For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures. GlobalFiler™ Express PCR Amplification Kit USER GUIDE Catalog Numbers 4476609 and 4474665 Publication Number 4477672
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...
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.
GlobalFilerTM 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
Date
G
13 October 2020
F
09 June 2020
E
21 December 2016
D
06 October 2016
C
May 2014
B
April 2014
A
October 2012
Description
Add the SeqStudioTM 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 GeneMapperTM IDX 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
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Kit overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Single-source sample types supported . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Substrate examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 About the primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Dyes used in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Loci amplified by the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Standards and controls that are required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Allelic ladder profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 DNA Control 007 profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Instrument and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
CHAPTER 2 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Optimize PCR cycle number (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Procedural guidelines when optimizing PCR cycle number . . . . . . . . . . . . . . . . . . . . . . 18 Select samples and prepare plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Determine optimum PCR conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Thaw reagents and prepare Master Mix (before first use of the kit) . . . . . . . . . . . . . . . 19
Treated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . 20 Sample preparation guidelines: treated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . 20 Prepare low-TE buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Prepare the amplification kit reactions: treated paper substrate . . . . . . . . . . . . . . . . . . 21
Untreated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . 22 Sample preparation guidelines: untreated paper substrate . . . . . . . . . . . . . . . . . . . . . . 22 Prepare the amplification kit reactions: untreated paper substrate . . . . . . . . . . . . . . . . 23
Swab substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Sample preparation guidelines: swab substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Prepare the sample lysate: room temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Prepare the sample lysate: heat protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
GlobalFilerTM Express PCR Amplification Kit User Guide
3
Contents
Prepare the reactions: swab substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Store the sample lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
CHAPTER 3 Perform electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Set up the SeqStudioTM instruments for electrophoresis (before first use of the kit) . . . . . . 30
Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . . 32 Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Obtain and run the HID Updater (v1 and v2 software only) . . . . . . . . . . . . . . . . . . . . . . 33 Modify 3500 QC protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) . . . . . . 36 Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Obtain and activate 6-dye license . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit) . . . . . . 38 Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Obtain and activate the 6-dye license . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Prepare samples for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
CHAPTER 4 Analyze data with GeneMapperTM IDX Software . . . . . . . . . . . . . . . . 42
Overview of GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Set up the GeneMapperTM IDX Software for analysis (before first use of the kit) . . . . . . . . 44
Workflow: Set up GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Check panel, bin, and stutter file versions on your computer . . . . . . . . . . . . . . . . . . . . 44 (If needed) Download newer versions of panel, bin, and stutter files . . . . . . . . . . . . . . 45 Import panels, bins, and marker stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 (Optional) Define custom table or plot settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Enter Analysis Method settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 About the GS600_LIZ_ (60460) size standard definition file . . . . . . . . . . . . . . . . . . . . 57 If you use POP-7TM polymer on a 3730 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Create a size standard definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Analyze and edit sample files with GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . 60
4
GlobalFilerTM Express PCR Amplification Kit User Guide
Contents
Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 For more information on using the GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . 61
CHAPTER 5 Experiments and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
SWGDAM guideline 2.2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 SWGDAM guideline 3.9.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 PCR components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Thermal cycling temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 SWGDAM guideline 3.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Accuracy observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Precision and size window description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Precision observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Causes of extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Extra peaks: Stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Extra peaks: Addition of 3' A nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Extra peaks: Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 SWGDAM guideline 3.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Loci in this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Nature of polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Genetic linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 SWGDAM Guideline 3.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Nonhuman study observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 SWGDAM guideline 3.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Sample collection factors that can affect DNA quantity . . . . . . . . . . . . . . . . . . . . . . . . 105 Effect of DNA quantity on results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Sensitivity observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
GlobalFilerTM Express PCR Amplification Kit User Guide
5
Contents
Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 SWGDAM guideline 3.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 DNA on FTATM cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 DNA on 4N6FLOQSwabsTM sample collectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 SWGDAM guideline 3.7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Population data overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Loci in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Population samples used in these studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Concordance studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Probability of Identity definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Probability of identity observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Probability of paternity exclusion observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
APPENDIX B Materials required but not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
STR kit required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Sample preparation required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Treated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Untreated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Swab substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Thermal cycler required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 VeritiTM Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 GeneAmpTM PCR System 9700 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Genetic analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 SeqStudioTM Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 3500 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 3130 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 3730 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 Analysis software required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Miscellaneous required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Plates and tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Laboratory supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
APPENDIX C Plate layouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Example PCR plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Example electrophoresis plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
6
GlobalFilerTM Express PCR Amplification Kit User Guide
Contents
APPENDIX D PCR work areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
References
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
GlobalFilerTM Express PCR Amplification Kit User Guide
7
1
Product information
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Instrument and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
IMPORTANT! Before using this product, read and understand the information in the "Safety" appendix in this document.
Product description
Kit overview
The Applied BiosystemsTM GlobalFilerTM 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 GlobalFilerTM 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 GlobalFilerTM 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.
8
GlobalFilerTM Express PCR Amplification Kit User Guide
1 Chapter 1 Product information Product description
Single-source sample types supported
The GlobalFilerTM 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 PrepnGoTM
Buffer. · Buccal samples collected on swab substrates and treated with PrepnGoTM Buffer.
Substrate examples
· Treated paper: NUCLEIC-CARDTM system or Whatman FTATM cards · Untreated paper: Bode Buccal DNA CollectorTM or 903 paper · Swab: FLOQSwabsTM 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 GlobalFilerTM Express PCR Amplification Kit primers are manufactured using the same synthesis and purification improvements as the primers in the NGM SElectTM and the IdentifilerTM Plus kits. These improvements enhance the assay signaltonoise 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 SElectTM kit and the IdentifilerTM 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 SElectTM kit (which are different from the IdentifilerTM Plus kit).
The GlobalFilerTM 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
9
1 Chapter 1 Product information Product description
Dyes used in the kit
Dye 6FAMTM
VICTM NEDTM TAZTM SIDTM LIZTM
Color Blue Green Yellow Red Purple Orange
Label Samples, allelic ladders, and controls
GeneScanTM 600 LIZTM Size Standard v2.0
Loci amplified by the kit
Table 1 GlobalFilerTM Express PCR Amplification Kit loci and alleles
Locus designation
D3S1358 vWA
D16S539 CSF1PO
TPOX Y indel Amelogenin
D8S1179 D21S11
D18S51
DYS391 D2S441 D19S433
TH01
Chromosome location
Alleles included in Allelic Ladder
3p21.31 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
12p13.31
11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24
16q24.1 5, 8, 9, 10, 11, 12,13, 14, 15
5q33.3-34 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
2p23-2per 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
Yq11.221 1, 2
X: p22.1-22.3 X, Y Y: p11.2
8q24.13 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
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, 36, 37, 38
18q21.33
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
Yq11.21 7, 8, 9, 10, 11, 12, 13
2p14
8, 9, 10, 11, 11.3, 12, 13, 14, 15, 16, 17
19q12
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
11p15.5 4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3
Dye label 6-FAMTM
VICTM
NEDTM
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
GlobalFilerTM Express PCR Amplification Kit User Guide
1 Chapter 1 Product information Product description
Table 1 GlobalFiler Express PCR Amplification Kit loci and alleles (continued)
Locus
Chromosome
designation
location
Alleles included in Allelic Ladder
Dye label
FGA
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
NEDTM
D22S1045
22q12.3 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
TAZTM
D5S818
5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18
D13S317
13q22-31 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
D7S820
7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
SE33
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
D10S1248
10q26.3 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
SIDTM
D1S1656
1q42.2
9, 10, 11, 12, 13, 14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18.3, 19.3, 20.3
D12S391
12p13.2
14, 15, 16, 17, 18, 19, 19.3, 20, 21, 22, 23, 24, 25, 26, 27
D2S1338
2q35
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28
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 GlobalFilerTM 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 GlobalFilerTM Express Allelic Ladder. DNA Control 007 is present in the kit. See "DNA Control 007 profile" on page 13.
· GeneScanTM 600 LIZTM Size Standard v2.0--Used for obtaining sizing results. This standard, which has been evaluated as an internal size standard, yields precise sizing results for PCR products. Order the GeneScanTM 600 LIZTM Size Standard v2.0 (Cat. No. 4408399) separately.
· GlobalFilerTM 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
11
1
Chapter 1 Product information Product description
Allelic ladder profile
Figure 1 GeneMapperTM IDX Software plot of the GlobalFilerTM Express Allelic Ladder
12
GlobalFilerTM Express PCR Amplification Kit User Guide
DNA Control 007 profile
1 Chapter 1 Product information Product description
Figure 2 DNA Control 007 (1 ng) amplified with the GlobalFilerTM Express PCR Amplification Kit and analyzed on an Applied BiosystemsTM 3500xL Genetic Analyzer (Y-axis scale 0 to 8,000 RFU).
GlobalFilerTM Express PCR Amplification Kit User Guide
13
1 Chapter 1 Product information Contents and storage
Contents and storage
The GlobalFilerTM 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 L/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 GlobalFilerTM Express Master Mix
Master Mix Additive
GlobalFilerTM Express Primer Set
GlobalFilerTM Express Allelic Ladder
Description
200 reactions (Cat. No. 4476609)
1,000 reactions (Cat. No. 4474665)
Storage
Contains enzyme, salts, dNTPs, bovine serum albumin, enzyme, and 0.05% sodium azide in buffer and salt.
1 × 1.13 mL
1 × 5.64 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).
Reagent for one-time addition to the GlobalFilerTM Express Master Mix following first thaw.
1 × 0.1 mL
1 × 0.45 mL
-25°C to -15°C on receipt.
Discard the tube after adding to the master mix.
Contains forward and reverse primers to amplify human DNA targets.
1 × 1.2 mL
1 × 6 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.
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, up to the expiration date stated on the kit.
Store protected from light.
14
GlobalFilerTM 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
· ProFlexTM 96well PCR System (Cat. No. 4484075) · ProFlexTM 2 × 96well PCR System (Cat. No. 4484076) · VeritiTM 96Well Thermal Cycler (Cat. No. 4479071) · GeneAmpTM PCR System 9700, 96-Well Silver (Cat. No. N8050001) · GeneAmpTM PCR System 9700, 96-Well Gold-Plated (Cat. No. 4314878)
IMPORTANT! GlobalFilerTM Express PCR Amplification Kit is NOT validated for use with:
· VeritiTM Fast 96Well Thermal Cycler (Cat. No. 4375305) · GeneAmpTM PCR System 9700, 96-Well Aluminum (Cat. No. 4314879)
GlobalFilerTM 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 (WindowsTM Vista operating system) and HID Updater 3500 Data Collection Software v2 (Cat. No. 4480670) · 3500 Series Data Collection Software 2 (WindowsTM 7 operating system) and HID Updater 3500 Data Collection Software v2 (Cat. No. 4480670) · 3500 Series Data Collection Software 3 (WindowsTM 7 operating system) · 3500 Series Data Collection Software 3.1 (WindowsTM 7 operating system) · 3500 Series Data Collection Software 4 (WindowsTM 10 operating system) · 3500 Series HID Data Collection Software v4.0.1 (WindowsTM 10 operating system)
3130/3130xl Genetic Analyzer · 3130 Series Data Collection Software 4 (WindowsTM 7 operating system) · 3130/3730 Data Collection 4 6-Dye Module v1
3730/3730xl DNA Analyzer · 3730 Series Data Collection Software 4 (WindowsTM 7 operating system) · 3730 Series Data Collection Software 4 6-Dye Module v1 · 3730xl Data Collection Software 5 (WindowsTM 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)
SeqStudioTM Genetic Analyzer · SeqStudioTM Data Collection Software v1.2 · SeqStudioTM Data Collection Software v1.2.1
Analysis software
GeneMapperTM IDX Software v1.4 or later WindowsTM XP, WindowsTM 7, or WindowsTM 10 operating system
[1] We conducted validation studies using the 3130xl, 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
GlobalFilerTM Express PCR Amplification Kit User Guide
1 Chapter 1 Product information Workflow
Workflow
Perform PCR on treated or untreated paper substrates
Perform PCR on swab substrates
"Prepare the amplification kit reactions: treated paper substrate" on page 21 or
"Prepare the amplification kit reactions: untreated paper substrate" on page 23
"Prepare the reactions: swab substrate" on page 26
Obtain punch with Harris Manual Punch or BSD SemiAutomated Dried Sample Punch Instrument
Untreated paper only: Process with PrepnGoTM Buffer
Lyse in PrepnGoTM Buffer
Process with the GlobalFilerTM Express PCR Amplification Kit
Process with the GlobalFilerTM Express PCR Amplification Kit
Amplify with a recommended thermal cycler
Perform electrophoresis "Set up the SeqStudioTM 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 GeneMapperTM IDX 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 GeneMapperTM IDX Software" on page 60
"Examine or edit a project" on page 61
GlobalFilerTM Express PCR Amplification Kit User Guide
17
2
Perform PCR
Optimize PCR cycle number (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Treated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Untreated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . 22 Swab substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Optimize PCR cycle number (before first use of the kit)
Before using the GlobalFilerTM 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 instrumenttoinstrument and sampletosample 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 96well plate. This minimizes the impact of runtorun 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 instrumenttoinstrument 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
GlobalFilerTM Express PCR Amplification Kit User Guide
2 Chapter 2 Perform PCR Before you begin
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 offscale allele peaks:
Instrument 3500 Series 3130 Series 3730 Series SeqStudioTM Genetic Analyzer
Heterozygous peak height 3,00012,000 RFU 1,0003,000 RFU 3,00012,000 RFU 3,00012,000 RFU
When amplifying singlesource, unpurified samples, you will see greater sampletosample 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.
GlobalFilerTM 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
GlobalFilerTM 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.
If this is the first time you are using the kit, follow the instructions in "Thaw reagents and prepare Master Mix (before first use of the kit)" on page 19 before proceeding.
1. Add samples to the MicroAmpTM Optical 96well 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 L of Control DNA 007 2 L of Control DNA 007 1 L 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 L 6.0 L 3.0 L
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-L 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 MicroAmpTM Optical 96-Well Reaction Plate.
GlobalFilerTM 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 MicroAmpTM Clear Adhesive Film (Cat. No. 4306311) or MicroAmpTM Optical Adhesive Film (Cat. No. 4311971).
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 GeneAmpTM PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmpTM 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 GeneAmpTM 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 CollectorTM, make
1
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.
· 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 CollectorTM
22
GlobalFilerTM 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.
If this is the first time you are using the kit, follow the instructions in "Thaw reagents and prepare Master Mix (before first use of the kit)" on page 19 before proceeding.
1. Add PrepnGoTM Buffer (Cat. No. 4467079) to the MicroAmpTM Optical 96-Well Reaction Plate:
To these wells ... Negative control Test samples Positive control
Add...
3 L of PrepnGoTM Buffer
3 L of PrepnGoTM Buffer
For 25 and 26 cycles 0 L of PrepnGoTM Buffer
For 27 cycles
1 L of PrepnGoTM Buffer
For 28 cycles
2 L of PrepnGoTM 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 L of Control DNA 007 2 L of Control DNA 007 1 L 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 PrepnGoTM 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 L 6.0 L
GlobalFilerTM 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-L 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 MicroAmpTM Optical 96-Well Reaction Plate. The final volume in each well is 15 µL (reaction mix plus PrepnGoTM Buffer and sample or positive control).
8. Seal the plate with MicroAmpTM Clear Adhesive Film (Cat. No. 4306311) or MicroAmpTM Optical Adhesive Film (Cat. No. 4311971). 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 GeneAmpTM PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmpTM 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 GeneAmpTM 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 (VWRTM Scientific Select dry heat block or similar) PrepFilerTM 96-Well Processing Plates (Cat. No. A47010) Robbins ScientificTM Model 400 Hybridization Incubator or similar
24
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR Swab substrates: prepare the amplification kit reactions
2
AgilentTM Benchtop Rack for 200 L 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 PrepnGoTM Buffer (Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a PrepFilerTM 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 deepwell 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 PrepnGoTM Buffer (for buccal swabs, Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a PrepFilerTM 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 deepwell 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 deepwell 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.
GlobalFilerTM 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.
If this is the first time you are using the kit, follow the instructions in "Thaw reagents and prepare Master Mix (before first use of the kit)" on page 19 before proceeding.
1. Add PrepnGoTM Buffer (Cat. No. 4471406) to the control wells in the MicroAmpTM Optical 96-Well Reaction Plate:
To these wells ... Negative control Positive control
Add...
3 L of PrepnGoTM Buffer
For 25 and 26 cycles 0 L of PrepnGoTM Buffer
For 27 cycles
1 L of PrepnGoTM Buffer
For 28 cycles
2 L of PrepnGoTM 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 L 6.0 L
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-L 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 L of the reaction mix into each reaction well of a MicroAmpTM Optical 96-Well Reaction Plate. The final volume in each well is 15 L (reaction mix plus PrepnGoTM Buffer or sample lysate or positive control).
26
GlobalFilerTM 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 MicroAmpTM Optical 96Well Reaction Plate...
Add...
Test samples
3 L of sample lysate
Positive control
For 25 and 26 cycles
3 L of Control DNA 007
For 27 cycles
2 L of Control DNA 007
For 28 cycles
1 L 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 MicroAmpTM Clear Adhesive Film (Cat. No. 4306311) or MicroAmpTM Optical Adhesive Film (Cat. No. 4311971).
