentration.
GlobalFiler ™ Express PCR Amplification Kit User Guide 63
5 Chapter 5 Experiments and results
Developmental validation
64
Figure 6 Buccal swab lysate amplified with the GlobalFiler
(Y-axis scale 0 to 28,000 RFU).
™ Express PCR Amplification Kit in the presence of varying concentrations of magnesium chloride and analyzed on a 3500xL Genetic Analyzer
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Developmental validation
5
PCR cycle number
Reactions were amplified for 24, 25, 26, and 27 cycles on the Veriti ™ Thermal Cycler using a buccal swab lysate. As expected, the amount of PCR product increased with the number of cycles. A full profile was generated for all numbers of thermal cycles (24–27) and off-scale data were collected for
several allele peaks at 27 cycle (Figure 7).
None of the cycle numbers tested produced nonspecific peaks.
Figure 7 Representative GlobalFiler scale 0 to 25,000 RFU).
™ Express PCR Amplification Kit profiles obtained from amplification of buccal swab lysates using 24, 25, 26, and 27 cycles, analyzed on a 3500xL Genetic Analyzer (Y-axis
Thermal cycling temperatures
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, peakheight balance, and resistance to PCR inhibitors.
For example, annealing/extension temperatures of 58, 59, 60, 61, and 62°C were tested using a Veriti ™
Thermal Cycler (Figure 8). The PCR products were analyzed using a 3500xL Genetic Analyzer.
Of the tested annealing temperatures, 59°C to 61°C produced robust profiles. At 58°C, many smaller amplicons were preferentially amplified relative to the larger amplicons, generating a ski-slope-like
STR profile. At 62°C, the yield of most loci was reduced, and the yield of Amelogenin and D7S820 was significantly affected. The optimal combination of specificity, sensitivity, and resistance to PCR
GlobalFiler ™ Express PCR Amplification Kit User Guide 65
5 Chapter 5 Experiments and results
Accuracy, precision, and reproducibility inhibition was observed at 60°C. Thermal cycler temperature is critical to assay performance; therefore, routine, regularly scheduled thermal cycler calibration is recommended.
Figure 8 Electropherograms obtained from amplification of blood sample on an FTA
RFU).
™ card at annealing temperatures of 58, 59, 60, 61, and 62°C, analyzed on a 3500xL Genetic Analyzer (Y-axis scale 0 to 20,000
Accuracy, precision, and reproducibility
SWGDAM guideline 3.5
“Precision and accuracy of the assay should be demonstrated: Precision characterizes the degree of mutual agreement among a series of individual measurements, values and/or results. Precision depends only on the distribution of random errors and does not relate to the true value or specified value. The measure of precision is usually expressed in terms of imprecision and computed as a
66 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Accuracy, precision, and reproducibility
5 standard deviation of the test results. Accuracy is the degree of conformity of a measured quantity to its actual (true) value. Accuracy of a measuring instrument is the ability of a measuring instrument to give responses close to a true value.” (SWGDAM, December 2012)
Accuracy observation
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 profiles have been determined from various sample types.
The following four figures show the size differences that are typically observed between sample alleles and allelic ladder alleles on the 3130 xl , 3500, and 3500xL Genetic Analyzers with POP-4 and the 3730 Genetic Analyzer with POP-7 ™ Polymer. The X-axis in the following figures represents the nominal nucleotide sizes for the GlobalFiler ™
™ Polymer
Express 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, irrespective of the capillary electrophoresis platforms.
Figure 9 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3130 xl Genetic Analyzer.
Size and ladder sizing for the GlobalFiler
GeneScan ™ 600 LIZ ™ Size Standard v2.0.
™ Express PCR Amplification Kit were calculated using the
GlobalFiler ™ Express PCR Amplification Kit User Guide 67
5 Chapter 5 Experiments and results
Accuracy, precision, and reproducibility
Figure 10 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3500 Genetic Analyzer. Size and ladder sizing for the GlobalFiler
Standard v2.0.
™ Express kit were calculated using the GeneScan ™ 600 LIZ ™ Size
68
Figure 11 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3500xL Genetic Analyzer.
Size and ladder sizing for the GlobalFiler
Size Standard v2.0.
™ Express kit were calculated using the GeneScan ™ 600 LIZ ™
GlobalFiler ™ Express PCR Amplification Kit User Guide
Common Callouts and Arrows
1. Copy-paste a callout or arrow to use in this SVG.
Note: If you need more advanced callouts or arrows
use the TechComm_Inkscape_Callout&Arrow_Libary.
2. Edit number and/or line-length, as needed.
3. Delete this text, this rectangle, and unused
callouts, arrows, or other SVG elements
before adding this SVG to the repository.
1 1 1
1
Chapter 5 Experiments and results
Accuracy, precision, and reproducibility
5
Figure 12 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on an 3730 Genetic Analyzer. Size and ladder sizing for the GlobalFiler
Standard v2.0.
™ Express kit were calculated using the GeneScan ™ 600 LIZ ™ Size
Precision and size window description
Sizing precision enables the determination of accurate and reliable genotypes. The recommended method for genotyping is to use a ±0.5-nt “window” around the size obtained for each allele in the allelic ladder. A ±0.5-nt window allows for the detection and correct assignment of alleles. Any sample allele that sizes outside the specified window could be either:
• An “off-ladder” allele, that is, an allele of a size that is not represented in the allelic ladder.
• An allele that does correspond to an allele in the allelic ladder, but whose size is just outside a window because of measurement error.
The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times. Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument.
GlobalFiler ™ Express PCR Amplification Kit User Guide 69
5 Chapter 5 Experiments and results
Accuracy, precision, and reproducibility
Precision observation
Table 2 lists typical precision results obtained from multiple runs of the GlobalFiler
using the GeneScan ™ 600 LIZ ™
™ Express Allelic Ladder
Size Standard v2.0. The results were obtained within a set of injections on a single capillary array. The number of repeated injections for each genetic analyzer platform is shown in the following table:
CE platform
3130 xl
3500
3500xL
3730
Capillaries
16/injection
8/injection
24/injection
48/injection
Number of injections
5
12
4
4
Sizing method
Local Southern, 60– 460 bp
Local Southern, 60– 460 bp
Local Southern, 60– 460 bp
3rd Order Least Square
The mean sizes and the standard deviation for the allele sizing were calculated for all the alleles in each run
across multiple runs.
Sample alleles can occasionally size outside of the ±0.5
‑ nt window for a respective Allelic Ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems with the
clustering of allele sizes obtained on the Applied Biosystems ™ genetic analyzers, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5
‑ nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith, 1995).
For sample alleles that do not size within a ±0.5
‑ nt window, the PCR product must be rerun to distinguish between a true off–ladder allele versus measurement error of a sample allele that corresponds to an allele in the Allelic Ladder. Repeat analysis, when necessary, provides an added level of confidence in the final allele assignment.
GeneMapper ™ ID ‑ X Software automatically flags sample alleles that do not size within the prescribed window around an allelic ladder allele by labeling the allele as OL (off ‑ ladder).
Maximum sizing precision is obtained within the same set of capillary injections. Cross–platform sizing differences occur due to several factors including type and concentration of polymer, run temperature, and electrophoresis conditions. Variations in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors.
IMPORTANT! To minimize the variation in sizing between runs and to ensure accurate genotyping, follow the
guidelines in “Allelic ladder requirements for data analysis” on page 43
and use allelic ladders obtained from the same run as samples to analyze the samples.
For more information on precision and genotyping, see Lazaruk et al ., 1998 and Mansfield et al ., 1998.
70 GlobalFiler ™ Express PCR Amplification Kit User Guide
Table 2 Precision results of multiple runs of the GlobalFiler ™ Express Allelic Ladder
12
13
14
15
10
11
8
9
3130 xl 3500
Allele
Mean
Standard deviation
Mean
AMEL
X
Y
CSF1PO
98.86–98.89
0.022–0.027
98.63–98.68
104.90–104.93
0.025–0.037
104.71–104.77
6
7
282.58–282.63
0.022–0.042
283.17–283.26
286.54–286.59
0.026–0.046
287.12–287.21
Standard deviation
Mean
3500xL
Standard deviation
0.031–0.051
98.58–98.62
0.042–0.047
0.019–0.050
104.67–104.69
0.030–0.043
0.017–0.062
283.17–283.26
0.046–0.059
0.026–0.049
287.13–287.25
0.035–0.054
290.49–290.54
294.44–294.49
298.38–298.42
302.28–302.33
0.029–0.045
0.026–0.039
0.038–0.052
0.025–0.043
291.09–291.18
295.04–295.14
298.99–299.09
302.91–302.99
0.031–0.066
0.030–0.061
0.006–0.059
0.016–0.062
291.10–291.22
295.07–295.18
299.01–299.12
302.91–303.03
0.044–0.056
0.032–0.055
0.032–0.063
0.035–0.054
306.19–306.25
310.15–310.20
314.18–314.23
318.40–318.45
D10S1248
8 85.31–85.33
9
10
11
89.40–89.43
93.47–93.49
97.54–97.57
0.038–0.044
0.029–0.043
0.038–0.042
0.034–0.039
0.025–0.036
0.025–0.035
0.030–0.037
0.026–0.040
306.84–306.91
310.81–310.91
314.87–314.96
319.10–319.18
85.39–85.46
89.53–89.58
93.62–93.68
97.73–97.79
0.025–0.063
306.84–306.97
0.040–0.063
0.042–0.064
310.83–310.96
0.045–0.058
0.005–0.078
314.88–315.01
0.037–0.047
0.004–0.063
319.10–319.25
0.045–0.061
0.024–0.051
0.022–0.055
0.035–0.057
0.006–0.054
85.37–85.40
89.49–89.52
93.58–93.63
97.70–97.73
0.031–0.046
0.033–0.045
0.040–0.053
0.045–0.050
Mean
3730
Standard deviation
100.47–100.49
106.20–106.22
282.49–282.63
286.48–286.62
290.47–290.62
294.47–294.62
298.49–298.61
302.46–302.63
306.46–306.62
310.46–310.61
314.46–314.61
318.45–318.62
86.20–86.26
90.15–90.22
94.07–94.17
98.04–98.11
0.065–0.069
0.050–0.061
0.060–0.068
0.062–0.075
0.066–0.075
0.057–0.069
0.048–0.079
0.066–0.079
0.065–0.070
0.061–0.081
0.072–0.076
0.061–0.082
0.070–0.080
0.064–0.091
0.058–0.074
0.064–0.081
16
17
18
19
3130 xl 3500
Allele
Mean
Standard deviation
Mean
101.62–101.65
0.029–0.036
101.84–101.90
12
13
14
15
16
17
18
19
D12S391
105.70–105.73
0.029–0.037
105.96–106.03
109.75–109.79
0.030–0.038
110.02–110.09
113.74–113.79
0.027–0.039
114.05–114.13
117.65–117.69
0.026–0.035
117.95–118.02
121.52–121.56
0.029–0.043
121.85–121.91
125.40–125.45
0.028–0.041
125.77–125.85
129.32–129.37
0.027–0.045
129.73–129.78
3500xL
Standard deviation
Mean
Standard deviation
0.017–0.053
101.80–101.86
0.041–0.045
0.034–0.052
105.90–105.96
0.037–0.043
0.014–0.065
109.98–110.05
0.038–0.048
0.001–0.058
114.01–114.08
0.019–0.048
0.001–0.046
117.90–117.97
0.038–0.050
0.004–0.054
121.81–121.86
0.032–0.048
0.022–0.062
125.73–125.79
0.036–0.048
0.028–0.049
129.68–129.75
0.040–0.053
14
15
216.01–216.08
220.06–220.12
0.040–0.040
0.040–0.045
216.47–216.57
220.52–220.59
0.019–0.061
0.039–0.067
216.54–216.61
220.61–220.67
0.040–0.060
0.040–0.055
224.08–224.14
228.02–228.08
231.99–232.07
235.95–236.01
0.040–0.047
0.029–0.047
0.033–0.043
0.030–0.044
224.56–224.64
228.53–228.62
232.53–232.60
236.50–236.55
0.018–0.081
0.030–0.095
0.017–0.070
0.026–0.076
224.66–224.72
228.61–228.69
232.58–232.68
236.55–236.64
0.050–0.059
0.052–0.065
0.044–0.057
0.050–0.058
19.3
239.02–239.08
0.029–0.046
239.52–239.62
20 239.95–240.03
0.028–0.054
240.51–240.61
21
22
23
244.03–244.09
247.99–248.04
251.99–252.05
0.033–0.045
0.033–0.042
0.024–0.039
244.55–244.65
248.51–248.60
252.50–252.58
0.001–0.071
239.60–239.70
0.048–0.065
0.001–0.068
240.58–240.68
0.036–0.061
0.025–0.061
244.62–244.72
0.047–0.055
0.039–0.063
248.57–248.69
0.046–0.055
0.028–0.056
252.57–252.66
0.051–0.056
215.51–215.66
219.47–219.59
223.48–223.60
227.36–227.48
231.30–231.42
235.20–235.32
238.19–238.32
239.21–239.33
243.07–243.20
246.88–247.01
250.84–250.97
Mean
101.96–102.07
105.90–106.00
109.85–109.95
113.77–113.88
117.70–117.81
121.64–121.75
125.56–125.69
129.47–129.60
3730
Standard deviation
0.048–0.074
0.064–0.068
0.065–0.074
0.064–0.068
0.059–0.070
0.069–0.083
0.066–0.082
0.061–0.071
0.054–0.071
0.054–0.074
0.062–0.078
0.063–0.072
0.063–0.076
0.064–0.083
0.062–0.075
0.061–0.086
0.067–0.081
0.057–0.088
0.065–0.074
Allele
24
25
26
27
3130 xl 3500
Mean
Standard deviation
Mean
255.97–256.02
0.031–0.047
256.50–256.58
3500xL
Standard deviation
Mean
Standard deviation
0.014–0.055
256.54–256.64
0.038–0.053
259.86–259.90
0.033–0.042
260.41–260.52
263.80–263.84
0.032–0.039
264.37–264.46
267.90–267.95
0.033–0.049
268.46–268.55
0.036–0.076
0.033–0.058
0.028–0.064
260.46–260.56
264.41–264.52
268.54–268.63
0.037–0.059
0.045–0.061
0.042–0.052
10
11
12
13
D13S317
5
6
7
198.77–198.81
0.023–0.035
198.95–199.01
202.77–202.82
0.024–0.043
202.92–202.97
0.004–0.055
198.96–198.99
0.045–0.055
0.035–0.065
202.94–202.97
0.039–0.048
206.75–206.79
0.022–0.034
206.87–206.93
206.87–206.93
206.89–206.94
0.036–0.046
8
9
210.82–210.87
214.85–214.89
0.027–0.037
0.028–0.037
210.93–210.99
214.95–214.99
0.009–0.065
0.008–0.054
210.96–210.98
214.97–215.00
0.044–0.050
0.032–0.049
218.95–218.97
223.00–223.02
227.11–227.13
231.05–231.09
0.022–0.033
0.022–0.041
0.024–0.040
0.029–0.040
219.04–219.09
223.08–223.15
227.17–227.25
231.15–231.20
0.032–0.054
0.010–0.064
0.031–0.061
0.020–0.058
219.08–219.09
223.13–223.16
227.24–227.25
231.19–231.21
0.028–0.053
0.045–0.054
0.052–0.067
0.043–0.050
14
15
16
235.01–235.05
0.029–0.036
235.11–235.17
239.05–239.08
0.031–0.039
239.15–239.21
243.18–243.21
0.025–0.036
243.26–243.31
0.020–0.063
235.15–235.18
0.042–0.057
0.005–0.062
239.19–239.22
0.023–0.055
0.005–0.046
243.29–243.32
0.035–0.049
Mean
254.90–255.01
258.86–258.97
262.77–262.90
266.86–266.98
199.47–199.56
203.51–203.58
207.55–207.64
211.70–211.78
215.74–215.81
219.76–219.83
223.81–223.87
227.90–227.96
231.87–231.92
235.80–235.86
239.85–239.90
243.89–243.93
3730
Standard deviation
0.064–0.075
0.067–0.089
0.064–0.079
0.069–0.076
0.046–0.055
0.047–0.056
0.044–0.053
0.046–0.053
0.045–0.057
0.042–0.058
0.045–0.059
0.047–0.054
0.040–0.053
0.043–0.051
0.041–0.056
0.048–0.051
3130 xl 3500
Allele
Mean
Standard deviation
Mean
D16S539
5 227.17–227.21
0.031–0.038
227.46–227.54
8
9
10
11
12
13
14
15
239.33–239.37
243.46–243.51
247.59–247.63
251.65–251.68
255.61–255.65
259.55–259.59
263.55–263.60
267.59–267.63
0.028–0.044
0.029–0.038
0.025–0.045
0.027–0.039
0.025–0.037
0.022–0.036
0.025–0.038
0.027–0.041
239.66–239.73
243.77–243.87
247.90–247.98
251.95–252.03
255.93–256.00
259.89–259.98
263.91–264.00
267.93–268.04
Standard deviation
Mean
3500xL
Standard deviation
0.029–0.063
227.32–227.40
0.044–0.057
0.001–0.057
239.49–239.58
0.042–0.051
0.005–0.049
0.001–0.049
247.76–247.84
0.039–0.047
0.008–0.057
251.79–251.89
0.045–0.055
0.006–0.050
255.78–255.86
0.039–0.045
0.001–0.059
0.015–0.059
263.75–263.86
0.050–0.054
0.025–0.056
243.63–243.72
259.72–259.84
267.79–267.89
0.027–0.046
0.051–0.057
0.043–0.055
D18S51
7 261.06–261.11
0.024–0.038
261.26–261.37
9
10
269.20–269.24
273.27–273.32
