Thermo Fisher Scientific VeriFiler Express PCR Amplification Kit USER GUIDE

The VeriFiler Express PCR Amplification Kit is a 6-dye, short tandem repeat (STR) multiplex assay for the amplification of single-source human genomic DNA. This kit is optimized for paternity and single-source samples. The kit amplifies the following loci within the read region of 75– 465 nt: 23 autosomal STR loci, 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel) and Amelogenin (sex determining marker).

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VeriFiler

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Express PCR Amplification Kit

USER GUIDE

Catalog Numbers A32014, A32070, A33032

Publication Number 100043588

Revision B

For Research, Forensic, or Paternity Use Only.

For licensing and limited use restrictions visit thermofisher.com/HIDlicensing.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,

INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR

USE OF IT.

Revision history: Pub. No. 100043588

Revision

Date

14 November 2016

04 October 2016

Description

Add representative electropherograms for several types of substrate.

New document

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Windows and Windows Vista are trademarks of Microsoft Corporation. EasiCollect , Whatman, and FTA are trademarks of Whatman Limited. NUCLEIC-CARD and Copan are trademarks of Copan Italia S.P.A., and used by Life Technologies under their permission. Bode Buccal DNA Collector is a trademark of Bode

Technology Group, Inc. Harris Micro-Punch is a trademark of Harris, Joel S. TA Shunderson Communications. VWR Scientific is a trademark of VWR

International, Inc. Robbins Scientific is a trademark of Molecular Bioproducts, Inc. Agilent is a trademark of Agilent Technologies, Inc. Adobe, Acrobat, and Reader are trademarks of Adobe Systems Incorporated.

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CHAPTER 1 Product information ....................................... 8

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Kit overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Single-source sample types supported . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Substrate examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

About the primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Dyes used in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Loci amplified by the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Standards and controls that are required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Allelic ladder profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

DNA Control 007 profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Instrument and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■

CHAPTER 2 Perform PCR ............................................. 16

Optimize PCR cycle number (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Procedural guidelines Optimize PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Select samples and prepare plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Determine optimum PCR conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Thaw reagents (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Treated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . 18

Sample preparation guidelines: treated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . 18

Prepare low-TE buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Prepare the reactions: treated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Untreated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . 20

Sample preparation guidelines: untreated paper substrate . . . . . . . . . . . . . . . . . . . . . 20

Prepare the reactions: untreated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

VeriFiler

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Express PCR Amplification Kit User Guide

Contents

Swab substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Sample preparation guidelines: swab substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Prepare the sample lysate: room temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Prepare the sample lysate: heat protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Prepare the reactions: swab substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Store the sample lysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

■

CHAPTER 3 Perform electrophoresis ................................. 27

Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . 28

Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Obtain and run the HID Updater (v1 and v2 software only) . . . . . . . . . . . . . . . . . . . . . . . 28

Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . 30

Set up the 3130/3130 xl instruments for electrophoresis (before first use of the kit) . . . . . 31

Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Obtain and activate 6-dye license . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . 33

Set up the 3730 instruments for electrophoresis (before first use of the kit) . . . . . . . . . . . 34

Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Obtain and activate the 6-dye license . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Prepare samples for electrophoresis (3730 instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

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CHAPTER 4 Analyze data with GeneMapper

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ID ‑X

Software ....... 38

Overview of GeneMapper

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‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Set up the GeneMapper

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‑X Software for analysis (before use of the kit) . . . . . . . . . . . . . 40

Workflow: Set up GeneMapper

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‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Check panel, bin, and stutter file versions on your computer . . . . . . . . . . . . . . . . . . . . 40

Download newer versions of panel bin and stutter files if needed . . . . . . . . . . . . . . . . 40

Import panels, bins, and marker stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

(Optional) Define custom table or plot settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Enter Analysis Method settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

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Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

About the GS600_LIZ_ (60– 460) size standard definition file . . . . . . . . . . . . . . . . . . . . 52

If you use POP-7

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polymer on a 3730 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Create a size standard definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Analyze and edit sample files with GeneMapper

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‑X Software . . . . . . . . . . . . . . . . . . . . . 55

Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

For more information on using the GeneMapper

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‑X Software . . . . . . . . . . . . . . . . . . . . . 56

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CHAPTER 5 Experiments and results ................................. 57

Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

SWGDAM guideline 2.2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

SWGDAM guideline 3.9.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

PCR components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Thermal cycling temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Representative data for common sample and substrate types . . . . . . . . . . . . . . . . . . . . . . . . 62

Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

SWGDAM guideline 3.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Accuracy observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Precision and size window description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Precision and size window observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Causes of extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Extra peaks: Stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Extra peaks: Addition of 3' A nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Extra peaks: Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

SWGDAM guideline 3.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Loci in this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Nature of polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Genetic linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

SWGDAM Guideline 3.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Nonhuman studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

VeriFiler

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Express PCR Amplification Kit User Guide

Contents

Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

SWGDAM guideline 3.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Sample collection factors that can affect DNA quantity . . . . . . . . . . . . . . . . . . . . . . . . . 83

Effect of DNA quantity on results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

SWGDAM guideline 3.7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Population data overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Loci in this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Population samples used in these studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Concordance studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Probability of Identity definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Probability of paternity exclusion observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

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APPENDIX A Troubleshooting ........................................ 110

■

APPENDIX B Materials required but not supplied .................. 114

STR kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Sample preparation required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Treated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Untreated paper substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Swab substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Thermal cycler required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

ProFlex

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PCR System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

GeneAmp

™

PCR System 9700 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

Genetic analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

3500 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

3130 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

3730 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Analysis software required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

GeneMapper

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‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Miscellaneous required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Plates and tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Laboratory supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

■

APPENDIX C Plate layouts ............................................ 120

Example PCR plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Example electrophoresis plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

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APPENDIX D PCR work areas ........................................ 122

Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

■

Safety ..................................................................... 124

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

■

APPENDIX E Documentation and support .......................... 127

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

References

Index ..................................................................... 135

VeriFiler

™

Express PCR Amplification Kit User Guide

Product information

■

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

■

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■

Instrument and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

IMPORTANT! Before using this product, read and understand the information in the

“Safety” appendix in this document.

Product description

Kit overview

The Applied Biosystems

™

VeriFiler

™

Express PCR Amplification Kit is a 6-dye, short tandem repeat (STR) multiplex assay for the amplification of single-source human genomic DNA.

The kit is part of a fully integrated and validated forensic DNA workflow backed by best-in-class global training, service, and support. This kit is optimized for paternity and single-source samples.

The kit amplifies the following loci within the read region of 75– 465 nt:

• 23 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179,

D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317,

D7S820, D10S1248, D1S1656, D12S391, D2S1338, D6S1043, Penta D, Penta E)

• 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel)

• Amelogenin (sex determining marker)

The VeriFiler

™

Express kit delivers a 25-locus multiplex with the highest discrimination power of any Thermo Fisher Scientific Human Identification Kit. The highly optimized buffer formulation allows completion of amplification in

~45 minutes.

The VeriFiler

™

Express kit uses the same improved process for synthesis and purification of the amplification primers developed for other next-generation Thermo

Fisher Scientific STR chemistries. The improved amplification primers deliver clean electrophoretic backgrounds that assist interpretation.

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 1 Product information

Product description

Single-source sample types supported

Substrate examples

The VeriFiler

™

Express kit is optimized to allow direct amplification from the following types of single-source samples without the need for sample purification:

• Blood and buccal samples on treated paper substrates.

• Blood and buccal samples collected on untreated paper substrates and treated with Prep-n-Go

™

Buffer.

• Buccal samples collected on swab substrates and treated with Prep-n-Go

™

Buffer.

• Treated paper: NUCLEIC-CARD

™

system or Whatman FTA

™

cards

• Untreated paper: Bode Buccal DNA Collector

™

or 903 paper

• Swab: cotton swabs

Note: Our testing does not include blood samples on swab substrates. This sample type is not typically used for the collection of reference samples.

About the primers

The VeriFiler

™

Express kit primers are manufactured using the same synthesis and purification improvements as the primers in the NGM SElect

™

, the Sinofiler

™

, and the

Identifiler

™

Plus kits. These improvements enhance the assay signal-to-noise ratio and simplify the interpretation of results.

The VeriFiler

™

Express kit includes the same primers as the GlobalFiler

™

Express PCR

Amplification Kit with the following primer additions and modifications:

• Addition of new primers for Penta D, Penta E, and D6S1043.

• Addition of 1 new SNP-specific primer for D19S433 loci to address a SNP in the primer binding site.

Non-nucleotide linkers are used in primer synthesis for the following loci: D19S433, vWA, CSF1PO, D2S441, TH01, FGA, and D12S391. For these primers, non-nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis (Butler 2005, Grossman et al., 1994). Non-nucleotide linkers enable reproducible positioning of the alleles to facilitate interlocus spacing. The combination of a 6-dye fluorescent system and the use of non-nucleotide linkers allows simultaneous amplification and efficient separation of all 25 markers during automated DNA fragment analysis.

Dyes used in the kit

Label

Samples, allelic ladders, and controls

Dye

6 ‑FAM ™

VIC ™

NED ™

TAZ ™

SID ™

LIZ ™

Color

Blue

Green

Yellow

Red

Purple

Orange GeneScan ™ 600 LIZ ™ Size Standard v2.0

VeriFiler

™

Express PCR Amplification Kit User Guide

1 Chapter 1 Product information

Product description

Loci amplified by the kit

Table 1 VeriFiler ™ Express kit loci and alleles

Locus designation

D3S1358 vWA

D16S539

CSF1PO

TPOX

Y indel Yq11.221

1, 2

Amelogenin X: p22.1-22.3 Y: p11.2

X, Y

D8S1179 8q24.13

5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19

D21S11

D18S51

Penta E

D2S441

D19S433

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,

18q21.33

15q26.2

2p14

19q12

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

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

20, 21, 22, 23, 24, 25, 26

8, 9, 10, 11, 11.3, 12, 13, 14, 15, 16, 17

6, 7, 8, 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15,

15.2, 16, 16.2, 17, 17.2, 18.2, 19.2

TH01

FGA

D22S1045

D5S818

D13S317

D7S820

D6S1043

D10S1248

D1S1656

Chromosome location

3p21.31

Alleles included in Allelic Ladder

12p13.31

16q24.1

5q33.3-34

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20

11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24

5, 8, 9, 10, 11, 12,13, 14, 15

6, 7, 8, 9, 10, 11, 12, 13, 14, 15

2p23-2per 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15

11p15.5

4q28

22q12.3

5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18

13q22-31 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16

7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15

6q16.1

10q26.3

1q42.2

4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

26.2, 27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2,

43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19

9, 10, 11, 12, 13, 14, 14.3, 15, 15.3, 16, 16.3, 17,

17.3, 18.3, 19.3, 20.3

Dye label

6-FAM ™

VIC ™

NED ™

TAZ ™

SID ™

DNA Control

007

15, 16

14, 16

9, 10

11, 12

8, 8

X, Y

12, 13

28, 31

11, 16

11, 11

7, 12

12, 14

12, 15

13, 16

12, 15

7, 12

14, 15

7, 9.3

24, 26

VeriFiler

™

Express PCR Amplification Kit User Guide

Common Callouts and Arrows

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows

use the TechComm_Inkscape_Callout&Arrow_Libary.

2. Edit number and/or line-length, as needed.

3. Delete this text, this rectangle, and unused

callouts, arrows, or other SVG elements

before adding this SVG to the repository.

1 1 1

Chapter 1 Product information

Product description

Locus designation

D12S391

D2S1338

Penta D

Standards and controls that are required

Chromosome location

12p13.2

2q35

21q22.3

Alleles included in Allelic Ladder

14, 15, 16, 17, 18, 19, 19.3, 20, 21, 22, 23, 24, 25,

26, 27

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

25, 26, 27, 28

2.2, 3.2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17

Dye label

SID ™

DNA Control

007

18, 19

20, 23

11, 12

For the VeriFiler

™

Express kit, the panel of standards needed for PCR amplification,

PCR product sizing, and genotyping are:

• DNA Control 007—A positive control for evaluating the efficiency of the amplification step and STR genotyping using the VeriFiler

™

Express kit Allelic

Ladder. DNA Control 007 is present in the kit. See “DNA Control 007 profile“ on page 12.

• GeneScan

™

600 LIZ

™

Size Standard v2.0—Used for obtaining sizing results. This standard, which has been evaluated as an internal size standard, yields precise sizing results for PCR products. Order the GeneScan

™

600 LIZ

™

Size Standard v2.0 (Cat. No. 4408399) separately.

• VeriFiler

™

Express Allelic Ladder—Developed for accurate characterization of the alleles amplified by the kit. The Allelic Ladder is present in the kit, and allows automatic genotyping of most of the reported alleles for the loci in the kit. See

“Allelic ladder profile“ on page 11.

Allelic ladder profile

Figure 1 GeneMapper ™

ID ‑X Software plot of the VeriFiler

™ Express Allelic Ladder

VeriFiler

™

Express PCR Amplification Kit User Guide

Common Callouts and Arrows

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows

use the TechComm_Inkscape_Callout&Arrow_Libary.

2. Edit number and/or line-length, as needed.

3. Delete this text, this rectangle, and unused

callouts, arrows, or other SVG elements

before adding this SVG to the repository.

1 1 1

1 Chapter 1 Product information

Contents and storage

DNA Control 007 profile

Figure 2 DNA Control 007 (2 ng) amplified at 27 PCR cycles with the VeriFiler

™

Applied Biosystems

™

3500xL Genetic Analyzer (Y-axis scale 0 to 14,000 RFU).

Express kit and analyzed on an

Contents and storage

Table 2 VeriFiler ™ Express PCR Amplification Kit (Cat. No. A32014; 200 reactions)

Contents

VeriFiler ™ Express Master Mix

Contains MgCl

, dATP, dGTP, dCTP, and dTTP, bovine serum albumin, enzyme, and 0.05% sodium azide in buffer and salt.

VeriFiler ™ Express Primer Set

Contains locus-specific 6-FAM ™ , VIC ™ , NED ™ ,TAZ ™ , and SID ™ dye-labeled and unlabeled primers in buffer. The primers amplify the STR loci D3S1358, vWA, D16S539, CSF1PO,TPOX, D8S1179,

D21S11, D18S51, Penta E, D2S441, D19S433, TH01, FGA,

D22S1045, D5S818, D13S317, D7S820, D6S1043, D10S1248,

D1S1656, D12S391, D2S1338, Penta D, and the sex-determining markers, Y indel and Amelogenin.

Amount Storage

2 tubes, 1.0 mL –25°C to –15°C on receipt.

2°C to 8°C after first use, for up to 6 months or up to the expiration date stated on the kit (whichever comes first).

2 tubes, 1.0 mL –25°C to –15°C on receipt.

2°C to 8°C after first use, for up to 6 months or up to the expiration date stated on the kit (whichever comes first).

Store protected from light.

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 1 Product information

Contents and storage

Contents

VeriFiler ™ Express Allelic Ladder

Contains the following amplified alleles:

• 6-FAM ™ dye (blue): D3S1358 9–20; vWA 11–24; D16S539 5,

8–15; CSF1PO 6–15; TPOX 5–15.

• VIC

™

dye (green): Y indel 1, 2; Amelogenin X, Y; D8S1179

5-19; D21S11 24, 24.2, 25–28, 28.2, 29, 29.2, 30, 30.2, 31,

31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38; D18S51 7, 9,

10, 10.2, 11–13, 13.2, 14, 14.2, 15–27; DYS391 7–13; Penta E

5–26.

• NED ™ dye (yellow): D2S441 8–11, 11.3, 12–17; D19S433 6–

12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2, 18.2,

19.2; TH01 4–9, 9.3, 10, 11, 13.3; FGA 13–26, 26.2, 27–30,

30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2,

50.2, 51.2.

• TAZ

™

dye (red): D22S1045 8–19; D5S818 7–18; D13S317 5–

16; D7S820 6–15; D6S1043 9–25.

• SID ™ dye (purple): D10S1248 8–19; D1S1656 9–14, 14.3, 15,

15.3, 16, 16.3, 17, 17.3, 18.3, 19.3, 20.3; D12S391 14–19, 19.3,

20–27; D2S1338 11–28; Penta D 2.2, 3.2, 5–17.

DNA Control 007

Contains 2.0 ng/μL human male genomic DNA in 0.05% sodium azide and buffer.

The profile of this male DNA is D3S1358 15,16; vWA 14, 16;

D16S539 9, 10; CSF1PO 11,12; TPOX 8; D8S1179 12, 13; D21S11

28, 31; D18S51 12, 15; Penta E 7, 12; D2S441 14,15; D19S433

14,15; TH01 7, 9.3; FGA 24, 26; D22S1045 11, 16; D5S818 11;

D13S317 11; D7S820 7,12; D6S1043 12, 14; D10S1248 12, 15;

D1S1656 13, 16; D12S391 18, 19; D2S1338 20, 23; Penta D 11, 12.

Amount Storage

1 tube, 0.05 mL –25°C to –15°C on receipt.

2°C to 8°C after first use, up to the expiration date stated on the kit.

Store protected from light.

IMPORTANT!

The allelic ladder contains PCR products. Do not amplify. To avoid contamination, store the allelic ladder separate from the other kit components and unamplified DNA.

1 tube, 0.05 mL –25°C to –15°C on receipt.

2°C to 8°C after first use, up to the expiration date stated on the kit.

VeriFiler

™

Express PCR Amplification Kit User Guide

1 Chapter 1 Product information

Required materials not supplied

See Appendix B, “Materials required but not supplied“.

Instrument and software compatibility

Instrument type

Thermal cyclers

Validated models

• ProFlex ™ 96 ‑well PCR System (Cat. No. 4484075)

• GeneAmp ™ PCR System 9700, 96-Well Silver (Cat. No. N8050001)

• GeneAmp ™ PCR System 9700, 96-Well Gold-Plated (Cat. No. 4314878)

IMPORTANT! VeriFiler ™ Express kit is NOT validated for use with:

Veriti ™ 96 ‑Well Thermal Cycler (Cat. No. 4479071)

Veriti ™ 96 ‑Well Fast Thermal Cycler (Cat. No. 4375305)

GeneAmp ™ PCR System 9700, 96-Well Aluminum (Cat. No. 4314879)

Genetic analyzers

[1]

• 3500/3500xL Genetic Analyzer with any of the following:

– 3500 Data Collection Software v1 (Windows ™ Vista operating system) and HID Updater

3500 Data Collection Software v2 (Cat. No. 4480670)

– 3500 Data Collection v2 Software (Windows ™ 7 operating system) and HID Updater 3500

Data Collection Software v2 (Cat. No. 4480670)

– 3500 Data Collection v3 Software (Windows ™ 7 operating system)

• 3130/3130 xl Genetic Analyzer with:

– Data Collection Software v4 (Windows ™ 7 operating system)

– 3130/3730 Data Collection v4 6-Dye Module v1

• 3730 Genetic Analyzer

– Data Collection Software v4 (Windows ™ 7 operating system)

– 3130/3730 Data Collection v4 6-Dye Module v1

Analysis software

GeneMapper ™

ID ‑X Software v1.4 or later

Windows ™ XP or Windows ™ 7 operating system

[1]

We conducted validation studies using the 3130 xl, 3500, and 3500xL configurations.

