Thermo Fisher Scientific AmpFLSTR NGM SElect Express PCR Amplification Kit User Guide
Thermo Fisher Scientific AmpFLSTR NGM SElect Express PCR Amplification Kit is a powerful tool for forensic and paternity testing. This STR multiplex assay is optimized for direct amplification of single-source samples, including blood and buccal samples obtained from treated or untreated paper substrates or buccal swabs. This kit allows for efficient and accurate analysis of DNA profiles using multiple STR loci and the sex-determining marker Amelogenin.
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USER GUIDE AmpFlSTR® NGM SElect™ Express PCR Amplification Kit for use with: 200 reaction kit (Part no. 4472193) Publication Part Number 4474109 Rev. C Revision Date June 2014 For Forensic or Paternity Use Only. For Forensic or Paternity Use Only. Information in this document is subject to change without notice. LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses. TRADEMARKS Windows and Windows Vista are registered trademarks of Microsoft Corporation. FTA is a registered trademark of Whatman International Ltd. Whatman is a registered trademark of GE Healthcare Companies. Bode Buccal DNA Collector is a trademark of Bode Technology Group, Inc. NUCLEIC-CARD is a trademark of and FLOQSwabs is a registered trademark of Copan Diagnostics Inc. Adobe, Acrobat and Reader are registered trademarks of Adobe Systems Incorporated. © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Contents About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 ■ CHAPTER 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substrate examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About the primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Loci amplified by the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allelic ladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 11 11 11 12 12 13 Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Instrument and software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data collection and analysis software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrument and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About multicomponent analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How multicomponent analysis works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 16 16 16 16 Materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Standards for samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 ■ CHAPTER 2 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Optimize PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Select samples and prepare plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Determine optimum conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Treated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample prep guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prepare low-TE buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 21 21 21 Untreated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Sample prep guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Swab substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample prep guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prepare the sample lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Store the sample lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 26 26 27 29 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 3 Contents ■ CHAPTER 3 Perform Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Allelic ladder requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Section 3.1 3100/3100-Avant and 3130/3130xl instruments . . . . . . . . . . . . . . . . . . . . . . . 33 Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis . . . . . . . . . . . . . . . . 33 Reagents and parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments . . . . . . 34 Section 3.2 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Set up the 3500/3500xL instruments for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Reagents and parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Electrophoresis software setup and reference documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Prepare samples for electrophoresis on the 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . 35 ■ CHAPTER 4 Analyze Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Section 4.1 GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Overview of GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Set up GeneMapper® ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . File names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Before using the software for the first time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Import panels and bins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allele tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Detector tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Quality tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quality Flags tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 38 38 38 42 42 43 44 45 46 47 Analyze and edit sample files with GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Section 4.2 GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Overview of GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Set up GeneMapper® ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Panel, bin, and stutter file version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Before using the software for the first time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Check panel, bin, and stutter file version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Import panels, bins, and marker stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 51 51 51 51 52 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Contents Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allele tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Detector tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peak Quality tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SQ & GQ tab settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Create a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 57 58 59 60 61 61 Analyze and edit sample files with GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 ■ CHAPTER 5 Experiments and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiments using the NGM SElect™ Express Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 65 65 65 Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 1.2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.10.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thermal cycler parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 66 66 66 67 68 Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Precision and size windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 69 69 70 Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Causes of extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nature of the polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 88 88 89 89 Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 SWGDAM Guideline 2.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blood on treated paper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buccal cells on treated or untreated paper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buccal swab lysate in Prep-n-Go™ Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of DNA quantity on results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 91 91 91 91 92 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 SWGDAM guideline 2.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 5 Contents DNA on FTA® Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 DNA on Copan 4N6 FLOQSwabs® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SWGDAM guideline 2.7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concordance studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 95 95 96 Mutation rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Estimating germ-line mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Additional mutation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Probability of paternity exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 ■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 ■ APPENDIX B Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Equipment and materials not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 ■ APPENDIX C Plate Layouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Example PCR plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Example electrophoresis plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 ■ APPENDIX D PCR Work Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 ■ APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Obtain SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Limited Product Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 6 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Contents Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 7 Contents 8 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide About This Guide IMPORTANT! Before using this product, read and understand the information the “Safety” appendix in this document. Revision history Revision Date Description A February 2012 New document. B May 2012 Add Experiments and Results chapter. C June 2014 Add Master Mix Additive information. Purpose The AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide provides information about the Life Technologies instruments, chemistries, and software associated with the AmpFlSTR® NGM SElect™ Express PCR Amplification Kit. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 9 About This Guide Purpose 10 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 1 Overview ■ Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 ■ Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 ■ Instrument and software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 ■ Materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Product overview Purpose The AmpFlSTR® NGM SElect™ Express PCR Amplification Kit is a short tandem repeat (STR) multiplex assay optimized to allow direct amplification of single-source samples: • Blood and buccal samples on treated paper substrates without the need for sample purification. • Blood and buccal samples collected on untreated paper substrates and treated with Applied Biosystems® Prep-n-Go™ Buffer. • Buccal samples collected on swab substrates and treated with Applied Biosystems® Prep-n-Go™ Buffer. The NGM SElect™ Express Kit amplifies 16 autosomal STR loci (D10S1248, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656, D12S391 and SE33) and the sex-determining marker, Amelogenin, in a single PCR reaction. Substrate examples • Treated paper: Copan NUCLEIC-CARD™ system or Whatman FTA® cards • Untreated paper: Bode Buccal DNA Collector™ or 903 paper • Swab: Copan FLOQSwabs™ Product description The NGM SElect™ Express Kit contains all the necessary reagents for the amplification of human genomic DNA. The reagents are designed for use with the following instruments: • Applied Biosystems® 3100/3100-Avant Genetic Analyzer • Applied Biosystems® 3130/3130xl Genetic Analyzer • Applied Biosystems® 3500/3500xL Genetic Analyzer • Veriti® 96-Well Thermal Cycler (Part no. 4375786) • GeneAmp® PCR System 9700 with the Silver 96-Well Block • GeneAmp® PCR System 9700 with the Gold-plated Silver 96-Well Block AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 11 1 Chapter 1 Overview Product overview IMPORTANT! The NGM SElect™ Express Kit is validated for use with the Veriti® 96-well Thermal Cycler (Part no. 4375786) and the GeneAmp® PCR System 9700 with the silver block (Part no. N8050251) or the gold-plated silver block (Part no. 4314443). It is not verified for use with the Veriti® 96-Well Fast Thermal Cycler (Part no. 4375305) or the GeneAmp® PCR System 9700 with the aluminium block (Part no. 4314879). About the primers The NGM SElect™ Express Kit uses the same primer sequences as the AmpFlSTR® NGM SElect™ Kit and benefits from the same primer synthesis and purification improvements. These improvements enhance the assay signal-to-noise ratio and simplify the interpretation of results. Non-nucleotide linkers are used in primer synthesis for the following loci: D19S433, TH01, FGA, D3S1358, D1S1656, D12S391, SE33. 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 inter-locus spacing. The combination of a five-dye fluorescent system and the inclusion of non-nucleotide linkers allows for simultaneous amplification and efficient separation of the 16 STR loci and Amelogenin during automated DNA fragment analysis. Loci amplified by the kit Table 1 shows the loci amplified, their chromosomal locations, and the corresponding fluorescent marker dyes. The AmpFlSTR® NGM SElect™ Express Allelic Ladder is used to genotype the analyzed samples. The alleles contained in the allelic ladder, and the genotype of the AmpFlSTR® Control DNA 007, are also listed in the table. Table 1 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit loci and alleles Locus designation Chromosome location Alleles included in Allelic Ladder Dye label 6-FAM™ Control DNA 007 12, 15 D10S1248 10q26.3 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 vWA 12p13.31 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 14, 16 D16S539 16q24.1 5, 8, 9, 10, 11, 12, 13, 14, 15 9, 10 D2S1338 2q35 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 20, 23 Amelogenin X: p22.1-22.3 X, Y VIC® X, Y Y: p11.2 D8S1179 8q24.13 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 12, 13 D21S11 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 28, 31 D18S51 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 12, 15 12 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 1 Overview Product overview Chromosome location Locus designation Alleles included in Allelic Ladder Dye label NED™ Control DNA 007 D22S1045 22q12.3 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 D19S433 19q12 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2 14, 15 TH01 11p15.5 4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3 7, 9.3 FGA 4q28 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 NED™ 24, 26 D2S441 2p14 9, 10, 11, 11.3, 12, 13, 14, 15, 16 PET® 14, 15 D3S1358 3p21.31 12, 13, 14, 15, 16, 17, 18, 19 15, 16 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 13, 16 D12S391 12p13.2 14, 15, 16, 17, 18, 19, 19.3, 20, 21, 22, 23, 24, 25, 26, 27 18, 19 SE33 6 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 17, 25.2 Allelic ladder 1 11, 16 Figure 1 shows the allelic ladder for the NGM SElect™ Express Kit. See “Allelic ladder requirements” on page 32 for information on ensuring accurate genotyping. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 13 1 Chapter 1 Overview Product overview Figure 1 GeneMapper® ID-X Software plot of the AmpFlSTR® NGM SElect™ Express Allelic Ladder 14 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 1 Overview Workflow overview 1 Perform PCR Harris Manual Punch Obtain samples BSD600 Semi-Automated Dried Sample Punch Instrument Prep-n-Go™ Buffer (untreated paper only) AmpFlSTR® NGM SElect™ Express PCR Amplification Kit Swab substrates Prepare samples Treated or untreated paper substrates Lyse in Prep-n-Go™ Buffer Prepare reactions Prepare reactions Prepare samples Perform PCR Obtain samples Workflow overview AmpFlSTR® NGM SElect™ Express PCR Amplification Kit GeneAmp® PCR System 9700 Cycler Veriti® 96-Well Thermal Cycler Perform electrophoresis 3100/3100-Avant Genetic Analyzer 3130/3130xl Genetic Analyzer 3500/3500xL Genetic Analyzer Analyze data GeneMapper® ID-X Software AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide GeneMapper® ID Software 15 1 Chapter 1 Overview Instrument and software overview Instrument and software overview This section provides information about the data collection and analysis software versions required to run the this kit on specific instruments. Data collection and analysis software Instrument and software compatibility The data collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time. As the instrument measures sample fluorescence with its detection system, the data collection software collects the data and stores it. The data collection software stores information about each sample in a sample file (.fsa files for 31xx instruments and .hid files for 3500 instruments), which is then analyzed by the analysis software. Instrument 3100/3100Avant Operating system Windows® NT Data collection software 1.1 (3100) 1.0 (3100-Avant) Windows® 2000 2.0 3130/3130xl† Windows XP 3.0 3500/3500xL† • Windows® XP or 3500 Series Data Collection Software v1.0 • Windows Vista® Analysis software • GeneMapper® ID Software v3.2.1 • GeneMapper® ID-X Software v1.0.1 or higher GeneMapper® ID-X Software v1.2 or higher † We conducted validation studies for the NGM SElect™ Express Kit using these configurations. About multicomponent analysis Life Technologies fluorescent multi-color dye technology allows the analysis of multiple loci, including loci that have alleles with overlapping size ranges. Alleles for overlapping loci are distinguished by labeling locus-specific primers with different colored dyes. Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components. The four dyes used in the NGM SElect™ Express Kit to label samples are 6-FAM™, VIC®, NED™, and PET® dyes. The fifth dye, LIZ®, is used to label the GeneScan™ 600 LIZ® Size Standard v2.0. How multicomponent analysis works Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength. During data collection on the Life Technologies instruments, the fluorescence signals are separated by a diffraction grating according to their wavelengths and projected onto a charge-coupled device (CCD) camera in a predictably spaced pattern. The 6-FAM™ dye emits at the shortest wavelength and is displayed as blue, followed by the VIC® dye (green), NED™ dye (yellow), PET® dye (red), and LIZ® dye (orange). Although each of these dyes emits its maximum fluorescence at a different wavelength, there is some overlap in the emission spectra between the dyes (Figure 2). The goal of multicomponent analysis is to correct for spectral overlap. 16 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 1 Overview Materials and equipment 1 Figure 2 Emission spectra of the five dyes used in the NGM SElect™ Express Kit Dyes Normalized Emission 6-FAM VIC NED PET LIZ 100 80 60 40 20 0 500 550 600 650 700 Wavelength (nm) Materials and equipment Kit contents and storage The NGM SElect™ Express Kit contains sufficient quantities to perform 200 (Part no. 4472193) reactions at 25 µL/reaction: IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Keep freeze-thaw cycles to a minimum. Table 2 Kit contents and storage Component Description 200 reaction Storage AmpFlSTR® NGM SElect™ Express Master Mix Contains enzyme, salts, dNTPs, carrier protein, and 0.05% sodium azide 2 tubes, 1 mL each –15 to –25°C upon receipt, 2 to 8°C after addition of Additive for up to 6 months or the expiration date stated on the kit (whichever comes first). Master Mix Additive Reagent for one-time addition to each Master Mix tube prior to first use. 2 tubes, 0.1 mL each –15 to –25°C upon receipt. Discard the tube after additions to the Master Mix tube. AmpFlSTR® NGM SElect™ Express Primer Set Contains forward and reverse primers to amplify human DNA targets. 