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AmpFLSTR Yfiler PCR Amplification Kit User`s Manual (PN 4358101C)

1. 6 2 Developmental Validation 6 3 Accuracy Precision and Reproducibility 6 7 Extra Peaks in the Electropherogram 6 18 Characterization of Loci 6 25 Species Specificity 6 27 Sensitivity 6 29 Stability 6 31 Mixture Studies 6 34 Population Data 6 38 Analyzing the Population Data 6 40 Mutation Rate 6 41 vi AmpFlSTR Yfiler PCR Amplification Kit User s Manual Appendix A Troubleshooting Troubleshooting A 2 Troubleshooting Automated Genotyping
2. 5 44 Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder 5 53 Section 5 5 Interpretation of Haplotype Data 5 59 Overview 5 60 Searching the Database 5 62 Reviewing Results 5 69 Chapter 5 Analyzing Data 5 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Data Analysis Overview Section 5 1 Data Analysis Overview AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 3 Analyzing Data Data Analysis Overview Section 5 1 Data Analysis Overview This section covers Overview 5 4 Chapter 5 Analyzing Data 5 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Data Analysis Overview Overview After electrophoresis the Data Collection software stores information for each sample in an fsa file Using analysis software you can then analyze and interpret the data Extract and quantify DNA PCR amplify DNA Perform elect
3. 5 7 Analyzing Sample Files With GeneMapper ID Software 5 21 Examining and Editing GeneMapper ID Software Results 5 23 Section 5 3 Using GeneScan Analysis Software to Analyze Yfiler Kit Data 5 25 Analyzing Sample Files Using GeneScan Software 5 26 Viewing GeneScan Software Results 5 33 AmpFlSTR Yfiler PCR Amplification Kit User s Manual v Section 5 4 Using Genotyper Software to Analyze Yfiler Kit Data 5 35 Overview 5 36 Understanding the AmpFlSTR Yfiler Kit Template 5 37 Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping 5 44 Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder 5 53 Section 5 5 Interpretation of Haplotype Data 5 59 Overview 5 60 Searching the Database 5 62 Reviewing Results 5 69 Chapter 6 Experiments and Results Overview
4. A 6 Bibliography Index DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual vii Preface How to Use This Guide Purpose of This Guide The AmpFlSTR Yfiler PCR Amplification Kit User s Manual provides information about the Applied Biosystems instruments chemistries and software associated with the AmpFlSTR Yfiler PCR Amplification Kit Yfiler kit Text Conventions This guide uses the following conventions Bold indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu For example Select File gt Open gt Spot Set Right click the sample row then select View Filter gt View All Runs Double brackets are used to indicate a field on a software screen for example Click the arrow beside lt Collection Setting gt Pull Out Chapters This guide is designed to allow users to pull out Chapters 3 4 and 5 The pull out chapters have title and back pages which indicate the chapter number and title DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm Preface viii AmpFlSTR Yfiler PCR Amplification Kit User s Manual User Attention
5. 1 4 Instrument and Software Overview 1 5 Materials and Equipment 1 7 Chapter 2 Extracting and Quantifying DNA Extracting DNA 2 2 Quantifying DNA 2 4 Chapter 3 PCR Amplification PCR Work Areas 3 2 Required User Supplied Materials and Reagents 3 4 Preparing the Reactions 3 6 Performing PCR 3 8 Amplification Using Bloodstained FTA Cards 3 9 iv AmpFlSTR Yfiler PCR Amplification Kit User s Manual Chapter 4 Performing Electrophoresis Section 4 1 ABI PRISM 3100 3100 Avant Genetic Analyzer Setup 4 3 Overview 4 4 Setting up the 3100 3100 Avant Instrument 4 7 Performing a Spectral Calibration 4 9 Preparing Samples
6. 1 5 Materials and Equipment 1 7 Chapter 1 Overview 1 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Product Overview Purpose The AmpFlSTR Yfiler PCR Amplification Kit is a short tandem repeat STR multiplex assay that amplifies 17 Y STR loci in a single PCR reaction The kit amplifies the loci in the European minimal haplotype DYS19 DYS385a b DYS389I II DYS390 DYS391 DYS392 DYS393 Scientific Working Group DNA Analysis Methods SWGDAM recommended Y STR panel European minimal haplotype plus DYS438 and DYS439 Additional highly polymorphic loci DYS437 DYS448 DYS456 DYS458 DYS635 Y GATA C4 and Y GATA H4 Product Description The Yfiler kit contains all the necessary reagents for the amplification of human male specific DNA The reagents are designed and optimized for use with the following Applied Biosystems instruments ABI PRISM 3100 3100 Avant Genetic Analyzer ABI PRISM 310 Genetic Analyzer GeneAmp PCR System 9600 Silver 96 Well GeneAmp PCR System 9700 Gold plated silver block GeneAmp PCR System 9700 Loci Amplified by the Kit The following table shows the loci amplified by the Yfiler kit and the corresponding dyes used The AmpFlSTR Yfiler Kit Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the
7. DYS389II 13 85 DYS458 12 2 DYS19 11 4 10 21 2 bp DYS385 13 9 DYS393 12 58 DYS391 11 62 Chapter 5 Analyzing Data 5 40 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software The peak filtering included in the Kazam macro is intended only as a tool and guideline Final conclusions should be based on careful examination of the STR profiles For instructions on filtering stutter peaks refer to Examining and Editing Data on page 5 47 Viewing the Kazam Macro Steps To view the Kazam macro steps click Kazam in the Macro list then select View gt Show Step Window Calculating Offsets The Calculate locus Offsets macros which are automatically called by the Kazam macros calculate offsets by Identifying the first allele peak in each allelic ladder Comparing reference sizes to allelic ladder sizes and determining the offset value Applying the offset value to each allele Applying the offset value to off ladder and virtual alleles DYS439 11 18 DYS635 YGATAC4 10 75 DYS392 16 22 7 9 3 bp Y GATA H4 11 08 DYS438 4 28 DYS437 8 59 DYS448 4 96 Table 5 5 Kazam macro stutter filter percentages for Yfiler loci continued Locus Stutter bp stutter plus or minus Understanding the AmpFlSTR Yfiler Kit Template AmpFlSTR Yfiler PCR Amplification Kit User
8. 4 Click Save Make sure to save the file in the following location C AppliedBio Shared Analysis SizeCaller Params Table 5 3 GeneScan analysis parameters continued Parameter Procedure Analyzing Sample Files Using GeneScan Software AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 31 Analyzing Data GeneScan Software ABI PRISM GeneScan Analysis version 3 1 User s Manual Macintosh PN 4306157 Defining a Size Standard While GeneScan software is shipped with several built in size standard definitions it is sometimes useful or necessary to create your own size standard definition For Yfiler kits you create a size standard definition based on the GeneScan 500 LIZ size standard without the 250 bp peak To define the size standard for Yfiler kits 1 In the Size Standard column of the Analysis Control window click the arrow beside lt Collection Setting gt for any of the sample files 2 Click Define New The software launches the size standard definition utility 3 Define the known size standard peaks for the GeneScan 500 LIZ Size Standard 75 100 139 150 160 200 250 300 340 350 400 IMPORTANT Do not assign the 250 bp peak skip a row or assign a size of 0 This peak can be used as an indicator of precision within a run 4 Save the size standard definition in C AppliedBio Shared Analysis SizeCaller SizeStandards Chapter 5 Analyzing Data 5 32 AmpF
9. Section 4 1 ABI PRISM 3100 3100 Avant Genetic Analyzer Setup 4 3 Overview 4 4 Setting up the 3100 3100 Avant Instrument 4 7 Performing a Spectral Calibration 4 9 Preparing Samples for Electrophoresis 4 14 Setting Up the Electrophoresis Run 4 16 Performing Electrophoresis 4 24 Section 4 2 ABI PRISM 310 Genetic Analyzer Setup 4 27 Overview 4 28 Setting Up the 310 Genetic Analyzer 4 31 Creating a Matrix File for the 310 Genetic Analyzer 4 34 Setting Up the Electrophoresis Run 4 38 Preparing Samples for Electrophoresis 4 45 Performing Electrophoresis 4 46 Chapter 4 Performing Electrophoresis 4 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup DRAFT May 25 2005 10 28 am 3100 fm Section 4 1 ABI
10. 61 C 62 C 63 C DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 6 AmpFlSTR Yfiler PCR Amplification Kit User s Manual PCR Cycle Number AmpFlSTR Yfiler kit reactions were amplified for 28 29 30 31 and 32 cycles on the Silver 96 Well GeneAmp PCR System 9700 using 1 0 ng of three male DNA samples As expected PCR product increased with the number of cycles A full profile was generated at 28 cycles off scale data were collected for several allele peaks at 32 cycles While none of the cycle numbers tested produced nonspecific peaks 30 cycles was found to give optimal sensitivity when the amplified products were examined on ABI PRISM 3100 Genetic Analyzers At 30 cycles high ratios of female to male DNA amplify reliably and specifically following the conditions outlined in this user manual Figure 6 15 on page 6 35 Figure 6 3 Yfiler profiles obtained from amplification of 1 ng DNA template using 28 29 30 31 and 32 cycles analyzed on the ABI PRISM 3100 Genetic Analyzer 28 29 30 31 32 DRAFT August 7 2006 8 20 am ExperimentsResults fm Accuracy Precision and Reproducibility AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 7 Accuracy Precision and Reproducibility SWGDAM Guideline 2 9 The extent to which a given set of measurements of the same sample agree with their mean and the extent to which these measurements match the actua
11. I Import Panels dialog box 5 8 IMPORTANT description ix Information Development department contacting xiv instrument protocol creating 4 17 italic text when to use vii L loci and alleles 1 2 loci characterization 6 25 M manuals See documentation related marker displaying Bin view of 5 11 materials and equipment 1 7 materials not included with Quantifiler kits 1 8 materials user supplied 3 4 matrix file creating for the 310 4 34 menu commands conventions for describing vii MSDSs description x obtaining x xv DRAFT May 25 2005 9 53 am 7x9_MultiChapter_IX fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual Index 3 referring to x xi mutation rate 6 41 N navigation pane displaying list of panels 5 11 Panel Manager 5 8 O off ladder alleles 5 6 P Panel Manager 5 8 panels viewing 5 11 PCR Setup 3 2 PCR performing 3 4 plate assembly preparing 4 24 population data 6 38 preparing the plate assembly 4 24 Q quantifying DNA methods 2 5 R radioactive waste handling xii reactions preparing 3 6 results group creating 4 17 S safety biological hazards chemical waste xi safety alert words CAUTIONS ix DANGERS ix description ix IMPORTANTS ix WARNINGS ix Searching for a haplotype 5 62 Services and Support obtaining xv software setup viewing imported panels 5 11 spec
12. Microvariants whose sizes are outside the size range for the locus are designated as 0 This designation may be changed to lt m where m is the smallest allele in the ladder range for a locus or gt n where n is the largest allele in the ladder range for a locus 5 Resave the amended version of the table to be searched in txt format 1 In the Search tool select Input Haplotype s from your file Chapter 5 Analyzing Data 5 68 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Haplotype Database 2 Select Genotyper File Format or GeneMapper File Format and browse to find the file 3 Select Upload to input the table haplotypes Selection of the haplotype ID will populate the manual entry screen with the selected haplotype 4 Select Search to search the population database for matching haplotypes Reviewing Results AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 69 Analyzing Data Haplotype Database Reviewing Results Select Allele Search Results An example search result is shown in Figure 5 5 Figure 5 5 Search result example The table provides information about the frequency of the specified haplotype within individual populations and within the entire database a Indicates that for the specified loci one match was found in the Caucasian population b Indicates the frequency of the specified haplotype within the Caucasian populati
13. 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum Table 4 1 User supplied Materials Material Source AmpFlSTR Yfiler PCR Amplification Kit 4359513 3100 3100 Avant Analyzer materials 96 Well Plate Septa 4315933 3100 Capillary Array 36 cm 4315931 36 cm 3100 Avant Capillary Array 4333464 3100 Performance Optimized Polymer 4 POP 4 4316355 Autosampler 96 well Plate Kit 4316471 GeneScan 500 LIZ Size Standard 4322682 10 Genetic Analyzer Buffer with EDTA 402824 DS 33 Dye Set G5 Matrix Standard Kit for 3100 3100 Avant analyzers 4345833 MicroAmp Optical 96 Well Reaction Plate N801 0560 3100 Instrument Consumable Reservoir Septa 4315932 Array fill syringe 250 L glass syringe 4304470 Polymer reserve syringe 5 0 mL glass syringe 628 3731 For a complete list of parts and accessories for the 3100 3100 Avant instrument refer to Appendix B of the ABI PRISM 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide PN 4335393 DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 8 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup Setting up the 3100 3100 Avant Instrument Following is a summary of the tasks involved in setting up the 3100 3100 Avant instrument for use with Data Collection Software v2 0 For detailed information about
14. A nucleotide The AmpFlSTR Yfiler PCR Amplification Kit is designed so that most PCR products contain the non templated 3 A nucleotide Alleles have been named following the nomenclature used in the NIST Standard Reference Material 2395 for Human Y Chromosome DNA Profiling Standard The nomenclature for DYS635 was based on Kayser et al 2004 The number of complete four base pair repeat units observed is designated by an integer Variant alleles that contain a partial repeat are designated by a decimal followed by the number of bases in the partial repeat For example a DYS385 14 2 allele contains 14 complete four base pair repeat units and a partial repeat unit of two base pairs Additional variation has been seen at some loci where alleles that differ from integer allele lengths by one or three base pairs exist For example DYS385 allele 16 3 contains 16 complete repeat units and a partial 3 bp unit Schoske et al 2004 The table below lists the loci included in the AmpFlSTR Yfiler Allelic Ladder Table 5 6 AmpFlSTR Yfiler Kit loci and alleles Locus Designation Alleles Included in Yfiler Kit Allelic Laddera Dye Label DNA 007 Genotype DYS456 13 18 6 FAM 15 DYS389I 10 15 13 DYS390 18 27 24 DYS389II 24 34 29 Chapter 5 Analyzing Data 5 54 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software DYS458 14 20 VIC 17
15. Instrument and Software Overview AmpFlSTR Yfiler PCR Amplification Kit User s Manual 1 5 Instrument and Software Overview This section provides information about the data collection and analysis software versions required to run the Yfiler kit on specific instruments Data Collection and Analysis Software The data collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time As the instrument records sample fluorescence on the detection system hardware the data collection software collects the data and stores it The data collection software stores information about each sample in a sample file fsa which is then analyzed by the analysis software Instrument and Software Compatibility About Multicomponent Analysis Applied Biosystems fluorescent multi color dye technology allows the analysis of multiple loci including loci that have alleles with overlapping size ranges Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes Instrument Operating System Data Collection Software Analysis Software 3100 3100 Avant Windows NTa a Applied Biosystems conducted validation studies for YFiler using these configurations 1 1 3100 1 0 3100 Avant GeneMapper ID 3 2 GeneScan 3 7 1 GenoTyper 3 7 Windows 2000a 2 0 GeneMapper ID 3 2 310 Win
16. Performing Electrophoresis Preparing the Plate Assembly Running the Plate on the 3100 3100 Avant Instrument 5 Click OK to save then close the plate record IMPORTANT After you click OK in the Plate Editor the Data Collection Software stores the completed plate record in the Plate Manager database Once the plate is in the Plate Manager database you can search for edit delete or export a plate record To set up data collection software for electrophoresis continued To prepare the plate assembly 1 Place the reaction plate into the plate base provided with the instrument 2 Align the septa strip on the reaction plate 3 Snap the plate retainer onto the reaction plate and plate base 4 Verify that the holes of the plate retainer and septa strip are aligned 5 Place the plate assembly on the autosampler To run the plate on the 3100 3100 Avant instrument 1 Search for your plate record For more information about searching for plate records refer to Chapter 6 of the ABI PRISM 3100 3100 Avant Genetic Analyzer User Guide PN 4347102 DRAFT May 25 2005 9 53 am 3100_part2 fm Performing Electrophoresis AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 25 3100 3100 Avant Electrophoresis Setup Viewing Run Data You can view data both during a run and after a run Refer to ABI PRISM 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin PN 4350218
17. Reviewing Results on page 5 69 To search for a haplotype continued Searching the Database AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 65 Analyzing Data Haplotype Database To upload haplotypes from allele tables generated in GeneMapper ID Software v3 2 1 Under the Tools menu select the Table Setting Editor 2 In the Genotypes tab select the following Column Settings Sample Name Marker Allele 3 In the Samples tab hide all columns Note The table settings may be saved by creating a new table Using the pulldown menu adjacent to Table Setting select New and name the table for example Yfiler Upload Table Select settings and click OK to save Chapter 5 Analyzing Data 5 66 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Haplotype Database 4 While in the Samples tab click the File menu and select Export Combined Table Select the One line per sample option under Merge Name the file and save as Tab and Comma delimited text txt csv 5 Using a text editor such as Notepad or Wordpad modify the text table for upload by deleting allelic ladder samples and other samples that do not need to be included in the search such as known reference or control samples 6 Check the table for microvariants Microvariants whose sizes are within the size range for the locus are designated as OL
18. Select the appropriate sample sheet from the Sample Sheet popup menu at the top left of the Injection List window To create a sample sheet and injection list continued Column Value Sample Name Enter a name for the sample This column indicates which sample is in which tube of the sample tray Standard Click in the column beside O The software displays a diamond to indicate that orange is the size standard Sample Info Copy the information from the Sample Name column TIP Complete the Sample Name column then copy and paste the information into the Sample Info column DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 42 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup 7 For autoanalysis complete the following information Note If you edited the default sample sheet and injection list defaults as described in Setting GeneScan Software Sample Sheet and Injection List Defaults on page 4 39 you do not need to perform this step Note If you are performing a manual analysis you only need to complete the run module field 8 Save the injection list By default the injection list is saved in the Run folder To create a sample sheet and injection list continued Setting Value Analysis Parameters Select Module gt Module GS STR POP4 1 mL G5 for every injection Matrix Select the matrix file for the injections from the
19. AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup Preparing Samples for Electrophoresis Required Materials Refer to Kit Contents and Storage on page 4 7 for a list of materials Preparing the Samples Prepare the samples for electrophoresis immediately prior to loading To prepare samples for electrophoresis 1 Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The amount of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results experiments CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 2 Pipette the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly Reagent Volume L per reaction GeneScan 500 LIZ Size Standard 0 3 Hi Di Formamide 8 7 DRAFT May 25 2005 9 53 am 3100_part2 fm Preparing Samples for Electrophoresis AmpFlSTR Yfiler PCR Amplification Kit Us
20. PN 4317588 PCR Amplification MicroAmp 96 Well Trays for Tubes with Caps N801 0541 MicroAmp Reaction Tubes with Caps 0 2 mL N801 0540 MicroAmp 8 Strip Reaction Tubes N801 0580 Table 1 4 User supplied materials continued Material Source Materials and Equipment AmpFlSTR Yfiler PCR Amplification Kit User s Manual 1 11 MicroAmp Caps 8 Caps Strip N801 0535 MicroAmp 96 Well Tray Retainer Sets 403081 MicroAmp 96 Well Support Base N801 0531 MicroAmp Optical 96 Well Reaction Plate N801 0560 Other user supplied materials Hi Di Formamide 25mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Tris HCL pH 8 0 MLS 0 5 M EDTA MLS Vortex MLS Table 1 4 User supplied materials continued Material Source Chapter 1 Overview 1 12 AmpFlSTR Yfiler PCR Amplification Kit User s Manual AmpFlSTR Yfiler PCR Amplification Kit User s Manual 2 1 2 Extracting and Quantifying DNA 2 This chapter covers Extracting DNA 2 2 Quantifying DNA 2 4 Chapter 2 Extracting and Quantifying DNA 2 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Extracting DN