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 1 Product information

Workflow

Perform PCR on treated or untreated paper substrates

Perform PCR on swab substrates

“Prepare the reactions: treated paper substrate“ on page 19 or

“Prepare the reactions: untreated paper substrate“ on page 21

▼

Obtain punch with Harris Manual Punch or BSD

Semi-Automated Dried Sample Punch Instrument

“Prepare the reactions: swab substrate“ on page 24

▼

Untreated paper only: Process with Prep ‑n‑Go ™

Buffer

▼

Process with VeriFiler

™

Express kit

Lyse in Prep ‑n‑Go ™ Buffer

▼

Process with VeriFiler

™

Express kit

▼ ▼

Amplify with the GeneAmp ™ PCR System 9700 or ProFlex ™ 96 ‑well PCR System

▼

Perform electrophoresis

“Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)“ on page 28 or

“Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)“ on page 31 or

“Set up the 3730 instruments for electrophoresis (before first use of the kit)“ on page 34

▼

“Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)“ on page 30 or

“Prepare samples for electrophoresis (3730 instruments)“ on page 36

▼

Analyze data

“Set up the GeneMapper ™ ID ‑X Software for analysis (before use of the kit)“ on page 40

“Create an analysis method“ on page 45

“Create a size standard definition file“ on page 53

“Analyze and edit sample files with GeneMapper ™ ID ‑X Software“ on page 55

“Examine or edit a project“ on page 56

VeriFiler

™

Express PCR Amplification Kit User Guide

Perform PCR

■

Optimize PCR cycle number (before first use of the kit) . . . . . . . . . . . . . . . . . . . . 16

■

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■

Treated paper substrates: prepare the amplification kit reactions . . . . . . . . . . . . 18

■

Untreated paper substrates: prepare the amplification kit reactions . . . . . . . . . 20

■

Swab substrates: prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . 23

■

Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Optimize PCR cycle number (before first use of the kit)

Before using the VeriFiler

™

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 kit. This experiment accounts for instrument-to-instrument and sample-to-sample variations. If you are processing multiple sample type and substrate combinations (for example, buccal samples on treated paper and buccal samples on swabs), perform separate sensitivity experiments for each sample type and substrate to be used for testing.

Procedural guidelines

Optimize PCR cycle number

Select samples and prepare plates

• Use 19 samples so that you can complete electrophoresis using a single 96-well plate. This minimizes the impact of run-to-run variation on the results.

Additional samples may be used for a more comprehensive sensitivity experiment. Examples of PCR and electrophoresis plate layouts are provided on

page “Example PCR plate layout“ on page 120.

• To maximize result quality, prepare and amplify Plate 1, then repeat for Plates 2,

3, and 4. Do not prepare all 4 plates before amplification.

• To minimize the effect of instrument-to-instrument variation, use the same thermal cycler to amplify all 4 plates.

1. Select 19 of each sample+substrate type. Ensure that the selected samples represent a "typical" range of samples analyzed in your laboratory.

2. Prepare the samples and the reactions as described in the appropriate protocols later in this chapter. Prepare sufficient PCR reagents to complete amplification of three replicate plates.

3. Create the first of 4 identical PCR plates (see page “Example PCR plate layout“ on page 120 for a suggested plate layout).

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 2 Perform PCR

Before you begin

Determine optimum PCR conditions

4. Amplify each plate using a different cycle number to determine the optimum conditions for use in your laboratory.

Suggested cycle numbers for different sample type and substrate combinations are listed in the following table.

Sample type

Blood

Buccal

Substrate

Treated paper Untreated paper

25, 26, 27, 28 cycles 25, 26, 27, 28 cycles

Swab

N/A

25, 26, 27, 28 cycles 25, 26, 27, 28 cycles 25, 26, 27. 28 cycles

1. Run the PCR products on the appropriate CE platform using the recommended

protocol that is described in Chapter 3, “Perform electrophoresis“.

2. Based on the results of the sensitivity study, select the appropriate PCR cycle number for future experiments.

Our studies indicate the optimum PCR cycle number should generate profiles with the following heterozygote peak heights, with no instances of allelic dropout and minimal occurrence of off-scale allele peaks:

Instrument

3500 Series

3130 Series

3730

Heterozygous peak height

3,000–12,000 RFU

1,000–3,000 RFU

3,000–12,000 RFU

When amplifying single-source, unpurified samples, you will see greater sample-to-sample variation in peak height than you see with purified samples.

Careful optimization of the cycle number helps to minimize this variation.

Before you begin

Thaw reagents

(before first use of the kit)

Thaw the Master Mix and Primer Set.

IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.

IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2°C to 8°C and do not require subsequent thawing. Do not refreeze the reagents.

VeriFiler

™

Express PCR Amplification Kit User Guide

2 Chapter 2 Perform PCR

Treated paper substrates: prepare the amplification kit reactions

Sample preparation guidelines: treated paper substrate

Prepare low-TE buffer

• Do not add water to the wells on the reaction plate before adding the punches. If you observe static issues with the paper discs, you can prepare and dispense the

25-µL reaction mix into the wells of the reaction plate before adding the punches.

Alternatively, dispense 3 µL of low-TE Buffer into each sample and negative amplification control well (not the positive amplification control wells) before adding the punches.

• Make the punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification.

• For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the punch in to the appropriate well on the reaction plate.

• For automated punching: See the User Guide of your automated or semiautomated disc punch instrument for proper guidance.

For optimal results, we recommend using low-TE buffer for sample preparation.

Prepare it as described in this procedure or buy it from Teknova (Cat. No. T0223).

1. Mix together:

• 10 mL of 1 M Tris-HCl, pH 8.0

• 0.2 mL of 0.5 M EDTA, pH 8.0

• 990 mL glass-distilled or deionized water

Note: Adjust the volumes accordingly for specific needs.

2. Aliquot, then autoclave the solutions.

3. Store the aliquots at room temperature.

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 2 Perform PCR

Treated paper substrates: prepare the amplification kit reactions

Prepare the reactions: treated paper substrate

IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.

If this is the first time you are using the kit, follow the instructions in “Thaw reagents

(before first use of the kit)“ on page 17 before proceeding.

1. Add samples to the MicroAmp

™

Optical 96-well Reaction Plate:

To these wells ...

Negative control

Test samples

Positive control

IMPORTANT!

Do not add a blank disc to the positive control well.

Add...

1.2 mm blank disc

1.2 mm sample disc

For 25 and 26 cycles 3 μL of Control DNA 007

For 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 can be adjusted if peak heights are too high or too low for your optimized cycle number.

2. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.

3. Pipet the required volumes of components into an appropriately sized polypropylene tube.

Reaction component

Master Mix

Primer Set

Low-TE buffer

Volume per reaction

10.0 μL

5.0 μL

Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.

IMPORTANT! This kit is optimized for a 25-µL PCR volume to overcome the

PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.

4. Vortex the reaction mix for 3 seconds, then centrifuge briefly.

5. Dispense 25 µL of the reaction mix into each reaction well of a MicroAmp

™

Optical 96-Well Reaction Plate.

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2 Chapter 2 Perform PCR

Untreated paper substrates: prepare the amplification kit reactions

6. Seal the plate with MicroAmp

™

Clear Adhesive Film (Cat. No. 4306311) or

MicroAmp

™

Optical Adhesive Film (Cat. No. 4311971).

IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across all wells.

IMPORTANT! If you are using the GeneAmp

™

PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp

™

Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.

7. Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.

8. Amplify the samples as described in Chapter 2, “Perform PCR“.

IMPORTANT! This kit is not validated for use with the GeneAmp

™

PCR System

9700 with the aluminum 96-well block. Use of this thermal cycling platform may adversely affect performance of this kit.

Untreated paper substrates: prepare the amplification kit reactions

Sample preparation guidelines: untreated paper substrate

• Make a 1.2 mm punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification.

• If you are using a Bode

Buccal DNA Collector

™

, make a 1.2 mm punch as close as possible to the tip of the DNA collector to ensure optimum peak intensity. A larger punch may cause inhibition during PCR amplification.

1 Location of punch with a Bode Buccal DNA Collector

™

• For automated punching: See the User Guide of your automated or semiautomated disc punch instrument for proper guidance.

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Chapter 2 Perform PCR

Untreated paper substrates: prepare the amplification kit reactions

Prepare the reactions: untreated paper substrate

IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.

If this is the first time you are using the kit, follow the instructions in “Thaw reagents

(before first use of the kit)“ on page 17 before proceeding.

1. Add Prep-n-Go

™

Buffer (Cat. No. 4467079) to the MicroAmp

™

Optical 96-Well

Reaction Plate:

To these wells ...

Negative control

Test samples

Positive control

For 27 cycles

For 28 cycles

Add...

5 μL of Prep ‑n‑Go ™ Buffer

For 25 and 26 cycles

2 μL of Prep ‑n‑Go ™ Buffer

3 μL of Prep ‑n‑Go ™ Buffer

4 μL of Prep

‑n‑Go ™

Buffer

2. Add samples to the reaction plate:

To these wells ...

Negative control

Test samples

Positive control

IMPORTANT!

Do not add a blank disc to the positive control well.

Add...

1.2 mm blank disc

1.2 mm sample disc

For 25 and 26 cycles 3 μL of Control DNA 007

For 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.

3. Centrifuge the plate to ensure that the punches are immersed in the Prep-n-Go

™

Buffer.

4. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.

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2 Chapter 2 Perform PCR

Untreated paper substrates: prepare the amplification kit reactions

5. Pipet the required volumes of components into an appropriately sized polypropylene tube.

Reaction component

Master Mix

Primer Set

Volume per reaction

10.0 μL

Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.

IMPORTANT! This kit is optimized for a 25-µL PCR volume to overcome the

PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.

6. Vortex the reaction mix for 3 seconds, then centrifuge briefly.

7. Dispense 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 plus Prep-n-Go

™

Buffer and sample or positive control).

8. Seal the plate with MicroAmp

™

Clear Adhesive Film (Cat. No. 4306311) or

MicroAmp

™

Optical Adhesive Film (Cat. No. 4311971).

IMPORTANT! If you are using the GeneAmp

™

PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp

™

Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.

9. Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.

10. Amplify the samples as described in Chapter 2, “Perform PCR“.

IMPORTANT! This kit is not validated for use with the GeneAmp

™

PCR System

9700 with the aluminum 96-well block. Use of this thermal cycling platform may adversely affect performance of this kit.

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 2 Perform PCR

Swab substrates: prepare the amplification kit reactions

Sample preparation guidelines: swab substrate

Prepare the sample lysate: room temperature

Prepare the sample lysate: heat protocol

• Detach each buccal swab head from the swab shaft before lysis.

• If you are using the heated lysis protocol, perform lysis in either of the following formats:

– 1.5-mL tubes with a heat block (VWR

™

Scientific Select dry heat block or similar)

– PrepFiler

™

96-Well Processing Plates (Cat. No. 4392904)

– Robbins Scientific

™

Model 400 Hybridization Incubator or similar

– Agilent

™

Benchtop Rack for 200 µL Tubes/V Bottom Plates (metal) or similar

(Cat. No. 410094)

IMPORTANT! Do not use a plastic plate adaptor.

• For optimum performance, lyse the entire swab. If you need to preserve the sample, use half of the lysate prepared from the entire swab.

This protocol may improve the performance for challenging or aged samples.

1. Add 400 µL Prep-n-Go

™

Buffer (Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a PrepFiler

™

96-Well Processing Plate (Cat. No. 4392904).

2. Into each tube or well, put the entire head of each swab, then let stand for

20 minutes at room temperature (20°C to 25°C) to lyse the sample.

3. After 20 minutes, transfer the sample lysate out of the sample plate into tubes or plates for storage, then discard the deep-well plate containing the swab heads.

Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate.

4. Go to “Prepare the reactions: swab substrate“ on page 24 or “Store the sample

lysate“ on page 25.

This protocol may improve the performance for challenging or aged samples.

1. Preheat the heat block to 90°C or the oven with metal plate adaptor to 99°C.

2. Add 400 µL Prep-n-Go

™

Buffer (for buccal swabs, Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a PrepFiler

™

96-Well Processing Plate

(Cat. No. 4392904).

3. Into each tube or well, put the entire head of each swab. If you are using tubes, cap the tubes. Let the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample.

4. After 20 minutes, remove the tubes or the deep-well plate from the heat block or oven.

5. Let the lysate stand at room temperature for at least 15 minutes to cool the lysate

(for accurate pipetting).

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2 Chapter 2 Perform PCR

Swab substrates: prepare the amplification kit reactions

Prepare the reactions: swab substrate

6. Transfer the sample lysate out of the 1.5-mL tubes or sample plate into tubes or plates for storage. Discard the 1.5-mL tubes or deep-well plate containing the swab heads.

Note:

To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate.

7. Go to “Prepare the reactions: swab substrate“ on page 24 or “Store the sample

lysate“ on page 25.

IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.

If this is the first time you are using the kit, follow the instructions in “Thaw reagents

(before first use of the kit)“ on page 17 before proceeding.

1. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.

2. Pipet the required volumes of components into an appropriately sized polypropylene tube.

Reaction component

Master Mix

Primer Set

Low-TE buffer

Volume per reaction

10.0 μL

3.0 μL

Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers.

IMPORTANT! This kit is optimized for a 25-µL PCR volume to overcome the

PCR inhibition that is expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles.

3. Vortex the reaction mix for 3 seconds, then centrifuge briefly.

4. Dispense 23 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 plus Prep-n-Go

™

Buffer or sample lysate).

VeriFiler

™

Express PCR Amplification Kit User Guide

Store the sample lysate

Chapter 2 Perform PCR

Swab substrates: prepare the amplification kit reactions

5. Add samples to the MicroAmp

™

Optical 96-Well Reaction Plate:

To these wells ...

Test samples

Positive control

Add...

2 μL of sample lysate

For 25 and 26 cycles

For 27 cycles

3 μL of Control

DNA 007

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.

6. Seal the plate with MicroAmp

™

Clear Adhesive Film (Cat. No. 4306311) or

MicroAmp

™

Optical Adhesive Film (Cat. No. 4311971).

IMPORTANT! If you are using the GeneAmp

™

PCR System 9700 with silver or gold-plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp

™

Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. Other validated thermal cyclers do not require a compression pad.

7. Vortex the reaction mix at medium speed for 3 seconds.

8. Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.

9. Amplify the samples as described in Chapter 2, “Perform PCR“.

IMPORTANT! This kit is not validated for use with the GeneAmp

™ PCR System

9700 with the aluminum 96-well block or the Veriti ™ Thermal Cyclers. Use of these thermal cycling platforms may adversely affect performance of this kit.

1. Cap the sample lysate storage tubes or seal the sample lysate storage plate with

MicroAmp

™

Clear Adhesive Film.

2. Store the sample lysate as needed:

If you are storing the sample lysate...

<2 weeks

>2 weeks

Then place at...

2°C to 8°C

–25°C to –15°C

Note: The effects of multiple freeze/thaw cycles on the lysate have not been fully evaluated. Therefore, multiple freeze/thaw cycles are not recommended.

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2 Chapter 2 Perform PCR

Perform PCR

IMPORTANT! This kit is validated for use with the validated thermal cyclers listed in

“Instrument and software compatibility“ on page 14

1. Program the thermal cycling conditions.

IMPORTANT! If you are using the GeneAmp

™

PCR System 9700, select the Max ramping mode. If you are using the ProFlex

™

96-well PCR System, select the

GeneAmp

™

PCR System 9700 simulation mode.

Initial incubation step

HOLD

95°C,

1 minute

Optimum cycle number

[1]

Denature Anneal Extend

94°C,

3 seconds

CYCLE

59°C,

16 seconds

65°C,

29 seconds

Final extension

HOLD

60°C,

5 minutes

Final hold

HOLD

4°C, up to 24 hours

[2]

[1]

[2]

See “Optimize PCR cycle number (before first use of the kit)“ on page 16.

The infinity (∞) setting allows an unlimited hold time.

2. Load the plate into the thermal cycler, close the heated cover, then start the run.

IMPORTANT! If you are using adhesive clear film instead of caps to seal the plate wells, be sure to place a MicroAmp

™

Optical Film Compression Pad

(Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. The ProFlex

™

96-well PCR System does not require a compression pad.

3. When the run is complete, store the amplified DNA.

If you are storing the DNA...

<2 weeks

>2 weeks

Then place at...

2°C to 8°C

–25°C to –15°C

IMPORTANT! Protect the amplified DNA from light.

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Perform electrophoresis

■

Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

■

Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■

Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■

Prepare samples for electrophoresis (3500 Series and 3130

Series instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

■

Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

■

Prepare samples for electrophoresis (3500 Series and 3130

Series instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

■

Set up the 3730 instruments for electrophoresis (before first use of the kit) . . . 34

■

Prepare samples for electrophoresis (3730 instruments) . . . . . . . . . . . . . . . . . . . . 36

Allelic ladder requirements for electrophoresis

To accurately genotype samples, you must run an allelic ladder with the samples.

Instrument

3500

3500xL

3130

3130 xl

3730

Number of allelic ladders to run

One injection equals

1 per 3 injections 8 samples

1 per injection 24 samples

1 per 4 injections 4 samples

1 per injection 16 samples

3 per injection 48 samples

Number of samples per allelic ladder(s)

23 samples + 1 allelic ladder

15 samples + 1 allelic ladder

IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between runs. Follow the guidelines in the preceding table, which should account for normal variation in run speed. Perform internal validation studies to verify the required allelic ladder injection frequency, to ensure accurate genotyping of all samples in your laboratory environment.

It is critical to genotype using an allelic ladder run under the same conditions as the samples. Size values obtained for the same sample can differ between instrument platforms, because of different polymer matrices and electrophoretic conditions.

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3 Chapter 3 Perform electrophoresis

Materials required for electrophoresis

Appendix B, “Materials required but not supplied“ lists the required materials that

are not supplied with this kit.

IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use.

Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)

Electrophoresis software setup

Genetic

Analyzer

3500

3500xL

Operating

System

Windows

Vista

™

The following table lists the data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures,

see the documents listed in Appendix E, “Documentation and support“.

Note: We conducted validation studies for the kit using the 3130xl, 3500, or 3500xL configurations.

Data

Collection

Software

3500 Data

Collection

Software v1

Additional software

HID Updater 3500

DC v2

(Cat. No. 4480670)

Plate templates, assays, run modules, and conditions (installed with the HID Updater)

Plate templates: 6dye_36_POP4 (and _xl)

Assays: GF+Norm_POP4 (and _xl) and

GF_POP4 (and _xl), which contain instrument protocol HID36_POP4 (and_xl)_J6_NT3200 with the following conditions:

• Run module: HID36_POP4

• Injection conditions: 1.2 kV/15 sec (24 sec for xl)

• Run conditions: 13 kV/1550 sec

• Dye Set J6

3500

3500xL

3500

3500xL

Windows ™ 7

3500 Data

Collection

Software v2

3500 Data

Collection

Software v3

HID Updater 3500

DC v2

(Cat. No. 4480670)

None

Same as 3500 Data Collection Software v1 listed above

Obtain and run the

HID Updater (v1 and v2 software only)

Perform this procedure if you are using 3500 Series Software v1 or v2.

You can run 6-dye samples on 3500 Data Collection Software. Before running on either system for the first time, run the HID Updater 3500 DC v2 (Cat. No. 4480670).

The HID Updater installs plate templates, assays, and instrument protocols that can

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Express PCR Amplification Kit User Guide

Perform spectral calibration

Chapter 3 Perform electrophoresis

Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) be used to run VeriFiler

™

Express kit samples. For more information, refer to the release notes provided with the Updater.

Note:

If you have a new instrument installed by a Thermo Fisher Scientific representative, the updater may have been run during installation.

1. Shut down the 3500/3500xL Data Collection Software.

2. Download the updater from www.thermofisher.com/us/en/home/

technical-resources/software-downloads/

3500-Series-Genetic-Analyzers-for-Human-Identification.html.

3. Open the Read me file and review the software release notes.

4. Click the updater .exe file.

5. Follow the on-screen prompts.

6. Restart the computer.

Perform a spectral calibration using the DS-36 Matrix Standard Kit (6-Dye) (J6 Dye

Set) (Cat. No. 4425042).

The following figure is an example of a passing 6-dye spectral calibration.

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3 Chapter 3 Perform electrophoresis

Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)

This procedure applies to the 3500 Series and 3130 Series instruments.

Prepare the samples for electrophoresis immediately before loading.

1. Pipet the required volumes of components into an appropriately sized polypropylene tube:

Reagent

GeneScan ™ 600 LIZ ™ Size Standard v2.0

Hi ‑Di ™ Formamide

Volume per reaction

0.4 μL

9.6 μL

Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers.

IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results.

2. Vortex the tube, then centrifuge briefly.

3. Into each well of a MicroAmp

™

Optical 96-Well Reaction Plate, add:

• 10 µL of the formamide/size standard mixture

• 1 µL of PCR product or Allelic Ladder

Note: For blank wells, add 10 µL of Hi-Di

™

Formamide.

4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom.

5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.

6. Immediately place the plate on ice for 3 minutes.

7. Place the sample tray on the autosampler, then start the electrophoresis run.

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Chapter 3 Perform electrophoresis

Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)

Set up the 3130/3130 use of the kit)

xl

instruments for electrophoresis (before first

Electrophoresis software setup

Genetic

Analyzer

3130

3130 xl

Operating

System

Windows ™ 7

The following table lists the data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures,

see the documents listed in Appendix E, “Documentation and support“.

Note: We conducted validation studies for the VeriFiler

3500, or 3500xL configurations.

™

Express kit using the 3130xl,

Data

Collection

Software

Data

Collection

Software v4

[1]

Additional software

3130/3730 DC v4

6 ‑Dye Module v1

Run modules and conditions

• HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/5 sec

• Run conditions: 15 kV/1500 sec

• Dye Set J6

• HIDFragmentAnalysis36_POP4_1 Injection conditions: 3 kV/10 sec

• Run conditions: 15 kV/1500 sec

• Dye Set J6

[1]

Requires activation of 6 ‑dye license.

Obtain and activate 6-dye license

1. Confirm that you are running Data Collection Software v4 (Help4About).

2. Obtain a 3130 DC v4 6-Dye Module v1 License key. Contact your local Human

Identification representative for information.

3. Ensure that all network cards in the computer are enabled.

IMPORTANT! You can run the 3130 Series Data Collection Software v4 using only the network cards that are enabled when you activate the software license.

For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled.

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3 Chapter 3 Perform electrophoresis

Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)

4. Select Tools4License Manager to display the Software Activation dialog box.

5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you.

6. Obtain the software license file from your email.

7. Make a copy of the software license file and keep it in a safe location.

8. Copy the software license file to the desktop of the Data Collection Software v4 computer.

9. If the Software Activation dialog box has closed, select Tools4License Manager.

10. Click Browse, then navigate to the software license file saved on your computer.

11. Click Install and Validate License.

A message is displayed when the license is installed and validated.

12. Click Close.

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Chapter 3 Perform electrophoresis

Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)

Perform spectral calibration

Perform a spectral calibration using the DS-36 Matrix Standard Kit (6-Dye)

(Cat. No. 4425042). Select the J6 dye set for the 3130 instrument, or the J6-RCT dye set for the 3730 instrument.

The following figure is an example of a passing 6-dye spectral calibration.

Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)

This procedure applies to the 3500 Series and 3130 Series instruments.

Prepare the samples for electrophoresis immediately before loading.

1. Pipet the required volumes of components into an appropriately sized polypropylene tube:

Reagent

GeneScan ™ 600 LIZ ™ Size Standard v2.0

Hi ‑Di ™ Formamide

Volume per reaction

0.4 μL

9.6 μL

Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers.

IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results.

2. Vortex the tube, then centrifuge briefly.

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3 Chapter 3 Perform electrophoresis

Set up the 3730 instruments for electrophoresis (before first use of the kit)

3. Into each well of a MicroAmp

™

Optical 96-Well Reaction Plate, add:

• 10 µL of the formamide/size standard mixture

• 1 µL of PCR product or Allelic Ladder

Note: For blank wells, add 10 µL of Hi-Di

™

Formamide.

4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom.

5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.

6. Immediately place the plate on ice for 3 minutes.

7. Place the sample tray on the autosampler, then start the electrophoresis run.

Set up the 3730 instruments for electrophoresis (before first use of the kit)

Electrophoresis software setup

Genetic

Analyzer

3730

Operating

System

Windows ™ 7

The following table lists the data collection software and the run modules that you can use to analyze PCR products generated by this kit. For details on the procedures,

see the documents listed in Appendix E, “Documentation and support“.

Data

Collection

Software

Data

Collection

Software v4

[1]

Additional software

3130/3730 DC v4 6-

Dye Module v1

Run modules and conditions

• GeneMapper36_POP7_1 Injection conditions: 2 kV/10 sec

• Run conditions: 15 kV/1200 sec

• Dye Set J6

[1]

Requires activation of 6 ‑dye license.

Obtain and activate the 6-dye license

1. Confirm that you are running Data Collection Software v4 (Help4About)

2. Obtain a 3730 DC v4 6-Dye Module v1 License key. Contact Thermo Fisher

Scientific for information.

3. Ensure that all network cards in the computer are enabled.

IMPORTANT! You can run the 3730 Series Data Collection Software v4 using only the network cards enabled when you activate the software license. For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled.

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Chapter 3 Perform electrophoresis

Set up the 3730 instruments for electrophoresis (before first use of the kit)

4. Select Tools4License Managerto display the Software Activation dialog box.

5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you.

6. Obtain the software license file from your email.

7. Make a copy of the software license file and keep in a safe location.

8. Copy the software license file to the desktop of the Data Collection Software v4 computer.

9. If the Software Activation dialog box has closed, select Tools4License Manager.

10. Click Browse, then navigate to the software license file saved on your computer.

11. Click Install and Validate License.

A message is displayed when the license is installed and validated.

12. Click Close.

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3 Chapter 3 Perform electrophoresis

Prepare samples for electrophoresis (3730 instruments)

Perform spectral calibration

Perform a spectral calibration using the DS-36 Matrix Standard Kit (6-Dye)

(Cat. No. 4425042). Select the J6 dye set for the 3130 instrument, or the J6-RCT dye set for the 3730 instrument.

The following figure is an example of a passing 6-dye spectral calibration.

Prepare samples for electrophoresis (3730 instruments)

Prepare the samples for electrophoresis immediately before loading.

1. Pipet the required volumes of components into an appropriately sized polypropylene tube:

Reagent

GeneScan ™ 600 LIZ ™ Size Standard v2.0

Hi ‑Di ™ Formamide

Volume per reaction

0.4 μL

9.6 μL

Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers.

IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results.

2. Vortex the tube, then centrifuge briefly.

3. Into each well of a MicroAmp

™

Optical 96-Well Reaction Plate, add:

• 10 µL of the formamide: size standard mixture

• 1 µL of PCR product or Allelic Ladder

Note: For blank wells, add 10 µL of Hi-Di

™

Formamide.

VeriFiler

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Express PCR Amplification Kit User Guide

Chapter 3 Perform electrophoresis

Prepare samples for electrophoresis (3730 instruments)

4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom.

5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes.

6. Immediately place the plate on ice for 3 minutes.

7. Place the sample tray on the autosampler, then start the electrophoresis run.

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Analyze data with GeneMapper

™

‑X

Software

■

Overview of GeneMapper

™

ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

■

Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

■

File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

■

Set up the GeneMapper

™

ID-X Software for analysis (before use of the kit) . . 40

■

Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■

Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

■

Analyze and edit sample files with GeneMapper

™

ID-X Software . . . . . . . . . . 55

■

Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

■

For more information on using the GeneMapper

™

ID-X Software . . . . . . . . . . 56

Overview of GeneMapper

™

ID

‑X

Software

GeneMapper

™

ID‑X Software is an automated genotyping software application for forensic casework, databasing, and paternity data analysis.

GeneMapper

™

ID‑X Software v1.4 or later analyzes 4-dye, 5-dye, and 6-dye data and is required to correctly analyze data that is generated using the VeriFiler

™

Express kit.

After electrophoresis, the data collection software stores information for each sample in a .fsa or .hid file. The GeneMapper

™

ID‑X Software v1.4 or later allows you to analyze and interpret the data from the .fsa or .hid files.

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ID ‑X

Software

Allelic ladder requirements for data analysis

• HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies before you use multiple allelic ladder samples in an analysis.

For multiple allelic ladder samples, the GeneMapper

™

ID‑X Software calculates allelic bin offsets by using an average of all allelic ladders that use the same panel in a run folder.

• Allelic ladder samples in an individual run folder are considered to be from a single run. When the software imports multiple run folders into a project, only the ladders in their respective run folders are used for calculating allelic bin offsets and subsequent genotyping.

• Allelic ladder samples must be labeled as "Allelic Ladder" in the Sample Type column in a project. Analysis will fail if the Allelic Ladder Sample Type is not specified.

• Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples, to ensure proper allele calling.

• Alleles that are not in the Allelic Ladders do exist. Off-ladder (OL) alleles can contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the ±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.

File names and versions used in this section

The file names and version numbers of panel, bin, and stutter files that are shown in this section may differ from the file names that you see when you download or import files.

If you need help to determine the correct files to use, contact your local Human

Identification representative, or go to thermofisher.com/support.

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ID ‑X

Software

Set up the GeneMapper

™

‑X Software for analysis (before use of the kit)

Set up the GeneMapper

™

the kit)

ID

‑X

Software for analysis (before use of

Workflow: Set up

GeneMapper

™

ID ‑X Software

Before you use GeneMapper

™

ID‑X Software to analyze data for the first time, you must do the following:

“Check panel, bin, and stutter file versions on your computer“ on page 40

▼

“Download newer versions of panel bin and stutter files if needed“ on page 40

▼

“Import panels, bins, and marker stutter“ on page 41

▼

“(Optional) Define custom table or plot settings“ on page 45

Check panel, bin, and stutter file versions on your computer

Download newer versions of panel bin and stutter files if needed

1. Start the GeneMapper

™

ID‑X Software , then log in with the appropriate user name and password.

2. Select Tools4Panel Manager.

3. Check the version of files that are currently available in the Panel Manager: a.

Select Panel Manager in the navigation pane.

Expand the Panel Manager folder and any subfolders to identify the analysis file version that is already installed for your kit choice.

4. Check the version of files available for import into the Panel Manager: a.

Select Panel Manager, then select File4Import Panels to open the Import

Panels dialog box.

Navigate to, then open the Panels folder, then check the version of panel, bin, and stutter files installed.

5. Check for newer versions of the files as described in the next procedure.

1. Go to thermofisher.com/us/en/home/technical-resources/software-downloads/

genemapper-id-x-software.html.

2. If the file versions listed are newer than the versions on your computer, download the file VeriFilerExpress Analysis Files.

Note: When downloading new versions of analysis files, see the associated Read

Me file for details of changes between software file versions. Perform the appropriate internal validation studies before using new file versions for analysis.

3. Unzip the file.

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Import panels, bins, and marker stutter

Chapter 4 Analyze data with GeneMapper

™

ID ‑X

Software

Set up the GeneMapper

™

‑X Software for analysis (before use of the kit)

Common Callouts and Arrows

To import the latest panel, bin set, and marker stutter from the website into the

GeneMapper ID‑X Software database:

Note: If you need more advanced callouts or arrows

1. Start the GeneMapper name and password.

™

ID‑X Software, then log in with the appropriate user

3. Find, then open the folder containing the panels, bins, and marker stutter:

1 a.

Select Panel Manager, then select File4Import Panels to open the Import

Panels dialog box.

1 b.

Navigate to, then open the VeriFilerExpress Analysis Files folder that you unzipped in the previous procedure.

4. Select VeriFilerExpress_Panels.txt , then click Import.

Note:

Importing this file creates a new folder in the navigation pane of the Panel

Manager, VeriFilerExpress_Panels. This folder contains the panel and associated markers.

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4 Chapter 4 Analyze data with GeneMapper

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ID ‑X

Software

Set up the GeneMapper

™

‑X Software for analysis (before use of the kit)

5. Import the bins file: a.

Select the VeriFilerExpress_Panels folder in the navigation pane.

Select File4Import Bin Set to open the Import Bin Set dialog box.

Navigate to, then open the VeriFilerExpress Analysis Files folder.

Select VeriFilerExpress_Bins.txt, then click Import.

Note: Importing this file associates the bin set with the panels in the

VeriFilerExpress_Panels folder.

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ID ‑X

Software

Set up the GeneMapper

™

‑X Software for analysis (before use of the kit)

6. (Optional) View the imported panels and bins in the navigation pane: Doubleclick the VeriFilerExpress_Panels folder.

The panel information is displayed in the right pane and the markers are displayed below it.

7. Import the stutter file: a.

Select the VeriFilerExpress_Panels folder in the navigation panel.

Select File4Import Marker Stutter to open the Import Marker Stutter dialog box.

Navigate to, then open the VeriFilerExpress Analysis Files folder.

Select VeriFilerExpress_Stutter.txt, then click Import.

Note: Importing this file associates the marker stutter ratio with the bin set in the VeriFilerExpress_Panels folder and overwrites any existing stutter ratios associated with the panels and bins in that folder.

8. View the imported marker stutters in the navigation pane: a.

Double-click the VeriFilerExpress_Panels folder to display the folder.

Double-click the folder to display its list of markers below it.

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ID ‑X

Software

Set up the GeneMapper

™

‑X Software for analysis (before use of the kit) c.

Double-click a marker to display the Stutter Ratio & Distance view for the marker in the right pane.

9. Click Apply, then click OK to add the panel, bin set, and marker stutter to the

GeneMapper

™

ID‑X Software database.

IMPORTANT! If you close the Panel Manager without clicking Apply, the panels, bin sets, and marker stutter are not imported into the GeneMapper

™

ID‑X

Software database.

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Chapter 4 Analyze data with GeneMapper

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ID ‑X

Software

Create an analysis method

(Optional) Define custom table or plot settings

Default views for table and plot settings are provided with the software. For information on defining custom views, see GeneMapper

™

ID‑X Software Getting Started

Guide— Basic Features.

Create an analysis method

IMPORTANT! Analysis methods are version-specific, so you must create an analysis method for each version of the software. For example, an analysis method that is created in GeneMapper

Software v3.2.1.

™

ID‑X Software version 1.2 is not compatible with analysis methods that are created in earlier versions of software, or with GeneMapper

™

1. Select Tools4GeneMapper

ID-X Manager to open the GeneMapper ID-X

Manager.

Common Callouts and Arrows

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows

use the TechComm_Inkscape_Callout&Arrow_Libary.

2. Edit number and/or line-length, as needed.

3. Delete this text, this rectangle, and unused

callouts, arrows, or other SVG elements

before adding this SVG to the repository.

1 1 1

2. Click the Analysis Methods tab, then click New to open the Analysis Method

Editor with the General tab selected.

3. Enter the settings shown in the figures on the following pages.

Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs.

4. After you enter the settings on all tabs, click Save.

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Create an analysis method

ID ‑X

Software

Enter Analysis

Method settings

Enter General tab settings

1. Enter a Name and select the Security Group appropriate for your software configuration.

2. (Optional) Enter a Description and Instrument.

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Chapter 4 Analyze data with GeneMapper

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ID ‑X

Software

Create an analysis method

Enter Allele tab settings

IMPORTANT! Perform appropriate internal validation studies to determine the appropriate settings to use.

1. Select the VeriFilerExpress_Bins_v1X bin set.

Figure 3 Settings used in developmental validation of the kit

2. (Optional) To apply the stutter ratios contained in the

VeriFilerExpress_Stutter.txt, select the Use marker-specific stutter ratio and distance if available checkbox (selected by default).

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Create an analysis method

ID ‑X

Software

3. If using GeneMapper

™

ID‑X Software v1.4 or later, enter values for the 4 Marker

Repeat Types.

Note:

For paternity and database applications, we recommended using a 20%

Global Cut-off Value for the Tri, Tetra, and Penta loci as shown in Figure 3.

4. Enter the appropriate filter settings.

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ID ‑X

Software

Create an analysis method

Enter Peak Detector tab settings

IMPORTANT! Perform appropriate internal validation studies to determine the appropriate settings to use.

Enter the appropriate values:

Figure 4 Settings used in developmental validation of the kit

Note: When using the 3730 DNA Analyzer with POP-7 ™ polymer only; a Smoothing setting of None and the Peak Window Size of 9 pts, the instances of spacing failures for the D2S441 and D1S1656 markers in some allelic ladder samples are significantly reduced. With the default Smoothing setting of Light, failures of base-pair spacing quality assessment are observed.