2 tubes, 1 mL each –15 to –25°C upon receipt. 2 to 8°C after initial use for up to 6 months or the expiration date stated on the kit (whichever comes first). Store protected from light. AmpFlSTR® NGM SElect™ Express Allelic Ladder Contains amplified alleles. 1 tube, 50 µL See Table 1 on page 12 for a list of alleles included in the allelic ladder. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide –15 to –25°C upon receipt. 2 to 8°C after initial use up to the expiration date stated on the kit. Store protected from light. 17 1 Chapter 1 Overview Materials and equipment Component AmpFlSTR® Control DNA 007 Description Contains 2 ng/μL human cell line DNA in 0.05% sodium azide and buffer.† 200 reaction 1 tube, 50 µL Storage –15 to –25°C upon receipt. 2 to 8°C after initial use up to the expiration date stated on the kit. See Table 1 on page 12 for profile. † The Control DNA 007 is included at a concentration appropriate to its intended use as an amplification control (i.e., to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype). The Control DNA 007 is not designed to be used as a DNA quantitation control and you may see variation from the labelled concentration when quantitating aliquots of the Control DNA 007. Standards for samples For the NGM SElect™ Express Kit, the panel of standards needed for PCR amplification, PCR product sizing, and genotyping are: • AmpFlSTR® Control DNA 007 – A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpFlSTR® NGM SElect™ Express Allelic Ladder. • GeneScan™ 600 LIZ® Size Standard v2.0 – Used for obtaining sizing results. This standard, which has been evaluated as an internal size standard, yields precise sizing results for NGM SElect™ Express Kit PCR products. Order the GeneScan™ 600 LIZ® Size Standard v2.0 (Part no. 4408399) separately. • AmpFlSTR® NGM SElect™ Express Allelic Ladder – Developed for accurate characterization of the alleles amplified by the NGM SElect™ Express Kit. The Allelic Ladder contains most of the alleles reported for the 16 autosomal loci. See page 12 for a list of the alleles included in the Allelic Ladder. 18 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 2 Perform PCR ■ Optimize PCR cycle number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Treated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 ■ Untreated paper substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 ■ Swab substrates: prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 ■ Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Optimize PCR cycle number Before using the NGM SElect™ Express 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 NGM SElect™ Express Kit. This experiment accounts for instrument-to-instrument and sample-to-sample variations. If you are processing multiple sample type and substrate combinations (for example, buccal samples on treated paper and buccal samples on swabs), perform separate sensitivity experiments for each sample type and substrate to be used for testing. Select samples and prepare plates 1. Select 26 of each sample type and substrate combination. Ensure the selected samples represent a “typical” range of samples analyzed in your laboratory. IMPORTANT! The number of samples recommended for this study has been chosen to allow you to complete electrophoresis using a single 96-well plate, thus minimizing the impact of run-to-run variation on the results. Examples of PCR and electrophoresis plate layouts are provided on page 117. 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 three identical PCR plates (see page 117 for a suggested plate layout). Note: To maximize result quality, prepare and amplify Plate 1 then repeat for Plates 2 and 3. Do not prepare all three plates before amplification. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 19 2 Chapter 2 Perform PCR Optimize PCR cycle number 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 below. Sample type Substrate Treated paper Untreated paper Swab Blood 25, 26, 27 cycles 25, 26, 27 cycles N/A Buccal 26, 27, 28 cycles 26, 27, 28 cycles 25, 26, 27 cycles Note: Our testing has not included blood samples on swab substrates. This sample type is not frequently used for the collection of database or casework reference samples. Note: To minimize the effect of instrument-to-instrument variation, use the same thermal cycler to amplify all three plates. Determine optimum conditions 1. Run the PCR products on the appropriate CE platform using the recommended protocol (see Chapter 3, “Perform Electrophoresis” on page 31). 2. Based on the results of the sensitivity study, select the appropriate PCR cycle number for future experiments. Our studies indicate the optimum PCR cycle number should generate profiles with the following heterozygote peak heights, with no instances of allelic dropout and minimal occurrence of off-scale allele peaks: Instrument Heterozygous peak height 31xx 1000–3000 RFU 3500 Series 3000–12,000 RFU The NGM SElect™ Express Kit is optimized to amplify unpurified: • Single-source blood samples on treated paper or untreated paper • Buccal samples on treated paper, untreated paper, or swabs When amplifying single-source, unpurified samples using the NGM SElect™ Express Kit, you should expect to see greater variation in peak height from sample to sample than is expected with purified samples. Careful optimization of the cycle number will help to minimize this variation. 20 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 2 Perform PCR Treated paper substrates: prepare reactions 2 Treated paper substrates: prepare reactions Sample prep guidelines • Do not add water to the wells on the reaction plate before adding the punches. If your laboratory is experiencing static issues with the paper discs, you may prepare and dispense the 25 µL reaction mix into the wells of the reaction plate before adding the punches. • 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. • 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: Refer to the User Guide of your automated or semiautomated disc punch instrument for proper guidance. Prepare low-TE buffer You can prepare the buffer as described below or order it from Teknova (Cat # T0223). To prepare low-TE buffer: 1. Mix together: • 10 mL of 1 M Tris-HCl, pH 8.0 • 0.2 mL of 0.5 M EDTA, pH 8.0 • 990 mL glass-distilled or deionized water Note: Adjust the volumes accordingly for specific needs. 2. Aliquot and autoclave the solutions. 3. Store at room temperature. Prepare the reactions 1. Add samples to the reaction plate: To these well(s) of a MicroAmp® Optical 96-Well Reaction Plate... Add: Negative control 1.2 mm blank disc Test samples 1.2 mm sample disc Positive control • For 25 cycles 3 µL of Control DNA 007 • For 26 and 27 cycles 2 µL of Control DNA 007 • For 28 cycles 1 µL of Control DNA 007 IMPORTANT! Do not add a blank disc to the positive control well. 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. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 21 2 Chapter 2 Perform PCR Treated paper substrates: prepare reactions 2. Prepare reagents: a. Thaw the Master Mix, Master Mix Additive, and Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes. b. Prepare the Master Mix (required before first use only): • Add 66 µL of Master Mix Additive to each tube of Master Mix prior to first use. • Gently invert the Master Mix tube 10 times and centrifuge briefly. • Mark the cap of the Master Mix tube with a (+) to indicate that the Master Mix Additive has been added. • Discard the Master Mix Additive tube. IMPORTANT! Thawing is required only during first use of the reagents. After first use, reagents are stored at 2 to 8 °C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 3. Calculate the volume of each component needed to prepare the reactions using the table below. Reaction component Volume per reaction Master Mix 10.0 µL Primer Set 10.0 µL Low-TE buffer 5.0 µL Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! This kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition 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. Pipet the required volumes of components into an appropriately sized polypropylene tube. 5. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 6. Dispense 25 µL of the reaction mix into each reaction well of a MicroAmp® Optical 96-Well Reaction Plate. 7. Seal the plate with MicroAmp® Clear Adhesive Film or MicroAmp® Optical Adhesive Film. IMPORTANT! If you are using the 9700 thermal cycler with a silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp® compression pad (Part no. 4312639) on top of the plate to prevent evaporation during thermal cycling. The Veriti® Thermal Cycler does not require a compression pad. 8. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 22 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 2 Perform PCR Treated paper substrates: prepare reactions 2 9. Amplify the samples in a Veriti® 96-well Thermal Cycler or PCR System 9700 with the silver or gold-plated silver 96-well block as described in “Perform PCR” on page 29. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 23 2 Chapter 2 Perform PCR Untreated paper substrates: prepare reactions Untreated paper substrates: prepare reactions Sample prep guidelines • Make a 1.2 mm punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. • If you are using a Bode Buccal DNA Collector™, Bode Buccal make a 1.2 mm punch as close as possible to the tip DNA Collector™ of the DNA collector to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. Take punch as close to the tip as possible • 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: Please refer to the User Guide of your automated or semi-automated disc punch instrument for proper guidance. Prepare the reactions 1. Add Prep-n-Go™ Buffer (Part no. 4467079) to the reaction plate: To these well(s) of a MicroAmp® Optical 96-Well Reaction Plate... Add: Negative control 5 µL of Prep-n-Go™ Buffer Test samples 5 µL of Prep-n-Go™ Buffer Positive control • For 25 cycles 2 µL of Prep-n-Go™ Buffer • For 26 and 27 cycles 3 µL of Prep-n-Go™ Buffer • For 28 cycles 4 µL of Prep-n-Go™ Buffer 2. Add samples to the reaction plate: To these well(s) of a MicroAmp® Optical 96-Well Reaction Plate... Add: Negative control 1.2 mm blank disc Test samples 1.2 mm sample disc Positive control • For 25 cycles 3 µL of Control DNA 007 • For 26 and 27 cycles 2 µL of Control DNA 007 • For 28 cycles 1 µL of Control DNA 007 IMPORTANT! Do not add a blank disc to the positive control well. 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 the punches are immersed in the Prep-n-Go™ Buffer. 24 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 2 Perform PCR Untreated paper substrates: prepare reactions 2 4. Prepare reagents: a. Thaw the Master Mix, Master Mix Additive, and Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes. b. Prepare the Master Mix (required before first use only): • Add 66 µL of Master Mix Additive to each tube of Master Mix prior to first use. • Gently invert the Master Mix tube 10 times and centrifuge briefly. • Mark the cap of the Master Mix tube with a (+) to indicate that the Master Mix Additive has been added. • Discard the Master Mix Additive tube. IMPORTANT! Thawing is required only during first use of the reagents. After first use, reagents are stored at 2 to 8 °C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 5. Calculate the volume of each component needed to prepare the reactions using the table below. Reaction component Volume per reaction Master Mix 10.0 µL Primer Set 10.0 µL Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! This kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition 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. Pipet the required volumes of components into an appropriately sized polypropylene tube. 7. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 8. Dispense 20 µL of the reaction mix into each reaction well of a MicroAmp® Optical 96-Well Reaction Plate. The final volume in each well is 25 µL (reaction mix and Prep-n-Go™ Buffer plus sample disc, negative control disc, or positive control). 9. Seal the plate with MicroAmp® Clear Adhesive Film or MicroAmp® Optical Adhesive Film. IMPORTANT! If using the 9700 thermal cycler with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp® compression pad (Part no. 4312639) on top of the plate to prevent evaporation during thermal cycling. The Veriti® Thermal Cycler does not require a compression pad. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 25 2 Chapter 2 Perform PCR Swab substrates: prepare reactions 10. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 11. Amplify the samples in a Veriti® 96-well Thermal Cycler or PCR System 9700 with the silver or gold-plated silver 96-well block as described in “Perform PCR” on page 29. Swab substrates: prepare reactions Sample prep guidelines • Detach each buccal swab head from the swab shaft before lysis. • Lysis is performed using Prep-n-Go™ buffer (Part no. 4471406 for buccal swabs) and can be conducted in 1.5 mL tubes or in a 96-well deep-well plate (Part no. 4392904) as described below. • For optimum performance, lysis of a whole swab is recommended. To preserve the sample, evaluate lysis of a half swab. Prepare the sample lysate 1. Add 400 µL Prep-n-Go™ Buffer (Part. no. 4471406) to 1.5 mL tubes or the appropriate wells of a 96-well deep-well plate (Part no. 4392904). 2. Into each tube or well, put the entire head of each swab and let stand for 20 minutes at room temperature to lyse the sample. 3. After 20 minutes, transfer the sample lysate out of the sample plate into tubes or plates for storage, then discard the deep-well plate containing the swab heads. Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate. 4. Proceed to the next section to prepare the reactions or see “Store the sample lysate” on page 29. 26 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 2 Perform PCR Swab substrates: prepare reactions Prepare the reactions 2 1. Add Prep-n-Go™ Buffer (Part no. 4471406) to the control wells in the reaction plate: To these well(s) of a MicroAmp® Optical 96-Well Reaction Plate... Add: Negative control 5 µL of Prep-n-Go™ Buffer Positive control • For 25 cycles 2 µL of Prep-n-Go™ Buffer • For 26 and 27 cycles 3 µL of Prep-n-Go™ Buffer • For 28 cycles 4 µL of Prep-n-Go™ Buffer 2. Prepare reagents: a. Thaw the Master Mix, Master Mix Additive, and Primer Set, then vortex for 3 seconds and centrifuge briefly before opening the tubes. b. Prepare the Master Mix (required before first use only): • Add 66 µL of Master Mix Additive to each tube of Master Mix prior to first use. • Gently invert the Master Mix tube 10 times and centrifuge briefly. • Mark the cap of the Master Mix tube with a (+) to indicate that the Master Mix Additive has been added. • Discard the Master Mix Additive tube. IMPORTANT! Thawing is required only during first use of the reagents. After first use, reagents are stored at 2 to 8 °C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 3. Calculate the volume of each component needed to prepare the reactions using the table below. Reaction component Volume per reaction Master Mix 10.0 µL Primer Set 10.0 µL Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! This kit has been optimized for a 25-µL PCR reaction volume to overcome the PCR inhibition 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. Pipet the required volumes of components into an appropriately sized polypropylene tube. 5. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 6. Dispense 20 µL of the reaction mix into each reaction well of a MicroAmp® Optical 96-Well Reaction Plate. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 27 2 Chapter 2 Perform PCR Swab substrates: prepare reactions 7. Add samples to the reaction plate: To these well(s) of a MicroAmp® Optical 96-Well Reaction Plate... Add: Test samples 5 µL of sample lysate Positive control • For 25 cycles 3 µL of Control DNA 007 • For 26 and 27 cycles 2 µL of Control DNA 007 • For 28 cycles 1 µL of Control DNA 007 Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. The final volume in each well is 25 µL (reaction mix and Prep-n-Go™ Buffer plus sample lysate or positive control). 8. Seal the plate with MicroAmp® Clear Adhesive Film or MicroAmp® Optical Adhesive Film. IMPORTANT! If using the 9700 thermal cycler with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp® compression pad (Part no. 4312639) on top of the plate to prevent evaporation during thermal cycling. The Veriti® Thermal Cycler does not require a compression pad. 9. Vortex the reaction mix at medium speed for 3 seconds. 10. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 11. Amplify the samples in a Veriti® 96-well Thermal Cycler or PCR System 9700 with the silver or gold-plated silver 96-well block as described in “Perform PCR” on page 29. 28 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 2 Perform PCR Perform PCR Store the sample lysate 2 Cap the sample lysate storage tubes or seal the sample lysate storage plate with MicroAmp® Clear Adhesive Film. Store the sample lysate as needed: If you are storing the sample lysate... Then place at... <2 weeks 2 to 8°C >2 weeks –15 to –25°C These storage recommendations are preliminary pending the results of ongoing stability studies. 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! The NGM SElect™ Express Kit is validated for use with the Veriti® 96-well Thermal Cycler (Part no. 4375786) and the GeneAmp® PCR System 9700 with the silver block (Part no. N8050251) or the gold-plated silver block (Part no. 4314443). It is not verified for use with the Veriti® 96-Well Fast Thermal Cycler (Part no. 4375305) or the GeneAmp® PCR System 9700 with the aluminium block (Part no. 4314879). 1. Program the thermal cycling conditions. IMPORTANT! When using the GeneAmp PCR System 9700, select the Max ramping mode. When using the Veriti® Thermal Cycler, select the 100% ramping rate. DO NOT use 9600 emulation mode. Initial incubation step Optimum cycle number† Denature HOLD 95°C 1 min Anneal Extend Final extension Final hold HOLD HOLD 60°C 5 min 4°C ∞ CYCLE 94°C 3 sec 59°C 16 sec 65°C 29 sec † Determine the optimum cycle number for your laboratory according to the instructions on page 19. 2. Load the plate into the thermal cycler and close the heated cover. 3. Start the run. 4. On completion of the run, store the amplified DNA. If you are storing the DNA... Then place at... <2 weeks 2 to 8°C >2 weeks –15 to –25°C IMPORTANT! Protect the amplified products from light. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 29 2 30 Chapter 2 Perform PCR Perform PCR AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 3 Perform Electrophoresis Allelic ladder requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 ■ Section 3.1 3100/3100-Avant and 3130/3130xl instruments . . . . . . . . . . . . . . . . . . 33 Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis . . . 33 Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34 ■ Section 3.2 3500/3500xL instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Set up the 3500/3500xL instruments for electrophoresis . . . . . . . . . . . . . . . . . . . . . 