21. if the 310 instrument is ready to use for electrophoresis Condition Task Have you checked the electrode The electrode must not be bent and must be clean For more information refer to Chapter 3 of the ABI PRISM 310 Genetic Analyzer User Guide PN 4317588 for Windows Chapter 4 of the ABI PRISM 310 Genetic Analyzer User Manual PN 903565 for Macintosh Have you cleaned the pump block The pump block must be clean Refer to the appropriate user manual listed above for more information about cleaning the pump block Has the capillary been replaced within the last 100 runs A new capillary may be required if you noticed the following conditions in a previous run Poor sizing precision or allele calling Poor resolution and or decreased signal intensity Refer to the appropriate user manual listed above for more information about replacing the capillary Have you checked the syringe Two o rings should be present the ferrule firmly seated and the syringe should be clean For more information refer to the appropriate user manual listed above Have the syringes been replaced within the last three months Replace the syringes as described in the appropriate user manual listed above Have you replenished the electrophoresis reagents Replenish the reagents as described in the appropriate user manual listed above Do you have a valid matrix The 310
22. in the calling region may or may not be reproducible Figure 6 10 demonstrates examples of baseline noise and artifacts in an electropherogram while using the AmpFlSTR Yfiler kit You should consider possible noise and artifacts when interpreting data from the AmpFlSTR Yfiler kit on the ABI PRISM 3100 Genetic Analyzer DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 24 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Figure 6 10 Examples of baseline noise and reproducible artifacts in data produced on the ABI PRISM 3100 Genetic Analyzer Genotyping may result in the detection of these artifacts as off ladder alleles or OL Alleles Note A high degree of magnification y axis is used in this figure to illustrate these artifacts data produced on capillary electrophoresis instrument platforms DRAFT August 7 2006 8 20 am ExperimentsResults fm Characterization of Loci AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 25 Characterization of Loci SWGDAM Guideline 2 1 The basic characteristics of a genetic marker must be determined and documented SWGDAM July 2003 This section describes basic characteristics of the 17 loci that are amplified with the AmpFlSTR Yfiler kit These loci have been extensively characterized by other laboratories Gusmao et al 1999 Butler et al 2002 Gonzalez Neira et al 2001 Hall and Ballantyne 2003 Redd et al 2002 Schosk
23. or Chapter 6 of the ABI PRISM 3100 3100 Avant Genetic Analyzer User Guide PN 4347102 for more information about viewing run data 2 Select the plate record you want to run then click the plate position indicator that corresponds to the plate you are linking Note The 3100 Avant instrument has only one plate position to link to a plate record The plate position indicator changes from yellow to green when linked and the green run button is active 3 Verify that the active spectral calibration matches your dye set and capillary array length 4 Verify that the Autoanalysis Manager is running 5 Click the green run button then click OK in the Processing Plates dialog box To run the plate on the 3100 3100 Avant instrument continued DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 26 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup DRAFT May 25 2005 9 59 am 310 fm Section 4 2 ABI PRISM 310 Genetic Analyzer Setup AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 27 3100 3100 Avant Electrophoresis SetupData Analysis Overview Section 4 2 ABI PRISM 310 Genetic Analyzer Setup This section covers Overview 4 28 Setting Up the 310 Genetic Analyzer 4 31 Creating a Matrix File for the 310
24. or safe use of a chemical Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices Indicates a potentially hazardous situation that if not avoided could result in death or serious injury Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Chemical Hazard Warning CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm Preface x AmpFlSTR Yfiler PCR Amplification Kit User s Manual Chemical Safety Guidelines To minimize the hazards of chemicals Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines
25. partial profiles Degraded DNA As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced due to the reduced number of intact templates in the size range necessary for amplification Degraded DNA was prepared to examine the potential for preferential amplification of loci High molecular weight DNA was incubated with the enzyme DNase I for varying amounts of time The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each time point Two nanograms of degraded DNA or 1 ng undegraded DNA was amplified using the AmpFlSTR Yfiler kit As the DNA became increasingly degraded the loci became undetectable according to size Preferential amplification was not observed The loci failed to robustly amplify in the order of decreasing size as the extent of degradation progressed DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 32 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Figure 6 13 Multiplex amplification of DNA samples incubated with DNAseI The top panel corresponds to 1 ng of DNA with no DNAseI added panels 2 6 correspond to 2 ng of DNA incubated with DNAseI for 1 2 4 8 and 12 minutes respectively Effect of Inhibitors Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis et al 1988 Akane et al 1994 It i
26. 1 bp c Scroll through the tables to verify correct GeneScan 500 LIZ peak assignments d Check the GeneScan 500 LIZ Size Standard peaks in the remaining samples taking note of which samples if any have incorrect peak assignments Viewing GeneScan Software Results AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 33 Analyzing Data GeneScan Software Viewing GeneScan Software Results After sample files have been analyzed use the Results Control window to display the results from each injection into a capillary The Results Control window displays the newly analyzed sample files and allows the user to specify the format of the results Selecting both the Electropherogram and Tabular Data icons is recommended for reviewing results 6 View AmpFlSTR Yfiler kit results using the Results Control window Refer to the ABI PRISM GeneScan Analysis Software v3 7 for the Windows NT Platform PN 4308923 and the ABI PRISM GeneScan Analysis version 3 1 User s Manual Macintosh PN 4306157 for printing options To analyze the sample files continued Chapter 5 Analyzing Data 5 34 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneScan Software Section 5 4 Using Genotyper Software to Analyze Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 35 Analyzing Data Genotyper Software Section 5 4 Using Genotyper Software to A
27. 28 269 42 0 08 29 273 36 0 06 30 277 63 0 07 31 281 76 0 09 32 285 78 0 07 33 289 93 0 05 34 293 94 0 06 DYS458 14 130 98 0 05 15 134 87 0 06 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder continued ABI PRISM 310 Genetic Analyzer Allele Mean Standard Deviation DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 12 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 16 138 81 0 03 17 142 95 0 05 18 147 31 0 05 19 151 72 0 05 20 155 94 0 04 DYS19 10 176 06 0 07 11 179 98 0 05 12 183 84 0 05 13 187 76 0 03 14 191 64 0 05 15 195 49 0 05 16 199 32 0 05 17 203 20 0 06 18 207 09 0 07 19 211 02 0 06 DYS385 a b 7 242 79 0 05 8 246 89 0 07 9 250 94 0 04 10 254 98 0 07 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder continued ABI PRISM 310 Genetic Analyzer Allele Mean Standard Deviation DRAFT August 7 2006 8 20 am ExperimentsResults fm Accuracy Precision and Reproducibility AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 13 11 259 04 0 08 12 263 08 0 06 13 267 24 0 05 14 271 38 0 06 15 275 47 0 10 16 279 56 0 08 17 283 70 0 07 18 287 79 0 05 19 292 06 0 06 20 296 19 0 07 21 300 42 0 06 22 305 06 0 12 23
28. 309 50 0 07 24 313 99 0 10 25 318 39 0 05 DYS393 8 100 26 0 05 9 104 19 0 04 10 108 05 0 04 11 112 04 0 04 12 115 98 0 04 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder continued ABI PRISM 310 Genetic Analyzer Allele Mean Standard Deviation DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 14 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 13 119 89 0 04 14 123 89 0 04 15 127 80 0 05 16 131 95 0 04 DYS391 7 150 88 0 08 8 155 27 0 06 9 159 67 0 06 10 163 83 0 05 11 167 94 0 07 12 172 00 0 07 13 176 03 0 06 DYS439 8 197 84 0 05 9 201 70 0 03 10 205 68 0 05 11 209 46 0 04 12 213 47 0 03 13 217 41 0 03 14 221 42 0 05 15 225 17 0 04 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder continued ABI PRISM 310 Genetic Analyzer Allele Mean Standard Deviation DRAFT August 7 2006 8 20 am ExperimentsResults fm Accuracy Precision and Reproducibility AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 15 DYS635 Y GATA C4 20 246 43 0 07 21 250 49 0 06 22 254 45 0 06 23 258 49 0 03 24 262 45 0 06 25 266 56 0 06 26 270 56 0 03 DYS392 7 291 38 0 05 8 294 39 0 07 9 297 44 0 06 10 300 30 0 06 11 303 91 0 07 12 3
29. 50 L tube 15 to 25 C AmpFlSTR Control DNA 9947A 10 ng L human female cell line DNA in 0 05 sodium azide and buffer 1 tube 25 L 2 to 8 C Chapter 1 Overview 1 8 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Standards for Samples For the Yfiler kit the panel of standards needed for PCR amplification PCR product base pair sizing and genotyping are Control DNA 007 A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpFlSTR Yfiler Kit Allelic Ladder GeneScan 500 LIZ Size Standard Used for obtaining base pair sizing results The GeneScan 500 LIZ Size Standard is designed for sizing DNA fragments in the 35 500 bp range and it contains 16 single stranded fragments of 35 50 75 100 139 150 160 200 250 300 340 350 400 450 490 and 500 bases This standard has been evaluated as an internal lane size standard and it yields precise sizing results for AmpFlSTR Yfiler kit PCR products Order the GeneScan 500 LIZ Size Standard PN 4322682 separately AmpFlSTR Yfiler Kit Allelic Ladder Developed by Applied Biosystems for accurate characterization of the alleles amplified by the AmpFlSTR Yfiler kit The AmpFlSTR Yfiler Allelic Ladder contains the majority of alleles reported for the 17 loci Refer to Loci Amplified by the Kit on page 1 2 for a list of the alleles included in the AmpFlSTR Yfiler kit Equipment and M
30. DYS19 10 19 15 DYS385 a b 7 25 11 14 DYS393 8 16 NED 13 DYS391 7 13 11 DYS439 8 15 12 DYS635 20 26 24 DYS392 7 18 13 Y GATA H4 8 13 PET 13 DYS437 13 17 15 DYS438 8 13 12 DYS448 17 24 19 a See About the AmpFlSTR Yfiler Kit Allelic Ladder on page 5 53 for more information about the Yfiler Kit allelic ladder Table 5 6 AmpFlSTR Yfiler Kit loci and alleles continued Locus Designation Alleles Included in Yfiler Kit Allelic Laddera Dye Label DNA 007 Genotype Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 55 Analyzing Data Genotyper Software A Genotyper software electropherogram of the AmpFlSTR Yfiler Allelic Ladder listing the designation for each allele is shown in Figure 5 3 This electropherogram indicates the designation for each allele Results were obtained on an ABI PRISM 3100 instrument Figure 5 3 Genotyper software plot of the AmpFlSTR Yfiler Allelic Ladder Genotyping Based on the AmpFlSTR Yfiler Allelic Ladder Genotyper software assigns genotypes to sample alleles by comparing their sizes to those obtained for the known alleles in the AmpFlSTR Yfiler Allelic Ladder Genotypes not sizes are used for comparison of data between runs instruments and laboratories Applied Biosystems strongly recommends that laboratories use an AmpFlSTR Yfiler Allelic
31. Genetic Analyzer 4 34 Setting Up the Electrophoresis Run 4 38 Preparing Samples for Electrophoresis 4 45 Performing Electrophoresis 4 46 DRAFT May 25 2005 9 59 am 310 fm Chapter 4 Performing Electrophoresis 4 28 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Electrophoresis Setup Overview Flowchart Matrix file created Set up instrument Install and clean pump block Install or replace capillary Prepare syringes Clean electrode Recalibrate autosampler Fill buffer reservoir Create matrix file Display and check matrix file Not sure Yes No Set up the instrument Prepare the samples Load samples Select injection list Start run Perform electrophoresis If using GeneScan Software v3 7 1 Preheat the instrument Set up the sample sheet and injection list defaults Create the sample sheet and injection list If using GeneMapper ID Software v3 2 Preheat the instrument Create the sample sheet and injection list Set up Data Collection Software v3 0 Prepare formamide size standard cocktail 0 5 L GeneScan 500 LIZ Size Standard 24 5 L Hi Di Formamide per sample Vortex Into each 0 2 mL or 0 5 mL sample tube add 25 L formamide size standard mixt
32. Ladder from the specific electrophoresis run to convert the allele sizes to genotypes because The size values obtained for the same sample can differ between instrument platforms because of differences in the type and concentration of the gel polymer matrices and in electrophoretic conditions The size values obtained for the same sample can differ between protocols for the same instrument platform because of differences in gel or polymer concentration run temperature gel or capillary thickness and well to read length Chapter 5 Analyzing Data 5 56 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software Slight procedural and reagent variations between gels or between single and multiple capillaries result in greater size variation than that found between samples on the same gel or between samples injected in the same capillary in a single run About the Size Standard and Sizing Method The Genotyper software uses the internal lane size standard included in every sample that is both PCR products and allelic ladder samples to normalize lane to lane or injection to injection migration differences Running an internal size standard ensures sizing precision within a gel or within a set of capillary injections Because the common alleles for all AmpFlSTR Yfiler kit loci are less than 400 base pairs you can use the GeneScan 500 LIZ Size Standard Applied
33. Manual 4 39 310 Analyzer Setup Setting GeneScan Software Sample Sheet and Injection List Defaults When you create a new sample sheet the data collection software automatically fills in portions of the sample sheet based on settings specified in the Preferences dialog This section provides information on changing the default preferences To preheat the instrument to the run temperature 1 Make sure the instrument doors are closed 2 Launch the Data Collection software 3 Set the temperature a Select Window gt Manual Control b Select Temperature Set from the popup menu c Set the temperature to 60 C d Click Execute Note It takes up to 30 min for the instrument to reach the 60 C run temperature To set sample sheet and injection list defaults 1 If necessary launch the Data Collection Software 2 Select Windows gt Preferences 3 In the Preferences dialog box select GeneScan Injection Sample Sheet Defaults 4 Set the size standard color for 5 Dye to orange O 5 In the Preferences dialog box select GeneScan Injection List Defaults The software displays the default settings as shown in the following figure DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 40 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup Creating a Sample Sheet and Injection List for the Run You can prepare the sample sheet at
34. Matrix file popup menu IMPORTANT Use only matrix files created using DS 33 6 FAM VIC NED PET and LIZ dyes and the Filter Set G5v2 module To perform autoanalysis this matrix file must be located in the GeneScan GSMatrix folder Size Standard Select the size standard from the Size Standard popup menu DRAFT May 25 2005 9 53 am 310_part2 fm Setting Up the Electrophoresis Run AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 43 310 Analyzer Setup Setup for Data Collection Software 3 0 with GeneMapper ID Software Overview Setting up the electrophoresis run involves three tasks 1 Preheating the instrument optional 2 Creating a Sample Sheet and Injection List for the Run Preheating the Instrument Preheat the instrument as described in Preheating the Instrument on page 4 38 Creating a Sample Sheet and Injection List for the Run You can prepare the sample sheet at any time before the preparation of samples and save it in the Sample Sheet folder for later use To create a sample sheet and injection list 1 If necessary launch the Data Collection Software 2 Select File gt New then click GeneScan Sample Sheet 3 Complete the sample sheet Column Value Sample Name Enter a name for the sample This column indicates which sample is in which tube of the sample tray Standard Click in the column beside O The software displays a diamond to indicate that o
35. Name column of row A enter a sample name then click the next cell The value 100 is automatically displayed in the Priority column b In the Comments column of row A enter any additional comments or notations for the sample at the corresponding position of the plate c In the Instrument Protocol 1 column of row A select the protocol you created in step 4 on page 4 11 d Highlight the entire row row A e Select Edit gt Fill Down Special The software automatically fills in the appropriate well numbers for a single run f Click OK 7 Link your reaction plate and start the run 8 View the status after the run To perform a spectral calibration for 3100 3100 Avant Data Collection Software v2 0 continued DRAFT May 25 2005 9 53 am 3100_part2 fm Performing a Spectral Calibration AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 13 3100 3100 Avant Electrophoresis Setup For more information about performing spectral calibrations refer to ABI PRISM 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin PN 4350218 or Chapter 3 of the ABI PRISM 3100 3100 Avant Genetic Analyzer User Guide PN 4347102 9 Review and evaluate the spectral calibration profile for each capillary To perform a spectral calibration for 3100 3100 Avant Data Collection Software v2 0 continued DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 14
36. PCR Amplification Kit User s Manual Figure 6 6 Stutter percentages for the DYS458 DYS19 and DYS385 loci The 4 bp and 2 bp stutter percentages for DYS19 are shown in blue and green respectively Figure 6 7 Stutter percentages for the DYS393 DYS391 DYS439 DYS635 DYS392 loci The DYS392 3 bp and 3 bp stutter percentages are shown in blue and grey respectively DRAFT August 7 2006 8 20 am ExperimentsResults fm Extra Peaks in the Electropherogram AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 21 Figure 6 8 Stutter percentages for the Y GATA H4 DYS437 DYS438 and DYS448 loci Addition of 3 A Nucleotide AmpliTaq Gold enzyme like many other DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 Magnuson et al 1996 This non template addition results in a PCR product that is one base pair longer than the actual target sequence and the PCR product with the extra nucleotide is referred to as the A form The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The AmpFlSTR Yfiler kit includes two main design features that promote maximum A addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 80 min DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experi
37. PRISM 3100 3100 Avant Genetic Analyzer Setup AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 3 3100 3100 Avant Electrophoresis SetupData Analysis Overview Section 4 1 ABI PRISM 3100 3100 Avant Genetic Analyzer Setup This section covers Overview 4 4 Setting up the 3100 3100 Avant Instrument 4 7 Performing a Spectral Calibration 4 9 Preparing Samples for Electrophoresis 4 14 Setting Up the Electrophoresis Run 4 16 Performing Electrophoresis 4 24 DRAFT May 25 2005 10 28 am 3100 fm Chapter 4 Performing Electrophoresis 4 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setupverview Overview Flowchart Perform electrophoresis ABI PRISM 3100 3100 Avant Spatial calibration done Set up instrument Prepare syringes Install capillary array Add change polymer Fill reservoirs Perform calibration Display and check calibration Not sure Yes No Spectral calibration done Perform calibration Not sure Yes No Set up the instrument Prepare formamide size standard cocktail 0 3 L GeneScan 500 LIZ Size Standar
38. Polymerase for 3 to 5 seconds and centrifuge briefly CHEMICAL HAZARD AmpliTaq Gold DNA Polymerase may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 3 Pipette the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the PCR master mix for 3 to 5 seconds then centrifuge briefly 5 Dispense 15 L of the PCR master mix into each reaction well Component Volume Per Reaction L AmpFlSTR Yfiler Kit PCR Reaction Mix 9 2 AmpFlSTR Yfiler Kit Primer Set 5 0 AmpliTaq Gold DNA Polymerase 0 8 Preparing the Reactions AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3 7 6 Add 10 L of sample or control 0 1ng L to the appropriate wells using MicroAmp Reaction Tubes or a MicroAmp Optical 96 Well Reaction Plate The final reaction volume should be 25 L 7 Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles Note If a tabletop centrifuge with 96 well plate adapters is not available you can omit this step 8 Amplify the DNA in a GeneAmp PCR System 9600 or a Silver 96 Well GeneAmp PCR System 9700 or a Gold plated silver block GeneAmp PCR System 9700 To prepare the reactions continued Chapter 3 PCR Amplification 3 8 AmpFlS
39. Show Table Window to view the table in full screen mode 4 Open and view the plot Note For all tables except the Make Allele Table clicking in a cell of the table causes the corresponding sample electropherogram to appear in the plot window Click any cell in the table to display the locus region of the corresponding electropherogram in the Plot window for that sample Zoom out Ctrl H to view all loci for a particular dye color for the corresponding sample 5 To edit the cells of the table a Click a cell of the table that contains an allele designation b Select Edit gt Edit Cell c Type the desired information in the box and click OK 6 Print the table by selecting File gt Print 7 Optional Select Table gt Export to File to save the table as a file that can be opened in Microsoft Excel 8 Select File gt Save to save the template file with data Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 53 Analyzing Data Genotyper Software Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder About the AmpFlSTR Yfiler Kit Allelic Ladder The AmpFlSTR Yfiler Kit Allelic Ladder contains the most common alleles for each locus The macro size ranges include the actual number of nucleotides contained in the smallest and largest allelic ladder alleles for each locus The size range also includes the 3