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4 Chapter 4 Analyze data with GeneMapper

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Create an analysis method

ID ‑X

Software

Enter Peak Quality tab settings

IMPORTANT! Perform the appropriate internal validation studies to determine the heterozygous and homozygous minimum peak height thresholds, maximum peak height threshold, and the minimum peak height ratio threshold for interpretation of data.

Enter the following values:

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Chapter 4 Analyze data with GeneMapper

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ID ‑X

Software

Create an analysis method

Enter SQ and GQ tab settings

IMPORTANT! The values that are shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use.

Enter the following values:

Note: Set the ACC GQ Weighting according to the values you determine during internal validation studies of the ACC PQV. For example, set the ACC GQ Weighting to 0.3 or higher to flag samples in which the Amelogenin result is anything other than

X, X or X, Y, or does not agree with the results for the Y indel marker.

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Create a size standard definition file if needed

ID ‑X

Software

Create a size standard definition file if needed

If you cannot use the default settings that are provided, create a new size standard definition file.

About the

GS600_LIZ_ (60–

460) size standard definition file

If you use POP-7

™

polymer on a 3730 instrument

The GS600_LIZ_(60– 460) size standard definition that is provided with GeneMapper

™

ID‑X Software and used with the Local Southern size calling method contains the following peaks: 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280,

300, 314, 320, 340, 360, 380, 400, 414, 420, 440, and 460.

This size standard definition has been validated for use with this kit on the genetic

analyzers listed in “Instrument and software compatibility“ on page 14. If you need to

create your own size standard definition, see “Create a size standard definition file“ on page 53.

The 60 bp size-standard peak may occasionally be obscured by the primer peak. The issue can be addressed by either of the following steps:

• Re-inject samples in which the 60 base-pair peak is not recognized.

• Use the 80– 460 bp size-standard definition after performing appropriate validation studies (as a general rule, the 60 base-pair peak is not required for accurate fragment sizing with the 3rd Order Least Squares sizing method).

For more information, see the GeneMapper

DNA Analyzer sizing failures”.

™

ID‑X Software v1.4 New Features and

Installation Procedures User Bulletin (Pub. No. 4477684 Rev. B), “Known issues: 3730

VeriFiler

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Express PCR Amplification Kit User Guide

Create a size standard definition file

Chapter 4 Analyze data with GeneMapper

™

ID ‑X

Software

Create a size standard definition file if needed

1. Select Tools4GeneMapper ID-X Manager to open the GeneMapper ID-X

Manager.

2. Click the Size Standards tab, then click New.

Common Callouts and Arrows

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows

use the TechComm_Inkscape_Callout&Arrow_Libary.

2. Edit number and/or line-length, as needed.

3. Delete this text, this rectangle, and unused

callouts, arrows, or other SVG elements

before adding this SVG to the repository.

1 1 1

3. Specify settings in the Size Standard Editor: a.

Enter a name as shown in the following figure or enter a new name.

In the Security Group field, select the Security Group appropriate for your software configuration.

In the Size Standard Dye field, select Orange.

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4 Chapter 4 Analyze data with GeneMapper

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Create a size standard definition file if needed

ID ‑X

Software d.

In the Size Standard Table, enter the peak sizes that correspond to your size standard.

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™

ID ‑X

Software

Analyze and edit sample files with GeneMapper

™

‑X Software

Analyze and edit sample files with GeneMapper

™

ID

‑X

Software

1. In the Project window, select Edit4Add Samples to Project, then navigate to the disk or directory that contains the sample files.

2. Apply analysis settings to the samples in the project.

Parameter

Sample Type

Analysis Method

Panel

Size Standard

Settings

Select the sample type.

Select VeriFilerExpress_AnalysisMethod (or the name of the analysis method you created).

Select VeriFiler_Express.

Use a size range of 60– 460 bp for Local Southern size calling method or a size range of 80– 460 bp for 3rd Order Least

Squares size-calling method.

[1]

The VeriFiler ™ Express kit was originally validated with the GeneScan of this size standard with the VeriFiler ™ Express kit.

™ 600 LIZ ™ Size Standard v2.0 . If you use a different size standard, perform the appropriate internal validation studies to support the use

3. Click Analyze, enter a name for the project (in the Save Project dialog box), then click OK to start analysis.

• The status bar displays the progress of analysis as a completion bar.

• The table displays the row of the sample currently being analyzed in green

(or red if analysis failed for the sample).

• The Analysis Summary tab is displayed, and the Genotypes tab is available when the analysis is complete.

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4 Chapter 4 Analyze data with GeneMapper

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Examine or edit a project

ID ‑X

Software

Examine or edit a project

Display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data.

For more information on using the GeneMapper

™

ID

‑X

Software

VeriFiler

™

Express PCR Amplification Kit User Guide

Experiments and results

■

Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

■

Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

■

Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . 58

■

Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

■

Representative data for common sample and substrate types . . . . . . . . . . . . . . . 62

■

Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

■

Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

■

Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

■

Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

■

Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

■

Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Importance of validation

Validation of a DNA typing procedure for human identification applications is an evaluation of the efficiency, reliability, and performance characteristics of the procedure. By challenging the procedure with samples that are commonly encountered in forensic and parentage laboratories, the validation process uncovers attributes and limitations that are critical for sound data interpretation (Sparkes,

Kimpton, Watson, 1996; Sparkes, Kimpton, Gilbard, 1996; Wallin, 1998).

Experiment conditions

We conducted developmental validation experiments according to the updated and revised guidelines from the Scientific Working Group on DNA Analysis Methods

(SWGDAM, December 2012). Based on these guidelines, we conducted experiments that comply with guidelines 2.0 and 3.0 and its associated subsections. This DNA methodology is not novel. (Moretti et al., 2001; Frank et al., 2001; Wallin et al., 2002; and

Holt et al., 2000).

We used conditions that produced optimum PCR product yield and that met reproducible performance standards. It is our opinion that while these experiments are not exhaustive, they are appropriate for a manufacturer of STR kits intended for forensic and/or parentage testing use.

Note: All results were analyzed on a 3500xL Genetic Analyzer in accordance with the

GeneMapper

™

ID-X Software“.

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5 Chapter 5 Experiments and results

Laboratory requirements for internal validation

Each laboratory using this kit must perform internal validation studies. Performance of this kit is supported when used according to the following developmentally validated parameters. Modifications to the protocol should be accompanied by appropriate validation studies performed by the laboratory.

Developmental validation

Except where noted, all developmental validation studies were performed using the

GeneAmp

™

PCR System 9700 96-Well thermal cycler according to the protocol described in the Perform PCR chapter.

SWGDAM guideline 2.2.1

SWGDAM guideline 3.9.2

“Developmental validation is the acquisition of test data and determination of conditions and limitations of a new or novel DNA methodology for use on forensic, database, known or casework reference samples.” (SWGDAM, December 2012)

“The reaction conditions needed to provide the required degree of specificity and robustness should be determined. These include, but are not limited to, thermal cycling parameters, the concentration of primers, magnesium chloride, DNA polymerase, and other critical reagents.” (SWGDAM, December 2012)

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Chapter 5 Experiments and results

Developmental validation

PCR components

We examined the concentration of each component of the 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 were amplified in the presence of varying concentrations of magnesium chloride, and

the results were analyzed on a 3500xL Genetic Analyzer (Figure 5). The performance

of the multiplex is most robust within ±20% of the optimal magnesium chloride concentration.

Figure 5 Blood sample on FTA

(Y-axis scale 0 to 28,000 RFU).

™ card with the VeriFiler ™ Express kit in the presence of varying concentrations of magnesium chloride and analyzed on a 3500xL Genetic Analyzer

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5 Chapter 5 Experiments and results

Developmental validation

PCR cycle number

Reactions were amplified for 24, 25, and 26 cycles on the GeneAmp

™

PCR System

9700 using a blood sample on treated paper. As expected, the amount of PCR product increased with the number of cycles. A full profile was generated for all numbers of thermal PCR cycles (24–26) and off-scale data were collected for several allele peaks at

26 cycles (Figure 6).

The data demonstrates that for this workflow 25 PCR cycles optimizes signal peak height and minimizes artifactual or other undesirable peaks.

Figure 6 Representative VeriFiler ™ Express kit profiles obtained from amplification of blood sample on untreated paper using 24, 25, and 26 cycles, analyzed on a 3500xL Genetic

Analyzer (Y-axis scale 0 to 25,000 RFU).

VeriFiler

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Express PCR Amplification Kit User Guide

Thermal cycling temperatures

Chapter 5 Experiments and results

Developmental validation

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/extension temperatures of 58, 59, and 60°C were tested using a GeneAmp

™

PCR System 9700 (Figure 7). The PCR products were analyzed using an

3500xL Genetic Analyzer.

All of the tested annealing temperatures, 58 to 60°C produced robust profiles. 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.

Figure 7 Electropherograms obtained from amplification of a blood sample on an FTA

™ card at annealing temperatures of 58, 59, and 60°C, analyzed on a 3500xL Genetic Analyzer

(Y-axis scale 0 to 5,000 RFU).

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5 Chapter 5 Experiments and results

Representative data for common sample and substrate types

For all the representative electropherograms shown (Figure 8 through Figure 12),

peak heights at the D22S1045 marker may be elevated as compared to the other markers in the TAZ

™

dye channel. This result is typical to the assay and has no impact on the ability to generate a full STR profile.

Figure 8 Electropherogram of a blood sample on an FTA

™

VeriFiler

™

(Y-axis scale 0 to 12,000 RFU).

card amplified with the

Express kit at a customer test site and analyzed on a 3500xL Genetic Analyzer

Figure 9 Electropherogram of a blood sample on a NUCLEIC-CARD

™ amplified with the VeriFiler

™

Genetic Analyzer (Y-axis scale 0 to 25,000 RFU).

Collection Device

Express kit at a customer test site and analyzed on a 3500xL

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Representative data for common sample and substrate types

Figure 10 Electropherogram of a buccal sample on an FTA ™

VeriFiler ™

(Y-axis scale 0 to 7,000 RFU).

card amplified with the

Express kit at a customer test site and analyzed on a 3500xL Genetic Analyzer

Figure 11 Electropherogram of a buccal sample on a NUCLEIC-CARD

™ amplified with the VeriFiler

™

Genetic Analyzer (Y-axis scale 0 to 7,000 RFU).

Collection Device

Express kit at a customer test site and analyzed on a 3500xL

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™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Accuracy, precision, and reproducibility

Figure 12 Electropherogram of a buccal sample on a cotton swab (with lysate) extracted using Prep ‑n‑Go ™ Buffer, and amplified with the VeriFiler ™ Express kit at a customer test site and analyzed on a 3500xL Genetic Analyzer (Y-axis scale 0 to 25,000 RFU).

Accuracy, precision, and reproducibility

SWGDAM guideline 3.5

Accuracy observation

“Precision and accuracy of the assay should be demonstrated: Precision characterizes the degree of mutual agreement among a series of individual measurements, values and/or results. Precision depends only on the distribution of random errors and does not relate to the true value or specified value. The measure of precision is usually expressed in terms of imprecision and computed as a standard deviation of the test results. Accuracy is the degree of conformity of a measured quantity to its actual

(true) value. Accuracy of a measuring instrument is the ability of a measuring instrument to give responses close to a true value.” (SWGDAM, December 2012)

Laser-induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology (Holt et al., 2000; and Wallin et al., 2002). However, accuracy and reproducibility of profiles have been determined from various sample types.

The following three figures show the size differences that are typically observed between sample alleles and allelic ladder alleles on the 3130xl, and 3500xL Genetic

Analyzers with POP-4

Polymer. The X-axis in the following figures represents the nominal nucleotide sizes for the VeriFiler

™

Polymer and the 3730xl Genetic Analyzer with POP-7

™

Express Allelic Ladder. The dashed lines parallel to the X-axis represent the ±0.25-nt windows. The y-axis represents the deviation of each sample allele size from the corresponding Allelic Ladder allele size. All sample alleles are

VeriFiler

™

Express PCR Amplification Kit User Guide

Common Callouts and Arrows

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows

use the TechComm_Inkscape_Callout&Arrow_Libary.

2. Edit number and/or line-length, as needed.

3. Delete this text, this rectangle, and unused

callouts, arrows, or other SVG elements

before adding this SVG to the repository.

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Chapter 5 Experiments and results

Accuracy, precision, and reproducibility

5 within ±0.5 nt from a corresponding allele in the Allelic Ladder, irrespective of the capillary electrophoresis platforms.

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Figure 13 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3130 xl Genetic

Analyzer. Size and ladder sizing for the VeriFiler

GeneScan ™ 600 LIZ ™ Size Standard v2.0.

™ Express kit were calculated using the

Figure 14 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on a 3500xL

Genetic Analyzer. Size and ladder sizing for the VeriFiler using the GeneScan ™ 600 LIZ ™ Size Standard v2.0.

™ Express kit were calculated

VeriFiler

™

Express PCR Amplification Kit User Guide

Common Callouts and Arrows

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Chapter 5 Experiments and results

Accuracy, precision, and reproducibility

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Precision and size window description

Figure 15 Allele Size vs. Allelic Ladder Sizing for 84 samples analyzed on an 3730 xl

Genetic Analyzer. Size and ladder sizing for the VeriFiler using the GeneScan ™ 600 LIZ ™ Size Standard v2.0.

™ Express kit were calculated

Sizing precision enables the determination of accurate and reliable genotypes. The recommended method for genotyping is to use a ±0.5-nt “window” around the size obtained for each allele in the allelic ladder. A ±0.5-nt window allows for the detection and correct assignment of alleles. Any sample allele that sizes outside the specified window could be either:

• An “off-ladder” allele, that is, an allele of a size that is not represented in the allelic ladder.

• An allele that does correspond to an allele in the allelic ladder, but whose size is just outside a window because of measurement error.

The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times. Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument.

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Accuracy, precision, and reproducibility

Precision and size window observation

Figure 16 lists typical precision results obtained from multiple runs of the VeriFiler

™

Express Allelic Ladder using the GeneScan

™

600 LIZ

™

Size Standard v2.0. The results were obtained within a set of injections on a single capillary array. The number of repeated injections for each genetic analyzer platform is shown in the following table:

CE platform

3130 xl

3500

3500xL

3730

Capillaries

16/injection

8/injection

24/injection

48/injection

# Injections

Sizing method

3rd Order Least Square, 60–

460 bp

3rd Order Least Square, 60–

460 bp

3rd Order Least Square, 60–

460 bp

3rd Order Least Square

The mean sizes and the standard deviation for the allele sizing were calculated for all

the alleles in each run (Figure 16). The mean range and the standard deviation range

show the lowest and highest values obtained across multiple runs.

Sample alleles can occasionally size outside of the ±0.5-nt window for a respective

Allelic Ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems with the smallest standard deviations in

sizing. The figures in “Accuracy observation“ on page 64 illustrate the tight clustering

of allele sizes obtained on the Applied Biosystems

™

genetic analyzers, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5-nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith,

1995).

Note: The read region for the VeriFiler

™

Express kit is 75–465 nt.

For sample alleles that do not size within a ±0.5-nt window, the PCR product must be rerun to distinguish between a true off–ladder allele versus measurement error of a sample allele that corresponds to an allele in the Allelic Ladder. Repeat analysis, when necessary, provides an added level of confidence in the final allele assignment.

GeneMapper

™

ID‑X Software automatically flags sample alleles that do not size within the prescribed window around an allelic ladder allele by labeling the allele as

OL (off-ladder).

Maximum sizing precision is obtained within the same set of capillary injections.

Cross–platform sizing differences occur due to several factors including type and concentration of polymer, run temperature, and electrophoresis conditions. Variations in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors.

IMPORTANT! To minimize the variation in sizing between runs and to ensure

accurate genotyping, follow the guidelines in “Allelic ladder requirements for data analysis“ on page 39 and use allelic ladders obtained from the same run as samples to

analyze the samples.

For more information on precision and genotyping, see Lazaruk et al., 1998 and

Mansfield et al., 1998.

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Accuracy, precision, and reproducibility

In the following figure, the sizes for all the alleles across multiple injections of the

VeriFiler

™

Express Allelic Ladder were calculated. Each box represents the 25th through the 75th percentile of the observed values, with the center line indicating the median. Standard error bars indicate the range of the data, with presumed outliers

(points that are different from the mean by more than twice the pooled standard

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Accuracy, precision, and reproducibility

5 deviation) shown as black dots. Panel B shows the distribution of the standard deviations for all the markers combined for each CE instrument platform.

Figure 16 Precision results of multiple runs of the VeriFiler

™

Express Allelic Ladder

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Extra peaks in the electropherogram

Causes of extra peaks

Extra peaks:

Stutter

Peaks other than the target alleles may be detected on the electropherogram. Causes for the appearance of extra peaks include stutter products, incomplete 3´ A nucleotide addition (at the n-1 position), dye artifacts, and mixed DNA samples (see DNA

Advisory Board (DAB) Standard 8.1.2.2).

Stutter definition

Stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller than the target STR allele product (minus stutter), or less frequently, one repeat larger (plus stutter) (Butler, 2005; Mulero et al., 2006). Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the minus stutter product is missing a single tetranucleotide core repeat unit relative to the main allele (Walsh et al., 1996). Although plus-stutter is normally much less significant than minus-stutter in STR loci with tetranucleotide repeats, the incidence of plus-stutter may be more significant in trinucleotide repeat-containing loci.

Contact HID Support for more information on plus stutter.

The proportion of the stutter product relative to the main allele (percent stutter) is measured by dividing the height of the stutter peak by the height of the main allele peak.

Stutter observations

The level of stutter in this kit is normal and as expected for STR chemistries that are designed to overcome inhibitors and obtain robust performance with single source reference samples. Note the following:

Peak heights were measured for amplified samples at the loci used in the kit. All data were generated on the 3500xL Genetic Analyzer. Some conclusions from these measurements and observations are:

• For each locus, the stutter percentage generally increases with allele length.

• Smaller alleles display a lower level of stutter relative to the longer alleles within each locus.

• Each allele within a locus displays a consistent stutter percentage.

• Peaks in the stutter position that are above the stutter filter percentage specified in the software are not filtered (stutter filter percentage is calculated as the mean stutter for the locus plus three standard deviations). Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated.

• The measurement of stutter percentage for allele peaks that are off-scale may be unusually high due to artificial truncation of the main allele peak.

Marker-specific stutter observed in the population study with the VeriFiler

™

Express

kit is shown in Figure 17 through Figure 23 and listed in Table 3.