35 Prepare samples for electrophoresis on the 3500/3500xL instruments. . . . . . . . . . 35 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 31 3 Chapter 3 Perform Electrophoresis Allelic ladder requirements Allelic ladder requirements To accurately genotype samples, you must run an allelic ladder sample along with the unknown samples. Instrument Number of allelic ladders to run One injection equals 3100-Avant or 3130 1 per 4 injections 4 samples 15 samples + 1 allelic ladder 3100 or 3130xl 1 per injection 16 samples 15 samples + 1 allelic ladder 3500 1 per 3 injections 8 samples 23 samples + 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples + 1 allelic ladder Number of samples per allelic ladder(s) IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single- and multiple-capillary runs (with larger size variations seen between samples injected in multiple-capillary runs). We recommend the above frequency of allelic ladder injections, which should account for normal variation in run speed. However, during internal validation studies, verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment. It is critical to genotype using an allelic ladder run under the same conditions as the samples, because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions. 32 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 3.1 3100/3100-Avant and 3130/3130xl instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis 3 Section 3.1 3100/3100-Avant and 3130/3130xl instruments Reagents and parts Appendix B, “Ordering Information” on page 113 lists the required materials 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. Keep freeze-thaw cycles to a minimum. Electrophoresis software setup and reference documents Genetic Analyzer The following table lists data collection software and the run modules that can be used to analyze PCR products generated by this kit. For details on the procedures, refer to the documents listed in the table. Data Collection Software Applied Biosystems® 3100-Avant 1.0 Applied Biosystems® 3100 2.0 Operating System Run modules and conditions References Windows® NT • GeneScan36Avb_DyeSetG5Module Injection condition: 3 kV/5sec 3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4332345) • GS600v2.0Analysis.gsp Windows® 2000 • HIDFragmentAnalysis36_POP4_1 Injection condition: 3kV/10 sec • Dye Set G5 1.1 Windows® NT • GeneScan36vb_DyeSetG5Module Injection condition: 3kV/10 sec • GS600v2.0Analysis.gsp Applied Biosystems® 3130/3130xl† 3.0 Windows® XP • HIDFragmentAnalysis36_POP4_1 Injection conditions: – 3130 = 3 kV/5 sec – 3130xl = 3 kV/10 sec • Dye Set G5 3100/3100-Avant Genetic Analyzers Using Data Collection Software v2.0, Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4350218) 3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4332345) Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0, Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin (Part no. 4363787) † We conducted validation studies for the NGM SElect™ Express Kit using the 3130xl or the 3500xL configurations. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 33 3100/3100-Avant and 3130/3130xl Instruments Set up the 3100/3100-Avant and 3130/3130xl instruments for electrophoresis 3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Prepare samples for electrophoresis on the 3100/3100-Avant or 3130/3130xl instruments Prepare the samples for electrophoresis immediately before loading. 1. Calculate the volume of Hi-Di™ Formamide and size standard needed to prepare the samples: Reagent Volume per reaction GeneScan™ 600 LIZ® Size Standard v2.0 0.5 µL Hi-Di™ 9.5 µL Formamide Note: Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. 2. Pipet the required volumes of components into an appropriately sized polypropylene tube. 3. Vortex the tube, then centrifuge briefly. 4. Into each well of a MicroAmp® Optical 96-Well Reaction Plate, add: • 10 µL of the formamide:size standard mixture • 1 µL of PCR product or Allelic Ladder Note: For blank wells, add 10 µL of Hi-Di™ Formamide. 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the reaction plate in a thermal cycler for 3 minutes at 95°C. 7. Immediately place the plate on ice for 3 minutes. 8. Prepare the plate assembly on the autosampler. 9. Start the electrophoresis run. 34 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 3 3500/3500xL Instruments Set up the 3500/3500xL instruments for electrophoresis 3 Section 3.2 3500/3500xL instruments Set up the 3500/3500xL instruments for electrophoresis Appendix B, “Ordering Information” on page 113 lists the required materials 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. Keep freeze-thaw cycles to a minimum. Electrophoresis software setup and reference documents Genetic Analyzer Applied Biosystems® 3500 Applied Biosystems® 3500xL† The following table lists data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures, refer to the documents listed in the table. Data Collection Software 1.0 Operating System Run modules and conditions Windows® XP • HID36_POP4 Injection conditions: 1.2kV/15 sec or • Dye Set G5 Windows Vista ® • HID36_POP4 Injection conditions: 1.2kV/24 sec References Applied Biosystems® 3500/ 3500xL Genetic Analyzer User Guide (Part no. 4401661) 3500 and 3500xL Genetic Analyzers Quick Reference Card (Part no. 4401662) • Dye Set G5 † We conducted validation studies for the NGM SElect™ Express Kit using the 3130xl configuration or the 3500xL configuration. Prepare samples for electrophoresis on the 3500/3500xL instruments Prepare the samples for electrophoresis immediately before loading. 1. Calculate the volume of Hi-Di™ Formamide and GeneScan™ 600 LIZ® Size Standard v2.0 needed to prepare the samples: Reagent Volume per reaction GeneScan™ 600 LIZ® Size Standard v2.0 0.5 µL Hi-Di™ 9.5 µL Formamide Note: Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 35 3500/3500 xL Instruments Reagents and parts 3 Chapter 3 3500/3500xL Instruments Prepare samples for electrophoresis on the 3500/3500xL instruments IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. 2. Pipet the required volumes of components into an appropriately sized polypropylene tube. 3. Vortex the tube, then centrifuge briefly. 4. Into each well of a MicroAmp® Optical 96-Well Reaction Plate, add: • 10 µL of the formamide:size standard mixture • 1 µL of PCR product or Allelic Ladder Note: For blank wells, add 10 µL of Hi-Di™ Formamide. 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the plate in a thermal cycler for 3 minutes at 95°C. 7. Immediately place the plate on ice for 3 minutes. 8. Place the sample tray on the autosampler. 9. Start the electrophoresis run. 36 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 4 Analyze Data ■ Section 4.1 GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Overview of GeneMapper® ID Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Set up GeneMapper® ID Software for data analysis . . . . . . . . . . . . . . . . . . . . . . . 38 Analyze and edit sample files with GeneMapper® ID Software . . . . . . . . . . . . . 48 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 For more information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 ■ Section 4.2 GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Overview of GeneMapper® ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Set up GeneMapper® ID-X Software for data analysis . . . . . . . . . . . . . . . . . . . . . 51 Analyze and edit sample files with GeneMapper® ID-X Software . . . . . . . . . . . 63 Examine and edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 For more information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Section 4.1 GeneMapper® ID Software Overview of GeneMapper® ID Software GeneMapper® ID Software is an automated genotyping software for forensic casework, databasing, and paternity data analysis. After electrophoresis, the Data Collection Software stores information for each sample in an .fsa file. Using GeneMapper® ID Software v3.2.1 software, you can then analyze and interpret the data from the .fsa files. Instruments Refer to “Instrument and software overview” on page 16 for a list of compatible instruments. Before you start When using GeneMapper® ID Software v3.2.1 to perform human identification (HID) analysis with AmpFlSTR® kits, be aware that: • HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis. For multiple ladder samples, the GeneMapper® ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder. • Allelic ladder samples in an individual run folder are considered to be from a single run. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 37 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis When the software imports multiple run folders into a project, only the ladder(s) within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping. • Allelic ladder samples must be labeled as “Allelic Ladder” in the Sample Type column in a project. Failure to apply this setting for ladder samples results in failed analysis. • Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples, to ensure proper allele calling. • Alleles that are not in the AmpFlSTR® Allelic Ladders do exist. Off-ladder (OL) alleles may contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the ±0.5-nt bin window of any known allelic ladder allele or virtual bin. Note: If a sample allele peak is called as an off-ladder allele, verify the sample result according to your laboratory’s protocol. Set up GeneMapper® ID Software for data analysis File names The file names shown in this section may differ from the file names you see when you download or import files. If you need help determining the correct files to use, contact your local Life Technologies Human Identification representative, or go to www.lifetechnologies.com/supportSoftware, Patches & UpdatesGeneMapper® ID Software. Before using the software for the first time To analyze sample files (.fsa) using GeneMapper® ID Software v3.2.1 for the first time: • Import panels and bins into the Panel Manager, as explained in “Import panels and bins” on page 38. • Create an analysis method, as explained in “Create an analysis method” on page 42. • Create a size standard, as explained in “Create a size standard” on page 47. • Define custom views of analysis tables. Refer to the GeneMapper® ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no. 4335523) for more information. • Define custom views of plots. Refer to the GeneMapper® ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no. 4335523) for more information. Import panels and bins To import the latest panel and bin set from the our web site into the GeneMapper® ID Software v3.2.1 database: 1. Download and open the file containing panels and bins: a. From the Support menu of www.lifetechnologies.com/supportSoftware, Patches & UpdatesGeneMapper® ID Software, and download the file NGMSElectExpress Analysis Files GMID. b. Unzip the file. 38 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 2. Start the GeneMapper® ID Software, then log in with the appropriate user name and password. IMPORTANT! For logon instructions, refer to the GeneMapper® ID Software Version 3.1 Human Identification Analysis User Guide (Part no. 4338775). 3. Select ToolsPanel Manager. GeneMapper® ID Software 4. Find, then open the folder containing the panels and bins: a. Select Panel Manager in the navigation pane. b. Select FileImport Panels to open the Import Panels dialog box. c. Navigate to, then open the NGMSElectExpress Analysis Files GMID folder that you unzipped in step 1 on page 38. 5. Select NGMSElectExpress_panel_v1.txt, then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager, AmpFLSTR_NGMSElectExpress_v1. This folder contains the panel and associated markers. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 39 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis 6. Import the bins file: a. Select the AmpFLSTR_NGMSElectExpress_v1 folder in the navigation pane. b. Select FileImport Bin Set to open the Import Bin Set dialog box. c. Navigate to, then open the NGMSElectExpress Analysis Files GMID folder. d. Select NGMSElectExpress_bins_v1.txt, then click Import. Note: Importing this file associates the bin set with the panels in the NGMSElectExpress_panel_v1 folder. 7. View the imported panels in the navigation pane: a. Double-click the AmpFLSTR_NGMSElectExpress_v1 folder to view the NGMSElectExpress_panel_v1 folder. 40 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 b. Double-click the NGMSElectExpress_panel_v1 folder to display the panel information in the right pane. GeneMapper® ID Software 8. Select D10S1248 to display the Bin view for the marker in the right pane. 9. Click Apply, then OK to add the panel and bin set to the GeneMapper® ID Software database. IMPORTANT! If you close the Panel Manager without clicking OK, the panels and bins are not imported into the GeneMapper® ID Software database. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 41 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis Create an analysis method Use the following procedure to create an analysis method. 1. Select ToolsGeneMapper Manager to open the GeneMapper Manager. 2. Select the Analysis Methods tab, then click New to open the New Analysis Method dialog box. 3. Select HID and click OK to open the Analysis Method Editor with the General tab selected. 4. 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. 5. After you enter settings in all tabs, click Save. General tab settings In the Name field, either type the name as shown for consistency with files supplied with other AmpFlSTR® kits, or enter a name of your choosing. The Description and Instrument fields are optional. 42 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 Allele tab settings GeneMapper® ID Software • In the Bin Set field, select the NGMSElectExpress_bins_v1 bin set imported previously and configure the stutter distance parameters as shown. • GeneMapper® ID Software v3.2.1 allows you to specify four types of marker repeat motifs: tri, tetra, penta, and hexa. You can enter parameter values for each type of repeat in the appropriate column. • Specify the peak filter ratios: – To apply the stutter ratios listed in the Allele tab for single-source data, deselect the “Use marker-specific stutter ratio if available” check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. Note: Applying global stutter ratios may reduce the editing required for single-source sample data. – To apply the stutter ratios contained in the NGMSElectExpress_panel_v1 file, select the “Use marker-specific stutter ratio if available” check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. • To specify an Amelogenin Cutoff Ratio, enter the appropriate ratio into the Amelogenin Cutoff field. Note: Do not use an Amelogenin cutoff for data that may contain mixtures of male and female DNA AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 43 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis Peak Detector tab settings Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the peak amplitude thresholds for interpretation of data. Fields include: • Peak amplitude thresholds – The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks. Although GeneMapper® ID Software displays peaks that fall below the specified amplitude in electropherograms, the software does not label or determine the genotype of these peaks. • Size calling method – This kit has been validated using the Local Southern sizing method. Before using alternative sizing methods, perform the appropriate internal validation studies. 44 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis 4 Peak Quality tab settings GeneMapper® ID Software Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of data. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 45 4 Chapter 4 Analyze Data Set up GeneMapper® ID Software for data analysis Quality Flags tab settings IMPORTANT! The values shown are the software defaults and are the values we used during developmental validation. Perform the appropriate internal validation studies to determine the appropriate values to use in your laboratory. 46 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Set up GeneMapper® ID Software for data analysis Create a size standard 4 The GeneScan™ 600 LIZ® Size Standard v2.0 contains the following size standard peaks: GeneScan™ 600 LIZ® Size Standard v2.0 peak sizes 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 GeneMapper® ID Software To create the size standard definition file: 1. Select ToolsGeneMapper Manager to open the GeneMapper Manager. 2. Select the Size Standards tab, then click New. 3. Enter a name as shown below or enter a name of your choosing. In the Size Standard Dye field, select Orange. In the Size Standard Table, enter the sizes specified above. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 47 4 Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper® ID Software Analyze and edit sample files with GeneMapper® ID Software 1. In the Project window, select FileAdd Samples to Project, then navigate to the disk or directory containing the sample files. 2. Apply analysis settings to the samples in the project. The names of the settings shown are the names suggested in the sections above. If you named the settings differently, select the names you specified. Parameter Settings Sample Type Select the sample type. Analysis Method NGMSElectExpress_AnalysisMethod_v1 (or the name of the analysis method you created) Panel NGMSElectExpress_panel_v1 Size Standard CE_G5_NGMSElectExpress_GS600v2 (or the name of the size standard you created) For more information about how the Size Caller works, refer to the GeneScan® Analysis Software for the Windows NT® Operating System Overview of the Analysis Parameters and Size Caller User Bulletin (Part no. 4335617). For additional information about size standards, refer to the GeneMapper® ID Software Version 3.1 Human Identification Analysis User Guide (Part no. 4338775). 3. Click (Analyze), enter a name for the project (in the Save Project dialog box), then click OK to start analysis. During analysis: • The status bar displays the progress of analysis as both: – A completion bar extending to the right with the percentage completed indicated – Text messages on the left • The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample). • The Genotypes tab becomes available after analysis. 