0.025–0.041
0.026–0.043
269.40–269.49
273.45–273.53
0.005–0.060
0.028–0.056
0.024–0.049
261.21–261.29
269.33–269.39
273.41–273.45
0.033–0.048
0.038–0.054
0.048–0.054
0.014–0.050
275.42–275.45
0.034–0.045
10.2
275.27–275.32
0.028–0.040
275.43–275.52
11 277.35–277.37
0.022–0.035
277.50–277.59
12
13
281.39–281.44
285.42–285.46
0.026–0.037
0.027–0.038
281.55–281.64
285.58–285.66
13.2
14
287.38–287.42
289.43–289.46
0.028–0.037
0.033–0.037
287.54–287.62
289.60–289.67
0.009–0.058
0.008–0.054
0.018–0.056
277.48–277.52
281.51–281.54
285.52–285.55
0.043–0.059
0.050–0.053
0.042–0.047
0.025–0.052
287.49–287.53
0.049–0.053
0.025–0.058
289.52–289.57
0.042–0.051
Mean
228.05–228.18
240.14–240.26
244.18–244.30
248.20–248.32
252.23–252.34
256.26–256.38
260.28–260.40
264.30–264.43
268.32–268.43
263.60–263.73
271.85–271.99
276.01–276.11
277.97–278.10
280.14–280.26
284.28–284.40
288.40–288.53
290.40–290.51
292.56–292.68
3730
Standard deviation
0.059–0.073
0.067–0.077
0.060–0.073
0.059–0.070
0.068–0.073
0.059–0.074
0.063–0.069
0.062–0.076
0.055–0.077
0.047–0.064
0.048–0.057
0.045–0.064
0.053–0.060
0.050–0.058
0.054–0.058
0.044–0.064
0.051–0.059
0.048–0.062
8
9
6
7
10
21
22
23
24
17
18
19
20
3130 xl 3500
Allele
Mean
Standard deviation
Mean
14.2
291.41–291.43
0.027–0.035
291.56–291.64
Standard deviation
Mean
3500xL
Standard deviation
0.023–0.060
291.50–291.54
0.041–0.052
15
16
293.43–293.47
297.44–297.47
0.022–0.032
0.024–0.039
293.57–293.66
297.58–297.69
0.035–0.063
0.008–0.055
293.53–293.57
297.54–297.57
0.041–0.045
0.034–0.048
301.40–301.43
305.36–305.39
309.38–309.41
313.44–313.48
0.025–0.035
0.028–0.046
0.021–0.039
0.030–0.040
301.55–301.64
305.51–305.60
309.50–309.60
313.55–313.66
0.009–0.058
0.028–0.065
0.019–0.065
0.000–0.065
301.49–301.55
305.47–305.52
309.47–309.54
313.53–313.59
0.052–0.056
0.043–0.045
0.037–0.063
0.033–0.063
25
26
27
D19S433
317.69–317.71
322.00–322.01
326.04–326.05
330.13–330.15
334.18–334.22
338.22–338.26
0.027–0.040
0.028–0.044
0.020–0.036
0.024–0.035
0.030–0.037
0.025–0.049
317.78–317.91
322.07–322.20
326.12–326.23
330.21–330.30
334.27–334.36
338.29–338.41
342.29–342.34
0.026–0.038
342.37–342.47
0.021–0.065
317.78–317.83
0.038–0.057
0.012–0.067
322.06–322.10
0.026–0.056
0.030–0.060
326.11–326.15
0.052–0.063
0.030–0.055
330.19–330.25
0.036–0.058
0.027–0.072
334.28–334.30
0.046–0.057
0.008–0.052
338.30–338.35
0.036–0.053
0.011–0.058
342.38–342.42
0.045–0.063
118.28–118.35
0.024–0.047
118.50–118.61
122.03–122.10
0.024–0.045
122.28–122.39
125.82–125.89
0.022–0.050
126.11–126.21
129.64–129.68
0.031–0.049
129.94–130.05
133.46–133.52
0.033–0.047
133.81–133.93
0.026–0.068
118.52–118.55
0.042–0.048
0.042–0.085
122.33–122.34
0.040–0.052
0.025–0.063
126.15–126.16
0.046–0.061
0.040–0.067
129.99–130.01
0.051–0.054
0.034–0.073
133.86–133.89
0.039–0.058
120.32–120.46
124.04–124.19
127.78–127.91
131.52–131.67
135.27–135.42
Mean
294.55–294.65
296.70–296.82
300.86–300.95
304.98–305.09
309.12–309.23
313.27–313.38
317.41–317.52
321.56–321.65
325.74–325.85
329.85–329.97
333.99–334.10
338.14–338.26
342.29–342.41
346.45–346.57
3730
Standard deviation
0.054–0.059
0.051–0.060
0.049–0.056
0.051–0.060
0.048–0.054
0.052–0.062
0.050–0.058
0.057–0.062
0.050–0.061
0.044–0.055
0.051–0.061
0.050–0.056
0.052–0.063
0.047–0.057
0.056–0.091
0.056–0.089
0.064–0.092
0.063–0.107
0.068–0.089
3130 xl 3500
Allele
Mean
Standard deviation
Mean
137.33–137.38
0.036–0.048
137.69–137.82
11
12 141.22–141.27
0.021–0.044
141.63–141.76
12.2
143.21–143.27
0.030–0.051
143.63–143.75
3500xL
Standard deviation
Mean
Standard deviation
0.044–0.072
137.74–137.78
0.047–0.058
0.044–0.068
141.67–141.73
0.042–0.058
0.042–0.084
143.69–143.74
0.038–0.055
13 145.17–145.22
0.028–0.047
145.60–145.73
13.2
147.16–147.23
0.031–0.04
147.61–147.73
0.032–0.068
0.037–0.065
145.65–145.71
147.67–147.71
0.044–0.058
0.034–0.060
14 149.11–149.17
0.036–0.045
149.56–149.69
14.2
151.11–151.18
0.029–0.051
151.58–151.71
15 153.07–153.13
0.029–0.053
153.56–153.67
15.2
155.06–155.15
0.030–0.040
155.57–155.68
0.041–0.076
0.035–0.074
0.032–0.072
0.036–0.075
149.64–149.69
151.63–151.71
153.61–153.67
155.63–155.69
0.047–0.062
0.046–0.068
0.047–0.056
0.050–0.057
16
16.2
17
17.2
18.2
19.2
157.02–157.11
159.03–159.12
160.98–161.07
162.97–163.04
166.99–167.07
170.89–170.97
0.032–0.052
0.038–0.050
0.038–0.048
0.036–0.047
0.030–0.045
0.028–0.044
157.53–157.66
159.57–159.67
161.49–161.61
163.50–163.62
167.55–167.66
171.45–171.56
D1S1656
9 159.91–159.94
0.018–0.037
159.94–160.03
0.031–0.077
157.60–157.66
0.046–0.063
0.036–0.071
159.61–159.68
0.030–0.060
0.007–0.072
161.56–161.62
0.040–0.070
0.015–0.073
163.57–163.63
0.051–0.061
0.024–0.067
167.61–167.68
0.052–0.060
0.034–0.074
171.52–171.61
0.038–0.059
10
11
12
163.93–163.97
167.94–167.97
171.94–171.98
0.023–0.036
0.026–0.036
0.025–0.033
163.97–164.04
167.97–168.05
172.02–172.07
0.000–0.054
159.98–160.00
0.030–0.052
0.016–0.054
163.99–164.02
0.036–0.043
0.028–0.059
168.03–168.03
0.031–0.046
0.018–0.057
172.03–172.05
0.034–0.048
160.85–160.91
164.81–164.88
168.78–168.84
172.72–172.80
Mean
139.00–139.17
142.78–142.93
144.68–144.85
146.52–146.71
148.45–148.61
150.30–150.48
152.22–152.40
154.08–154.25
156.00–156.17
157.86–158.04
159.79–159.96
161.63–161.82
163.58–163.75
167.48–167.67
171.28–171.46
3730
Standard deviation
0.062–0.085
0.072–0.091
0.064–0.085
0.065–0.084
0.072–0.077
0.059–0.082
0.066–0.093
0.062–0.092
0.057–0.089
0.070–0.086
0.069–0.087
0.070–0.094
0.064–0.100
0.065–0.094
0.068–0.095
0.046–0.058
0.047–0.058
0.051–0.061
0.050–0.059
3130 xl 3500
Allele
Mean
Standard deviation
Mean
175.95–175.99
0.023–0.033
176.01–176.08
13
14 180.02–180.06
0.031–0.038
180.11–180.16
14.3
183.09–183.12
0.022–0.041
183.17–183.21
3500xL
Standard deviation
Mean
Standard deviation
0.012–0.056
176.05–176.07
0.040–0.044
0.004–0.059
180.14–180.16
0.046–0.055
0.032–0.056
183.19–183.22
0.027–0.051
15 184.00–184.04
0.029–0.035
184.10–184.16
15.3
187.15–187.18
0.020–0.034
187.23–187.30
0.032–0.052
0.024–0.059
184.12–184.15
187.28–187.30
0.035–0.054
0.042–0.049
16 188.05–188.08
0.021–0.030
188.16–188.22
16.3
191.19–191.24
0.017–0.035
191.30–191.36
17 192.10–192.14
0.030–0.039
192.22–192.30
17.3
195.23–195.26
0.021–0.038
195.37–195.42
0.031–0.068
0.023–0.063
0.021–0.063
0.011–0.060
188.19–188.22
191.35–191.37
192.26–192.28
195.40–195.42
0.027–0.045
0.032–0.054
0.038–0.050
0.035–0.055
18.3
19.3
20.3
D21S11
24 182.84–182.89
0.020–0.038
183.09–183.14
24.2
184.92–184.98
0.029–0.033
185.17–185.23
25
26
186.94–187.00
191.04–191.10
0.027–0.038
0.019–0.033
187.21–187.26
191.33–191.37
27
28
199.28–199.31
203.24–203.27
207.19–207.22
195.16–195.20
199.20–199.24
0.020–0.035
0.025–0.038
0.029–0.034
0.025–0.036
0.029–0.039
199.40–199.47
203.37–203.46
207.32–207.39
195.45–195.52
199.49–199.57
28.2
201.21–201.26
0.027–0.039
201.48–201.53
0.036–0.056
199.44–199.47
0.023–0.049
0.013–0.057
203.40–203.41
0.035–0.047
0.022–0.057
207.34–207.38
0.041–0.049
0.018–0.056
0.026–0.055
0.021–0.057
0.024–0.055
182.98–183.05
185.06–185.14
187.11–187.17
191.21–191.29
0.024–0.049
0.039–0.055
0.041–0.054
0.037–0.059
0.014–0.058
195.35–195.41
0.033–0.051
0.007–0.059
199.39–199.46
0.021–0.056
0.012–0.058
201.38–201.45
0.042–0.054
183.41–183.58
185.42–185.58
187.45–187.60
191.47–191.63
195.55–195.69
199.51–199.66
201.52–201.66
Mean
176.69–176.76
180.71–180.80
183.70–183.78
184.61–184.67
187.65–187.72
188.56–188.63
191.62–191.69
192.52–192.58
195.56–195.64
199.54–199.59
203.50–203.56
207.46–207.52
3730
Standard deviation
0.046–0.066
0.049–0.060
0.049–0.062
0.050–0.055
0.049–0.053
0.056–0.060
0.047–0.055
0.052–0.063
0.053–0.060
0.050–0.067
0.044–0.060
0.051–0.064
0.060–0.075
0.051–0.076
0.053–0.074
0.060–0.072
0.059–0.067
0.063–0.070
0.053–0.072
3130 xl 3500
Allele
29
Mean
203.16–203.22
Standard deviation
0.022–0.037
Mean
203.46–203.55
29.2
205.22–205.27
0.022–0.034
205.49–205.56
3500xL
Standard deviation
Mean
Standard deviation
0.004–0.059
203.34–203.41
0.029–0.044
0.008–0.067
205.38–205.45
0.039–0.054
30 207.19–207.25
0.022–0.038
207.48–207.55
30.2
209.17–209.23
0.025–0.041
209.45–209.51
0.008–0.060
0.020–0.060
207.36–207.44
209.34–209.41
0.039–0.047
0.038–0.051
31 211.20–211.24
0.021–0.039
211.48–211.54
31.2
213.17–213.23
0.021–0.041
213.45–213.53
32 215.22–215.27
0.020–0.036
215.52–215.57
32.2
217.26–217.31
0.020–0.034
217.54–217.59
0.012–0.061
0.004–0.054
0.035–0.055
0.031–0.052
211.37–211.45
213.33–213.41
215.40–215.48
217.44–217.51
0.048–0.051
0.051–0.057
0.031–0.044
0.026–0.053
33 219.33–219.38
0.025–0.041
219.61–219.71
33.2
221.30–221.34
0.024–0.037
221.60–221.68
34 223.44–223.48
0.027–0.038
223.75–223.81
34.2
225.37–225.41
0.033–0.040
225.68–225.76
35 227.46–227.50
0.032–0.043
227.79–227.86
35.2
229.41–229.44
0.025–0.042
229.74–229.80
36
37
38
231.40–231.45
235.50–235.54
239.48–239.53
0.037–0.045
0.025–0.044
0.026–0.042
231.76–231.83
235.84–235.93
239.83–239.89
0.006–0.056
0.039–0.061
0.038–0.058
0.038–0.072
0.028–0.067
0.024–0.058
0.026–0.065
0.019–0.065
0.001–0.059
219.54–219.60
221.52–221.58
223.65–223.72
225.59–225.66
227.68–227.75
229.64–229.72
231.66–231.75
235.75–235.84
239.71–239.82
0.044–0.060
0.028–0.046
0.050–0.059
0.048–0.063
0.045–0.058
0.043–0.068
0.041–0.054
0.042–0.054
0.047–0.059
Mean
203.48–203.64
205.58–205.74
207.54–207.68
209.55–209.70
211.54–211.71
213.56–213.71
215.56–215.70
217.57–217.72
219.58–219.75
221.53–221.70
223.71–223.86
225.62–225.75
227.70–227.84
229.63–229.76
231.53–231.66
235.63–235.77
239.53–239.69
3730
0.061–0.075
0.063–0.074
0.053–0.074
0.067–0.075
0.062–0.074
0.069–0.076
0.065–0.077
0.056–0.075
0.061–0.076
0.057–0.078
Standard deviation
0.055–0.070
0.065–0.073
0.065–0.067
0.062–0.086
0.066–0.083
0.059–0.063
0.057–0.074
11
12
13
14
3130 xl 3500
Allele
Mean
Standard deviation
Mean
D22S1045
8
9
10
11
12
88.49–88.51
91.47–91.48
94.46–94.47
97.44–97.46
0.024–0.027
0.023–0.032
0.027–0.037
0.027–0.033
88.33–88.40
91.33–91.39
94.31–94.38
97.30–97.37
100.44–100.46
0.018–0.031
100.31–100.36
13
14
15
16
17
18
19
D2S1338
103.47–103.49
106.49–106.50
109.48–109.49
112.46–112.48
115.39–115.41
118.28–118.30
0.018–0.035
0.022–0.034
0.028–0.039
0.032–0.041
0.023–0.034
0.025–0.028
103.36–103.39
106.36–106.43
109.36–109.41
112.34–112.39
115.27–115.34
118.15–118.23
121.15–121.17
0.029–0.037
121.06–121.10
Standard deviation
0.006–0.045
0.006–0.047
0.004–0.051
0.006–0.050
88.29–88.33
91.29–91.32
94.28–94.31
97.28–97.29
0.029–0.038
0.026–0.043
0.034–0.042
0.030–0.040
0.032–0.053
100.28–100.29
0.027–0.043
0.011–0.070
103.31–103.33
0.028–0.050
0.001–0.054
106.32–106.34
0.034–0.041
0.014–0.053
109.33–109.34
0.040–0.049
0.006–0.065
0.001–0.051
115.24–115.26
0.024–0.037
0.001–0.052
Mean
3500xL
112.30–112.33
118.10–118.13
Standard deviation
0.044–0.051
0.014–0.033
0.014–0.051
120.98–121.00
0.029–0.048
15
16
280.99–281.04
284.94–284.99
288.88–288.95
292.82–292.88
296.70–296.76
300.63–300.69
0.027–0.035
0.031–0.042
0.028–0.036
0.027–0.045
0.033–0.041
0.026–0.042
281.61–281.74
285.60–285.67
289.53–289.60
293.46–293.53
297.27–297.34
301.22–301.30
0.009–0.060
0.031–0.059
0.026–0.062
0.019–0.060
0.037–0.057
0.009–0.061
281.68–281.78
285.60–285.71
289.53–289.66
293.46–293.58
297.29–297.41
301.26–301.37
0.049–0.055
0.048–0.052
0.037–0.050
0.046–0.051
0.034–0.060
0.045–0.052
Mean
90.00–90.01
92.94–92.96
95.88–95.91
98.83–98.87
101.79–101.83
104.76–104.78
107.71–107.77
110.67–110.72
113.63–113.68
116.60–116.64
119.56–119.62
122.53–122.60
281.21–281.39
285.24–285.42
289.29–289.45
293.33–293.50
297.42–297.56
301.44–301.59
3730
Standard deviation
0.062–0.066
0.058–0.069
0.060–0.075
0.060–0.075
0.059–0.064
0.060–0.072
0.057–0.063
0.056–0.069
0.059–0.065
0.051–0.068
0.051–0.055
0.058–0.065
0.050–0.059
0.052–0.059
0.054–0.062
0.048–0.059
0.046–0.064
0.044–0.059
Allele
21
22
23
24
17
18
19
20
3130 xl 3500
Mean
Standard deviation
Mean
304.53–304.58
0.029–0.041
305.12–305.20
3500xL
Standard deviation
Mean
Standard deviation
0.027–0.064
305.14–305.27
0.038–0.054
308.46–308.52
312.46–312.51
316.62–316.67
320.81–320.86
0.024–0.036
0.029–0.042
0.020–0.037
0.025–0.043
309.05–309.14
313.06–313.16
317.21–317.30
321.40–321.49
25
26
27
28
324.89–324.95
0.029–0.045
325.44–325.53
328.91–328.99
0.029–0.038
329.48–329.58
332.95–333.00
0.025–0.034
333.53–333.61
336.94–337.00
0.024–0.035
337.51–337.59
340.96–341.00
345.05–345.09
349.37–349.42
D2S441
8
9
10
11
11.3
12
13
76.79–76.81
80.93–80.95
85.04–85.08
89.16–89.18
92.32–92.35
93.25–93.27
97.18–97.20
0.026–0.037
0.023–0.036
0.026–0.038
0.019–0.034
0.026–0.034
0.018–0.033
0.023–0.031
0.025–0.033
0.020–0.029
0.022–0.031
341.50–341.57
345.58–345.67
349.79–349.89
76.57–76.62
80.74–80.77
84.87–84.91
89.01–89.05
92.19–92.24
93.12–93.17
97.07–97.11
0.015–0.064
0.008–0.061
0.026–0.065
0.042–0.064
0.016–0.058
0.031–0.066
0.018–0.056
0.016–0.065
0.009–0.060
0.023–0.057
0.032–0.062
0.001–0.046
0.000–0.047
0.006–0.052
0.015–0.048
0.018–0.049
0.015–0.050
0.005–0.048
309.11–309.22
313.12–313.25
317.27–317.39
321.45–321.58
325.50–325.63
329.57–329.67
333.58–333.69
337.57–337.67
341.57–341.68
345.64–345.74
349.90–349.95
76.55–76.60
80.70–80.75
84.84–84.88
88.97–89.02
92.16–92.18
93.09–93.12
97.03–97.06
0.040–0.061
0.043–0.059
0.047–0.057
0.038–0.063
0.049–0.075
0.050–0.062
0.049–0.060
0.050–0.058
0.033–0.061
0.047–0.054
0.043–0.050
0.026–0.047
0.035–0.048
0.029–0.036
0.036–0.043
0.032–0.040
0.033–0.041
0.032–0.046
Mean
305.48–305.65
309.50- 309.67
313.54–313.72
317.60–317.76
321.65–321.82
325.72–325.88
329.76–329.93
333.80–333.99
337.85–338.05
341.91–342.10
345.98–346.18
350.21–350.38
3730
Standard deviation
0.062–0.074
0.057–0.066
0.062–0.066
0.057–0.064
0.061–0.074
0.053–0.068
0.057–0.077
0.060–0.073
0.057–0.065
0.060–0.079
0.060–0.067
0.050–0.063
78.52–78.54
82.51–82.55
86.55–86.57
90.56–90.59
93.68–93.70
94.57–94.59
98.44–98.47
0.070–0.078
0.067–0.072
0.061–0.066
0.050–0.068
0.058–0.064
0.052–0.068
0.047–0.061
Allele
14
15
16
17
3130 xl 3500
Mean
Standard deviation
Mean
101.30–101.32
0.025–0.033
101.18–101.23
3500xL
Standard deviation
Mean
Standard deviation
0.011–0.053
101.17–101.18
0.011–0.045
105.43–105.45
0.028–0.033
105.33–105.38
109.55–109.56
0.027–0.037
109.46–109.51
113.74–113.76
0.031–0.038
113.67–113.72
0.001–0.055
0.030–0.055
0.005–0.052
105.32–105.34
109.45–109.46
113.65–113.68
0.038–0.047
0.031–0.046
0.048–0.051
17
18
19
20
13
14
15
16
D3S1358
9
10
96.60–96.64
0.022–0.033
96.49–96.55
100.76–100.79
0.026–0.037
100.66–100.71
11
12
104.95–104.97
108.98–109.00
0.026–0.037
0.024–0.039
104.81–104.90
108.83–108.92
113.23–113.25
117.21–117.24
121.09–121.12
125.20–125.24
0.032–0.037
0.024–0.041
0.023–0.034
0.030–0.036
113.08–113.17
117.09–117.14
120.96–121.04
125.09–125.15
129.33–129.37
0.023–0.038
129.22–129.29
133.36–133.40
0.020–0.035
133.26–133.32
137.35–137.39
0.026–0.036
137.24–137.31
141.66–141.71
0.027–0.036
141.56–141.62
0.008–0.047
0.004–0.052
0.016–0.051
0.023–0.046
0.000–0.061
0.015–0.047
0.004–0.053
0.024–0.061
0.013–0.063
0.018–0.055
0.014–0.058
0.005–0.054
96.47–96.49
100.63–100.65
104.80–104.83
108.83–108.85
113.07–113.09
117.06–117.07
120.92–120.94
125.05–125.06
129.18–129.20
133.22–133.23
137.21–137.21
141.52–141.54
0.039–0.049
0.044–0.051
0.040–0.048
0.040–0.044
0.047–0.052
0.015–0.035
0.031–0.050
0.033–0.047
0.037–0.047
0.044–0.052
0.041–0.048
0.038–0.046
Mean
102.46–102.49
106.50–106.53
110.51–110.56
114.64–114.70
98.32–98.38
102.48–102.54
106.63–106.71
110.66–110.74
114.94–115.00
119.07–119.13
123.11–123.19
127.36–127.41
131.58–131.63