40. Tubes 401956 Matrix Standard Set DS 33 6FAM VIC NED PET and LIZ dyes for 310 377 systems 4318159 MicroAmp 8 strip Reaction Tubes N801 0580 MicroAmp 96 Well Support Base holds 0 2 mL reaction tubes N801 0531 MicroAmp 96 Well Full Plate Cover N801 0550 MicroAmp 96 Well Tray Retainer Sets 403081 DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 32 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup POP 4 Performance Optimized Polymer 402838 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the ABI PRISM 310 Genetic Analyzer User Guide PN 4317588 Material Source DRAFT May 25 2005 9 53 am 310_part2 fm Setting Up the 310 Genetic Analyzer AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 33 310 Analyzer Setup Setting Up the 310 Instrument Following is a summary of the tasks involved in setting up the 310 instrument For detailed information about these tasks refer to Chapter 3 of the ABI PRISM 310 Genetic Analyzer User Guide PN 4317588 for Windows Chapter 4 of the ABI PRISM 310 Genetic Analyzer User Manual PN 903565 for Macintosh Installing the G5V2 Run Module The G5v2 run module is required for the 310 instrument to analyze information from Yfiler kits To set up the 310 instrument 1 Power on the computer and log on power on the 310 i
41. and degraded DNA How it works The DNA quantification assay combines two 5 nuclease assays A target specific human DNA or human male DNA assay which consists of two primers for amplifying human or human male DNA and one TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence An internal PCR control IPC assay which consists of an IPC template DNA a synthetic sequence not found in nature two primers for amplifying the IPC template DNA and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC DNA Quantifiler Human DNA Quantification Kits User s Manual PN 4344790 Quantifiler Human DNA Quantification Kit PN 4343895 QuantiBlot Human DNA Quantitation Kit PN N808 0114 Properties High specificity for human DNA Detects single stranded or degraded DNA How it works A biotinylated probe specific for the human D17Z1 sequence is hybridized to sample DNA that has been immobilized via slot blot onto a nylon membrane The subsequent binding of horseradish peroxidase streptavidin enzyme conjugate HRP SA to the bound probe allows for either colorimetric or chemiluminescent detection QuantiBlot Human DNA Quantitation Kit Product Inserta QuantiBlot Human DNA Quantitation Kit Human Identification Product Bulletin PN 112PB04 02 a Contact your Applied Biosystems sales representative to obtain a copy of the product inse
42. by the height of the main allele peak Peak heights have been measured for amplified samples at the loci used in the AmpFlSTR Yfiler kit All data were generated on the ABI PRISM 3100 Genetic Analyzer Some of the general conclusions from these measurements and observations are as follows For each AmpFlSTR Yfiler kit locus the percent stutter generally increases with allele length as shown in Figures 6 5 to Figures 6 8 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus Each allele within a locus displays a percent stutter that is consistent DRAFT August 7 2006 8 20 am ExperimentsResults fm Extra Peaks in the Electropherogram AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 19 The highest observed percent stutter for each locus is included as the filtering step in GeneMapper ID v3 2 and Genotyper softwares Peaks in the stutter position that are above the highest observed percent stutter will not be filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated For evaluation of mixed samples see Figure 6 16 on page 6 37 The measurement of percent stutter for peaks that are off scale may be unusually high Figure 6 5 Stutter percentages for the DYS456 DYS389I DYS390 and DYS389II loci DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 20 AmpFlSTR Yfiler
43. for Electrophoresis 4 14 Setting Up the Electrophoresis Run Data Collection Software v2 0 4 16 Performing Electrophoresis 4 24 Section 4 2 ABI PRISM 310 Genetic Analyzer Setup 4 27 Overview 4 28 Setting Up the 310 Genetic Analyzer 4 31 Creating a Matrix File for the 310 Genetic Analyzer 4 34 Setting Up the Electrophoresis Run 4 38 Preparing Samples for Electrophoresis 4 45 Performing Electrophoresis 4 46 Chapter 5 Analyzing Data Section 5 1 Data Analysis Overview 5 3 Overview 5 4 Section 5 2 Using GeneMapper ID Software v3 2 to Analyze AmpFlSTR Yfiler Kit Data 5 5 Overview 5 6 Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data
44. of specificity and robustness must be determined These include thermocycling parameters the concentration of primers magnesium chloride DNA polymerase and other critical reagents SWGDAM July 2003 PCR Components The concentration of each component of the AmpFlSTR Yfiler kit was examined The PCR components are Tris HCl pH 8 3 KCl dNTPs primers AmpliTaq Gold DNA polymerase MgCl2 bovine serum albumin and sodium azide The concentration for a particular component was established to be in the window that meets the reproducible performance characteristics of specificity and sensitivity Various magnesium chloride concentrations were tested on the ABI PRISM 3100 Genetic Analyzer The amplification of 1 ng of male genomic DNA is shown in Figure 6 1 The performance of the multiplex is most robust within a 20 window of magnesium chloride DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Figure 6 1 1 ng genomic DNA amplified with the AmpFlSTR Yfiler Kit in the presence of varying concentrations of magnesium chloride 1 12 mM 1 28 mM 1 44 mM 1 6 mM 1 76 mM 1 92 mM and 2 08 mM analyzed on the ABI PRISM 3100 Genetic Analyzer Thermal Cycler Parameters Thermal cycling parameters were established for amplification of the AmpFlSTR Yfiler Kit Thermal cycling times and temperatures of GeneAmp PCR systems were verified Varyi
45. of Blood Banks 6th edition 2004 The sensitivity of the AmpFlSTR Yfiler PCR Amplification Kit and other PCR based tests enables amplification of minute quantities of DNA necessitating precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 Also take care while handling and processing samples to prevent chance 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 Applied Biosystems does not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology PCR Setup Work Area IMPORTANT These items should never leave the PCR Setup Work Area Calculator Gloves disposable Marker pen permanent Microcentrifuge Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipet tips sterile disposable hydrophobic filter plugged Pipettors Tube decapper autoclavable Vortex PCR Work Areas AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3 3 Amplified DNA Work Area IMPORTANT The GeneAmp PCR Systems should be placed in the Amplified DNA Work Area You can use the following systems Silver 96 Well GeneAmp PCR System 9700 Gold plated silver block GeneAmp PCR System 9
46. peaks in a range of 7 bp around the reference size for the indicated allele Each Calculate locus Offsets macro applies a percentage filter to all peaks in the 7 bp range in the allelic ladder avoiding the first stutter peak in each allelic ladder Consequently the first allele peak is identified as the leftmost peak Chapter 5 Analyzing Data 5 42 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software Comparing Reference Sizes to Allelic Ladder Sizes The base pair size indicated in each offset category is a reference size The Calculate locus Offsets macros offset the reference sizes relative to the sizes obtained for the alleles in the allelic ladder After the macros run the calculated offset values are indicated in parentheses near the end of each category line in the Categories window For example the sequenced size for allele 10 of locus DYS392 is 303 bp The size obtained on a 3100 Genetic Analyzer for the allele for a particular injection was 300 39 bp The offset value is calculated as 300 39 303 2 61 The category size used for allele assignment is 300 39 or 303 2 61 That is the category sizes used for genotyping are equivalent to the allele sizes obtained in the lane or injection of allelic ladder Applying the Appropriate Offset Value to Each Allele Once the leftmost allele peak in each allelic ladder is identified the offs
47. such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Figure 6 4 on page 6 8 illustrates the tight clustering of allele sizes obtained on the ABI PRISM 3100 Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 bp The instance of a sample allele sizing outside of the 0 5 bp window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 bp or less Smith 1995 For sample alleles that do not size within a 0 5 bp window the PCR product must be rerun to distinguish between a true off ladder allele versus measurement error of a sample allele that corresponds with an allele in the allelic ladder Repeat analysis when necessary provides an added level of confidence to the final allele assignment GeneMapper ID v3 2 and Genotyper softwares automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele It is important to note that while the precision within a gel or set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences arise from a number of parameters including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can also be found between runs on the same instrument and between runs on different instruments because of these paramete
48. the template file simultaneously 2 Under Edit select Set Preferences to import raw data and Blue Green Yellow Red and Orange data 3 Import the GeneScan Software sample files a Select File gt Import GeneScan File s b Select the project file and click Import 4 If each sample does not already have Sample Info completed in the sample sheet enter a sample description a Select Views gt Show Dye lanes b Click the first sample row to select it c Click the Sample Info box at the top of the window and type the sample designation or description d Repeat steps b and c to enter a sample description for every dye lane in the list Enter the same sample description for all dye colors of a single sample 5 From the Macro list at the bottom left of the Main window select Check GS500 then select Macro gt Run Macro In the plot window that opens scroll through each sample to verify that each GeneScan 500 peak from 75 400 bp was assigned the correct size in the GeneScan Analysis Software Chapter 5 Analyzing Data 5 46 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software About the Plot Window 6 From the Macro list at the bottom left of the Main window select Kazam then select Macro gt Run Macro This macro may take a few minutes to run When it is finished a plot window opens with the blue allelic ladder DYS456 DYS38
49. to convert sample files created on one platform to files that can be read by the other platform Refer to Chapter 4 of the ABI PRISM GeneScan Analysis Software v3 7 for the Windows NT Platform PN 4308923 for detailed information about this process Defining Analysis Parameters Analysis parameters are defined in the Analysis Settings dialog box Figure 5 2 shows the default analysis parameter settings for GeneScan analysis software v3 7 1 on the Windows NT operating system 4 Click Finish when you have added all relevant sample files The sample files appear in the Analysis Control window 5 For sample files generated by ABI PRISM 310 and 377 Analyzers install and apply a matrix to the sample files a Click the Sample column to select all the sample files in the project b Click Sample gt Install New Matrix c Navigate to the location that contains the matrix file then select it Note Data Collection Software on the ABI PRISM 3100 3100 Avant Analyzers performs multicomponenting you do not need to perform this step when analyzing sample files from those instruments To create a new project continued Chapter 5 Analyzing Data 5 28 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneScan Software Figure 5 2 Analysis Parameters dialog box with default settings 400 This range is set by the user Analyzing Sample Files Using GeneScan Software AmpF
50. too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in the following Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem for two reasons 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 which results in poor spectral separation pull up Incomplete A nucleotide addition When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile Quantifying DNA AmpFlSTR Yfiler PCR Amplification Kit User s Manual 2 5 Methods for Quantifying DNA Applied Biosystems provides several kits for accurately quantifying DNA in samples Detailed information about how the kits work kit specificity and sensitivity and other frequently asked questions are provided in the cited references Product Description References Quantifiler Y Human Male DNA Quantification Kit PN 4343906 Properties Both Quantifiler kits have high specificity for human DNA The Quantifiler Y kit is highly specific for human male DNA Able to detect single stranded
51. two alleles of different sizes doublets DYS385 haplotypes containing three alleles triplets Note Occasionally three or more alleles may be detected in loci other than DYS385 and four or more alleles may be detected for DYS385 This type of variant cannot be searched and a wild type value should be substituted To search for a haplotype 1 Go to the Yfiler Haplotype Database Search Tool www appliedbiosystems com yfilerdatabase Chapter 5 Analyzing Data 5 64 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Haplotype Database Searching for a Haplotype by Uploading Analysis Files You can upload information from allele tables created from GeneMapper ID and Genotyper software 2 Specify allele values for the loci that you want to include in the search Use the lt toggle icon gt to switch between modes If using Default Mode select a value for the allele using the drop down list see Table 5 8 on page 5 62 If using Custom Entry Mode enter up to three comma separated values including noninteger values for example 18 2 You can specify values for one to up to all 17 loci If you do not specify a value for a locus that locus is omitted from the search In a single search query you can use Default Mode for some loci and Custom Entry Mode for other loci as required by your data 3 Click Search 4 Review the results as explained in
52. 0 ng L human female cell line DNA in 0 05 sodium azide and buffer 1 tube 25 L 2 to 8 C Required User Supplied Materials and Reagents AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3 5 User supplied Reagents In addition to the reagents supplied with the Yfiler kit it is recommended that you use low TE buffer You can prepare the buffer as described in the following table or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together 10 mL of 1 M Tris HCl pH 8 0 0 2 mL of 0 5 M EDTA 990 mL glass distilled or deionized water CHEMICAL HAZARD EDTA Exposure causes eye irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature Chapter 3 PCR Amplification 3 6 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Preparing the Reactions To prepare the reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers 2 Prepare the reagents a Thaw the PCR reaction mix and primer set then vortex 3 to 5 seconds and centrifuge briefly before opening the tubes b Vortex the AmpliTaq Gold DNA
53. 07 44 0 07 13 310 64 0 08 14 313 74 0 07 15 317 12 0 11 16 320 45 0 08 17 323 54 0 09 18 326 79 0 10 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder continued ABI PRISM 310 Genetic Analyzer Allele Mean Standard Deviation DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 16 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Y GATA H4 8 122 01 0 06 9 125 98 0 06 10 129 97 0 07 11 134 01 0 04 12 138 09 0 03 13 142 37 0 05 DYS437 13 182 53 0 05 14 186 45 0 07 15 190 40 0 04 16 194 25 0 04 17 198 07 0 03 DY438 8 223 69 0 06 9 228 68 0 06 10 233 63 0 07 11 238 59 0 06 12 243 63 0 05 13 248 66 0 05 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder continued ABI PRISM 310 Genetic Analyzer Allele Mean Standard Deviation DRAFT August 7 2006 8 20 am ExperimentsResults fm Accuracy Precision and Reproducibility AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 17 DYS448 17 280 49 0 04 18 286 58 0 03 19 292 70 0 05 20 298 92 0 05 21 305 51 0 04 22 312 25 0 06 23 318 60 0 10 24 324 88 0 08 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder continued ABI PRISM 310 Genetic Analyzer Allele Mean Standard D
54. 17019 DYS385 AC022486 Z93950 DYS389 AC011289 AF140635 DYS390 AC011289 DYS391 G09613 AC011302 DYS392 G09867 AC06152 DYS393 G09601 AC06152 DYS437 AC002992 DYS438 AC002531 DYS439 AC002992 DYS448 AC025227 6 DYS456 AC010106 2 DYS458 AC010902 4 DYS635 G42676 AC011751 and Y GATA C4 G42673 DRAFT August 7 2006 8 20 am ExperimentsResults fm Species Specificity AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 27 Species Specificity SWGDAM Guideline 2 2 For techniques designed to type human DNA the potential to detect DNA from forensically relevant nonhuman species should be evaluated SWGDAM July 2003 The AmpFlSTR Yfiler kit provides the required degree of specificity for primates Other species do not amplify for the loci tested Nonhuman Studies Nonhuman DNA may be present in forensic casework samples The AmpFlSTR Yfiler kit provides the required degree of specificity for the species tested The following experiments were conducted to investigate interpretation of AmpFlSTR Yfiler kit results from nonhuman DNA sources Figure 6 11 Representative electropherograms from a species specificity study including positive and non template control NTC Male control Chimpanzee Cat Dog Microbial pool NTC DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 28 AmpFlSTR Yfiler PCR Amplification Kit User s Manual The t
55. 20 stutter filter to all loci Use this macro To apply a single filter value for all loci When a high level of filtering specificity is not required as in the typing of single source samples such as database samples To view the Kazam 20 Filter macro steps click Kazam 20 Filter in the Macro list then select View gt Show Step Window The Kazam 20 Filter macro does not take into account the size in bp of the filtered peak relative to higher peaks In fact it removes labels from all peaks that are less than a specified percentage by default 20 of the highest peak observed anywhere in the locus range However you can specify a different filter value Chapter 5 Analyzing Data 5 44 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software Make Table Macros The Yfiler Template includes three macros for making tables Make Allele Table 310 Analyzer Make Table 377 Analyzer Make Table For more information about the Make Table macros refer to Chapter 10 of the AmpFlSTR Profiler Plus PCR Amplification Kit User s Manual PN 4303501 Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping Installing the Yfiler Template The AmpFlSTR Yfiler Kit Template 9 is the Genotyper software template file that contains macros specifically written for use with the AmpFlSTR Yfiler PCR Amplification Kit This template is provi
56. 700 GeneAmp PCR System 9600 Chapter 3 PCR Amplification 3 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Required User Supplied Materials and Reagents Kit Contents and Storage Each Yfiler kit contains materials sufficient to perform 100 reactions at a 25 L reaction volume IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpFlSTR Yfiler Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum Table 3 1 Yfiler kit contents Reagent Contents Quantity Storage AmpFlSTR Yfiler Primer Set Forward and reverse primers to amplify human male DNA target 1 tube 0 55 mL 2 to 8 C AmpFlSTR Yfiler PCR Reaction Mix MgCl2 dNTPs and bovine serum albumin in buffer with 0 05 sodium azide 1 tube 1 1 mL tube 2 to 8 C AmpFlSTR Yfiler Allelic Ladder Allelic ladder containing amplified alleles refer to Loci Amplified by the Kit on page 1 2 for a list of alleles included in the ladder 1 tube 50 L 2 to 8 C AmpFlSTR Control DNA 007 0 10 ng L human male genomic DNA in 0 05 sodium azide and buffer refer to Loci Amplified by the Kit on page 1 2 for profile 1 tube 0 3 mL 2 to 8 C AmpliTaq Gold DNA Polymerase DNA polymerase 5 U L 2 tubes 50 L tube 15 to 25 C AmpFlSTR Control DNA 9947A 1
57. 9I DYS390 and DYS389II and sample allele peaks labeled 7 Examine data edit peaks then print the electropherograms a In the Main Window click the green G button at the top left b Select Views gt Show Plot Window c Examine the data edit the peaks then print the electropherograms by selecting File gt Print Examining and Editing Data on page 5 47 provides more information about inspecting data d Repeat steps a through c for the yellow Y button in the Main Window and red R button in the Main Window data To use the AmpFlSTR Yfiler Kit Template continued To zoom in and out on regions of the plot window 1 In the Plot window click and drag in a region of an electropherogram to draw a box around the desired size range the vertical size of the box is not important 2 Type Ctrl R hold down the Ctrl key and type the letter R to zoom in 3 Type Ctrl H to zoom out completely Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 47 Analyzing Data Genotyper Software Examining and Editing Data You examine Yfiler Kit data by checking peaks Consider the following guidelines when examining peaks Refer to Figure 5 3 on page 5 55 Genotyper software plot of the AmpFlSTR Yfiler Allelic Ladder Peaks in the allelic ladder should be labeled correctly Scroll through the samples below the allelic ladder to exam
58. A Overview Extraction procedures can be classified as organic or nonorganic Depending on the material received scientists should determine which procedure is appropriate for each piece of evidence DNA extracted from fresh or frozen whole blood peripheral blood lymphocytes blood stains sperm cells paraffin blocks teeth hair tissue bone and other biological samples can be PCR amplified and analyzed using the AmpFlSTR YFiler PCR Amplification Kit The quality of the DNA degree of degradation its purity and its quantity in a sample influence the efficiency of PCR amplification Decreased amplification is usually caused by highly degraded DNA the presence of PCR inhibitors insufficient DNA quantity or any combination of these factors DNA Extraction Methods Many DNA extraction procedures including phenol chloroform Chelex and FTA paper are currently in use Regardless of which method you use handle all samples carefully to prevent sample to sample contamination or contamination by extraneous DNA When possible process evidence samples separately from reference samples Phenol Chloroform Method This method removes proteins and other cellular components from nucleic acids resulting in relatively pure DNA preparations Double stranded DNA extracted by this method is suitable for use with AmpFlSTR YFiler kit amplifications provided it is not significantly degraded The phenol chloroform method is ofte