48 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.1 GeneMapper® ID Software Examine and edit a project 4 The following figure shows the analysis summary window after analysis. GeneMapper® ID Software Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data. These procedures start with the Samples tab of the Project window (assuming the analysis is complete). For more information For details about GeneMapper® ID Software features, allele filters, peak detection algorithms, and project editing, refer to: • GeneMapper® ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial (Part no. 4335523) • GeneMapper® ID Software Version 3.1 Human Identification Analysis User Guide (Part no. 4338775) • Installation Procedures and New Features for GeneMapper® ID Software Software Version v3.2 User Bulletin (Part no. 4352543) AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 49 4 Chapter 4 GeneMapper® ID-X Software Overview of GeneMapper® ID-X Software Section 4.2 GeneMapper® ID-X Software Overview of GeneMapper® ID-X Software GeneMapper® ID-X Software is an automated genotyping software for forensic casework, databasing, and paternity data analysis. After electrophoresis, the data collection software stores information for each sample in a .fsa or .hid file. Using GeneMapper® ID-X Software, you can then analyze and interpret the data from the .fsa files (GeneMapper® ID-X Software v1.0.1 or higher) or .hid files (GeneMapper® ID-X Software v1.2 or higher). Instruments Refer to “Instrument and software overview” on page 16 for a list of compatible instruments. Before you start When using GeneMapper® ID-X Software v1.0.1 or higher to perform human identification (HID) analysis with AmpFlSTR® kits, be aware that: • HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis. For multiple ladder samples, the GeneMapper® ID-X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder. • Allelic ladder samples in an individual run folder are considered to be from a single run. When the software imports multiple run folders into a project, only the ladder(s) within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping. • Allelic ladder samples must be labeled as “Allelic Ladder” in the Sample Type column in a project. Failure to apply this setting for ladder samples results in failed analysis. • Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling. • Alleles that are not in the AmpFlSTR® Allelic Ladders do exist. Off-ladder (OL) alleles may contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the ±0.5-nt bin window of any known allelic ladder allele or virtual bin. Note: If a sample allele peak is called as an off-ladder allele, verify the sample result according to your laboratory protocol. 50 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 Set up GeneMapper® ID-X Software for data analysis Panel, bin, and stutter file version The instructions and examples in this section refer to the latest version of panel, bin, and stutter file available at the time of publication. Before using the software for the first time Before you use GeneMapper® ID-X Software (v1.0.1 or higher for .fsa files, v1.2 or higher for .hid files) to analyze data for the first time, you must do the following: 1. Check the version of panel, bin, and stutter files installed with the GeneMapper® ID-X Software as explained in “Check panel, bin, and stutter file version” below. 2. Check www.lifetechnologies.com/supportSoftware, Patches & UpdatesGeneMapper® ID-X Software to determine if newer files are available. 3. If updated files are available, download and import the files into the GeneMapper® ID-X Software, as explained in “Import panels, bins, and marker stutter” on page 52. Note: When downloading new versions of analysis files, refer to the associated Read Me file for details of changes between software file versions. If you have validated previous file versions for data analysis, conduct the appropriate internal verification studies before using new file versions for operational analysis. 4. Create an analysis method, as explained in “Create an analysis method” on page 56. 5. Define custom views of analysis tables. Refer to Chapter 1 of the GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574) for more information. 6. Define custom views of plots. Refer to Chapter 1 of the GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574) for more information. Check panel, bin, and stutter file version 1. Start the GeneMapper® ID-X Software, then log in with the appropriate user name and password. IMPORTANT! For logon instructions, refer to the GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574). 2. Select ToolsPanel Manager. 3. Check the version of files imported into the Panel Manager: a. Select Panel Manager in the navigation pane. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 51 GeneMapper® ID-X Software The file names shown in this section may differ from the file names you see when you download or import files. If you need help determining the correct files to use, contact your local Life Technologies Human Identification representative, or go to www.lifetechnologies.com/supportSoftware, Patches & UpdatesGeneMapper® ID-X Software. 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis b. Expand the Panel Manager folder and any sub-folders to identify the analysis file version 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 FileImport Panels to open the Import Panels dialog box. b. Navigate to, then open the Panels folder and check the version of panel, bin, and stutter files installed. 5. If newer versions are available on the website, download and import as described below. Import panels, bins, and marker stutter To import the latest panel, bin set, and marker stutter from the our web site into the GeneMapper® ID-X Software database: 1. Download and open the file containing panels, bins, and marker stutter: a. Go to www.lifetechnologies.com/supportSoftware, Patches & UpdatesGeneMapper® ID-X Software. Download the file NGMSElectExpress Analysis Files GMIDX. b. Unzip the file. 2. Start the GeneMapper® ID-X Software, then log in with the appropriate user name and password. IMPORTANT! For logon instructions, refer to the GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574). 3. Select ToolsPanel Manager. 4. Find, then open the folder containing the panels, bins, and marker stutter: a. Select Panel Manager in the navigation pane. b. Select FileImport Panels to open the Import Panels dialog box. c. Navigate to, then open the NGMSElectExpress Analysis Files GMIDX folder that you unzipped above. 52 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 5. Select NGMSElectExpress_panel_v1X.txt, then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager, AmpFLSTR_NGMSElectExpress_v1X. This folder contains the panel and associated markers. GeneMapper® ID-X Software 6. Import the bins file: a. Select the AmpFLSTR_NGMSElectExpress_v1X folder in the navigation pane. b. Select File Import Bin Set to open the Import Bin Set dialog box. c. Navigate to, then open the NGMSElectExpress Analysis Files GMIDX folder. d. Select NGMSElectExpress_bins_v1X.txt, then click Import. Note: Importing this file associates the bin set with the panels in the AmpFLSTR_NGMSElectExpress_v1X folder. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 53 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 7. View the imported panels in the navigation pane: a. Double-click the AmpFLSTR_NGMSElectExpress_v1X folder. b. Double-click the NGMSElectExpress_panel_v1X folder to display the panel information in the right pane and the markers below it. 8. Select D10S1248 to display the Bin view for the marker in the right pane. 54 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 9. Import the stutter file: a. Select the AmpFLSTR_NGMSElectExpress_v1X folder in the navigation panel. c. Navigate to, then open the NGMSElectExpress Analysis Files GMIDX folder. d. Select NGMSElectExpress_stutter_v1X.txt, then click Import. Note: Importing this file associates the marker stutter ratio with the bin set in the AmpFLSTR_NGMSElectExpress_v1X folder. 10. View the imported marker stutters in the navigation pane: a. Select the AmpFLSTR_NGMSElectExpress_v1X folder to display the folder NGMSElectExpress_panel_v1X. b. Double-click the NGMSElectExpress_panel_v1X folder to display its list of markers below it. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 55 GeneMapper® ID-X Software b. Select FileImport Marker Stutter to open the Import Marker Stutter dialog box. 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis c. Double-click D22S1045 to display the Stutter Ratio & Distance view for the marker in the right pane. 11. Click Apply, then OK to add the panel, bin set, and marker stutter to the GeneMapper® ID-X Software database. IMPORTANT! If you close the Panel Manager without clicking Apply, the panels, bin sets, and marker stutter will not be imported into the GeneMapper® ID-X Software database. Create an analysis method Use the following procedure to 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 created for GeneMapper® ID-X Software version 1.2 is not compatible with earlier versions of GeneMapper® ID-X Software, or with GeneMapper® ID Software version 3.2.1. 1. Select ToolsGeneMapper® ID-X Manager to open the GeneMapper® ID-X Manager. 2. Select the Analysis Methods tab, then click New to open the Analysis Method Editor with the General tab selected. 56 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 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. GeneMapper® ID-X Software General tab settings In the Name field, either type the name as shown or enter a name of your choosing. In the Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. The Description and Instrument fields are optional. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 57 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis Allele tab settings • In the Bin Set field, select the NGMSElectExpress_bins_v1X bin set imported previously and configure the parameters as shown. • GeneMapper® ID-X Software allows you to specify 4 types of marker repeat motifs: tri, tetra, penta and hexa. You can enter parameter values for each type of repeat in the appropriate column. • Specify the peak filter ratios: – To apply the stutter ratios listed in the Allele tab for single-source data, deselect the “Use marker-specific stutter ratio if available” check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. Note: Applying global stutter ratios may reduce the editing required for single-source sample data. – To apply the stutter ratios contained in the NGMSElectExpress_stutter_v1X.txt file, select the “Use marker-specific stutter ratio if available” check box (selected by default). Perform appropriate internal validation studies to determine the appropriate filter setting to use. • To specify an Amelogenin Cutoff Ratio, enter the appropriate ratio into the Amelogenin Cutoff field. Note: Do not use an Amelogenin cutoff for data that may contain mixtures of male and female DNA 58 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 Peak Detector tab settings GeneMapper® ID-X Software Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of data. Fields include: • Peak amplitude thresholds – The software uses these parameters to specify the minimum peak height, in order to limit the number of detected peaks. Although GeneMapper® ID-X Software displays peaks that fall below the specified amplitude in electropherograms, the software does not label or determine the genotype of these peaks. • Size calling method – This kit has been validated using the Local Southern sizing method. Before using alternative sizing methods, perform the appropriate internal validation studies. • Normalization (v1.2 or higher) – For use with 3500 data. Perform internal validation studies to determine whether to use the Normalization feature for analysis of data. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 59 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis Peak Quality tab settings Perform internal validation studies to determine settings IMPORTANT! Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds, maximum peak height threshold, and the minimum peak height ratio threshold for interpretation of data. 60 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 4 SQ & GQ tab settings GeneMapper® ID-X Software IMPORTANT! The values shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use. Create a size standard The size standard for the GeneScan™ 600 LIZ® Size Standard v2.0 contains the following peaks: GeneScan™ 600 LIZ® Size Standard v2.0 peak sizes 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 Use the following procedure to create the size standard definition file: 1. Select ToolsGeneMapper® ID-X Manager to open the GeneMapper® ID-X Manager. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 61 4 Chapter 4 GeneMapper® ID-X Software Set up GeneMapper® ID-X Software for data analysis 2. Select the Size Standards tab, then click New. 3. Complete the Name field as shown below or with a name of your choosing. In the Security Group field, select the Security Group appropriate to your software configuration from the drop-down list. In the Size Standard Dye field, select Orange. In the Size Standard Table, enter the sizes specified on page 61. 62 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Section 4.2 GeneMapper® ID-X Software Analyze and edit sample files with GeneMapper® ID-X Software 4 Analyze and edit sample files with GeneMapper® ID-X Software 1. In the Project window, select FileAdd Samples to Project, then navigate to the disk or directory containing the sample files. 2. Apply analysis settings to the samples in the project. The names of the settings Parameter Settings Sample Type Select the sample type. Analysis Method NGMSElectExpress_AnalysisMethod_v1X (or the name of the analysis method you created) Panel NGMSElectExpress_panel_v1X Size Standard CE_G5_NGMSElectExpress_GS600v2 (or the name of the size standard you created) For more information about how the Size Caller works, or about size standards, refer to the GeneMapper® ID-X Software v1.2 Reference Guide (Part no. 4426481). 3. Click (Analyze), enter a name for the project (in the Save Project dialog box), then click OK to start analysis. During analysis: • The status bar displays the progress of analysis as a completion bar extending to the right with the percentage completed indicated. • The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample). • The Analysis Summary tab is displayed upon completion of the analysis. The following figure shows the analysis summary window after analysis. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 63 GeneMapper® ID-X Software shown are the names suggested in the sections above. If you named the settings differently, select the names you specified. 4 Chapter 4 GeneMapper® ID-X Software Examine and edit a project Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data. These procedures start with the Analysis Summary tab of the Project window (assuming the analysis is complete). For more information For more information about any of these tasks, refer to: • GeneMapper® ID-X Software Version 1.0 Getting Started Guide (Part no. 4375574) • GeneMapper® ID-X Software Version 1.0 Quick Reference Guide (Part no. 4375670) • GeneMapper® ID-X Software Version 1.0 Reference Guide (Part no. 4375671) • GeneMapper® ID-X Software Version 1.1(Mixture Analysis) Getting Started Guide (Part no. 4396773) • GeneMapper® ID-X Software Version 1.2 Reference Guide (Part no. 4426481) • GeneMapper® ID-X Software Version 1.2 Quick Reference Guide (Part no. 4426482) 64 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 5 Experiments and Results ■ Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 ■ Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 ■ Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 ■ Extra peaks in the electropherogram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 ■ Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 ■ Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 ■ Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 ■ Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 ■ Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 ■ Mutation rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 ■ Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 ■ Probability of paternity exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Overview Experiments using the NGM SElect™ Express Kit This chapter provides results of the developmental validation experiments we performed using the AmpFlSTR® NGM SElect™ Express PCR Amplification Kit. Importance of validation Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure’s efficiency, reliability, and performance characteristics. By challenging the procedure with samples commonly encountered in forensic and parentage laboratories, the validation process uncovers attributes and limitations that are critical for sound data interpretation in casework (Sparkes, Kimpton, Watson et al., 1996; Sparkes, Kimpton, Gilbard et al., 1996; Wallin et al., 1998). Experiment conditions The experiments to evaluate the performance of the NGM SElect™ Express Kit were performed according to the revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, July 10, 2003). Based on these guidelines, we conducted experiments that comply with guidelines 1.0 and 2.0 and its associated subsections. This DNA methodology is not novel. (Moretti et al., 2001; Frank et al., 2001; Wallin et al., 2002; and Holt et al., 2000). AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 65 5 Chapter 5 Experiments and Results Developmental validation This chapter discusses many of the experiments we performed and provides examples of results obtained. We chose conditions that produced optimum PCR product yield and that met reproducible performance standards. It is our opinion that while these experiments are not exhaustive, they are appropriate for a manufacturer of STR kits intended for forensic and/or parentage testing use. IMPORTANT! Each laboratory using the NGM SElect™ Express Kit must perform internal validation studies. Developmental validation SWGDAM guideline 1.2.1 “Developmental validation is the demonstration of the accuracy, precision, and reproducibility of a procedure by the manufacturer, technical organization, academic institution, government laboratory, or other party.” (SWGDAM, July 2003) SWGDAM guideline 2.10.1 “The reaction conditions needed to provide the required degree of specificity and robustness must be determined. These include thermocycling parameters, the concentration of primers, magnesium chloride, DNA polymerase, and other critical reagents.” (SWGDAM, July 2003) PCR components We examined the concentration of each component of the NGM SElect™ Express Kit and established that the concentration of each component was within the range where data indicated that the amplification met the required performance criteria for specificity, sensitivity, and reproducibility. For example, blood and buccal samples on treated paper substrates or swab sample lysates were amplified in the presence of varying concentrations of magnesium chloride, and the results were analyzed on an Applied Biosystems® 3130xl or 3500xL Genetic Analyzer. Results are shown in Figure 3. The performance of the multiplex is robust within ±20% of the optimal magnesium chloride concentration. 66 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Developmental validation 5 Figure 3 Buccal swab sample lysate amplified with the NGM SElect™ Express Kit in the presence of varying concentrations of magnesium chloride and analyzed on an Applied Biosystems® 3500xL Genetic Analyzer. +30% +20% +10% Optimal –10% –20% –30% Thermal cycler parameters Thermal cycling parameters were optimized using a Design of Experiments (DOE) approach that attempts to identify the combination of temperatures and hold times that produce the best assay performance. Optimal assay performance was determined through evaluation of assay sensitivity, peak-height balance and resistance to PCR inhibitors. For example, annealing temperatures of 57, 58, 59, 60, 61, and 62 °C were tested using a Veriti® 96-Well Thermal Cycler (Figure 4). The PCR products were analyzed using an Applied Biosystems® 3130xl Genetic Analyzer. Of the tested annealing temperatures, 58 to 60 °C produced robust profiles. At 62 °C the yield of certain loci was significantly reduced. The optimal combination of specificity, sensitivity, and resistance to PCR inhibition was observed at 59 °C. Thermal cycler temperature is critical to assay performance; therefore, routine, regularly scheduled thermal cycler calibration is strongly recommended. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 67 5 Chapter 5 Experiments and Results Developmental validation Figure 4 Electropherograms obtained from amplification of a buccal swab sample lysate at annealing temperatures of 57, 58, 59, 60, 61, and 62 °C, analyzed on an Applied Biosystems® 3130xl Genetic Analyzer (y-axis 6000 RFU). 57°C 58°C 59°C 60°C 61°C 62°C PCR cycle number NGM SElect™ Express Kit reactions were amplified for 23, 24, 25, 26, and 27 cycles on a Veriti® 96-Well Thermal Cycler using a buccal swab sample 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 (23–27) and off-scale data were collected for several allele peaks at 27 cycles (Figure 5). None of the cycle numbers tested produced non-specific peaks. 68 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Figure 5 Representative NGM SElect™ Express Kit profiles obtained from amplification of a buccal swab sample lysate using 23, 24, 25, 26, and 27 cycles, analyzed on an Applied Biosystems® 3130xl Genetic Analyzer (y-axis 8000 RFU) 23 cycles 24 cycles 25 cycles 26 cycles 27 cycles Accuracy, precision, and reproducibility SWGDAM guideline 2.9 “The extent to which a given set of measurements of the same sample agree with their mean and the extent to which these measurements match the actual values being measured should be determined.” (SWGDAM, July 2003) Accuracy Laser-induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology (Holt et al., 2000; and Wallin et al., 2002). However, accuracy and reproducibility of NGM SElect™ Express Kit profiles have been determined from various sample types. Figure 6 shows the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems® 3130xl Genetic Analyzer with POP-4® polymer. The x axis in Figure 6 represents the nominal nucleotide sizes for the AmpFlSTR® NGM SElect™ Express Allelic Ladder. The dashed lines parallel to the x axis represent ±0.25-nt windows. The y axis represents the deviation of each sample allele size from the corresponding Allelic Ladder allele size. All sample alleles are within ±0.5 nt from a corresponding allele in the Allelic Ladder. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 69 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Figure 6 Allele Size vs. Allelic Ladder Sizing for 42 samples analyzed on an Applied Biosystems® 3130xl Genetic Analyzer. Size and ladder sizing for the NGM SElect™ Express Kit were calculated using the GeneScan™ 600 LIZ™ v2.0 Size Standard. Precision and size windows Sizing precision enables the determination of accurate and reliable genotypes. Sizing precision was measured on an Applied Biosystems® 3130xl Genetic Analyzer. The recommended method for genotyping is to employ a ±0.5-nt “window” around the size obtained for each allele in the AmpFlSTR® NGM SElect™ Express Allelic Ladder. A ±0.5-nt window allows for the detection and correct assignment of alleles. Any sample allele that sizes outside the specified window could be: • An “off-ladder” allele, that is, an allele of a size that is not represented in the AmpFlSTR® NGM SElect™ Express Allelic Ladder or • An allele that does correspond to an Allelic Ladder allele, but whose size is just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times. Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument. Table 3 on page 71 shows typical precision results obtained from five runs (16 capillaries/run) of the AmpFlSTR® NGM SElect™ Express Allelic Ladder on an Applied Biosystems® 3130xl Genetic Analyzer (36-cm capillary and POP-4® polymer), using the GeneScan™ 600 LIZ™ v2.0 Size Standard. The results were obtained within a set of injections on a single capillary array. 70 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility 5 Sample alleles may occasionally size outside of the ±0.5-nt window for a respective Allelic Ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing. Figure 6 on page 70 illustrates the tight clustering of allele sizes obtained on the Applied Biosystems® 3130xl Genetic Analyzer, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5-nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith, 1995). For sample alleles that do not size within a ±0.5-nt window, the PCR product must be rerun to distinguish between a true off-ladder allele versus measurement error of a sample allele that corresponds with an allele in the Allelic Ladder. Repeat analysis, when necessary, provides an added level of confidence in the final allele assignment. GeneMapper® ID Software and GeneMapper® ID-X Software automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling the allele as OL (off-ladder). Maximum sizing precision is obtained within the same set of capillary injections. Cross-platform sizing differences occur due to a number of factors including type and concentration of polymer, run temperature, and electrophoresis conditions. Variations in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors. We strongly recommend that the allele sizes be compared to the sizes obtained for known alleles in the AmpFlSTR® NGM SElect™ Express Allelic Ladder from the same run and then be converted to genotypes as described in “Before you start” on pages 37 (GeneMapper® ID Software) and 50 (GeneMapper® ID-X Software). See Table 3 for the results of five runs of the AmpFlSTR® NGM SElect™ Express Allelic Ladder on an Applied Biosystems® 3130xl Genetic Analyzer. For more information on precision and genotyping, see Lazaruk et al., 1998 and Mansfield et al., 1998. In Table 3, the mean sizes for all the alleles in each run (16 capillaries) were calculated. The mean range shown in the table represents the lowest and highest mean size values obtained across all five runs. Similarly, the standard deviation for the allele sizing was calculated for all the alleles in each run. The standard deviation range shown in Table 3 represents the lowest and highest standard deviation values obtained across all five runs. Table 3 Precision results of five runs (16 capillaries/run) of the AmpFlSTR® NGM SElect™ Express Allelic Ladder Allele Mean Standard Deviation X 98.96–98.98 0.024–0.038 Y 104.99–105.02 0.027–0.039 8 74.74–74.83 0.030–0.044 9 78.97–79.06 0.026–0.043 10 83.17–83.26 0.028–0.044 11 87.34–87.45 0.031–0.053 12 91.49–91.60 0.028–0.042 AMEL D10S1248 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 71 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Allele Mean Standard Deviation 13 95.64–95.76 0.037–0.045 14 99.76–99.86 0.024–0.043 15 103.93–104.05 0.032–0.052 16 108.06–108.18 0.028–0.044 17 112.14–112.26 0.033–0.054 18 116.14–116.25 0.026–0.046 14 228.77–228.81 0.034–0.051 15 232.73–232.75 0.036–0.049 16 236.74–236.76 0.033–0.044 17 240.68–240.71 0.035–0.041 18 244.72–244.76 0.031–0.045 19 248.71–248.75 0.037–0.046 19.3 251.71–251.75 0.038–0.041 20 252.66–252.70 0.035–0.047 21 256.51–256.54 0.025–0.039 22 260.26–260.31 0.036–0.044 23 264.22–264.27 0.034–0.043 24 268.24–268.30 0.034–0.045 25 272.22–272.27 0.030–0.047 26 276.16–276.21 0.031–0.049 27 280.22–280.26 0.022–0.040 5 227.39–227.51 0.050–0.060 8 239.52–239.63 0.040–0.055 9 243.66–243.78 0.040–0.051 10 247.78–247.89 0.042–0.055 11 251.82–251.92 0.037–0.055 12 255.79–255.89 0.045–0.049 13 259.73–259.83 0.040–0.049 14 263.74–263.85 0.035–0.059 15 267.76–267.89 0.034–0.049 7 261.17–261.26 0.027–0.051 9 269.29–269.40 0.036–0.051 10 273.36–273.48 0.032–0.054 10.2 275.38–275.46 0.028–0.049 11 277.44–277.53 0.037–0.043 D12S391 D16S539 D18S51 72 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Allele Mean Standard Deviation 12 281.46–281.57 0.030–0.043 13 285.48–285.59 0.031–0.050 13.2 287.45–287.57 0.031–0.046 14 289.52–289.62 0.034–0.038 14.2 291.48–291.59 0.033–0.051 15 293.53–293.62 0.032–0.050 16 297.53–297.63 0.031–0.051 17 301.51–301.60 0.028–0.047 18 305.47–305.55 0.031–0.041 19 309.49–309.56 0.034–0.050 20 313.55–313.62 0.033–0.042 21 317.81–317.89 0.031–0.038 22 322.07–322.18 0.033–0.051 23 326.12–326.21 0.037–0.050 24 330.22–330.30 0.039–0.047 25 334.30–334.38 0.029–0.045 26 338.33–338.40 0.033–0.049 27 342.41–342.48 0.028–0.042 9 125.98–126.02 0.033–0.040 10 129.78–129.82 0.031–0.038 11 133.60–133.65 0.027–0.044 12 137.45–137.50 0.026–0.038 12.2 139.41–139.47 0.024–0.043 13 141.35–141.38 0.032–0.037 13.2 143.34–143.38 0.031–0.040 14 145.29–145.32 0.029–0.038 14.2 147.27–147.31 0.023–0.039 15 149.22–149.25 0.030–0.040 15.2 151.21–151.26 0.026–0.043 16 153.18–153.22 0.027–0.035 16.2 155.16–155.21 0.027–0.037 17 157.12–157.18 0.031–0.042 17.2 159.12–159.18 0.024–0.036 9 173.48–173.54 0.027–0.047 10 177.41–177.48 0.033–0.048 11 181.37–181.43 0.031–0.044 5 D19S433 D1S1656 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 73 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Allele Mean Standard Deviation 12 185.36–185.41 0.027–0.036 13 189.38–189.43 0.024–0.042 14 193.45–193.49 0.024–0.036 14.3 196.44–196.50 0.030–0.047 15 197.33–197.36 0.026–0.042 15.3 200.40–200.46 0.024–0.043 16 201.25–201.30 0.022–0.045 16.3 204.31–204.37 0.030–0.039 17 205.16–205.21 0.027–0.041 17.3 208.21–208.28 0.025–0.044 18.3 212.13–212.20 0.030–0.034 19.3 216.11–216.16 0.029–0.039 20.3 220.12–220.16 0.030–0.042 24 182.86–182.97 0.030–0.054 24.2 184.92–185.04 0.032–0.055 25 186.95–187.07 0.026–0.051 26 191.05–191.17 0.031–0.051 27 195.18–195.28 0.030–0.044 28 199.22–199.34 0.032–0.047 28.2 201.20–201.30 0.035–0.037 29 203.17–203.29 0.024–0.043 29.2 205.23–205.33 0.032–0.046 30 207.19–207.29 0.033–0.040 30.2 209.18–209.28 0.032–0.046 31 211.18–211.29 0.031–0.045 31.2 213.17–213.29 0.033–0.047 32 215.21–215.32 0.031–0.048 32.2 217.24–217.36 0.035–0.048 33 219.31–219.44 0.036–0.055 33.2 221.27–221.41 0.039–0.050 34 223.41–223.55 0.043–0.053 34.2 225.34–225.47 0.035–0.050 35 227.42–227.55 0.035–0.052 35.2 229.37–229.51 0.039–0.053 36 231.38–231.51 0.033–0.049 37 235.46–235.61 0.038–0.058 38 239.44–239.56 0.032–0.055 D21S11 74 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Allele Mean Standard Deviation 5 78.18–78.21 0.031–0.043 6 81.29–81.32 0.032–0.040 7 84.36–84.39 0.031–0.047 8 87.45–87.48 0.027–0.043 9 90.52–90.56 0.028–0.042 10 93.59–93.63 0.021–0.038 11 96.66–96.70 0.028–0.036 12 99.74–99.77 0.022–0.038 13 102.83–102.87 0.025–0.039 14 105.91–105.97 0.024–0.045 15 108.98–109.04 0.030–0.046 16 112.04–112.09 0.034–0.041 15 288.31–288.45 0.034–0.051 16 292.31–292.45 0.042–0.056 17 296.27–296.42 0.041–0.045 18 300.19–300.32 0.036–0.050 19 304.11–304.25 0.035–0.059 20 308.06–308.20 0.045–0.051 21 312.06–312.20 0.034–0.051 22 316.20–316.34 0.038–0.054 23 320.41–320.54 0.027–0.056 24 324.52–324.64 0.040–0.050 25 328.58–328.71 0.036–0.049 26 332.62–332.74 0.037–0.053 27 336.67–336.77 0.035–0.054 28 340.93–341.03 0.033–0.049 9 78.49–78.50 0.030–0.037 10 82.63–82.66 0.027–0.037 11 86.76–86.79 0.023–0.038 12 89.94–89.97 0.024–0.039 13 90.87–90.88 0.025–0.044 14 94.82–94.84 0.027–0.036 15 98.92–98.94 0.025–0.034 16 103.05–103.08 0.026–0.038 5 D22S1045 D2S1338 D2S441 D3S1358 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 75 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Allele Mean Standard Deviation 12 133.60–133.70 0.026–0.039 13 137.62–137.71 0.027–0.050 14 141.52–141.62 0.031–0.048 15 145.40–145.49 0.025–0.050 16 149.51–149.61 0.030–0.041 17 153.62–153.72 0.027–0.040 18 157.61–157.71 0.036–0.044 19 161.51–161.61 0.031–0.053 8 122.53–122.56 0.033–0.040 9 126.53–126.56 0.027–0.039 10 130.55–130.57 0.028–0.042 11 134.60–134.63 0.023–0.037 12 138.67–138.71 0.026–0.040 13 142.90–142.92 0.023–0.041 14 147.03–147.06 0.027–0.036 15 151.18–151.20 0.028–0.040 16 155.33–155.37 0.023–0.040 17 159.49–159.52 0.008–0.040 18 163.59–163.62 0.024–0.039 19 167.67–167.70 0.027–0.038 17 232.00–232.06 0.026–0.047 18 235.84–235.90 0.030–0.047 19 239.67–239.72 0.036–0.047 20 243.58–243.64 0.032–0.045 21 247.49–247.55 0.036–0.049 22 251.34–251.40 0.023–0.053 23 255.12–255.17 0.030–0.047 24 258.85–258.92 0.034–0.047 25 262.64–262.73 0.036–0.050 26 266.48–266.58 0.034–0.057 26.2 268.35–268.46 0.029–0.051 27 270.39–270.50 0.036–0.057 28 274.24–274.35 0.034–0.053 29 278.05–278.17 0.031–0.048 30 281.80–281.92 0.032–0.051 30.2 284.05–284.19 0.033–0.046 D8S1179 FGA 76 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Allele Mean Standard Deviation 31.2 287.85–287.98 0.035–0.047 32.2 291.64–291.77 0.035–0.046 33.2 295.44–295.57 0.034–0.053 42.2 330.53–330.68 0.040–0.055 43.2 334.39–334.54 0.037–0.055 44.2 338.38–338.53 0.041–0.061 45.2 342.30–342.45 0.044–0.067 46.2 345.90–346.07 0.045–0.057 47.2 349.74–349.92 0.046–0.073 48.2 353.77–353.93 0.046–0.063 50.2 361.42–361.59 0.053–0.074 51.2 365.26–365.44 0.037–0.070 4.2 311.01–311.05 0.030–0.039 6.3 320.41–320.46 0.031–0.062 8 325.55–325.58 0.031–0.061 9 329.62–329.66 0.035–0.057 11 337.65–337.71 0.034–0.047 12 341.73–341.79 0.039–0.054 13 345.82–345.87 0.038–0.046 14 349.89–349.93 0.028–0.050 15 353.93–353.96 0.032–0.041 16 358.04–358.09 0.032–0.057 17 362.12–362.18 0.029–0.055 18 366.17–366.23 0.031–0.048 19 370.10–370.16 0.032–0.049 20 374.14–374.20 0.021–0.041 20.2 376.13–376.18 0.026–0.047 21 378.13–378.19 0.025–0.043 21.2 380.11–380.18 0.024–0.043 22.2 384.19–384.25 0.035–0.048 23.2 388.29–388.34 0.027–0.043 24.2 392.33–392.38 0.031–0.045 25.2 396.31–396.36 0.032–0.050 26.2 400.39–400.44 0.029–0.048 27.2 404.38–404.41 0.028–0.056 28.2 408.32–408.37 0.033–0.061 29.2 412.30–412.35 0.035–0.050 5 SE33 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 77 5 Chapter 5 Experiments and Results Accuracy, precision, and reproducibility Allele Mean Standard Deviation 30.2 416.38–416.44 0.033–0.040 31.2 420.40–420.45 0.020–0.048 32.2 424.52–424.59 0.035–0.057 33.2 428.55–428.60 0.032–0.053 34.2 432.53–432.58 0.030–0.047 35 434.53–434.61 0.029–0.050 35.2 436.53–436.62 0.032–0.055 36 438.56–438.61 0.027–0.052 37 442.58–442.65 0.039–0.059 4 179.32–179.35 0.032–0.050 5 183.33–183.39 0.024–0.041 6 187.37–187.42 0.034–0.050 7 191.40–191.45 0.029–0.042 8 195.42–195.46 0.031–0.043 9 199.45–199.49 0.031–0.052 9.3 202.45–202.49 0.030–0.043 10 203.36–203.41 0.028–0.045 11 207.31–207.34 0.021–0.043 13.3 218.29–218.33 0.027–0.049 11 151.26–151.34 0.031–0.042 12 155.35–155.44 0.026–0.040 13 159.51–159.57 0.034–0.037 14 163.78–163.84 0.028–0.042 15 167.71–167.77 0.031–0.043 16 171.76–171.84 0.033–0.049 17 175.80–175.89 0.028–0.039 18 179.79–179.87 0.027–0.051 19 183.90–184.00 0.025–0.050 20 187.99–188.08 0.031–0.050 21 192.05–192.13 0.031–0.048 22 196.11–196.19 0.035–0.049 23 200.08–200.15 0.027–0.041 24 204.40–204.47 0.030–0.043 TH01 vWA 78 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 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 DAB Standard 8.1.2.2). Stutter products Stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller (or less frequently, one repeat larger) than the major STR product (Butler, 2005; Mulero et al., 2006). Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele (Walsh et al., 1996). The proportion of the stutter product relative to the main allele (stutter percent) is measured by dividing the height of the stutter peak by the height of the main allele peak. Peak heights were measured for amplified samples at the loci used in the NGM SElect™ Express Kit: • Treated paper workflow: 349 blood samples on FTA® Classic Card and 172 buccal samples on Indicating FTA® Cards • Buccal swab workflow: 147 buccal swab samples lysed in Prep-n-Go™ Buffer All data were generated on the Applied Biosystems® 3130xl Genetic Analyzer. Some conclusions from these measurements and observations are: • For each NGM SElect™ Express Kit locus, the stutter percentage generally increases with allele length, as shown in Figure 7 to Figure 12 on pages 80 to 82. • 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. • Stutter filter sets in GeneMapper® ID and GeneMapper® ID-X Software, calculated as the mean stutter for the locus plus three standard deviations (N = 668), are shown in Table 4 on page 83. Peaks in the stutter position that are above the stutter filter percentage specified in the software are not filtered. Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated. • 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. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 79 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Figure 7 Stutter percentages for D10S1248, D12S391 and D16S539 loci. (Blue and red colors indicate loci labeled with FAM™ and PET® dyes, respectively.) Figure 8 Stutter percentages for the D18S51, D19S433 and D1S1656 loci. (Green, black, and red colors indicate loci labeled with VIC®, NED™ and PET® dyes, respectively.) 80 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Figure 9 Stutter percentages for the D21S11, D22S1045 and D2S1338 loci. (Green, black/gray, and blue colors indicate loci labeled with VIC®, NED™ and FAM™ dyes, respectively. Black and gray data points associated with the D22S1045 locus indicate minus and plus stutter, respectively.) Figure 10 Stutter percentages for the FGA and TH01 loci. (Black data points indicate loci labeled with NED™ dye.) AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 81 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Figure 11 Stutter percentages for the D2S441, D3S1358, D8S1179, and vWA loci. (Red, green, and blue colors indicate loci labeled with and PET®, VIC®, and FAM™ dyes, respectively.) Figure 12 Stutter percentages for the SE33 locus. (Red data points indicate loci labeled with PET® dye.) 82 AmpFlSTR® NGM SElect™ 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 NGM SElect™ Express Kit loci Locus† % Stutter D10S1248 12.65 D12S391 16.02 D16S539 10.81 D18S51 13.78 D19S433 11.52 D1S1656 D1S1656 (–2 13.83 nt)‡ 3.63 D21S11 11.99 D22S1045 17.41 D22S1045 (+3 nt) 7.68 D2S1338 14.14 D2S441 10.69 D3S1358 13.12 D8S1179 10.91 FGA 11.93 SE33 SE33 (–2 14.82 nt)‡ 4.51 TH01 6.14 vWA 12.96 † These percentages are used as stutter filters in GeneMapper® ID v3.2.1 NGMSElectExpress_panel_v1 and GeneMapper® ID-X NGMSElectExpress_stutter_v1X. ‡ The –2nt stutter filters are not included in GeneMapper® ID NGMSElectExpress_panel v1 due to functional limitations of the software. IMPORTANT! The values shown are the values we determined during developmental validation studies. We recommend that laboratories perform internal validation studies to determine the appropriate values to use. Addition of 3´ A nucleotide 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. The efficiency of +A addition is related to the particular sequence of the DNA at the 3´ end of the PCR product. The NGM SElect™ Express Kit includes two main design features that promote maximum +A addition: • The primer sequences have been optimized to encourage +A addition. • The new, highly robust PCR chemistry allows complete +A addition with a short final incubation at 60°C for 5 minutes. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 83 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram This final extension step gives the DNA polymerase additional time to complete +A addition to all double-stranded PCR products. Figure 13 shows examples of incomplete and normal +A addition. Figure 13 Omitting the final extension step results in shoulders on main allele peaks due to incomplete A nucleotide addition. Examples shown are D2S441 (red) and FGA (black) data from an Applied Biosystems® 3130xl Genetic Analyzer using the NGM SElect™ Express Kit. 0 minute final extension 5 minute final extension 0 minute final extension 5 minute final extension Due to improved PCR buffer chemistry, the lack of +A addition is generally less of an issue with the NGM SElect™ Express Kit than with earlier generation kits. However, “shouldering” of allele peaks may still be observed if the amount of input DNA is greater than that recommended by the NGM SElect™ Express Kit protocol. Amplification of excess input DNA may also result in offscale data. Artifacts 84 Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible while anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise). Due to improvements in PCR primer manufacturing processes, the incidence of artifacts has been greatly reduced in the NGM SElect™ Express Kit. Kit electropherograms are essentially free of reproducible dye artifacts within the kit's read region of 70– 452 nt. Figure 14 on page 86 shows the very low baseline level fluorescence of a typical negative control PCR using the NGM SElect™ Express Kit. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Most STR loci produce minus-stutter peaks as a by-product of PCR amplification. A process of “slippage” has been proposed as a molecular mechanism for stutter, where the Taq DNA polymerase enzyme “slips” on the template DNA during replication and produces a minority PCR product that is shorter than the template strand, usually by one repeat unit. The stutter process may also occur in the opposite direction to produce amplicon DNA that is usually one repeat unit longer than the template strand, termed plus-stutter. While plus-stutter is normally much less significant than minus-stutter in STR loci with tetranucleotide repeats, the incidence of plus-stutter may be more significant in trinucleotide repeat-containing loci. The D22S1045 locus in the NGM SElect™ Express Kit is a trinucleotide repeat locus, and shows an elevated level of plus-stutter. For example, Figure 16 on page 87 is an electropherogram of the D22S1045 locus showing plus stutter. GeneMapper® ID-X analysis files supplied for use with the NGM SElect™ Express Kit contain a plus-stutter filter to prevent these peaks from being called in normal profiles. Figure 15 on page 86 shows an example of a non-standard (minus 2-nt) stutter that may be observed in certain STR loci such as SE33 and D1S1656 that exhibit more complex nucleotide sequences including regions of dinucleotide repeats. In cases where these stutter peaks exceed the peak amplitude threshold (e.g., 50 RFU), they may be detected by analysis software as additional alleles in the profile. It is important to consider possible noise and artifacts when interpreting data from the NGM SElect™ Express Kit on the Applied Biosystems® 3500/3500xL, 3130/3130xl, and 3100/3100-Avant Genetic Analyzers. Note that a high degree of magnification is used in the sample electropherograms shown in Figure 14 through Figure 16 on pages 86 to 87. Using a cycle number that is too high for the DNA input amount may cause artifacts, including: • Shoulder peaks due to incomplete +A addition. • Pull-up peaks caused by poor spectral separation when fluorescence signals exceeds the linear dynamic range for detection by the instrument. • Grass-like artifacts between 50 bp and 72 bp, which are outside the read region of the kit. Figure 17 on page 88 shows examples of shoulder peaks due to incomplete +A addition and grass-like artifacts between 50 bp and 72 bp. The grass-like artifacts are most commonly observed in the NED™ dye channel. These artifacts can be reduced by using a lower cycle number for samples with high DNA input amount. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 85 5 Chapter 5 Experiments and Results Extra peaks in the electropherogram Figure 14 Examples of fluorescence background in data produced on an Applied Biosystems® 3130xl Genetic Analyzer (y-axis 70 RFU). Figure 15 Example of a –2 nt reproducible artifact at the SE33 locus. Data produced on an Applied Biosystems® 3130xl Genetic Analyzer. 86 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra peaks in the electropherogram 5 Figure 16 NGM SElect™ Express Kit electropherogram showing plus stutter associated with the D22S1045 STR locus. Data produced on an Applied Biosystems® 3130xl Genetic Analyzer. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 87 5 Chapter 5 Experiments and Results Characterization of loci Figure 17 Comparison of the grass-like artifacts and shoulder peaks for a blood sample on treated paper amplified at 25 cycles (top) and 28 cycles (bottom). The artifacts are highlighted for illustrative purposes. The artifacts are greatly reduced by using a lower cycle number as shown in the top panel. Characterization of loci SWGDAM guideline 2.1 “The basic characteristics of a genetic marker must be determined and documented.” (SWGDAM, July 2003) This section describes basic characteristics of the 16 loci and the sex-determining marker, Amelogenin, which are amplified with the NGM SElect™ Express Kit. Most of these loci have been extensively characterized by other laboratories. Nature of the polymorphisms The primers for the Amelogenin locus flank a 6-nucleotide deletion within intron 1 of the X homolog. Amplification results in 104-nt and 110-nt products from the X and Y chromosomes, respectively. (Sizes are the actual nucleotide size according to sequencing results, including 3´ A nucleotide addition, and size may not correspond exactly to allele mobility observed on capillary electrophoresis platforms.) With the sole exception of D22S1045, a trinucleotide STR, the remaining NGM SElect™ Express Kit loci are tetranucleotide short tandem repeat (STR) loci. The length differences among alleles of a particular locus result from differences in the number of repeat units. All the alleles in the AmpFlSTR® NGM SElect™ Express Allelic Ladder, including microvariants, have been subjected to sequencing at Applied Biosystems®. In addition, other groups in the scientific community have sequenced alleles at some of these loci (Nakahori et al., 1991; Puers et al., 1993; Möller et al., 1994; Barber et al., 1995; Möller and Brinkmann, 1995; Barber et al., 1996; Barber and Parkin, 1996; Brinkmann et al., 1998; Momhinweg et al., 1998; Watson et al., 1998). Among the various sources of sequence data on the NGM SElect™ Express Kit loci, there is consensus on the repeat patterns and structure of the STRs. 88 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Species specificity 5 Mapping The NGM SElect™ Express 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). Genetic linkage Two sets of STR loci in the NGM SElect™ Express Kit are located on the same chromosomes. vWA and D12S391 are located approximately 6.3 million bp apart on the p arm of chromosome 12, while D2S1338 and D2S441 are located approximately 150 million bp apart on opposite arms of chromosome 2. Linkage disequilibrium analysis was conducted on 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 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 shared between the NGM™ and NGM SElect™ Kits were independent at the population level (note that the SE33 locus was not part of this analysis). Therefore, to calculate the rarity of a profile for comparison to single-source and mixture samples, the frequencies of all loci including vWA and D12S391 could be multiplied. However, the analysis of the CEPH pedigree families demonstrated a degree of linkage between vWA and D12S391 that does not support the assumption of independence for the purpose of kinship analysis. Table 5 GenePop LD Result (p value for pairwise analysis of loci) Chromosome Map Position† Chromosome Nuclear Coordinates† (million bp) vWA p13.31 5.9 D12S391 p13.2 12.2 D2S441 p14 68 D2S1338 q35 218 Locus AfricanAmerican (N = 350) Caucasian Hispanic (N = 350) (N = 293) 0.86 0.29 0.27 0.11 0.32 0.19 † STR locus mapping data was obtained from the NCBI Map Viewer http://www.ncbi.nlm.nih.gov/projects/ mapview/map_search.cgi?taxid=9606 or the UCSC Genome Browser (http://genome.ucsc.edu/). GenePop LD analysis probability results (p values) greater than 0.05 were considered to indicate that linkage disequilibrium between the loci within the population tested was not statistically significant. Species specificity SWGDAM Guideline 2.2 “For techniques designed to type human DNA, the potential to detect DNA from forensically relevant nonhuman species should be evaluated.” (SWGDAM, July 2003) The NGM SElect™ Express Kit provides the required specificity for detecting human alleles. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 89 5 Chapter 5 Experiments and Results Species specificity Nonhuman studies The following species were tested (in the specified amounts) using standard PCR and capillary electrophoresis conditions for the NGM SElect™ Express 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 assessed for the presence of any amplified peaks that would indicate cross-reactivity of the NGM SElect™ Express Kit with any of these non-human species. Figure 18 on page 90 shows example electropherogram results from the species specificity tests. All the primate DNA samples produced fragments within the read region (gorilla and macaque data not shown). Figure 18 Representative electropherograms for some species tested in a species specificity study including positive and non-template controls (NTC) Human Control DNA 007 Chimpanzee Dog Cat Horse Rat Microbes Negative Control 90 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Sensitivity 5 Among the non-primate species, most produced no peaks over a threshold of 50 RFU. Horse yielded reproducible VIC® -dye labeled peaks at 94 bp (less than 200 RFU) due to Amelogenin cross-reactivity. Individual replicate PCRs of cat DNA yielded small (less than 100 RFU) peaks at the 140 bp position. This cross-reactivity was not detectable when the cat DNA was amplified in the presence of human blood on an FTA® disc (data not shown). Sensitivity SWGDAM guideline 2.3 “When appropriate, the range of DNA quantities able to produce reliable typing results should be determined.” (SWGDAM, July 2003) Blood on treated paper The NGM SElect™ Express Kit has been optimized at 25 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying blood samples directly from unpurified 1.2 mm treated paper discs. Depending on the volume of blood spotted onto the treated paper, DNA quantities present on the 1.2 mm disc may vary from laboratory to laboratory. It is essential for your laboratory to optimize the PCR conditions based on the types of blood samples received or based on your standard operating protocol used in the spotting of blood onto treated paper. See page 19 for instructions on PCR optimization. Buccal cells on treated or untreated paper The NGM SElect™ Express Kit has been optimized at 25 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying buccal cells directly from unpurified 1.2 mm treated or untreated paper discs. Depending on the collecting devices used, the collection methods applied, and the swab-to-paper transfer protocol employed, DNA quantities present on the 1.2 mm disc may vary from sample to sample and from laboratory to laboratory. It is essential for your laboratory to optimize the PCR conditions based on the types of buccal samples received or based on your standard operating protocol used in transferring saliva from a buccal swab onto treated or untreated paper. Refer to page 19 for instructions on PCR optimization. Buccal swab lysate in Prep-n-Go™ Buffer The NGM SElect™ Express Kit has been optimized at 25 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying 5 µL buccal swab samples lysed in Prep-n-Go™ buffer. Depending on the collecting devices used and the collection methods applied, DNA quantities present in the lysates may vary from sample to sample and from laboratory to laboratory. It is essential for your laboratory to optimize the PCR conditions based on the types of buccal samples received and/or on your standard operating protocol. See page 19 for instructions on PCR optimization. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 91 5 Chapter 5 Experiments and Results Sensitivity 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 NGM SElect™ Express Kit, optimize PCR cycle number according to instructions on page 19. When the total number of allele copies added to the PCR is extremely low, unbalanced amplification of the alleles may occur because of stochastic fluctuation. Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA. 92 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Stability 5 Figure 19 Effect of amplifying varying amounts of white blood cells (WBCs) lysed in Prep-n-Go™ Buffer ~400 cells (2.4 ng) ~200 cells (1.2 ng) ~100 cells (0.6 ng) ~50 cells (0.3 ng) ~25 cells (0.15 ng) ~12 cells (0.07 ng) Negative control Note that the y-axis scale is magnified for the lower amounts of DNA, analyzed using the Applied Biosystems® 3130xl 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. Stability SWGDAM guideline 2.4 “The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults has been extensively documented. In most instances, assessment of the effects of these factors on new forensic DNA procedures is not required. However, if substrates and/or environmental and/or chemical insults could potentially affect the analytical process, then the process should be evaluated using known samples to determine the effects of such factors.” (SWGDAM, July 2003) AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 93 5 Chapter 5 Experiments and Results Stability DNA on FTA® Cards Aged samples collected from various individuals were prepared to examine the sample-on-substrate stability: • Finger-prick blood spotted onto FTA® Classic Cards stored for 150 days • Buccal cells collected with the EasiCollect™ device, stored for 110 days Aged FTA® samples were amplified with the NGM SElect™ Express Kit in a Veriti® 96-Well Thermal Cycler, then electrophoresed and detected using an Applied Biosystems® 3130xl Genetic Analyzer. The results of the aged blood on FTA® Classic Card are shown in Figure 20 and the results of the aged buccal cells on Indicating FTA® Card are shown in Figure 21. The analysis revealed that the age of the FTA® samples did not impact the performance of the NGM SElect™ Express Kit. Figure 20 Amplification of blood on FTA® card stored for various amounts of time at room temperature (y-axis 5000 RFU) Sample A 40 days Sample A 150 days Sample B 40 days Sample B 150 days Figure 21 Amplification of buccal cells on Indicating FTA® card stored for various amounts of time at room temperature (y-axis 4000 to 8000 RFU) Sample C 0 days Sample C 110 days Sample D 0 days Sample D 110 days 94 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data DNA on Copan 4N6 FLOQSwabs® 5 Aged buccal cell samples on Copan 4N6 FLOQSwabs® were also prepared to verify their respective sample-on-substrate stability. Buccal swabs were collected from 12 individuals over the course of three months. The aged Copan 4N6 FLOQSwabs® samples were lysed in Prep-n-Go™ Buffer, amplified using the NGM SElect™ Express Kit in a Veriti® 96-Well Thermal Cycler, and electrophoresed and detected using an Applied Biosystems® 3130xl Genetic Analyzer. The results of the aged buccal samples collected on Copan 4N6 FLOQSwabs® are shown in Figure 22. The analysis revealed that buccal samples on Copan 4N6 FLOQSwabs®, air-dried immediately after collection, and aged up to three months at room temperature produce acceptable profiles when amplified with the NGM SElect™ Express Kit. Figure 22 Amplification of buccal cells on Copan 4N6 FLOQSwabs® stored for various amounts of time at room temperature and lysed in Prep-n-Go™ Buffer (y-axis 3000 RFU) Sample E 0 days Sample E 3 months Sample F 0 days Sample F 3 months Population data SWGDAM guideline 2.7 “The distribution of genetic markers in populations should be determined in relevant population groups.” (SWGDAM, July 2003) 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 a suspects reference sample, 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 a suspects reference sample, then the suspect is included as a possible source of the evidence sample. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 95 5 Chapter 5 Experiments and Results Mutation rate The probability that another, unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant population(s). The NGM SElect™ Express Kit contains loci for which extensive population data are available. For additional information on 11 loci shared between many of the AmpFlSTR® kits, see the population data and additional studies section of the AmpFlSTR® SGM Plus™ PCR Amplification Kit User Guide (Part no. 44309589). Population samples used in these studies The NGM SElect™ 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 – 344 samples • Caucasian – 346 samples • Hispanic – 390 samples DNA was extracted using a 6100 Nucleic Acid PrepStation. In addition to the alleles that were observed and recorded in the Life Technologies 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 We compared allele calls between the NGM SElect™ Kit and the NGM SElect™ Express Kit. Genotype data from 200 blood samples on FTA® Classic Card showed 100% concordance between the two kits. Mutation rate Estimating germline mutations Estimation of spontaneous or induced germ-line mutation at genetic loci can be achieved by comparing the genotypes of offspring to those of their parents. From such comparisons the number of observed mutations are counted directly. Genotyping analysis of three CEPH families (1333, 1340, and 1345) was conducted using the NGM SElect™ Kit. These three-generation families, comprising 58 meioses altogether, showed three cases of apparent STR allele repeat number mutations, one in each family. Family 1333 had a mutation at the D12S391 locus that resulted in a change of allele 25 in a grandmother (individual 7341) to allele 24 in her daughter (individual 6987). Family 1340 had a mutation at the D8S1179 locus that resulted in a change of allele 10 in a grandfather (individual 6994) to allele 11 in his son (individual 7029). Family 1345 had a mutation at the SE33 locus that resulted in a change of allele 16 in a mother (individual 7348) to allele 15 in one of her sons (individual 7352). In all cases, the apparent mutation events resulted in either a gain or loss of a single repeat unit. In previous studies, genotypes of ten STR loci that were amplified by the AmpFlSTR SGM Plus® PCR Amplification Kit were determined for a total of 146 parent-offspring allelic transfers (meioses) at the Forensic Science Service, Birmingham, England. One length-based STR mutation was observed at the D18S11 locus; mutations were not detected at any of the other nine STR loci. The D18S11 mutation was represented by an increase of one 4-nt repeat unit, allele 17 was inherited as allele 18 (single-step mutation). The maternal/paternal source of this mutation could not be distinguished. 