135.68–135.75
139.73–139.80
144.02–144.09
3730
Standard deviation
0.053–0.067
0.047–0.061
0.053–0.061
0.053–0.056
0.061–0.073
0.060–0.072
0.060–0.064
0.054–0.067
0.055–0.063
0.051–0.066
0.049–0.069
0.053–0.065
0.051–0.061
0.051–0.065
0.049–0.055
0.058–0.061
8
9
6
7
3130 xl 3500
Allele
Mean
Standard deviation
Mean
D5S818
7 138.53–138.55
0.025–0.039
138.60–138.68
8
9
10
11
12
13
14
15
16
17
18
D7S820
142.59–142.60
146.68–146.71
150.82–150.83
154.91–154.93
159.00–159.03
163.04–163.09
167.07–167.10
171.08–171.12
175.09–175.13
179.10–179.14
183.16–183.20
0.027–0.034
0.013–0.034
0.022–0.032
0.029–0.039
0.025–0.038
0.019–0.032
0.026–0.034
0.021–0.032
0.029–0.032
0.021–0.039
0.021–0.035
142.66–142.71
146.75–146.82
150.90–150.97
155.00–155.06
159.10–159.17
163.16–163.21
167.16–167.24
171.20–171.26
175.22–175.27
179.25–179.31
183.32–183.39
Standard deviation
0.004–0.050
138.58–138.60
0.038–0.056
0.034–0.053
142.63–142.66
0.040–0.051
0.017–0.048
146.74–146.75
0.036–0.048
0.020–0.054
150.89–150.90
0.030–0.042
0.015–0.057
0.038–0.056
159.09–159.12
0.037–0.053
0.015–0.051
163.13–163.17
0.037–0.053
0.030–0.055
167.17–167.20
0.040–0.044
0.025–0.051
0.038–0.062
175.22–175.25
0.038–0.055
0.005–0.052
Mean
3500xL
154.98–155.02
171.20–171.22
179.25–179.27
Standard deviation
0.041–0.052
0.034–0.056
0.005–0.050
0.026–0.062
183.30–183.37
0.027–0.049
10
11
262.40–262.42
266.41–266.43
270.41–270.43
274.43–274.45
278.44–278.46
282.43–282.45
0.025–0.040
0.027–0.044
0.025–0.033
0.029–0.034
0.026–0.032
0.023–0.032
262.54–262.60
266.56–266.62
270.57–270.63
274.59–274.64
278.61–278.69
282.62–282.67
0.034–0.057
0.020–0.058
0.027–0.068
0.027–0.051
0.007–0.060
0.011–0.066
262.55–262.60
266.55–266.61
270.58–270.62
274.62–274.64
278.63–278.66
282.60–282.64
0.035–0.051
0.043–0.052
0.037–0.053
0.035–0.050
0.038–0.059
0.035–0.048
Mean
139.69–139.76
143.72–143.77
147.69–147.77
151.67–151.76
155.65–155.74
159.64–159.70
163.61–163.69
167.57–167.66
171.56–171.64
175.54–175.61
179.50–179.59
183.49–183.58
262.97–263.01
266.96–266.99
270.92–270.98
274.92–274.97
278.92–278.97
282.91–282.96
3730
Standard deviation
0.049–0.058
0.050–0.057
0.050–0.057
0.052–0.060
0.051–0.058
0.043–0.068
0.051–0.061
0.052–0.060
0.045–0.063
0.047–0.052
0.045–0.064
0.043–0.055
0.050–0.061
0.056–0.064
0.055–0.064
0.052–0.061
0.054–0.066
0.053–0.070
Allele
12
13
14
15
3130 xl 3500
Mean
Standard deviation
Mean
286.40–286.41
0.028–0.036
286.58–286.64
3500xL
Standard deviation
Mean
Standard deviation
0.033–0.057
286.57–286.60
0.042–0.054
290.35–290.38
0.026–0.032
290.54–290.61
294.30–294.33
0.027–0.037
294.49–294.57
298.25–298.26
0.026–0.051
298.46–298.54
0.025–0.057
0.015–0.062
0.006–0.060
290.54–290.58
294.51–294.54
298.46–298.49
0.036–0.045
0.033–0.054
0.042–0.052
16
17
18
19
12
13
14
15
10
11
8
9
D8S1179
5 114.45–114.46
0.029–0.036
114.21–114.27
6
7
118.42–118.43
122.38–122.40
0.024–0.035
0.022–0.036
118.20–118.25
122.17–122.21
126.39–126.41
130.41–130.43
134.45–134.49
138.52–138.57
0.024–0.031
0.030–0.038
0.024–0.035
0.028–0.032
126.18–126.23
130.21–130.25
134.25–134.30
138.32–138.36
142.66–142.70
146.89–146.93
151.05–151.08
155.21–155.24
159.37–159.40
163.46–163.49
167.54–167.57
171.62–171.64
0.027–0.040
0.017–0.039
0.025–0.035
0.025–0.038
0.028–0.035
0.020–0.035
0.022–0.037
0.025–0.034
142.45–142.50
146.71–146.74
150.85–150.89
155.00–155.04
159.16–159.19
163.25–163.28
167.32–167.36
171.39–171.42
0.032–0.052
0.000–0.054
0.034–0.057
0.008–0.052
0.018–0.062
0.015–0.049
0.004–0.054
0.012–0.053
0.023–0.054
0.025–0.053
0.015–0.057
0.004–0.057
0.031–0.051
0.031–0.048
0.024–0.063
114.15–114.18
118.12–118.16
122.09–122.12
126.08–126.12
130.11–130.13
134.17–134.19
138.24–138.27
142.37–142.39
146.61–146.64
150.76–150.78
154.90–154.93
159.07–159.08
163.16–163.19
167.23–167.26
171.30–171.33
0.031–0.046
0.029–0.044
0.035–0.050
0.035–0.049
0.039–0.047
0.037–0.054
0.037–0.050
0.032–0.040
0.034–0.042
0.034–0.040
0.036–0.046
0.032–0.046
0.030–0.048
0.037–0.050
0.036–0.051
Mean
286.91–286.96
290.91–290.95
294.89–294.94
298.89–298.94
116.07–116.10
120.19–120.24
124.33–124.37
128.45–128.50
132.58–132.61
136.68–136.75
140.81–140.86
144.93–144.98
149.11–149.16
153.21–153.26
157.31–157.36
161.39–161.45
165.50–165.54
169.59–169.62
173.66–173.72
3730
Standard deviation
0.051–0.068
0.052–0.059
0.050–0.053
0.058–0.060
0.052–0.064
0.056–0.062
0.057–0.065
0.049–0.070
0.051–0.059
0.048–0.062
0.053–0.068
0.051–0.060
0.053–0.060
0.053–0.067
0.046–0.059
0.052–0.066
0.054–0.062
0.050–0.062
0.057–0.062
20
21
22
23
16
17
18
19
FGA
13
14
15
3130 xl 3500
Allele
Mean
Standard deviation
Mean
DYS391
7 364.73–364.78
0.028–0.044
365.17–365.27
8
9
10
11
12
13
368.80–368.85
372.81–372.85
376.80–376.86
380.85–380.89
384.90–384.95
389.00–389.04
0.027–0.045
0.027–0.036
0.029–0.039
0.030–0.042
0.030–0.038
0.023–0.046
369.25–369.35
373.25–373.34
377.24–377.34
381.26–381.34
385.24–385.35
389.33–389.43
Standard deviation
0.028–0.062
365.11–365.19
0.045–0.066
0.025–0.060
369.18–369.28
0.043–0.061
0.022–0.064
373.18–373.28
0.046–0.057
0.011–0.067
377.17–377.27
0.044–0.063
0.005–0.057
0.024–0.064
385.21–385.29
0.049–0.059
0.027–0.065
Mean
3500xL
381.21–381.30
389.30–389.37
Standard deviation
0.044–0.054
0.046–0.056
223.48–223.53
227.48–227.55
231.52–231.59
0.028–0.038
0.037–0.043
0.024–0.041
223.35–223.42
227.38–227.46
231.40–231.48
235.55–235.62
0.031–0.039
235.43–235.49
0.038–0.080
0.029–0.079
0.034–0.073
0.024–0.077
223.43–223.50
227.47–227.53
231.50–231.56
235.53–235.59
0.036–0.060
0.045–0.061
0.050–0.065
0.047–0.062
239.58–239.64
243.70–243.75
247.82–247.87
0.017–0.036
0.027–0.035
0.022–0.036
239.45–239.52
243.55–243.64
247.63–247.71
251.86–251.89
0.023–0.034
251.68–251.75
0.004–0.071
0.039–0.087
0.011–0.065
0.004–0.070
239.55–239.60
243.65–243.72
247.76–247.81
251.78–251.83
0.025–0.052
0.042–0.051
0.053–0.056
0.024–0.046
255.81–255.84
259.73–259.77
263.74–263.78
0.027–0.039
0.031–0.039
0.028–0.041
255.61–255.70
259.55–259.61
263.55–263.62
0.006–0.070
0.000–0.072
0.031–0.078
255.74–255.79
259.66–259.71
263.68–263.72
0.032–0.047
0.024–0.056
0.050–0.056
Mean
367.09–367.32
371.22–371.44
375.29–375.52
379.37–379.59
383.46–383.67
387.45–387.65
391.61–391.80
226.22–226.30
230.28–230.34
234.36–234.41
238.42–238.47
242.50–242.54
246.55–246.61
250.63–250.67
254.71–254.76
258.77–258.84
262.85–262.91
266.92–266.98
3730
Standard deviation
0.053–0.065
0.054–0.068
0.043–0.063
0.050–0.066
0.056–0.067
0.055–0.074
0.055–0.070
0.050–0.069
0.051–0.073
0.059–0.078
0.057–0.073
0.053–0.080
0.051–0.073
0.053–0.074
0.057–0.074
0.060–0.074
0.061–0.073
0.060–0.079
3130 xl 3500
Allele
Mean
Standard deviation
Mean
267.74–267.79
0.022–0.029
267.55–267.62
24
25
26
271.79–271.84
0.031–0.038
271.58–271.66
275.80–275.87
0.024–0.032
275.61–275.67
26.2
277.84–277.88
0.026–0.035
277.62–277.70
3500xL
Standard deviation
Mean
Standard deviation
0.025–0.067
267.65–267.73
0.038–0.055
0.024–0.067
271.72–271.77
0.041–0.053
0.027–0.063
275.73–275.80
0.044–0.054
0.009–0.064
277.76–277.84
0.044–0.059
27
28
279.82–279.85
283.81–283.84
0.028–0.038
0.029–0.038
279.59–279.66
283.59–283.65
0.040–0.059
0.008–0.064
279.70–279.78
283.68–283.74
0.025–0.060
0.042–0.053
29
30
287.79–287.84
0.030–0.048
287.57–287.63
291.80–291.85
0.034–0.046
291.57–291.63
30.2
293.58–293.63
0.027–0.038
293.35–293.42
31.2
297.55–297.61
0.034–0.037
297.31–297.37
0.027–0.048
0.029–0.065
0.019–0.057
0.008–0.055
287.67–287.75
291.69–291.75
293.46–293.53
297.42–297.50
0.040–0.055
0.044–0.051
0.043–0.050
0.044–0.053
32.2
301.49–301.54
0.032–0.044
301.24–301.33
33.2
305.42–305.46
0.028–0.040
305.17–305.25
42.2
342.26–342.29
0.028–0.049
341.96–342.00
43.2
346.34–346.37
0.033–0.042
346.01–346.09
44.2
350.42–350.45
0.025–0.047
350.10–350.16
45.2
354.49–354.55
0.022–0.041
354.18–354.24
46.2
358.43–358.46
0.027–0.053
358.12–358.18
47.2
362.45–362.49
0.030–0.043
362.13–362.19
48.2
366.52–366.54
0.027–0.041
366.21–366.24
0.006–0.066
0.028–0.064
0.008–0.060
0.022–0.071
0.027–0.077
0.028–0.077
0.008–0.064
0.046–0.066
0.017–0.065
301.38–301.44
305.31–305.37
342.11–342.18
346.17–346.27
350.24–350.34
354.34–354.42
358.27–358.37
362.29–362.37
366.37–366.45
0.031–0.057
0.036–0.056
0.036–0.052
0.036–0.060
0.042–0.047
0.050–0.062
0.028–0.060
0.051–0.058
0.044–0.060
Mean
270.94–271.01
275.08–275.15
279.16–279.20
281.25–281.29
283.20–283.26
287.29–287.33
291.39–291.45
295.53–295.60
297.22–297.28
301.32–301.38
305.43–305.49
309.52–309.57
346.61–346.66
350.71–350.78
354.79–354.84
358.91–358.96
362.98–363.02
367.09–367.14
371.22–371.30
3730
Standard deviation
0.058–0.081
0.060–0.081
0.055–0.074
0.059–0.081
0.059–0.075
0.051–0.082
0.062–0.082
0.063–0.083
0.063–0.072
0.056–0.085
0.062–0.078
0.059–0.085
0.060–0.086
0.063–0.085
0.061–0.078
0.065–0.089
0.060–0.097
0.072–0.094
0.062–0.092
3130 xl 3500
Allele
Mean
Standard deviation
Mean
50.2
374.50–374.53
0.026–0.038
374.16–374.21
Standard deviation
Mean
3500xL
Standard deviation
0.029–0.074
374.32–374.41
0.037–0.062
51.2
378.51–378.57
0.027–0.040
378.13–378.21
0.042–0.065
378.32–378.41
0.047–0.066
SE33
4.2
6.3
8
9
11
12
13
14
15
16
17
18
19
20
307.08–307.10
0.024–0.037
307.12–307.19
316.31–316.34
0.028–0.036
316.31–316.39
321.56–321.58
0.024–0.037
321.54–321.62
325.67–325.70
0.024–0.038
325.63–325.72
333.77–333.80
0.029–0.039
333.74–333.83
337.85–337.87
0.030–0.040
337.83–337.88
341.90–341.93
0.029–0.038
341.85–341.93
346.01–346.03
0.032–0.048
345.93–346.02
350.07–350.09
0.031–0.041
349.99–350.10
354.17–354.20
0.030–0.043
354.11–354.20
358.26–358.29
0.031–0.038
358.20–358.29
362.32–362.33
0.026–0.042
362.26–362.35
366.28–366.29
0.026–0.041
366.20–366.31
370.32–370.34
0.026–0.036
370.24–370.33
20.2
372.32–372.33
0.028–0.041
372.23–372.31
21 374.33–374.34
0.027–0.036
374.25–374.33
21.2
376.34–376.35
0.025–0.035
376.23–376.32
0.031–0.080
0.018–0.057
0.006–0.065
0.017–0.063
0.032–0.053
0.016–0.060
0.011–0.062
0.026–0.055
0.022–0.055
0.026–0.060
0.008–0.060
0.010–0.065
0.030–0.064
0.031–0.065
0.026–0.054
0.026–0.065
0.033–0.064
307.19–307.22
316.40–316.43
321.65–321.67
325.76–325.77
333.86–333.88
337.93–337.94
341.98–342.00
346.07–346.09
350.11–350.14
354.22–354.25
358.34–358.36
362.41–362.43
366.34–366.36
370.38–370.40
372.37–372.39
374.39–374.40
376.39–376.41
0.038–0.053
0.036–0.051
0.041–0.067
0.047–0.062
0.041–0.049
0.048–0.054
0.025–0.052
0.044–0.053
0.037–0.051
0.043–0.060
0.055–0.065
0.024–0.059
0.042–0.052
0.036–0.050
0.048–0.059
0.041–0.049
0.047–0.051
Mean
379.46–379.51
383.56–383.62
310.30–310.35
319.58–319.65
324.71–324.77
328.83–328.88
337.06–337.12
341.22–341.28
345.34–345.43
349.47–349.55
353.55–353.63
357.75–357.82
361.90–361.97
366.01–366.09
370.09–370.16
374.26–374.33
376.32–376.39
378.37–378.44
380.47–380.51
3730
Standard deviation
0.068–0.098
0.063–0.095
0.047–0.057
0.045–0.063
0.047–0.057
0.056–0.062
0.051–0.063
0.052–0.061
0.053–0.061
0.051–0.063
0.050–0.058
0.054–0.067
0.057–0.062
0.059–0.065
0.059–0.068
0.059–0.069
0.051–0.067
0.059–0.072
0.059–0.070
3130 xl 3500
Allele
Mean
Standard deviation
Mean
22.2
370.32–370.34
0.026–0.036
380.22–380.30
Standard deviation
Mean
3500xL
Standard deviation
0.040–0.065
370.38–370.40
0.036–0.050
23.2
372.32–372.33
0.028–0.041
384.30–384.41
24.2
374.33–374.34
0.027–0.036
388.36–388.45
0.034–0.074
0.023–0.059
372.37–372.39
374.39–374.40
0.048–0.059
0.041–0.049
25.2
376.34–376.35
0.025–0.035
392.34–392.42
26.2
380.33–380.36
0.028–0.044
396.47–396.54
27.2
384.45–384.47
0.024–0.039
400.48–400.57
28.2
388.50–388.51
0.032–0.043
404.41–404.50
0.033–0.062
0.010–0.072
0.041–0.065
0.021–0.067
376.39–376.41
380.38–380.40
384.48–384.52
388.52–388.57
0.047–0.051
0.003–0.055
0.050–0.056
0.042–0.059
29.2
392.49–392.52
0.030–0.046
408.37–408.49
30.2
396.61–396.65
0.035–0.043
412.39–412.49
31.2
400.64–400.67
0.036–0.050
416.40–416.49
32.2
404.58–404.60
0.037–0.046
420.56–420.65
33.2
408.56–408.60
0.033–0.050
424.57–424.68
34.2
412.58–412.60
0.024–0.049
428.62–428.72
35 416.59–416.60
0.019–0.042
430.63–430.72
35.2
420.74–420.77
0.037–0.051
432.62–432.72
36
37
424.76–424.81
0.030–0.055
434.62–434.74
428.79–428.82
0.030–0.050
438.57–438.71
0.037–0.076
0.010–0.071
0.026–0.058
0.006–0.063
0.027–0.077
0.033–0.063
0.033–0.074
0.038–0.068
0.031–0.072
0.039–0.070
392.51–392.54
396.62–396.66
400.65–400.70
404.58–404.61
408.55–408.60
412.57–412.58
416.57–416.60
420.71–420.74
424.73–424.78
428.75–428.79
0.046–0.056
0.030–0.055
0.047–0.054
0.050–0.061
0.049–0.056
0.043–0.057
0.048–0.057
0.042–0.066
0.048–0.066
0.054–0.059
Mean
384.54–384.60
388.72–388.78
392.84–392.88
396.89–396.93
401.09–401.14
405.22–405.25
409.28–409.32
413.39–413.45
417.54–417.60
421.59–421.65
425.81–425.85
429.91–429.96
434.03–434.07
436.09–436.13
438.11–438.15
440.17–440.20
444.19–444.23
3730
0.062–0.075
0.064–0.077
0.060–0.074
0.060–0.077
0.063–0.075
0.064–0.085
0.069–0.094
0.069–0.082
0.068–0.084
0.068–0.084
Standard deviation
0.042–0.072
0.061–0.069
0.061–0.072
0.061–0.071
0.062–0.075
0.062–0.081
0.061–0.070
10
11
8
9
12
3130 xl 3500
Allele
Mean
Standard deviation
Mean
TH01
4
5
6
7
8
9
9.3
10
11
178.72–178.81
0.022–0.042
179.17–179.24
182.76–182.83
0.030–0.047
183.21–183.29
186.80–186.87
0.031–0.042
187.28–187.36
190.85–190.92
0.023–0.042
191.34–191.43
194.88–194.96
0.026–0.040
195.39–195.49
198.91–198.99
0.029–0.043
199.44–199.53
201.95–202.04
0.025–0.041
202.46–202.55
202.86–202.94
0.027–0.044
203.38–203.48
206.83–206.90
0.031–0.041
207.31–207.45
13.3
217.84–217.91
0.029–0.042
218.35–218.43
Standard deviation
Mean
3500xL
Standard deviation
0.001–0.080
179.17–179.24
0.046–0.059
0.034–0.070
183.21–183.28
0.045–0.054
0.034–0.068
187.29–187.36
0.043–0.054
0.025–0.064
191.35–191.42
0.045–0.052
0.031–0.070
195.39–195.46
0.042–0.047
0.000–0.080
199.43–199.50
0.050–0.057
0.010–0.067
202.47–202.53
0.047–0.053
0.013–0.065
203.36–203.44
0.039–0.053
0.026–0.071
207.32–207.40
0.047–0.057
0.017–0.088
218.37–218.44
0.041–0.052
TPOX
5
6
7
337.43–337.50
341.43–341.54
345.60–345.67
0.024–0.046
0.030–0.052
0.030–0.050
338.35–338.51
342.41–342.59
346.57–346.71
349.60–349.66
0.035–0.042
350.55–350.76
0.008–0.061
0.012–0.065
0.021–0.053
0.021–0.061
338.30–338.51
342.35–342.56
346.49–346.69
350.51–350.72
0.049–0.060
0.055–0.065
0.050–0.059
0.040–0.055
353.66–353.71
357.69–357.76
361.72–361.77
365.71–365.78
0.026–0.048
0.035–0.051
0.031–0.045
0.035–0.056
354.66–354.79
358.71–358.85
362.73–362.88
366.73–366.89
0.028–0.078
0.006–0.061
0.011–0.066
0.028–0.075
354.58–354.79
358.64–358.84
362.66–362.89
366.68–366.89
0.046–0.064
0.051–0.068
0.047–0.075
0.055–0.075
Mean
180.08–180.29
184.03–184.25
187.99–188.19
191.95–192.15
195.91–196.13
199.90–200.08
202.92–203.11
203.84–204.04
207.81–208.00
218.72–218.92
338.62–338.92
342.67–342.96
346.76–347.04
350.74–351.05
354.78–355.08
358.81–359.12
362.85–363.16
366.88–367.18
3730
Standard deviation
0.056–0.075
0.053–0.081
0.056–0.078
0.049–0.071
0.065–0.069
0.054–0.074
0.060–0.078
0.052–0.078
0.058–0.075
0.057–0.068
0.078–0.089
0.071–0.095
0.081–0.092
0.075–0.102
0.084–0.110
0.082–0.101
0.082–0.098
0.085–0.094
14
15
16
17 vWA
11
12
13
18
19
20
21
22
Allele
13
14
15
Y indel
1
2
3130 xl 3500
Mean
Standard deviation
Mean
369.72–369.79
0.035–0.046
370.76–370.92
3500xL
Standard deviation
Mean
Standard deviation
0.030–0.062
370.70–370.91
0.054–0.066
373.71–373.79
0.035–0.052
374.77–374.93
377.72–377.79
0.037–0.050
378.78–378.94
0.022–0.071
374.70–374.91
0.055–0.077
0.005–0.065
378.70–378.92
0.059–0.067
81.26–81.31
86.53–86.58
0.022–0.035
0.028–0.034
81.08–81.17