59. AmpFlSTR Yfiler PCR Amplification Kit User s Manual DRAFT August 7 2006 7 59 am 7x9_Title fm Copyright 2006 Applied Biosystems All rights reserved For Research Forensic or Paternity Use Only Not for use in diagnostic procedures Printed in the U S A NOTICE TO PURCHASER LIMITED LICENSE Use of the AmpFlSTR Yfiler PCR Amplification Kit is covered by one or more of the following US patents and corresponding patent claims outside the US 5 079 352 5 789 224 5 618 711 6 127 155 5 677 152 claims 1 23 only and 5 773 258 claims 1 and 6 only and claims outside the US corresponding to US Patent No 4 889 818 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in forensic and paternity testing including reporting results of purchaser s activities for a fee or other commercial consideration and also for the purchaser s own internal research No right under any other patent claim is conveyed expressly by implication or by estoppel Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA The AmpFlSTR Yfiler PCR Amplification Kit is covered by U S Patent No 5 364 759 owned by Baylor College of Medicine and is sold under license from Baylor College of Medicine Not for re s
60. Biosystems recommends that you use the Local Southern sizing method which uses two internal lane size standard peaks larger than each allele and two smaller than each allele to be sized Note When defining size standard peaks during routine analysis you should include the 350 and 400 bp peaks of the size standard Manual Genotyping The Kazam macros automatically assign allelic ladder sizes and determine sample genotype However you can perform both tasks manually as explained in the following procedure To perform manual genotyping 1 Size the AmpFlSTR Yfiler Allelic Ladder alleles Compare the base pair sizes of one lane or injection of allelic ladder to those obtained for the other lanes or injections of allelic ladder All corresponding peaks peaks at the same position in the allelic ladder should be within 0 5 bp of each other If one or more corresponding peaks are not within 0 5 bp of each other check the GeneScan 500 LIZ Size Standard peaks in all allelic ladder lanes or injections to confirm that all GeneScan 500 LIZ Size Standard peaks have been assigned the correct size and or that all peaks are clearly resolved Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 57 Analyzing Data Genotyper Software The AmpFlSTR Yfiler Allelic Ladder contains most alleles for the DYS19 DYS385 DYS389 I DYS389 II DYS390 DYS391 DYS392 DYS393 D
61. C x 100 percentage value For example if peak C 149 RFU and peak B 1886 RFU then the percentage value is calculated as follows 1886 149 149 x 100 1166 In this example the label will be removed from peak C provided that the filter option specifies a threshold of 1166 and that peak B is within 2 25 to 3 75 bp of peak C Conventionally percent stutter is calculated peak C peak B x 100 percent stutter The percentage value that is used in the Genotyper software filtering option F can be derived from the conventional percent stutter expression S F 10 000 S 100 For example if the desired stutter percent threshold for DYS392 is 7 9 then the percentage value that should be used in the Genotyper software filtering option is F 10 000 7 9 100 1166 4 To use a filter value different than 1166 for DYS392 enter another value then click Replace To filter plus stutter peaks continued Chapter 5 Analyzing Data 5 52 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software Making Tables IMPORTANT Before making a table examine all electropherograms and edit their peaks as described in the previous section To create and use tables 1 From the Macro list at the bottom of the Genotyper software Main Window click one of the three table macros 2 Select Macro gt Run Macro 3 Select Views gt
62. Control DNA 007 are listed in the table Table 1 1 AmpFlSTR Yfiler Kit loci and alleles Locus Designation Alleles Included in Yfiler Kit Allelic Laddera Dye Label DNA 007 Genotype DYS456 13 18 6 FAM 15 DYS389I 10 15 13 DYS390 18 27 24 Product Overview AmpFlSTR Yfiler PCR Amplification Kit User s Manual 1 3 DYS389II 24 34 29 DYS458 14 20 VIC 17 DYS19 10 19 15 DYS385 a b 7 25 11 14 DYS393 8 16 NED 13 DYS391 7 13 11 DYS439 8 15 12 DYS635 20 26 24 DYS392 7 18 13 Y GATA H4 8 13 PET 13 DYS437 13 17 15 DYS438 8 13 12 DYS448 17 24 19 a See About the AmpFlSTR Yfiler Kit Allelic Ladder on page 5 53 for more information about the Yfiler Kit allelic ladder Table 1 1 AmpFlSTR Yfiler Kit loci and alleles continued Locus Designation Alleles Included in Yfiler Kit Allelic Laddera Dye Label DNA 007 Genotype Chapter 1 Overview 1 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Procedural Overview Y STR Workflow Extract and Quantify DNA PCR Amplify DNA Perform Electrophoresis Analyze Data GeneAmp PCR System 9600 Thermal Cycler GeneAmp PCR System 9700 Thermal Cycler ABI PRISM 3100 3100 Avant Analyzer ABI PRISM 310 Analyzer GeneScan v 3 7 1 Genotyper Software AmpF STR YFiler PCR Amplification kit GeneMapper ID Software v 3 2 Quantifiler Quantiblot
63. DYS385a b 0 951 0 855 0 931 DYS393 0 619 0 412 0 507 DYS391 0 423 0 54 0 52 DYS439 0 629 0 663 0 665 DYS635 0 701 0 682 0 71 DYS392 0 419 0 615 0 671 Y GATA H4 0 599 0 604 0 575 DYS437 0 495 0 624 0 583 DYS438 0 528 0 622 0 712 DYS448 0 685 0 651 0 726 Table 6 2 AmpFlSTR Yfiler Kit gene diversity across three different U S populations continued Locus1 U S African American N 333 U S Caucasian N 254 U S Hispanic N 175 D n 1 pi 2 n 1 DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 40 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Analyzing the Population Data In addition to the alleles that were observed and recorded in the Applied Biosystems databases other known alleles have been published or reported to us by other laboratories Some of these alleles occur at a low frequency and include several microvariants Furedi et al 1999 Schoske et al 2004 Discriminatory Capacity of Haplotypes Table 6 3 shows the discriminatory capacity DC and the number of unique haplotypes UH for each Y STR marker combination listed The discriminatory capacity was determined by dividing the number of different haplotypes by the number of samples in that population Schoske et al 2004 A unique haplotype is defined as one that occurs only once in a given populati
64. DYS437 15 16 DYS438 11 10 DYS439 12 13 DYS448 19 21 DYS456 17 15 DYS458 18 16 DYS635 23 22 Y GATA H4 12 12 DRAFT August 7 2006 8 20 am ExperimentsResults fm Mixture Studies AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 37 Figure 6 16 Mixtures of two male DNA samples A and B were amplified in various ratios using a total of 1 ng input DNA The top panel shows Sample A and the bottom panel shows Sample B The ratios of Sample A to Sample B A B ratios shown are 10 1 3 1 and 1 1 in panels 2 3 and 4 respectively For the mixture samples the alleles attributable to the minor component even when the major component shares an allele are highlighted Sample A 10 1 3 1 1 1 Sample B DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 38 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Population Data SWGDAM Guideline 2 7 The distribution of genetic markers in populations should be determined in relevant population groups SWGDAM July 2003 Overview To interpret the significance of a match between genetically typed samples it is necessary to 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 suspects s reference sample then the suspect is excluded as the donor of the biological evidence tested An exclusion is in
65. Mutation Rate 6 41 DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Overview Experiments Using AmpFlSTR Yfiler PCR Amplification Kit This chapter provides results of the developmental validation experiments performed by Applied Biosystems using the AmpFlSTR Yfiler PCR Amplification Kit Importance of Validation Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure s efficiency reliability and performance characteristics By challenging the procedure with samples commonly encountered in forensic and parentage laboratories the validation process uncovers attributes and limitations which are critical for sound data interpretation in casework Sparkes et al 1996 Sparkes et al 1996 Wallin et al 1998 Experiments Experiments to evaluate the performance of the AmpFlSTR Yfiler PCR Amplification Kit were performed at Applied Biosystems These experiments were performed according to the DNA Advisory Board DAB Quality Assurance Standards effective October 1 1998 DNA Advisory Board 1998 The DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory The DAB defines a laboratory as a fa
66. TANT The amount of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results experiments CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 2 Pipette the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly Reagent Volume Per Reaction L GeneScan 500 LIZ Internal Size Standard 0 5 Hi Di Formamide 24 5 DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 46 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup Performing Electrophoresis Preparing the Plate Assembly and Loading the Samples 4 Into each 0 2 mL or 0 5 mL sample tube add 25 L of the formamide size standard mixture 1 5 L of PCR product or Allelic Ladder 5 Seal the tubes with the appropriate septa then briefly centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom 6 Heat the tubes in a thermal cycler for 3 min at 95 C 7 Immediately place the tubes on ice for 3 min To prepare the samples for electrophoresis continued To load the samples 1 Open the instrument
67. TR Yfiler PCR Amplification Kit User s Manual Performing PCR To run PCR 1 Program the thermal cycling conditions IMPORTANT If using the Gold plated Silver or Silver 96 Well GeneAmp PCR System 9700 select the 9600 Emulation Mode 2 Load the plate into the thermal cycler PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Keep hands away from the heated cover and sample block 3 Close the heated cover PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Before performing the procedure keep hands away until the heated cover and sample block reach room temperature 4 Start the run Initial Incubation Step Cycle 30 cycles Final Extension Final Hold De nature Anneal Extend HOLD CYCLE HOLD HOLD 95 C 11 min 94 C 1 min 61 C 1 min 72 C 1 min 60 C 80 min 4 C Amplification Using Bloodstained FTA Cards AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3 9 Amplification Using Bloodstained FTA Cards FTA treated DNA collection cards can be useful for the collection storage and processing of biological samples A small punch of the bloodstained card can be placed directly into an amplification tube p
68. This designation may be changed based on confirmation of the microvariant designation e g 17 2 Microvariants whose sizes are outside the size range for the locus are designated as 0 no value This designation may be changed to lt m where m is the smallest allele in the ladder range for a locus or gt n where n is the largest allele in the ladder range for a locus Searching the Database AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 67 Analyzing Data Haplotype Database Note Opening the text file in Excel and resaving as a txt file will result in a format which is not compatible with the upload program To upload haplotypes from allele tables generated in Genotyper Software To upload tables into the Search tool 7 Resave the amended version of the table to be searched as a txt file 1 Run the Make Allele Table macro 2 Under the Table menu select Export to File Save the file as Name txt 3 Open the file in Excel and follow the onscreen instructions to convert the tab delimited file to an Excel table Delete the allelic ladder sample and other samples that do not need to be included in the search such as known reference or control samples 4 Check the table for microvariants Microvariants whose sizes are within the size range for the locus are designated as OL allele This designation may be changed based on confirmation of the microvariant designation e g 17 2
69. Words Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical Examples of the user attention words appear below Note The size of the column affects the run time Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid Oracle user ID and password IMPORTANT You must create a separate Sample Entry Spreadsheet for each 96 well plate Safety Alert Words Safety alert words also appear in user documentation For more information see Safety Alert Words on page ix DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm Safety AmpFlSTR Yfiler PCR Amplification Kit User s Manual ix Safety Safety Alert Words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use
70. YS437 DYS438 DYS439 DYS448 DYS456 DYS458 DYS635 Y GATA C4 and Y GATA H4 loci However alleles not found in the AmpFlSTR Yfiler Allelic Ladder do exist These off ladder alleles may contain full and or partial repeat units An off ladder allele should flag itself by not falling inside the 0 5 bp window of any known allelic ladder allele Note If a sample allele peak is found to be 0 5 bp from the corresponding allelic ladder peak the sample must be rerun to verify the result 2 Select one lane or injection of allelic ladder to use for genotyping Applied Biosystems studies have shown that it does not matter which lane or injection of allelic ladder is selected if the alleles in the allelic ladder samples are within 0 5 bp of each other 3 Compare the base pair size obtained for each sample allele peak to the sizes obtained for the allelic ladder peaks 4 Assign genotypes to those sample allele peaks falling within 0 5 bp of the corresponding allelic ladder peak The allele designation for each allelic ladder peak is given in Figure 5 3 on page 5 55 To perform manual genotyping continued Chapter 5 Analyzing Data 5 58 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software Section 5 5 Interpretation of Haplotype Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 59 Analyzing Data Haplotype Database Section 5 5 Int
71. ZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves c Vortex thoroughly to mix then spin briefly in a microcentrifuge d Heat the tube at 95 C for 3 min to denature the DNA then e Immediately place the tube on ice for 3 min Reagent Volume L 3100 Analyzer Volume L 3100 Avant Analyzer Matrix Standard Set DS 33 5 2 Hi Di Formamide 195 78 Final Volume 200 80 DRAFT May 25 2005 9 53 am 3100_part2 fm Performing a Spectral Calibration AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 11 3100 3100 Avant Electrophoresis Setup 2 Dispense the appropriate amount of denatured standard into the wells of a reaction plate 3 Prepare the plate assembly and place the plate assembly onto the autosampler 4 Create a spectral instrument protocol a In the Tree pane of the Data Collection Software v2 0 click GA Instruments gt ga3100 or ga3100 Avant gt Protocol Manager b In the Instrument Protocols pane click New to open the Protocol Editor dialog box c Complete the Protocol Editor dialog box Type Spectral Dye Set G5 Polymer POP4 Array Length 36 cm Chemistry Matrix Standard Run Module Spect36_POP4_1 Note If using DC v1 1 or 1 0 select Spe
72. a 5 12 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software Creating HID Analysis Methods Two analysis methods are suitable for HID analysis using Yfiler kits HID_Classic Provides users with the same analysis parameters and produces results similar to those obtained for data analyzed with GeneScan Software v3 1 2 for the Macintosh OS This algorithm allows laboratories that have optimized analysis parameter settings on the Macintosh OS to use GeneMapper ID software to analyze their data HID_Advanced Provides users with the same analysis parameters available in GeneScan Software v3 7 1 for the Windows OS 9 Add the Yfiler_vl panel to the project window by clicking Apply then OK Note If you close the Panel Manager without clicking OK the panels and bins will not be available for analysis 10 In the first sample row under the column labeled Panel double click None open the folder to display a list of panels and double click on the panel you want to assign a panel set To import panels and bin sets continued To create an analysis methods for the HID Classic Mode 1 Select Tools gt GeneMapper Manager to open the GeneMapper Manager Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 13 Analyzing Data GeneMapper ID Software 2 Create an
73. a new Genotyper software project and run the Kazam macro again DRAFT May 25 2005 9 53 am 7x9_Bibliography fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual Bibliography 1 Bibliography Akane A Matsubara K Nakamura H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 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 58 Butler J M Schoske R Vallone P M Kline M C Redd A J Hammer M F 2002 A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers Forensic Sci Int 129 10 24 Butler J M 2001 Forensic DNA Typing San Diego CA Academic Press Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 86 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 Frank W Llewel
74. al 5 1 5 Analyzing Data 5 This chapter covers Section 5 1 Data Analysis Overview 5 3 Overview 5 4 Section 5 2 Using GeneMapper ID Software v3 2 to Analyze AmpFlSTR Yfiler Kit Data 5 5 Overview 5 6 Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data 5 7 Analyzing Sample Files With GeneMapper ID Software 5 21 Examining and Editing GeneMapper ID Software Results 5 23 Section 5 3 Using GeneScan Analysis Software to Analyze Yfiler Kit Data 5 25 Analyzing Sample Files Using GeneScan Software 5 26 Viewing GeneScan Software Results 5 33 Section 5 4 Using Genotyper Software to Analyze Yfiler Kit Data 5 35 Overview 5 36 Understanding the AmpFlSTR Yfiler Kit Template 5 37 Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping
75. al input and input of allele tables generated in GeneMapper ID v3 2 and Genotyper In addition the user may search profiles containing microvariant alleles and display resulting matches Allele Representation Table 5 7 Allele representation in the Haplotype Database Type Example Singlet 7 or 8 2 Doublet all except DYS385 11 12 or 13 14 5 Each allele in the doublet must have a different value Either or both values can be microvariants Doublet DYS385 11 12 or 14 14 Alleles can have different values or they can have the same value Either or both values can be microvariants Triplet DYS385 only 13 15 17 Overview AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 61 Analyzing Data Haplotype Database Microvariant 7 5 gt n lt m n largest ladder allele m smallest ladder allele Wild card Indicates that the locus will be omitted from the search Table 5 7 Allele representation in the Haplotype Database Type Example Chapter 5 Analyzing Data 5 62 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Haplotype Database Searching the Database Searching for a Haplotype by Entering Allele Information The Yfiler Kit Haplotype Database Search Tool provides two modes for searching the haplotype database Default Mode Allows you to specify a single allele value for
76. ale TRADEMARKS AB Design ABI PRISM AmpFlSTR Applied Biosystems COfiler Genotyper LIZ MicroAmp PET and VIC are registered trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries 6 FAM Applera GeneScan Hi Di NED POP 4 Profiler Plus and Yfiler are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries AmpliTaq AmpliTaq Gold GeneAmp QuantiBlot and TaqMan are registered trademarks of Roche Molecular Systems Inc Mac and Macintosh are registered trademarks of Apple Computer Inc Windows NT is a registered trademark of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4358101 Rev C 08 2006 Contents AmpFlSTR Yfiler PCR Amplification Kit User s Manual iii Preface How to Use This Guide vii Safety ix How to Obtain More Information xiii How to Obtain Support xv Chapter 1 Overview Product Overview 1 2 Procedural Overview
77. amined One nanogram of DNA from each sample was amplified using the AmpFlSTR Yfiler and AmpFlSTR Identifiler kits followed by analysis using an ABI PRISM 3100 Genetic Analyzer The families examined included 1333 9 offspring 7 males 1340 7 offspring 5 males and 1345 7 offspring 5 males representing 23 meiotic divisions The Identifiler results confirmed that the loci are inherited according to DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 26 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Mendelian rules as expected The AmpFlSTR Yfiler results confirmed that the loci were inherited according to a Y linked father to son transmission In no case was the maternal grandfather s Y haplotype found in the offspring In family 1345 one son 1345 7356 had a DYS458 18 allele while the rest of his male relatives had a DYS458 17 allele In family 1340 one son 1340 7342 had a DYS458 16 allele while the rest of his male relatives had DYS458 17 Calculation of a mutation rate based on this small population size would be inaccurate due to the small sample size The samples were reamplified and reinjected to confirm the allele call Mapping The AmpFlSTR Yfiler kit loci have been mapped and the chromosomal location on the Y chromosome is known based on the nucleotide sequence of the Y chromosome The Genbank accession numbers for representative sequences are DYS19 X77751 AC0