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 GeneAmpTM PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmpTM 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 GeneAmpTM 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 MicroAmpTM Clear Adhesive Film.
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
GlobalFilerTM 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! This kit is validated for use with the thermal cyclers listed in "Instrument and software compatibility" on page 15.
1. Program the thermal cycling conditions.
IMPORTANT! If you are using the GeneAmpTM PCR System 9700, select the Max ramping mode. If you are using the ProFlexTM 96well PCR System, select the GeneAmpTM PCR System 9700 simulation mode. If you are using the VeritiTM Thermal Cycler, select the 100% ramping rate. Do not use 9600 emulation mode.
Initial incubation step
HOLD 95°C, 1 minute
Optimum cycle number[1]
Denature
Anneal/Extend
CYCLE
94°C, 3 seconds 60°C, 30 seconds
Final extension
Final hold
HOLD
HOLD
60°C, 8 minutes 4°C, up to 24 hours[2]
[1] See "Optimize PCR cycle number (before first use of the kit)" on page 18. [2] 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 MicroAmpTM Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. The VeritiTM Thermal Cycler, ProFlexTM 96well PCR System, and ProFlexTM 2 × 96well PCR System do not require a compression pad.
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
GlobalFilerTM Express PCR Amplification Kit User Guide
3
Perform electrophoresis
Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Set up the SeqStudioTM 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 3130/3130xl instruments for electrophoresis (before first use of the kit) . . . . . . . . . . . . 36 Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit) . . . . . . . . . . . . 38 Prepare samples for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Allelic ladder requirements for electrophoresis
To accurately genotype samples, you must run an allelic ladder with the samples.
Instrument
3500 3500xl 3130 3130xl 3730/3730xl, 48capillary SeqStudioTM
Number of allelic ladders to run
1 per 3 injections 1 per injection
1 per 4 injections 1 per injection 3 per injection
One injection equals
8 samples 24 samples 4 samples 16 samples 48 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
1 per 6 injections
4 samples
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.
GlobalFilerTM Express PCR Amplification Kit User Guide
29
3
Chapter 3 Perform electrophoresis Materials required for electrophoresis
Materials required for electrophoresis
Appendix B, "Materials required but not supplied" lists the required materials that are not supplied with this kit.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.
Set up the SeqStudioTM 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
SeqStudioTM
Data Collection Software
SeqStudioTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 3 Perform electrophoresis Set up the SeqStudioTM instruments for electrophoresis (before first use of the kit)
3
Perform spectral calibration
Perform a spectral calibration using the DS36 Matrix Standard Kit (Dye set J6, 6dye) (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.
GlobalFilerTM 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 3130xl, 3500, or 3500xL configurations. Subsequent validation studies for the kit were performed using the 3730xl 48-capillary configuration and the SeqStudioTM Genetic Analyzer.
Genetic analyzer
3500 3500xL
3500 3500xL
3500 3500xL
3500 3500xL
Operating system
WindowsTM 10
WindowsTM 7 WindowsTM 7 WindowsTM
Vista
3500 Data Collection Software
v4.0.1
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
Plate templates: 6dye_36_POP4 (and _xl)
HID Updater 3500 DC v2 (Cat. No. 4480670)
HID Updater 3500 DC v2 (Cat. No. 4480670)
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
GlobalFilerTM 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 GlobalFilerTM 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 GlobalFilerTM Express PCR Amplification Kit has been validated with data that was analyzed using both the 3rd Order Least Squares method (80460 base pairs) and Local Southern method (60460 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
Setting
GS600_LIZ_(60-460)
Partial 60 bp 460 bp
GlobalFilerTM 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
3. Add the QC protocol to the HID assay. 34
GlobalFilerTM 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 DS36 Matrix Standard Kit (Dye set J6, 6dye) (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.
GlobalFilerTM 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 3130/3130xl instruments for electrophoresis (before first use of the kit)
Electrophoresis software setup
The following table lists the data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures, see the documents listed in "Documentation and support" on page 151.
Note: We conducted original validation studies for the GlobalFilerTM Express PCR Amplification Kit using the 3130xl, 3500, or 3500xL configurations. Subsequent validation studies for the kit were performed using the 3730xl 48-capillary configuration and the SeqStudioTM Genetic Analyzer.
Genetic Analyzer
3130
Operating System
Data Collection Software
Additional software
WindowsTM 7
Data Collection Software v4[1]
3130/3730 DC v4 6Dye Module v1
Run modules and conditions
· HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/5 sec
· Run conditions: 15 kV/1500 sec · Dye Set J6
3130 xl
· HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/10 sec
· Run conditions: 15 kV/1500 sec
· Dye Set J6
[1] Requires activation of 6dye license.
Obtain and activate 6-dye license
1. Confirm that you are running Data Collection Software v4 (Help4About).
2. Obtain a 3130 DC v4 6-Dye Module v1 License key. Contact your local Human Identification representative for information.
3. Ensure that all network cards in the computer are enabled.
IMPORTANT! You can run the 3130 Series Data Collection Software v4 using only the network cards that are enabled when you activate the software license. For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled.
36
GlobalFilerTM 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 Tools4License Manager to display the Software Activation dialog box.
5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you.
6. Obtain the software license file from your email. 7. Make a copy of the software license file and keep it in a safe location. 8. Copy the software license file to the desktop of the Data Collection Software v4 computer. 9. If the Software Activation dialog box has closed, select Tools4License Manager. 10. Click Browse, then navigate to the software license file saved on your computer. 11. Click Install and Validate License.
A message is displayed when the license is installed and validated. 12. Click Close.
GlobalFilerTM 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 DS36 Matrix Standard Kit (Dye set J6, 6dye) (Cat. No. 4425042). The following figure is an example of a passing 6-dye spectral calibration.
Set up the 3730/3730xl 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
Operating System
Data Collection Software
Additional software
3730xl
WindowsTM 10
Data Collection Software v5
None
3730
WindowsTM 7
Data Collection Software v4[1]
3130/3730 DC v4 6-Dye Module v1
[1] Requires activation of 6dye license.
Run modules and conditions
· GeneMapper36_POP7_1 Injection conditions: 2 kV/10 sec
· Run conditions: 15 kV/1,200 sec · Dye Set J6
38
GlobalFilerTM 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 (Help4About)
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 Tools4License Managerto display the Software Activation dialog box.
GlobalFilerTM 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 Tools4License Manager.
10. Click Browse, then navigate to the software license file saved on your computer.
11. Click Install and Validate License. A message is displayed when the license is installed and validated.
12. Click Close.
Perform spectral calibration
Perform a spectral calibration using the DS36 Matrix Standard Kit (Dye set J6, 6dye) (Cat. No. 4425042). The following figure is an example of a passing 6-dye spectral calibration.
40
GlobalFilerTM 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 GeneScanTM 600 LIZTM Size Standard v2.0 HiDiTM Formamide
Volume per reaction 0.5 L 9.5 L
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 MicroAmpTM 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 HiDiTM 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
41
4
Analyze data with GeneMapperTM IDX Software
Overview of GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Set up the GeneMapperTM IDX Software for analysis (before first use of the kit) . . . . . . . . . . . . . . 44 Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Analyze and edit sample files with GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 For more information on using the GeneMapperTM IDX Software . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Overview of GeneMapperTM IDX Software
GeneMapperTM IDX Software is an automated genotyping software application for forensic casework, databasing, and paternity data analysis. GeneMapperTM IDX 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 GlobalFilerTM Express PCR Amplification Kit. After electrophoresis, the data collection software stores information for each sample in a .fsa or .hid file. The GeneMapperTM IDX Software v1.4 or later allows you to analyze and interpret the data from the .fsa or .hid files.
42
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX 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 GeneMapperTM IDX 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
43
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Set up the GeneMapperTM IDX Software for analysis (before first use of the kit)
Set up the GeneMapperTM IDX Software for analysis (before first use of the kit)
Workflow: Set up GeneMapperTM IDX Software
Before you use GeneMapperTM IDX Software to analyze data for the first time, you must do the following:
"Check panel, bin, and stutter file versions on your computer" on page 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 GeneMapperTM IDX Software , then log in with the appropriate user name and password.
2. Select Tools4Panel 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 File4Import Panels to open the Import Panels dialog box.
b. Navigate to, then open the Panels folder, then check the version of panel, bin, and stutter files installed.
5. Check for newer versions of the files as described in the next procedure.
44
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX Software Set up the GeneMapperTM IDX 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 GeneMapperTM IDX Software database:
1. Start the GeneMapperTM IDX Software, then log in with the appropriate user name and password.
2. Select Tools4Panel Manager.
3. Find, then open the folder containing the panels, bins, and marker stutter:
a. Select Panel Manager, then select File4Import 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
45
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Set up the GeneMapperTM IDX Software for analysis (before first use of the kit)
5. Import the bins file: a. Select the AmpFLSTR_Panels folder in the navigation pane.
b. Select File4Import 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.
46
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX Software Set up the GeneMapperTM IDX 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 File4Import Marker Stutter to open the Import Marker Stutter dialog box.
c. Navigate to, then open the AmpFLSTR Analysis Files folder.
GlobalFilerTM Express PCR Amplification Kit User Guide
47
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Set up the GeneMapperTM IDX 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 GeneMapperTM IDX Software database. IMPORTANT! If you close the Panel Manager without clicking Apply, the panels, bin sets, and marker stutter are not imported into the GeneMapperTM IDX 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 GeneMapperTM IDX Software Getting Started Guide-- Basic Features.
48
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX Software Create an analysis method
4
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 GeneMapperTM IDX Software version 1.2 is not compatible with analysis methods that are created in earlier versions of software, or with GeneMapperTM Software v3.2.1.
1. Select Tools4GeneMapper® ID-X Manager to open the GeneMapper ID-X Manager.
Arrows
o use in this SVG.
ced callouts or arrows Callout&Arrow_Libary.
as needed.
and unused elements epository.
1
2. Click the Analysis Methods tab, then click New to open the Analysis Method Editor with the General tab selected.
1
3. Enter the settings shown in the figures on the following pages.
Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs.
4. After you enter the settings on all tabs, click Save.
GlobalFilerTM Express PCR Amplification Kit User Guide
49
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Create an analysis method
Enter Analysis Method settings
Enter General tab settings
1. Enter a Name and select the Security Group appropriate for your software configuration.
2. (Optional) Enter a Description and Instrument.
50
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX 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 GeneMapperTM IDX Software v1.4 or later, enter values for the 4 Marker Repeat Types.
4. Enter the appropriate filter settings.
GlobalFilerTM Express PCR Amplification Kit User Guide
51
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Create an analysis method
Enter Peak Detector tab settings
Figure 4 shows the Peak Detector tab settings that are used in the developmental validation of the kit. 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
Size Calling Method
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.
Enter the values shown in Figure 4, or adjust as needed dependent on the polymer you are using. 3730/3730xl DNA Analyzer with POP-7TM 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.
Additional information --
The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks. Although GeneMapperTM IDX 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 GeneMapperTM IDX Software v1.4 New Features and Installation Procedures User Bulletin (Pub. No. 4477684 Rev. B), "Known issues: 3730 DNA Analyzer allelic ladder failures".
--
Normalization (Optional) Select the Normalization checkbox.
A Normalization checkbox is available on this tab in GeneMapperTM IDX Software for use in conjunction with data run on the 3500 Series Genetic Analyzers.
52
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX Software Create an analysis method
4
Figure 4 Settings used in developmental validation of the kit
GlobalFilerTM Express PCR Amplification Kit User Guide
53
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Create an analysis method
Figure 5 Settings for use on the 3730/3730xl DNA Analyzer.
54
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX 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:
GlobalFilerTM Express PCR Amplification Kit User Guide
55
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Create an analysis method
Enter SQ and GQ tab settings
IMPORTANT! The values that are shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use.
Enter the following values:
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.
56
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX 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 GeneMapperTM IDX 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-7TM 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 80460 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 GeneMapperTM IDX Software v1.4 New Features and Installation Procedures User Bulletin (Pub. No. 4477684 Rev. B), "Known issues: 3730 DNA Analyzer sizing failures".
GlobalFilerTM Express PCR Amplification Kit User Guide
57
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Create a size standard definition file if needed
Create a size standard definition file
1. Select Tools4GeneMapper ID-X Manager to open the GeneMapper ID-X Manager. 2. Click the Size Standards tab, then click New.
ows
e in this SVG. allouts or arrows ut&Arrow_Libary.
eeded.
unused ents itory.
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
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
59
4
Chapter 4 Analyze data with GeneMapperTM IDX Software Analyze and edit sample files with GeneMapperTM IDX Software
Analyze and edit sample files with GeneMapperTM IDX Software
1. In the Project window, select Edit4Add Samples to Project, then navigate to the disk or directory that contains the sample files.
2. Apply analysis settings to the samples in the project.
Parameter
Settings
Sample Type
Select the sample type.
Analysis Method
Select GlobalFilerExpress_AnalysisMethod (or the name of the analysis method you created).
Panel
Select GlobalFiler_Express.
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 GlobalFilerTM Express PCR Amplification Kit was originally validated with the GeneScanTM 600 LIZTM Size Standard v2.0. If you use a different size standard, perform the appropriate internal validation studies to support the use of this size standard with the GlobalFilerTM Express PCR Amplification Kit.
3. Click Analyze, enter a name for the project (in the Save Project dialog box), then click OK to start analysis.
· The status bar displays the progress of analysis as a completion bar.
· The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample).
· The Analysis Summary tab is displayed, and the Genotypes tab is available when the analysis is complete.
60
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 4 Analyze data with GeneMapperTM IDX 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 GeneMapperTM IDX Software
See "Related documentation" on page 151 for a list of available documents.
GlobalFilerTM Express PCR Amplification Kit User Guide
61
5
Experiments and results
Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
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.
62
GlobalFilerTM 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 VeritiTM Thermal Cycler according to the protocol described in the Perform PCR chapter.
SWGDAM guideline 2.2.1
"Developmental validation is the acquisition of test data and determination of conditions and limitations of a new or novel DNA methodology for use on forensic, database, known or casework reference samples." (SWGDAM, December 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 treatedpaper substrates or swabsample 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
63
5 Chapter 5 Experiments and results Developmental validation
Figure 6 Buccal swab lysate amplified with the GlobalFilerTM Express PCR Amplification Kit in the presence of varying concentrations of magnesium chloride and analyzed on a 3500xL Genetic Analyzer (Y-axis scale 0 to 28,000 RFU).
64
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Developmental validation
PCR cycle number
Reactions were amplified for 24, 25, 26, and 27 cycles on the VeritiTM 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 (2427) 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 GlobalFilerTM 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 scale 0 to 25,000 RFU).
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 VeritiTM 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
GlobalFilerTM 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 FTATM 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 RFU).
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
GlobalFilerTM 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 3130xl, 3500, and 3500xL Genetic Analyzers with POP-4TM Polymer and the 3730 Genetic Analyzer with POP-7TM Polymer. The X-axis in the following figures represents the nominal nucleotide sizes for the GlobalFilerTM Express Allelic Ladder. The dashed lines parallel to the X-axis represent the ±0.25-nt windows. The yaxis 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 3130xl Genetic Analyzer. Size and ladder sizing for the GlobalFilerTM Express PCR Amplification Kit were calculated using the GeneScanTM 600 LIZTM Size Standard v2.0.
GlobalFilerTM 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 GlobalFilerTM Express kit were calculated using the GeneScanTM 600 LIZTM Size Standard v2.0.
Figure 11 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3500xL Genetic Analyzer. Size and ladder sizing for the GlobalFilerTM Express kit were calculated using the GeneScanTM 600 LIZTM Size Standard v2.0.
68
GlobalFilerTM Express PCR Amplification Kit User Guide
eded.
nused nts ory.
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 GlobalFilerTM Express kit were calculated using the GeneScanTM 600 LIZTM Size Standard v2.0.
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.
GlobalFilerTM 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 GlobalFilerTM Express Allelic Ladder using the GeneScanTM 600 LIZTM 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
3130xl 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 (Table 2). The mean range and the standard deviation range show the lowest and highest values obtained across multiple runs.
Sample alleles can occasionally size outside of the ±0.5nt window for a respective Allelic Ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems with the smallest standard deviations in sizing. The figures in "Accuracy observation" on page 67 illustrate the tight clustering of allele sizes obtained on the Applied BiosystemsTM genetic analyzers, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5nt 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 ±0.5nt window, the PCR product must be rerun to distinguish between a true offladder 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.
GeneMapperTM IDX Software automatically flags sample alleles that do not size within the prescribed window around an allelic ladder allele by labeling the allele as OL (offladder).