86.38–86.43
0.015–0.049
0.011–0.060
81.05–81.10
86.34–86.35
0.033–0.045
0.028–0.046
156.50–156.52
160.56–160.59
164.61–164.65
168.81–168.86
172.75–172.77
176.76–176.80
180.81–180.83
184.84–184.89
188.93–188.95
193.00–193.02
197.02–197.05
201.03–201.06
0.023–0.033
0.019–0.036
0.021–0.035
0.023–0.032
0.026–0.033
0.021–0.034
0.019–0.033
0.025–0.037
0.024–0.034
0.028–0.039
0.029–0.037
0.026–0.035
156.60–156.65
160.67–160.71
164.73–164.77
168.95–169.00
172.86–172.91
176.91–176.95
180.95–180.99
185.02–185.06
189.10–189.16
193.19–193.24
197.23–197.28
201.25–201.31
0.015–0.048
0.005–0.053
0.011–0.047
0.029–0.052
0.017–0.055
0.008–0.049
0.004–0.056
0.010–0.050
0.027–0.054
0.023–0.059
0.008–0.060
0.035–0.056
156.55–156.59
160.64–160.66
164.68–164.72
168.93–168.97
172.84–172.87
176.87–176.90
180.90–180.96
184.97–185.01
189.08–189.12
193.16–193.20
197.19–197.24
201.21–201.26
0.040–0.048
0.038–0.055
0.037–0.052
0.040–0.047
0.037–0.040
0.037–0.049
0.045–0.053
0.038–0.053
0.043–0.052
0.036–0.057
0.046–0.052
0.044–0.054
Mean
370.94–371.22
374.96–375.25
379.02–379.30
82.91–82.99
88.23–88.31
157.91–158.01
161.82–161.93
165.83–165.93
169.96–170.07
173.90–174.01
177.90–177.99
181.87–181.98
185.78–185.91
189.77–189.88
193.76–193.86
197.67–197.79
201.66–201.76
3730
Standard deviation
0.089–0.101
0.087–0.103
0.076–0.097
0.067–0.077
0.064–0.072
0.042–0.064
0.054–0.059
0.053–0.059
0.049–0.057
0.054–0.064
0.051–0.060
0.054–0.063
0.056–0.062
0.055–0.056
0.049–0.055
0.054–0.066
0.058–0.076
Allele
23
24
3130 xl 3500
Mean
Standard deviation
Mean
204.91–204.95
0.024–0.036
205.12–205.19
209.23–209.26
0.024–0.040
209.43–209.50
3500xL
Standard deviation
Mean
Standard deviation
0.015–0.047
205.08–205.13
0.041–0.048
0.028–0.063
209.40–209.44
0.041–0.053
Mean
205.41–205.53
209.89–209.99
3730
Standard deviation
0.048–0.065
0.051–0.061
Chapter 5 Experiments and results
Extra peaks in the electropherogram
5
Extra peaks in the electropherogram
Causes of extra peaks
Peaks other than the target alleles may be detected on the electropherogram. Causes for the appearance of extra peaks include stutter products, incomplete 3´ A nucleotide addition (at the n-1 position), dye artifacts, and mixed DNA samples (see DNA Advisory Board (DAB) Standard 8.1.2.2).
Extra peaks: Stutter
Stutter definition
Stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller than the target STR allele product (minus stutter), or less frequently, one repeat larger (plus stutter) (Butler, 2005; Mulero et al ., 2006). Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the minus stutter product is missing a single tetranucleotide core repeat unit relative to the main allele (Walsh et al.
, 1996). Although plus-stutter is normally much less significant than minus-stutter in STR loci with tetranucleotide repeats, the incidence of plus-stutter may be more significant in trinucleotide repeat-containing loci.
Contact HID Support for more information on plus stutter.
The proportion of the stutter product relative to the main allele (percent stutter) is measured by dividing the height of the stutter peak by the height of the main allele peak.
Stutter observations
Peak heights were measured for amplified samples at the loci used in the kit. All data were generated on the 3500xL Genetic Analyzer. Some conclusions from these measurements and observations are:
• For each locus, the stutter percentage generally increases with allele length.
• Smaller alleles display a lower level of stutter relative to the longer alleles within each locus.
• Each allele within a locus displays a consistent stutter percentage.
• Peaks in the stutter position that are above the stutter filter percentage specified in the software are not filtered (stutter filter percentage is calculated as the mean stutter for the locus plus three standard deviations). Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated.
• The measurement of stutter percentage for allele peaks that are off-scale may be unusually high due to artificial truncation of the main allele peak.
Marker-specific plus stutter observed in the population study with the GlobalFiler ™
Amplification Kit is shown in Figure 13 through Figure 19 and listed in Table 3.
Express PCR
Additional marker-specific plus stutter observed in the population study with the GlobalFiler ™ Express
peaks at two loci are shown in “Example of non-standard stutter peaks observed at the D22S1045 and
GlobalFiler ™ Express PCR Amplification Kit User Guide 91
5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
Workflow
Treated paper
Untreated paper
Buccal swab
Number of samples
284 blood samples on FTA ™ Classic Cards
272 buccal samples on Indicating FTA ™ Cards
45 blood samples on FTA ™ Bloodstain Cards
90 buccal samples on Bode Buccal DNA Collector ™ Device
45 buccal swab samples lysed in Prep ‑ n ‑ Go ™ Buffer
Figure 13 Stutter percentages for D1S1656, D2S441, D2S1338, and D3S1358 loci (Blue=FAM ™ black=NED ™ dye, purple=SID ™ dye)
dye,
92 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Extra peaks in the electropherogram
5
Figure 14 Stutter percentages for D5S818, D7S820, D8S1179, and D10S1248 (Green=VIC ™ red=TAZ ™ dye, purple=SID ™ dye)
dye,
Figure 15 Stutter percentages for D12S391, D13S317, and D16S539 loci (Blue=FAM ™ dye, purple=SID ™ dye)
dye, red=TAZ ™
GlobalFiler ™ Express PCR Amplification Kit User Guide 93
5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
Figure 16 Stutter percentages for D18S51, D19S433, and D21S11 loci (Green=VIC ™ dye)
dye, black=NED ™
94
Figure 17 Stutter percentages for D22S1045, DYS391, CSF1PO, and TH01 loci (Blue=FAM ™ green=VIC ™ dye, black=NED ™ dye, red=TAZ ™
dye,
dye). Red and orange data points associated with
D22S1045 locus indicate minus and plus stutter, respectively.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Extra peaks in the electropherogram
5
Figure 18 Stutter percentages for FGA and TPOX loci (Blue=FAM ™ dye, black=NED ™ dye)
Figure 19 Stutter percentages for SE33 and vWA loci (Blue=FAM ™ dye, red=TAZ ™ dye)
GlobalFiler ™ Express PCR Amplification Kit User Guide 95
5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
Table 3 Marker-specific plus stutter observed in the population study with the GlobalFiler that showed negligible plus stutter are omitted.
™ Express PCR
Amplification Kit. Data produced on a 3500xL Genetic Analyzer using a 50-RFU threshold cutoff. Markers
Loci
D10S1248
D12S391
D16S539
D18S51
D19S433
D1S1656
D21S11
D22S1045
D2S1338
D2S441
D3S1358
D8S1179
FGA
TH01 vWA
Observations
28
259
214
156
109
16
80
31
448
264
787
156
107
185
137
Mean
1.593
0.9106
1.282
1.2453
1
1.42
1.069
1.2437
0.8623
0.859
1.0282
1.944
0.9966
1.0044
3.68075
1.53
1.75
0.6228
1.707
1.1121
1.157
1.5
1.027
Standard deviation
1.0038
0.9123
0.7155
0.7157
1.322
0.6624
0.5295
Minimum
0.19
0.31
0.28
0.36
0.32
0.24
0.2
0.27
0.3
0.42
0.61
0.17
0.22
0.19
0.25
Maximum
7.13
4.65
10.3
7.15
8.82
6.09
5.01
5.2
7.26
4.66
11.06
4.58
5.91
6.4
5.9
Example of non-standard stutter peaks observed at the D22S1045 and SE33 loci
STR loci such as D1S1656 (Figure 20) and SE33 (Figure 21) include more complex nucleotide
sequences including regions of dinucleotide repeats which can yield additional stutter peaks. If these stutter peaks exceed the peak amplitude threshold (typically 175 RFU), they may be detected as additional alleles in the profile. The stutter file that is provided with the GeneMapper analysis of GlobalFiler ™
™ ID ‑ X Software for
kit data contain a minus 2−nt stutter filter for SE33 and D1S1656 to prevent
96 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Extra peaks in the electropherogram
5
Figure 20 GlobalFiler ™ Express PCR Amplification Kit electropherogram showing plus stutter associated with the D22S1045 STR locus. Data produced on a 3500xL Genetic Analyzer.
Figure 21 Example of a –2 nt reproducible artifact at the SE33 locus. Data produced on a 3500xL
Genetic Analyzer.
GlobalFiler ™ Express PCR Amplification Kit User Guide 97
5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
98
Stutter percentage filter settings that are provided with the GeneMapper
™
Software
ID ‑ X
The settings in Table 4 were derived using the data shown earlier in this section. The proportion of
the stutter product relative to the main allele (stutter percent) is measured by dividing the height of the stutter peak by the height of the main allele peak.
IMPORTANT! The values that are shown in the table are the values that were determined during developmental validation studies using specific data sets. Always perform internal validation studies to determine the appropriate values to use for your applications.
Table 4 Marker-specific stutter filter percentages for GlobalFiler ™ Express kit loci
Locus [1]
CSF1PO
D10S1248
D12S391
D13S317
D16S539
D18S51
D19S433
D1S1656
D1S1656 (−2 nt)
D21S11
D22S1045
D22S1045 (+3 nt)
D2S1338
D2S441
D3S1358
D5S818
D7S820
D8S1179
DYS391
FGA
SE33
10.58
13.08
1.79
11.42
17.30
8.27
13.14
8.75
% Stutter
11.40
12.50
15.08
9.98
10.17
13.47
12.45
10.84
10.21
10.20
8.54
11.96
14.42
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Extra peaks in the electropherogram
5
Table 4 Marker-specific stutter filter percentages for GlobalFiler Express kit loci (continued)
Locus [1] % Stutter
SE33 (−2 nt) 4.97
TH01
TPOX vWA
[1] These percentages are used as stutter filters in AmpFLSTR_Stutter.txt
5.24
5.43
12.33
Extra peaks: Addition of 3' A nucleotide
3 ′ A nucleotide addition definition
Many DNA polymerases can catalyze the addition of a single nucleotide (predominantly adenosine) to the 3 ′ ends of double-stranded PCR products (Clark, 1988; Magnuson et al ., 1996). This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence. The
PCR product with the extra nucleotide is referred to as the “+A” form.
3 ′ A observations
The efficiency of +A addition is related to the particular sequence of the DNA at the 3´ end of the PCR product.
The GlobalFiler ™ Express PCR Amplification Kit includes two main design features that promote maximum +A addition:
• The primer sequences have been optimized to encourage +A addition.
• The PCR chemistry allows complete +A addition with a short final incubation at The new, highly robust PCR chemistry allows complete +A addition with a short final incubation at 60°C for
8 minutes. .
This final extension step gives the DNA polymerase additional time to complete +A addition to all
double-stranded PCR products. Figure 22 shows examples of incomplete and normal +A addition. Final
extension incubation for longer than the recommended time can result in double +A addition, in which two nontemplate adenosine residues are added to the PCR product. Double +A addition can cause
"shoulders" on the right side of main allele peaks, and is therefore to be avoided.
GlobalFiler ™ Express PCR Amplification Kit User Guide 99
5 Chapter 5 Experiments and results
Extra peaks in the electropherogram
100 GlobalFiler ™ Express PCR Amplification Kit User Guide
Figure 22 Omitting the final extension step results in shoulders on main allele peaks due to incomplete
A nucleotide addition. Examples shown are the smaller amplicons of FAM data from a 3500xL Genetic Analyzer using the GlobalFiler ™
™ , NED ™ , and SID
Express PCR Amplification Kit.
™ dye channel
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 of an issue with the
GlobalFiler ™ Express PCR Amplification Kit than with earlier generation kits. However, "shouldering" of allele peaks can still be observed if the amount of input DNA is greater than recommended concentration. Amplification of excess input DNA can also result in off-scale data.
Extra peaks: Artifacts
Artifact definition
Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible.
Anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise).
Artifact observation
Due to improvements in PCR primer manufacturing processes, the incidence of artifacts has been greatly reduced in the GlobalFiler ™ Express kit. Kit electropherograms are free of reproducible dye
the low baseline ‑ level fluorescence that is observed in a typical negative control PCR.
However, it is important to consider noise and other amplification-related artifacts when interpreting data.
Figure 23 Examples of fluorescence background in data produced on a 3500xL Genetic Analyzer (Y-axis scale 0 to 200 RFU).
GlobalFiler ™ Express PCR Amplification Kit User Guide 101
5 Chapter 5 Experiments and results
Characterization of loci
Characterization of loci
SWGDAM guideline 3.1
“The basic characteristics of a genetic marker should be determined and documented.” (SWGDAM,
December 2012)
Loci in this kit
This section describes basic characteristics of the 21 autosomal STR loci, Y STR locus, Y indel locus, and sex-determining marker (Amelogenin), that are amplified with the GlobalFiler ™ Express PCR
Amplification Kit. Most of these loci have been extensively characterized by other laboratories.
Nature of polymorphisms
The primers for the Amelogenin locus flank a 6-nucleotide deletion in intron 1 of the X homolog.
Amplification generates 99 ‑ nt and 105 ‑ 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.)
Except for D22S1045, a trinucleotide STR, the remaining loci are tetranucleotide short tandem repeat
(STR) loci. The length differences among alleles of a particular locus are caused by differences in the number of repeat units.
We have sequenced all the alleles in the GlobalFiler ™ Express PCR Amplification Kit Allelic Ladder, including microvariants. In addition, other groups in the scientific community have sequenced alleles at some of these loci (Nakahori et al ., 1991; Puers et al ., 1993; Möller et al ., 1994; Barber et al ., 1995;
Möller and Brinkmann, 1995; Barber et al.
, 1996; Barber and Parkin, 1996; Brinkmann et al ., 1998;
Momhinweg et al ., 1998; Watson et al ., 1998). Among the various sources of sequence data on the loci, there is consensus on the repeat patterns and structure of the STRs.
Inheritance
The Centre d'Etude du Polymorphisme Humain (CEPH) has collected DNA from families of Utah
Mormon, French Venezuelan, and Amish descent. These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci.
Each family set contains three generations, generally including four grandparents, two parents, and several offspring. Consequently, the CEPH family DNA sets are ideal for studying inheritance patterns
(Begovich et al ., 1992).
Mapping
The GlobalFiler ™ Express PCR Amplification 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; and Lareu, et al.
, 1996).
102 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Species specificity
5
Genetic linkage
Two sets of STR loci in the GlobalFiler ™ Express PCR Amplification 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; D2S1338 and D2S441 are located approximately 150 million bp apart on opposite arms of chromosome 2. Linkage disequilibrium analysis was conducted on the genotype results from
1,034 individuals of three ethnic groups (350 African American, 349 Caucasian, and 335 Hispanic).
STR locus genotype results from the population study were analyzed using the Linkage Disequilibrium
module of GenePop software version 4.0.10 (Raymond and Rousset, 1995; Rousset, 2008). See Table 5
for results.
The relatively high probability values indicate that there is no statistically significant linkage disequilibrium found between the pairs of loci that are located on the same chromosome.