78. an automated genotyping software solution for forensic paternity and database data analysis and other genotyping needs The GeneMapper ID software combines GeneScan Analysis Software and the Genotyper Software functionality in a single rules based analysis package Instruments Refer to Instrument and Software Compatibility on page 1 5 for a list of compatible instruments Before You Start When using GeneMapper ID Software version 3 2 to perform Human Identification HID analysis with AmpFlSTR kits consider the following HID analysis requires the presence of at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in a single run folder are considered to be from a single run When the software imports multiple run folders into a project only ladders within a single run folder are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples need to be identified as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Allelic bin definitions are stored in the AmpFlSTR_Yfiler pan
79. analysis method for HID Classic a Select the Analysis Methods tab and click New to open the New Analysis Method dialog box b Select HID and click OK to open the Analysis Method Editor with the General tab selected c In the General tab enter an analysis name for the method such as AmpFlSTR_Yfiler_ClassicMode 3 Select the settings shown in Table 5 1 HID Classic analysis method settings IMPORTANT You must select your settings on all the tabs before you Click OK to save the analysis method and return to GeneMapper Manager To create an analysis methods for the HID Classic Mode Chapter 5 Analyzing Data 5 14 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software HID Classic Settings Table 5 1 HID Classic analysis method settings Tab Settings General Name AmpFlSTR_Yfiler_ClassicMode Allele Note GeneMapper ID Software v3 2 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameters for each type of repeat in the appropriate column Note Select Use marker specific stutter ratio if available If the box is selected the software applies the stutter ratio filters specific to the Yfiler panel defined by Applied Biosystems Note For more information about allele filters refer to Chapter 3 of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Gui
80. and species studies Int J Legal Med 109 186 94 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 DRAFT May 25 2005 9 53 am 7x9_Bibliography fm Bibliography 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 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 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 70 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 12 DRAFT May 25 2005 9 53 am 7x9_MultiChapter_IX fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual Index 1 Index Numerics 310 Analyzer setting up 4 31 3100 3100 Avant setup 4 5 A allelic bin definitions 5 6 offsets 5 6 allelic ladder analysis method for 5 6 sample type 5 6 AmpFlSTR Allelic Ladders using to determine genotypes 5 57 AmpFlSTR Yfiler Kit Template making tables 5 52 trou
81. any time before the preparation of samples and save it in the Sample Sheet folder for later use 6 Select the following settings 7 Click OK to save your changes To set sample sheet and injection list defaults continued Setting Value 5 Dye Module GS STR POP4 1 mL G5v2 Matrix File Select the appropriate matrix file Note A valid matrix file created using DS 33 and Filter Set G5v2 module must be available in the GeneScan GSMatrix folder Autoanalyze With Select AnalyzeGSSample bat if you want to send data automatically to GeneScan software Note You can select any saved Analysis Parameters file AnalyzeGSSample bat contains typical analysis parameter settings For more information about setting analysis parameters see Defining Analysis Parameters on page 5 27 Select none if you do not want to use the autoanalysis feature Size Standard Assign the appropriate size standard To create a sample sheet and injection list 1 If necessary launch the Data Collection Software 2 Select File gt New then click GeneScan Sample Sheet DRAFT May 25 2005 9 53 am 310_part2 fm Setting Up the Electrophoresis Run AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 41 310 Analyzer Setup 3 Complete the sample sheet 4 Click File gt Save As to save the sample sheet in the Sample Sheets folder 5 Select File gt New then click GeneScan Injection List 6
82. ata 5 24 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software For more information about any of these tasks refer to the following documents New Features and Installation Procedures for GeneMapper ID Software Version 3 2 User Bulletin PN 4352543 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 4 Close the Samples Plot window and save the project To examine and edit GeneMapper ID Software results continued Section 5 3 Using GeneScan Analysis Software to Analyze Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 25 Analyzing Data GeneScan Software Section 5 3 Using GeneScan Analysis Software to Analyze Yfiler Kit Data This section covers Analyzing Sample Files Using GeneScan Software 5 26 Viewing GeneScan Software Results 5 33 Chapter 5 Analyzing Data 5 26 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneScan Software Analyzing Sample Files Using GeneScan Software Summary Analyzing sample files using GeneScan software involves the following steps 1 Creating a project and if necessary installing and applying a matrix 2 Defining analy
83. aterials Not Included Tables 1 3 through 1 4 list required and optional equipment and materials not supplied with the Yfiler kit Unless otherwise noted many of the items are available from major laboratory suppliers MLS Table 1 3 Equipment Equipment Source ABI PRISM 3100 3100 Avant Genetic Analyzer Contact your local Applied Biosystems sales representative ABI PRISM 310 Genetic Analyzer GeneAmp PCR System 9600 GeneAmp PCR System 9700 with the Silver 96 Well block N8050001 GeneAmp PCR System 9700 with the Gold plated silver block 4314878 Silver 96 Well sample block N8050251 Gold plated Silver 96 Well sample block 4314443 Materials and Equipment AmpFlSTR Yfiler PCR Amplification Kit User s Manual 1 9 Tabletop centrifuge with 96 well plate adapters optional Major Laboratory Supplier MLS Table 1 4 User supplied materials Material Source AmpFlSTR Yfiler PCR Amplification Kit 4359513 3100 3100 Avant Analyzer materials 96 Well Plate Septa 4315933 3100 Capillary Array 36 cm 4315931 36 cm 3100 Avant Capillary Array 4333464 3100 Performance Optimized Polymer 4 POP 4 4316355 Autosampler 96 well Plate Kit 4316471 GeneScan 500 LIZ Size Standard 4322682 10 Genetic Analyzer Buffer with EDTA 402824 DS 33 Dye Set G5 Matrix Standard Kit for 3100 3100 Avant analyzers 4345833 MicroAmp Optical 96 Well Reaction Plate N801 0560 3100 Instrumen
84. ation Analysis Tutorial PN 4335523 for more information 5 Optionally view and set HID analysis options in Options tab Analysis options allow you to automatically set values for plate record fields Refer to Chapter 1 of the GeneMapper ID Software versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 for more information 6 If necessary convert any GeneScan software sample files generated on the Macintosh platform to the fsa format using the Mac to Win AppleScript software provided with GeneMapper ID software Conversion is described in the GeneMapper ID Software version 3 1 Human Identification Analysis User Guide PN 4338775 Note For more detailed information about GeneMapper features refer to the GeneMapper ID Software version 3 1 Human Identification Analysis User Guide PN 4338775 and the GeneMapper ID Software versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 Refer to the GeneMapper ID Software version 3 2 Human Identification Analysis User Bulletin PN 4352543 Chapter 5 Analyzing Data 5 8 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software Importing Panels and Bins Use this procedure to import panels and bin sets into the GeneMapper software database for subsequent analysis and to view imported panels markers and bins Import the panels and bin sets the first time you use the softwar
85. ation rate was estimated at 2 80 x 10 3 In two confirmed father son pairs mutation at two Y STRs were observed Additional studies need to be performed for other loci in order to estimate their average mutation rate DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 42 AmpFlSTR Yfiler PCR Amplification Kit User s Manual DRAFT May 25 2005 9 53 am 7x9_Appendix fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual A 1 A Troubleshooting A In This Appendix Follow the recommended actions for the observations described in this appendix to understand and eliminate problems you experience during analysis Troubleshooting A 2 Troubleshooting Automated Genotyping A 6 DRAFT May 25 2005 9 53 am 7x9_Appendix fm Appendix A Troubleshooting A 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Troubleshooting Table A 1 Troubleshooting causes and recommended actions Observation Possible Causes Recommended Actions Faint or no signal from both the AmpFlSTR Control DNA 007 and the DNA test samples at all loci Incorrect volume or absence of either AmpFlSTR PCR Reaction Mix AmpFlSTR Yfiler Primer Set or AmpliTaq Gold DNA Polymerase Repeat amplification No activation of AmpliTaq Gold DNA Polymerase Repeat amplification making sure to hol
86. bleshooting genotyping A 6 understanding the template kit A 6 AmpFlSTR_Panels_v3 folder 5 10 Amplification Using Bloodstained FTA Cards 3 9 analysis method for allelic ladders 5 6 Analysis Method Editor 5 13 5 17 Applied Biosystems contacting xiv customer feedback on documentation xiv Information Development department xiv Services and Support xv Technical Support xv artifacts in data 6 23 automated genotyping about the software 5 36 AmpFlSTR Yfiler Kit Template making tables 5 47 troubleshooting genotyping A 6 understanding the template kit A 6 using the kit 5 44 before running Genotyper 5 36 B bin sets importing 5 10 viewing 5 11 Bin view displaying for a marker 5 11 biohazard warning xii biohazardous waste handling xii biological hazard safety See biohazard warning bold text when to use vii C calibration spectral 4 9 CAUTION description ix characterization of loci 6 25 chemical safety guidelines x chemical waste hazards xi safety guidelines xi contents of kit 1 7 conventions bold text vii for describing menu commands vii IMPORTANTS viii in this guide vii italic text vii Notes viii user attention words viii Creating an Instrument Protocol 4 17 customer feedback on Applied Biosystems documents xiv D DANGER description ix DRAFT May 25 2005 9 53 am 7x9_MultiChapter_IX fm Index 2 A
87. butors should be considered when interpreting the results We recommend that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures Male Female Mixture Studies Evidence samples that contain body fluids and or tissues originating from more than one individual are an integral component of forensic casework Therefore it is essential to ensure that the DNA typing system is able to detect DNA mixtures In the case of Y STRs the female DNA component is not amplified by the Y chromosome specific primers Male female mixture studies were performed up to a ratio of 1 2000 using three different female DNAs The amount of female DNA was kept constant at 500 ng and the amount of male control DNA was changed The female DNA did not cause any interference with the interpretation of the male Y STR profile as shown in Figure 6 15 Low level artifacts with female DNA have been occasionally observed in the black 136 bp and red 291 bp dye In general these artifacts peaks will not affect interpretation due to their intensity DRAFT August 7 2006 8 20 am ExperimentsResults fm Mixture Studies AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 35 Figure 6 15 Amplification of Male Control DNA 007 in the presence of Female DNA 9947A Profiles shown in the panels from top
88. can36Avb_POP4DyeSetG5Module Note Before the first run on 3100 3100 Avant instruments running Data Collection Software v1 1 or v1 0 you must edit the default module parameters Refer to page 34 of ABI PRISM 3100 and 3100 Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products PN 4332345 Analysis Module G500Analysis gsp Polymer 3100 Performance Optimized Polymer 4 POP4 7 mL PN 4316355 Capillary Array 3100 Capillary Array PN 4315931 36 cm 3100 Avant Capillary Array PN 4333464 Running Buffer 10 Genetic Analyzer Buffer with EDTA 25 mL PN 402824 DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 6 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup Before You Begin Before using the instrument use the following checklist to determine if regular maintenance tasks have been performed for the 3100 3100 Avant instrument Condition Task Have the syringes been replaced within the last three months Replace the syringes as described in Chapter 1 of the ABI PRISM 3100 3100 Avant Genetic Analyzers User Guide Have the capillary arrays been replaced within the last 100 runs Replace the capillary arrays as described in Chapter 7 of the ABI PRISM 3100 3100 Avant Genetic Analyzers User Guide New capillary arrays may be required if you noticed the following conditions in a prev
89. cation Kit User s Manual 5 23 Analyzing Data GeneMapper ID Software Examining and Editing GeneMapper ID Software Results You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Samples tab of the Project window assuming the analysis is complete To examine and edit GeneMapper ID Software results 1 Examine the size standard a Assess whether samples pass the sizing criteria Examine the flags in the SQ column to assess sizing quality A green square indicates that a sample has passed the sizing criteria b Check the size standards for any samples that do not pass the sizing criteria Note Beginning with v3 2 GeneMapper ID software automatically labels size standards Refer to the New GeneMapper ID Software Version 3 2 User Bulletin PN 4352543 2 Examine the allelic ladder calls a In the Samples View tab find the plots for all allelic ladders b Display AmpFlSTR Genotyping Plot Setting c Verify that the allelic ladder is called correctly for each marker Note Deselecting Controls to Top will display each color within the allelic ladder d Close the Samples View window 3 Examine data a Select low quality samples indicated by red octagons or yellow triangles b It is recommended that a user carefully review all PQVs that display a yellow triangle or red octagon Chapter 5 Analyzing D
90. chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm Preface xii AmpFlSTR Yfiler PCR Amplification Kit User s Manual Waste Disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological Hazard Safety BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protecti
91. cility in which forensic DNA testing is performed Additional validation was performed according to the revised guidelines from the Scientific Working Group on DNA Analysis Methods SWGDAM July 10 2003 Based on these guidelines Applied Biosystems has conducted experiments that comply with guidelines 1 0 and 2 0 and its associated subsections This DNA methodology is not novel Moretti et al 2001 Frank et al 2001 Wallin et al 2002 and Holt et al 2001 This chapter discusses many of the experiments performed by Applied Biosystems and provides examples of results obtained Conditions were chosen which produced maximum PCR product yield and a window in which reproducible performance characteristics were met It is our opinion that while these experiments are not exhaustive they are appropriate for a manufacturer Each laboratory using the AmpFlSTR Yfiler PCR Amplification Kit should perform internal validation studies DRAFT August 7 2006 8 20 am ExperimentsResults fm Developmental Validation AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 3 Developmental Validation SWGDAM Guideline 1 2 1 Developmental validation is the demonstration of the accuracy precision and reproducibility of a procedure by the manufacturer technical organization academic institution government laboratory or other party SWGDAM July 2003 SWGDAM Guideline 2 10 1 The reaction conditions needed to provide the required degree
92. consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining MSDSs You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems This service is free and available 24 hours a day To obtain MSDSs 1 Go to https docs appliedbiosystems com msdssearch html 2 In the Search field type in the chemical name part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm Safety AmpFlSTR Yfiler PCR Amplification Kit Us
93. ct36vb_POP4_DefaultModule as the run module For more information about performing spectral calibrations for Data Collection Software v1 1 or 1 0 refer to ABI PRISM 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin PN 4332345 d Click OK to save the protocol To perform a spectral calibration for 3100 3100 Avant Data Collection Software v2 0 continued Instrument Plate Type Volume Wells 3100 96 Well 10 L A1 to H2 3100 Avant 96 Well 10 L A1 B1 C1 D1 DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 12 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup 5 Create a plate record a In the Tree pane of the Data Collection Software v2 0 click GA Instruments gt ga3100 or ga3100 Avant gt instrument name gt Run Scheduler b In the Run Scheduler view enter a new plate name in the Scan or Type Plate ID field then click Search c In the Create new plate dialog box click Yes d Complete the New Plate Dialog box Name lt Enter a name for the plate gt Application Spectral Calibration Plate Type 96 Well Owner Name lt Enter a name for the owner gt Operator Name lt Enter a name for the operator gt e Click OK 6 In the Spectral Calibration Plate Editor dialog box enter the following information a In the Sample
94. d 8 7 L Hi Di Formamide per sample Vortex Add to wells of 96 well plate 9 L formamide size standard mixture 1 L allelic ladder or sample 10 L formamide for blank Heat 95 C for 3 then place on ice for 3 Prepare the samples Assemble plate and place on autosampler Link plate to plate record Start run View and archive data Perform electrophoresis Create a results group Create a GeneMapper ID Software plate record Create an instrument protocol Set up Data Collection Software v2 0 Display and check calibration Extract and quantify data PCR amplify data Analyze data ABI PRISM 310 Analyzer DRAFT May 25 2005 9 53 am 3100_part2 fm Overview AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 5 3100 3100 Avant Electrophoresis Setup 3100 3100 Avant Analyzer Quick Reference Table The following table provides information users familiar with the 3100 3100 Avant instruments can utilize to get started analyzing samples from Yfiler kits Condition Setting Dye Set Matrix Standard Set DS 33 PN 4345833 Filter Set G5vb Size Standard GeneScan 500 LIZ Size Standard PN 4322682 Run Module For Data Collection Software v2 0 HIDFragmentAnalysis36_POP4_1 For Data Collection Software v1 1 3100 instrument GeneScan36vb_POP4DyeSetG5Module For Data Collection Software v1 0 3100 Avant instrument GeneS
95. d Chapter 5 Analyzing Data 5 10 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software 6 Import AmpFLSTR_Yfiler_Binset_v1 a Select the AmpFLSTR_Yfiler_Panel_v1 folder in the navigation pane b Select File gt Import Bin Set to open the Import Bin Set dialog box c Select AmpFLSTR_Yfiler_Binset_v1 then click Import Note Importing this file associates the bin set with the panels in the AmpFlSTR_Yfiler_Panel_v1 folder To import panels and bin sets continued Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 11 Analyzing Data GeneMapper ID Software 7 View the imported panels a Select the AmpFLSTR_Yfiler_Panel_v1 folder in the navigation pane to view the panel contained in this folder in the right pane b Double click the AmpFLSTR_Yfiler_Panel_v1 folder in the navigation pane to display the list of panels below it 8 View the markers and display the Bin view a Select the Yfiler_v1 folder in the navigation pane to display the list of markers it contains in the right pane b Double click the Yfiler_v1 folder in the navigation pane to display the list of markers below it c Select DYS389II in the navigation pane to display the Bin view for the marker in the right pane To import panels and bin sets continued Chapter 5 Analyzing Dat
96. d ladder in the Sample Info column The Kazam macro uses the first lane or injection of ladder to determine the sizes in the allele categories that will be used for genotyping You can skip the first lane or injection of allelic ladder and use the second lane or injection of allelic ladder for genotyping instead To do so remove the word ladder from the Sample Info column in all four sample dye colors for the first lane or injection of allelic ladder in the Dye lanes window after importing the sample files but before running the Kazam macro Make sure that the word ladder is entered for Sample Info in the second lane or injection of allelic ladder See step 4 on page 5 45 for a description of how to access the Sample Info column in the Dye lanes window GeneScan Analysis Software Peak Recognition Requirements All allele peaks in the allelic ladder for each locus must be recognized labeled in the GeneScan Analysis Software that is each allele peak must have an entry in the GeneScan table All allele peaks in each allelic ladder must have a peak height value in relative fluorescence units RFU greater than the Peak Amplitude Threshold PAT specified in the GeneScan software Analysis Parameters All allele peaks in each allelic ladder must be resolved For example the DYS439 12 13 and 14 alleles must be resolved so that each peak has an entry in the GeneScan software table Sample allele peak heigh
97. d reactions initially at 95 C for 11 min PCR Master Mix not vortexed thoroughly before aliquoting Vortex PCR Master Mix thoroughly AmpFlSTR Yfiler Primer Set exposed to too much light Store Primer Set protected from light GeneAmp PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters Tubes not seated tightly in the thermal cycler during amplification Push reaction tubes firmly into contact with block after first cycle Repeat test GeneAmp PCR System 9600 heated cover misaligned Align GeneAmp 9600 heated cover properly so that white stripes align after twisting the top portion clockwise Wrong PCR reaction tube Use Applied Biosystems MicroAmp Reaction Tubes with Caps for the GeneAmp 9600 and 9700 MicroAmp Base used with tray retainer set and tubes in GeneAmp 9600 and 9700 Remove MicroAmp Base from tray retainer set and repeat test DRAFT May 25 2005 9 53 am 7x9_Appendix fm Troubleshooting AmpFlSTR Yfiler PCR Amplification Kit User s Manual A 3 Faint or no signal from both the AmpFlSTR Control DNA 007 and the DNA test samples at all loci continued Insufficient PCR product electrokinetically injected For ABI PRISM 310 runs Mix 1 5 L of PCR product and 25 L of Hi Di Formamide GeneScan 500 LIZ solution CHEMICAL HAZARD For