96 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Probability of identity Additional mutation studies 5 Additional studies (Edwards et al., 1991; Edwards et al., 1992; Weber and Wong, 1993; Hammond et al., 1994; Brinkmann et al., 1995; Chakraborty et al., 1996; Chakraborty et al., 1997; Brinkmann et al., 1998; Momhinweg et al., 1998; Szibor et al., 1998) of direct mutation rate counts produced: • Larger sample sizes for some of the NGM SElect™ Express Kit loci. • Methods for modifications of these mutation rates (to infer mutation rates indirectly for those loci where the rates are not large enough to be measured directly and/or to account for those events undetectable as Mendelian errors). Probability of identity Table 6 shows the allele frequencies at NGM SElect™ Express Kit loci by population group. Table 7 shows the Probability of identity (PI) values of the NGM SElect™ Express Kit loci individually and combined. The PI value is the probability that two individuals selected at random will have an identical NGM SElect™ Express Kit genotype (Sensabaugh, 1982). The PI values for the populations described in this section are then 8.12 ✕ 10 – 22 (African-American), 2.35 ✕ 10 – 21 (U.S. Caucasian) and 3.16 ✕ 10 – 21 (U.S. Hispanic). Table 6 Allele frequencies by population group for NGM SElect™ Express Kit STR loci. (Dashes indicate alleles not detected or, where values appear in parentheses, alleles not detected in significant quantities.) African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 6 -- (0.15) -- -- 7 -- (0.15) -- -- 8 -- -- -- (0.13) 9 -- (0.15) -- -- (0.13) 10 -- (0.15) -- -- (0.13) 11 3.63 -- (0.58) -- (0.26) 12 13.95 3.47 4.23 13 22.67 29.05 25.51 14 28.2 29.77 36.03 15 18.6 19.65 22.95 16 9.88 13.44 8.08 17 2.18 3.76 2.56 18 -- (0.29) -- (0.29) -- 19 -- -- -- 20 -- -- -- 13 -- -- -- (0.13) 14 -- -- -- (0.13) Allele D10S1248 D12S391 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 97 5 Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 15 7.12 4.19 3.97 15.1 -- (0.15) -- -- 16 5.09 3.47 5.13 16.1 -- (0.15) -- -- 17 15.7 10.55 7.31 17.1 -- (0.44) -- -- (0.26) 17.3 -- (0.58) 2.02 1.15 18 25.73 16.18 20 18.3 1.16 2.17 2.05 19 14.1 12.28 18.59 19.1 -- (0.58) -- -- 19.3 -- (0.29) -- (0.58) 1.15 20 11.92 9.83 17.31 20.3 -- -- -- 21 6.98 13.73 8.72 21.3 -- (0.15) -- (0.14) -- 22 5.09 10.69 6.92 23 3.34 8.09 3.72 24 -- (0.58) 3.61 1.79 25 0.87 2.02 1.28 26 -- -- (0.29) -- (0.26) 27 -- -- (0.14) -- (0.13) 28 -- -- -- 5 -- -- -- 6 -- -- -- (0.13) 7 -- -- -- 8 3.49 1.45 1.92 9 22.24 12.43 10.38 10 11.63 4.05 15.77 11 29.07 32.23 31.92 12 19.33 30.78 24.49 12.2 -- -- -- 13 12.94 17.2 13.97 14 1.31 1.73 1.15 15 -- -- (0.14) -- (0.26) 16 -- -- -- Allele D16S539 98 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 7 -- -- -- 8 -- -- -- 9 -- -- -- (0.13) 9.2 -- -- -- 10 -- (0.29) 1.16 0.64 10.2 -- (0.15) -- -- 11 -- (0.44) 0.87 1.15 11.2 -- -- -- 12 6.25 14.74 10.77 12.2 -- -- -- 13 4.07 11.85 11.54 13.2 -- (0.29) -- -- 14 5.81 17.49 15.77 14.2 -- (0.44) -- -- (0.13) 15 17.3 15.32 12.31 15.2 -- -- -- (0.13) 16 18.31 11.85 12.82 16.2 -- -- -- 17 15.7 10.98 17.05 17.2 -- -- -- 18 14.1 8.53 8.46 18.2 -- -- -- 19 9.16 4.34 3.46 19.2 -- -- -- 20 4.22 1.3 1.79 20.2 -- (0.15) -- -- 21 2.03 1.01 2.31 21.2 -- -- -- 22 1.02 -- (0.29) 0.64 22.2 -- -- -- 23 -- (0.29) -- (0.29) -- (0.51) 23.2 -- -- -- 24 -- -- -- (0.26) 25 -- -- -- (0.13) 26 -- -- -- 27 -- -- -- Allele 5 D18S51 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 99 5 Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 9 -- (0.29) -- -- 9.2 -- -- -- 10 1.16 -- (0.14) -- (0.38) 10.2 -- (0.15) -- -- 11 9.74 -- 1.54 11.2 -- (0.29) -- -- (0.26) 12 10.76 7.23 8.46 12.1 -- -- (0.14) -- 12.2 3.78 -- (0.14) 1.41 13 28.63 27.46 18.72 13.2 5.23 1.59 7.05 14 18.31 34.68 30.38 14.2 5.81 2.17 4.49 15 6.25 16.18 12.69 15.2 4.22 3.47 7.31 16 1.45 5.92 4.1 16.2 3.34 -- (0.29) 2.31 17 -- -- (0.29) -- (0.51) 17.2 -- (0.58) -- (0.14) -- (0.38) 18 -- -- (0.14) -- 18.2 -- -- -- 9 -- (0.15) -- -- (0.13) 10 1.31 -- (0.29) -- (0.38) 11 5.52 5.92 3.85 12 7.99 16.04 9.49 13 11.19 6.65 7.18 14 24.27 6.36 11.03 14.3 1.02 -- (0.29) -- (0.26) 15 17.44 15.17 16.03 15.3 1.89 8.53 2.95 16 10.32 9.97 15.26 16.1 -- -- -- (0.26) 16.3 7.27 4.91 5.26 17 2.62 4.91 6.92 17.1 -- -- (0.29) -- Allele D19S433 D1S1656 100 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 17.3 5.96 12.72 15.26 18 -- (0.44) -- (0.29) 0.77 18.3 1.89 5.92 4.36 19 -- (0.15) -- -- 19.3 -- (0.58) 1.73 0.64 20.3 -- -- -- 23.2 -- -- -- 24 -- -- -- 24.2 -- -- -- (0.26) 25 -- -- -- 25.2 -- -- -- 26 -- (0.29) -- (0.58) -- (0.38) 26.2 -- -- -- 27 5.52 2.75 1.41 27.1 -- (0.15) -- -- 27.2 -- -- -- 28 25.29 16.91 11.03 28.2 -- -- -- (0.13) 29 15.7 23.55 21.15 29.2 -- -- (0.14) -- 29.3 -- (0.15) -- (0.14) -- 30 20.93 23.41 27.95 30.2 1.6 2.75 1.67 31 8.72 6.79 5 31.2 4.94 8.67 11.28 32 1.31 2.31 1.28 32.2 6.98 9.54 12.44 33 0.87 -- -- (0.13) 33.2 3.2 2.02 5.26 34 -- (0.15) -- -- 34.2 -- -- (0.43) -- (0.13) 35 3.49 -- -- (0.26) 35.2 -- -- -- 36 0.73 -- -- (0.13) 36.2 -- -- -- 37 -- -- -- Allele 5 D21S11 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 101 5 Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 37.2 -- -- -- 38 -- -- -- (0.13) 38.2 -- -- -- 39 -- -- -- 5 -- -- -- 6 -- -- -- 7 -- -- -- 8 0.73 -- -- 9 -- -- -- 10 4.07 -- (0.43) 0.64 11 14.53 13.58 7.82 12 5.96 -- (0.58) 1.03 13 -- (0.29) 1.01 1.03 14 8.58 3.47 2.18 15 23.55 36.56 42.56 16 19.91 36.27 35.64 17 20.35 7.51 7.95 18 2.03 -- (0.58) 1.03 20 -- -- -- (0.13) 13 -- (0.15) -- (0.14) -- 14 -- -- (0.14) -- 15 -- (0.29) -- (0.14) -- 16 5.23 4.19 3.59 17 10.03 18.79 17.69 18 4.8 8.38 6.54 19 15.99 14.31 17.82 20 10.03 15.46 13.72 21 12.79 2.75 3.59 22 12.65 1.73 6.28 23 9.3 10.12 14.87 24 8.58 9.97 8.85 25 6.98 11.85 5.38 26 2.47 1.73 1.41 27 0.73 -- (0.29) -- (0.13) 28 -- -- -- (0.13) Allele D22S1045 D2S1338 102 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) -- -- -- 8 -- (0.15) -- -- 9 -- -- (0.58) -- (0.26) 10 8.87 19.8 31.15 11 35.03 33.82 31.67 11.3 3.34 5.06 4.36 12 20.06 4.05 3.97 12.3 -- (0.15) -- (0.29) -- (0.38) 13 3.63 3.18 1.92 13.3 -- -- -- 14 26.89 28.32 22.56 14.3 -- -- -- 15 1.89 4.48 3.33 16 -- -- (0.43) -- (0.38) 17 -- -- -- 9 -- (0.29) -- -- (0.13) 11 -- -- (0.29) -- 12 -- (0.15) -- -- (0.13) 13 -- (0.58) -- (0.14) -- (0.38) 14 9.16 15.17 9.49 15 28.34 27.31 34.49 15.2 -- (0.29) -- -- 16 32.85 23.99 26.54 16.2 -- -- -- 17 22.09 19.8 17.95 17.2 -- -- -- 18 5.81 11.85 10.13 18.2 -- -- -- 19 -- (0.44) 1.45 0.77 20 -- -- -- 7 -- -- -- 8 -- (0.44) 2.02 0.64 9 -- (0.29) 1.3 -- (0.26) 10 3.34 10.84 9.49 Allele 29 5 D2S441 D3S1358 D8S1179 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 103 5 Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 11 5.81 6.65 4.87 12 11.05 15.03 12.44 13 18.31 33.53 33.33 14 36.05 18.64 23.46 15 17.73 8.67 11.54 16 5.96 2.89 3.33 17 1.02 -- (0.43) 0.64 18 -- -- -- 19 -- -- -- 20 -- -- -- 16 -- -- (0.14) -- 16.1 -- (0.29) -- -- 16.2 -- -- -- 17 -- -- (0.14) -- 17.2 -- -- -- 18 0.87 1.16 0.64 18.2 0.73 -- -- 19 6.83 5.35 7.82 19.2 -- (0.44) -- -- 20 6.69 15.61 8.72 20.2 -- (0.29) -- (0.43) -- (0.26) 21 12.06 18.35 13.59 21.2 -- (0.15) -- (0.29) -- 22 18.17 18.93 14.1 22.2 -- (0.15) 0.87 0.64 23 16.86 14.6 12.69 23.2 -- -- (0.43) -- (0.38) 23.3 -- (0.29) -- -- 24 18.75 14.31 15.9 24.2 -- -- -- 25 9.3 6.79 14.1 25.2 -- -- -- 26 3.92 1.88 6.79 26.2 -- -- -- 27 2.62 -- (0.58) 2.95 27.2 -- -- -- Allele FGA 104 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 28 1.16 -- (0.14) 0.9 28.2 -- -- -- 29 -- -- -- (0.38) 29.2 -- -- -- 30 -- (0.15) -- -- (0.13) 30.2 -- (0.15) -- -- 31 -- -- -- 31.2 -- -- -- 32 -- -- -- 32.2 -- -- -- 33.2 -- -- -- 34.2 -- (0.15) -- -- 42.2 -- -- -- 43.2 -- -- -- 44.2 -- -- -- 45.2 -- -- -- 46.2 -- -- -- 47.2 -- -- -- 48.2 -- -- -- 49.2 -- -- -- 50.2 -- -- -- 51.2 -- -- -- 4.2 -- -- -- 5 -- -- -- 5.2 -- (0.15) -- -- 6 -- -- -- 6.3 -- -- (0.14) -- 7 -- -- -- 8 -- -- -- 8.2 -- -- -- 9 -- -- -- 9.2 -- -- -- 10 -- -- -- 10.2 -- -- -- 11 -- -- -- 11.2 0.73 -- -- (0.13) Allele 5 SE33 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 105 5 Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 12 -- (0.15) -- (0.43) -- (0.13) 12.1 -- -- (0.14) -- 12.2 -- (0.29) -- (0.14) -- (0.13) 13 1.31 0.87 1.15 13.2 -- (0.44) -- -- (0.13) 14 3.63 3.76 1.92 14.2 -- -- 0.77 14.3 -- -- (0.14) -- 15 3.92 3.61 4.74 15.2 -- (0.29) -- (0.14) -- (0.13) 16 6.98 5.2 5.51 16.2 -- (0.29) -- -- (0.26) 16.3 -- -- -- (0.13) 17 7.7 6.5 8.72 17.2 -- (0.15) -- -- 17.3 -- -- (0.14) -- 18 11.05 8.24 10 18.2 -- (0.15) -- -- (0.26) 19 15.26 8.09 7.95 19.2 -- (0.29) -- (0.29) -- 19.3 -- -- (0.43) -- 20 9.3 4.91 4.49 20.2 0.87 0.87 0.77 21 5.81 2.02 3.33 21.1 -- -- -- 21.2 1.02 1.16 1.28 22 1.74 -- (0.58) 1.03 22.2 1.45 3.32 2.18 23 -- (0.29) -- -- 23.2 -- (0.58) 2.6 2.69 23.3 -- -- -- (0.13) 24 -- (0.29) -- (0.14) -- (0.13) 24.2 1.6 4.48 2.18 25 -- -- -- 25.2 2.33 3.9 3.46 26 -- (0.15) -- -- (0.26) 26.2 5.67 4.19 6.92 Allele 106 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 27 -- -- -- 27.2 5.81 7.08 6.79 27.3 -- -- -- (0.13) 28 -- -- -- 28.2 4.07 7.8 5.9 29 -- -- -- 29.2 2.47 7.8 6.28 30 -- -- -- 30.2 1.45 4.91 3.97 31 -- -- -- (0.13) 31.2 1.02 2.89 2.18 32 -- -- (0.43) -- 32.2 0.73 1.59 2.05 33 -- -- (0.29) -- (0.38) 33.2 -- (0.44) -- (0.14) -- (0.51) 34 -- -- (0.29) -- (0.38) 34.2 -- (0.15) -- -- (0.26) 35 -- -- (0.14) -- 35.2 -- -- -- 36 -- -- (0.14) -- 36.2 -- -- -- 37 -- -- -- (0.13) 37.2 -- -- -- 38 -- -- -- 3 -- -- -- 4 -- -- -- 5 -- (0.44) -- (0.14) -- 5.3 -- -- -- 6 15.41 21.68 27.95 6.1 -- (0.15) -- -- 6.3 -- -- -- 7 37.06 17.77 31.92 7.3 -- -- -- 8 21.22 11.42 8.46 8.3 -- -- -- 9 15.84 17.05 12.56 Allele 5 TH01 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 107 5 Chapter 5 Experiments and Results Probability of identity African American Caucasian Hispanic (N = 344) (N = 346) (N = 390) 9.3 8.43 31.07 17.69 10 1.45 0.87 1.41 10.3 -- -- -- 11 -- -- -- 12 -- -- -- 13 -- -- -- 13.3 -- -- -- 10 -- -- -- 11 -- (0.44) -- -- (0.13) 12 -- -- -- (0.26) 13 0.73 -- (0.14) -- (0.13) 14 7.41 8.38 6.41 15 20.93 12.14 9.87 15.2 -- -- -- 16 27.47 22.69 30 17 19.48 27.31 27.18 17.3 -- -- -- (0.13) 18 13.81 18.06 18.46 18.2 -- -- -- 19 6.98 9.97 6.67 20 2.03 1.3 0.77 21 -- (0.58) -- -- 22 -- -- -- 23 -- (0.15) -- -- 24 -- -- -- 25 -- -- -- Allele vWA Table 7 Probability of identity (PI) values for the NGM SElect™ Express Kit STR loci African-American Caucasian Hispanic N = 344 N = 346 N = 390 D10S1248 0.0695 0.0937 0.1114 D12S391 0.0392 0.0232 0.0319 D16S539 0.0717 0.1042 0.0816 D18S51 0.0314 0.0312 0.0279 D19S433 0.0396 0.0852 0.0478 Locus 108 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Chapter 5 Experiments and Results Probability of paternity exclusion African-American Caucasian Hispanic N = 344 N = 346 N = 390 D1S1656 0.0335 0.0224 0.0247 D21S11 0.0454 0.0520 0.0502 D22S1045 0.0556 0.1326 0.1611 D2S1338 0.0226 0.0316 0.0318 D2S441 0.1014 0.0980 0.1068 D3S1358 0.0998 0.0749 0.0952 D8S1179 0.0755 0.0635 0.0682 FGA 0.0327 0.0387 0.0283 SE33 0.0121 0.0085 0.0079 TH01 0.0944 0.0798 0.0905 vWA 0.0617 0.0654 0.0913 Combined 8.12 x e –22 2.35 x e –21 3.16 x e –21 Locus 5 Probability of paternity exclusion Table 8 shows the Probability of paternity exclusion (PE) values of the NGM SElect™ Express Kit STR loci individually and combined. Table 8 Probability of paternity exclusion values for the NGM SElect™ Express Kit STR loci African-American Caucasian Hispanic N = 344 N = 346 N = 390 D10S1248 0.6590 0.5680 0.4690 D12S391 0.7450 0.7990 0.6620 D16S539 0.5560 0.5790 0.5570 D18S51 0.7860 0.7580 0.7180 D19S433 0.6360 0.5070 0.6470 D1S1656 0.7450 0.8110 0.7230 D21S11 0.7270 0.7290 0.6920 D22S1045 0.6980 0.4550 0.3970 D2S1338 0.7980 0.7520 0.7380 D2S441 0.5250 0.4970 0.4770 D3S1358 0.4760 0.5220 0.4820 D8S1179 0.5870 0.6160 0.5570 FGA 0.7270 0.6660 0.7080 SE33 0.8630 0.9120 0.8690 TH01 0.5150 0.5020 0.5390 vWA 0.6030 0.6440 0.6380 Locus AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 109 5 Chapter 5 Experiments and Results Probability of paternity exclusion African-American Caucasian Hispanic N = 344 N = 346 N = 390 PEi 9.451788e–09 1.455231e–08 8.163126e–08 Combined 0.9999999905 0.9999999854 0.9999999184 Locus 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 NGM SElect™ Express Kit STR loci (Chakraborty, Stivers, and Zhong, 1996). 110 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide A Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis. Table 9 Troubleshooting Observation Faint or no signal from both the Control DNA 007 and the DNA test samples at all loci Possible causes Recommended actions Incorrect volume or absence of Master Mix or Primer Set Repeat amplification. No activation of DNA Polymerase Repeat amplification, making sure to hold reactions initially at 95°C for 1 minute. Master Mix not vortexed thoroughly before aliquoting Vortex the Master Mix thoroughly. Primer Set exposed to too much light Store the Primer Set protected from light. PCR System malfunction Refer to the thermal cycler user’s manual and check instrument calibration. Use of incorrect thermal cycling parameters Check the protocol for correct thermal cycling parameters. MicroAmp® Base used with tray/ retainer set and tubes in GeneAmp® 9700 Remove MicroAmp Base from tray/retainer set and repeat test. Insufficient PCR product electrokinetically injected Prepare PCR product as described in Chapter 3, “Perform Electrophoresis” on page 31. Degraded formamide Check the storage of formamide; do not thaw and refreeze multiple times. Try Hi-Di™ Formamide. Sample punch location was not optimal 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 spot based on past experiences. For buccal samples collected with the Bode Buccal DNA Collector™, punch from near the tip of the collector. Insufficient lysis of the swab head AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Ensure swab heads are incubated for 20 minutes in 400 µL Prep-n-Go™ Buffer. 111 A Appendix A Troubleshooting Observation More than two alleles present at a locus Possible causes Recommended actions Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling. Amplification of stutter product (–1 repeat unit position) See “Experiments and Results” on page 65. Incomplete 3´ A base addition (n-1 nt position) See “Experiments and Results” on page 65. Be sure to include the final extension step of 60°C for 5 minutes in the PCR. Signal exceeds dynamic range of instrument (off-scale data) Ensure cycle number is optimized according to instructions on page 19. Repeat PCR amplification using fewer PCR cycles or use your laboratory’s SOP to analyze off-scale data. Poor spectral separation (bad matrix) Follow the steps for creating a spectral file. Confirm that Filter Set G5 modules are installed and used for analysis. Some but not all loci visible on electropherogram of DNA Test Samples STR profiles contain many off-scale alleles 112 Contamination carried over from the disc punching tool Clean the disc punching tool thoroughly. If necessary, include a blank punch step in between the sample punches. Incomplete denaturation of double stranded DNA Use recommended amount of Hi-Di™ Formamide and perform heat denaturation step according to the instructions in Chapter 3, “Perform Electrophoresis”. Disc size used in the amplification reaction was greater than 1.2 mm Repeat amplification using a use 1.2 mm punch size. Insufficient volume of swab lysate added to the reaction Repeat amplification using the recommended lysate input volume. Less than 25 µL of PCR reaction volume was used Repeat amplification using the recommended PCR reaction volume of 25 µL. PCR cycle number was too high Perform sensitivity experiment (page 19) to determine the optimal PCR cycle number based on the sample type. For blood samples: Too much liquid blood was spotted onto paper substrate. Spot <100 µL of liquid blood per sample area. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide B Ordering Information Equipment and materials not included Table 10 Equipment Equipment Applied Biosystems® 3100/3100-Avant Genetic Analyzer Applied Biosystems® 3500/3500xL Genetic Analyzer Applied Biosystems® 3130/3130xl Genetic Analyzer Veriti® 96-Well Thermal Cycler GeneAmp® Source Contact your local Life Technologies sales representative 4375786 PCR System 9700 with the Silver 96-Well Block N8050001 GeneAmp® PCR System 9700 with the Gold-plated Silver 96-Well Block 4314878 Silver 96-Well Sample Block Gold-plated Silver 96-Well Sample Block Tabletop centrifuge with 96-Well Plate Adapters (optional) Harris Manual Punch, 1.2 mm BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a 1.2 mm punch head BSD1000-GenePunch Automated Dried Sample Punch Instrument with a 1.2 mm punch head Copan NUCLEIC-CARD™ system N8050251 4314443 MLS (major laboratory supplier) MLS Contact your local Life Technologies support representative for information. Contact your local Life Technologies support representative for information. This product is not available for sale in the US. 96 well, deep well plate AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 4392904 113 B Appendix B Ordering Information Equipment and materials not included Table 11 User-supplied materials Item† Source AmpFlSTR® NGM SElect™ Express PCR Amplification Kit, 200 reaction 4467831 Prep-n-Go™ Buffer (untreated paper) 4467079 Prep-n-Go™ Buffer (buccal swab) 4471406 3100 Analyzer materials 96-Well Plate Septa 4315933 Reservoir Septa 4315932 3100/3130xl Genetic Analyzer Capillary Array, 36-cm 4315931 POP-4™ Polymer for 3100/3100-Avant Genetic Analyzers 4316355 3100/3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well 4316471 GeneScan™ 600 LIZ® Size Standard v2.0 4408399 Running Buffer, 10✕ 402824 Hi-Di™ 4311320 Formamide DS-33 Matrix Standard Kit (Dye Set G5) MicroAmp® 4345833 Optical 96-Well Reaction Plate N8010560 250-µL Glass Syringe (array-fill syringe) 4304470 5.0-mL Glass Syringe (polymer-reserve syringe) 628-3731 For a complete list of parts and accessories for the 3100 instrument, refer to Appendix B of the 3100 Genetic Analyzer and 3100-Avant Genetic Analyzer User Reference Guide (Part no. 4335393). 3130xl Analyzer materials 96-Well Plate Septa 4315933 Reservoir Septa 4315932 3100/3130xl Genetic Analyzer Capillary Array, 36-cm 4315931 POP-4® Polymer for 3130/3130xl Genetic Analyzers 4352755 3100/3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well 4316471 GeneScan™ 4408399 600 LIZ® Size Standard v2.0 Running Buffer, 10✕ 402824 DS-33 Matrix Standard Kit (Dye Set G5) 4345833 MicroAmp® Optical 96-Well Reaction Plate Hi-Di™ N8010560 Formamide 4311320 For a complete list of parts and accessories for the 3130xl instrument, refer to Appendix A of the Applied Biosystems® 3130/ 3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (Part no. 4352716). 