Maximum sizing precision is obtained within the same set of capillary injections. Crossplatform 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
GlobalFilerTM Express PCR Amplification Kit User Guide
71
GlobalFilerTM Express PCR Amplification Kit User Guide
Table 2 Precision results of multiple runs of the GlobalFilerTM Express Allelic Ladder
Allele
3130xl
Mean
Standard deviation
AMEL
X
98.8698.89 0.0220.027
Y 104.90104.93 0.0250.037
CSF1PO
6
282.58282.63 0.0220.042
7
286.54286.59 0.0260.046
8
290.49290.54 0.0290.045
9
294.44294.49 0.0260.039
10 298.38298.42 0.0380.052
11 302.28302.33 0.0250.043
12 306.19306.25 0.0380.044
13 310.15310.20 0.0290.043
14 314.18314.23 0.0380.042
15 318.40318.45 0.0340.039
D10S1248
8
85.3185.33 0.0250.036
9
89.4089.43 0.0250.035
10
93.4793.49 0.0300.037
11
97.5497.57 0.0260.040
3500
Mean
Standard deviation
98.6398.68 104.71104.77
0.0310.051 0.0190.050
283.17283.26 287.12287.21 291.09291.18 295.04295.14 298.99299.09 302.91302.99 306.84306.91 310.81310.91 314.87314.96 319.10319.18
0.0170.062 0.0260.049 0.0310.066 0.0300.061 0.0060.059 0.0160.062 0.0250.063 0.0420.064 0.0050.078 0.0040.063
85.3985.46 89.5389.58 93.6293.68 97.7397.79
0.0240.051 0.0220.055 0.0350.057 0.0060.054
3500xL
Mean
Standard deviation
98.5898.62 104.67104.69
0.0420.047 0.0300.043
283.17283.26 287.13287.25 291.10291.22 295.07295.18 299.01299.12 302.91303.03 306.84306.97 310.83310.96 314.88315.01 319.10319.25
0.0460.059 0.0350.054 0.0440.056 0.0320.055 0.0320.063 0.0350.054 0.0400.063 0.0450.058 0.0370.047 0.0450.061
85.3785.40 89.4989.52 93.5893.63 97.7097.73
0.0310.046 0.0330.045 0.0400.053 0.0450.050
Mean
3730
Standard deviation
100.47100.49 106.20106.22
0.0650.069 0.0500.061
282.49282.63 286.48286.62 290.47290.62 294.47294.62 298.49298.61 302.46302.63 306.46306.62 310.46310.61 314.46314.61 318.45318.62
0.0480.079 0.0660.079 0.0650.070 0.0610.081 0.0720.076 0.0610.082 0.0700.080 0.0640.091 0.0580.074 0.0640.081
86.2086.26 90.1590.22 94.0794.17 98.0498.11
0.0600.068 0.0620.075 0.0660.075 0.0570.069
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
72
Allele
3130xl
Mean
Standard deviation
12 101.62101.65 0.0290.036
13 105.70105.73 0.0290.037
14 109.75109.79 0.0300.038
15 113.74113.79 0.0270.039
16 117.65117.69 0.0260.035
17 121.52121.56 0.0290.043
18 125.40125.45 0.0280.041
19 129.32129.37 0.0270.045
D12S391
14 216.01216.08 0.0400.040
15 220.06220.12 0.0400.045
16 224.08224.14 0.0400.047
17 228.02228.08 0.0290.047
18 231.99232.07 0.0330.043
19 235.95236.01 0.0300.044
19.3 239.02239.08 0.0290.046
20 239.95240.03 0.0280.054
21 244.03244.09 0.0330.045
22 247.99248.04 0.0330.042
23 251.99252.05 0.0240.039
3500
Mean
Standard deviation
101.84101.90 0.0170.053
105.96106.03 0.0340.052
110.02110.09 0.0140.065
114.05114.13 0.0010.058
117.95118.02 0.0010.046
121.85121.91 0.0040.054
125.77125.85 0.0220.062
129.73129.78 0.0280.049
216.47216.57 220.52220.59 224.56224.64 228.53228.62 232.53232.60 236.50236.55 239.52239.62 240.51240.61 244.55244.65 248.51248.60 252.50252.58
0.0190.061 0.0390.067 0.0180.081 0.0300.095 0.0170.070 0.0260.076 0.0010.071 0.0010.068 0.0250.061 0.0390.063 0.0280.056
3500xL
Mean
Standard deviation
101.80101.86 0.0410.045
105.90105.96 0.0370.043
109.98110.05 0.0380.048
114.01114.08 0.0190.048
117.90117.97 0.0380.050
121.81121.86 0.0320.048
125.73125.79 0.0360.048
129.68129.75 0.0400.053
216.54216.61 220.61220.67 224.66224.72 228.61228.69 232.58232.68 236.55236.64 239.60239.70 240.58240.68 244.62244.72 248.57248.69 252.57252.66
0.0400.060 0.0400.055 0.0500.059 0.0520.065 0.0440.057 0.0500.058 0.0480.065 0.0360.061 0.0470.055 0.0460.055 0.0510.056
3730
Mean
Standard deviation
101.96102.07
0.0480.074
105.90106.00
0.0640.068
109.85109.95
0.0650.074
113.77113.88
0.0640.068
117.70117.81
0.0590.070
121.64121.75
0.0690.083
125.56125.69
0.0660.082
129.47129.60
0.0610.071
215.51215.66 219.47219.59 223.48223.60 227.36227.48 231.30231.42 235.20235.32 238.19238.32 239.21239.33 243.07243.20 246.88247.01 250.84250.97
0.0540.071 0.0540.074 0.0620.078 0.0630.072 0.0630.076 0.0640.083 0.0620.075 0.0610.086 0.0670.081 0.0570.088 0.0650.074
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
73
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
3130xl
Mean
Standard deviation
24 255.97256.02 0.0310.047
25 259.86259.90 0.0330.042
26 263.80263.84 0.0320.039
27 267.90267.95 0.0330.049
D13S317
5
198.77198.81 0.0230.035
6
202.77202.82 0.0240.043
7
206.75206.79 0.0220.034
8
210.82210.87 0.0270.037
9
214.85214.89 0.0280.037
10 218.95218.97 0.0220.033
11 223.00223.02 0.0220.041
12 227.11227.13 0.0240.040
13 231.05231.09 0.0290.040
14 235.01235.05 0.0290.036
15 239.05239.08 0.0310.039
16 243.18243.21 0.0250.036
3500
Mean
Standard deviation
256.50256.58 0.0140.055
260.41260.52 0.0360.076
264.37264.46 0.0330.058
268.46268.55 0.0280.064
3500xL
Mean
Standard deviation
256.54256.64 0.0380.053
260.46260.56 0.0370.059
264.41264.52 0.0450.061
268.54268.63 0.0420.052
198.95199.01 0.0040.055 198.96198.99 202.92202.97 0.0350.065 202.94202.97 206.87206.93 206.87206.93 206.89206.94 210.93210.99 0.0090.065 210.96210.98 214.95214.99 0.0080.054 214.97215.00 219.04219.09 0.0320.054 219.08219.09 223.08223.15 0.0100.064 223.13223.16 227.17227.25 0.0310.061 227.24227.25 231.15231.20 0.0200.058 231.19231.21 235.11235.17 0.0200.063 235.15235.18 239.15239.21 0.0050.062 239.19239.22 243.26243.31 0.0050.046 243.29243.32
0.0450.055 0.0390.048 0.0360.046 0.0440.050 0.0320.049 0.0280.053 0.0450.054 0.0520.067 0.0430.050 0.0420.057 0.0230.055 0.0350.049
3730
Mean
Standard deviation
254.90255.01
0.0640.075
258.86258.97
0.0670.089
262.77262.90
0.0640.079
266.86266.98
0.0690.076
199.47199.56 203.51203.58 207.55207.64 211.70211.78 215.74215.81 219.76219.83 223.81223.87 227.90227.96 231.87231.92 235.80235.86 239.85239.90 243.89243.93
0.0460.055 0.0470.056 0.0440.053 0.0460.053 0.0450.057 0.0420.058 0.0450.059 0.0470.054 0.0400.053 0.0430.051 0.0410.056 0.0480.051
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
74
Allele
3130xl
Mean
Standard deviation
D16S539
5
227.17227.21 0.0310.038
8
239.33239.37 0.0280.044
9
243.46243.51 0.0290.038
10 247.59247.63 0.0250.045
11 251.65251.68 0.0270.039
12 255.61255.65 0.0250.037
13 259.55259.59 0.0220.036
14 263.55263.60 0.0250.038
15 267.59267.63 0.0270.041
D18S51
7
261.06261.11 0.0240.038
9
269.20269.24 0.0250.041
10 273.27273.32 0.0260.043
10.2 275.27275.32 0.0280.040
11 277.35277.37 0.0220.035
12 281.39281.44 0.0260.037
13 285.42285.46 0.0270.038
13.2 287.38287.42 0.0280.037
14 289.43289.46 0.0330.037
3500
Mean
Standard deviation
227.46227.54 239.66239.73 243.77243.87 247.90247.98 251.95252.03 255.93256.00 259.89259.98 263.91264.00 267.93268.04
0.0290.063 0.0010.057 0.0050.049 0.0010.049 0.0080.057 0.0060.050 0.0010.059 0.0150.059 0.0250.056
261.26261.37 269.40269.49 273.45273.53 275.43275.52 277.50277.59 281.55281.64 285.58285.66 287.54287.62 289.60289.67
0.0050.060 0.0280.056 0.0240.049 0.0140.050 0.0090.058 0.0080.054 0.0180.056 0.0250.052 0.0250.058
3500xL
Mean
Standard deviation
227.32227.40 239.49239.58 243.63243.72 247.76247.84 251.79251.89 255.78255.86 259.72259.84 263.75263.86 267.79267.89
0.0440.057 0.0420.051 0.0270.046 0.0390.047 0.0450.055 0.0390.045 0.0510.057 0.0500.054 0.0430.055
261.21261.29 269.33269.39 273.41273.45 275.42275.45 277.48277.52 281.51281.54 285.52285.55 287.49287.53 289.52289.57
0.0330.048 0.0380.054 0.0480.054 0.0340.045 0.0430.059 0.0500.053 0.0420.047 0.0490.053 0.0420.051
Mean
3730
Standard deviation
228.05228.18 240.14240.26 244.18244.30 248.20248.32 252.23252.34 256.26256.38 260.28260.40 264.30264.43 268.32268.43
0.0590.073 0.0670.077 0.0600.073 0.0590.070 0.0680.073 0.0590.074 0.0630.069 0.0620.076 0.0550.077
263.60263.73 271.85271.99 276.01276.11 277.97278.10 280.14280.26 284.28284.40 288.40288.53 290.40290.51 292.56292.68
0.0470.064 0.0480.057 0.0450.064 0.0530.060 0.0500.058 0.0540.058 0.0440.064 0.0510.059 0.0480.062
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
75
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
3130xl
Mean
Standard deviation
14.2 291.41291.43 0.0270.035
15 293.43293.47 0.0220.032
16 297.44297.47 0.0240.039
17 301.40301.43 0.0250.035
18 305.36305.39 0.0280.046
19 309.38309.41 0.0210.039
20 313.44313.48 0.0300.040
21 317.69317.71 0.0270.040
22 322.00322.01 0.0280.044
23 326.04326.05 0.0200.036
24 330.13330.15 0.0240.035
25 334.18334.22 0.0300.037
26 338.22338.26 0.0250.049
27 342.29342.34 0.0260.038
D19S433
6
118.28118.35 0.0240.047
7
122.03122.10 0.0240.045
8
125.82125.89 0.0220.050
9
129.64129.68 0.0310.049
10 133.46133.52 0.0330.047
3500
Mean
Standard deviation
291.56291.64 0.0230.060
293.57293.66 0.0350.063
297.58297.69 0.0080.055
301.55301.64 0.0090.058
305.51305.60 0.0280.065
309.50309.60 0.0190.065
313.55313.66 0.0000.065
317.78317.91 0.0210.065
322.07322.20 0.0120.067
326.12326.23 0.0300.060
330.21330.30 0.0300.055
334.27334.36 0.0270.072
338.29338.41 0.0080.052
342.37342.47 0.0110.058
118.50118.61 122.28122.39 126.11126.21 129.94130.05 133.81133.93
0.0260.068 0.0420.085 0.0250.063 0.0400.067 0.0340.073
3500xL
Mean
Standard deviation
291.50291.54 0.0410.052
293.53293.57 0.0410.045
297.54297.57 0.0340.048
301.49301.55 0.0520.056
305.47305.52 0.0430.045
309.47309.54 0.0370.063
313.53313.59 0.0330.063
317.78317.83 0.0380.057
322.06322.10 0.0260.056
326.11326.15 0.0520.063
330.19330.25 0.0360.058
334.28334.30 0.0460.057
338.30338.35 0.0360.053
342.38342.42 0.0450.063
118.52118.55 122.33122.34 126.15126.16 129.99130.01 133.86133.89
0.0420.048 0.0400.052 0.0460.061 0.0510.054 0.0390.058
3730
Mean
Standard deviation
294.55294.65
0.0540.059
296.70296.82
0.0510.060
300.86300.95
0.0490.056
304.98305.09
0.0510.060
309.12309.23
0.0480.054
313.27313.38
0.0520.062
317.41317.52
0.0500.058
321.56321.65
0.0570.062
325.74325.85
0.0500.061
329.85329.97
0.0440.055
333.99334.10
0.0510.061
338.14338.26
0.0500.056
342.29342.41
0.0520.063
346.45346.57
0.0470.057
120.32120.46 124.04124.19 127.78127.91 131.52131.67 135.27135.42
0.0560.091 0.0560.089 0.0640.092 0.0630.107 0.0680.089
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
76
Allele
3130xl
Mean
Standard deviation
11 137.33137.38 0.0360.048
12 141.22141.27 0.0210.044
12.2 143.21143.27 0.0300.051
13 145.17145.22 0.0280.047
13.2 147.16147.23 0.0310.04
14 149.11149.17 0.0360.045
14.2 151.11151.18 0.0290.051
15 153.07153.13 0.0290.053
15.2 155.06155.15 0.0300.040
16 157.02157.11 0.0320.052
16.2 159.03159.12 0.0380.050
17 160.98161.07 0.0380.048
17.2 162.97163.04 0.0360.047
18.2 166.99167.07 0.0300.045
19.2 170.89170.97 0.0280.044
D1S1656
9
159.91159.94 0.0180.037
10 163.93163.97 0.0230.036
11 167.94167.97 0.0260.036
12 171.94171.98 0.0250.033
3500
Mean
Standard deviation
137.69137.82 0.0440.072
141.63141.76 0.0440.068
143.63143.75 0.0420.084
145.60145.73 0.0320.068
147.61147.73 0.0370.065
149.56149.69 0.0410.076
151.58151.71 0.0350.074
153.56153.67 0.0320.072
155.57155.68 0.0360.075
157.53157.66 0.0310.077
159.57159.67 0.0360.071
161.49161.61 0.0070.072
163.50163.62 0.0150.073
167.55167.66 0.0240.067
171.45171.56 0.0340.074
159.94160.03 163.97164.04 167.97168.05 172.02172.07
0.0000.054 0.0160.054 0.0280.059 0.0180.057
3500xL
Mean
Standard deviation
137.74137.78 0.0470.058
141.67141.73 0.0420.058
143.69143.74 0.0380.055
145.65145.71 0.0440.058
147.67147.71 0.0340.060
149.64149.69 0.0470.062
151.63151.71 0.0460.068
153.61153.67 0.0470.056
155.63155.69 0.0500.057
157.60157.66 0.0460.063
159.61159.68 0.0300.060
161.56161.62 0.0400.070
163.57163.63 0.0510.061
167.61167.68 0.0520.060
171.52171.61 0.0380.059
159.98160.00 163.99164.02 168.03168.03 172.03172.05
0.0300.052 0.0360.043 0.0310.046 0.0340.048
3730
Mean
Standard deviation
139.00139.17
0.0620.085
142.78142.93
0.0720.091
144.68144.85
0.0640.085
146.52146.71
0.0650.084
148.45148.61
0.0720.077
150.30150.48
0.0590.082
152.22152.40
0.0660.093
154.08154.25
0.0620.092
156.00156.17
0.0570.089
157.86158.04
0.0700.086
159.79159.96
0.0690.087
161.63161.82
0.0700.094
163.58163.75
0.0640.100
167.48167.67
0.0650.094
171.28171.46
0.0680.095
160.85160.91 164.81164.88 168.78168.84 172.72172.80
0.0460.058 0.0470.058 0.0510.061 0.0500.059
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
77
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
3130xl
Mean
Standard deviation
13 175.95175.99 0.0230.033
14 180.02180.06 0.0310.038
14.3 183.09183.12 0.0220.041
15 184.00184.04 0.0290.035
15.3 187.15187.18 0.0200.034
16 188.05188.08 0.0210.030
16.3 191.19191.24 0.0170.035
17 192.10192.14 0.0300.039
17.3 195.23195.26 0.0210.038
18.3 199.28199.31 0.0200.035
19.3 203.24203.27 0.0250.038
20.3 207.19207.22 0.0290.034
D21S11
24 182.84182.89 0.0200.038
24.2 184.92184.98 0.0290.033
25 186.94187.00 0.0270.038
26 191.04191.10 0.0190.033
27 195.16195.20 0.0250.036
28 199.20199.24 0.0290.039
28.2 201.21201.26 0.0270.039
3500
Mean
Standard deviation
176.01176.08 0.0120.056
180.11180.16 0.0040.059
183.17183.21 0.0320.056