98. de PN 4338775 and User Bulletin New Features and Installation Procedures for GeneMapper ID Software Version 3 2 PN 4352543 Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 15 Analyzing Data GeneMapper ID Software Peak Detector The software uses the peak detection parameters to specify the minimum peak height to limit the number of peaks detected Although GeneMapper ID software displays peaks that fall below the specified height in electropherograms the software does not label or determine the genotype of these peaks The analysis range is set by the user based on location of the primer peak and size standard peaks Note For more information on peak detection algorithms refer to Appendix A of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 and User Bulletin New Features and Installation Procedures for GeneMapper ID Software Version 3 2 PN 4352543 Table 5 1 HID Classic analysis method settings continued Tab Settings 400 Chapter 5 Analyzing Data 5 16 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software Peak Quality Quality Flags Quality flag settings PQV thresholds Table 5 1 HID Classic analysis method settings continued Tab Settings Setting Up GeneMapper ID Software v3 2 for Analy
99. ded in the CD that ships with this manual This CD AmpFlSTR Yfiler Kit Template 9 CD part number 4360913 can be ordered separately if lost The template file can also be downloaded from the following website http www appliedbiosystems com support software genotyper temp lates cfm To edit the filter value 1 Select View gt Show Step Window 2 Click the first step of the macro Remove labels from peaks whose height is less than 20 of the highest peak in a category s range 3 Select Macro gt Edit Step Note This macro uses the second filter option of the 4 filter options in the Filter Labels window 4 Change the value then click Replace Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 45 Analyzing Data Genotyper Software You must have Genotyper Software v3 7 or higher and Windows NT 4 0 with Service Pack 4 or 5 operating system to run the AmpFlSTR Yfiler Kit Template Install the template onto your computer following the instructions in the READ_ME file Note The AmpFlSTR Yfiler Kit Template file is a read only file When using the Template you must save the file under a different name to ensure that the original template file is not overwritten Automatically Assigning Genotypes To use the AmpFlSTR Yfiler Kit Template 1 Double click the Yfiler icon to launch the Genotyper software application and open
100. dependent of the frequency of the two genotypes in the population If the suspect and evidence samples have the same genotype 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 population s Population Samples Used in These Studies The AmpFlSTR Yfiler PCR Amplification Kit was used to generate the population data provided in this section Samples were collected from individuals throughout the United States with no geographical preference African American 333 samples were provided U S Caucasian 254 samples were provided Hispanic 175 samples were provided AmpFlSTR Yfiler Kit Gene Diversity Values Table 6 2 AmpFlSTR Yfiler Kit gene diversity across three different U S populations Locus1 U S African American N 333 U S Caucasian N 254 U S Hispanic N 175 DYS456 0 598 0 703 0 663 DYS389I 0 538 0 575 0 492 DYS390 0 635 0 713 0 694 DYS389II 0 744 0 703 0 727 DRAFT August 7 2006 8 20 am ExperimentsResults fm Population Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 39 1The gene diversity D for each locus was computed using the formula where n represents the sample size and pi is the allele frequency Johnson et al 2003 DYS458 0 755 0 808 0 77 DYS19 0 748 0 541 0 645
101. ding the AmpFlSTR Yfiler Kit Template AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 39 Analyzing Data Genotyper Software Kazam Macro The Kazam macro automatically determines genotypes relative to the allelic ladder For each locus the Kazam macro calls the Calculate locus Offsets macros which label peaks and filter remove labels from the stutter peaks Consequently the Kazam Macro allows a different stutter threshold to be calculated for each locus and thus provides maximum flexibility for customizing the filter that is used for each locus Removing Labels from Stutter Peaks by Applying Percentage Filters The Kazam macro includes a step that removes labels from stutter peaks by applying a percentage filter Labels are removed from peaks that are followed by a specified percent difference higher labeled peak within 2 25 to 3 75 bp for a trinucleotide repeat marker 3 25 to 4 75 bp for a tetranucleotide repeat marker 4 25 to 5 75 bp for a pentanucleotide repeat marker and 5 25 to 6 75 bp for a hexanucleotide repeat marker An additional filter for 2 bp stutter has been added for the DYS19 locus 1 50 to 2 50 bp and a 3 bp stutter filter for the DYS392 locus 2 25 to 3 75 bp The specified filter percentages for the loci are listed in Table 5 5 Table 5 5 Kazam macro stutter filter percentages for Yfiler loci Locus Stutter bp stutter plus or minus DYS456 13 21 DYS389I 11 79 DYS390 10 4
102. displayed in the Matrix Result table e Click OK If you are using GeneScan analysis software a Select File gt New then click the Matrix icon b In the Make New Matrix dialog box Indicate the number of dyes by selecting 5 in the Number of Dyes dropdown Select the sample file that corresponds to each dye by clicking a button B G Y R or O then selecting the appropriate sample file Enter the starting point for each file The Start At point should be after the primer peak c Click OK A successful matrix opens an untitled Matrix Values window with a 5x5 matrix of numerical values 5 Use the Save As command to name and save the matrix file Choose a name that reflects the chemistry and run conditions Save the matrix file in the ABI folder D AppliedBio Shared Analysis SizeCaller Matrix To create a matrix file for the 310 instrument Data Collection Software v3 0 continued DRAFT May 25 2005 9 53 am 310_part2 fm Creating a Matrix File for the 310 Genetic Analyzer AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 37 310 Analyzer Setup Verifying the Accuracy of the Matrix File If this verification test does not pass then the capillary may not have been aligned properly in the instrument during the run To correct this problem a Tape the capillary to the heat plate so that the capillary is immobilized during the run b Repeat the experiment making sure that
103. door and press the Tray button to present the autosampler DRAFT May 25 2005 9 53 am 310_part2 fm Performing Electrophoresis AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 47 310 Analyzer Setup Running the Samples on the 310 Instrument 2 Load the sample tubes on a 48 well or 96 well sample tray as shown in the following illustration For 48 well sample tray For 96 well sample tray 3 Place the sample tray on the autosampler 4 Press the Tray button to retract the autosampler 5 Close the instrument door To load the samples continued 2 1 3 1 2 3 To run the samples on the 310 instrument 1 If necessary launch the data collection software 2 Open the GeneScan Software Injection list that you saved earlier 3 Click Run If you did not preheat the instrument the run module brings the instrument up to the 60 C run temperature This process can take up to 30 minutes The run begins once the run temperature is reached DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 48 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup 08 2006 Part Number 4358101 Rev C DRAFT May 25 2005 9 53 am DataAnalysisTitle fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual Chapter 5 Analyzing Data I DRAFT May 25 2005 9 53 am DataAnalysisTitle fm AmpFlSTR Yfiler PCR Amplification Kit User s Manu
104. dows 2000 3 0 GeneMapper ID 3 2 GeneScan 3 7 1 GenoTyper 3 7 Macintosh OS 9 0 2 1 GeneMapper ID 3 2 GeneScan 3 1 2 GenoTyper 2 5 2 Chapter 1 Overview 1 6 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components The four dyes used in the AmpFlSTR Yfiler PCR Amplification Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ is used to label the GeneScan 500 Size Standard How Multicomponent Analysis Works Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on the ABI PRISM instruments the fluorescent signals are separated by a diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern 6 FAM dye emits at the shortest wavelength and is displayed as blue followed by the VIC dye green NED dye yellow PET dye red and LIZ dye orange Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 1 1 The goal of multicomponent analysis is to effectively correct for spectral overlap Figure 1 1 Emission spectra of the five dyes used in the AmpFlSTR Yfiler PCR Amplification Kit Normalized Emi
105. e and when updated versions of panels and bin sets are provided To import panels and bin sets 1 Start the GeneMapper ID software a Select Start gt Programs gt Applied Biosystems gt GeneMapper gt GeneMapper ID b In the login box that appears make the following selections and click OK User Name type in a unique user name Password type in a password of 6 10 characters 2 Create a new password The first time you start the software you are prompted to change the password When the password dialog box opens Leave the Old Password box blank Type a the New Password Type the new password again to verify it The GeneMapper Project window opens with a blank untitled project 3 Select Tools gt Panel Manager to open the Panel Manager 4 Locate and open the folder containing the panels and bins a Select Panel Manager in the navigation pane b Select File gt Import Panels to open the Import Panels dialog box Highlight this Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 9 Analyzing Data GeneMapper ID Software 5 Select AmpFLSTR_Yfiler_Panel_v1 then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFlSTR_Yfiler_Panel_v1 This folder contains the panels and associated markers To import panels and bin sets continue
106. e et al 2004 Nature of the Polymorphisms DYS392 is a trinucleotide repeat DYS438 is a pentanucleotide repeat and DYS448 is a hexanucleotide repeat Their allele differences result from differences in the number of repeat units 3 bp 5 bp and 6 bp respectively The remaining AmpFlSTR Yfiler kit loci are tetranucleotide short tandem repeat STR loci The length differences among alleles of these particular loci result from differences in the number of 4 bp repeat units All the alleles in the AmpFlSTR Yfiler Allelic Ladder have been subjected to DNA sequencing at Applied Biosystems In addition other groups in the scientific community have sequenced alleles at some of these loci Redd et al 2002 www cstl nist gov biotech strbase y strs htm Among the various sources of sequence data on the AmpFlSTR Yfiler kit loci there is consensus on the repeat patterns and structure of the STRs Inheritance The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from 39 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 Three CEPH family DNA sets were ex
107. each of the 17 loci included in the AmpFlSTR Yfiler kit by selecting from a drop down list of the most common alleles for each locus Table 5 8 Types of allele values for Default Mode Drop down List Values Description Integers representing the most common alleles for the loci For example the dropdown list includes the numbers 16 to 24 for the DYS448 locus Wildcard Indicates that the locus will be omitted from the search lt m where m is the smallest allele in the allele range Indicates that alleles smaller than m will be included in the search For example in Figure 5 4 below alleles smaller than 16 will be included gt n where n is the largest allele in the allele range Indicates that alleles larger than n will be included in the search For example in Figure 5 4 below alleles larger than 24 will be included Searching the Database AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 63 Analyzing Data Haplotype Database Figure 5 4 Different Custom Entry Options Custom Entry Mode Allows you to enter up to three comma separated values for DYS385 and two comma separated values for all other loci Use the Custom Entry Mode to search for Alleles that fall within the size range for the locus but that are not included in the dropdown list microvariants An example would be if the size range for a locus is 14 to 20 and the allele you want to search for is 18 2 Loci that have
108. efect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves IMPORTANT Do not include the GeneScan 500 LIZ Size Standard when preparing matrix standards c Vortex thoroughly to mix then spin briefly in a microcentrifuge d Heat the tube at 95 C for 5 min to denature the DNA e Immediately place the tube on ice for 3 min f Place the tubes in the appropriate sample tray 2 Set up the run a In the Data Collection Software select File gt New then click GeneScan Smpl Sheet 48 Tube or GeneScan Smpl Sheet 96 Tube as appropriate b Complete the sample sheet then save and close it c Select File gt New then click GeneScan Injection List d Complete the injection list making sure to select the sample sheet you set up in step b Module GS STR POP4 1mL G5v2 Matrix File none 3 Click Run to run the matrix samples DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 36 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup 4 Create the matrix file using GeneMapper ID or GeneScan analysis software If you are using GeneMapper ID analysis software a Navigate to the GeneMapper Manager and click the Matrices tab b Click the New tab and the Matrix editor is displayed c In the Matrix editor enter the appropriate values d Click Create and the values are
109. els in the Panel Manager Lanes or injections containing the allelic ladder should be analyzed with the same analysis method and parameters used for samples Alleles not found in the AmpFlSTR Allelic Ladders do exist These off ladder alleles may contain full and or partial repeat units An off ladder allele is defined as an allele falling outside of the 0 5 bp bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample should be rerun to verify the result Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 7 Analyzing Data GeneMapper ID Software Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data Perform the following tasks before you analyze sample fsa files for the first time 1 Import panels and bins into the Panel Manager as explained in Importing Panels and Bins on page 5 8 2 Create an analysis method with the appropriate bin set option as explained in Creating HID Analysis Methods on page 5 12 3 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID Software versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 for more information 4 Define custom views of plots Refer to Chapter 1 of the GeneMapper ID Software versions 3 1 and 3 2 Human Identific
110. er s Manual 4 15 3100 3100 Avant Electrophoresis Setup 4 Into each well of a MicroAmp Optical 96 Well reaction plate add 9 L of the formamide size standard mixture 1 L of PCR product or Allelic Ladder Note For blank wells add 10 L of Hi Di formamide 5 Seal the reaction plate with appropriate septa then briefly centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom 6 Heat the reaction plate in a thermal cycler for 3 min at 95 C 7 Immediately place the plate on ice for 3 min To prepare samples for electrophoresis continued DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 16 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup Setting Up the Electrophoresis Run Compatible Data Collection and Analysis Software The following table lists data collection and analysis software that you can use to analyze YFiler data Software Setup Summary Setting up the Data Collection Software v2 0 involves the following three tasks Creating an Instrument Protocol on page 4 17 Creating a Results Group on page 4 17 Creating a GeneMapper ID Software Plate Record for Autoanalysis on page 4 21 Note Before the first run on 3100 3100 Avant instruments running Data Collection Software v1 1 or v1 0 you must edit the default module parameters Ref
111. er s Manual xi Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose 4 To have a copy of a document sent by fax or e mail select Fax or Email to the left of the document title in the Search Results page then click RETRIEVE DOCUMENTS at the end of the document list 5 After you enter the required information click View Deliver Selected Documents Now Chemical Waste Hazard CHEMICAL WASTE HAZARD Some wastes produced by the operation of the instrument or system are potentially hazardous and can cause injury illness or death Chemical Waste Safety Guidelines To minimize the hazards of chemical waste Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide 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 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of
112. er to page 34 of ABI PRISM 3100 and 3100 Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products PN 4332345 Operating System Data Collection Software Analysis Software References Windows NTa 1 1 3100 Analyzer 1 0 3100 Avant Analyzer GeneMapper ID 3 2 GeneScan 3 7 1 GenoTyper 3 7 ABI PRISM 3100 Genetic Analyzer User Manual Data Collection Software v1 1 PN 4315834 ABI PRISM 3100 Avant Genetic Analyzer User Guide Data Collection Software v1 0 PN 4333549 ABI PRISM 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin PN 4332345 Windows 2000a 2 0 GeneMapper ID 3 2 This section a Applied Biosystems conducted validation studies for Yfiler using these configurations DRAFT May 25 2005 9 53 am 3100_part2 fm Setting Up the Electrophoresis Run AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 17 3100 3100 Avant Electrophoresis Setup Creating an Instrument Protocol You must create an instrument protocol before the first run for any AmpFlSTR PCR Amplification Kit You can use the same instrument protocol for all subsequent runs Creating a Results Group Results groups are used to analyze name sort and deliver samples from a run Typically you modify the results group for each fragment analysis run To create an instrument protocol 1 In the Tree pane of Da
113. erpretation of Haplotype Data This section covers Overview 5 60 Searching the Database 5 62 Reviewing Results 5 69 Chapter 5 Analyzing Data 5 60 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Haplotype Database Overview The Yfiler Haplotype Database online search tool allows you to estimate the frequency of a given Y chromosome haplotype in specified populations Frequency calculations are based on the haplotype data generated from the 17 loci included in the AmpFlSTR Yfiler Kit This data was compiled from more than 2 000 samples from a range of populations The Yfiler Haplotype Database can be used to search complete or partial profiles generated with the Yfiler Kit and enables comparison of the discrimination capacity of the Yfiler Kit relative to other combinations of Y STR loci e g European Minimal Haplotype and SWGDAM loci Functions of the Web Based Search Tool The Yfiler Kit Haplotype Database online search tool has been designed to allow searches of haplotypes generated using the AmpFlSTR Yfiler Kit The tool will allow the user to estimate the frequency of occurrence of a haplotype in a number of reference populations The tool allows manu
114. et value determined for this allele is applied to the relevant allele s in the allele categories For example assume that the offset value determined by the 7 os category in the DYS392 os group is 2 67 for a particular lane or injection of allelic ladder This offset value is then applied to the allele 7 category in the DYS392 group thus setting the correct offset value for allele 7 In order for the software to find the next allele peak in the DYS392 allelic ladder allele 8 the offset value for the 7 os allele is also applied to the 8 os category The result of this operation is that the 8 os category size will be 3 bp longer than the 7 os category In other words allele 8 is expected to be found at a size that is 3 bp longer than allele 7 To maximize the ease of peak recognition the size width for most offset categories is 1 bp as compared to the allele categories which have a width of 0 5 bp Once allele 8 is recognized in the DYS392 allelic ladder the correct offset value is calculated and assigned to the appropriate categories This process of peak recognition offset calculation and offset assignment is carried out for each of the alleles in each of the allelic ladders Understanding the AmpFlSTR Yfiler Kit Template AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 43 Analyzing Data Genotyper Software Off Ladder Alleles and Virtual Alleles In the previous example the 7 os offset value 2 67 is al
115. eviation DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 18 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Extra Peaks in the Electropherogram Causes of Extra Peaks Peaks other than the target alleles may be detected on the electropherogram Causes for the appearance of extra peaks include stutter products incomplete 3 A nucleotide addition at the n 1 position dye artifacts and mixed DNA samples see DAB Standard 8 1 2 2 Stutter Products A stutter is a well characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller or less frequently one repeat larger than the major STR product Butler 2001 Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 It has been reported that the DYS19 tetranucleotide repeat locus displays the typical 4 bp stutter but also a 2 bp stutter Prinz et al 2001 Gusmao et al 1999 The DYS392 trinucleotide repeat locus displays the typical 3 bp stutter but also a smaller 3 bp stutter Sequence analysis of this 3 bp stutter revealed that the product contains an additional repeat unit relative to the true allele peak The proportion of the stutter product relative to the main allele percent stutter is measured by dividing the height of the stutter peak