The stutter filter settings that are derived from this data are listed in “Stutter percentage filter settings provided with GeneMapper

™

ID-X Software“ on page 77.

Workflow

Treated paper

Untreated paper

Number of samples

49 blood samples on FTA

™

Classic Cards

425 blood samples on FTA ™ Bloodstain Cards

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Extra peaks in the electropherogram

Figure 17 Stutter percentages for D1S1656, D2S441, D2S1338, and D3S1358 loci

(Blue=FAM

™

dye, black=NED

™

dye, purple=SID

™

dye)

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Extra peaks in the electropherogram

Figure 18 Stutter percentages for D5S818, D7S820, D8S1179, and D10S1248 (Green=VIC

™ dye, red=TAZ

™

dye, purple=SID

™

dye)

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Extra peaks in the electropherogram

Figure 19 Stutter percentages for D12S391, D13S317, and D16S539 loci (Blue=FAM

™

dye, red=TAZ

™

dye, purple=SID

™

dye)

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Extra peaks in the electropherogram

Figure 20 Stutter percentages for D18S51, D19S433, and D21S11 loci (Green=VIC

™

dye, black=NED

™

dye)

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™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Extra peaks in the electropherogram

Figure 21 Stutter percentages for D22S1045, CSF1PO, TH01, and vWA loci (Blue=FAM

™ dye, green=VIC

™

dye, black=NED

™

dye, red=TAZ

™

dye). Red and orange data points associated with D22S1045 locus indicate minus and plus stutter, respectively.

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Extra peaks in the electropherogram

Figure 22 Stutter percentages for FGA and TPOX loci (Blue=FAM

™

dye, black=NED

™

dye)

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Extra peaks in the electropherogram

Figure 23 Stutter percentages for D6S1043, Penta D, and Penta E loci (Red=TAZ

™

dye, purple=SID

™

dye, green=VIC

™

dye)

Stutter percentage filter settings provided with GeneMapper ™

‑X

Software

The settings in Table 3 were derived using the data shown earlier in this section. The

proportion of the stutter product relative to the main allele (stutter percent) is measured by dividing the height of the stutter peak by the height of the main allele peak.

IMPORTANT! The values that are shown in the table are the values that were determined during developmental validation studies using specific data sets. Always perform internal validation studies to determine the appropriate values to use for your applications.

Table 3 Marker-specific stutter filter percentages for VeriFiler ™ Express kit loci

Locus

[1]

CSF1PO

D10S1248

D12S391

D13S317

D16S539

D18S51

% Stutter

10.54

12.27

14.44

10.10

10.76

14.15

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Extra peaks in the electropherogram

Extra peaks:

Addition of 3' A nucleotide

Locus

[1]

D19S433

D1S1656

D21S11

D22S1045

D22S1045 (+3 nt)

D2S1338

D2S441

D3S1358

D5S818

D7S820

D8S1179

FGA

Penta D

Penta E

TH01

8.31

4.93

TPOX 5.20

vWA

[1]

These percentages are used as stutter filters in VeriFilerExpress_Stutter.txt.

13.18

% Stutter

10.77

13.62

12.24

8.05

18.0

13.29

9.40

12.16

10.53

9.39

10.85

12.27

4.09

3′ A definition

Many DNA polymerases can catalyze the addition of a single nucleotide

(predominantly adenosine) to the 3′ ends of double-stranded PCR products (Clark,

1988; Magnuson et al., 1996). This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence. The PCR product with the extra nucleotide is referred to as the “+A” form.

3′ A observations

The efficiency of +A addition is related to the particular sequence of the DNA at the 3´ end of the PCR product.

The VeriFiler

™

Express kit includes two main design features that promote maximum

+A addition:

• The primer sequences have been optimized to encourage +A addition.

• The PCR chemistry allows complete +A addition with a short final incubation at

60°C for 5 minutes .

This final extension step gives the DNA polymerase additional time to complete +A

addition to all double-stranded PCR products. Figure 24 shows examples of

incomplete and normal +A addition. Final extension incubation for longer than the

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Extra peaks in the electropherogram

5 recommended time can result in double +A addition, in which two nontemplate adenosine residues are added to the PCR product. Double +A addition can cause

"shoulders" on the right side of main allele peaks, and is therefore to be avoided.

Extra peaks:

Artifacts

Figure 24 Omitting the final extension step results in shoulders on main allele peaks due to incomplete A nucleotide addition. Examples shown are the smaller amplicons of FAM

™

NED

™

, and SID

™

dye channel data from a 3500xL Genetic Analyzer using the VeriFiler

™

Express kit.

Due to improved PCR buffer chemistry, the lack of +A addition is generally less of an issue with the VeriFiler

™

Express kit than with earlier generation kits. However,

"shouldering" of allele peaks can still be observed if the amount of input DNA is greater than recommended concentration. Amplification of excess input DNA can also result in off-scale data.

Artifact definition

Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible. Anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise).

Artifact observation

Due to improvements in PCR primer manufacturing processes, the incidence of artifacts has been greatly reduced in the VeriFiler

™

Express kit. Kit electropherograms are free of reproducible dye artifacts in the kit read region of 75– 465 nt for commonly

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Extra peaks in the electropherogram

used analytical thresholds. Figure 25 shows the low baseline-level fluorescence that is

observed in a typical negative control PCR.

Figure 25 Examples of fluorescence background in data produced on a 3500xL Genetic

Analyzer (Y-axis scale 0 to 200 RFU).

Additional reproducible DNA-dependent artifacts have been characterized and

documented in Table 5. It is important to consider noise and other amplification-

related artifacts when interpreting data.

Table 4 DNA-dependent artifacts observed in the population study with the VeriFiler

™

Express kit.

Artifact

TAZ60–80

SID130

SID202

SID402–411

Color

Red

Purple

Size

60–80 nt

130–131 nt

202– 203 nt

402– 411 nt

Type

PCR artifact

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™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Characterization of loci

SWGDAM guideline 3.1

Loci in this kit

“The basic characteristics of a genetic marker should be determined and documented.” (SWGDAM, December 2012)

Nature of polymorphisms

Inheritance

Mapping

Genetic linkage

This section describes basic characteristics of the 23 autosomal STR loci, one Y indel locus, and the sex-determining marker (Amelogenin), which are amplified with the

VeriFiler

™

Express kit. Most of these loci have been extensively characterized by other laboratories.

The primers for the Amelogenin locus flank a 6-nucleotide deletion in intron 1 of the

X homolog. Amplification generates 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.) Except for

D22S1045, a trinucleotide STR locus, and Penta D and Penta E, pentanucleotide STR loci (Bacher and Schumm, 1998), the remaining loci are tetranucleotide short tandem repeat (STR) loci. The length differences among alleles of a particular locus are caused by differences in the number of repeat units.

We have sequenced all the alleles in the VeriFiler

™

Express kit Allelic Ladder, including microvariants. In addition, other groups in the scientific community have sequenced alleles at some of these loci (Nakahori et al., 1991; Puers et al., 1993; Möller

et al., 1994; Barber et al., 1995; Möller and Brinkmann, 1995; Barber et al., 1996; Barber and Parkin, 1996; Brinkmann et al., 1998; Momhinweg et al., 1998; Watson et al., 1998).

Among the various sources of sequence data on the loci, there is consensus on the repeat patterns and structure of the STRs.

The Centre d'Etude du Polymorphisme Humain (CEPH) has collected DNA from families of Utah Mormon, French Venezuelan, and Amish descent. These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci. Each family set contains three generations, generally including four grandparents, two parents, and several offspring. Consequently, the CEPH family DNA sets are ideal for studying inheritance patterns (Begovich et al., 1992).

The VeriFiler

™

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; and Lareu, et al., 1996).

Two sets of STR loci in the VeriFiler

™

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; 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.

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™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Species specificity

The relatively high probability values indicate that there is no statistically significant linkage disequilibrium found between the pairs of loci that are located on the same chromosome.

An independent analysis of data from the same collection of population samples

(Budowle, et al., 2010) also concluded that the 15 STR loci that are shared between the

NGM

™

and NGM SElect

™

kits were independent at the population level (note that the

SE33 locus was not part of this analysis). Therefore, to calculate the rarity of a profile for comparison to single-source and mixture samples, the frequencies of all loci including vWA and D12S391 could be multiplied. However, the analysis of the CEPH pedigree families demonstrated a degree of linkage between vWA and D12S391 that does not support the assumption of independence for kinship analysis.

Table 5 GenePop LD Result (p ‑value for pairwise analysis of loci)

Locus

Chromosome map position

[1]

Chromosome

Nuclear

Coordinates

[1]

(million bp)

African-

American

(n = 350)

Caucasian

(n = 350)

Hispanic

(n = 293)

vWA

D12S391

12p13.31

12p13.2

5.9

12.2

0.86

0.29

0.27

D2S441 2p14 68

0.11

0.32

0.19

D2S1338 2q35 218

[1]

STR locus mapping data was obtained from the NCBI Map Viewer http://www.ncbi.nlm.nih.gov/projects/ mapview/map_search.cgi?taxid=9606 or the UCSC Genome Browser (http://genome.ucsc.edu/). GenePop LD analysis probability results (p values) greater than 0.05 were considered to indicate that linkage disequilibrium between the loci within the population tested was not statistically significant.

Species specificity

SWGDAM

Guideline 3.2

“The ability to detect genetic information from non-targeted species (e.g., detection of microbial DNA in a human assay) should be determined. The detection of genetic information from non-targeted species does not necessarily invalidate the use of the assay, but may help define the limits of the assay.” (SWGDAM, December 2012)

Nonhuman studies

The VeriFiler

™

Express kit provides the required specificity for detecting human alleles. Species specificity testing was performed to ensure that there is no crossreactivity with nonhuman DNA that may be present in forensic casework samples.

The following species were tested (in the specified amounts) using standard PCR and capillary electrophoresis conditions for the VeriFiler

™

Express kit kit:

• Primates: chimpanzee, gorilla, and orangutang (1.0 ng each)

• Non-primates: cow, chicken, dog, horse, mouse, pig, rabbit, rat, sheep (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).)

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™

Express PCR Amplification Kit User Guide

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Note: If you need more advanced callouts or arrows

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2. Edit number and/or line-length, as needed.

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Chapter 5 Experiments and results

Sensitivity

Results were evaluated for the presence of any amplified peaks that would indicate cross reactivity of the VeriFiler

™

Express kit with any of these non-human species.

“Nonhuman studies“ on page 82 shows example electropherogram results from the

species specificity tests. The chimpanzee, gorilla, and orangutang DNA samples produced partial profiles in the 75–465 nucleotide region.

Figure 26 Representative electropherograms for some species tested in a species specificity study. Data produced on a 3500xL Genetic Analyzer.

Among the non-primate species, most produced no peaks over a threshold of

175 RFU. Horse yielded reproducible VIC

™

dye peaks at 94 bp (<100 RFU) due to

Amelogenin cross-reactivity. Pig yielded reproducible TAZ

™

(<50 RFU), non-reproducible peaks.

dye peaks at 424 bp (

<200 RFU). Individual replicate PCRs of dog, mouse, and chicken yielded single, small

Sensitivity

SWGDAM guideline 3.3

Sample collection factors that can affect DNA quantity

“The ability to obtain reliable results from a range of DNA quantities, to include the upper and lower limits of the assay, should be evaluated.” (SWGDAM, December

2012)

The VeriFiler

™

Express kit has been optimized at 25 µL PCR reaction volume to overcome the PCR inhibition expected when amplifying:

• Blood samples that are obtained directly from unpurified 1.2 mm treated paper discs

• Buccal cells that are obtained directly from unpurified 1.2 mm treated paper discs

(with the addition of Prep-n-Go

™

Buffer )

• Buccal swab sample lysate is prepared using Prep-n-Go

™

Buffer

VeriFiler

™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Population data

Effect of DNA quantity on results

Depending on the following conditions, DNA quantities present on the 1.2 mm disc may vary from laboratory to laboratory:

• Volume of blood that is spotted onto the treated paper

• Collecting devices that are used

• Collection methods that are applied

• Swab-to-paper transfer protocol that is used

It is essential to optimize the PCR conditions for types of blood samples and spotting

protocol. See “Optimize PCR cycle number (before first use of the kit)“ on page 16.

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 VeriFiler

™

Express PCR

Amplification Kit , optimize PCR cycle number according to instructions in the

Perform PCR chapter.

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.

Population data

SWGDAM guideline 3.7

Population data overview

“The distribution of genetic markers in populations should be determined in relevant population groups.” (SWGDAM, December 2012)

To interpret the significance of a match between genetically typed samples, you must know the population distribution of alleles at each locus in question. If the genotype of the relevant evidence sample is:

• Different from the genotype of the reference sample for a suspect, then the suspect is excluded as the donor of the biological evidence that was tested. An exclusion is independent of the frequency of the two genotypes in the population.

• The same as the genotype of the reference sample for a suspect, then the suspect is included as a possible source of the evidence sample.

The probability that another, unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant populations.

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™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

Loci in this kit

Population samples used in these studies

Concordance studies

Probability of

Identity definition

Probability of identity

This section describes basic characteristics of the 23 autosomal STR loci, one Y indel locus, and the sex-determining marker (Amelogenin), which are amplified with the

VeriFiler

™

Express kit. Most of these loci have been extensively characterized by other laboratories.

Population data for loci contained in theVeriFiler

™

Express PCR Amplification Kit was obtained from data published by (Hill et. al., 2013). See the original data at

www.cstl.nist.gov/biotech/strbase/NISTpop.htm. Ethnicities of sample donors were:

• African-American—342 samples

• Asian—97 samples

• Caucasian—361 samples

• Hispanic—236 samples

The VeriFiler

™

Express kit contains loci for which extensive population data are available.

The primer sequences used in the VeriFiler

™

GlobalFiler

™

Express PCR Amplification Kit and

Express kit are identical except for the D6S1043, Penta E, and Penta D loci which are not shared between the two kits.

We compared allele calls between the VeriFiler the PowerPlex the PowerPlex

™

Express PCR Amplification Kit and

21 System. Genotype data from 200 blood samples on FTA

™

21 System at the D5S818 and Penta E loci. These alleles were accurately genotyped by the VeriFiler

™

Express PCR Amplification Kit.

Classic

Cards showed 99.6% concordance between the two kits. Null alleles were found with

The P

value is the probability that two individuals selected at random will have an identical genotype (Sensabaugh, 1982).

Table 6 shows the Autosomal STR allele frequencies at VeriFiler

™ population group.

Express kit loci by

Table 7 shows the Probability of identity (P

I individually and combined.

) values of the VeriFiler

™

Express kit loci

Table 6 Autosomal allele frequencies by population group for VeriFiler ™

Kit STR loci. (*=Alleles not detected)

Express kit

Allele

African

American

(n = 342)

Asian (n = 97)

U.S. Caucasian

(n = 361)

U.S. Hispanic

(n = 236)

CSF1PO

5.0

6.0

7.0

7.3

8.0

8.1

0.0556

0.0206

0.0055

0.0127

0.0042

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™

Express PCR Amplification Kit User Guide

5 Chapter 5 Experiments and results

Population data

Allele

D10S1248

7.0

8.0

9.0

10.0

11.0

12.1

12.3

13.0

14.0

15.0

16.0

12.0

13.0

14.0

15.0

16.0

17.0

10.3

11.0

11.1

11.2

11.3

12.0

9.0

9.1

10.0

10.1

10.2

0.1301

0.2339

0.2763

0.1974

0.0877

0.0249

0.0029

0.0073

0.0351

African

American

(n = 342)

0.0395

0.2500

0.2485

0.2953

0.0468

0.0088

0.2165

0.3866

0.0670

0.2010

0.0876

0.0155

0.0052

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.3089

0.3601

0.0139

0.2202

0.0817

0.0097

U.S. Hispanic

(n = 236)

0.2797

0.3750

0.0233

0.2373

0.0593

0.0064

0.0021

0.0876

0.3196

0.2526

0.2062

0.0979

0.0309

0.0052

0.0319

0.3075

0.2978

0.1967

0.1330

0.0277

0.0014

0.0424

0.2733

0.3390

0.2119

0.0996

0.0254

0.0042

VeriFiler

™

Express PCR Amplification Kit User Guide

Allele

20.0

20.1

20.3

21.0

21.1

22.0

18.1

18.2

18.3

19.0

19.1

19.3

18.0

19.0

D12S391

13.0

14.0

15.0

16.0

17.0

17.1

17.3

18.0

22.3

23.0

24.0

24.3

25.0

26.0

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

African

American

(n = 342)

0.0015

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0014

0.0028

U.S. Hispanic

(n = 236)

0.0021

0.0643

0.0365

0.0292

0.0132

0.1477

0.0088

0.0044

0.1038

0.0029

0.0088

0.0044

0.2529

0.0015

0.0044

0.0015

0.0775

0.0673

0.1667

0.0979

0.0567

0.0258

0.0103

0.1753

0.0052

0.1959

0.0052

0.0155

0.0103

0.2629

0.0412

0.0103

0.0825

0.1288

0.0956

0.0693

0.0471

0.1247

0.0042

0.1108

0.0166

0.0028

0.0208

0.1717

0.0249

0.0319

0.0222

0.1274

0.1123

0.0678

0.0572

0.0169

0.1886

0.0021

0.0064

0.1547

0.0064

0.0169

0.1780

0.0127

0.0445

0.0424

0.0763

5 Chapter 5 Experiments and results

Population data

Allele

D16S539

4.0

5.0

15.0

16.0

17.0

11.3

12.0

12.3

13.0

13.3

14.0

27.0

D13S317

5.0

6.0

7.0

9.0

10.0

11.0

7.1

8.0

8.1

8.3

9.0

9.3

6.0

7.0

8.0

0.1443

0.1031

0.2680

0.2062

0.2216

0.0567

0.3557

African

American

(n = 342)

Asian (n = 97)

0.0052

U.S. Caucasian

(n = 361)

0.0014

U.S. Hispanic

(n = 236)

0.0064

0.0336

0.0307

0.3099

0.4181

0.0278

0.1404

0.0395

0.0015

0.0322

0.1827

0.0776

0.0471

0.3255

0.2687

0.1205

0.1163

0.0429

0.0014

0.0180

0.1066

0.1653

0.0996

0.2182

0.2352

0.1102

0.1059

0.0614

0.0042

0.0191

0.1398

VeriFiler

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Express PCR Amplification Kit User Guide

Allele

D18S51

6.0

7.0

8.0

9.0

9.2

10.0

10.2

11.0

11.2

12.0

12.2

12.3

13.0

13.1

13.2

13.3

14.0

12.2

13.0

13.3

14.0

15.0

16.0

10.0

11.0

11.3

12.0

12.1

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

0.0044

0.0015

0.0760

0.0029

0.0409

0.0044

0.0716

African

American

(n = 342)