184.10184.16 0.0320.052
187.23187.30 0.0240.059
188.16188.22 0.0310.068
191.30191.36 0.0230.063
192.22192.30 0.0210.063
195.37195.42 0.0110.060
199.40199.47 0.0360.056
203.37203.46 0.0130.057
207.32207.39 0.0220.057
183.09183.14 185.17185.23 187.21187.26 191.33191.37 195.45195.52 199.49199.57 201.48201.53
0.0180.056 0.0260.055 0.0210.057 0.0240.055 0.0140.058 0.0070.059 0.0120.058
3500xL
Mean
Standard deviation
176.05176.07 0.0400.044
180.14180.16 0.0460.055
183.19183.22 0.0270.051
184.12184.15 0.0350.054
187.28187.30 0.0420.049
188.19188.22 0.0270.045
191.35191.37 0.0320.054
192.26192.28 0.0380.050
195.40195.42 0.0350.055
199.44199.47 0.0230.049
203.40203.41 0.0350.047
207.34207.38 0.0410.049
182.98183.05 185.06185.14 187.11187.17 191.21191.29 195.35195.41 199.39199.46 201.38201.45
0.0240.049 0.0390.055 0.0410.054 0.0370.059 0.0330.051 0.0210.056 0.0420.054
3730
Mean
Standard deviation
176.69176.76
0.0460.066
180.71180.80
0.0490.060
183.70183.78
0.0490.062
184.61184.67
0.0500.055
187.65187.72
0.0490.053
188.56188.63
0.0560.060
191.62191.69
0.0470.055
192.52192.58
0.0520.063
195.56195.64
0.0530.060
199.54199.59
0.0500.067
203.50203.56
0.0440.060
207.46207.52
0.0510.064
183.41183.58 185.42185.58 187.45187.60 191.47191.63 195.55195.69 199.51199.66 201.52201.66
0.0600.075 0.0510.076 0.0530.074 0.0600.072 0.0590.067 0.0630.070 0.0530.072
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
78
Allele
29 29.2 30 30.2 31 31.2 32 32.2 33 33.2 34 34.2 35 35.2 36 37 38
3130xl
Mean
Standard deviation
203.16203.22 0.0220.037
205.22205.27 0.0220.034
207.19207.25 0.0220.038
209.17209.23 0.0250.041
211.20211.24 0.0210.039
213.17213.23 0.0210.041
215.22215.27 0.0200.036
217.26217.31 0.0200.034
219.33219.38 0.0250.041
221.30221.34 0.0240.037
223.44223.48 0.0270.038
225.37225.41 0.0330.040
227.46227.50 0.0320.043
229.41229.44 0.0250.042
231.40231.45 0.0370.045
235.50235.54 0.0250.044
239.48239.53 0.0260.042
3500
Mean
Standard deviation
203.46203.55 0.0040.059
205.49205.56 0.0080.067
207.48207.55 0.0080.060
209.45209.51 0.0200.060
211.48211.54 0.0120.061
213.45213.53 0.0040.054
215.52215.57 0.0350.055
217.54217.59 0.0310.052
219.61219.71 0.0060.056
221.60221.68 0.0390.061
223.75223.81 0.0380.058
225.68225.76 0.0380.072
227.79227.86 0.0280.067
229.74229.80 0.0240.058
231.76231.83 0.0260.065
235.84235.93 0.0190.065
239.83239.89 0.0010.059
3500xL
Mean
Standard deviation
203.34203.41 0.0290.044
205.38205.45 0.0390.054
207.36207.44 0.0390.047
209.34209.41 0.0380.051
211.37211.45 0.0480.051
213.33213.41 0.0510.057
215.40215.48 0.0310.044
217.44217.51 0.0260.053
219.54219.60 0.0440.060
221.52221.58 0.0280.046
223.65223.72 0.0500.059
225.59225.66 0.0480.063
227.68227.75 0.0450.058
229.64229.72 0.0430.068
231.66231.75 0.0410.054
235.75235.84 0.0420.054
239.71239.82 0.0470.059
3730
Mean
Standard deviation
203.48203.64
0.0550.070
205.58205.74
0.0650.073
207.54207.68
0.0650.067
209.55209.70
0.0620.086
211.54211.71
0.0660.083
213.56213.71
0.0590.063
215.56215.70
0.0570.074
217.57217.72
0.0610.075
219.58219.75
0.0630.074
221.53221.70
0.0530.074
223.71223.86
0.0670.075
225.62225.75
0.0620.074
227.70227.84
0.0690.076
229.63229.76
0.0650.077
231.53231.66
0.0560.075
235.63235.77
0.0610.076
239.53239.69
0.0570.078
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
79
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
3130xl
Mean
Standard deviation
D22S1045
8
88.4988.51 0.0240.027
9
91.4791.48 0.0230.032
10
94.4694.47 0.0270.037
11
97.4497.46 0.0270.033
12 100.44100.46 0.0180.031
13 103.47103.49 0.0180.035
14 106.49106.50 0.0220.034
15 109.48109.49 0.0280.039
16 112.46112.48 0.0320.041
17 115.39115.41 0.0230.034
18 118.28118.30 0.0250.028
19 121.15121.17 0.0290.037
D2S1338
11 280.99281.04 0.0270.035
12 284.94284.99 0.0310.042
13 288.88288.95 0.0280.036
14 292.82292.88 0.0270.045
15 296.70296.76 0.0330.041
16 300.63300.69 0.0260.042
3500
Mean
Standard deviation
88.3388.40 91.3391.39 94.3194.38 97.3097.37 100.31100.36 103.36103.39 106.36106.43 109.36109.41 112.34112.39 115.27115.34 118.15118.23 121.06121.10
0.0060.045 0.0060.047 0.0040.051 0.0060.050 0.0320.053 0.0110.070 0.0010.054 0.0140.053 0.0060.065 0.0010.051 0.0010.052 0.0140.051
281.61281.74 285.60285.67 289.53289.60 293.46293.53 297.27297.34 301.22301.30
0.0090.060 0.0310.059 0.0260.062 0.0190.060 0.0370.057 0.0090.061
3500xL
Mean
Standard deviation
88.2988.33 91.2991.32 94.2894.31 97.2897.29 100.28100.29 103.31103.33 106.32106.34 109.33109.34 112.30112.33 115.24115.26 118.10118.13 120.98121.00
0.0290.038 0.0260.043 0.0340.042 0.0300.040 0.0270.043 0.0280.050 0.0340.041 0.0400.049 0.0440.051 0.0240.037 0.0140.033 0.0290.048
281.68281.78 285.60285.71 289.53289.66 293.46293.58 297.29297.41 301.26301.37
0.0490.055 0.0480.052 0.0370.050 0.0460.051 0.0340.060 0.0450.052
Mean
3730
Standard deviation
90.0090.01 92.9492.96 95.8895.91 98.8398.87 101.79101.83 104.76104.78 107.71107.77 110.67110.72 113.63113.68 116.60116.64 119.56119.62 122.53122.60
0.0570.063 0.0560.069 0.0590.065 0.0510.068 0.0510.055 0.0580.065 0.0500.059 0.0520.059 0.0540.062 0.0480.059 0.0460.064 0.0440.059
281.21281.39 285.24285.42 289.29289.45 293.33293.50 297.42297.56 301.44301.59
0.0620.066 0.0580.069 0.0600.075 0.0600.075 0.0590.064 0.0600.072
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
80
Allele
3130xl
Mean
Standard deviation
17 304.53304.58 0.0290.041
18 308.46308.52 0.0240.036
19 312.46312.51 0.0290.042
20 316.62316.67 0.0200.037
21 320.81320.86 0.0250.043
22 324.89324.95 0.0290.045
23 328.91328.99 0.0290.038
24 332.95333.00 0.0250.034
25 336.94337.00 0.0240.035
26 340.96341.00 0.0260.037
27 345.05345.09 0.0230.036
28 349.37349.42 0.0260.038
D2S441
8
76.7976.81 0.0190.034
9
80.9380.95 0.0260.034
10
85.0485.08 0.0180.033
11
89.1689.18 0.0230.031
11.3 92.3292.35 0.0250.033
12
93.2593.27 0.0200.029
13
97.1897.20 0.0220.031
3500
Mean
Standard deviation
305.12305.20 0.0270.064
309.05309.14 0.0150.064
313.06313.16 0.0080.061
317.21317.30 0.0260.065
321.40321.49 0.0420.064
325.44325.53 0.0160.058
329.48329.58 0.0310.066
333.53333.61 0.0180.056
337.51337.59 0.0160.065
341.50341.57 0.0090.060
345.58345.67 0.0230.057
349.79349.89 0.0320.062
76.5776.62 80.7480.77 84.8784.91 89.0189.05 92.1992.24 93.1293.17 97.0797.11
0.0010.046 0.0000.047 0.0060.052 0.0150.048 0.0180.049 0.0150.050 0.0050.048
3500xL
Mean
Standard deviation
305.14305.27 0.0380.054
309.11309.22 0.0400.061
313.12313.25 0.0430.059
317.27317.39 0.0470.057
321.45321.58 0.0380.063
325.50325.63 0.0490.075
329.57329.67 0.0500.062
333.58333.69 0.0490.060
337.57337.67 0.0500.058
341.57341.68 0.0330.061
345.64345.74 0.0470.054
349.90349.95 0.0430.050
76.5576.60 80.7080.75 84.8484.88 88.9789.02 92.1692.18 93.0993.12 97.0397.06
0.0260.047 0.0350.048 0.0290.036 0.0360.043 0.0320.040 0.0330.041 0.0320.046
3730
Mean
Standard deviation
305.48305.65
0.0620.074
309.50- 309.67 0.0570.066
313.54313.72
0.0620.066
317.60317.76
0.0570.064
321.65321.82
0.0610.074
325.72325.88
0.0530.068
329.76329.93
0.0570.077
333.80333.99
0.0600.073
337.85338.05
0.0570.065
341.91342.10
0.0600.079
345.98346.18
0.0600.067
350.21350.38
0.0500.063
78.5278.54 82.5182.55 86.5586.57 90.5690.59 93.6893.70 94.5794.59 98.4498.47
0.0700.078 0.0670.072 0.0610.066 0.0500.068 0.0580.064 0.0520.068 0.0470.061
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
81
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
3130xl
Mean
Standard deviation
14 101.30101.32 0.0250.033
15 105.43105.45 0.0280.033
16 109.55109.56 0.0270.037
17 113.74113.76 0.0310.038
D3S1358
9
96.6096.64 0.0220.033
10 100.76100.79 0.0260.037
11 104.95104.97 0.0260.037
12 108.98109.00 0.0240.039
13 113.23113.25 0.0320.037
14 117.21117.24 0.0240.041
15 121.09121.12 0.0230.034
16 125.20125.24 0.0300.036
17 129.33129.37 0.0230.038
18 133.36133.40 0.0200.035
19 137.35137.39 0.0260.036
20 141.66141.71 0.0270.036
3500
Mean
Standard deviation
101.18101.23 0.0110.053
105.33105.38 0.0010.055
109.46109.51 0.0300.055
113.67113.72 0.0050.052
96.4996.55 100.66100.71 104.81104.90 108.83108.92 113.08113.17 117.09117.14 120.96121.04 125.09125.15 129.22129.29 133.26133.32 137.24137.31 141.56141.62
0.0080.047 0.0040.052 0.0160.051 0.0230.046 0.0000.061 0.0150.047 0.0040.053 0.0240.061 0.0130.063 0.0180.055 0.0140.058 0.0050.054
3500xL
Mean
Standard deviation
101.17101.18 0.0110.045
105.32105.34 0.0380.047
109.45109.46 0.0310.046
113.65113.68 0.0480.051
96.4796.49 100.63100.65 104.80104.83 108.83108.85 113.07113.09 117.06117.07 120.92120.94 125.05125.06 129.18129.20 133.22133.23 137.21137.21 141.52141.54
0.0390.049 0.0440.051 0.0400.048 0.0400.044 0.0470.052 0.0150.035 0.0310.050 0.0330.047 0.0370.047 0.0440.052 0.0410.048 0.0380.046
3730
Mean
Standard deviation
102.46102.49
0.0530.067
106.50106.53
0.0470.061
110.51110.56
0.0530.061
114.64114.70
0.0530.056
98.3298.38 102.48102.54 106.63106.71 110.66110.74 114.94115.00 119.07119.13 123.11123.19 127.36127.41 131.58131.63 135.68135.75 139.73139.80 144.02144.09
0.0610.073 0.0600.072 0.0600.064 0.0540.067 0.0550.063 0.0510.066 0.0490.069 0.0530.065 0.0510.061 0.0510.065 0.0490.055 0.0580.061
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
82
Allele
3130xl
Mean
Standard deviation
D5S818
7
138.53138.55 0.0250.039
8
142.59142.60 0.0270.034
9
146.68146.71 0.0130.034
10 150.82150.83 0.0220.032
11 154.91154.93 0.0290.039
12 159.00159.03 0.0250.038
13 163.04163.09 0.0190.032
14 167.07167.10 0.0260.034
15 171.08171.12 0.0210.032
16 175.09175.13 0.0290.032
17 179.10179.14 0.0210.039
18 183.16183.20 0.0210.035
D7S820
6
262.40262.42 0.0250.040
7
266.41266.43 0.0270.044
8
270.41270.43 0.0250.033
9
274.43274.45 0.0290.034
10 278.44278.46 0.0260.032
11 282.43282.45 0.0230.032
3500
Mean
Standard deviation
138.60138.68 142.66142.71 146.75146.82 150.90150.97 155.00155.06 159.10159.17 163.16163.21 167.16167.24 171.20171.26 175.22175.27 179.25179.31 183.32183.39
0.0040.050 0.0340.053 0.0170.048 0.0200.054 0.0150.057 0.0380.056 0.0150.051 0.0300.055 0.0250.051 0.0380.062 0.0050.052 0.0260.062
262.54262.60 266.56266.62 270.57270.63 274.59274.64 278.61278.69 282.62282.67
0.0340.057 0.0200.058 0.0270.068 0.0270.051 0.0070.060 0.0110.066
3500xL
Mean
Standard deviation
138.58138.60 142.63142.66 146.74146.75 150.89150.90 154.98155.02 159.09159.12 163.13163.17 167.17167.20 171.20171.22 175.22175.25 179.25179.27 183.30183.37
0.0380.056 0.0400.051 0.0360.048 0.0300.042 0.0410.052 0.0370.053 0.0370.053 0.0400.044 0.0340.056 0.0380.055 0.0050.050 0.0270.049
262.55262.60 266.55266.61 270.58270.62 274.62274.64 278.63278.66 282.60282.64
0.0350.051 0.0430.052 0.0370.053 0.0350.050 0.0380.059 0.0350.048
Mean
3730
Standard deviation
139.69139.76 143.72143.77 147.69147.77 151.67151.76 155.65155.74 159.64159.70 163.61163.69 167.57167.66 171.56171.64 175.54175.61 179.50179.59 183.49183.58
0.0510.061 0.0520.060 0.0450.063 0.0470.052 0.0450.064 0.0430.055 0.0500.061 0.0560.064 0.0550.064 0.0520.061 0.0540.066 0.0530.070
262.97263.01 266.96266.99 270.92270.98 274.92274.97 278.92278.97 282.91282.96
0.0490.058 0.0500.057 0.0500.057 0.0520.060 0.0510.058 0.0430.068
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
83
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
3130xl
Mean
Standard deviation
12 286.40286.41 0.0280.036
13 290.35290.38 0.0260.032
14 294.30294.33 0.0270.037
15 298.25298.26 0.0260.051
D8S1179
5
114.45114.46 0.0290.036
6
118.42118.43 0.0240.035
7
122.38122.40 0.0220.036
8
126.39126.41 0.0240.031
9
130.41130.43 0.0300.038
10 134.45134.49 0.0240.035
11 138.52138.57 0.0280.032
12 142.66142.70 0.0270.040
13 146.89146.93 0.0170.039
14 151.05151.08 0.0250.035
15 155.21155.24 0.0250.038
16 159.37159.40 0.0280.035
17 163.46163.49 0.0200.035
18 167.54167.57 0.0220.037
19 171.62171.64 0.0250.034
3500
Mean
Standard deviation
286.58286.64 0.0330.057
290.54290.61 0.0250.057
294.49294.57 0.0150.062
298.46298.54 0.0060.060
114.21114.27 118.20118.25 122.17122.21 126.18126.23 130.21130.25 134.25134.30 138.32138.36 142.45142.50 146.71146.74 150.85150.89 155.00155.04 159.16159.19 163.25163.28 167.32167.36 171.39171.42
0.0320.052 0.0000.054 0.0340.057 0.0080.052 0.0180.062 0.0150.049 0.0040.054 0.0120.053 0.0230.054 0.0250.053 0.0150.057 0.0040.057 0.0310.051 0.0310.048 0.0240.063
3500xL
Mean
Standard deviation
286.57286.60 0.0420.054
290.54290.58 0.0360.045
294.51294.54 0.0330.054
298.46298.49 0.0420.052
114.15114.18 118.12118.16 122.09122.12 126.08126.12 130.11130.13 134.17134.19 138.24138.27 142.37142.39 146.61146.64 150.76150.78 154.90154.93 159.07159.08 163.16163.19 167.23167.26 171.30171.33
0.0310.046 0.0290.044 0.0350.050 0.0350.049 0.0390.047 0.0370.054 0.0370.050 0.0320.040 0.0340.042 0.0340.040 0.0360.046 0.0320.046 0.0300.048 0.0370.050 0.0360.051
3730
Mean
Standard deviation
286.91286.96
0.0510.068
290.91290.95
0.0520.059
294.89294.94
0.0500.053
298.89298.94
0.0580.060
116.07116.10 120.19120.24 124.33124.37 128.45128.50 132.58132.61 136.68136.75 140.81140.86 144.93144.98 149.11149.16 153.21153.26 157.31157.36 161.39161.45 165.50165.54 169.59169.62 173.66173.72