116. for peak window size is 15 Polynomial Degree The default parameter for polynomial degree is 3 Slope Threshold for Peak Start The default parameter for slope threshold for peak start is 0 0 Chapter 5 Analyzing Data 5 30 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneScan Software For additional information about analysis parameters refer to ABI PRISM GeneScan Analysis Software v3 7 for the Windows NT Platform PN 4308923 Slope Threshold for Peak End The default parameter for slope threshold for peak end is 0 0 Size Call Rangea Click the This Range Base Pairs radio button and enter the values of 75 for Min and 400 for Max Size Calling Methoda Click the Local Southern Method radio button for sizing of the AmpFlSTR products This method determines the sizes of fragments by using the reciprocal relationship between fragment length and mobility Baseline Window Size Refer to the user bulletin P N 4335617 for more information Auto Analysis Only Refer to the user bulletin P N 4335617 for more information a Same for Windows v3 7 1 and Macintosh v3 1 2 To define analysis parameters 1 In the Parameters column of the Analysis Control window click on the arrow beside lt Analysis Parameters gt for any of the sample files 2 Select Define New The Analysis Settings dialog box opens 3 Modify the default settings as necessary
117. g the multicomponenting process Perform a spectral calibration When you use a new dye set on the instrument When you change the capillary array length or polymer type After the laser or CCD camera has been realigned by a service engineer If you begin to see a decrease in spectral separation pull up and or pull down peaks For more information about performing spectral calibrations refer to ABI PRISM 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin PN 4350218 For information about performing spectral calibrations when using other Data Collection Software versions refer to the appropriate user manual Related Documentation on page xiii provides a list of related documentation 8 Prepare the syringes 9 Perform spatial and spectral calibration if necessary To set up the 3100 3100 Avant instrument continued DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 10 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup To perform a spectral calibration for 3100 3100 Avant Data Collection Software v2 0 1 Prepare the spectral calibration matrix standards for Dye Set G5 a Thaw and thoroughly mix the contents of the Matrix Standard Set DS 33 tube then spin briefly in a microcentrifuge b Combine the following in a labeled 1 5 mL microcentrifuge tube CHEMICAL HA
118. h allele in the AmpFlSTR Yfiler Allelic Ladder A 0 5 bp window allows for the detection and correct assignment of alleles Any sample allele that sizes outside a window could be either of the following An off ladder allele i e an allele of a size that is not represented in the AmpFlSTR Yfiler Allelic Ladder An allele that does correspond to an allelic ladder allele but whose size is just outside a window because of measurement error DRAFT August 7 2006 8 20 am ExperimentsResults fm Accuracy Precision and Reproducibility AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 9 The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument or in several lanes of one gel Table 6 1 indicates typical precision results obtained from the nine injections of the AmpFlSTR Yfiler Allelic Ladder analyzed on the ABI PRISM 310 Genetic Analyzer 47 cm capillary and POP 4 polymer The internal lane size standard used was GeneScan 500 LIZ Size Standard These results were obtained within a set of injections on a single capillary As indicated above sample alleles may occasionally size outside of the 0 5 bp window for a respective allelic ladder allele because of measurement error The frequency of
119. heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 Repeat test Table A 1 Troubleshooting causes and recommended actions continued Observation Possible Causes Recommended Actions DRAFT May 25 2005 9 53 am 7x9_Appendix fm Appendix A Troubleshooting A 6 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Troubleshooting Automated Genotyping To Troubleshoot Automated Genotyping Observation Probable Cause Recommended Action Warning message Could not complete Run Macro command because no dye lanes are selected The word ladder is not in Sample Info for the lane or injection of allelic ladder Type the word ladder in the Sample Info column for each dye color Blue Green Yellow and Red for the AmpFlSTR Yfiler Allelic Ladder sample Warning message Could not complete Run Macro command because the labeled peak could not be found One or more peaks in the allelic ladder are below the Peak Amplitude Threshold that was specified in the GeneScan software Analysis Parameters Use another allelic ladder in the project or 1 In the GeneScan Analysis Software lower the Peak Amplitude Threshold values for Blue Green Yellow and Red dye colors in the Analysis Parameters 2 Reanalyze the sample file s containing the allelic ladder 3 Import all sample files into
120. ication Kit User s Manual 4 21 3100 3100 Avant Electrophoresis Setup Creating a GeneMapper ID Software Plate Record for Autoanalysis Refer to ABI PRISM 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin PN 4350218 and Chapter 5 of the ABI PRISM 3100 3100 Avant Genetic Analyzers User Guide PN 4347102 for more information about creating these files 6 continued Note If you choose elements from the Format lists that do not create unique Sample file or Run folder names the following warning message appears below the Example line INVALID NAME Filename does not have a unique identifier in it You can proceed to start a run without removing the warning message If you want to remove the warning message select an additional Format element that distinguishes one file from another for example the capillary number is unique while the instrument name is not 7 Select the Automated Processing tab 8 Click OK to save the Results Group To create a results group continued If you select Then Use with Setting from Analysis tab page 4 18 Only when the result group is complete Samples are analyzed after all samples using the same results group have been run Do Autoanalysis and Results Entry Group Complete When every run completes Samples are analyzed after each run of 16 or 4 samples Do Autoanalysis To set up data collection software for elect
121. ication using a system such as the Quantifiler Human DNA and Quantifiler Y Human Male DNA Quantification Kit P N 4343895 and 4343906 The final DNA concentration should be in the range of 0 05 0 10 ng L so that 0 5 1 0 ng of DNA will be added to the PCR reaction in a volume of 10 L If the sample contains degraded DNA amplification of additional DNA may be beneficial Effect of DNA Quantity on Results If too much DNA is added to the PCR reaction the increased amount of PCR product that is generated can result in the following Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem for two reasons 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 The sample can be re amplified using less DNA When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the alleles may occur due to stochastic fluctuation Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instrumen
122. ies specificity 6 27 spectral calibration 4 9 standards for samples 1 8 storage recommendations for kits 1 7 stutter filter percentages 5 39 T Technical Support contacting xv text conventions vii training information on xiv troubleshooting automated genotyping A 6 troubleshooting causes and actions A 2 U user attention words described viii V validation developmental 6 3 W WARNING description ix waste disposal guidelines xii Y Yfiler Haplotype Database overview 5 60 Yfiler Kit Template examining data 5 47 DRAFT May 25 2005 9 53 am 7x9_MultiChapter_IX fm Index 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 www appliedbiosystems com 08 2006 Part Number 4358101 Rev C
123. ine the peak labels in each electropherogram Clicking a labeled peak removes the label Clicking the same peak again defaults to the placement of bp size of that peak To customize the peak label select Analysis gt Set Click Options type the allele designation and or desired text then click OK Allele categories which appear as dark gray bars in the Plot window are defined to be 0 5 bp wide Peaks that size within 0 5 bp of an allele category has a label indicating the allele designation Peaks that do not size within an allele category have a label indicating OL Allele off ladder allele A sample allele peak must be recognized by GeneScan software before it can be recognized by Genotyper software Sample allele peaks that are below the PAT that was specified in the GeneScan software Analysis Parameters cannot be labeled by Genotyper software Also because no information is imported for peaks that are not recognized by GeneScan software such peaks will not align exactly by size relative to the x axis size scale in the Genotyper software plot window To view electropherograms from more than one dye color in the Plot window 1 Select Views gt Show Dye Lanes Window 2 Click the desired Dye lane rows Note Hold down the Shift key on the keyboard to select multiple adjacent Dye lane rows Hold down the Ctrl key to select Dye lane rows that are not adjacent 3 Select Views gt Show Plo
124. ing Data GeneMapper ID Software Peak Quality Quality Flags Quality flag settings PQV thresholds Table 5 2 HID_Advanced analysis method settings continued Tab Settings Analyzing Sample Files With GeneMapper ID Software AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 21 Analyzing Data GeneMapper ID Software Analyzing Sample Files With GeneMapper ID Software To analyze a project 1 From the Project window select File gt Add Samples to Project to navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project Parameter Analysis Method Advanced Method Classic Method Table Setting Select AmpFlSTR Table from the drop down list Select AmpFlSTR Table from the drop down list Sample Type Select the sample type Select the sample type Analysis Method AmpFlSTR_Yfiler_ AdvancedMode AmpFlSTR_Yfiler_ ClassicMode Panel AmpFlSTR_Yfiler_ Panel_v1 AmpFlSTR_Yfiler_ Panel_v1 Size Standarda a For more information about how the Size Caller works refer to the ABI PRISM GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin PN 4335617 CE_G5_HID_ GS500b c b The following fragments are defined for the CE_G5_HID_GS500 size standard provided with the AmpFlSTR kits 75 100 139 150 160 200 300 340 350 400 450 For additional informat
125. instrument uses a matrix file to correct for the overlapping of fluorescence emission spectra of the dyes For information about creating a matrix file refer to Creating a Matrix File for the 310 Genetic Analyzer on page 4 34 and Chapter 6 of ABI PRISM GeneScan Analysis Software v3 7 for the Windows NT Platform PN 4308923 DRAFT May 25 2005 9 53 am 310_part2 fm Setting Up the 310 Genetic Analyzer AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 31 310 Analyzer Setup Setting Up the 310 Genetic Analyzer Kit Contents and Storage Each Yfiler kit contains materials sufficient to perform 100 reactions at a 25 L reaction volume IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpFlSTR Yfiler Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum Table 4 2 User supplied materials Material Source 310 Analyzer materials 310 Capillaries 47 cm 50 m i d internally uncoated green 402839 0 5 mL Sample Tray 5572 96 Well Tray Adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 10 Genetic Analyzer Buffer with EDTA 402824 Genetic Analyzer Retainer Clips 96 Tube Tray Septa Clips 402866 Genetic Analysis Sample Tubes 0 5 mL 401957 Genetic Analysis Septa for 0 5 mL Sample
126. ion about size standards refer to Appendix A of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 c The 250 bp peak is not included in the size standard definition because this peak can be used as an indicator of precision within a run Define a new size standard d d Refer to Chapter 2 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 Matrix Select a matrix for 310 instruments only Select a matrix for 310 instruments only Chapter 5 Analyzing Data 5 22 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software Figure 5 1 Project Window For more information about any of these tasks refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 3 Click Analyze type a name for the project in the Save Project dialog then click OK to initiate analysis The status bar displays progress of analysis As a completion bar extending to the right with the percentage indicated With text messages on the left The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample See Figure 5 1 The genotypes tab becomes available after analysis To analyze a project continued Examining and Editing GeneMapper ID Software Results AmpFlSTR Yfiler PCR Amplifi
127. ious run Poor sizing precision or allele calling Poor resolution and or decreased signal intensity Have you replenished the electrophoresis reagents Replenish the reagents as described in Chapter 1 of the ABI PRISM 3100 3100 Avant Genetic Analyzers User Guide PN 4347102 Have you performed a spatial calibration Perform a spatial calibration each time you Install or replace a capillary Temporarily remove the capillary array from the detection block Refer to Chapter 2 of the ABI PRISM 3100 3100 Avant Genetic Analyzers User Guide for information about performing spatial calibration Have you performed a spectral calibration Perform a spectral calibration A spectral calibration creates a matrix to correct for the overlapping of fluorescence emission spectra of the dyes See Performing a Spectral Calibration on page 4 9 for information DRAFT May 25 2005 9 53 am 3100_part2 fm Setting up the 3100 3100 Avant Instrument AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 7 3100 3100 Avant Electrophoresis Setup Setting up the 3100 3100 Avant Instrument Kit Contents and Storage Each Yfiler kit contains materials sufficient to perform 100 reactions at a 25 L reaction volume IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpFlSTR Yfiler Allelic Ladder and GeneScan
128. l values being measured should be determined SWGDAM July 2003 Laser induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2001 and Wallin et al 2002 However accuracy and reproducibility of AmpFlSTR Yfiler kit profiles have been determined from various sample types Figure 6 4 illustrates the size differences that are typically observed between sample alleles and allelic ladder alleles on the ABI PRISM 3100 Genetic Analyzer with POP 4 polymer The x axis in Figure 6 4 represents the nominal base pair sizes for the AmpFlSTR Yfiler Allelic Ladder and the dashed lines parallel to the x axis represent the 0 5 bp windows The y axis is the deviation of each sample allele size from the corresponding allelic ladder allele size All sample alleles are within 0 5 bp of a corresponding allele in an allelic ladder DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 8 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Figure 6 4 Size deviation of 78 samples analyzed on the ABI PRISM 3100 Genetic Analyzer Precision and Size Windows Sizing precision allows for determining accurate and reliable genotypes Sizing precision was measured on the ABI PRISM 310 Genetic Analyzer As indicated in the Automated Genotyping section the recommended method for genotyping is to employ a 0 5 bp window around the size obtained for eac
129. lSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneScan Software Performing Data Analysis 5 In the Analysis Control window apply the size standard definition to all sample files in the project To define the size standard for Yfiler kits continued To analyze the sample files 1 In the Analysis Control window select the blue green yellow red and orange columns The Analyze button is activated 2 Confirm that the orange dye LIZ is assigned to the size standard as indicated by a diamond symbol 3 If the diamond symbol does not appear in the orange boxes Ctrl Click or click to place a diamond in the box 4 Click Analyze 5 After the analysis is complete confirm that the sizes for the peaks in the GeneScan 500 LIZ Size Standard have been correctly assigned a Select Window gt Results Control and examine the orange GeneScan 500 LIZ Size Standard peaks in overlapping groups of 16 samples Quick Tile Off Be sure to select View gt Align By Size b While the samples are tiled check the 250 bp peaks sized as approximately 246 bp in the enlarged view window Remember that this peak was not defined in the size standard The tiled 250 bp peaks should size consistently they should all overlap In a typical run the 250 bp peaks all fall within a size window of approximately 1 bp Temperature fluctuations in the laboratory may cause variations gt
130. lSTR Yfiler PCR Amplification Kit User s Manual 5 29 Analyzing Data GeneScan Software Table 5 3 lists the individual parameters and explains how to set them Table 5 3 GeneScan analysis parameters Parameter Procedure Analysis Rangea 1 Click the This Range Data Points radio button 2 Look at the raw data and enter the values that are appropriate for all sample files in the project These data points affect data in the results display 3 Enter Start and Stop data point numbers in the entry fields Select the Start data point just before the first peak of interest the 75 bp size standard peak At a minimum select the Stop data point just after the last peak of interest the 400 bp size standard peak See Figure 5 2 on page 5 28 Smooth Options The default parameter for Smooth Options is light Peak Amplitude Thresholdsa 1 Select a Peak Amplitude Threshold PAT for each dye color 2 Use the active scroll bar to enter the PATs for each of the five colors 3 After analysis the GeneScan table contains data for all peaks with a height above that specified by the PAT Note Applied Biosystems suggests that you determine the PATs appropriate for your analysis Conduct sensitivity experiments in your laboratory with each instrument to evaluate the PATs used for analysis Min Peak Half Width The Min Peak Half Width for use with the AmpFlSTR products is 2 Pts Peak Window Size The default parameter
131. ling M A Sajantila A Tyler Smith C 2004 A comprehensive survey of human Y chromosomal microsatellites Am J Hum Genet 2004 74 1183 97 Kayser M Sajantila A Mutations at Y STR loci implications for paternity testing and forensic analysis 2001 Forensic Sci Int 118 116 21 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 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 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 non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Biotechniques 21 700 9 DRAFT May 25 2005 9 53 am 7x9_Bibliography fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual Bibliography 3 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 1998 19 101 7 Moretti T Baumstark A Defenbaugh D Keys K Smerick J and Budo
132. lyn B Fish P et al 2001 Validation of the AmpFlSTR Profiler Plus PCR Amplification Kit for use in forensic casework J Forensic Sci 46 3 642 646 Furedi S Woller J Padar Z Angyal M 1999 Y STR haplotyping in two Hungarian populations Int J Legal Med 113 38 42 DRAFT May 25 2005 9 53 am 7x9_Bibliography fm Bibliography 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Gonzalez Neira A Elmoznino M Lareu M V Sanchez Diz P Gusmao L Prinz M Carracedo A 2001 Sequence structure of 12 novel Y chromosome microsatellites and PCR amplification strategies Forensic Sci Int 122 19 26 Gusmao L Gonzalez Neira A Pestoni C Brion M Lareu M V Carracedo A 1999 Robustness of the Y STRs DYS19 DYS389 I and II DYS390 and DYS393 optimization of a PCR pentaplex Forensic Sci Int 106 163 72 Hall A and Ballantyne J 2003 The development of an 18 locus Y STR system for forensic casework Anal Bioanal Chem 376 1234 46 Holt C Stauffer C Wallin J et al 2000 Practical applications of genotypic surveys for forensic STR testing Forensic Sci Int 112 2 3 91 109 Johnson C L Warren J H Giles R C Staub R W 2003 Validation and uses of a Y chromosome STR 10 plex for forensic and paternity laboratories J Forensic Sci 2003 48 6 1260 8 Kayser M Kittler R Erler A Hedman M Lee A C Mohyuddin A Mehdi S Q Rosser Z Stoneking M Job
133. mamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide CHEMICAL HAZARD Formamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Table A 1 Troubleshooting causes and recommended actions continued Observation Possible Causes Recommended Actions DRAFT May 25 2005 9 53 am 7x9_Appendix fm Appendix A Troubleshooting A 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Positive signal from AmpFlSTR Control DNA 007 but no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantitate DNA and add 0 5 1 0 ng of DNA Repeat test Test sample contains PCR inhibitor e g heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 Repeat test Test sample DNA is degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded re amplify with an increased amount of DNA Dilution of test sample DNA in H2O or w
134. mation and number of matching haplotypes as for manual database queries Chapter 5 Analyzing Data 5 72 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Haplotype Database 08 2006 Part Number 4358101 Rev C DRAFT August 7 2006 8 20 am ExperimentsResults fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 1 6 Experiments and Results 6 This chapter covers Overview 6 2 Developmental Validation 6 3 Accuracy Precision and Reproducibility 6 7 Extra Peaks in the Electropherogram 6 18 Characterization of Loci 6 25 Species Specificity 6 27 Sensitivity 6 29 Stability 6 31 Mixture Studies 6 34 Population Data 6 38 Analyzing the Population Data 6 40
135. ments and Results 6 22 AmpFlSTR Yfiler PCR Amplification Kit User s Manual This final extension step gives the AmpliTaq Gold DNA polymerase additional time to complete A addition to all double stranded PCR products especially in mixtures containing high levels of female DNA and low levels of male DNA STR systems that have not been optimized for A addition may have split peaks where each allele is represented by two peaks one base pair apart Figure 6 9 Omission of the final extension step resulted in split peaks due to incomplete A nucleotide addition These data were generated on the ABI PRISM 310 Genetic Analyzer using another AmpFlSTR kit DRAFT August 7 2006 8 20 am ExperimentsResults fm Extra Peaks in the Electropherogram AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 23 Lack of complete A nucleotide addition may be observed in AmpFlSTR Yfiler kit results when the amount of input DNA is greater than the recommended protocols because more time is needed for AmpliTaq Gold DNA Polymerase to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA may also result in off scale data Artifacts Artifacts or anomalies have been seen in data produced on genetic analyzers when using the AmpFlSTR Yfiler kit In amplified samples artifacts in the non calling region may appear in the green 88 bp black 80 and 95 bp and red 80 bp dye Low level artifacts