0.1170

0.3143

0.2047

0.1228

0.0249

0.0979

0.0155

0.1649

0.1907

0.1753

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.1634

0.0263

0.0568

0.3144

U.S. Hispanic

(n = 236)

0.1335

0.0127

0.0021

0.1504

0.2648

0.2775

0.0361

0.2165

0.2371

0.0083

0.0097

0.1136

0.1233

0.1343

0.0021

0.0148

0.1144

0.1229

0.1610

5 Chapter 5 Experiments and results

Population data

Allele

21.0

21.1

21.2

22.0

22.2

23.0

18.1

18.2

19.0

19.2

20.0

20.2

23.2

24.0

25.0

26.0

27.0

28.0

16.1

16.2

17.0

17.2

17.3

18.0

14.2

15.0

15.2

15.3

16.0

0.0102

0.0015

0.0073

0.0044

0.0994

0.0629

African

American

(n = 342)

0.1652

0.0015

0.1711

0.1520

0.1213

0.0015

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0097

0.0069

0.0402

0.0180

0.0014

0.1385

0.0776

0.0014

0.1704

0.1468

0.0103

0.0155

0.0052

0.0412

0.0258

0.0052

0.0670

0.0309

0.1804

0.1289

U.S. Hispanic

(n = 236)

0.0085

0.0106

0.0466

0.0275

0.0021

0.1250

0.0784

0.0021

0.1589

0.1250

VeriFiler

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Express PCR Amplification Kit User Guide

Allele

13.2

14.0

14.2

14.3

15.0

15.2

11.2

12.0

12.1

12.2

13.0

13.1

D19S433

5.2

6.0

6.2

7.0

10.0

10.2

11.0

8.0

9.0

9.2

16.0

16.2

17.0

17.2

18.0

18.2

VeriFiler

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Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

African

American

(n = 342)

Asian (n = 97)

U.S. Caucasian

(n = 361)

U.S. Hispanic

(n = 236)

0.0804

0.0614

0.0044

0.0263

0.0365

0.2456

0.0526

0.2105

0.0746

0.0088

0.0029

0.0102

0.0629

0.1228

0.0619

0.1237

0.0103

0.0309

0.0258

0.2835

0.0206

0.2990

0.1031

0.0052

0.0361

0.1565

0.0360

0.0568

0.0152

0.0069

0.0014

0.2548

0.0069

0.3615

0.0235

0.0014

0.0055

0.0706

0.1356

0.0551

0.0254

0.0275

0.0127

0.2225

0.0445

0.3538

0.0381

0.0021

0.0148

0.0657

0.0021

5 Chapter 5 Experiments and results

Population data

Allele

16.3

17.0

17.1

17.3

18.0

18.3

14.2

14.3

15.0

15.3

16.0

16.1

19.0

19.2

D1S1656

8.0

9.0

10.0

11.0

12.0

13.0

13.3

14.0

19.0

19.3

20.0

20.3

21.0

African

American

(n = 342)

Asian (n = 97)

U.S. Caucasian

(n = 361)

U.S. Hispanic

(n = 236)

0.0497

0.0029

0.0234

0.0073

0.0292

0.1096

0.1023

0.0278

0.1009

0.2573

0.0073

0.1579

0.0146

0.0453

0.0643

0.0876

0.0155

0.0515

0.0052

0.2010

0.0155

0.0722

0.1340

0.0619

0.2784

0.0309

0.0464

0.1330

0.0055

0.0499

0.0152

0.0582

0.1357

0.0609

0.0471

0.0665

0.0789

0.0028

0.1496

0.0028

0.0776

0.1163

0.1483

0.0064

0.0254

0.0042

0.0508

0.1758

0.0508

0.0424

0.1144

0.1165

0.0042

0.1377

0.0064

0.0275

0.0890

VeriFiler

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Express PCR Amplification Kit User Guide

Allele

28.3

29.0

29.1

29.2

29.3

30.0

27.1

27.2

27.3

28.0

28.1

28.2

D21S11

23.2

24.0

24.2

24.3

25.0

25.2

25.3

26.0

26.2

27.0

30.1

30.2

30.3

31.0

31.1

31.2

VeriFiler

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Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

African

American

(n = 342)

Asian (n = 97)

U.S. Caucasian

(n = 361)

U.S. Hispanic

(n = 236)

0.0015

0.1696

0.0175

0.2456

0.2047

0.0789

0.0512

0.0015

0.0746

0.3299

0.0052

0.0103

0.0567

0.0052

0.2010

0.1237

0.0361

0.0028

0.2825

0.0291

0.1593

0.2022

0.0720

0.0983

0.0222

0.0014

0.0021

0.2733

0.0233

0.0996

0.2076

0.0763

0.0996

0.0021

0.0275

0.0021

5 Chapter 5 Experiments and results

Population data

Allele

D22S1045

7.0

8.0

9.0

10.0

11.0

36.2

37.0

37.2

38.0

38.2

39.0

34.2

35.0

35.1

35.2

36.0

36.1

33.0

33.1

33.2

33.3

34.0

34.1

31.3

32.0

32.1

32.2

0.0029

0.0015

0.0219

0.0088

African

American

(n = 342)

0.0088

0.0614

0.0044

0.0029

0.0351

0.0058

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0042

0.0014

0.0263

0.0055

0.0900

0.0155

0.0464

0.0567

0.1134

U.S. Hispanic

(n = 236)

0.0042

0.0021

0.0339

0.0021

0.0169

0.1271

0.0073

0.0409

0.1447

0.2010

0.1399

0.0148

0.0636

VeriFiler

™

Express PCR Amplification Kit User Guide

Allele

18.0

18.3

19.0

19.3

20.0

21.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

D2S1338

10.0

11.0

12.0

13.0

14.0

15.0

16.0

22.0

23.0

23.2

23.3

24.0

24.2

VeriFiler

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Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

0.1038

0.1360

0.1374

0.1038

0.0833

0.0015

0.0556

0.1009

0.0424

0.1389

African

American

(n = 342)

0.0541

0.0029

0.0775

0.2515

0.1915

0.2091

0.0146

0.0058

0.0052

0.0103

0.3093

0.2268

0.2216

0.0206

0.0052

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0125

0.0069

0.0568

0.3213

0.3823

0.0748

0.0055

U.S. Hispanic

(n = 236)

0.0127

0.0085

0.0275

0.4258

0.3496

0.0911

0.0064

0.1598

0.0155

0.0515

0.1649

0.1289

0.0206

0.0567

0.1340

0.1804

0.1565

0.0374

0.0346

0.1053

0.1150

0.0014

0.0374

0.1856

0.0734

0.1205

0.1271

0.0318

0.0572

0.1398

0.0763

0.0297

0.1695

0.0805

0.1928

5 Chapter 5 Experiments and results

Population data

Allele

D3S1358

8.0

9.0

15.0

16.0

17.0

12.3

13.0

13.3

14.0

14.1

14.3

10.1

11.0

11.1

11.3

12.0

12.2

D2S441

8.0

8.1

9.0

9.1

10.0

25.0

26.0

27.0

28.0

29.0

0.0052

0.0258

0.0825

0.0103

0.3505

0.0309

0.2165

0.0052

0.2680

0.0058

0.0439

0.0029

0.2675

0.0015

0.0190

0.3626

0.0439

0.1652

0.0029

0.0848

African

American

(n = 342)

0.0775

0.0146

0.0044

Asian (n = 97)

0.0464

0.0361

0.0052

U.S. Caucasian

(n = 361)

0.1025

0.0305

U.S. Hispanic

(n = 236)

0.0784

0.0169

0.0042

0.0291

0.2410

0.0596

0.0014

0.3435

0.0609

0.0471

0.0014

0.2105

0.0021

0.0233

0.2055

0.0487

0.0021

0.2987

0.0445

0.0360

0.3369

VeriFiler

™

Express PCR Amplification Kit User Guide

Allele

20.1

21.0

D5S818

6.0

7.0

8.0

17.1

17.2

18.0

18.2

19.0

20.0

9.0

10.0

10.1

11.0

11.1

11.3

14.0

15.0

15.2

16.0

16.2

17.0

10.0

11.0

12.0

13.0

13.2

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

0.0015

0.0468

0.0322

0.0731

0.2339

African

American

(n = 342)

0.0044

0.0029

0.0906

0.3085

0.0015

0.3187

0.2120

0.0570

0.0044

0.0258

0.3660

0.3299

0.2010

0.0670

0.0103

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.1066

0.2729

0.2382

0.2105

0.0014

0.1510

0.0166

0.0014

U.S. Hispanic

(n = 236)

0.0784

0.3220

0.2797

0.1843

0.0064

0.1229

0.0042

0.0021

0.0155

0.0052

0.0979

0.2268

0.2680

0.0028

0.0055

0.0416

0.0554

0.3560

0.0339

0.0085

0.0530

0.0572

0.3898

5 Chapter 5 Experiments and results

Population data

Allele

D6S1043

7.0

8.0

9.0

10.0

11.0

12.0

12.3

13.0

14.0

15.0

16.0

17.0

17.2

17.3

18.0

18.1

18.3

14.3

15.0

16.0

17.0

18.0

19.0

12.0

12.1

12.3

13.0

14.0

0.2237

0.0965

0.0585

0.0541

0.0161

0.0015

0.0058

0.1535

0.0570

0.1067

0.0015

African

American

(n = 342)

0.3699

0.2237

0.0161

0.0029

0.2062

0.1649

0.0155

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0014

0.3878

0.1427

0.0069

U.S. Hispanic

(n = 236)

0.0021

0.3390

0.1081

0.0085

0.1237

0.0052

0.1237

0.1546

0.0361

0.0052

0.0309

0.1495

0.0722

0.1598

0.2368

0.0859

0.0554

0.0125

0.0042

0.0014

0.0166

0.2964

0.0609

0.0886

0.2055

0.1017

0.1356

0.0297

0.0021

0.0042

0.1780

0.0487

0.1081

0.0021

VeriFiler

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Express PCR Amplification Kit User Guide

Allele

24.0

24.3

25.0

26.0

D7S820

5.0

7.0

7.1

7.3

5.2

6.0

6.3

8.3

9.0

9.1

8.0

8.1

8.2

21.0

21.3

22.0

22.3

23.0

23.3

19.0

19.2

19.3

20.0

20.3

VeriFiler

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Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

0.2281

0.1155

0.0015

0.0117

African

American

(n = 342)

0.1126

0.0015

0.0731

0.0175

0.0015

0.0102

0.0058

0.0015

0.0052

0.0928

0.0412

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0097

0.0014

0.0983

0.0319

U.S. Hispanic

(n = 236)

0.0064

0.0403

0.0127

0.0021

0.0763

0.0021

0.0318

0.0127

0.1289

0.0464

0.0052

0.1440

0.0014

0.1676

0.0277

0.1208

0.0911

0.0106

5 Chapter 5 Experiments and results

Population data

Allele

D8S1179

4.0

5.0

6.0

7.0

8.0

13.0

13.1

14.0

14.1

15.0

16.0

9.0

10.0

10.2

11.0

12.0

12.3

11.0

11.1

11.3

12.0

12.1

12.3

9.2

9.3

10.0

10.1

10.3

100

0.0044

0.0307

0.0526

0.1301

0.0073

African

American

(n = 342)

0.3363

0.2032

0.0877

0.0146

0.0015

0.3608

0.1753

0.2577

0.0258

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.2050

0.1593

0.2562

0.0346

0.0042

U.S. Hispanic

(n = 236)

0.2775

0.1547

0.3072

0.0021

0.0360

0.1237

0.1186

0.0055

0.1025

0.0762

0.1676

0.0139

0.0064

0.0932

0.0530

0.1292

0.0148

VeriFiler

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Express PCR Amplification Kit User Guide

Allele

FGA

16.0

17.0

18.0

19.0

20.0

13.0

13.3

14.0

15.0

15.3

16.1

16.2

17.0

17.2

18.0

18.2

12.3

13.0

14.0

15.0

15.2

16.0

19.0

19.2

19.3

20.0

20.1

20.2

VeriFiler

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Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

0.0015

0.0175

0.0512

0.0029

0.0541

African

American

(n = 342)

0.2193

0.2939

0.1901

0.0643

0.0044

0.0029

0.0928

0.0103

0.0052

0.2010

0.1289

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0332

0.0014

0.3296

0.1662

0.1039

U.S. Hispanic

(n = 236)

0.0318

0.0042

0.0021

0.2733

0.2627

0.1292

0.0103

0.0258

0.0567

0.0876

0.0249

0.0499

0.1233

0.0021

0.0127

0.0805

0.0847

101

5 Chapter 5 Experiments and results

Population data

Allele

25.1

25.2

25.3

26.0

26.1

26.2

23.3

24.0

24.1

24.2

24.3

25.0

27.0

27.2

28.0

28.2

29.0

29.1

22.1

22.2

22.3

23.0

23.1

23.2

20.3

21.0

21.2

21.3

22.0

102

0.0015

0.0702

0.1330

0.1184

African

American

(n = 342)

0.1228

0.1988

0.0044

0.0015

0.1696

0.0015

0.0234

0.0146

0.0058

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0263

0.1343

0.0014

0.0789

0.0042

0.0125

0.1524

0.0028

0.1787

0.0055

0.2050

0.0309

0.1495

0.0052

0.0722

0.0052

0.2062

0.1031

0.2423

U.S. Hispanic

(n = 236)

0.0614

0.1419

0.1186

0.0445

0.0021

0.0042

0.1208

0.0042

0.1525

0.1653

VeriFiler

™

Express PCR Amplification Kit User Guide

Allele

Penta D

2.2

3.2

49.2

50.2

51.2

43.2

44.2

45.2

46.2

47.2

48.2

7.4

8.0

9.0

5.0

6.0

7.0

32.2

33.2

34.2

41.2

42.2

29.2

30.0

30.2

31.0

31.2

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

0.1140

0.0088

0.0439

0.0102

0.0439

0.1082

0.1681

African

American

(n = 342)

0.0015

0.0052

Asian (n = 97)

U.S. Caucasian

(n = 361)

U.S. Hispanic

(n = 236)

0.0021

0.0103

0.0052

0.3144

0.0042

0.0208

0.2216

0.0169

0.0021

0.0064

0.0021

0.0191

0.2415

103

5 Chapter 5 Experiments and results

Population data

Allele

15.0

16.0

17.0

18.0

Penta E

4.0

8.0

9.0

10.0

5.0

6.0

7.0

11.0

11.4

12.0

13.0

14.0

14.2

12.0

12.2

13.0

13.4

14.0

14.1

9.2

9.4

10.0

11.0

11.2

104

0.0643

0.1287

0.1038

0.0687

0.0950

0.0015

0.1038

0.1667

0.0512

0.0468

African

American

(n = 342)

0.0994

0.1798

0.1082

0.0833

0.0015

0.0249

0.0044

0.0015

0.1804

0.1031

0.0103

0.1856

0.1804

0.0103

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.2327

0.1967

0.0609

0.1150

0.1260

0.0097

0.0028

0.0014

U.S. Hispanic

(n = 236)

0.1631

0.1441

0.0720

0.1568

0.0106

0.0042

0.0021

0.1598

0.0928

0.0515

0.0722

0.0052

0.0258

0.0619

0.0873

0.1994

0.0859

0.0623

0.0762

0.1690

0.0139

0.0125

0.0859

0.0742

0.1737

0.0932

0.0720

0.0360

0.1186

0.0254

0.0169

0.0847

VeriFiler

™

Express PCR Amplification Kit User Guide

Allele

TH01

19.4

20.0

21.0

22.0

23.0

24.0

25.0

26.0

27.0

16.4

17.0

17.4

18.0

18.4

19.0

15.0

15.2

15.4

16.0

16.2

3.0

4.0

5.0

5.3

6.0

6.3

7.0

7.3

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

0.0044

0.1316

0.4079

0.0044

0.0015

African

American

(n = 342)

0.0556

0.0409

0.0439

0.0161

0.0073

0.0103

0.0206

0.0309

0.0206

0.0052

0.0825

0.0773

0.0412

0.1134

0.0567

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0097

0.0028

0.0014

0.0485

0.0332

0.0152

0.0429

0.0014

0.0512

U.S. Hispanic

(n = 236)

0.0212

0.0064

0.0021

0.0064

0.0042

0.0551

0.0339

0.0212

0.0911

0.0021

0.0614

0.1701

0.2680

0.0014

0.2355

0.1939

0.2394

0.2966

105

5 Chapter 5 Experiments and results

Population data

Allele

8.0

9.0

10.0

10.1

10.3

11.0

7.0

7.1

7.3

4.0

5.0

6.0

10.3

11.0

12.0

TPOX

13.0

13.3

8.0

8.3

9.0

9.3

10.0

12.0

13.0

14.0

15.0

16.0

0.3670

0.1959

0.0863

0.2164

0.0892

0.0175

0.0263

0.0015

African

American

(n = 342)

0.1959

0.1594

0.0965

0.0044

0.0722

0.4433

0.0412

0.0052

Asian (n = 97)

U.S. Caucasian

(n = 361)

0.0014

0.0956

0.1191

0.3449

0.0083

U.S. Hispanic

(n = 236)

0.0911

0.1462

0.2182

0.0085

0.5515

0.0773

0.0258

0.2990

0.0464

0.5249

0.1274

0.0499

0.2521

0.0014

0.0416

0.0014

0.4852

0.0932

0.0487

0.2542

0.0085

0.0064

0.1038

106

VeriFiler

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Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

Allele

African

American

(n = 342)

Asian (n = 97)

U.S. Caucasian

(n = 361)

U.S. Hispanic

(n = 236) vWA

16.0

17.0

18.0

18.2

19.0

20.0

10.0

11.0

12.0

13.0

14.0

15.0

15.2

21.0

22.0

23.0

24.0

25.0

0.2500

0.2354

0.1491

0.0629

0.0161

0.0029

0.0015

0.0088

0.0804

0.1915

0.0015

0.1392

0.3144

0.2062

0.1082

0.0155

0.1959

0.0206

0.2008

0.2839

0.2022

0.1039

0.0069

0.0014

0.0928

0.1053

0.0014

Table 7 Probability of identity (PI) values for the VeriFiler ™ Express kit STR loci

Locus

CSF1PO

D10S1248

D12S391

D13S317

D16S539

D18S51

D19S433

African

American

(n = 342)

0.0828

0.0666

0.0346

0.1339

0.0706

0.0268

0.0358

Asian (n = 97)

0.0984

0.0863

0.0444

0.0686

0.0873

0.0456

0.0676

Caucasian

(n = 361)

0.1291

0.0979

0.0219

0.0767

0.0948

0.0284

0.0844

Hispanic

(n = 236)

0.1271

0.1010

0.0268

0.0495

0.0742

0.0266

0.0661

0.2839

0.2458

0.1801

0.0508

0.0106

0.0021

0.0805

0.1441

0.0021

VeriFiler

™

Express PCR Amplification Kit User Guide

107

5 Chapter 5 Experiments and results

Population data

Probability of paternity exclusion observation

Locus

D1S1656

D21S11

D22S1045

D2S1338

D2S441

D3S1358

D5S818

D6S1043

D7S820

D8S1179

FGA

Penta D

Penta E

TH01

TPOX vWA

Combined

African

American

(n = 342)

0.0310

0.0395

0.0538

0.0212

0.0937

0.1067

0.1012

0.0266

0.0863

0.0650

0.0289

0.0257

0.0169

0.1017

0.0892

0.0615

2.546 × 10

−30

Asian (n = 97)

0.1350

0.0721

0.0266

0.0992

0.0405

0.0403

0.0424

0.0566

0.0967

0.0307

0.1027

0.0745

0.0124

0.1405

0.2233

0.0759

4.693 × 10

−28

Caucasian

(n = 361)

0.0768

0.1437

0.0517

0.0597

0.0607

0.0356

0.0188

0.0497

0.1221

0.0257

0.0872

0.0542

0.0211

0.0919

0.1783

0.0639

1.092 × 10

−28

The following table shows the Probability of paternity exclusion (PE) values of the

VeriFiler

™

Express kit STR loci individually and combined.