0.0520.064 0.0560.062 0.0570.065 0.0490.070 0.0510.059 0.0480.062 0.0530.068 0.0510.060 0.0530.060 0.0530.067 0.0460.059 0.0520.066 0.0540.062 0.0500.062 0.0570.062
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
84
Allele
3130xl
Mean
Standard deviation
DYS391
7
364.73364.78 0.0280.044
8
368.80368.85 0.0270.045
9
372.81372.85 0.0270.036
10 376.80376.86 0.0290.039
11 380.85380.89 0.0300.042
12 384.90384.95 0.0300.038
13 389.00389.04 0.0230.046
FGA
13 223.48223.53 0.0280.038
14 227.48227.55 0.0370.043
15 231.52231.59 0.0240.041
16 235.55235.62 0.0310.039
17 239.58239.64 0.0170.036
18 243.70243.75 0.0270.035
19 247.82247.87 0.0220.036
20 251.86251.89 0.0230.034
21 255.81255.84 0.0270.039
22 259.73259.77 0.0310.039
23 263.74263.78 0.0280.041
3500
Mean
Standard deviation
365.17365.27 369.25369.35 373.25373.34 377.24377.34 381.26381.34 385.24385.35 389.33389.43
0.0280.062 0.0250.060 0.0220.064 0.0110.067 0.0050.057 0.0240.064 0.0270.065
223.35223.42 227.38227.46 231.40231.48 235.43235.49 239.45239.52 243.55243.64 247.63247.71 251.68251.75 255.61255.70 259.55259.61 263.55263.62
0.0380.080 0.0290.079 0.0340.073 0.0240.077 0.0040.071 0.0390.087 0.0110.065 0.0040.070 0.0060.070 0.0000.072 0.0310.078
3500xL
Mean
Standard deviation
365.11365.19 369.18369.28 373.18373.28 377.17377.27 381.21381.30 385.21385.29 389.30389.37
0.0450.066 0.0430.061 0.0460.057 0.0440.063 0.0440.054 0.0490.059 0.0460.056
223.43223.50 227.47227.53 231.50231.56 235.53235.59 239.55239.60 243.65243.72 247.76247.81 251.78251.83 255.74255.79 259.66259.71 263.68263.72
0.0360.060 0.0450.061 0.0500.065 0.0470.062 0.0250.052 0.0420.051 0.0530.056 0.0240.046 0.0320.047 0.0240.056 0.0500.056
Mean
3730
Standard deviation
367.09367.32 371.22371.44 375.29375.52 379.37379.59 383.46383.67 387.45387.65 391.61391.80
0.0530.065 0.0540.068 0.0430.063 0.0500.066 0.0560.067 0.0550.074 0.0550.070
226.22226.30 230.28230.34 234.36234.41 238.42238.47 242.50242.54 246.55246.61 250.63250.67 254.71254.76 258.77258.84 262.85262.91 266.92266.98
0.0500.069 0.0510.073 0.0590.078 0.0570.073 0.0530.080 0.0510.073 0.0530.074 0.0570.074 0.0600.074 0.0610.073 0.0600.079
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
85
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
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
3130xl
Mean
Standard deviation
267.74267.79 0.0220.029
271.79271.84 0.0310.038
275.80275.87 0.0240.032
277.84277.88 0.0260.035
279.82279.85 0.0280.038
283.81283.84 0.0290.038
287.79287.84 0.0300.048
291.80291.85 0.0340.046
293.58293.63 0.0270.038
297.55297.61 0.0340.037
301.49301.54 0.0320.044
305.42305.46 0.0280.040
342.26342.29 0.0280.049
346.34346.37 0.0330.042
350.42350.45 0.0250.047
354.49354.55 0.0220.041
358.43358.46 0.0270.053
362.45362.49 0.0300.043
366.52366.54 0.0270.041
3500
Mean
Standard deviation
267.55267.62 0.0250.067
271.58271.66 0.0240.067
275.61275.67 0.0270.063
277.62277.70 0.0090.064
279.59279.66 0.0400.059
283.59283.65 0.0080.064
287.57287.63 0.0270.048
291.57291.63 0.0290.065
293.35293.42 0.0190.057
297.31297.37 0.0080.055
301.24301.33 0.0060.066
305.17305.25 0.0280.064
341.96342.00 0.0080.060
346.01346.09 0.0220.071
350.10350.16 0.0270.077
354.18354.24 0.0280.077
358.12358.18 0.0080.064
362.13362.19 0.0460.066
366.21366.24 0.0170.065
3500xL
Mean
Standard deviation
267.65267.73 0.0380.055
271.72271.77 0.0410.053
275.73275.80 0.0440.054
277.76277.84 0.0440.059
279.70279.78 0.0250.060
283.68283.74 0.0420.053
287.67287.75 0.0400.055
291.69291.75 0.0440.051
293.46293.53 0.0430.050
297.42297.50 0.0440.053
301.38301.44 0.0310.057
305.31305.37 0.0360.056
342.11342.18 0.0360.052
346.17346.27 0.0360.060
350.24350.34 0.0420.047
354.34354.42 0.0500.062
358.27358.37 0.0280.060
362.29362.37 0.0510.058
366.37366.45 0.0440.060
3730
Mean
Standard deviation
270.94271.01
0.0580.081
275.08275.15
0.0600.081
279.16279.20
0.0550.074
281.25281.29
0.0590.081
283.20283.26
0.0590.075
287.29287.33
0.0510.082
291.39291.45
0.0620.082
295.53295.60
0.0630.083
297.22297.28
0.0630.072
301.32301.38
0.0560.085
305.43305.49
0.0620.078
309.52309.57
0.0590.085
346.61346.66
0.0600.086
350.71350.78
0.0630.085
354.79354.84
0.0610.078
358.91358.96
0.0650.089
362.98363.02
0.0600.097
367.09367.14
0.0720.094
371.22371.30
0.0620.092
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
86
Allele
50.2 51.2 SE33 4.2 6.3
8 9 11 12 13 14 15 16 17 18 19 20 20.2 21 21.2
3130xl
Mean
Standard deviation
374.50374.53 0.0260.038
378.51378.57 0.0270.040
307.08307.10 316.31316.34 321.56321.58 325.67325.70 333.77333.80 337.85337.87 341.90341.93 346.01346.03 350.07350.09 354.17354.20 358.26358.29 362.32362.33 366.28366.29 370.32370.34 372.32372.33 374.33374.34 376.34376.35
0.0240.037 0.0280.036 0.0240.037 0.0240.038 0.0290.039 0.0300.040 0.0290.038 0.0320.048 0.0310.041 0.0300.043 0.0310.038 0.0260.042 0.0260.041 0.0260.036 0.0280.041 0.0270.036 0.0250.035
3500
Mean
Standard deviation
374.16374.21 0.0290.074
378.13378.21 0.0420.065
307.12307.19 316.31316.39 321.54321.62 325.63325.72 333.74333.83 337.83337.88 341.85341.93 345.93346.02 349.99350.10 354.11354.20 358.20358.29 362.26362.35 366.20366.31 370.24370.33 372.23372.31 374.25374.33 376.23376.32
0.0310.080 0.0180.057 0.0060.065 0.0170.063 0.0320.053 0.0160.060 0.0110.062 0.0260.055 0.0220.055 0.0260.060 0.0080.060 0.0100.065 0.0300.064 0.0310.065 0.0260.054 0.0260.065 0.0330.064
3500xL
Mean
Standard deviation
374.32374.41 0.0370.062
378.32378.41 0.0470.066
307.19307.22 316.40316.43 321.65321.67 325.76325.77 333.86333.88 337.93337.94 341.98342.00 346.07346.09 350.11350.14 354.22354.25 358.34358.36 362.41362.43 366.34366.36 370.38370.40 372.37372.39 374.39374.40 376.39376.41
0.0380.053 0.0360.051 0.0410.067 0.0470.062 0.0410.049 0.0480.054 0.0250.052 0.0440.053 0.0370.051 0.0430.060 0.0550.065 0.0240.059 0.0420.052 0.0360.050 0.0480.059 0.0410.049 0.0470.051
3730
Mean
Standard deviation
379.46379.51
0.0680.098
383.56383.62
0.0630.095
310.30310.35 319.58319.65 324.71324.77 328.83328.88 337.06337.12 341.22341.28 345.34345.43 349.47349.55 353.55353.63 357.75357.82 361.90361.97 366.01366.09 370.09370.16 374.26374.33 376.32376.39 378.37378.44 380.47380.51
0.0470.057 0.0450.063 0.0470.057 0.0560.062 0.0510.063 0.0520.061 0.0530.061 0.0510.063 0.0500.058 0.0540.067 0.0570.062 0.0590.065 0.0590.068 0.0590.069 0.0510.067 0.0590.072 0.0590.070
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
87
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
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
3130xl
Mean
Standard deviation
370.32370.34 0.0260.036
372.32372.33 0.0280.041
374.33374.34 0.0270.036
376.34376.35 0.0250.035
380.33380.36 0.0280.044
384.45384.47 0.0240.039
388.50388.51 0.0320.043
392.49392.52 0.0300.046
396.61396.65 0.0350.043
400.64400.67 0.0360.050
404.58404.60 0.0370.046
408.56408.60 0.0330.050
412.58412.60 0.0240.049
416.59416.60 0.0190.042
420.74420.77 0.0370.051
424.76424.81 0.0300.055
428.79428.82 0.0300.050
3500
Mean
Standard deviation
380.22380.30 0.0400.065
384.30384.41 0.0340.074
388.36388.45 0.0230.059
392.34392.42 0.0330.062
396.47396.54 0.0100.072
400.48400.57 0.0410.065
404.41404.50 0.0210.067
408.37408.49 0.0370.076
412.39412.49 0.0100.071
416.40416.49 0.0260.058
420.56420.65 0.0060.063
424.57424.68 0.0270.077
428.62428.72 0.0330.063
430.63430.72 0.0330.074
432.62432.72 0.0380.068
434.62434.74 0.0310.072
438.57438.71 0.0390.070
3500xL
Mean
Standard deviation
370.38370.40 0.0360.050
372.37372.39 0.0480.059
374.39374.40 0.0410.049
376.39376.41 0.0470.051
380.38380.40 0.0030.055
384.48384.52 0.0500.056
388.52388.57 0.0420.059
392.51392.54 0.0460.056
396.62396.66 0.0300.055
400.65400.70 0.0470.054
404.58404.61 0.0500.061
408.55408.60 0.0490.056
412.57412.58 0.0430.057
416.57416.60 0.0480.057
420.71420.74 0.0420.066
424.73424.78 0.0480.066
428.75428.79 0.0540.059
3730
Mean
Standard deviation
384.54384.60
0.0420.072
388.72388.78
0.0610.069
392.84392.88
0.0610.072
396.89396.93
0.0610.071
401.09401.14
0.0620.075
405.22405.25
0.0620.081
409.28409.32
0.0610.070
413.39413.45
0.0620.075
417.54417.60
0.0640.077
421.59421.65
0.0600.074
425.81425.85
0.0600.077
429.91429.96
0.0630.075
434.03434.07
0.0640.085
436.09436.13
0.0690.094
438.11438.15
0.0690.082
440.17440.20
0.0680.084
444.19444.23
0.0680.084
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
88
Allele
3130xl
Mean
Standard deviation
TH01
4
178.72178.81 0.0220.042
5
182.76182.83 0.0300.047
6
186.80186.87 0.0310.042
7
190.85190.92 0.0230.042
8
194.88194.96 0.0260.040
9
198.91198.99 0.0290.043
9.3 201.95202.04 0.0250.041
10 202.86202.94 0.0270.044
11 206.83206.90 0.0310.041
13.3 217.84217.91 0.0290.042
TPOX
5
337.43337.50 0.0240.046
6
341.43341.54 0.0300.052
7
345.60345.67 0.0300.050
8
349.60349.66 0.0350.042
9
353.66353.71 0.0260.048
10 357.69357.76 0.0350.051
11 361.72361.77 0.0310.045
12 365.71365.78 0.0350.056
3500
Mean
Standard deviation
179.17179.24 183.21183.29 187.28187.36 191.34191.43 195.39195.49 199.44199.53 202.46202.55 203.38203.48 207.31207.45 218.35218.43
0.0010.080 0.0340.070 0.0340.068 0.0250.064 0.0310.070 0.0000.080 0.0100.067 0.0130.065 0.0260.071 0.0170.088
338.35338.51 342.41342.59 346.57346.71 350.55350.76 354.66354.79 358.71358.85 362.73362.88 366.73366.89
0.0080.061 0.0120.065 0.0210.053 0.0210.061 0.0280.078 0.0060.061 0.0110.066 0.0280.075
3500xL
Mean
Standard deviation
179.17179.24 183.21183.28 187.29187.36 191.35191.42 195.39195.46 199.43199.50 202.47202.53 203.36203.44 207.32207.40 218.37218.44
0.0460.059 0.0450.054 0.0430.054 0.0450.052 0.0420.047 0.0500.057 0.0470.053 0.0390.053 0.0470.057 0.0410.052
338.30338.51 342.35342.56 346.49346.69 350.51350.72 354.58354.79 358.64358.84 362.66362.89 366.68366.89
0.0490.060 0.0550.065 0.0500.059 0.0400.055 0.0460.064 0.0510.068 0.0470.075 0.0550.075
Mean
3730
Standard deviation
180.08180.29 184.03184.25 187.99188.19 191.95192.15 195.91196.13 199.90200.08 202.92203.11 203.84204.04 207.81208.00 218.72218.92
0.0560.075 0.0530.081 0.0560.078 0.0490.071 0.0650.069 0.0540.074 0.0600.078 0.0520.078 0.0580.075 0.0570.068
338.62338.92 342.67342.96 346.76347.04 350.74351.05 354.78355.08 358.81359.12 362.85363.16 366.88367.18
0.0780.089 0.0710.095 0.0810.092 0.0750.102 0.0840.110 0.0820.101 0.0820.098 0.0850.094
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
89
GlobalFilerTM Express PCR Amplification Kit User Guide
Allele
3130xl
Mean
Standard deviation
13 369.72369.79 0.0350.046
14 373.71373.79 0.0350.052
15 377.72377.79 0.0370.050
Y indel
1
81.2681.31 0.0220.035
2
86.5386.58 0.0280.034
vWA
11 156.50156.52 0.0230.033
12 160.56160.59 0.0190.036
13 164.61164.65 0.0210.035
14 168.81168.86 0.0230.032
15 172.75172.77 0.0260.033
16 176.76176.80 0.0210.034
17 180.81180.83 0.0190.033
18 184.84184.89 0.0250.037
19 188.93188.95 0.0240.034
20 193.00193.02 0.0280.039
21 197.02197.05 0.0290.037
22 201.03201.06 0.0260.035
3500
Mean
Standard deviation
370.76370.92 0.0300.062
374.77374.93 0.0220.071
378.78378.94 0.0050.065
81.0881.17 86.3886.43
0.0150.049 0.0110.060
156.60156.65 160.67160.71 164.73164.77 168.95169.00 172.86172.91 176.91176.95 180.95180.99 185.02185.06 189.10189.16 193.19193.24 197.23197.28 201.25201.31
0.0150.048 0.0050.053 0.0110.047 0.0290.052 0.0170.055 0.0080.049 0.0040.056 0.0100.050 0.0270.054 0.0230.059 0.0080.060 0.0350.056
3500xL
Mean
Standard deviation
370.70370.91 0.0540.066
374.70374.91 0.0550.077
378.70378.92 0.0590.067
81.0581.10 86.3486.35
0.0330.045 0.0280.046
156.55156.59 160.64160.66 164.68164.72 168.93168.97 172.84172.87 176.87176.90 180.90180.96 184.97185.01 189.08189.12 193.16193.20 197.19197.24 201.21201.26
0.0400.048 0.0380.055 0.0370.052 0.0400.047 0.0370.040 0.0370.049 0.0450.053 0.0380.053 0.0430.052 0.0360.057 0.0460.052 0.0440.054
3730
Mean
Standard deviation
370.94371.22
0.0890.101
374.96375.25
0.0870.103
379.02379.30
0.0760.097
82.9182.99 88.2388.31
0.0670.077 0.0640.072
157.91158.01 161.82161.93 165.83165.93 169.96170.07 173.90174.01 177.90177.99 181.87181.98 185.78185.91 189.77189.88 193.76193.86 197.67197.79 201.66201.76
0.0420.064 0.0540.059 0.0530.059 0.0490.057 0.0540.064 0.0510.060 0.0540.063 0.0560.062 0.0550.056 0.0490.055 0.0540.066 0.0580.076
5
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
GlobalFilerTM Express PCR Amplification Kit User Guide
90
Allele
23 24
3130xl
Mean
Standard deviation
204.91204.95 0.0240.036
209.23209.26 0.0240.040
3500
Mean
Standard deviation
205.12205.19 0.0150.047
209.43209.50 0.0280.063
3500xL
Mean
Standard deviation
205.08205.13 0.0410.048
209.40209.44 0.0410.053
3730
Mean
Standard deviation
205.41205.53
0.0480.065
209.89209.99
0.0510.061
Chapter 5 Experiments and results Accuracy, precision, and reproducibility
5
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 GlobalFilerTM Express PCR Amplification Kit is shown in Figure 13 through Figure 19 and listed in Table 3.