An independent analysis of data from the same collection of population samples (Budowle, et al ., 2010) also concluded that the 15 STR loci that are shared between the NGM ™ and NGM SElect ™ kits were independent at the population level (note that the SE33 locus was not part of this analysis). Therefore, to calculate the rarity of a profile for comparison to single-source and mixture samples, the frequencies of all loci including vWA and D12S391 could be multiplied. However, the analysis of the CEPH pedigree families demonstrated a degree of linkage between vWA and D12S391 that does not support the assumption of independence for kinship analysis.
Table 5 GenePop software LD Result (p ‑ value for pairwise analysis of loci)
Locus
Chromosome map position [1]
Chromosome
Nuclear
Coordinates [1]
(million bp)
African-
American
(n = 350)
Caucasian
(n = 350)
Hispanic
(n = 293) vWA
D12S391
12p13.31
12p13.2
5.9
12.2
0.86
0.29
0.27
D2S441 2p14 68
0.11
0.32
0.19
D2S1338 2q35 218
[1] 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.
Species specificity
SWGDAM Guideline 3.2
“The ability to detect genetic information from non-targeted species (e.g., detection of microbial DNA in a human assay) should be determined. The detection of genetic information from non-targeted species does not necessarily invalidate the use of the assay, but may help define the limits of the assay.”
(SWGDAM, December 2012)
GlobalFiler ™ Express PCR Amplification Kit User Guide 103
5 Chapter 5 Experiments and results
Species specificity
Nonhuman study observation
The GlobalFiler ™ Express PCR Amplification Kit provides the required specificity for detecting human alleles. Species specificity testing was performed to ensure that there is no cross-reactivity with nonhuman DNA that 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 GlobalFiler ™ Express PCR Amplification Kit kit:
• Primates: gorilla, chimpanzee, and macaque (1.0 ng each)
• Non-primates: mouse, dog, sheep, pig, rabbit, cat, horse, hamster, rat, chicken, and cow (10.0 ng each)
• Microorganisms: Candida albicans , Enterococcus faecalis , Escherichia coli , Fusobacterium nucleatum , Lactobacillus casei , Staphylococcus aureus , Streptococcus mitis , Streptococcus mutans , Streptococcus salivarius , and Streptococcus viridans (equivalent to 105 copies) (These microorganisms are commonly found in the oral cavity (Suido et al.
, 1986; Guthmiller et al ., 2001).)
Results were evaluated for the presence of any amplified peaks that would indicate cross reactivity of the GlobalFiler ™ Express PCR Amplification Kit with any of these non-human species.
Figure 24 shows example electropherogram results from the species specificity tests. The chimpanzee
and gorilla DNA samples produced partial profiles in the 70– 400 nucleotide region (gorilla data not shown). Macaque DNA produced an Amelogenin X peak, a 6-FAM ™ peak at 278 bp, and two small SID ™
dye peak at 359 bp, a NED
dye peaks at 304 bp and 328 bp.
™ dye
Figure 24 Representative electropherograms for some species tested in a species specificity study. Data produced on a 3500xL Genetic Analyzer.
Among the non-primate species, most produced no peaks over a threshold of 175 RFU. Horse yielded reproducible VIC ™ yielded reproducible TAZ ™
dye peaks at 94 bp (<100 RFU) due to Amelogenin cross ‑ reactivity. Pig
dye peaks at 424 bp (<200 RFU). Individual replicate PCRs of dog, mouse, and chicken yielded single, small (<50 RFU), non-reproducible peaks. These non-reproducible
104 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Sensitivity
5 cross ‑ reactivities were not detectable when the dog, mouse, or chicken DNA were amplified in the presence of human blood or buccal samples on an FTA ™ card (data not shown).
Sensitivity
SWGDAM guideline 3.3
“The ability to obtain reliable results from a range of DNA quantities, to include the upper and lower limits of the assay, should be evaluated.” (SWGDAM, December 2012)
Sample collection factors that can affect DNA quantity
The GlobalFiler ™ Express PCR Amplification Kit has been optimized at 15 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying:
• Blood samples that are obtained directly from unpurified 1.2 mm treated paper discs
• Buccal cells that are obtained directly from unpurified 1.2 mm treated paper discs (with the addition of Prep ‑ n ‑ Go ™ Buffer )
• Buccal swab sample lysate is prepared using Prep ‑ n ‑ Go ™ Buffer
Depending on the following conditions, DNA quantities present on the 1.2 mm disc may vary from laboratory to laboratory:
• Volume of blood that is spotted onto the treated paper
• Collecting devices that are used
• Collection methods that are applied
• Swab‐to‐paper transfer protocol that is used
It is essential to optimize the PCR conditions for types of blood samples and spotting protocol. See
“Optimize PCR cycle number (before first use of the kit)” on page 18 .
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 is a problem because:
– Quantitation (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 ensure minimal occurrence of offscale data when using the GlobalFiler ™ Express PCR Amplification
Kit , optimize PCR cycle number according to instructions in the Perform PCR chapter.
GlobalFiler ™ Express PCR Amplification Kit User Guide 105
5 Chapter 5 Experiments and results
Sensitivity
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.
106 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Sensitivity
5
Sensitivity observation
the lower amounts of DNA. All data was collected using the 3500xL Genetic Analyzer. The amount of
DNA was calculated based on the assumptions of 100% cell lysis efficiency and that each cell contains
6 pg of DNA.
To determine an appropriate minimum peak height threshold for your instruments and data, perform internal validation studies.
GlobalFiler ™ Express PCR Amplification Kit User Guide 107
5 Chapter 5 Experiments and results
Sensitivity
108
Figure 25 Effect of amplifying varying amounts of white blood cells (WBCs) lysed in Prep ‑ n ‑ Go ™
Samples were amplified for 25 PCR cycles.
Buffer.
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Stability
5
Stability
SWGDAM guideline 3.4
“The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults should be evaluated. In most instances, assessment of the effects of these factors on new forensic DNA procedures is not required. However, if substrates and/or environmental and/or chemical insults could potentially affect the analytical process, then the process should be evaluated to determine the effects of such factors.”
(SWGDAM, December 2012)
DNA on FTA
™
cards
The following aged samples were prepared to examine the sample-on-substrate stability:
• Finger-prick blood that was spotted onto FTA ™ Classic Cards stored for 210 days
• Buccal cells that were collected with the EasiCollect ™ device, stored for 120 days
Aged FTA ™ samples were amplified with the GlobalFiler ™ Express kit in a Veriti ™ Thermal Cycler, then the PCR products were collected and detected using a 3500xL Genetic Analyzer. The analysis shows that the age of the FTA ™ samples did not impact the performance of the GlobalFiler ™ Express kit
Figure 26 Amplification of blood on FTA ™ card stored for various amounts of time at room temperature
(Y-axis scale 0 to 19,000 RFU or 0 to 23,000 RFU).
GlobalFiler ™ Express PCR Amplification Kit User Guide 109
5 Chapter 5 Experiments and results
Stability
Figure 27 Amplification of buccal cells on Indicating FTA ™ room temperature (Y-axis scale 0 to 12,000 RFU).
card stored for various amounts of time at
DNA on 4N6FLOQSwabs
™
sample collectors
Aged buccal cell samples on 4N6FLOQSwabs ™ sample collectors were prepared to verify their sample-on-substrate stability. Buccal swabs were collected from 12 individuals over the course of
4 months. The aged 4N6FLOQSwabs ™ samples were lysed in Prep ‑ n ‑ Go ™ the GlobalFiler ™ Express kit in a Veriti ™
Buffer and amplified using
Thermal Cycler. The PCR products were run on a 3500xL
Genetic Analyzer.
The results from the aged buccal samples collected on 4N6FLOQSwabs when amplified with the GlobalFiler ™ Express kit.
™ collectors are shown
in Figure 28. The analysis revealed that buccal samples on 4N6FLOQSwabs
™ collectors, air-dried immediately after collection and aged up to 4 months at room temperature, produce acceptable profiles
110 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
Figure 28 Amplification of buccal cells on 4N6FLOQSwabs ™ amounts of time at room temperature and lysed in Prep ‑ n ‑ Go
sample collectors stored for various
™ Buffer (Y-axis scale 0 to 5,000 RFU).
Population data
SWGDAM guideline 3.7
“The distribution of genetic markers in populations should be determined in relevant population groups.” (SWGDAM, December 2012)
Population data overview
To interpret the significance of a match between genetically typed samples, you must know the population distribution of alleles at each locus in question. If the genotype of the relevant evidence sample is:
• Different from the genotype of the reference sample for a suspect, then the suspect is excluded as the donor of the biological evidence that was tested. An exclusion is independent of the frequency of the two genotypes in the population.
• The same as the genotype of the reference sample for a suspect, then the suspect is included as a possible source of the evidence sample.
The probability that another, unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant populations.
GlobalFiler ™ Express PCR Amplification Kit User Guide 111
5 Chapter 5 Experiments and results
Population data
Loci in the kit
The GlobalFiler ™ Express PCR Amplification Kit contains loci for which extensive population data are available. For additional information on the loci shared between many of the AmpFℓSTR ™ kits, see the population data and additional studies section of the AmpFℓSTR ™ NGM SElect ™
Kit User Guide (Pub. No. 4458841) and the AmpFℓSTR ™ Identifiler ™
PCR Amplification Kit
Plus PCR Amplification Kit User
Guide (Pub. No. 4440211).
Population samples used in these studies
The GlobalFiler ™ Express PCR Amplification Kit was used to generate the population data provided in this section. Whole blood samples, provided by the Interstate Blood Bank (Memphis, Tennessee) and Boca Biolistics (Coconut Creek, Florida), were collected in the United States (with no geographical preference) from randomly selected individuals of known ethnicities. Ethnicities of sample donors were:
• African-American—330 samples
• Asian—153 samples
• Caucasian—343 samples
• Hispanic—368 samples
DNA was extracted with a 6100 Nucleic Acid Prep Station.
The GlobalFiler ™ available.
Express PCR Amplification Kit contains loci for which extensive population data are
In addition to the alleles that we 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 ).
Concordance studies
The primer sequences used in the GlobalFiler
kit genotypes of the above population data were also compared against the genotypes generated using the Identifiler and the NGM SElect ™
™ kit and GlobalFiler ™ Express kit are identical. We compared allele calls between the two kits. Genotype data from 200 blood samples on FTA
Cards showed 100% concordance between the two kits. The GlobalFiler ™
™
™ Classic
Plus kit
kit. The few discordant genotypes observed were exclusively found in loci where degenerate primers were added in the GlobalFiler ™ binding sites.
kit to rescue known SNPs found in the primer
Probability of Identity definition
The P
I
value is the probability that two individuals selected at random will have an identical genotype
(Sensabaugh, 1982).
112 GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
Probability of identity observation
Table 6 shows the Autosomal STR allele frequencies at GlobalFiler
™ kit loci by population group.
‑ specific allele frequencies by population group for GlobalFiler ™ Express PCR
Amplification Kit DYS391 and Y indel loci. The Y ‑ specific allele frequencies were not included in the probability of identity calculation.
Table 8 shows the Probability of identity (P
I loci individually and combined.
) values of the GlobalFiler ™ Express PCR Amplification Kit
Table 6 Autosomal allele frequencies by population group for GlobalFiler ™ Express PCR
Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities)
Allele
African American
(n = 330)
Asian (n = 153)
U.S. Caucasian
(n = 343)
U.S. Hispanic
(n = 368)
CSF1PO
5
6
7
10
11
8
9
D10S1248
7
15
16
11.1
12
13
14
10
11
12
8
9
3.79
26.21
23.33
*
*
0.15*
3.79
9.39
27.88
4.39
1.06
*
*
0.15*
*
0.15*
*
3.64
14.09
*
*
*
0.33*
2.61
26.14
21.9
*
38.56
9.8
0.65*
*
*
*
*
*
0.33*
*
10.78
*
*
*
0.29*
2.77
27.99
31.78
*
30.76
5.98
0.44*
*
*
*
*
*
*
0.58*
3.5
*
0.14*
0.14*
0.14*
0.27*
4.48
*
*
0.95
0.54*
2.58
25.14
27.45
0.14*
37.91
4.62
0.54*
0.14*
*
GlobalFiler ™ Express PCR Amplification Kit User Guide 113
5 Chapter 5 Experiments and results
Population data
114
18
19
D12S391
13
14
15
16
17
16.1
17
17.1
17.3
14
15
15.1
16
18
18.3
19
19.1
19.3
20
20.3
21
21.3
22
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
13
African American
(n = 330)
22.88
Asian (n = 153)
36.93
U.S. Caucasian
(n = 343)
29.45
U.S. Hispanic
(n = 368)
25.95
27.88
18.48
10.15
2.27
0.30*
*
22.55
22.55
5.23
1.63
*
*
29.74
19.39
13.41
3.64
0.29*
*
36.14
22.69
7.74
2.31
*
*
1.9
16.18
2.19
12.54
*
0.58*
9.77
0.15*
3.35
*
10.35
*
*
*
4.37
*
13.56
0.15*
10.79
*
28.43
*
23.86
*
*
16.99
*
0.98*
*
8.17
*
*
*
2.61
*
10.78
*
3.59
0.61*
24.55
1.21
13.94
0.61*
0.30*
11.52
*
5.15
0.15*
16.52
0.45*
*
*
7.58
0.15*
7.27
0.15*
5
1.22
19.7
2.17
18.75
*
1.22
17.12
*
5.03
*
7.34
0.27*
0.14*
0.14*
4.08
*
8.7
*
6.79
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
D16S539
4
16
17
12
13
14
15
D13S317
5
6
7
10
11
8
9
8
9
5
6
10
11
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
23
African American
(n = 330)
3.64
Asian (n = 153)
3.27
U.S. Caucasian
(n = 343)
8.16
U.S. Hispanic
(n = 368)
3.67
24
25
26
27
0.61*
0.61*
*
*
0.98*
0.33*
*
*
3.64
1.9
0.29*
0.15*
1.9
1.36
0.27*
0.14*
2.27
3.03
29.24
43.79
*
*
*
2.27
14.55
4.55
0.30*
*
*
*
*
*
3.33
21.67
11.52
30
*
*
*
29.74
12.09
13.73
25.49
14.38
3.92
0.33*
0.33*
*
*
31.05
14.05
20.59
*
*
*
*
*
*
0.15*
10.93
7.14
6.85
29.01
30.76
10.64
4.52
*
*
*
*
*
*
1.46
12.68
4.08
32.22
*
*
0.14*
2.04
10.19
15.76
31.79
16.3
9.65
22.83
27.45
*
*
*
8.97
10.05
4.76
*
*
*
GlobalFiler ™ Express PCR Amplification Kit User Guide 115
5 Chapter 5 Experiments and results
Population data
116
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
12
African American
(n = 330)
19.09
Asian (n = 153)
21.57
U.S. Caucasian
(n = 343)
30.9
U.S. Hispanic
(n = 368)
24.18
13
14
15
16
13.03
1.36
*
*
11.44
1.31*
*
*
16.76
1.75
0.15*
*
14.4
1.22
0.27*
*
D18S51
9
10
6
7
13.2
14
14.2
15
15.2
16
10.2
11
12
13
20.2
21
22
17
18
19
20
*
17.64
*
15.31
*
11.95
10.79
8.31
4.08
1.31
*
0.87
15.01
11.95
*
*
*
1.17
*
1.02
0.29*
6.54
4.9
5.23
1.96
*
22.88
*
16.99
*
12.42
*
1.31*
5.56
17.32
*
*
*
*
*
1.96
0.98*
0.30*
5.91
0.45*
16.52
*
18.18
16.36
14.09
9.7
4.7
0.15*
0.45*
6.21
3.94
*
*
*
0.15*
0.15*
1.82
0.61*
*
16.3
0.14*
12.23
0.14*
12.91
17.39
7.74
3.53
1.9
*
*
0.14*
0.68
*
1.22
10.46
11.41
*
2.17
0.68
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
28
D19S433
5.2
6
24
25
26
27
10.2
11
11.2
12
12.1
12.2
13
9
10
7
8
13.2
14
14.2
15
15.2
16
16.2
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
23
African American
(n = 330)
0.30*
Asian (n = 153)
0.98*
U.S. Caucasian
(n = 343)
0.29*
U.S. Hispanic
(n = 368)
0.54*
*
*
*
*
* 0.65*
*
0.33*
*
*
*
*
*
*
* 0.27*
0.14*
*
*
*
*
0.15*
*
*
*
*
*
*
*
7.29
0.15*
0.15*
27.26
1.6
35.13
2.04
16.18
3.5
5.69
0.29*
0.33*
*