The PE value is the probability, averaged over all possible mother-child pairs, that a random alleged father will be excluded from paternity after DNA typing using the

VeriFiler

™

Express kit STR loci (Chakraborty, Stivers, and Zhong, 1996).

Table 8 Probability of paternity exclusion values for the VeriFiler ™ loci

Express kit STR

Locus

CSF1PO

D10S1248

D12S391

D13S317

African

American

(n = 342)

0.5724

0.6138

0.7214

0.4687

Asian (n = 97)

0.5351

0.5619

0.6849

0.6068

Caucasian

(n = 361)

0.4708

0.5346

0.7794

0.5877

Hispanic

(n = 236)

0.4756

0.5286

0.7559

0.6657

Hispanic

(n = 236)

0.0942

0.1271

0.0285

0.0808

0.0611

0.0261

0.0242

0.0448

0.1533

0.0285

0.1035

0.0460

0.0149

0.0853

0.1476

0.0710

2.947 × 10

−29

108

VeriFiler

™

Express PCR Amplification Kit User Guide

Chapter 5 Experiments and results

Population data

D7S820

D8S1179

FGA

Penta D

Penta E

TH01

TPOX vWA

Combined

D16S539

D18S51

D19S433

D1S1656

D21S11

D22S1045

D2S1338

D2S441

D3S1358

D5S818

D6S1043

0.5615

0.6186

0.7457

0.7607

0.8069

0.5259

0.5557

0.6283

0.999999999984

527

0.6018

0.7555

0.7167

0.7356

0.7028

0.6524

0.7832

0.5471

0.5158

0.5293

0.7559

0.5580

0.6807

0.6135

0.6916

0.6440

0.5350

0.7382

0.5259

0.4613

0.5973

0.7562

0.5323

0.6976

0.6994

0.5908

0.8347

0.4535

0.6330

0.6302

0.7173

0.6509

0.7830

0.5476

0.3428

0.5872

0.3988

0.6206

0.999993438259

661

0.999989850701

339

0.5436

0.7481

0.5698

0.7957

0.6661

0.4888

0.7606

0.5616

0.5842

0.4503

0.6602

0.5761

0.6303

0.7585

0.6782

0.8184

0.5624

0.4452

0.6009

0.999985788729

133

0.5918

0.7563

0.6172

0.7679

0.6829

0.4388

0.7474

0.5250

0.5427

0.4825

0.7475

VeriFiler

™

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109

110

Troubleshooting

Observation

Faint or no signal from both the

DNA Control 007 and the DNA test samples at all loci

Possible cause

The incorrect volume of Master

Mix or Primer Set was used.

The DNA Polymerase was not activated.

The Master Mix was not vortexed thoroughly before aliquoting.

The Primer Set was exposed to too much light.

Evaporation.

Recommended action

Use the correct volume of Master Mix or

Primer Set.

Repeat the amplification with an initial hold at

95°C for 1 minute.

Vortex the Master Mix thoroughly.

Store the Primer Set protected from light.

Ensure that the plate is properly sealed with film and that a compression pad was used with the GeneAmp

™

PCR System 9700. (A compression pad is not needed with other validated thermal cyclers.)

The thermal cycler malfunctioned.

Incorrect thermal cycler conditions were used.

A MicroAmp

™

base was used with a tray/retainer set and tubes in GeneAmp

System 9700.

™ PCR

See the thermal cycler user manual and check the instrument calibration.

Use correct thermal cycler conditions.

Remove the MicroAmp

™

base.

The tubes or plate were not seated tightly in the thermal cycler during amplification.

The wrong PCR reaction tubes or plate were used.

Push the tubes or plate firmly into the block after first cycle.

Use MicroAmp

™

Reaction Tubes with Caps or the MicroAmp

™

Optical 96 ‑well Reaction Plate for the GeneAmp ™

Thermal Cycler.

PCR System 9700 or Veriti ™

Use correct genetic analyzer settings.

Insufficient PCR product was electrokinetically injected.

Degraded formamide was used. Check the storage of formamide. Do not thaw and refreeze multiple times. Try Hi ‑Di

™

Formamide.

VeriFiler

™

Express PCR Amplification Kit User Guide

Appendix A Troubleshooting

Population data

Observation

Faint no signal Control and samples all loci

Positive signal from DNA

Control 007 but partial or no signal from DNA test samples

Possible cause

The sample punch location was not optimal.

Insufficient lysis of the swab head occurred.

Proper low-TE buffer not used for treated paper substrates.

Recommended action

For blood samples on treated paper, punch in the center of the blood stain.

For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal location you have previously determined.

For buccal samples collected with the Bode

Buccal DNA Collector

™

device, punch from near the tip of the collector.

Ensure the swab heads are incubated for

20 minutes in 400 µL of Prep ‑n‑Go

™

Buffer.

Insufficient volume of swab lysate was added to the reaction.

Proper low-TE buffer not used for treated paper substrates.

The test sample was diluted in water or in the wrong buffer

(for example, a TE buffer with an incorrect EDTA concentration).

The sample punch location was not optimal.

Prepare low-TE buffer. See “Prepare low-TE buffer“ on page 18.

Redilute DNA using low-TE buffer (with 0.1 mM

EDTA).

For blood samples on treated paper, punch in the center of the blood stain.

For buccal samples on treated paper, punch in the center of the buccal transfer or punch in the optimal location you have previously determined.

For buccal samples collected with the Bode

Buccal DNA Collector

™

device, punch from near the tip of the collector.

Ensure the swab heads are incubated for

20 minutes in 400 µL of Prep

‑n‑Go

™ Buffer.

Prepare low-TE buffer.

VeriFiler

™

Express PCR Amplification Kit User Guide

111

A Appendix A Troubleshooting

Population data

More than two alleles present at a locus

Observation

Poor peak height balance

Some but not all loci visible on electropherogram of DNA Test

Samples

STR profiles contain many offscale alleles

Possible cause

Exogenous DNA is present in the sample.

Stutter product (–1 repeat unit position) was amplified.

Triallelic patterns occur.

Incomplete 3´ A base addition

(n−1 nt position) occured.

The signal exceeds the dynamic range of the instrument.

Poor spectral separation occurred.

The double-stranded DNA was not completely denatured.

Contamination was carried over from the disc punching tool.

Recommended action

Use appropriate techniques to avoid introducing foreign DNA during laboratory handling.

See Chapter 5, “Experiments and results“

Confirm the triallelic pattern per the laboratory's guidelines.

Include the final extension step of 60°C for

10 minutes in the PCR.

Incubate the amplification products at 60°C for an additional 10 minutes.

Ensure the cycle number is optimized. Use fewer PCR cycles or interpret the off-scale data according to your laboratory procedure.

Perform a spectral calibration.

Confirm that Filter Set J6 modules are installed and used for analysis.

Use the recommended amount of Hi ‑Di

™

Formamide and heat the sample plate at 95°C for 3 minutes.

Clean the disc punching tool thoroughly. If necessary, include a blank punch step in between the sample punches.

Use correct thermal cycler conditions.

Incorrect thermal cycler conditions were used.

Expected amplification pattern observed in the TAZ channel.

™ dye

Peak heights are typically elevated at

D22S1045 for buccal samples. Follow laboratory guidelines for evaluation of the generated results.

Use a 1.2 mm disc.

The punched disc you used was too large.

Insufficient lysis of the swab head occurred.

The PCR reaction volume you used is lower than the volume required for the amplification.

The PCR cycle number used was too high.

Blood samples: Too much liquid blood was spotted onto the paper substrate.

Ensure the swab heads are incubated for

20 minutes in 400 µL of Prep ‑n‑Go

™

Buffer.

Use the correct PCR reaction volume: 25 μL

Perform a sensitivity experiment to determine the optimal PCR cycle number based on the sample type.

Spot <100 µL of liquid blood per sample area.

112

VeriFiler

™

Express PCR Amplification Kit User Guide

Observation

Data collected on the 3730 instrument with POP-7

™ polymer fails sizing

Data collected on the 3730 instrument with POP-7

™ polymer: the D2S441 and

D1S1656 markers in some allelic ladder samples fail base-pair spacing quality assessment

Appendix A Troubleshooting

Population data

Possible cause

The 60-bp size-standard peak is occasionally obscured by the primer peak.

Data was analyzed using the

Light setting for Smoothing.

Recommended action

Reinject samples that fail to recognize the

60 base-pair peak.

Use the 80 to 460 bp size-standard definition after performing appropriate validation studies

(as a general rule, the 60 base-pair peak is not required for accurate fragment sizing using the

3rd Order Least Squares sizing method).

For more information, see the

GeneMapper

™

ID ‑X Software v1.4 New Features and

Installation Procedures User Bulletin

(Pub. No. 4477684), “Known issues: 3730 DNA

Analyzer sizing failures”.

Use the None setting for Smoothing after performing appropriate validation studies.

For more information, see the

GeneMapper

™

ID ‑X Software v1.4 New Features and

Installation Procedures User Bulletin

(Pub. No. 4477684), “Known issues: 3730 DNA

Analyzer sizing failures”.

VeriFiler

™

Express PCR Amplification Kit User Guide

113

Materials required but not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.

MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

STR kit

Item

VeriFiler ™ Express PCR Amplification Kit, 200-reaction kit

GeneScan ™ 600 LIZ ™ Size Standard v2.0, 2 × 200 µL

IMPORTANT!

Do not use GeneScan

GeneScan ™ 500 LIZ ™

™ 350 ROX ™ , GeneScan ™ 500 ROX

Size Standards with this kit.

™ , or

‑Di ™

Formamide, 25

‑mL

Source

A32014

4408399

4311320

Sample preparation required materials

Treated paper substrate

Item

Collection system: NUCLEIC-CARD ™ system or Whatman FTA ™

NUCLEIC-CARD ™ Sample Collection Device

NUCLEIC-CARD ™ matrix, 1 spot

NUCLEIC-CARD ™ COLOR matrix, 1 spot

Whatman ™ FTA ™ Classic Cards

Whatman ™ EasiCollect ™ system

Sample preparation:

Prep ‑n‑Go ™ Buffer (for use with untreated paper substrates)

Low-TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0)

Punch tool:

Harris Micro-Punch ™ tool, 1.2

‑mm

Source

4473980

4474001

4473974

MLS

4467079

Teknova T0223

MLS

114

VeriFiler

™

Express PCR Amplification Kit User Guide

Appendix B Materials required but not supplied

Sample preparation required materials

Item

BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a

1.2

‑mm punch head

BSD1000-GenePunch Automated Dried Sample Punch Instrument with a

1.2

‑mm punch head

Untreated paper substrate

Item

Collection system: Bode or paper

Buccal DNA Collector ™ Device

903 paper

Punch tool:

Harris Micro-Punch ™ tool, 1.2

‑mm

BSD600-Duet Semi-Automated Dried Sample Punch Instrument with a

1.2

‑mm punch head

BSD1000-GenePunch Automated Dried Sample Punch Instrument with a

1.2

‑mm punch head

Source

Contact your local sales office.

Source

Contact Bode Cellmark Forensics

MLS

Contact your local sales office.

Swab substrate

Item

96-well deep-well plate format

PrepFiler ™ 96-Well Processing Plates

Source

Sample preparation:

Prep ‑n‑Go ™ Buffer (for use with buccal swab substrates)

Heated lysis protocol only: 1.5 mL tube format or 96-well deep-well plate format

1.5 mL tube format

1.5 mL tubes

Oven

4471406

MLS

VWR

™

Scientific dry heat block or equivalent

4392904

MLS Robbins Scientific ™ Model 400 Hybridization Incubator or equivalent

Agilent

™

Benchtop Rack for 200 µL Tubes/V Bottom Plates (metal) or equivalent

Agilent Technologies 410094

IMPORTANT!

Do not use a plastic plate adaptor.

VeriFiler

™

Express PCR Amplification Kit User Guide

115

B Appendix B Materials required but not supplied

Thermal cycler required materials

ProFlex

™

PCR System

Item

ProFlex ™ 96 ‑well PCR System

ProFlex ™ 2 × 96 ‑well PCR System

ProFlex

™

3 × 32

‑Well PCR System

GeneAmp

™

PCR System 9700

Item

GeneAmp ™ PCR System 9700, 96-Well Silver

GeneAmp ™ PCR System 9700, 96-Well Gold-Plated

Silver 96-Well Sample Block

Gold-Plated 96-Well Block

Genetic analyzer required materials

3500 Series Genetic Analyzer

Item

3500/3500xL Data Collection Software v3 (RUO)

3500/3500xL Data Collection Software v2 (RUO)

HID Updater 3500 Data Collection Software v2

Anode buffer container (ABC)

Cathode buffer container (CBC)

POP-4 ™ Polymer (960 samples) for 3500/3500xL Genetic Analyzers

POP-4 ™ Polymer (384 samples) for 3500/3500xL Genetic Analyzers

DS-36 Matrix Standard Kit (Dye Set J6)

Conditioning reagent

8-Capillary array, 36 cm for 3500 Genetic Analyzers

24-Capillary array, 36 cm for 3500xL Genetic Analyzers

96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers

8-Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers

116

Source

4484075

4484076

4484073

Source

N8050001

4314878

N805021

4314443

Source

A26287

[1]

4475183

[2]

4480670

4393927

4408256

4393710

4393715

4425042

4393718

4404683

4404687

4410228

4410231

VeriFiler

™

Express PCR Amplification Kit User Guide

Item

8-Strip Septa for 3500/3500xL Genetic Analyzers

96-Well Septa for 3500/3500xL Genetic Analyzers

Septa Cathode Buffer Container, 3500 series

[1]

[2]

Contact your Thermo Fisher Scientific HID representative.

3130 Series Genetic Analyzer

Item

3130 Data Collection Software v4

3130 xl Data Collection Software‑v4

3130/3730 Data Collection Software ‑v4 6‑Dye Module v1

96 ‑Well Plate Septa

Reservoir Septa

3100/3130 xl Genetic Analyzer Capillary Array, 36‑cm

POP-4

™

Polymer for 3130/3130

xl Genetic Analyzers

Running Buffer, 10✕

DS ‑36 Matrix Standard Kit (Dye Set J6)

MicroAmp

™

Optical 96-Well Reaction Plate

3730 Series Genetic Analyzer

Item

3730/3730 xl Data Collection Software‑v4

3130/3730 Data Collection Software v4 6 ‑Dye Module v1

96 ‑Well Plate Septa

Reservoir Septa

3730 DNA Analyzer 48-Capillary Array, 36-cm

3730 xl DNA Analyzer 48-Capillary Array, 36-cm

POP-7 ™ Polymer for 3730/3730 xl DNA Analyzers

Running Buffer, 10✕

DS ‑36 Matrix Standard Kit (Dye Set J6)

MicroAmp ™ Optical 96-Well Reaction Plate

Appendix B Materials required but not supplied

Genetic analyzer required materials

Source

4410701

4412614

4410715

Source

4475154

4480670

4315933

4315932

4331247

4331244

4335611

4335613

4425042

N8010560

Source

4475105

4475126

4480670

4315933

4315932

4315931

4352755

402824

4425042

N8010560

VeriFiler

™

Express PCR Amplification Kit User Guide

117

B Appendix B Materials required but not supplied

Analysis software required materials

Item

250 ‑μL Glass Syringe (array-fill syringe)

5.0

‑mL Glass Syringe (polymer-reserve syringe)

Analysis software required materials

GeneMapper

™

ID ‑X Software

Item

GeneMapper ™ ID

‑X Software v1.5 Full Installation

GeneMapper ™

ID ‑X Software v1.5 Client Installation

GeneMapper ™

ID ‑X Software v1.4 Full Installation

GeneMapper ™ ID

‑X Software v1.4 Client Installation

Miscellaneous required materials

Plates and tubes

Item

MicroAmp ™ 96-Well Tray

MicroAmp ™ Reaction Tube with Cap, 0.2 mL

MicroAmp

™

8-Tube Strip, 0.2 mL

MicroAmp ™ Optical 8-Cap Strips

MicroAmp ™ 96-Well Tray/Retainer Set

MicroAmp

™

96-Well Base

MicroAmp ™ Clear Adhesive Film

MicroAmp ™ Optical Adhesive Film

MicroAmp

™

Optical 96-Well Reaction Plate

Source

4304470

628 ‑3731

Source

A27884

A27886

4479707

4479711

Source

N8010541

N8010540

N8010580

N8010535

403081

N8010531

4306311

4311971

N8010560

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Laboratory supplies

Various procedures

Aerosol resistant pipette tips

Microcentrifuge tubes

Pipettors

Tape, labeling

Tube, 50-mL Falcon ™

Tube decapper, autoclavable

Deionized water, PCR grade

Vortex

[1] Major laboratory supplier

Item

Appendix B Materials required but not supplied

Miscellaneous required materials

Source

MLS

[1]

MLS

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Common Callouts and Arrows

Plate layouts

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows

use the TechComm_Inkscape_Callout&Arrow_Libary.

2. Edit number and/or line-length, as needed.

■

Example PCR plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

■

Example electrophoresis plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

3. Delete this text, this rectangle, and unused

callouts, arrows, or other SVG elements

Example PCR plate layout

1 1 1

The following layout is recommended for use with the sensitivity experiment in the

Perform PCR chapter. Create 3 identical plates for amplification at 4 different cycle numbers.