Additional marker-specific plus stutter observed in the population study with the GlobalFilerTM Express PCR Amplification Kit is listed in "Stutter observations" on page 91. Examples of non-standard stutter peaks at two loci are shown in "Example of non-standard stutter peaks observed at the D22S1045 and SE33 loci" on page 96.
The stutter filter settings that are derived from this data are listed in "Stutter percentage filter settings that are provided with the GeneMapperTM IDX Software" on page 98.
GlobalFilerTM 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 FTATM Classic Cards 272 buccal samples on Indicating FTATM Cards 45 blood samples on FTATM Bloodstain Cards 90 buccal samples on Bode Buccal DNA CollectorTM Device 45 buccal swab samples lysed in PrepnGoTM Buffer
Figure 13 Stutter percentages for D1S1656, D2S441, D2S1338, and D3S1358 loci (Blue=FAMTM dye, black=NEDTM dye, purple=SIDTM dye)
92
GlobalFilerTM 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=VICTM dye, red=TAZTM dye, purple=SIDTM dye)
Figure 15 Stutter percentages for D12S391, D13S317, and D16S539 loci (Blue=FAMTM dye, red=TAZTM dye, purple=SIDTM dye)
GlobalFilerTM 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=VICTM dye, black=NEDTM dye)
Figure 17 Stutter percentages for D22S1045, DYS391, CSF1PO, and TH01 loci (Blue=FAMTM dye, green=VICTM dye, black=NEDTM dye, red=TAZTM dye). Red and orange data points associated with D22S1045 locus indicate minus and plus stutter, respectively.
94
GlobalFilerTM 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=FAMTM dye, black=NEDTM dye)
Figure 19 Stutter percentages for SE33 and vWA loci (Blue=FAMTM dye, red=TAZTM dye)
GlobalFilerTM 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 GlobalFilerTM Express PCR Amplification Kit. Data produced on a 3500xL Genetic Analyzer using a 50-RFU threshold cutoff. Markers that showed negligible plus stutter are omitted.
Loci
D10S1248 D12S391 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D8S1179
FGA TH01 vWA
Observations
156 107 185 137 31 448 264 787 28 259 214 156 109 16 80
Mean
1.2437 0.8623 0.859 1.0282 1.944 0.9966 1.0044 3.68075 1.593 0.9106 1.282 1.2453
1 1.42 1.069
Standard deviation
1.0038 0.9123 0.7155 0.7157 1.322 0.6624 0.5295
1.53 1.75 0.6228 1.707 1.1121 1.157 1.5 1.027
Minimum
0.17 0.22 0.19 0.25 0.27 0.3 0.42 0.61 0.19 0.31 0.28 0.36 0.32 0.24 0.2
Maximum
4.58 5.91 6.4 5.9 5.2 7.26 4.66 11.06 7.13 4.65 10.3 7.15 8.82 6.09 5.01
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 GeneMapperTM IDX Software for analysis of GlobalFilerTM kit data contain a minus 2-nt stutter filter for SE33 and D1S1656 to prevent these peaks from being called in normal profiles (see "Stutter percentage filter settings that are provided with the GeneMapperTM IDX Software" on page 98).
96
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results Extra peaks in the electropherogram
5
Figure 20 GlobalFilerTM 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
97
5
Chapter 5 Experiments and results Extra peaks in the electropherogram
Stutter percentage filter settings that are provided with the GeneMapperTM IDX Software
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 GlobalFilerTM Express kit loci
Locus [1]
% Stutter
CSF1PO
11.40
D10S1248
12.50
D12S391
15.08
D13S317
9.98
D16S539
10.17
D18S51
13.47
D19S433
10.58
D1S1656
13.08
D1S1656 (-2 nt)
1.79
D21S11
11.42
D22S1045
17.30
D22S1045 (+3 nt)
8.27
D2S1338
13.14
D2S441
8.75
D3S1358
12.45
D5S818
10.84
D7S820
10.21
D8S1179
10.20
DYS391
8.54
FGA
11.96
SE33
14.42
98
GlobalFilerTM 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
5.24
TPOX
5.43
vWA
[1] These percentages are used as stutter filters in AmpFLSTR_Stutter.txt
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 GlobalFilerTM 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
99
5
Chapter 5 Experiments and results Extra peaks in the electropherogram
100
GlobalFilerTM 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 FAMTM, NEDTM, and SIDTM dye channel
TM
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 GlobalFilerTM 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 GlobalFilerTM Express kit. Kit electropherograms are free of reproducible dye artifacts in the kit read region of 74 444 nt for commonly used analytical thresholds. Figure 23 shows the low baselinelevel 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).
GlobalFilerTM 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 GlobalFilerTM 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 99nt and 105nt 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 GlobalFilerTM 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 GlobalFilerTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
Chapter 5 Experiments and results Species specificity
5
Genetic linkage
Two sets of STR loci in the GlobalFilerTM 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 NGMTM and NGM SElectTM 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 (pvalue for pairwise analysis of loci)
Locus
Chromosome map position[1]
Chromosome Nuclear
Coordinates[1] (million bp)
AfricanAmerican
(n = 350)
Caucasian (n = 350)
Hispanic (n = 293)
vWA
12p13.31
5.9
D12S391
12p13.2
12.2
0.86
0.29
0.27
D2S441 D2S1338
2p14 2q35
68
0.11
0.32
0.19
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)
GlobalFilerTM Express PCR Amplification Kit User Guide
103
5 Chapter 5 Experiments and results Species specificity
Nonhuman study observation
The GlobalFilerTM 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 GlobalFilerTM 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 GlobalFilerTM 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-FAMTM dye peak at 359 bp, a NEDTM dye peak at 278 bp, and two small SIDTM dye peaks at 304 bp and 328 bp.
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 VICTM dye peaks at 94 bp (<100 RFU) due to Amelogenin crossreactivity. Pig yielded reproducible TAZTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Sensitivity
crossreactivities were not detectable when the dog, mouse, or chicken DNA were amplified in the presence of human blood or buccal samples on an FTATM 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 GlobalFilerTM 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 PrepnGoTM Buffer ) · Buccal swab sample lysate is prepared using PrepnGoTM 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 GlobalFilerTM Express PCR Amplification Kit , optimize PCR cycle number according to instructions in the Perform PCR chapter.
GlobalFilerTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Sensitivity
Sensitivity observation
Figure 25 shows the results of amplification of different input DNA amounts. The y-axis is magnified for 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
107
5 Chapter 5 Experiments and results Sensitivity
Figure 25 Effect of amplifying varying amounts of white blood cells (WBCs) lysed in PrepnGoTM Buffer. Samples were amplified for 25 PCR cycles.
108
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Stability
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 FTATM cards
The following aged samples were prepared to examine the sample-on-substrate stability: · Finger-prick blood that was spotted onto FTATM Classic Cards stored for 210 days · Buccal cells that were collected with the EasiCollectTM device, stored for 120 days
Aged FTATM samples were amplified with the GlobalFilerTM Express kit in a VeritiTM Thermal Cycler, then the PCR products were collected and detected using a 3500xL Genetic Analyzer. The analysis shows that the age of the FTATM samples did not impact the performance of the GlobalFilerTM Express kit (Figure 26 and Figure 27).
Figure 26 Amplification of blood on FTATM card stored for various amounts of time at room temperature (Y-axis scale 0 to 19,000 RFU or 0 to 23,000 RFU).
GlobalFilerTM Express PCR Amplification Kit User Guide
109
5 Chapter 5 Experiments and results Stability
Figure 27 Amplification of buccal cells on Indicating FTATM card stored for various amounts of time at room temperature (Y-axis scale 0 to 12,000 RFU).
DNA on 4N6FLOQSwabsTM sample collectors
Aged buccal cell samples on 4N6FLOQSwabsTM 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 4N6FLOQSwabsTM samples were lysed in PrepnGoTM Buffer and amplified using the GlobalFilerTM Express kit in a VeritiTM Thermal Cycler. The PCR products were run on a 3500xL Genetic Analyzer. The results from the aged buccal samples collected on 4N6FLOQSwabsTM collectors are shown in Figure 28. The analysis revealed that buccal samples on 4N6FLOQSwabsTM collectors, air-dried immediately after collection and aged up to 4 months at room temperature, produce acceptable profiles when amplified with the GlobalFilerTM Express kit.
110
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
Figure 28 Amplification of buccal cells on 4N6FLOQSwabsTM sample collectors stored for various amounts of time at room temperature and lysed in PrepnGoTM 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
111
5 Chapter 5 Experiments and results Population data
Loci in the kit
The GlobalFilerTM Express PCR Amplification Kit contains loci for which extensive population data are available. For additional information on the loci shared between many of the AmpFSTRTM kits, see the population data and additional studies section of the AmpFSTRTM NGM SElectTM PCR Amplification Kit Kit User Guide (Pub. No. 4458841) and the AmpFSTRTM IdentifilerTM Plus PCR Amplification Kit User Guide (Pub. No. 4440211).
Population samples used in these studies
The GlobalFilerTM 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 GlobalFilerTM Express PCR Amplification Kit contains loci for which extensive population data are available. 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 GlobalFilerTM kit and GlobalFilerTM Express kit are identical. We compared allele calls between the two kits. Genotype data from 200 blood samples on FTATMClassic Cards showed 100% concordance between the two kits. The GlobalFilerTM kit genotypes of the above population data were also compared against the genotypes generated using the IdentifilerTM Plus kit and the NGM SElectTM kit. The few discordant genotypes observed were exclusively found in loci where degenerate primers were added in the GlobalFilerTM kit to rescue known SNPs found in the primer binding sites.
Probability of Identity definition
The PI value is the probability that two individuals selected at random will have an identical genotype (Sensabaugh, 1982).
112
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
Probability of identity observation
Table 6 shows the Autosomal STR allele frequencies at GlobalFilerTM kit loci by population group.
Table 7 shows the Yspecific allele frequencies by population group for GlobalFilerTM Express PCR Amplification Kit DYS391 and Y indel loci. The Yspecific allele frequencies were not included in the probability of identity calculation.
Table 8 shows the Probability of identity (PI) values of the GlobalFilerTM Express PCR Amplification Kit loci individually and combined.
Table 6 Autosomal allele frequencies by population group for GlobalFilerTM Express PCR Amplification Kit STR loci. (*=Alleles not detected or not detected in significant quantities)
Allele
CSF1PO 5 6 7 8 9 10 11
11.1 12 13 14 15 16 D10S1248 7 8 9 10 11 12
African American (n = 330)
Asian (n = 153)
U.S. Caucasian (n = 343)
U.S. Hispanic (n = 368)
* 0.15* 3.79 9.39 3.79 26.21 23.33
* 27.88 4.39 1.06
* *
* * * 0.33* 2.61 26.14 21.9 * 38.56 9.8 0.65* * *
* * * 0.29* 2.77 27.99 31.78 * 30.76 5.98 0.44* * *
* * 0.95 0.54* 2.58 25.14 27.45 0.14* 37.91 4.62 0.54* 0.14* *
0.15* *
0.15* *
3.64 14.09
* * * 0.33* * 10.78
* * * * 0.58* 3.5
* 0.14* 0.14* 0.14* 0.27* 4.48
GlobalFilerTM Express PCR Amplification Kit User Guide
113
5 Chapter 5 Experiments and results Population data
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)
13
22.88
36.93
29.45
25.95
14
27.88
22.55
29.74
36.14
15
18.48
22.55
19.39
22.69
16
10.15
5.23
13.41
7.74
17
2.27
1.63
3.64
2.31
18
0.30*
*
0.29*
*
19
*
*
*
*
D12S391
13
*
*
*
0.14*
14
*
*
*
0.14*
15
7.58
2.61
4.37
4.08
15.1
0.15*
*
*
*
16
5.15
0.98*
3.35
5.03
16.1
0.15*
*
*
*
17
16.52
8.17
10.35
7.34
17.1
0.45*
*
*
0.27*
17.3
0.61*
*
1.9
1.22
18
24.55
28.43
16.18
19.7
18.3
1.21
*
2.19
2.17
19
13.94
23.86
12.54
18.75
19.1
0.61*
*
*
*
19.3
0.30*
*
0.58*
1.22
20
11.52
16.99
9.77
17.12
20.3
*
*
0.15*
*
21
7.27
10.78
13.56
8.7
21.3
0.15*
*
0.15*
*
22
5
3.59
10.79
6.79
114
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
23
3.64
3.27
8.16
3.67
24
0.61*
0.98*
3.64
1.9
25
0.61*
0.33*
1.9
1.36
26
*
*
0.29*
0.27*
27
*
*
0.15*
0.14*
D13S317
5
*
*
*
*
6
*
*
*
*
7
*
*
0.15*
*
8
2.27
29.74
10.93
8.97
9
2.27
12.09
7.14
16.3
10
3.03
13.73
6.85
9.65
11
29.24
25.49
29.01
22.83
12
43.79
14.38
30.76
27.45
13
14.55
3.92
10.64
10.05
14
4.55
0.33*
4.52
4.76
15
0.30*
0.33*
*
*
16
*
*
*
*
17
*
*
*
*
D16S539
4
*
*
*
*
5
*
*
*
*
6
*
*
*
0.14*
8
3.33
*
1.46
2.04
9
21.67
31.05
12.68
10.19
10
11.52
14.05
4.08
15.76
11
30
20.59
32.22
31.79
GlobalFilerTM Express PCR Amplification Kit User Guide
115
5 Chapter 5 Experiments and results Population data
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)
12
19.09
21.57
30.9
24.18
13
13.03
11.44
16.76
14.4
14
1.36
1.31*
1.75
1.22
15
*
*
0.15*
0.27*
16
*
*
*
*
D18S51
6
*
*
*
*
7
*
*
*
*
9
*
*
*
0.14*
10
0.15*
*
1.17
0.68
10.2
0.15*
*
*
*
11
0.45*
1.31*
0.87
1.22
12
6.21
5.56
15.01
10.46
13
3.94
17.32
11.95
11.41
13.2
0.30*
*
*
*
14
5.91
22.88
17.64
16.3
14.2
0.45*
*
*
0.14*
15
16.52
16.99