*
*
*
*
*
*
*
4.58
*
0.33*
28.1
2.61
23.2
9.48
7.52
20.26
0.33*
2.61
0.30*
1.21
0.15*
9.85
*
*
*
*
0.30*
10.45
*
3.94
27.88
5.3
18.94
5.3
6.67
4.39
1.52
3.18
*
0.41*
*
1.63
*
*
*
*
0.27*
8.42
*
1.49
18.48
6.93
30.71
4.62
13.04
6.79
4.08
2.17
GlobalFiler ™ Express PCR Amplification Kit User Guide 117
5 Chapter 5 Experiments and results
Population data
118
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
17
African American
(n = 330)
*
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
0.29*
U.S. Hispanic
(n = 368)
0.54*
17.2
18
18.2
19.2
0.61*
*
*
*
0.65*
*
*
*
0.15*
0.15*
*
*
0.41*
*
*
*
D1S1656
8
9
10
11
12
13
14
14.3
15
15.3
16
16.1
16.3
17
17.1
17.3
18
18.3
19
19.3
20
15.31
8.75
9.33
*
4.96
4.96
0.29*
12.68
0.29*
5.98
*
*
0.29*
6.27
15.74
7
6.27
0.29*
*
1.6
*
*
3.59
0.33*
1.63
20.26
*
31.05
*
0.65*
14.05
4.25
13.73
6.21
*
*
*
*
3.59
*
0.33*
0.33*
16.97
1.82
10
*
7.27
2.73
*
5.76
0.45*
1.82
8.48
11.06
25
0.91
*
0.15*
1.36
5.3
0.15*
0.61*
*
15.49
2.99
15.08
0.27*
5.16
6.79
*
15.76
0.82
4.48
*
0.14*
0.41*
3.94
9.38
7.07
11.28
0.27*
*
0.68
*
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
20.3
African American
(n = 330)
0.15*
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
*
U.S. Hispanic
(n = 368)
*
21 * * * *
0.65*
9.48
3.92
0.33*
2.61
26.8
*
*
30.72
*
*
4.9
0.65*
*
*
*
*
*
*
*
14.38
0.98*
4.58
*
1.67
8.79
4.55
*
1.36
15.61
*
0.15*
20.76
0.30*
5.91
25.15
*
*
*
*
*
7.12
0.91
3.18
0.15*
*
3.64
*
D21S11
23.2
24
24.2
25
26
27
28
30
30.2
31
31.2
31.3
32
28.2
29
29.2
29.3
32.2
33
33.2
34
34.2
35
35.2
2.77
6.85
8.89
*
2.33
23.76
0.15*
0.15*
23.18
0.58*
2.62
16.76
*
*
*
*
*
9.62
*
1.9
*
0.44*
*
*
21.06
*
*
27.17
1.77
5.16
11.14
*
1.36
*
*
0.27*
*
0.41*
1.49
11.41
0.14*
12.5
0.14*
5.3
*
0.14*
0.27*
*
GlobalFiler ™ Express PCR Amplification Kit User Guide 119
5 Chapter 5 Experiments and results
Population data
120
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
36
African American
(n = 330)
0.76
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
*
U.S. Hispanic
(n = 368)
0.14*
37
38
39
*
*
*
*
*
*
*
*
*
*
0.14*
*
18
19
20
D2S1338
10
14
15
16
17
D22S1045
7
8
9
10
11
12
13
11
12
13
14
15
16
23.33
20.3
20.45
2.12
*
*
14.7
6.21
0.30*
7.88
*
0.61*
*
4.09
*
*
*
0.15*
*
0.30*
5.3
33.66
23.86
24.18
1.96
*
*
15.36
0.33*
0.33*
0.33*
*
*
*
*
*
*
1.63
*
*
*
*
36.3
36.3
7.58
0.58*
*
*
*
*
*
0.44*
13.85
0.58*
1.02
3.35
*
*
*
0.15*
0.15*
0.15*
4.08
*
*
3.8
*
*
*
*
43.48
34.65
8.42
0.95
*
0.14*
7.61
0.95
1.09
2.04
*
*
*
0.68
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
D2S441
9.1
10
8
9
11
11.3
15
16
17
12
12.3
13
14
22
23
24
25
18
19
20
21
26
27
28
29
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
17
African American
(n = 330)
10
Asian (n = 153)
14.05
U.S. Caucasian
(n = 343)
18.37
U.S. Hispanic
(n = 368)
17.8
4.85
16.21
10.45
11.97
12.42
9.24
8.79
6.97
2.58
0.76
*
*
*
0.33*
0.98*
0.33*
13.07
16.67
8.82
2.94
5.88
18.3
11.11
5.88
1.6
0.29*
*
*
8.31
14.14
15.74
2.92
1.75
10.06
10.2
12.1
0.15*
*
*
9.09
35.61
2.88
20.45
0.15*
3.48
26.21
1.97
*
*
*
*
2.94
20.59
36.27
2.61
20.92
*
6.21
9.8
0.65*
*
*
*
0.58*
*
19.83
33.09
5.1
4.08
0.29*
3.35
28.86
4.37
0.44*
*
1.49
0.14*
0.14*
*
6.52
17.53
13.86
3.67
6.52
14.27
8.83
5.43
3.8
0.41*
1.9
23.1
*
0.14*
*
30.3
31.93
4.62
3.4
0.41*
*
GlobalFiler ™ Express PCR Amplification Kit User Guide 121
5 Chapter 5 Experiments and results
Population data
122
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
African American
(n = 330)
Asian (n = 153)
U.S. Caucasian
(n = 343)
U.S. Hispanic
(n = 368)
9
10
11
12
6
7
8
13
14
15
D3S1358
8
9
10
11
12
13
14
D5S818
18
19
20
21
15
15.2
16
17
*
0.30*
6.21
1.97
7.27
25
35.45
21.82
1.67
0.30*
0.30*
32.42
22.27
6.06
0.61*
*
*
0.15*
0.61*
9.09
28.18
*
0.30*
*
*
*
1.63
0.33*
9.15
22.22
28.76
24.51
12.75
0.65*
*
*
*
*
*
21.9
19.61
6.54
0.33*
*
2.61
49.02
*
*
*
*
*
*
0.73
5.39
5.54
33.82
37.61
14.87
1.75
0.29*
*
24.34
19.68
11.66
1.46
*
*
*
*
*
0.29*
*
0.15*
15.16
27.26
*
26.9
17.93
9.92
0.82
*
*
*
0.14*
*
*
0.14*
0.41*
9.1
34.65
*
5.3
1.49
5.03
4.35
38.18
30.16
14.54
0.95
*
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
16
African American
(n = 330)
*
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
*
U.S. Hispanic
(n = 368)
*
17
18
19
*
*
*
*
*
*
*
*
*
*
*
*
D7S820
9
9.1
10
10.3
7
8
5
6
11
11.3
12
13
13.1
14
15
16
D8S1179
4
5
6
7
8
19.85
*
13.03
2.27
0.15*
0.15*
*
*
*
0.30*
0.45*
21.67
11.67
*
30.45
*
*
*
*
*
0.30*
31.37
*
20.26
2.61
*
0.33*
*
*
*
*
0.33*
16.99
7.52
0.33*
20.26
*
*
*
*
*
*
20.99
*
14.58
2.33
*
0.29*
0.15*
*
*
*
1.31
16.47
16.62
*
27.26
*
*
*
*
*
2.04
*
*
*
*
0.68
29.35
0.14*
19.02
3.94
*
0.41*
*
*
8.29
*
25.14
0.14*
*
*
1.09
12.5
GlobalFiler ™ Express PCR Amplification Kit User Guide 123
5 Chapter 5 Experiments and results
Population data
124
18
18.2
19
19.2
20
20.2
21
12.2
13
14
15
16
16.1
17
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
9
African American
(n = 330)
0.30*
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
1.31
U.S. Hispanic
(n = 368)
0.27*
14
15
16
17
10
11
12
13
18
19
20
3.33
5.61
11.36
18.18
35.91
18.03
5.91
1.06
*
*
*
9.8
9.48
13.4
24.18
15.69
22.22
4.25
0.98*
*
*
*
18.8
9.04
2.77
0.44*
10.5
6.71
15.16
33.24
*
*
*
9.51
5.03
12.5
33.15
23.23
11.41
3.53
0.68
*
*
*
FGA
0.91
0.61*
6.97
0.45*
6.82
0.30*
11.67
*
*
*
*
*
0.30*
*
3.27
*
4.25
*
3.92
*
13.07
*
*
*
*
0.33*
*
0.33*
1.02
*
5.69
*
14.87
0.44*
18.22
*
*
*
*
0.15*
*
0.15*
0.68
*
7.61
*
8.7
0.27*
13.45
*
*
*
*
*
*
*
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
21.2
African American
(n = 330)
0.15*
Asian (n = 153)
0.33*
U.S. Caucasian
(n = 343)
0.29*
U.S. Hispanic
(n = 368)
*
*
0.15*
0.15*
*
4.09
*
2.58
1.21
*
*
0.15*
17.27
0.15*
17.27
*
0.30*
18.94
*
9.55
*
*
*
*
*
*
*
29
30
30.2
31.2
26
26.2
27
28
32.2
33.2
34.2
23.3
24
24.2
25
22
22.2
23
23.2
42.2
43.2
44.2
45.2
46.2
47.2
48.2
*
*
*
*
*
*
*
3.27
*
0.65*
*
*
18.3
0.33*
9.8
14.38
*
27.12
0.65*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
1.9
*
0.58*
0.15*
19.24
0.87
14.87
0.44*
*
14.43
*
6.71
*
*
*
*
*
*
*
0.41*
0.14*
*
*
*
*
*
7.07
*
3.12
0.95
14.4
0.54*
12.91
0.41*
*
15.62
*
13.72
*
*
*
*
*
*
*
GlobalFiler ™ Express PCR Amplification Kit User Guide 125
5 Chapter 5 Experiments and results
Population data
126
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
50.2
African American
(n = 330)
*
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
*
U.S. Hispanic
(n = 368)
*
51.2
* * * *
SE33
14
14.2
14.3
15
15.2
12.1
12.2
13
13.2
9
11
11.2
12
4.2
5.2
6.3
8
16
16.2
16.3
17
17.2
17.3
18
3.33
0.15*
*
4.24
0.15*
*
0.30*
1.36
0.45*
*
*
0.76
0.15*
*
0.15*
*
*
6.97
0.30*
*
7.73
0.15*
*
10.76
*
*
*
1.31*
*
*
*
*
*
*
*
*
0.33*
*
*
*
*
*
*
4.9
3.59
*
*
5.23
3.64
*
0.15*
3.64
0.15*
0.15*
0.15*
0.87
*
*
*
*
0.44*
*
*
0.15*
*
5.39
*
*
6.56
*
0.15*
7.87
1.77
0.82
*
4.89
0.14*
*
0.14*
1.22
0.14*
*
*
0.14*
0.14*
*
*
*
*
5.57
0.27*
0.14*
8.7
*
*
10.05
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
18.2
African American
(n = 330)
0.15*
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
*
U.S. Hispanic
(n = 368)
0.27*
0.30*
1.67
2.42
0.15*
1.36
0.30*
0.61*
*
5.61
5.91
*
0.91
5.76
0.91
1.97
15
0.30*
*
9.55
3.94
2.58
1.21
*
1.06
*
0.76
24
24.2
25.2
26
22.2
23
23.2
23.3
26.2
27.2
27.3
20.2
21
21.2
22
19
19.2
19.3
20
28.2
29.2
30.2
31
31.2
32
32.2
0.33*
6.54
7.19
*
2.29
*
2.61
*
7.52
3.59
*
0.33*
6.21
1.63
2.61
9.48
*
*
6.86
7.84
8.5
7.52
*
1.63
*
1.31*
0.15*
4.52
3.79
*
3.35
*
2.62
*
4.52
6.85
*
0.87
2.04
1.17
0.58*
8.31
0.29*
0.29*
5.25
7.73
7.87
4.66
*
2.77
0.44*
1.6
0.14*
2.31
3.12
0.27*
2.31
*
2.85
0.14*
6.39
7.07
0.14*
0.82
3.12
1.09
1.09
8.15
*
*
4.48
6.25
5.84
3.8
0.14*
2.31
*
2.04
GlobalFiler ™ Express PCR Amplification Kit User Guide 127
5 Chapter 5 Experiments and results
Population data
128
8
9
6.1
7
9.3
10
5
6
3
4
11
13.3
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
33
African American
(n = 330)
*
Asian (n = 153)
*
U.S. Caucasian
(n = 343)
0.29*
U.S. Hispanic
(n = 368)
0.41*
35.2
36
37
38
33.2
34
34.2
35
*
*
*
*
0.45*
*
0.15*
*
0.33*
*
*
*
0.33*
*
*
*
*
0.15*
*
*
0.15*
0.29*
*
0.15*
*
*
0.14*
*
0.54*
0.41*
0.27*
*
TH01
*
*
0.45*
15.45
0.15*
37.42
20.61
16.06
8.33
1.52
*
*
*
*
*
13.07
*
26.14
3.59
51.63
4.25
1.31*
*
*
*
*
0.15*
21.72
*
17.64
11.37
17.06
31.2
0.87
*
*
*
*
*
27.17
*
32.74
8.7
12.77
17.12
1.49
*
*
TPOX
6
7
4
5 *
*
8.03
2.27
*
*
*
0.98*
*
*
0.15*
*
*
*
0.54*
0.14*
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
23
24
25
19
20
21
22
14
15
16
17
17.3
18
10
11
12
13
Table 6 Autosomal allele frequencies by population group for GlobalFiler Express
PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities) (continued)
Allele
8
African American
(n = 330)
35.91
Asian (n = 153)
49.35
U.S. Caucasian
(n = 343)
50.15
U.S. Hispanic
(n = 368)
47.83
13
14
15
16
9
10
11
12
19.09
9.55
21.67
3.33
0.15*
*
*
*
*
*
*
*
13.07
3.59
29.74
3.27
*
*
*
*
12.97
4.66
28.28
3.79
0.14*
0.14*
*
*
8.02
6.11
26.36
10.73
vWA
6.52
1.97
0.61*
*
0.15*
*
*
7.27
20.91
27.58
19.85
*
13.79
*
0.45*
*
0.91
*
*
*
9.15
0.98*
*
*
23.53
1.63
15.36
29.74
*
19.61
*
*
*
*
*
*
*
10.06
1.31
*
*
*
*
*
0.15*
8.75
12.24
22.3
27.41
*
17.78
*
*
*
6.39
0.82
*
*
*
0.14*
0.27*
0.14*
6.52
9.78
30.57
27.17
0.14*
18.07
GlobalFiler ™ Express PCR Amplification Kit User Guide 129
5 Chapter 5 Experiments and results
Population data
130
Table 7 Y-specific frequencies by population group for GlobalFiler ™ Express PCR Amplification Kit
DYS391 and Y indel loci. (*=Alleles not detected or not detected in significant quantities)
Allele
African American
(n = 246)
Asian (n = 65)
U.S. Caucasian
(n = 233)
U.S. Hispanic
(n = 182)
DYS391
6
7
8
9
10
11
12
13
14
Y indel
1
2
71.54
26.42
0.41*
0.41*
*
*
*
*
1.22
1.22
98.78
83.08
13.85
*
*
*
*
*
*
3.08*
67.69
32.31
44.64
51.93
1.72
*
*
*
*
*
1.72
*
100
0.55*
99.45
52.75
36.26
3.3
1.10*
*
*
*
*
6.59
Table 8 Probability of identity (P
I loci
) values for the GlobalFiler ™ Express PCR Amplification Kit STR
Locus
CSF1PO
D10S1248
D12S391
D13S317
D16S539
D18S51
D19S433
D1S1656
D21S11
D22S1045
D2S1338
African American
(n = 330)
0.0850
0.0693
0.0377
0.1451
0.0727
0.0322
0.0388
0.0340
0.0453
0.0559
0.0225
Asian (n = 153)
0.1317
0.1045
0.0664
0.0817
0.0915
0.0402
0.0663
0.0564
0.0671
0.1073
0.0337
U.S. Caucasian
(n = 343)
0.1333
0.0943
0.0231
0.0761
0.1043
0.0311
0.0862
0.0223
0.0520
0.1309
0.0316
U.S. Hispanic
(n = 368)
0.1353
0.1131
0.0318
0.0564
0.0809
0.0281
0.0484
0.0247
0.0487
0.1604
0.0316
GlobalFiler ™ Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results
Population data
5
Table 8 Probability of identity (PI) values for the GlobalFiler Express PCR Amplification Kit STR loci (continued)
Locus
D2S441
African American
(n = 330)
0.1030
Asian (n = 153)
0.0961
U.S. Caucasian
(n = 343)
0.0976
U.S. Hispanic
(n = 368)
0.1079
D3S1358
D5S818
D7S820
D8S1179
FGA
SE33
TH01
TPOX vWA
Combined
0.0984
0.0968
0.0784
0.0762
0.0322
0.0118
0.0949
0.0833
0.0622
6.18 × 10 −27
0.1689
0.0883
0.0875
0.0527
0.0555
0.0139
0.1750
0.1788
0.0840
3.34 × 10 −24
0.0749
0.1341
0.0680
0.0631
0.0384
0.0085
0.0801
0.1757
0.0650
3.71 × 10 −26
0.0949
0.1122
0.0790
0.0661
0.0282
0.0081
0.0902
0.1456
0.0926
3.09 × 10 −26
Probability of paternity exclusion observation
Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match Probability (MP), and p ‑ value of each locus was calculated using a software program developed by Ge (Li et al ., 2013) and shown in the following table.
Departures from Hardy-Weinberg Equilibrium (HWE) expectations of each locus were derived using Arlequin
(Excoffier et al ., 2010). After Bonferroni correction (Weir, 1990), (p-value = 0.05/21 = 0.0024), no departures from HWE were observed at any locus.
The average observed heterozygosity across the 21 autosomal STR loci was 0.815 in the African American population, 0.779 in the Asian population, 0.804 in the U.S. Caucasian population, and 0.789 in the Hispanic population. The most heterozygous locus was SE33 (mean observed heterozygosity across all populations of
0.948), and the least heterozygous STR locus was TPOX (mean observed heterozygosity across all populations of 0.652). The cumulative match probability (including the Y chromosome loci) was 2.17 × 10 −27
American, 2.26 × 10 −25 for Asian, 5.27 × 10 −27 for Caucasian, and 5.0 × 10 −27 for Hispanics.
for African
GlobalFiler ™ Express PCR Amplification Kit User Guide 131
Table 9 Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match probability (MP), and p-value of STR loci
African American Asian U.S. Caucasian U.S. Hispanic
Marker
Y indel
DYS391
Ho
—
—
He
—
—
MP
0.976
0.540
pvalue
—
—
Ho
—
—
He
-
—
MP
0.556
0.709
pvalue
—
—
Ho
—
—
He
—
—
MP
1.000
0.472
pvalue
—
—
Ho
—
—
He
—
—
MP
1.000
0.409
pvalue
—
—
D3S1358 0.772
0.762
0.094
0.902
0.686
0.682
0.151
0.012
0.753
0.786
0.079
0.698
0.698
0.758
0.098
0.291
vWA 0.752
0.798
0.068
0.116
0.829
0.784
0.080
0.309
0.841
0.807
0.064
0.621
0.842
0.782
0.080
0.463
D16S539 0.772
0.797
0.071
0.026
0.800
0.764
0.092
0.457
0.801
0.749
0.103
0.047
0.770
0.774
0.084
0.835
CSF1PO 0.772
0.773
0.086
0.020
0.743
0.721
0.125
0.128
0.721
0.720
0.131
0.745
0.727
0.710
0.137
0.619
TPOX 0.693
0.755
0.093
0.579
0.671
0.665
0.168
0.770
0.633
0.644
0.181
0.598
0.669
0.667
0.153
0.102
D8S1179 0.782
0.793
0.068
0.662
0.814
0.828
0.052
0.795
0.793
0.797
0.065
0.122
0.755
0.802
0.064
0.518
D21S11 0.861
0.849
0.039
0.553
0.800
0.791
0.069
0.667
0.873
0.837
0.046
0.385
0.827
0.839
0.044
0.315
D18S51 0.931
0.868
0.031
0.324
0.829
0.853
0.038
0.572
0.873
0.872
0.030
0.962
0.849
0.870
0.031
0.945
D2S441 0.772
0.756
0.099
0.421
0.714
0.746
0.101
0.238
0.757
0.766
0.090
0.077
0.791
0.763
0.094
0.611
D19S433 0.812
0.825
0.051
0.663
0.714
0.802
0.064
0.130
0.785
0.774
0.083
0.859
0.820
0.834
0.046
0.446
TH01
FGA
0.762
0.782
0.747
0.866
0.102
0.033
0.418
0.082
0.614
0.900
0.656
0.841
0.171
0.044
0.381
0.240
0.753
0.829
0.783
0.857
0.081
0.037
0.326
0.337
0.770
0.842
0.767
0.882
0.091
0.025
0.845
0.127
D22S104
5
0.842
0.822
0.055
0.062
0.743
0.742
0.112
0.966
0.705
0.714
0.131
0.026
0.698
0.672
0.162
0.064
D5S818 0.752
0.761
0.094
0.799
0.800
0.786
0.079
0.430
0.761
0.716
0.127
0.197
0.727
0.727
0.115
0.219
D13S317 0.762
0.695
0.138
0.217
0.786
0.799
0.069
0.327
0.769
0.777
0.081
0.718
0.799
0.815
0.059
0.855
D7S820 0.842
0.786
0.077
0.404
0.814
0.778
0.081
0.062
0.789
0.805
0.067
0.278
0.748
0.789
0.076
0.830
SE33 0.960
0.929
0.009
0.776
0.943
0.936
0.008
0.526
0.968
0.947
0.005
0.532
0.921
0.941
0.007
0.597
Table 9 Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match probability (MP), and p-value of STR loci (continued)
African American Asian U.S. Caucasian U.S. Hispanic
Marker
D10S124
8
Ho He MP pvalue
Ho He MP pvalue
Ho He MP pvalue
Ho He MP pvalue
0.792
0.789
0.075
0.823
0.757
0.764
0.091
0.928
0.785
0.769
0.090
0.630
0.691
0.724
0.124
0.336
D1S1656 0.921
0.863
0.033
0.351
0.757
0.818
0.056
0.043
0.912
0.899
0.019
0.550
0.871
0.896
0.020
0.048
D12S391 0.861
0.864
0.032
0.190
0.771
0.808
0.063
0.650
0.904
0.896
0.020
0.450
0.842
0.874
0.028
0.071
D2S1338 0.911
0.894
0.020
0.763
0.871
0.872
0.030
0.356
0.880
0.878
0.027
0.230
0.906
0.877
0.027
0.929
5 Chapter 5 Experiments and results
Population data
D21S11
D22S1045
D2S1338
D2S441
D3S1358
D5S818
D7S820
D8S1179
CSF1PO
D10S1248
D12S391
D13S317
D16S539
D18S51
D19S433
D1S1656
FGA
SE33
TH01
TPOX vWA
PEi
Combined
The following table shows the Probability of paternity exclusion (PE) values of the GlobalFiler ™ Express PCR
Amplification Kit STR loci individually and combined.
The PE value is the probability, averaged over all possible mother-child pairs, that a random alleged father will be excluded from paternity after DNA typing using the GlobalFiler ™ Express PCR Amplification Kit STR loci
(Chakraborty, Stivers, and Zhong, 1996).