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Common Callouts and Arrows

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows

use the TechComm_Inkscape_Callout&Arrow_Libary.

2. Edit number and/or line-length, as needed.

3. Delete this text, this rectangle, and unused

callouts, arrows, or other SVG elements

before adding this SVG to the repository.

Example electrophoresis plate layout

1 1 1

Appendix C Plate layouts

Example electrophoresis plate layout

The following layout is recommended for use with the sensitivity experiment in the

Perform PCR chapter.

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PCR work areas

■

Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

■

PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

■

Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Work area setup and lab design

Many resources are available for the appropriate design of a PCR laboratory. If you are using this kit for:

• Forensic DNA testing, see "Forensic Laboratories: Handbook for Facility

Planning, Design, Construction, and Moving", National Institute of Justice, 1998

• Parentage DNA testing, see the "Guidance for Standards for Parentage

Relationship Testing Laboratories", American Association of Blood Banks, 7th edition, 2004

The sensitivity of this kit (and other PCR-based tests) enables amplification of minute quantities of DNA, necessitating precautions to avoid contamination of samples yet to be amplified (Kwok and Higuchi, 1989).

Process samples carefully to prevent contamination by human DNA. Wear gloves at all times and change them frequently. Close sample tubes when not in use. Limit aerosol dispersal by handling sample tubes and reagents carefully.

Note: We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology.

PCR setup work area materials

IMPORTANT! Do not remove these items from the PCR Setup Work Area.

• Calculator

• Gloves, disposable

• Marker pen, permanent

• Microcentrifuge

• Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate nuclease-free tube

(for master mix preparation)

• Microcentrifuge tube rack

• Pipette tips, sterile, disposable hydrophobic filter-plugged

• Pipettors

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Appendix D PCR work areas

Amplified DNA work area

• Tube decapper, autoclavable

• Vortex

Amplified DNA work area

IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area.

Use only the validated thermal cyclers listed in “Instrument and software compatibility“ on page 14.

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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.

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Safety

Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions:

Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and

Support” section in this document.

Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing).

Minimize the inhalation of chemicals. Do not leave chemical containers open.

Use only with 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.

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Safety

Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.

U.S. Department of Health and Human Services, Biosafety in Microbiological

and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)

21-1112, Revised December 2009; found at:

www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

World Health Organization, Laboratory Biosafety Manual, 3rd Edition,

WHO/CDS/CSR/LYO/2004.11; found at:

www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

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Documentation and support

■

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

■

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Customer and technical support

For support:

• In North America—Send an email to [email protected], or call 888-821-4443 option 1.

• Outside North America—Contact your local support office.

• For latest services and support information for all locations, go to

thermofisher.com/support.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life

Technologies' website at www.thermofisher.com/us/en/home/global/

terms-and-conditions.html. If you have any questions, please contact Life

Technologies at www.thermofisher.com/support.

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130

References

Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994.

Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.

J. Forensic Sci. 39:362–372.

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.

Bacher, J., Schumm, J.W., 1998. Development of Highly Polymorphic Pentanucleotide

Tandem Repeat Loci with Low Stutter. Profiles in DNA 2:3– 6.

Barber, M.D., McKeown, B.J. and Parkin, B.H. 1996. Structural variation in the alleles of a short tandem repeat system at the human alpha fibrinogen locus. Int. J. Leg. Med.

108:180–185.

Barber, M.D. and Parkin, B.H. 1996. Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179. Intl. J. Legal Med. 109:62–65.

Barber, M.D., Piercy, R.C., Andersen, J.F. and Parkin, B.H. 1995. Structural variation of novel alleles at the Hum vWA and Hum FES/FPS short tandem repeat loci. Int. J. Leg.

Med. 108:31–35.

Begovich A.B., McClure G.R., Suraj V.C., Helmuth R.C., Fildes N., Bugawan T.L.,

Erlich H.A., Klitz W. 1992. Polymorphism, recombination, and linkage disequilibrium within the HLA class II region. J. Immunol. 148:249–258.

Bender, K., Farfan, M.J., Schneider, P.M. 2004. Preparation of degraded human DNA under controlled conditions. Forensic Sci. Int. 139:134–140.

Brinkmann, B., Klintschar, M., Neuhuber, F., Huhne, J. and Rolf, B. 1998. Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat.

Am. J. Hum. Genet. 62:1408–1415.

Brinkmann, B., Möller, A. and Wiegand, P. 1995. Structure of new mutations in 2 STR systems. Intl. J. Legal Med. 107:201–203.

Budowle, B. et al. 2010. Population genetic analyses of the NGM

™

STR loci. Int. J. Legal

Med. e-publication www.springerlink.com/content/p713q3w5440674u3/

Butler, J.M. 2005. Forensic DNA Typing. Burlington, MA:Elsevier Academic Press.

Butler, J.M., Shen, Y., McCord, B.R. 2003. The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci. 48:1054–1064.

Chakraborty, R. Kimmel, M., Stivers, D., Davison, L., and Deka, R. 1997. Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc. Natl. Acad. Sci.

USA 94:1041–1046.

Chakraborty, R. and Stivers, D.N. 1996. Paternity exclusion by DNA markers: effects of paternal mutations. J. Forensic Sci. 41:671–677.

Chakraborty, R., Stivers, D., and Zhong, Y. 1996. Estimation of mutation rates from parentage exclusion data: applications to STR and VNTR loci. Mutat. Res. 354:41–48.

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References

Limited product warranty

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.

Drabek, J., Chung, D.T., Butler, J.M., McCord, B.R. 2004. Concordance study between

Miniplex assays and a commercial STR typing kit. J. Forensic Sci. 49:859–860.

Edwards, A., Civitello, A., Hammond, H., and Caskey, C. 1991. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet.

49:746–756.

Edwards, A., Hammond, H.A., Lin, J., Caskey, C.T., and Chakraborty, R. 1992. Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12:241–253.

Excoffier, L., Lischer, H.E.L. 2010. A new series of programs to perform population genetics analyses under Linux and Windows. Arleguin suite v. 3.5. Mol. Ecol. Res.

10:564–567.

Frank, W., Llewellyn, B., Fish, P., et al. 2001. Validation of the AmpFℓSTR

™

Plus

™

Profiler

PCR Amplification Kit for use in forensic casework. J. Forensic Sci. 46:642–646.

Glock, B., Dauber, E.M., Schwartz, D.W., Mayr W.R. 1997. Additional variability at the

D12S391 STR locus in an Austrian population sample: sequencing data and allele distribution. Forensic Sci. Int. 90:197–203.

Grossman, P.D., Bloch, W., Brinson, E., Chang, C.C., Eggerding, F.A., Fung, S.,

Iovannisci, D.M., Woo, S., Winn-Deen, E.S. 1994. High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

Nucleic Acids Res. 22:4527–4534.

Grubwieser, P. Muhlmann, R., Berger, B., Niederstatter, H., Palvic, M., Parson, W.

2006. A new “mini-STR-multiplex” displaying reduced amplicon lengths for the analysis of degraded DNA. Int. J. Legal Med. 120:115–120.

Guo, S.W. and Thompson, E.A. 1992. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48:361–372.

Hammond, H., Jin, L., Zhong, Y., Caskey, C., and Chakraborty, R. 1994. Evaluation of

13 short tandem repeat loci for use in personal identification applications. Am J. Hum.

Genet. 55:175–189.

Hill, C.R., Duewer, D.L., Kline, M.C., Coble, M.D., and Butler, J.M. 2013. U.S.

population data for 29 autosomal STR loci. Forensic Sci. Int. Genet. 7:e82– e83.

Holt, C., Stauffer, C., Wallin, J., et al. 2000. Practical applications of genotypic Surveys for forensic STR testing. Forensic Sci. Int. 112:91–109.

Kalinowski, S.T. 2006. HW-QuickCheck: an easy-to-use computer program for checking genotypes for agreement with Hardy-Weinberg expectations. Molecular

Ecology Notes 6:974–979.

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References

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132

Kimpton, C., Walton, A., and Gill, P. 1992. A further tetranucleotide repeat polymorphism in the vWF gene. Hum. Mol. Genet. 1:287. Kong, X., Murphy, K., Raj,

T., He, C., White, P.S., Matise, T.C. 2004. A combined linkage-physical map of the human genome. Am. J. Hum. Genet. 75:1143–1148.

Kwok, S., and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237–

238.

Lareu, M.V., Pestoni, M.C., Barros, F., Salas, A., Carracedo, A. 1996. Sequence variation of a hypervariable short tandem repeat at the D12S391 locus. Gene 182:151–153.

Lazaruk, K., Walsh, P.S., Oaks, F., Gilbert, D., Rosenblum, B.B., Menchen, S., Scheibler,

D., Wenz, H.M., Holt, C., Wallin, J. 1998. Genotyping of forensic short tandem repeat

(STR) systems based on sizing precision in a capillary electrophoresis instrument.

Electrophoresis 19:86–93.

Levene, H. 1949. On a matching problem in genetics. Ann. Math. Stat. 20:91–94.

Li, B., Ge, J., Wu, F., Ye, L., Budowle, B., Vhen, Y. 2013. Population genetic analyses of the STR loci of the AmpFℓSTR

™

NGM SElect

™

PCR Amplification Kit for Han population in Fujian Province, China. Int J. Legal Med. 127:345–346.

Li, H. Schmidt, L., Wei, M-H., Hustad, T. Leman, M.I., Zbar, B. and Tory, K. 1993.

Three tetranucleotide polymorphisms for loci:D3S1352; D3S1358; D3S1359. Hum. Mol.

Genet. 2:1327.

Magnuson, V.L., Ally, D.S., Nylund, S.J., Karanjawala, Z.E., Rayman, J.B., Knapp, J.I.,

Lowe, A.L., Ghosh, S., Collins, F.S. 1996. Substrate nucleotide-determined nontemplated addition of adenine by Taq DNA polymerase: implications for PCRbased genotyping and cloning. Biotechniques 21:700–709.

Mansfield, E.S., Robertson, J.M., Vainer, M., Isenberg, A.R., Frazier, R.R., Ferguson, K.,

Chow, S., Harris, D.W., Barker, D.L., Gill, P.D., Budowle, B., McCord, B.R. 1998.

Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis. Electrophoresis 19:101–107.

Mills, K.A., Even, D., and Murrau, J.C. 1992. Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus (FGA). Hum. Mol. Genet. 1:779. Möller, A. and

Brinkmann, B. 1994. Locus ACTBP2 (SE33): Sequencing data reveal considerable polymorphism. Int. J. Leg. Med. 106:262–267.

Möller, A. and Brinkmann, B. 1995. PCR-VNTRs (PCR-Variable Number of Tandem

Repeats) in forensic science. Cellular & Molec. Bio. 41(5):715-724. Momhinweg, E.,

Luckenbach, C., Fimmers, R., and Ritter, H. 1998. D3S1358: sequence analysis and gene frequency in a German population. Forensic Sci. Int. 95:173–178.

Moretti, T., Baumstark, A., Defenbaugh, D., Keys, K., Smerick, J., and Budowle, B.

2001. Validation of short tandem repeats (STRs) for forensic usage: Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. J. Forensic Sci. 46(3):647–660.

Mulero, J.J., Chang, C.W., and Hennessy, L.K. 2006. Characterization of N+3 stutter product in the trinucleotide repeat locus DYS392. J. Forensic Sci. 51:826–830.

Nakahori, Y., Takenaka, O., and Nakagome, Y. 1991. A human X-Y homologous region encodes amelogenin. Genomics 9:264–269.

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Sci. USA 70:3321–3323. Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590.

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References

Limited product warranty

Puers C., Hammond H.A., Jin L., Caskey C.T., Schumm J.W. 1993. Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus

HUMTH01[AATG]n and reassignment of alleles in population analysis by using a locus-specific allelic ladder. Am J. Hum. Genet. 53(4):953–958.

Raymond M. and Rousset F. 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86:248–249.

Rousset, F. 2008. Genepop'007: A complete reimplementation of the Genepop software for Windows and Linux. Molecular Ecology Resources 8:103–106.

Scientific Working Group on DNA Analysis Methods (SWGDAM). 2012. Validation

Guidelines for DNA Analysis Methods. Available at http://swgdam.org/

SWGDAM_Validation_Guidelines_APPROVED_Dec_2012.pdf. Accessed 29 July 2013.

Sensabaugh, G.F. 1982. Biochemical markers of individuality. In: Saferstein, R., ed.

Forensic Science Handbook. Prentice-Hall, Inc., New York, pp. 338–415.

Sharma, V. and Litt, M. 1992. Tetranucleotide repeat polymorphism at the D21S11 locus. Hum Mol. Genet. 1:67.

Shin, C.H., Jang, P., Hong, K.M., Paik, M.K. 2004. Allele frequencies of 10 STR loci in

Koreans. Forensic Sci. Int. 140:133–135.

Smith, R.N. 1995. Accurate size comparison of short tandem repeat alleles amplified by PCR. Biotechniques 18:122–128.

Sparkes, R., Kimpton, C., Watson, S., Oldroyd, N., Clayton, T., Barnett, L., Arnold, J.,

Thompson, C., Hale, R., Chapman, J., Urquhart, A., and Gill, P. 1996a. The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies. Int. J. Legal Med. 109:186–194.

Sparkes, R., Kimpton, C., Gilbard, S., Carne, P., Andersen, J., Oldroyd, N., Thomas, D.,

Urquhart, A., and Gill, P. 1996b. The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artifacts, casework studies and success rates. Int. J. Legal

Med. 109:195–204.

Straub, R.E., Speer, M.C., Luo, Y., Rojas, K., Overhauser, J., Ott, J., and Gilliam, T.C.

1993. A microsatellite genetic linkage map of human chromosome 18. Genomics 15:48–

56.

Szibor, R., Lautsch, S., Plate, I., Bender, K., Krause, D. 1998. Population genetic data of the STR HumD3S1358 in two regions of Germany. Int. J. Legal Med. 111(3):160–161.

Waiyawuth, W., Zhang, L., Rittner, C., Schneider, P.M. 1998. Genetic analysis of the short tandem repeat system D12S391 in the German and three Asian populations.

Forensic Sci. Int. 94:25–31.

Wallin, J.M., Buoncristiani, M.R., Lazaruk, K.D., Fildes, N., Holt, C.L., Walsh, P.S. 1998.

SWGDAM validation of the AmpFlSTR blue PCR amplification kit for forensic casework analysis. J. Forensic Sci. 43:854–870.

Wallin, J.M., Holt, C.L., Lazaruk, K.D., Nguyen, T.H., Walsh, P.S. 2002. Constructing universal multiplex PCR systems for comparative genotyping. J. Forensic Sci. 47:52–65.

Walsh, P.S., Fildes, N.J., Reynolds, R. 1996. Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res. 24:2807–

2812.

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References

Limited product warranty

Watson, S., Kelsey, Z., Webb, R., Evans, J., and Gill, P. 1998. The development of a third generation STR multiplex system (TGM). Olaisen, B., Brinkmann, B., and Lincoln, P.J., eds. Progress in Forensic Genetics 7: Proceedings of the 17th International ISFH Congress,

Oslo 2‑6 September 1997. Elsevier, Amsterdam, pp. 192–194.

Weber, J. and Wong, C. 1993. Mutation of human short tandem repeats. Hum. Mol.

Genet. 2:1123–1128.

Weir, B. 1990. Genetic Data Analysis. Sinauer Associates Sunderland, MA

Wiegand, P. and Kleiber, M. 2001. Less is more—length reduction of STR amplicons using redesigned primers. Int. J. Legal Med. 114:285–287.

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Index

+A 78

+A nucleotide addition 78

6-dye

license activation 31, 34

spectral calibration 29, 33, 36

600 LIZ Size Standard v2.0 52

accuracy and reproducibility 64

alleles, off-ladder 66

allelic ladder, requirements for electrophoresis 27

allelic ladder profile 11

allelic ladder, volume per reaction 36

Bode Buccal DNA Collector 20

buccal 16, 83

characterization of loci, validation 81

control DNA, 007 11

Copan, treated paper 9

developmental validation 58

DNA control profile 12

documentation, related 127

DS-36 matrix standard 29, 33, 36 dye set for 6-dye samples 29, 33, 36

electrophoresis

data collection software 28, 31

prepare samples 30, 33

references 28, 31 run module 28, 31

setup of the 3130 and 3130xl instruments 31

setup of the 3500 and 3500xL instruments 28

extra peaks 70

GeneScan 600 LIZ Size Standard v2.0 52

GeneScan size standard, about 11

HID updater 28

import panels, bins, and marker stutter 41

Index Term 12

instrument and software compatibility 14

limited product warranty 129

LIZ size standard

about 11

peak sizes 52

volume per reaction 30, 33

LIZ Size Standard v2.0 52

marker stutter, import 41

materials not supplied 114

panels

check version 40

import 41

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Index

PCR

conditions 26

optimize cycle number 16

perform 26

setup 122 work areas 122

plate layout, PCR 120

required materials 114

run module for electrophoresis, 3500 and 3500xL in-

struments 28

run module, electrophoresis, 3130 and 3130xl 31

safety, biohazard 126

sensitivity 83

size standard 53

spectral calibration 29, 33, 36

stutter 70

stutter file, import 41

substrates 9

swab

PCR 26

sample preparation guidelines 23

terms and conditions 129

thermal cyclers

for use with kit 14

programming 26

treated paper

PCR 26

sample preparation guidelines 18

troubleshoot 110

untreated paper, PCR 26

validation, importance 57

warranty 129

work area, PCR setup 122

136

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For support visit thermofisher.com/support or email [email protected]

thermofisher.com

14 November 2016

Key features

Single-source sample types supported
6-dye STR multiplex assay
Optimized for paternity and single-source samples
Amplifies 25 loci
Highly optimized buffer formulation
Improved amplification primers
Clean electrophoretic backgrounds
Part of a fully integrated and validated forensic DNA workflow

Frequently asked questions

The VeriFiler Express PCR Amplification Kit is a 6-dye, short tandem repeat (STR) multiplex assay for the amplification of single-source human genomic DNA. It is optimized for paternity and single-source samples.

The VeriFiler Express kit is optimized to allow direct amplification from the following types of single-source samples: Blood and buccal samples on treated paper substrates, Blood and buccal samples collected on untreated paper substrates and treated with Prep-n-Go™ Buffer, Buccal samples collected on swab substrates and treated with Prep-n-Go™ Buffer.

The kit amplifies the following loci within the read region of 75– 465 nt: 23 autosomal STR loci, 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel), and Amelogenin (sex determining marker).