15.31
12.23
15.2
*
*
*
0.14*
16
18.18
12.42
11.95
12.91
17
16.36
6.54
10.79
17.39
18
14.09
4.9
8.31
7.74
19
9.7
5.23
4.08
3.53
20
4.7
1.96
1.31
1.9
20.2
0.15*
*
*
*
21
1.82
1.96
1.02
2.17
22
0.61*
0.98*
0.29*
0.68
116
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
23
0.30*
0.98*
0.29*
0.54*
24
*
0.65*
*
0.27*
25
*
*
*
0.14*
26
*
0.33*
*
*
27
*
*
*
*
28
*
*
*
*
D19S433
5.2
*
*
*
*
6
*
*
*
*
7
*
*
*
*
8
*
*
*
*
9
0.30*
0.33*
*
*
10
1.21
*
0.15*
0.41*
10.2
0.15*
*
*
*
11
9.85
*
*
1.63
11.2
0.30*
*
*
0.27*
12
10.45
4.58
7.29
8.42
12.1
*
*
0.15*
*
12.2
3.94
0.33*
0.15*
1.49
13
27.88
28.1
27.26
18.48
13.2
5.3
2.61
1.6
6.93
14
18.94
23.2
35.13
30.71
14.2
5.3
9.48
2.04
4.62
15
6.67
7.52
16.18
13.04
15.2
4.39
20.26
3.5
6.79
16
1.52
0.33*
5.69
4.08
16.2
3.18
2.61
0.29*
2.17
GlobalFilerTM Express PCR Amplification Kit User Guide
117
5 Chapter 5 Experiments and results Population data
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)
17
*
*
0.29*
0.54*
17.2
0.61*
0.65*
0.15*
0.41*
18
*
*
0.15*
*
18.2
*
*
*
*
19.2
*
*
*
*
D1S1656
8
*
*
*
*
9
0.15*
*
*
0.14*
10
1.36
*
0.29*
0.41*
11
5.3
3.59
6.27
3.94
12
8.48
4.25
15.74
9.38
13
11.06
13.73
7
7.07
14
25
6.21
6.27
11.28
14.3
0.91
*
0.29*
0.27*
15
16.97
20.26
15.31
15.49
15.3
1.82
*
8.75
2.99
16
10
31.05
9.33
15.08
16.1
*
*
*
0.27*
16.3
7.27
0.65*
4.96
5.16
17
2.73
14.05
4.96
6.79
17.1
*
*
0.29*
*
17.3
5.76
3.59
12.68
15.76
18
0.45*
0.33*
0.29*
0.82
18.3
1.82
1.63
5.98
4.48
19
0.15*
*
*
*
19.3
0.61*
0.33*
1.6
0.68
20
*
0.33*
*
*
118
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
20.3
0.15*
*
*
*
21
*
*
*
*
D21S11
23.2
*
*
*
*
24
*
*
*
*
24.2
*
*
*
0.27*
25
*
*
*
*
26
0.30*
*
0.58*
0.41*
27
5.91
*
2.62
1.49
28
25.15
4.9
16.76
11.41
28.2
*
0.65*
*
0.14*
29
15.61
26.8
23.76
21.06
29.2
*
*
0.15*
*
29.3
0.15*
*
0.15*
*
30
20.76
30.72
23.18
27.17
30.2
1.67
0.65*
2.77
1.77
31
8.79
9.48
6.85
5.16
31.2
4.55
3.92
8.89
11.14
31.3
*
0.33*
*
*
32
1.36
2.61
2.33
1.36
32.2
7.12
14.38
9.62
12.5
33
0.91
0.98*
*
0.14*
33.2
3.18
4.58
1.9
5.3
34
0.15*
*
*
*
34.2
*
*
0.44*
0.14*
35
3.64
*
*
0.27*
35.2
*
*
*
*
GlobalFilerTM Express PCR Amplification Kit User Guide
119
5 Chapter 5 Experiments and results Population data
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)
36
0.76
*
*
0.14*
37
*
*
*
*
38
*
*
*
0.14*
39
*
*
*
*
D22S1045
7
*
*
*
*
8
0.61*
*
*
*
9
*
*
*
*
10
4.09
*
0.44*
0.68
11
14.7
15.36
13.85
7.61
12
6.21
0.33*
0.58*
0.95
13
0.30*
0.33*
1.02
1.09
14
7.88
0.33*
3.35
2.04
15
23.33
33.66
36.3
43.48
16
20.3
23.86
36.3
34.65
17
20.45
24.18
7.58
8.42
18
2.12
1.96
0.58*
0.95
19
*
*
*
*
20
*
*
*
0.14*
D2S1338
10
*
*
*
*
11
*
*
*
*
12
*
*
*
*
13
0.15*
*
0.15*
*
14
*
*
0.15*
*
15
0.30*
*
0.15*
*
16
5.3
1.63
4.08
3.8
120
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
17
10
14.05
18.37
17.8
18
4.85
13.07
8.31
6.52
19
16.21
16.67
14.14
17.53
20
10.45
8.82
15.74
13.86
21
11.97
2.94
2.92
3.67
22
12.42
5.88
1.75
6.52
23
9.24
18.3
10.06
14.27
24
8.79
11.11
10.2
8.83
25
6.97
5.88
12.1
5.43
26
2.58
*
1.6
1.49
27
0.76
0.33*
0.29*
0.14*
28
*
0.98*
*
0.14*
29
*
0.33*
*
*
D2S441
8
0.15*
*
*
*
9
*
*
0.58*
0.14*
9.1
*
2.94
*
*
10
9.09
20.59
19.83
30.3
11
35.61
36.27
33.09
31.93
11.3
2.88
2.61
5.1
4.62
12
20.45
20.92
4.08
3.8
12.3
0.15*
*
0.29*
0.41*
13
3.48
6.21
3.35
1.9
14
26.21
9.8
28.86
23.1
15
1.97
0.65*
4.37
3.4
16
*
*
0.44*
0.41*
17
*
*
*
*
GlobalFilerTM Express PCR Amplification Kit User Guide
121
5 Chapter 5 Experiments and results Population data
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)
D3S1358
8
*
*
*
*
9
0.30*
*
*
0.14*
10
*
*
*
*
11
*
*
0.29*
*
12
0.15*
0.33*
*
0.14*
13
0.61*
*
0.15*
0.41*
14
9.09
2.61
15.16
9.1
15
28.18
49.02
27.26
34.65
15.2
0.30*
*
*
*
16
32.42
21.9
24.34
26.9
17
22.27
19.61
19.68
17.93
18
6.06
6.54
11.66
9.92
19
0.61*
*
1.46
0.82
20
*
*
*
*
21
*
*
*
*
D5S818
6
*
*
*
*
7
0.30*
1.63
*
5.3
8
6.21
0.33*
0.73
1.49
9
1.97
9.15
5.39
5.03
10
7.27
22.22
5.54
4.35
11
25
28.76
33.82
38.18
12
35.45
24.51
37.61
30.16
13
21.82
12.75
14.87
14.54
14
1.67
0.65*
1.75
0.95
15
0.30*
*
0.29*
*
122
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
16
*
*
*
*
17
*
*
*
*
18
*
*
*
*
19
*
*
*
*
D7S820
5
*
*
*
*
6
0.30*
*
*
*
7
0.45*
0.33*
1.31
1.09
8
21.67
16.99
16.47
12.5
9
11.67
7.52
16.62
8.29
9.1
*
0.33*
*
*
10
30.45
20.26
27.26
25.14
10.3
*
*
*
0.14*
11
19.85
31.37
20.99
29.35
11.3
*
*
*
0.14*
12
13.03
20.26
14.58
19.02
13
2.27
2.61
2.33
3.94
13.1
0.15*
*
*
*
14
0.15*
0.33*
0.29*
0.41*
15
*
*
0.15*
*
16
*
*
*
*
D8S1179
4
*
*
*
*
5
*
*
*
*
6
*
*
*
*
7
*
*
*
*
8
0.30*
*
2.04
0.68
GlobalFilerTM Express PCR Amplification Kit User Guide
123
5 Chapter 5 Experiments and results Population data
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
0.30*
*
1.31
0.27*
10
3.33
9.8
10.5
9.51
11
5.61
9.48
6.71
5.03
12
11.36
13.4
15.16
12.5
13
18.18
24.18
33.24
33.15
14
35.91
15.69
18.8
23.23
15
18.03
22.22
9.04
11.41
16
5.91
4.25
2.77
3.53
17
1.06
0.98*
0.44*
0.68
18
*
*
*
*
19
*
*
*
*
20
*
*
*
*
FGA
12.2
*
*
*
*
13
*
*
*
*
14
*
*
*
*
15
*
*
*
*
16
*
0.33*
0.15*
*
16.1
0.30*
*
*
*
17
*
0.33*
0.15*
*
18
0.91
3.27
1.02
0.68
18.2
0.61*
*
*
*
19
6.97
4.25
5.69
7.61
19.2
0.45*
*
*
*
20
6.82
3.92
14.87
8.7
20.2
0.30*
*
0.44*
0.27*
21
11.67
13.07
18.22
13.45
124
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
21.2
0.15*
0.33*
0.29*
*
22
17.27
14.38
19.24
14.4
22.2
0.15*
*
0.87
0.54*
23
17.27
27.12
14.87
12.91
23.2
*
0.65*
0.44*
0.41*
23.3
0.30*
*
*
*
24
18.94
18.3
14.43
15.62
24.2
*
0.33*
*
*
25
9.55
9.8
6.71
13.72
26
4.09
3.27
1.9
7.07
26.2
*
*
*
*
27
2.58
0.65*
0.58*
3.12
28
1.21
*
0.15*
0.95
29
*
*
*
0.41*
30
0.15*
*
*
0.14*
30.2
0.15*
*
*
*
31.2
*
*
*
*
32.2
*
*
*
*
33.2
*
*
*
*
34.2
0.15*
*
*
*
42.2
*
*
*
*
43.2
*
*
*
*
44.2
*
*
*
*
45.2
*
*
*
*
46.2
*
*
*
*
47.2
*
*
*
*
48.2
*
*
*
*
GlobalFilerTM Express PCR Amplification Kit User Guide
125
5 Chapter 5 Experiments and results Population data
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)
50.2
*
*
*
*
51.2
*
*
*
*
SE33
4.2
*
*
*
*
5.2
0.15*
*
*
*
6.3
*
*
0.15*
*
8
*
*
*
*
9
*
*
*
*
11
*
*
*
*
11.2
0.76
*
*
0.14*
12
0.15*
0.33*
0.44*
0.14*
12.1
*
*
0.15*
*
12.2
0.30*
*
0.15*
0.14*
13
1.36
*
0.87
1.22
13.2
0.45*
*
*
0.14*
14
3.33
*
3.64
1.77
14.2
0.15*
*
*
0.82
14.3
*
*
0.15*
*
15
4.24
1.31*
3.64
4.89
15.2
0.15*
*
0.15*
0.14*
16
6.97
3.59
5.39
5.57
16.2
0.30*
*
*
0.27*
16.3
*
*
*
0.14*
17
7.73
5.23
6.56
8.7
17.2
0.15*
*
*
*
17.3
*
*
0.15*
*
18
10.76
4.9
7.87
10.05
126
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
18.2
0.15*
*
*
0.27*
19
15
9.48
8.31
8.15
19.2
0.30*
*
0.29*
*
19.3
*
*
0.29*
*
20
9.55
6.86
5.25
4.48
20.2
0.91
0.33*
0.87
0.82
21
5.76
6.21
2.04
3.12
21.2
0.91
1.63
1.17
1.09
22
1.97
2.61
0.58*
1.09
22.2
1.36
2.29
3.35
2.31
23
0.30*
*
*
*
23.2
0.61*
2.61
2.62
2.85
23.3
*
*
*
0.14*
24
0.30*
0.33*
0.15*
0.14*
24.2
1.67
6.54
4.52
2.31
25.2
2.42
7.19
3.79
3.12
26
0.15*
*
*
0.27*
26.2
5.61
7.52
4.52
6.39
27.2
5.91
3.59
6.85
7.07
27.3
*
*
*
0.14*
28.2
3.94
7.84
7.73
6.25
29.2
2.58
8.5
7.87
5.84
30.2
1.21
7.52
4.66
3.8
31
*
*
*
0.14*
31.2
1.06
1.63
2.77
2.31
32
*
*
0.44*
*
32.2
0.76
1.31*
1.6
2.04
GlobalFilerTM Express PCR Amplification Kit User Guide
127
5 Chapter 5 Experiments and results Population data
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)
33
*
*
0.29*
0.41*
33.2
0.45*
0.33*
0.15*
0.54*
34
*
*
0.29*
0.41*
34.2
0.15*
*
*
0.27*
35
*
*
0.15*
*
35.2
*
0.33*
*
*
36
*
*
0.15*
*
37
*
*
*
0.14*
38
*
*
*
*
TH01
3
*
*
*
*
4
*
*
*
*
5
0.45*
*
0.15*
*
6
15.45
13.07
21.72
27.17
6.1
0.15*
*
*
*
7
37.42
26.14
17.64
32.74
8
20.61
3.59
11.37
8.7
9
16.06
51.63
17.06
12.77
9.3
8.33
4.25
31.2
17.12
10
1.52
1.31*
0.87
1.49
11
*
*
*
*
13.3
*
*
*
*
TPOX
4
*
*
*
*
5
*
*
*
*
6
8.03
*
0.15*
0.54*
7
2.27
0.98*
*
0.14*
128
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
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)
8
35.91
49.35
50.15
47.83
9
19.09
13.07
12.97
8.02
10
9.55
3.59
4.66
6.11
11
21.67
29.74
28.28
26.36
12
3.33
3.27
3.79
10.73
13
0.15*
*
*
0.14*
14
*
*
*
0.14*
15
*
*
*
*
16
*
*
*
*
vWA
10
*
*
*
*
11
0.45*
*
*
0.14*
12
*
*
*
0.27*
13
0.91
*
0.15*
0.14*
14
7.27
23.53
8.75
6.52
15
20.91
1.63
12.24
9.78
16
27.58
15.36
22.3
30.57
17
19.85
29.74
27.41
27.17
17.3
*
*
*
0.14*
18
13.79
19.61
17.78
18.07
19
6.52
9.15
10.06
6.39
20
1.97
0.98*
1.31
0.82
21
0.61*
*
*
*
22
*
*
*
*
23
0.15*
*
*
*
24
*
*
*
*
25
*
*
*
*
GlobalFilerTM Express PCR Amplification Kit User Guide
129
5 Chapter 5 Experiments and results Population data
Table 7 Y-specific frequencies by population group for GlobalFilerTM Express PCR Amplification Kit DYS391 and Y indel loci. (*=Alleles not detected or not detected in significant quantities)
Allele
DYS391 6 7 8 9 10 11 12 13 14
Y indel 1 2
African American (n = 246)
* * * 1.22 71.54 26.42 0.41* 0.41* *
1.22 98.78
Asian (n = 65)
U.S. Caucasian U.S. Hispanic
(n = 233)
(n = 182)
* * * 3.08* 83.08 13.85 * * *
* * * 1.72 44.64 51.93 1.72 * *
* * * 6.59 52.75 36.26 3.3 1.10* *
67.69 32.31
*
0.55*
100
99.45
Table 8 Probability of identity (PI) values for the GlobalFilerTM Express PCR Amplification Kit STR loci
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
130
GlobalFilerTM Express PCR Amplification Kit User Guide
5 Chapter 5 Experiments and results Population data
Table 8 Probability of identity (PI) values for the GlobalFiler Express PCR Amplification Kit STR loci (continued)
Locus
African American (n = 330)
Asian (n = 153)
U.S. Caucasian U.S. Hispanic
(n = 343)
(n = 368)
D2S441
0.1030
0.0961
0.0976
0.1079
D3S1358
0.0984
0.1689
0.0749
0.0949
D5S818
0.0968
0.0883
0.1341
0.1122
D7S820
0.0784
0.0875
0.0680
0.0790
D8S1179
0.0762
0.0527
0.0631
0.0661
FGA
0.0322
0.0555
0.0384
0.0282
SE33
0.0118
0.0139
0.0085
0.0081
TH01
0.0949
0.1750
0.0801
0.0902
TPOX
0.0833
0.1788
0.1757
0.1456
vWA
0.0622
0.0840
0.0650
0.0926
Combined
6.18 × 10 -27
3.34 × 10 -24
3.71 × 10 -26
3.09 × 10 -26
Probability of paternity exclusion observation
Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match Probability (MP), and pvalue 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 for African American, 2.26 × 10-25 for Asian, 5.27 × 10-27 for Caucasian, and 5.0 × 10-27 for Hispanics.
GlobalFilerTM Express PCR Amplification Kit User Guide
131
Chapter 5 Experiments and results
5 Population data
132
Table 9 Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match probability (MP), and p-value of STR loci
African American
Marker Ho
He
MP
pvalue
Ho
Asian
U.S. Caucasian
U.S. Hispanic
He
MP
pvalue
Ho
He
MP
pvalue
Ho
He
MP
pvalue
Y indel
--
-- 0.976 --
--
-
0.556 --
--
-- 1.000 --
--
-- 1.000 --
DYS391 --
-- 0.540 --
--
-- 0.709 --
--
-- 0.472 --
--
-- 0.409 --
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 0.762 0.747 0.102 0.418 0.614 0.656 0.171 0.381 0.753 0.783 0.081 0.326 0.770 0.767 0.091 0.845
FGA 0.782 0.866 0.033 0.082 0.900 0.841 0.044 0.240 0.829 0.857 0.037 0.337 0.842 0.882 0.025 0.127
D22S104 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 5
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
GlobalFilerTM Express PCR Amplification Kit User Guide
GlobalFilerTM Express PCR Amplification Kit User Guide
Table 9 Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match probability (MP), and p-value of STR loci (continued)
African American
Marker Ho
He
MP
pvalue
Ho
Asian
U.S. Caucasian
U.S. Hispanic
He
MP
pvalue
Ho
He
MP
pvalue
Ho
He
MP
pvalue
D10S124 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 8
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
Chapter 5 Experiments and results
5 Population data
133
5 Chapter 5 Experiments and results Population data
The following table shows the Probability of paternity exclusion (PE) values of the GlobalFilerTM 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 GlobalFilerTM Express PCR Amplification Kit STR loci (Chakraborty, Stivers, and Zhong, 1996).
Table 10 Probability of paternity exclusion values for the GlobalFilerTM Express PCR Amplification Kit STR loci
Locus
CSF1PO D10S1248 D12S391 D13S317 D16S539
D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179
FGA SE33 TH01 TPOX vWA PEi Combined
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)
0.4904 0.5353 0.6310 0.6063 0.6063 0.6560 0.5238 0.5703 0.6063 0.4795 0.7463 0.5353 0.3976 0.5942 0.5942 0.6063 0.8397 0.8800 0.3424 0.3602 0.6186 2.7761 × 10-09 0.9999999972
Caucasian (n = 343) Hispanic (n = 368)
0.4507 0.5649 0.8032 0.5544 0.5915 0.7557 0.5135 0.8032 0.7264 0.4507 0.7498 0.4986 0.5338 0.4839 0.5808 0.6187 0.6632 0.9231 0.5036 0.3435 0.6576 4.1986 × 10-10 0.9999999996
0.4644 0.4644 0.6588 0.5770 0.5623 0.7121 0.6431 0.7338 0.7013 0.3970 0.7392 0.5051 0.4689 0.4959 0.5970 0.5381 0.7175 0.8781 0.5381 0.3620 0.6276 2.1709 × 10-09 0.9999999978
134
GlobalFilerTM 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.
Use the correct volume of Master Mix or Primer Set.
The DNA Polymerase was not activated.
Repeat the amplification with an initial hold at 95°C for 1 minute.
The Master Mix was not vortexed thoroughly before aliquoting.
Vortex the Master Mix thoroughly.
The Primer Set was exposed to Replace the Primer Set and store it protected
too much light.
from light.
Evaporation.
Ensure that the plate is properly sealed with film and that a compression pad was used with the GeneAmpTM PCR System 9700. (A compression pad should not be used with other validated thermal cyclers.)
The thermal cycler malfunctioned.
See the thermal cycler user manual and check the instrument calibration.
Incorrect thermal cycler conditions were used.
Use correct thermal cycler conditions.
A MicroAmpTM base was used with a tray/retainer set and tubes in GeneAmpTM PCR System 9700.