Table 10 Probability of paternity exclusion values for the GlobalFiler ™ loci
Express PCR Amplification Kit STR
Locus
African American
(n = 330)
0.5878
0.6623
0.7401
0.4521
0.5548
0.7892
0.6332
0.7462
0.7280
0.7038
0.8140
0.5228
0.4918
0.4717
0.5767
0.5990
0.7280
0.8639
0.5124
0.4817
0.6103
2.0564 × 10 −10
0.9999999998
Asian (n = 153)
0.6063
0.4795
0.7463
0.5353
0.3976
0.5942
0.5942
0.6063
0.4904
0.5353
0.6310
0.6063
0.6063
0.6560
0.5238
0.5703
0.8397
0.8800
0.3424
0.3602
0.6186
2.7761 × 10 −09
0.9999999972
Caucasian (n = 343) Hispanic (n = 368)
0.7264
0.4507
0.7498
0.4986
0.5338
0.4839
0.5808
0.6187
0.4507
0.5649
0.8032
0.5544
0.5915
0.7557
0.5135
0.8032
0.6632
0.9231
0.5036
0.3435
0.6576
4.1986 × 10 −10
0.9999999996
0.7013
0.3970
0.7392
0.5051
0.4689
0.4959
0.5970
0.5381
0.4644
0.4644
0.6588
0.5770
0.5623
0.7121
0.6431
0.7338
0.7175
0.8781
0.5381
0.3620
0.6276
2.1709 × 10 −09
0.9999999978
134 GlobalFiler ™ Express PCR Amplification Kit User Guide
A
Troubleshooting
Observation
Faint or no signal from both the
DNA Control 007 and the DNA test samples at all loci
Possible cause Recommended action
The incorrect volume of Master
Mix or Primer Set was used.
The DNA Polymerase was not activated.
The Master Mix was not vortexed thoroughly before aliquoting.
The Primer Set was exposed to too much light.
Evaporation.
Use the correct volume of Master Mix or Primer
Set.
Repeat the amplification with an initial hold at
95°C for 1 minute.
Vortex the Master Mix thoroughly.
Replace the Primer Set and store it protected from light.
Ensure that the plate is properly sealed with film and that a compression pad was used with the
GeneAmp ™ PCR System 9700. (A compression pad should not be used with other validated thermal cyclers.)
See the thermal cycler user manual and check the instrument calibration.
Use correct thermal cycler conditions.
The thermal cycler malfunctioned.
Incorrect thermal cycler conditions were used.
A MicroAmp ™ base was used with a tray/retainer set and tubes in GeneAmp
9700.
™ PCR System
The tubes or plate were not seated tightly in the thermal cycler during amplification.
The wrong PCR reaction tubes or plate were used.
Remove the MicroAmp ™ base.
Push the tubes or plate firmly into the block after first cycle.
Use MicroAmp ™ Reaction Tubes with Caps or the MicroAmp ™ Optical 96 ‑ well Reaction Plate for the GeneAmp ™ PCR System 9700 or Veriti ™
Thermal Cycler.
Insufficient PCR product was electrokinetically injected.
Use correct genetic analyzer settings.
Degraded formamide was used.
Check the storage of formamide. Do not thaw and refreeze multiple times. Try Hi ‑ Di ™
Formamide.
GlobalFiler ™ Express PCR Amplification Kit User Guide 135
A Appendix A Troubleshooting
Population data
Observation
Faint no signal Control and samples all loci
Possible cause
The sample punch location was not optimal.
Positive signal from DNA Control
007 but partial or no signal from
DNA test samples
Insufficient volume of swab lysate was added to the reaction.
Proper low-TE buffer not used for treated paper substrates.
The test sample was diluted in the wrong buffer (for example, a
TE buffer with an incorrect EDTA concentration).
The sample punch location was not optimal.
More than two alleles present at a locus
Insufficient lysis of the swab head occurred.
Proper low-TE buffer not used for treated paper substrates.
Exogenous DNA is present in the sample.
Stutter product (–1 repeat unit position) was amplified.
Triallelic patterns occur.
Incomplete 3´ A base addition
(n−1 nt position) occured.
The signal exceeds the dynamic range of the instrument and is causing signal "pull-up" into adjacent channels.
Recommended action
For blood samples on treated paper, punch in the center of the blood stain.
For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal location you have previously determined.
For buccal samples collected with the Bode
Buccal DNA Collector ™ device, punch from near the tip of the collector.
Ensure the swab heads are incubated for
20 minutes in 400 µL of Prep ‑ n ‑ Go ™ Buffer.
Prepare low-TE buffer. See “Prepare low-TE buffer” on page 20
.
Redilute DNA using low-TE buffer (with 0.1 mM
EDTA).
For blood samples on treated paper, punch in the center of the blood stain.
For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal location you have previously determined.
For buccal samples collected with the Bode
Buccal DNA Collector ™ device, punch from near the tip of the collector.
Ensure the swab heads are incubated for
20 minutes in 400 µL of Prep ‑ n ‑ Go ™ Buffer.
Prepare low-TE buffer.
Use appropriate techniques to avoid introducing foreign DNA during laboratory handling.
See Chapter 5, “Experiments and results”
Confirm the triallelic pattern per the laboratory's guidelines.
Include the final extension step of 60°C for
10 minutes in the PCR.
Include the final extension step of 60°C for
10 minutes in the PCR.
Ensure the cycle number is optimized. Use fewer PCR cycles or interpret the off-scale data according to your laboratory procedure.
136 GlobalFiler ™ Express PCR Amplification Kit User Guide
Appendix A Troubleshooting
Population data
A
Observation
More than two alleles present at a locus
(continued)
Poor peak height balance
Some but not all loci visible on electropherogram of DNA Test
Samples
STR profiles contain many offscale alleles
Data collected on the
3730 instrument with POP-7 polymer fails sizing
Data collected on the
3730 instrument with POP-7 polymer: the D2S441 and
D1S1656 markers in some
™
™ allelic ladder samples fail basepair spacing quality assessment
Possible cause
Poor spectral separation occurred.
The double-stranded DNA was not completely denatured.
Contamination was carried over from the disc punching tool.
Data was analyzed using the
Light setting for Smoothing.
Recommended action
Perform a spectral calibration.
Confirm that Filter Set J6 modules are installed and used for analysis.
Use the recommended amount of Hi ‑ Di ™
Formamide and heat the sample plate at 95°C for 3 minutes.
Clean the disc punching tool thoroughly. If necessary, include a blank punch step in between the sample punches.
Use correct thermal cycler conditions.
Incorrect thermal cycler conditions were used.
The punched disc you used was too large.
Insufficient lysis of the swab head occurred.
The PCR reaction volume you used is lower than the volume required for the amplification.
The PCR cycle number used was too high.
Use a 1.2 mm disc.
Ensure the swab heads are incubated for
20 minutes in 400 µL of Prep ‑ n ‑ Go ™ Buffer.
Use the correct PCR reaction volume: 15 μL
Perform a sensitivity experiment to determine the optimal PCR cycle number based on the sample type.
Spot <100 µL of liquid blood per sample area.
Blood samples: Too much liquid blood was spotted onto the paper substrate.
The 60-bp size-standard peak is occasionally obscured by the primer peak.
Reinject samples that fail to recognize the
60 base-pair peak.
Use the 80 to 460 bp size-standard definition after performing appropriate validation studies
(as a general rule, the 60 base-pair peak is not required for accurate fragment sizing using the
3rd Order Least Squares sizing method).
For more information, see the GeneMapper ™
ID ‑ X Software v1.4 New Features and Installation Procedures User Bulletin
(Pub. No. 4477684), “Known issues: 3730 DNA
Analyzer sizing failures”.
Use the None setting for Smoothing after performing appropriate validation studies.
For more information, see the GeneMapper ™
ID ‑ X Software v1.4 New Features and Installation Procedures User Bulletin
(Pub. No. 4477684), “Known issues: 3730 DNA
Analyzer sizing failures”.
GlobalFiler ™ Express PCR Amplification Kit User Guide 137
B
Materials required but not supplied
■
■
■
■
■
■
Unless otherwise indicated, all materials are available through thermofisher.com
. "MLS" indicates that the material is available from fisherscientific.com
or another major laboratory supplier.
STR kit required materials
Item
GlobalFiler ™ Express PCR Amplification Kit, 200-reaction kit
GlobalFiler ™ Express PCR Amplification Kit, 1,000-reaction kit
GeneScan ™ 600 LIZ ™ Size Standard v2.0, 2 × 200 µL
IMPORTANT! Do not use GeneScan or GeneScan ™ 500 LIZ ™
™ 350 ROX ™ , GeneScan ™ 500 ROX
Size Standards with this kit.
™ ,
Hi ‑ Di ™ Formamide, 25 ‑ mL
Source
4476609
4474665
4408399
4311320
Sample preparation required materials
Treated paper substrate
Item
Collection system: NUCLEIC-CARD ™ system or Whatman FTA ™
NUCLEIC-CARD ™ Sample Collection Device
NUCLEIC-CARD ™ matrix, 1 spot
NUCLEIC-CARD ™ COLOR matrix, 1 spot
Source
A32607
4474001
4473974
138 GlobalFiler ™ Express PCR Amplification Kit User Guide
Appendix B Materials required but not supplied
Sample preparation required materials
B
(continued)
Item
Whatman ™ FTA ™ Classic Cards
Whatman ™ EasiCollect ™ system
Sample preparation:
Prep ‑ n ‑ Go ™ Buffer (for use with untreated paper substrates)
Low-TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0)
Punch tool:
Harris Micro-Punch ™ tool, 1.2
‑ mm
BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a
1.2
‑ mm punch head
BSD1000-GenePunch Automated Dried Sample Punch Instrument with a
1.2
‑ mm punch head
Untreated paper substrate
Item
Collection system: Bode or paper
Buccal DNA Collector ™ Device
903 paper
Punch tool:
Harris Micro-Punch ™ tool, 1.2
‑ mm
BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a
1.2
‑ mm punch head
BSD1000-GenePunch Automated Dried Sample Punch Instrument with a
1.2
‑ mm punch head
Swab substrate
Item
Collection system
4N6FLOQSwabs ™ , regular tip
Sample preparation:
Prep ‑ n ‑ Go ™ Buffer (for use with buccal swab substrates)
Source
MLS
MLS
4467079
Teknova T0223
MLS
Contact your local sales office.
Source
Contact Bode Cellmark Forensics
MLS
MLS
Contact your local sales office.
Source
4473979
4471406
GlobalFiler ™ Express PCR Amplification Kit User Guide 139
B Appendix B Materials required but not supplied
Thermal cycler required materials
(continued)
Item
96-well deep-well plate format
PrepFiler ™ 96-Well Processing Plates
Robbins Scientific ™ Model 400 Hybridization Incubator or equivalent
Agilent ™ Benchtop Rack for 200 µL Tubes/V Bottom Plates (metal) or equivalent
Source
Heated lysis protocol only: 1.5 mL tube format or 96-well deep-well plate format
1.5 mL tube format
1.5 mL tubes
Oven
MLS
VWR ™ Scientific dry heat block or equivalent
A47010
MLS
Agilent Technologies 410094
IMPORTANT! Do not use a plastic plate adaptor.
Thermal cycler required materials
Veriti
™
Thermal Cycler
Item
Veriti ™ 96 ‑ Well Thermal Cycler
( Optional ) Tabletop centrifuge with 96-Well Plate Adapters
GeneAmp
™
PCR System 9700
Item
GeneAmp ™ PCR System 9700, 96-Well Silver
GeneAmp ™ PCR System 9700, 96-Well Gold-Plated
Silver 96-Well Sample Block
Gold-Plated 96-Well Block
Source
4479071
MLS
Source
N8050001
4314878
N8050251
4314443
140 GlobalFiler ™ Express PCR Amplification Kit User Guide
Appendix B Materials required but not supplied
Genetic analyzer required materials
B
Genetic analyzer required materials
SeqStudio
™
Genetic Analyzer
Item
SeqStudio ™ Data Collection Software v1.2
SeqStudio ™ Genetic Analyzer Cartridge v2
SeqStudio ™ Genetic Analyzer Cathode Buffer Container
Reservoir Septa (for SeqStudio ™ Cathode Buffer Container)
SeqStudio ™ Integrated Capillary Protector
MicroAmp ™ Optical 96-Well Reaction Plate
MicroAmp ™ Optical 96-Well Reaction Plate with Barcode
MicroAmp ™ Optical 8-Tube Strip, 0.2 mL
Septa for SeqStudio ™ Genetic Analyzer, 96 well
Septa for SeqStudio ™ Genetic Analyzer, 8 strip
DS-36 Matrix Standard Kit (Dye Set J6)
3500 Series Genetic Analyzer
Item
3500 Series HID Data Collection Software v4.0.1
3500 Series Data Collection Software 3.1
3500 Series Data Collection Software 3
3500 Series Data Collection Software 2
HID Updater 3500 Data Collection Software v2
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
DS-36 Matrix Standard Kit (Dye Set J6)
Conditioning reagent
8-Capillary array, 36 cm for 3500 Genetic Analyzers
Source
A46085
4405187 [1] , A26287
4405186 [1]
4475183 [1]
4480670
4393927
4408256
4393710
4393715
4425042
4393718
4404683
Source
A46168
A41331
A33401
A35640
A31923
4316813
4326659
4316567
A36541
A36543
4425042
GlobalFiler ™ Express PCR Amplification Kit User Guide 141
B Appendix B Materials required but not supplied
Genetic analyzer required materials
(continued)
Item
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
[1] Contact your Thermo Fisher Scientific HID representative.
3130 Series Genetic Analyzer
Item
3130 Data Collection Software v4
3130 xl Data Collection Software ‑ v4
3130/3730 Data Collection Software ‑ v4 6 ‑ Dye Module v1
96 ‑ Well Plate Septa
Reservoir Septa
3100/3130 xl Genetic Analyzer Capillary Array, 36 ‑ cm
POP-4 ™ Polymer for 3130/3130 xl Genetic Analyzers
Running Buffer, 10 ✕
DS ‑ 36 Matrix Standard Kit (Dye Set J6)
MicroAmp ™ Optical 96-Well Reaction Plate
3730 Series Genetic Analyzer
Item
3730xl Data Collection Software 5
3730/3730 xl Data Collection Software ‑ v4
3130/3730 Data Collection Software v4 6 ‑ Dye Module v1
96 ‑ Well Plate Septa
Reservoir Septa
3730 DNA Analyzer 48-Capillary Array, 36-cm
142
Source
4475105
4475126
4480670
4315933
4315932
4315931
4352755
402824
4425042
N8010560
Source
4475133
4475154
4480670
4315933
4315932
4331247
Source
4404687
4410228
4410231
4410701
4412614
4410715
GlobalFiler ™ Express PCR Amplification Kit User Guide
Appendix B Materials required but not supplied
Analysis software required materials
B
(continued)
Item
POP-7 ™ Polymer for 3730/3730 xl DNA Analyzers
Running Buffer, 10 ✕
DS ‑ 36 Matrix Standard Kit (Dye Set J6)
MicroAmp ™ Optical 96-Well Reaction Plate
250 ‑ μL Glass Syringe (array-fill syringe)
5.0
‑ mL Glass Syringe (polymer-reserve syringe)
Analysis software required materials
GeneMapper
™
ID
‑
X Software
Item
GeneMapper ™ ID ‑ X Software v1.6 Full Installation
GeneMapper ™ ID ‑ X Software v1.6 Client Installation
GeneMapper ™ ID ‑ X Software v1.5 Full Installation
GeneMapper ™ ID ‑ X Software v1.5 Client Installation
GeneMapper ™ ID ‑ X Software v1.4 Full Installation
GeneMapper ™ ID ‑ X Software v1.4 Client Installation
Miscellaneous required materials
Plates and tubes
Item
MicroAmp ™ 96-Well Tray
MicroAmp ™ Reaction Tube with Cap, 0.2 mL
MicroAmp ™ 8-Tube Strip, 0.2 mL
MicroAmp ™ Optical 8-Cap Strips
MicroAmp ™ 96-Well Tray/Retainer Set
MicroAmp ™ 96-Well Base
Source
N8010541
N8010540
N8010580
4323032
403081
N8010531
Source
A39975
A39976
A27884
A27886
4479707
4479711
Source
4335611
4335613
4425042
N8010560
4304470
628 ‑ 3731
GlobalFiler ™ Express PCR Amplification Kit User Guide 143
B Appendix B Materials required but not supplied
Miscellaneous required materials
(continued)
Item
MicroAmp ™ Clear Adhesive Film
MicroAmp ™ Optical Adhesive Film
MicroAmp ™ Optical 96-Well Reaction Plate
Laboratory supplies
Item
Various procedures
Aerosol resistant pipette tips
Microcentrifuge tubes
Pipettors
Tape, labeling
Tube, 50-mL Falcon ™
Tube decapper, autoclavable
Deionized water, PCR grade
Vortex
[1] Major laboratory supplier
Source
4306311
4311971
N8010560
Source
MLS [1]
MLS
MLS
MLS
MLS
MLS
MLS
MLS
144 GlobalFiler ™ Express PCR Amplification Kit User Guide
C
Plate layouts
Example PCR plate layout
The following layout is recommended for use with the sensitivity experiment in the Perform PCR chapter. Create 3 identical plates for amplification at 3 different cycle numbers.
Example electrophoresis plate layout
The following layout is recommended for use with the sensitivity experiment in the Perform PCR chapter.
GlobalFiler ™ Express PCR Amplification Kit User Guide 145
D
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 this kit for:
• Forensic DNA testing, see "Forensic Laboratories: Handbook for Facility Planning, Design,
Construction, and Moving", National Institute of Justice, 1998
• Parentage DNA testing, see the "Guidance for Standards for Parentage Relationship Testing
Laboratories", American Association of Blood Banks, 7th edition, 2004
The sensitivity of this kit (and other PCR-based tests) enables amplification of minute quantities of DNA, necessitating precautions to avoid contamination of samples yet to be amplified (Kwok and Higuchi,
1989).
Process samples carefully to prevent contamination by human DNA. Wear gloves at all times and change them frequently. Close sample tubes when not in use. Limit aerosol dispersal by handling sample tubes and reagents carefully.
Note: We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology.
PCR setup work area materials
IMPORTANT! Do not remove these items from the PCR Setup Work Area.
• Calculator
• Gloves, disposable
• Marker pen, permanent
• Microcentrifuge
• Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate nuclease-free tube (for master mix preparation)
• Microcentrifuge tube rack
• Pipette tips, sterile, disposable hydrophobic filter-plugged
• Pipettors
146 GlobalFiler ™ Express PCR Amplification Kit User Guide
Appendix D PCR work areas
Amplified DNA work area
D
• Tube decapper, autoclavable
• Vortex
Amplified DNA work area
IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area.
Use only the validated thermal cyclers listed in “Instrument and software compatibility” on page 15
.
GlobalFiler ™ Express PCR Amplification Kit User Guide 147
E
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.
· Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain
SDSs, see the “Documentation and Support” section in this document.
148 GlobalFiler ™ Express PCR Amplification Kit User Guide
Appendix E Safety
Chemical safety
E
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING.
To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with 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.
AVERTISSEMENT ! PRÉCAUTIONS GÉNÉRALES EN CAS DE MANIPULATION DE PRODUITS
CHIMIQUES.
Pour minimiser les risques, veiller à ce que le personnel du laboratoire lise attentivement et mette en œuvre les consignes de sécurité générales relatives à l’utilisation et au stockage des produits chimiques et à la gestion des déchets qui en découlent, décrites ci-dessous.
Consulter également la FDS appropriée pour connaître les précautions et instructions particulières à respecter :
· Lire et comprendre les fiches de données de sécurité (FDS) fournies par le fabricant avant de stocker, de manipuler ou d’utiliser les matériaux dangereux ou les produits chimiques. Pour obtenir les FDS, se reporter à la section « Documentation et support » du présent document.