3500/3500xL Analyzer materials Anode buffer container (ABC) 4393927 Cathode buffer container (CBC) 4408256 DS-33 Matrix Standard Kit (Dye Set G5) 4345833 POP-4® polymer (960 samples) for 3500/3500xL Genetic Analyzers 4393710 POP-4® polymer (384 samples) for 3500/3500xL Genetic Analyzers 4393715 114 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Appendix B Ordering Information Equipment and materials not included Item† B Source GeneScan™ 600 LIZ® Size Standard v2.0 4408399 Conditioning reagent 4393718 8-Capillary array, 36 cm for 3500 Genetic Analyzers 4404683 24-Capillary array, 36 cm for 3500xL Genetic Analyzers 4404687 96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers 4410228 8-Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers 4410231 8-Strip Septa for 3500/3500xL Genetic Analyzers 4410701 96-Well Septa for 3500/3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container, 3500 series 4410715 For a complete list of parts and accessories for the 3500/3500xL instrument, refer to the Applied Biosystems® 3500/3500xL Genetic Analyzer User Guide (PN 4401661). PCR Amplification MicroAmp® 96-Well Tray N8010541 MicroAmp® Reaction Tube with Cap, 0.2-mL N8010540 MicroAmp® 8-Tube Strip, 0.2-mL N8010580 MicroAmp® 8-Cap Strip N8010535 MicroAmp® 96-Well Tray/Retainer Set MicroAmp® 96-Well Base MicroAmp® Clear Adhesive Film 4306311 MicroAmp® Optical Adhesive Film 4311971 MicroAmp® Optical 96-Well Reaction Plate 403081 N8010531 N8010560 Other user-supplied materials Hi-Di™ Formamide, 25-mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape, labeling MLS Tube, 50-mL Falcon MLS Tube decapper, autoclavable MLS Deionized water, PCR grade MLS Vortex MLS † For the Safety Data Sheet (SDS) of any chemical not distributed by Life Technologies, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 115 B 116 Appendix B Ordering Information Equipment and materials not included AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide C Plate Layouts Example PCR plate layout The following layout is recommended for use with the sensitivity experiment on page 19. Create three identical plates for amplification at three different cycle numbers. Example electrophoresis plate layout The following layout is recommended for use with the sensitivity experiment on page 19. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 117 C 118 Appendix C Plate Layouts Example electrophoresis plate layout AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide D PCR Work Areas ■ Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 ■ PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 ■ Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory. If you are using an AmpFlSTR® Kit for: • Forensic DNA testing, refer to “Forensic Laboratories: Handbook for Facility Planning, Design, Construction and Moving,” National Institute of Justice, 1998 • Parentage DNA testing, refer to the “Guidance for Standards for Parentage Relationship Testing Laboratories,” American Association of Blood Banks, 7th edition, 2004 The sensitivity of AmpFlSTR® Kits (and other PCR-based tests) enables amplification of minute quantities of DNA, necessitating precautions to avoid contamination of samples yet to be amplified (Kwok and Higuchi, 1989). Also take care while handling and processing samples to prevent contamination by human DNA. Wear gloves at all times and change them frequently. Close sample tubes when not in use. Limit aerosol dispersal by handling sample tubes and reagents carefully. Note: We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology. PCR setup work area IMPORTANT! These items should never leave the PCR Setup Work Area. • Calculator • Gloves, disposable • Marker pen, permanent • Microcentrifuge • Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate clean tube (for Master Mix preparation) • Microcentrifuge tube rack • Pipette tips, sterile, disposable hydrophobic filter-plugged • Pipettors AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 119 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. You can use the following systems: • Veriti® 96-Well Thermal Cycler (Part no. 4375786) • GeneAmp® PCR System 9700 with the Silver 96-Well Block • GeneAmp® PCR System 9700 with the Gold-plated Silver 96-Well Block IMPORTANT! The NGM SElect™ Express Kit is validated for use with the Veriti® 96-well Thermal Cycler (Part no. 4375786) and the GeneAmp® PCR System 9700 with the silver block (Part no. N8050251) or the gold-plated silver block (Part no. 4314443). It is not verified for use with the Veriti® 96-Well Fast Thermal Cycler (Part no. 4375305) or the GeneAmp® PCR System 9700 with the aluminium block (Part no. 4314879). 120 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 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, etc). To obtain SDSs, see the “Documentation and Support” section in this document. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 121 E Appendix E Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: • Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document. • Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). • Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). • Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. • Handle chemical wastes in a fume hood. • Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) • After emptying a waste container, seal it with the cap provided. • Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. • Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. • IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. Specific chemical handling CAS 26628-22-8 122 Chemical Sodium Azide Phrase Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Appendix E Safety Biological hazard safety E Biological hazard safety WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards. WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: In the U.S.: • U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at: www.cdc.gov/biosafety • Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR§1910.1030), found at: www.access.gpo.gov/nara/cfr/waisidx_01/ 29cfr1910a_01.html • Your company’s/institution’s Biosafety Program protocols for working with/ handling potentially infectious materials. • Additional information about biohazard guidelines is available at: www.cdc.gov In the EU: Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization (WHO) Laboratory Biosafety Manual, third edition, found at: www.who.int/ csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/ AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 123 E 124 Appendix E Safety Biological hazard safety AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Bibliography Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J. Forensic Sci. 39:362–372. Bonferroni, C.E. 1936. Teoria statistica delle classi e calcolo Belle probabilita. Publicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8:3–62. Barber, M.D. and Parkin, B.H. 1996. Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179. Intl. J. Legal Med. 109:62–65. Bender, K., Farfan, M.J., Schneider, P.M. 2004. Preparation of degraded human DNA under controlled conditions. Forensic Sci. Int. 139:134–140. Brinkman, B., Klintschar, M., Neuhuber, F., Huhne, J. and Rolf, B. 1998. Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat. Am. J. Hum. Genet. 62:1408–1415. Brinkman, B., Moller, A. and Wiegand, P. 1995. Structure of new mutations in 2 STR systems. Intl. J. Legal Med. 107:201–203. Butler, J.M. 2005. Forensic DNA Typing. Burlington, MA:Elsevier Academic Press. Butler, J.M., Shen, Y., McCord, B.R. 2003. The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci. 48:1054–1064. Chakraborty, R. Kimmel, M., Stivers, D., Davison, L., and Deka, R. 1997. Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc. Natl. Acad. Sci. USA 94:1041–1046. Chakraborty, R., Stivers, D., and Zhong, Y. 1996. Estimation of mutation rates from parentage exclusion data: applications to STR and VNTR loci. Mutat. Res. 354:41–48. Chakraborty, R. and Stivers, D.N. 1996. Paternity exclusion by DNA markers: effects of paternal mutations. J. Forensic Sci. 41:671–677. Chung, D.T., Drabek, J., Opel, K.L., Butler, J.M. and McCord, B.R. 2004. A study of the effects of degradation and template concentration on the amplification efficiency of the Miniplex primer sets. J. Forensic Sci. 49:733–740. Clark J.M. 1988. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16:9677–9686. Coble, M.D. and Butler, J.M. 2005. Characterization of new miniSTR loci to aid analysis of degraded DNA. J. Forensic Sci. 50:43–53. DeFranchis, R., Cross, N.C.P., Foulkes, N.S., and Cox, T.M. 1988. A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA. Nucleic Acids Res. 16:10355. DNA Advisory Board, Federal Bureau of Investigation, U.S. Department of Justice. 1998. Quality assurance standards for forensic DNA testing laboratories. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 125 Bibliography Drabek, J., Chung, D.T., Butler, J.M., McCord, B.R. 2004. Concordance study between Miniplex assays and a commercial STR typing kit. J. Forensic Sci. 49:859–860. Edwards, A., Civitello, A., Hammond, H., and Caskey, C. 1991. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. 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Legal Med. 114:285–287. 128 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Documentation and Support Related documentation Part number Document title AmpFlSTR® NGM SElect™ Express PCR Amplification Kit Quick Reference – PCR Amplification and CE 4474110 AmpFlSTR® 4474108 NGM SElect™ Express PCR Amplification Kit Quick Reference – PCR Setup – Treated Paper Substrate AmpFlSTR® NGM SElect™ Express PCR Amplification Kit Quick Reference – PCR Setup – Untreated Paper Substrate 4475983 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit Quick Reference – PCR Setup – Swab Substrate 4475984 3100/3100-Avant Data Collection v2.0 User Guide 4347102 3100/3100-Avant Genetic Analyzers Using Data Collection Software v2.0 User Bulletin 4350218 3100 Genetic Analyzer User Manual (Data Collection v1.1) 4315834 3100/3100-Avant Genetic Analyzers Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products User Bulletin 4332345 Applied Biosystems® 3130/3100xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin 4363787 Applied Biosystems® 3130/3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems® 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide 4352716 Applied Biosystems® 3130/3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems® 3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 Applied Biosystems® 3130/3100xl DNA Analyzers User Guide 4331468 Applied Biosystems® 3500/3500xL Genetic Analyzer Quick Reference Card Applied Biosystems® 4401662 3500/3500xL Genetic Analyzer User Guide, Data Collection v1.0 Life Technologies 3500/3500xL Genetic Analyzer User Bulletin: Solutions to issues related to software, data, hardware, and consumables 4401661 4445098 Note: Additional user bulletins may be available at www.lifetechnologies.com Life Technologies 3730/3730xl Genetic Analyzer Getting Started Guide GeneAmp® 4359476 PCR System 9700 Base Module User’s Manual Quantifiler® N805-0200 Quantifiler® Kits: Human DNA Quantification Kit and Quantification Kit User’s Manual Quantifiler® Y Human Male DNA 4344790 GeneMapper® ID Software Version 3.1 Human Identification Analysis User Guide 4338775 GeneMapper® 4335523 ID Software Versions 3.1 and 3.2 Human Identification Analysis Tutorial Installation Procedures and New Features for GeneMapper® ID Software v3.2 User Bulletin GeneMapper® ID-X Software Version 1.0 Getting Started Guide AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 4352543 4375574 129 Documentation and Support Obtain SDSs Part number Document title GeneMapper® ID-X Software Version 1.0 Quick Reference Guide 4375670 GeneMapper® ID-X Software Version 1.0 Reference Guide 4375671 GeneMapper® ID-X Software Version 1.1 (Mixture Analysis) Getting Started Guide 4396773 GeneMapper® ID-X Software Version 1.1 (Mixture Analysis) Quick Reference Guide 4402094 GeneMapper® ID-X Software Version 1.2 Reference Guide 4426481 GeneMapper® ID-X Software Version 1.2 Quick Reference Guide 4426482 Portable document format (PDF) versions of this guide and the documents listed above are available at www.lifetechnologies.com. Note: To open the user documentation available from the our web site, use the Adobe® Acrobat® Reader® software available from www.adobe.com. Obtain SDSs Safety Data Sheets (SDSs) are available from www.lifetechnologies.com/support. Note: For the SDSs of chemicals not distributed by Life Technologies, contact the chemical manufacturer. Obtain support For HID support: • In North America – Send an email to [email protected], or call 888‐821‐4443 option 1. • Outside North America – Contact your local support office. For the latest services and support information for all locations, go to: www.lifetechnologies.com At the website, you can: • Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities • Search through frequently asked questions (FAQs) • Submit a question directly to Technical Support • Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents • Obtain information about customer training • Download software updates and patches 130 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Notices Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support. AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 131 Notices Limited Product Warranty 132 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Index Symbols .fsa sample files 38, 50 .hid sample files 50 +A nucleotide addition defined 83 efficiency of 83 Numerics 3100/3100-Avant instruments 33 3130/3130xl instruments 33 3500/3500xL instruments 35 A accuracy and reproducibility 69 alleles, off-ladder 70 allelic ladder profile 13 requirements for accurate genotyping 32 volume per reaction 34, 36 artifacts 84 B baseline noise, examples 86 bins check version 51 import 38, 52 biohazard safety 123 blood samples 11, 19, 21 Bode Buccal DNA Collector 11, 19, 24 buccal samples 11, 19, 21, 24 buccal swabs 26 C characterization of loci, validation 88 chemical safety 122 contents of kit 17 control DNA, about 18 Copan FLOQSwabs 95 cycle number, validation 68 D data accuracy, precision, reproducibility 69 for different populations 95 Data Collection Software 16 developmental validation 66 DNA effect of quantity, figure 93 negative control sample preparation 21, 24, 27 positive control sample preparation 21, 24, 27, 28 sensitivity 91 documentation, related 129 E electrophoresis Data Collection Software 33, 35 prepare samples 34, 35 references 33, 35 run module 33, 35 emission spectra 17 equipment, not included with kit 113 extra peaks, causes 79 F Fast model, Veriti thermal cycler, not supported 120 five-dye fluorescent system 12 fluorescent dyes 16 FTA cards 11, 19, 21 G GeneMapper ID Software analyze project 48 create analysis method 42 create size standard 47 examine and edit project 49 import panels and bins 38 overview 37 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 133 Index chromosomal location 12 dye label 12 linkage 89 mapping 89 low-TE buffer 21 lysate, prepare 26 set up 38 GeneMapper ID-X Software analyze project 63 check version of panels, bins, and stutter 51 create analysis method 56 create size standard 61 examine and edit project 64 import panels and bins 52 overview 50 GeneScan size standard about 18 dye label 16 volume per reaction 34, 35 master mix, volume per reaction 22, 25, 27 materials and equipment 17 multicomponent analysis 16 mutation studies 96 H O Hi-Di formamide, volume per reaction 34, 35 off-ladder alleles 70 operating systems 16, 33, 35 ordering information 113 I identity, probability of 97 instrumentation 3100/3100-Avant 33 3130/3130xl 33 3500/3500xL 35 software compatibility 16 K kit contents 17 description 11 fluorescent dyes 16 instruments for use with 11 loci amplified 12 master mix 17 primers 12, 17 purpose 11 reagents 17 storage 17 thermal cyclers for use with 120 L Limited Product Warranty 131 LIZ size standard about 18 volume per reaction 34, 35 loci characterization 88 134 M P panels check version 51 import 38, 52 part numbers 113 paternity exclusion, probability of 109 PCR components, validation of 66 cycle number, validation 68 optimize cycle number 19 perform 29 setup 119 PCR work areas 113, 119 percent stutter 79 plate layout examples 117 Prep-n-Go Buffer 11, 19, 24 primers about 12 Amelogenin 88 volume per reaction 22, 25, 27 probability of identity, definition 97 R reaction mix, for PCR 22, 25, 27 reactions, prepare for PCR 21, 24, 27 reproducibility 69 run module, electrophoresis 33, 35 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Index S U safety biohazard 123 chemical 122 Safety Data Sheets (SDSs), obtaining 130 sample files .fsa 38, 50 .hid 50 size deviation, sample alleles and ladder alleles 69, 70 sizing precision 70 software overview 50 species specificity 89, 94, 95 split peaks, +A nucleotide addition 84 standards for samples 18 STRBase 96 stutter check version 51 import 52 stutter peak or products 79 stutter percentages, marker-specific 83 substrates swab 26 treated paper 21 untreated paper 24 support, obtaining 130 swab PCR 29 prepare reactions 26 sample preparation guidelines 26 samples 11 validation 95 untreated paper 24 PCR 29 prepare reactions 24 sample preparation guidelines 24 T V validation accuracy, precision, reproducibility 69 annealing temperatures 67, 68 characterization of loci 88 conditions 65 developmental 66 effect of DNA quantity 92 loci characterization 88 mutation rate 96 PCR cycle number 68 population data 95 probability of identity 97 probability of paternity exclusion 109 sensitivity 91 size deviation, sample and ladder alleles 69, 70 species specificity 89 stutter 79 thermal cycling parameters 67 W warranty 131 work area amplified DNA 117, 120 PCR setup 119 setup and lab design 119 workflow overview 15 technical support 130 Terms and Conditions 131 thermal cyclers for use with kit 120 programming 29 Veriti Fast model not supported 120 training, information on 130 treated paper 21 PCR 29 prepare reactions 21 sample preparation guidelines 21 troubleshooting 111 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide 135 Index 136 AmpFlSTR® NGM SElect™ Express PCR Amplification Kit User Guide Headquarters 5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288 For support visit www.appliedbiosystems.com/support www.lifetechnologies.com 20 June 2014 ">

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Key features
- Direct amplification of single-source samples
- Amplifies 16 autosomal STR loci and Amelogenin
- Optimized for treated or untreated paper and swab substrates
- Validation for forensic and paternity testing
- Compatible with Applied Biosystems Genetic Analyzers
- Improved primer synthesis and purification
- Non-nucleotide linkers for efficient separation
- Five-dye fluorescent system for simultaneous amplification
Frequently asked questions
The kit is optimized for single-source samples, including blood and buccal samples collected on various substrates like treated or untreated paper, and buccal swabs.
The kit amplifies 16 autosomal STR loci (D10S1248, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656, D12S391, and SE33) and Amelogenin.
The NGM SElect™ Express Kit is compatible with Applied Biosystems® 3100/3100-Avant, 3130/3130xl, and 3500/3500xL Genetic Analyzers. It is also validated for use with Veriti® 96-Well Thermal Cycler and GeneAmp® PCR System 9700 with the silver or gold-plated silver blocks.