Remove the MicroAmpTM base.
The tubes or plate were not seated tightly in the thermal cycler during amplification.
Push the tubes or plate firmly into the block after first cycle.
The wrong PCR reaction tubes or plate were used.
Use MicroAmpTM Reaction Tubes with Caps or the MicroAmpTM Optical 96well Reaction Plate for the GeneAmpTM PCR System 9700 or VeritiTM
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 HiDiTM
Formamide.
GlobalFilerTM 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.
Recommended action
For blood samples on treated paper, punch in the center of the blood stain.
Positive signal from DNA Control 007 but partial or no signal from DNA test samples
More than two alleles present at a locus
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.
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.
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 CollectorTM device, punch from near the tip of the collector. Ensure the swab heads are incubated for 20 minutes in 400 µL of PrepnGoTM 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 CollectorTM device, punch from near the tip of the collector. Ensure the swab heads are incubated for 20 minutes in 400 µL of PrepnGoTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
A Appendix A Troubleshooting Population data
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-7TM polymer fails sizing
Data collected on the 3730 instrument with POP-7TM 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.
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.
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.
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 HiDiTM 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.
Use a 1.2 mm disc.
Ensure the swab heads are incubated for 20 minutes in 400 µL of PrepnGoTM Buffer. Use the correct PCR reaction volume: 15 L
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.
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 GeneMapperTM IDX 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 GeneMapperTM IDX Software v1.4 New Features and Installation Procedures User Bulletin (Pub. No. 4477684), "Known issues: 3730 DNA Analyzer sizing failures".
GlobalFilerTM Express PCR Amplification Kit User Guide
137
B
Materials required but not supplied
STR kit required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Sample preparation required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Thermal cycler required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Genetic analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Analysis software required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Miscellaneous required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
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 GlobalFilerTM Express PCR Amplification Kit, 200-reaction kit GlobalFilerTM Express PCR Amplification Kit, 1,000-reaction kit GeneScanTM 600 LIZTM Size Standard v2.0, 2 × 200 µL IMPORTANT! Do not use GeneScanTM 350 ROXTM, GeneScanTM 500 ROXTM, or GeneScanTM 500 LIZTM Size Standards with this kit.
HiDiTM Formamide, 25mL
Source 4476609 4474665 4408399
4311320
Sample preparation required materials
Treated paper substrate
Item Collection system: NUCLEIC-CARDTM system or Whatman FTATM NUCLEIC-CARDTM Sample Collection Device NUCLEIC-CARDTM matrix, 1 spot NUCLEIC-CARDTM COLOR matrix, 1 spot
Source
A32607 4474001 4473974
138
GlobalFilerTM Express PCR Amplification Kit User Guide
B Appendix B Materials required but not supplied Sample preparation required materials
(continued) Item
WhatmanTM FTATM Classic Cards WhatmanTM EasiCollectTM system Sample preparation: PrepnGoTM Buffer (for use with untreated paper substrates) Low-TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Punch tool: Harris Micro-PunchTM tool, 1.2mm BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a 1.2mm punch head BSD1000-GenePunch Automated Dried Sample Punch Instrument with a 1.2mm punch head
Untreated paper substrate
Item Collection system: Bode or paper Buccal DNA CollectorTM Device 903 paper Punch tool: Harris Micro-PunchTM tool, 1.2mm BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a 1.2mm punch head BSD1000-GenePunch Automated Dried Sample Punch Instrument with a 1.2mm punch head
Swab substrate
Item Collection system 4N6FLOQSwabsTM, regular tip Sample preparation: PrepnGoTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
139
B Appendix B Materials required but not supplied Thermal cycler required materials
(continued)
Item
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
MLS
Oven
VWRTM Scientific dry heat block or equivalent
96-well deep-well plate format
PrepFilerTM 96-Well Processing Plates
A47010
Robbins ScientificTM Model 400 Hybridization Incubator or equivalent
MLS
AgilentTM Benchtop Rack for 200 µL Tubes/V Bottom Plates (metal) or equivalent
IMPORTANT! Do not use a plastic plate adaptor.
Agilent Technologies 410094
Thermal cycler required materials
VeritiTM Thermal Cycler
Item VeritiTM 96Well Thermal Cycler (Optional) Tabletop centrifuge with 96-Well Plate Adapters
GeneAmpTM PCR System 9700
Item GeneAmpTM PCR System 9700, 96-Well Silver GeneAmpTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
B Appendix B Materials required but not supplied Genetic analyzer required materials
Genetic analyzer required materials
SeqStudioTM Genetic Analyzer
Item SeqStudioTM Data Collection Software v1.2 SeqStudioTM Genetic Analyzer Cartridge v2 SeqStudioTM Genetic Analyzer Cathode Buffer Container Reservoir Septa (for SeqStudioTM Cathode Buffer Container) SeqStudioTM Integrated Capillary Protector MicroAmpTM Optical 96-Well Reaction Plate MicroAmpTM Optical 96-Well Reaction Plate with Barcode MicroAmpTM Optical 8-Tube Strip, 0.2 mL Septa for SeqStudioTM Genetic Analyzer, 96 well Septa for SeqStudioTM 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-4TM Polymer (960 samples) for 3500/3500xL Genetic Analyzers POP-4TM 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 A46168 A41331 A33401 A35640 A31923 4316813 4326659 4316567 A36541 A36543 4425042
Source A46085 4405187[1], A26287 4405186[1] 4475183[1] 4480670 4393927 4408256 4393710 4393715 4425042 4393718 4404683
GlobalFilerTM 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 3130xl Data Collection Softwarev4 3130/3730 Data Collection Softwarev4 6Dye Module v1 96Well Plate Septa Reservoir Septa 3100/3130 xl Genetic Analyzer Capillary Array, 36cm POP-4TM Polymer for 3130/3130 xl Genetic Analyzers Running Buffer, 10 DS36 Matrix Standard Kit (Dye Set J6) MicroAmpTM Optical 96-Well Reaction Plate
3730 Series Genetic Analyzer
Item 3730xl Data Collection Software 5 3730/3730 xl Data Collection Softwarev4 3130/3730 Data Collection Software v4 6Dye Module v1 96Well Plate Septa Reservoir Septa 3730 DNA Analyzer 48-Capillary Array, 36-cm
Source 4404687 4410228 4410231 4410701 4412614 4410715
Source 4475105 4475126 4480670 4315933 4315932 4315931 4352755 402824 4425042 N8010560
Source 4475133 4475154 4480670 4315933 4315932 4331247
142
GlobalFilerTM Express PCR Amplification Kit User Guide
B Appendix B Materials required but not supplied Analysis software required materials
(continued) Item
POP-7TM Polymer for 3730/3730xl DNA Analyzers Running Buffer, 10 DS36 Matrix Standard Kit (Dye Set J6) MicroAmpTM Optical 96-Well Reaction Plate 250L Glass Syringe (array-fill syringe) 5.0mL Glass Syringe (polymer-reserve syringe)
Source 4335611 4335613 4425042 N8010560 4304470 6283731
Analysis software required materials
GeneMapperTM IDX Software
Item GeneMapperTM IDX Software v1.6 Full Installation GeneMapperTM IDX Software v1.6 Client Installation GeneMapperTM IDX Software v1.5 Full Installation GeneMapperTM IDX Software v1.5 Client Installation GeneMapperTM IDX Software v1.4 Full Installation GeneMapperTM IDX Software v1.4 Client Installation
Source A39975 A39976 A27884 A27886 4479707 4479711
Miscellaneous required materials
Plates and tubes
Item MicroAmpTM 96-Well Tray MicroAmpTM Reaction Tube with Cap, 0.2 mL MicroAmpTM 8-Tube Strip, 0.2 mL MicroAmpTM Optical 8-Cap Strips MicroAmpTM 96-Well Tray/Retainer Set MicroAmpTM 96-Well Base
Source N8010541 N8010540 N8010580 4323032
403081 N8010531
GlobalFilerTM Express PCR Amplification Kit User Guide
143
B Appendix B Materials required but not supplied Miscellaneous required materials
(continued) Item
MicroAmpTM Clear Adhesive Film MicroAmpTM Optical Adhesive Film MicroAmpTM Optical 96-Well Reaction Plate
Laboratory supplies
Various procedures Aerosol resistant pipette tips Microcentrifuge tubes Pipettors Tape, labeling Tube, 50-mL FalconTM Tube decapper, autoclavable Deionized water, PCR grade Vortex
[1] Major laboratory supplier
Item
Source 4306311 4311971 N8010560
Source
MLS[1] MLS MLS MLS MLS MLS MLS MLS
144
GlobalFilerTM 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
145
D
PCR work areas
Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
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
GlobalFilerTM Express PCR Amplification Kit User Guide
D Appendix D PCR work areas Amplified DNA work area
· 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.
GlobalFilerTM 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
GlobalFilerTM Express PCR Amplification Kit User Guide
Chemical safety
Appendix E Safety Chemical safety
E
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 oeuvre 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.
GlobalFilerTM 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-2009P.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
GlobalFilerTM Express PCR Amplification Kit User Guide
Documentation and support
Related documentation
Document title
STR kits GlobalFilerTM Express PCR Amplification Kit--PCR and CE Quick Reference GlobalFilerTM Express PCR Amplification Kit--PCR Setup--Swab Substrate Quick Reference GlobalFilerTM Express PCR Amplification Kit--PCR Setup--Treated Paper Substrate Quick Reference GlobalFilerTM Express PCR Amplification Kit--PCR Setup--Untreated Paper Substrate Quick Reference
Thermal cyclers VeritiTM Thermal Cycler User Guide GeneAmpTM PCR System 9700 Base Module User Manual SeqStudioTM Genetic Analyzer SeqStudioTM Genetic Analyzer Instrument and Software User Guide SeqStudioTM 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
GlobalFilerTM 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
GeneMapperTM IDX Software all versions
GeneMapperTM IDX Software Bin Overlap User Bulletin
GeneMapperTM IDX Software v1.3
GeneMapperTM IDX Software v1.3 Verification Experiments and Installation Procedures User Bulletin
GeneMapperTM IDX Software v1.4
GeneMapperTM IDX Software v1.4 New Features and Installation Procedures User Bulletin
GeneMapperTM IDX Software v1.5 GeneMapperTM IDX Software v1.5 New Features and Verification User Bulletin
GeneMapperTM IDX Software v1.5 Getting Started Guide-- Basic Features
GeneMapperTM IDX Software v1.5 Quick Reference-- Basic Features
GeneMapperTM IDX Software v1.5 Getting Started Guide-- Mixture Analysis Tool
GeneMapperTM IDX Software v1.5 Quick Reference-- Mixture Analysis Tool
GeneMapperTM IDX Software v1.5 Installation Guide
GeneMapperTM IDX Software v1.5 Administrator Guide
GeneMapperTM IDX Software v1.5 Reference Guide
GeneMapperTM IDX Software v1.6
GeneMapperTM IDX 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
GlobalFilerTM 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 HIDTechSupport@thermofisher.com, 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.
GlobalFilerTM Express PCR Amplification Kit User Guide
153
References
Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J. Forensic Sci. 39:362372.
Bonferroni, C.E. 1936. Teoria statistica delle classi e calcolo Belle probabilita. Publicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8:362.
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. 108:180185.
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. 109:6265.
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. 108:3135.
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. 148:249258.
Bender, K., Farfan, M.J., Schneider, P.M. 2004. Preparation of degraded human DNA under controlled conditions. Forensic Sci. Int. 139:134140.
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. 62:14081415.
Brinkmann, B., Möller, A. and Wiegand, P. 1995. Structure of new mutations in 2 STR systems. Intl. J. Legal Med. 107:201203.
Budowle, B. et al. 2010. Population genetic analyses of the NGMTM STR loci. Int. J. Legal Med. e-publication www.springerlink.com/content/p713q3w5440674u3/
Butler, J.M. 2005. Forensic DNA Typing. Burlington, MA:Elsevier Academic Press.
Butler, J.M., Shen, Y., McCord, B.R. 2003. The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci. 48:10541064.
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 94:10411046.
Chakraborty, R. and Stivers, D.N. 1996. Paternity exclusion by DNA markers: effects of paternal mutations. J. Forensic Sci. 41:671677.
Chakraborty, R., Stivers, D., and Zhong, Y. 1996. Estimation of mutation rates from parentage exclusion data: applications to STR and VNTR loci. Mutat. Res. 354:4148.
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. 49:733740.
154
GlobalFilerTM 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. 16:96779686.
Coble, M.D. and Butler, J.M. 2005. Characterization of new miniSTR loci to aid analysis of degraded DNA. J. Forensic Sci. 50:4353.
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. 49:859860.
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. 49:746756.
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 12:241253.
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. 10:564567.
Frank, W., Llewellyn, B., Fish, P., et al. 2001. Validation of the AmpFSTRTM Profiler PlusTM PCR Amplification Kit for use in forensic casework. J. Forensic Sci. 46:642646.
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. 90:197203.
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. 22:45274534.
Grubwieser, P. Muhlmann, R., Berger, B., Niederstatter, H., Palvic, M., Parson, W. 2006. A new "miniSTR-multiplex" displaying reduced amplicon lengths for the analysis of degraded DNA. Int. J. Legal Med. 120:115120.
Guo, S.W. and Thompson, E.A. 1992. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48:361372.
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. 55:175189.
Holt, C., Stauffer, C., Wallin, J., et al. 2000. Practical applications of genotypic Surveys for forensic STR testing. Forensic Sci. Int. 112:91109.
Kalinowski, S.T. 2006. HW-QuickCheck: an easy-to-use computer program for checking genotypes for agreement with Hardy-Weinberg expectations. Molecular Ecology Notes 6:974979.
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. 75:11431148.
Kwok, S., and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237238.
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 182:151153.
GlobalFilerTM 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 19:8693.
Levene, H. 1949. On a matching problem in genetics. Ann. Math. Stat. 20:9194.
Li, B., Ge, J., Wu, F., Ye, L., Budowle, B., Vhen, Y. 2013. Population genetic analyses of the STR loci of the AmpFSTRTM NGM SElectTM PCR Amplification Kit for Han population in Fujian Province, China. Int J. Legal Med. 127:345346.
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 21:700709.
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 19:101107.
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. 106:262267.
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. 95:173178.
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):647660.
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. 51:826830.
Nakahori, Y., Takenaka, O., and Nakagome, Y. 1991. A human X-Y homologous region encodes amelogenin. Genomics 9:264269.
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 89:583590.
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):953958.
Raymond M. and Rousset F. 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86:248249.
Rousset, F. 2008. Genepop'007: A complete reimplementation of the Genepop software for Windows and Linux. Molecular Ecology Resources 8:103106.
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
GlobalFilerTM 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. 338415.
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. 140:133135.
Smith, R.N. 1995. Accurate size comparison of short tandem repeat alleles amplified by PCR. Biotechniques 18:122128.
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. 109:186194.
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. 109:195204.
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):160161.
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. 94:2531.
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. 43:854870.
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. 47:5265.
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. 192194.
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. 114:285287.
GlobalFilerTM Express PCR Amplification Kit User Guide
157
Index
+A 99 +A nucleotide addition 99
3' A 99 3130 instrument 36 3130/3130xl instrument, catalog numbers 142 3500 instrument 32 3730 instrument 30, 38 3730/3730xl instrument, 6-dye license 36 4N6FLOQSwabs sample collectors 110 6-dye
license activation 39 license activation for 3730/3730xl instrument 36 spectral calibration 31, 35, 38, 40 600 LIZ Size Standard v2.0 57
A
accuracy and reproducibility 66 alleles, off-ladder 69 allelic ladder, requirements for electrophoresis 29 allelic ladder, volume per reaction 41 artifacts 101
B
bins, import 45 biohazard safety 150 blood 18, 105 Bode Buccal DNA Collector 22 buccal 18, 105
C
characterization of loci, validation 102 control DNA
007 11 profile 13 Copan, treated paper 9
D
developmental validation 63 DNA control profile 13
documentation, related 151 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
extra peaks 91
F
FTA cards 109
G
GeneScan 600 LIZ Size Standard v2.0 57 GeneScan size standard, about 11
H
HID updater 33
I
import panels, bins, and marker stutter 45 instrument and software compatibility 15
L
limited product warranty 153 LIZ size standard
about 11 peak sizes 57 LIZ Size Standard v2.0 57
M
marker stutter, import 45 materials not supplied 138
158
GlobalFilerTM Express PCR Amplification Kit User Guide
P
panels check version 44 import 45
PCR conditions 28 optimize cycle number 18 perform 28 setup 146 work areas 146
plate layout, PCR 145
R
required materials 138 run module for electrophoresis, 3500 and 3500xL in-
struments 32 run module, electrophoresis, 3130 and 3130xl 36
S
safety, biohazard 150 sensitivity 105 size standard 58 spectral calibration 31, 35, 38, 40 stutter, peaks 96 stutter file, import 45 substrates 9 swab
FLOQSwabs 9 PCR 28 sample preparation guidelines 24 swabs 110
T
terms and conditions 153 thermal cyclers
for use with kit 15 programming 28 treated paper PCR 28 sample preparation guidelines 20 troubleshooting 135
U
untreated paper, PCR 28
V
validation, importance 62
W
warranty 153 work area, PCR setup 146
Index
GlobalFilerTM 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
Antenna House PDF Output Library 7.0.1574