· Limiter les contacts avec les produits chimiques. Porter des équipements de protection appropriés lors de la manipulation des produits chimiques (par exemple : lunettes de sûreté, gants ou vêtements de protection).
· Limiter l’inhalation des produits chimiques. Ne pas laisser les récipients de produits chimiques ouverts. Ils ne doivent être utilisés qu’avec une ventilation adéquate (par exemple, sorbonne).
· Vérifier régulièrement l’absence de fuite ou d’écoulement des produits chimiques. En cas de fuite ou d’écoulement d’un produit, respecter les directives de nettoyage du fabricant recommandées dans la FDS.
· Manipuler les déchets chimiques dans une sorbonne.
GlobalFiler ™ Express PCR Amplification Kit User Guide 149
E Appendix E Safety
Biological hazard safety
· Veiller à utiliser des récipients à déchets primaire et secondaire. (Le récipient primaire contient les déchets immédiats, le récipient secondaire contient les fuites et les écoulements du récipient primaire. Les deux récipients doivent être compatibles avec les matériaux mis au rebut et conformes aux exigences locales, nationales et communautaires en matière de confinement des récipients.)
· Une fois le récipient à déchets vidé, il doit être refermé hermétiquement avec le couvercle fourni.
· Caractériser (par une analyse si nécessaire) les déchets générés par les applications, les réactifs et les substrats particuliers utilisés dans le laboratoire.
· Vérifier que les déchets sont convenablement stockés, transférés, transportés et éliminés en respectant toutes les réglementations locales, nationales et/ou communautaires en vigueur.
· IMPORTANT !
Les matériaux représentant un danger biologique ou radioactif exigent parfois une manipulation spéciale, et des limitations peuvent s’appliquer à leur élimination.
Biological hazard safety
WARNING! BIOHAZARD.
Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases.
Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL) , 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at: https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-
P.pdf
· World Health Organization, Laboratory Biosafety Manual , 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at: www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
150 GlobalFiler ™ Express PCR Amplification Kit User Guide
Documentation and support
Related documentation
Document title
STR kits
GlobalFiler ™ Express PCR Amplification Kit—PCR and CE Quick Reference
GlobalFiler ™ Express PCR Amplification Kit—PCR Setup—Swab Substrate Quick Reference
GlobalFiler ™ Express PCR Amplification Kit—PCR Setup—Treated Paper Substrate Quick
Reference
GlobalFiler ™
Reference
Express PCR Amplification Kit—PCR Setup—Untreated Paper Substrate Quick
Thermal cyclers
Veriti ™ Thermal Cycler User Guide
GeneAmp ™ PCR System 9700 Base Module User Manual
SeqStudio ™ Genetic Analyzer
SeqStudio ™ Genetic Analyzer Instrument and Software User Guide
SeqStudio ™ Genetic Analyzer for HID Instrument and Software v1.2.1 User Bulletin—New
Features and Developmental Validation
3500 Series Genetic Analyzer
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v1 User Guide
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v2 User Guide
HID Updater 3500 Data Collection Software v2.0 User Bulletin
3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software 3 User Guide
3500 Series Data Collection Software v3 User Bulletin: New Features and HID Validation
Summary
3500 Series Data Collection Software v3.1 User Bulletin: New Features and HID Validation
Summary
3500 Series Data Collection Software 4 User Bulletin: New Features and Developmental
Validation
Pub. No.
4480794
4477601
4480904
4480795
4375799
4303481
MAN0018646
100086084
4401661
4476988
NA
100025036
MAN0010812
MAN0014110
100075298
GlobalFiler ™ Express PCR Amplification Kit User Guide 151
Documentation and support
Related documentation
(continued)
Document title
3130 Series Genetic Analyzer
3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide
3130/3130xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin
3130/3130xl Genetic Analyzers Getting Started Guide
3130/3130xl Genetic Analyzers Quick Reference Card
3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide
3730 Series Genetic Analyzer
3730/3730xl Genetic Analyzer Getting Started Guide
3730xl Data Collection Software 5 for HID User Bulletin: New Features and Developmental
Validation
GeneMapper ™ ID ‑ X Software all versions
GeneMapper ™ ID ‑ X Software Bin Overlap User Bulletin
GeneMapper ™ ID ‑ X Software v1.3
GeneMapper ™ ID ‑ X Software v1.3 Verification Experiments and Installation Procedures User
Bulletin
GeneMapper ™ ID ‑ X Software v1.4
GeneMapper ™ ID ‑ X Software v1.4 New Features and Installation Procedures User Bulletin
GeneMapper ™ ID ‑ X Software v1.5
GeneMapper ™ ID ‑ X Software v1.5 New Features and Verification User Bulletin
GeneMapper ™ ID ‑ X Software v1.5 Getting Started Guide— Basic Features
GeneMapper ™ ID ‑ X Software v1.5 Quick Reference— Basic Features
GeneMapper ™ ID ‑ X Software v1.5 Getting Started Guide— Mixture Analysis Tool
GeneMapper ™ ID ‑ X Software v1.5 Quick Reference— Mixture Analysis Tool
GeneMapper ™ ID ‑ X Software v1.5 Installation Guide
GeneMapper ™ ID ‑ X Software v1.5 Administrator Guide
GeneMapper ™ ID ‑ X Software v1.5 Reference Guide
GeneMapper ™ ID ‑ X Software v1.6
GeneMapper ™ ID ‑ X Software v1.6 New Features and Software Verification User Bulletin
Pub. No.
4352716
4363787
4352715
4362825
4359472
4359476
MAN0019461
100029546
4470483
4477684
100031708
100031701
100031702
100031704
100031705
100031706
100031703
100031707
100073905
152 GlobalFiler ™ Express PCR Amplification Kit User Guide
Documentation and support
Customer and technical support
Customer and technical support
For support:
• In North America —Send an email to [email protected]
, or call
888-821-4443 option 1 .
• Outside North America —Contact your local support office.
For the latest services and support information for all locations, go to thermofisher.com/support to obtain the following information.
• Worldwide contact telephone numbers
• Product support
• Order and web support
• Safety Data Sheets (SDSs; also known as MSDSs)
Additional product documentation, including user guides and Certificates of Analysis, are available by contacting Customer Support.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the
Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/ global/terms-and-conditions.html
. If you have any questions, please contact Life Technologies at www.thermofisher.com/support .
GlobalFiler ™ Express PCR Amplification Kit User Guide 153
References
154
Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.
J. Forensic Sci.
39:362–372.
Bonferroni, C.E. 1936. Teoria statistica delle classi e calcolo Belle probabilita.
Publicazioni del R Istituto
Superiore di Scienze Economiche e Commerciali di Firenze 8:3–62.
Barber, M.D., McKeown, B.J. and Parkin, B.H. 1996. Structural variation in the alleles of a short tandem repeat system at the human alpha fibrinogen locus. Int. J. Leg. Med. 108:180–185.
Barber, M.D. and Parkin, B.H. 1996. Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179.
Intl. J. Legal Med.
109:62–65.
Barber, M.D., Piercy, R.C., Andersen, J.F. and Parkin, B.H. 1995. Structural variation of novel alleles at the Hum vWA and Hum FES/FPS short tandem repeat loci. Int. J. Leg. Med.
108:31–35.
Begovich A.B., McClure G.R., Suraj V.C., Helmuth R.C., Fildes N., Bugawan T.L., Erlich H.A., Klitz
W. 1992. Polymorphism, recombination, and linkage disequilibrium within the HLA class II region. J.
Immunol.
148:249–258.
Bender, K., Farfan, M.J., Schneider, P.M. 2004. Preparation of degraded human DNA under controlled conditions. Forensic Sci. Int.
139:134–140.
Brinkmann, B., Klintschar, M., Neuhuber, F., Huhne, J. and Rolf, B. 1998. Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat. Am. J. Hum. Genet.
62:1408–1415.
Brinkmann, B., Möller, A. and Wiegand, P. 1995. Structure of new mutations in 2 STR systems. Intl. J.
Legal Med.
107:201–203.
Budowle, B. et al. 2010. Population genetic analyses of the NGM ™ e-publication www.springerlink.com/content/p713q3w5440674u3/
STR loci. Int. J. Legal Med .
Butler, J.M. 2005. Forensic DNA Typing. Burlington, MA:Elsevier Academic Press.
Butler, J.M., Shen, Y., McCord, B.R. 2003. The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci.
48:1054–1064.
Chakraborty, R. Kimmel, M., Stivers, D., Davison, L., and Deka, R. 1997. Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc. Natl. Acad. Sci.
USA 94:1041–1046.
Chakraborty, R. and Stivers, D.N. 1996. Paternity exclusion by DNA markers: effects of paternal mutations. J. Forensic Sci.
41:671–677.
Chakraborty, R., Stivers, D., and Zhong, Y. 1996. Estimation of mutation rates from parentage exclusion data: applications to STR and VNTR loci. Mutat. Res.
354:41–48.
Chung, D.T., Drabek, J., Opel, K.L., Butler, J.M. and McCord, B.R. 2004. A study of the effects of degradation and template concentration on the amplification efficiency of the Miniplex primer sets. J.
Forensic Sci.
49:733–740.
GlobalFiler ™ Express PCR Amplification Kit User Guide
References
Limited product warranty
Clark J.M. 1988. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res.
16:9677–9686.
Coble, M.D. and Butler, J.M. 2005. Characterization of new miniSTR loci to aid analysis of degraded
DNA. J. Forensic Sci. 50:43–53.
DeFranchis, R., Cross, N.C.P., Foulkes, N.S., and Cox, T.M. 1988. A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA. Nucleic Acids Res.
16:10355. DNA Advisory Board,
Federal Bureau of Investigation, U.S. Department of Justice. 1998. Quality assurance standards for forensic DNA testing laboratories.
Drabek, J., Chung, D.T., Butler, J.M., McCord, B.R. 2004. Concordance study between Miniplex assays and a commercial STR typing kit. J. Forensic Sci.
49:859–860.
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.
49:746–756.
Edwards, A., Hammond, H.A., Lin, J., Caskey, C.T., and Chakraborty, R. 1992. Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12:241–253.
Excoffier, L., Lischer, H.E.L. 2010. A new series of programs to perform population genetics analyses under Linux and Windows. Arleguin suite v. 3.5. Mol. Ecol. Res.
10:564–567.
Frank, W., Llewellyn, B., Fish, P., et al. 2001. Validation of the AmpFℓSTR ™
Amplification Kit for use in forensic casework. J. Forensic Sci.
46:642–646.
Profiler Plus ™ PCR
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.
90:197–203.
Grossman, P.D., Bloch, W., Brinson, E., Chang, C.C., Eggerding, F.A., Fung, S., Iovannisci, D.M., Woo,
S., Winn-Deen, E.S. 1994. High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation. Nucleic Acids Res. 22:4527–4534.
Grubwieser, P. Muhlmann, R., Berger, B., Niederstatter, H., Palvic, M., Parson, W. 2006. A new “mini-
STR-multiplex” displaying reduced amplicon lengths for the analysis of degraded DNA. Int. J. Legal
Med.
120:115–120.
Guo, S.W. and Thompson, E.A. 1992. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48:361–372.
Hammond, H., Jin, L., Zhong, Y., Caskey, C., and Chakraborty, R. 1994. Evaluation of 13 short tandem repeat loci for use in personal identification applications. Am J. Hum. Genet.
55:175–189.
Holt, C., Stauffer, C., Wallin, J., et al. 2000. Practical applications of genotypic Surveys for forensic STR testing. Forensic Sci. Int.
112:91–109.
Kalinowski, S.T. 2006. HW-QuickCheck: an easy-to-use computer program for checking genotypes for agreement with Hardy-Weinberg expectations. Molecular Ecology Notes 6:974–979.
Kimpton, C., Walton, A., and Gill, P. 1992. A further tetranucleotide repeat polymorphism in the vWF gene. Hum. Mol. Genet. 1:287. Kong, X., Murphy, K., Raj, T., He, C., White, P.S., Matise, T.C. 2004. A combined linkage-physical map of the human genome. Am. J. Hum. Genet.
75:1143–1148.
Kwok, S., and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237–238.
Lareu, M.V., Pestoni, M.C., Barros, F., Salas, A., Carracedo, A. 1996. Sequence variation of a hypervariable short tandem repeat at the D12S391 locus. Gene 182:151–153.
GlobalFiler ™ Express PCR Amplification Kit User Guide 155
References
Limited product warranty
Lazaruk, K., Walsh, P.S., Oaks, F., Gilbert, D., Rosenblum, B.B., Menchen, S., Scheibler, D., Wenz, H.M.,
Holt, C., Wallin, J. 1998. Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument. Electrophoresis 19:86–93.
Levene, H. 1949. On a matching problem in genetics. Ann. Math. Stat.
20:91–94.
Li, B., Ge, J., Wu, F., Ye, L., Budowle, B., Vhen, Y. 2013. Population genetic analyses of the STR loci of the AmpFℓSTR ™ NGM SElect ™
Legal Med.
127:345–346.
PCR Amplification Kit for Han population in Fujian Province, China. Int J.
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. 2:1327.
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.
Möller, A. and Brinkmann, B. 1995. PCR-VNTRs (PCR-Variable Number of Tandem Repeats) in forensic science. Cellular & Molec. Bio. 41(5):715-724. Momhinweg, E., Luckenbach, C., Fimmers, R., and Ritter,
H. 1998. D3S1358: sequence analysis and gene frequency in a German population. Forensic Sci. Int.
95:173–178.
Moretti, T., Baumstark, A., Defenbaugh, D., Keys, K., Smerick, J., and Budowle, B. 2001. Validation of short tandem repeats (STRs) for forensic usage: Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. J. Forensic Sci. 46(3):647–660.
Mulero, J.J., Chang, C.W., and Hennessy, L.K. 2006. Characterization of N+3 stutter product in the trinucleotide repeat locus DYS392. J. Forensic Sci.
51:826–830.
Nakahori, Y., Takenaka, O., and Nakagome, Y. 1991. A human X-Y homologous region encodes amelogenin. Genomics 9:264–269.
Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA 70:3321–
3323. Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590.
Puers C., Hammond H.A., Jin L., Caskey C.T., Schumm J.W. 1993. Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTH01[AATG]n and reassignment of alleles in population analysis by using a locus-specific allelic ladder. Am J. Hum. Genet.
53(4):953–958.
Raymond M. and Rousset F. 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism.
J. Heredity 86:248–249.
Rousset, F. 2008. Genepop'007: A complete reimplementation of the Genepop software for Windows and Linux. Molecular Ecology Resources 8:103–106.
Scientific Working Group on DNA Analysis Methods (SWGDAM). 2012.
Validation Guidelines for DNA Analysis Methods. Available at http://swgdam.org/
SWGDAM_Validation_Guidelines_APPROVED_Dec_2012.pdf. Accessed 29 July 2013.
156 GlobalFiler ™ Express PCR Amplification Kit User Guide
References
Limited product warranty
Sensabaugh, G.F. 1982. Biochemical markers of individuality. In: Saferstein, R., ed. Forensic Science
Handbook. Prentice-Hall, Inc., New York, pp. 338–415.
Sharma, V. and Litt, M. 1992. Tetranucleotide repeat polymorphism at the D21S11 locus. Hum Mol.
Genet.
1:67.
Shin, C.H., Jang, P., Hong, K.M., Paik, M.K. 2004. Allele frequencies of 10 STR loci in Koreans. Forensic
Sci. Int. 140:133–135.
Smith, R.N. 1995. Accurate size comparison of short tandem repeat alleles amplified by PCR.
Biotechniques 18:122–128.
Sparkes, R., Kimpton, C., Watson, S., Oldroyd, N., Clayton, T., Barnett, L., Arnold, J., Thompson, C.,
Hale, R., Chapman, J., Urquhart, A., and Gill, P. 1996a. The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies. Int. J. Legal Med.
109:186–194.
Sparkes, R., Kimpton, C., Gilbard, S., Carne, P., Andersen, J., Oldroyd, N., Thomas, D., Urquhart, A., and Gill, P. 1996b. The validation of a 7-locus multiplex STR test for use in forensic casework. (II),
Artifacts, casework studies and success rates. Int. J. Legal Med.
109:195–204.
Straub, R.E., Speer, M.C., Luo, Y., Rojas, K., Overhauser, J., Ott, J., and Gilliam, T.C. 1993. A microsatellite genetic linkage map of human chromosome 18. Genomics 15:48– 56.
Szibor, R., Lautsch, S., Plate, I., Bender, K., Krause, D. 1998. Population genetic data of the STR
HumD3S1358 in two regions of Germany. Int. J. Legal Med.
111(3):160–161.
Waiyawuth, W., Zhang, L., Rittner, C., Schneider, P.M. 1998. Genetic analysis of the short tandem repeat system D12S391 in the German and three Asian populations. Forensic Sci. Int. 94:25–31.
Wallin, J.M., Buoncristiani, M.R., Lazaruk, K.D., Fildes, N., Holt, C.L., Walsh, P.S. 1998. SWGDAM validation of the AmpFlSTR blue PCR amplification kit for forensic casework analysis.
J. Forensic Sci.
43:854–870.
Wallin, J.M., Holt, C.L., Lazaruk, K.D., Nguyen, T.H., Walsh, P.S. 2002. Constructing universal multiplex
PCR systems for comparative genotyping. J. Forensic Sci. 47:52–65.
Walsh, P.S., Fildes, N.J., Reynolds, R. 1996. Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res. 24:2807– 2812.
Watson, S., Kelsey, Z., Webb, R., Evans, J., and Gill, P. 1998. The development of a third generation
STR multiplex system (TGM). Olaisen, B., Brinkmann, B., and Lincoln, P.J., eds. Progress in Forensic
Genetics 7: Proceedings of the 17th International ISFH Congress, Oslo 2-6 September 1997 . Elsevier,
Amsterdam, pp. 192–194.
Weber, J. and Wong, C. 1993. Mutation of human short tandem repeats. Hum. Mol. Genet. 2:1123–
1128.
Weir, B. 1990. Genetic Data Analysis. Sinauer Associates Sunderland, MA
Wiegand, P. and Kleiber, M. 2001. Less is more—length reduction of STR amplicons using redesigned primers. Int. J. Legal Med.
114:285–287.
GlobalFiler ™ Express PCR Amplification Kit User Guide 157
Index
3130/3130xl instrument, catalog numbers 142
3730/3730xl instrument, 6-dye license 36
4N6FLOQSwabs sample collectors 110
6-dye
license activation for 3730/3730xl instrument 36
spectral calibration 31, 35, 38, 40
A
accuracy and reproducibility 66
allelic ladder, requirements for electrophoresis 29
allelic ladder, volume per reaction 41
B
C
characterization of loci, validation 102
control DNA
D
DS-36 matrix standard 31, 35, 38, 40 dye set for 6-dye samples 31, 35, 38, 40
E electrophoresis
data collection software 32, 36 references 32, 36 run module 32, 36
setup of the 3130 and 3130xl instruments 36
setup of the 3500 and 3500xL instruments 32
F
G
GeneScan 600 LIZ Size Standard v2.0 57
GeneScan size standard, about 11
H
I
import panels, bins, and marker stutter 45
instrument and software compatibility 15
L
LIZ size standard
M
158 GlobalFiler ™ Express PCR Amplification Kit User Guide
P panels
PCR
R
run module for electrophoresis, 3500 and 3500xL in‐
run module, electrophoresis, 3130 and 3130xl 36
S
spectral calibration 31, 35, 38, 40
swab
sample preparation guidelines 24
T
thermal cyclers
treated paper
sample preparation guidelines 20
U
V
W
Index
GlobalFiler ™ Express PCR Amplification Kit User Guide 159
GlobalFiler Express UG_4477672-v8-GUID-81AE5413-DFFD-4C75-AD51-7EE6E666D84C-2020/10/07
23:49:44 en
01:23:14.332+01:00 thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com
13 October 2020
Advertisement