136. mpFlSTR Yfiler PCR Amplification Kit User s Manual data collection software 1 5 data stability 6 31 data accuracy precision and reproducibility of 6 7 data analysis 6 40 data artifacts 6 23 data for different populations 6 38 developmental validation 6 3 discriminatory capacity 6 40 DNA extraction methods 2 2 DNA sensitivity 6 29 DNA mixture studies 6 34 documentation related xiii E electropherogram causes of extra peaks 6 18 electrophoresis run setup for 310 Analyzer 4 38 electrophoresis performing with 310 Analyzer 4 46 electrophoresis preparing samples 4 14 electrophoresis preparing samples for 310 Analyzer 4 45 electrophoresis run 4 14 Electrophoresis setting up run for 3100 4 16 equipment not included with Quantifiler kits 1 8 experiments and results 6 1 extra peaks causes of in electropherogram 6 7 extracting DNA 2 2 F FTA paper extraction 2 3 G gene diversity values 6 38 GeneMapper ID Software Plate Record creating 4 21 GeneMapper ID Software setup for Data Collection Software 3 0 4 43 GeneMapper Manager 5 12 5 17 GeneScan 500 LIZ Internal Lane Size Standard 5 56 guidelines chemical safety x chemical waste safety xi waste disposal xii H haplotype data interpreting 5 59 haplotype searching for 5 62 hazards biological xii chemical waste xi HID_Classic analysis method creating 5 17
137. mpatible Data Collection and Analysis Software The following table lists data collection and analysis software that you can use to analyze YFiler Kit data Setup for Data Collection Software 3 0 with GeneScan Software Overview Setting up the electrophoresis run involves three tasks 1 Preheating the instrument optional 2 Setting Up GeneScan Sample Sheet and Injection List Defaults 3 Creating a Sample Sheet and Injection List for the Run Preheating the Instrument Setting the run temperature prior to starting a run is optional this heating step occurs automatically at the beginning of the GS STR POP4 1 mL G5v2 run module However preheating the instrument prior to a run saves time Operating System Data Collection Software Analysis Software References Windows 2000 3 0 GeneMapper ID 3 2 GeneScan 3 7 1 GenoTyper 3 7 Setup for Data Collection Software 3 0 with GeneMapper ID Software on page 4 43 Setup for Data Collection Software 3 0 with GeneScan Software on page 4 38 Macintosh OS 9 0 2 1 GeneMapper ID 3 2 GeneScan 3 1 2 GenoTyper 2 5 2 The GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Chapter 3 of the AmpFlSTR Identifiler PCR Amplification Kit User s Manual PN 4323291 DRAFT May 25 2005 9 53 am 310_part2 fm Setting Up the Electrophoresis Run AmpFlSTR Yfiler PCR Amplification Kit User s
138. n used for extracting DNA from large samples when the amount of DNA is expected to exceed 100 ng Chelex Method The Chelex method of DNA extraction involves fewer steps than the phenol chloroform method and consequently results in fewer opportunities for cross sample contamination The single stranded DNA extracted by this method is suitable for AmpFlSTR YFiler kit amplification Extracting DNA AmpFlSTR Yfiler PCR Amplification Kit User s Manual 2 3 FTA Paper Extraction The FTA paper extraction process begins as soon as blood is spotted on FTA paper Upon spotting cells are lysed and DNA is immobilized within the paper matrix DNA is then purified by a series of washes after which the DNA is ready for PCR amplification Refer to Figure 3 1 on page 3 10 for AmpFlSTR Yfiler kit results from a 1 2 mm FTA bloodstain punch Sample Storage and Chain of Custody The proper storage of samples and DNA specimens is essential to ensure that the DNA profiles obtained are accurate and meaningful Additionally the proper chain of custody is vital to maintaining the integrity of each particular specimen Chapter 2 Extracting and Quantifying DNA 2 4 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Quantifying DNA Importance of Quantitation By quantifying the DNA in a sample you ensure that there is enough DNA for adequate amplification You can determine the smallest volume necessary to obtain 0 5 to 1 0 ng of DNA If
139. nalyze Yfiler Kit Data This section covers Overview 5 36 Understanding the AmpFlSTR Yfiler Kit Template 5 37 Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping 5 44 Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder 5 53 Chapter 5 Analyzing Data 5 36 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software Overview Information in this Section This chapter describes the use of ABI PRISM Genotyper Software v3 7 in conjunction with the AmpFlSTR Yfiler Kit Template and the Microsoft Windows operating system to automatically genotype samples If you have not worked with the Yfiler Template before Understanding the AmpFlSTR Yfiler Kit Template on page 5 37 describes the macros in the template If you are familiar with the Yfiler Template Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping on page 5 44 provides instructions for using the template If you prefer to genotype samples manually Manual Genotyping Against the AmpFlSTR Yfiler Kit Allelic Ladder on page 5 53 explains how Instruments Refer to Instrument a
140. nalyzer User Guide Windows NT 4317588 Protocols for Processing AmpFlSTR PCR Amplification Kit Products with the ABI PRISM 377 DNA Sequencer and Windows NT OS User Bulletin 4340648 New Features and Installation Procedures for GeneMapper ID Software Version 3 2 User Bulletin 4352543 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 ABI PRISM GeneScan Analysis Software version 3 1 User s Manual Macintosh 4306157 ABI PRISM GeneScan Analysis Software v3 7 for the Windows NT Platform 4308923 DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm Preface xiv AmpFlSTR Yfiler PCR Amplification Kit User s Manual For additional documentation see How to Obtain Support below Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com GeneScan Software Reference Guide ABI PRISM 310 Analyzer 4303189 ABI PRISM GeneScan Analysis Software for the Windows NT Platform Overview of the Analysis Parameters and Size Caller User Bulletin 4335617 ABI PRISM Genotyper 2 5 Software User s Manual Macintosh 904648 ABI PRISM Genotyper 3 7 NT Software User s Manual 4309947 ABI PRISM Genotyper 3 7 NT Software Applications T
141. nd Software Compatibility on page 1 5 for a list of compatible instruments About the Software Genotyper software is used to automatically convert allele sizes obtained from ABI PRISM GeneScan Analysis Software into allele designations and to build tables containing the genotype information The software assigns genotypes by comparing the sizes obtained for the unknown sample alleles with the sizes obtained for the alleles in the allelic ladder Refer to the ABI PRISM Genotyper 3 7 NT Software User s Manual PN 4309947 and ABI PRISM Genotyper 3 7 NT Software Applications Tutorials PN 4309961 for more detailed information about the Genotyper software Before Running Genotyper Software GeneScan Analysis Software sample data particularly the allelic ladder must meet the requirements described in this section before you can use the macros in the AmpFlSTR Yfiler Kit Template Sample Info Column Requirements All samples must have a unique sample description in the Sample Info column of the GeneScan software sample sheet for the macros in the AmpFlSTR Yfiler Kit Template to build a table Samples with an empty Sample Info column are not incorporated into the table of genotypes Understanding the AmpFlSTR Yfiler Kit Template AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 37 Analyzing Data Genotyper Software Lanes or injections that contain the AmpFlSTR Yfiler Allelic Ladder must have the wor
142. ng annealing and denaturation temperature windows were tested to verify that a 1 0 C window produced a specific PCR product with the desired sensitivity of at least 1 ng of AmpFlSTR Control DNA 007 The denaturation temperatures tested were 92 5 C 94 C and 95 5 C all for 1 minute hold times on the same Silver 96 Well GeneAmp PCR System 9700 The annealing temperatures tested were 59 60 61 62 and 63 C Figure 6 2 also for 1 minute hold times in the Silver 96 Well GeneAmp PCR System 9700 The PCR products were analyzed using the ABI PRISM 3100 Genetic Analyzer 1 12 mM 1 28 mM 1 44 mM 1 60 mM standard concentration 1 76 mM 1 92 mM 2 08 mM DRAFT August 7 2006 8 20 am ExperimentsResults fm Developmental Validation AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 5 No preferential amplification was observed in the denaturation temperature experiments Of the tested annealing temperatures 59 62 C produced robust profiles At 63 C the yield of the majority of loci was significantly reduced Routine thermal cycler calibration is recommended when following the amplification protocol No preferential amplification was observed at the standard annealing temperature of 61 C Figure 6 2 Electropherograms obtained from amplification of 1 ng genomic DNA at annealing temperatures of 59 C 60 C 61 C 62 C and 63 C analyzed on the ABI PRISM 3100 Genetic Analyzer 59 C 60 C
143. nstrument then start the data collection software 2 If necessary install and clean the pump block install or replace the capillary prepare the syringes clean the electrode recalibrate the autosampler 3 Prime the pump block 4 Fill the buffer reservoirs 5 If necessary create a matrix file To install the G5v2 run module 1 Close all windows and applications 2 Insert the G5v2 Module Software CD PN 4339037 into the computer CD ROM drive Alternatively you can download the module file from https www appliedbiosystems com support software 31 0 modules cfm 3 Navigate to the 310 Data Collection Modules folder The default path is D AppliedBio 310 Modules DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 34 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup Creating a Matrix File for the 310 Genetic Analyzer Overview The precise spectral overlap between the five dyes is measured by analyzing DNA fragments labeled with each of the dyes 6 FAM VIC NED PET and LIZ in separate injections on a capillary These dye labeled DNA fragments are called matrix standard samples Instruments that do not perform multicomponenting such as the 310 and 377 instruments the analysis software GeneMapper ID or GeneScan analyzes the data from each of the five dye samples and creates a matrix file The matrix file c
144. on c Indicates the frequency of the specified haplotype within the entire database d Indicates that for the specified loci one match was found in the entire database Printable Search Results Figure 5 6 illustrates the printable search result which contains the haplotype loci specified in the search and the haplotype frequency results a b c d Chapter 5 Analyzing Data 5 70 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Haplotype Database Figure 5 6 Example of a printable search result Results for Uploaded Allele Information When you specify an analysis file and click Search the portal search tool compares each sample in the file against the database Results are displayed in two stages First the portal displays a table that lists the haplotypes from the uploaded file as shown in Table 5 9 Table 5 9 Results for uploaded allele Reviewing Results AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 71 Analyzing Data Haplotype Database The first column in Table 5 9 indicates the sample name The second column indicates the number of haplotype matches for each specified haplotype The succeeding columns indicate the allele values for each of the 17 AmpFlSTR Yfiler loci for the samples Clicking on the sample name in the ID column results in population of the custom entry fields in the Select Alleles tab and provides frequency infor
145. on The number of unique haplotypes is usually less than the number of different haplotypes in any given population Table 6 3 Discriminatory capacity and number of unique haplotypes for three U S populations a The minimal haplotype includes the markers DYS19 DYS385 a b DYS389 I II DYS390 DYS391 DYS392 DYS393 b The U S haplotype includes the minimal haplotype loci plus DYS438 and DYS439 Y STR marker combination African American N 333 U S Caucasian N 254 U S Hispanics N 175 DC UH DC UH DC UH Minimal haplotype a 84 6 249 74 8 162 85 1 136 U S haplotype b 91 3 286 83 8 196 90 3 146 U S haplotype DYS437 91 9 286 85 8 202 91 4 148 Yfiler haplotype 99 1 327 98 8 248 98 3 169 DRAFT August 7 2006 8 20 am ExperimentsResults fm Mutation Rate AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 41 Mutation Rate Estimating Germline Mutations Estimation of spontaneous or induced germline mutation at genetic loci may be achieved through comparison of the genotypes of offspring to those of their parents From such comparisons the number of observed mutations are counted directly In a previous study the haplotypes for 8 loci amplified by the AmpFlSTR Yfiler PCR Amplification Kit were determined for a total of 4999 parent son Kayser and Sajantila 2001 Fourteen mutations were identified and an overall average mut
146. ontains information about normalized fluorescence intensities that represent a mathematical description of multicomponent overlap that is observed between the five dyes Because matrix file values vary between different instruments and between different virtual filter sets and run conditions on a single instrument you must create a matrix file for each instrument and for a particular set of run conditions You can apply the appropriate matrix file to data on subsequent runs on the same instrument as long as the electrophoresis conditions are constant from run to run 4 Copy GS STR POP4 1 mL G5v2 md5 into the Modules folder 5 Close all files then eject the CD To install the G5v2 run module continued DRAFT May 25 2005 9 53 am 310_part2 fm Creating a Matrix File for the 310 Genetic Analyzer AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 35 310 Analyzer Setup Creating a Matrix File DC v3 0 To create a matrix file for the 310 instrument Data Collection Software v3 0 1 Prepare the matrix standards for Dye Set G5 a Thaw and thoroughly mix the contents of the five color matrix tubes then spin briefly in a microcentrifuge b For each matrix standard combine 1 L of standard 12 L Hi Di Formamide in labeled 0 5 mL Genetic Analyzer Sample tubes CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth d
147. op panel shows a 1 ng amplification of the male control DNA 007 panel 2 chimpanzee 1 ng panel 3 cat 10 ng panel 4 dog 10 ng panel 5 microbial DNA pool 5 ng each of Candida albicans Neisseria gonorrhoeae E coli 0157 H7 Bacillus subtilis and Lactobacillus rhamnosus and the negative control All samples were analyzed on an ABI PRISM 3100 Genetic Analyzer The extracted DNA samples were amplified with the AmpFlSTR Yfiler kit and analyzed using the ABI PRISM 3100 Genetic Analyzer Primates gorilla chimpanzee orangutan and macaque 1 0 ng each Non primates mouse dog pig cat horse chicken and cow 10 ng each Microorganisms Candida albicans Staphylococcus aureus Escherichia coli Neisseria gonorrhoeae Bacillus subtilis and Lactobacillus rhamnosus The chimpanzee and gorilla DNA samples produced partial profiles within the 100 330 base pair region The remaining species tested did not yield reproducible detectable products DRAFT August 7 2006 8 20 am ExperimentsResults fm Sensitivity AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 29 Sensitivity SWGDAM Guideline 2 3 When appropriate the range of DNA quantities able to produce reliable typing results should be determined SWGDAM July 2003 Importance of Quantitation The amount of input DNA added to the AmpFlSTR Yfiler PCR Amplification Kit should be between 0 5 and 1 0 ng The DNA sample should be quantitated prior to amplif
148. ophoresis continued Column Value Sample Namea a Required fields for both manual and autoanalysis Enter a name for the sample Comment Enter any additional comments or notations for the sample Priority The value 100 is automatically displayed Change the priority value if desired Sample Typeb b Additional required fields for autoanalysis From the drop down list select a sample type that corresponds to the sample in that well Size Standardb Select GS500LIZ Panelb Select the AmpFlSTR Yfiler panel from the drop down list Analysis Methodb Select the appropriate analysis method from the drop down list Refer to Creating HID Analysis Methods on page 5 12 SNP Set Leave blank User Defined columns 1 to 3 Enter any additional text as necessary Results Group 1a Select the Results Group that you created for the AmpFlSTR Yfiler kit see Creating a Results Group on page 4 17 Instrument Protocola Select the Instrument Protocol that you created for the AmpFlSTR Yfiler kit see Creating an Instrument Protocol on page 4 17 DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 24 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup For additional information about setting up Data Collection software for electrophoresis refer to the ABI PRISM 3100 3100 Avant Genetic Analyzers User Guide PN 4347102
149. p to step 6 custom location complete steps a to c DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 20 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup 6 Select the Naming tab Use the Naming table to customize the sample file and run folder names For more information about sample naming conventions refer to Chapter 5 of the ABI PRISM 3100 3100 Avant Genetic Analyzers User Guide PN 4347102 Typically plates are grouped by plate name by selecting Plate Name under Run Folder Name Format If you select Plate Name the software stores all sample files in one folder based on the plate name The software uses the value for Name in the New Plate Dialog box when creating run folders IMPORTANT Sample name run folder name and path name combined cannot exceed 250 characters IMPORTANT You must select at least one Format element for the Sample File and the Run Folder Name Formats in order to proceed within the Results Group Note The run folder is stored in the following path Applied Biosystems UDC Data Collection Data To create a results group continued If you select Plate Name as a run folder naming parameter the software uses the value of the Name field of the New Plate Dialog when creating run folders DRAFT May 25 2005 9 53 am 3100_part2 fm Setting Up the Electrophoresis Run AmpFlSTR Yfiler PCR Amplif
150. r s Manual Data Analysis Overview Analyzing Data Genotyper Software You can manually filter plus stutter peaks as explained in the following procedure To filter plus stutter peaks 1 In the Step Window for the Kazam macro scroll down to the line that reads Select category DYS392 2 Five rows below select the line that reads Remove labels from peaks preceded by an 1166 higher labeled peak within 2 25 to 3 75 bp 3 Select Macro gt Edit Step to open the Filter Labels window In the Filter Labels window there are four options check boxes for filtering In this example the filtering option for DYS392 is denoted in the third and last check box This third option is used to filter plus stutter peaks seen at DYS392 This filtering option includes another check box that reads higher by at least 1166 Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 51 Analyzing Data Genotyper Software 3 continued For each labeled peak e g peak C in the locus size range this filtering option examines the preceding i e smaller in bp size labeled peak peak B The label will be removed from peak C if peak B meets both of the specified criteria Peak B is higher by at least 1166 Peak B is within 2 25 to 3 75 bp The percentage value in this filtering option is calculated as follows peak B peak C peak
151. range is the size standard Sample Info Copy the information from the Sample Name column TIP Complete the Sample Name column then copy and paste the information into the Sample Info column DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 44 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup 4 Click File gt Save As to save the sample sheet in the Sample Sheets folder 5 Select File gt New then click GeneScan Injection List 6 Select the appropriate sample sheet from the Sample Sheet popup menu at the top left of the Injection List window 7 Save the injection list By default the injection list is saved in the Run folder To create a sample sheet and injection list continued DRAFT May 25 2005 9 53 am 310_part2 fm Preparing Samples for Electrophoresis AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 45 310 Analyzer Setup Preparing Samples for Electrophoresis Required Materials IMPORTANT Refer to Kit Contents and Storage on page 4 31 for a list of materials Preparing the Samples To prepare the samples for electrophoresis 1 Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPOR
152. rong buffer e g wrong EDTA concentration Re dilute DNA using TE Buffer with 0 1 mM EDTA More than one allele present at a locus except for DYS385 a b Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Too much DNA in reaction Use recommended amount of template DNA 0 5 1 0 ng Mixed sample See Stutter Products on page 6 18 Amplification of stutter product n 4 bp position Incomplete 3 A base addition n 1 bp position See Addition of 3 A Nucleotide on page 6 21 Be sure to include the final extension step of 60 C for 80 min in the PCR Signal exceeds dynamic range of instrument off scale data Quantitate DNA and re amplify sample adding 0 5 1 0 ng of DNA Poor spectral separation bad matrix Follow the steps for creating a matrix file Confirm that Filter Set G5 modules are installed and used for analysis Table A 1 Troubleshooting causes and recommended actions continued Observation Possible Causes Recommended Actions DRAFT May 25 2005 9 53 am 7x9_Appendix fm Troubleshooting AmpFlSTR Yfiler PCR Amplification Kit User s Manual A 5 Some but not all loci visible on electropherogram Test sample DNA is degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded re amplify with an increased amount of DNA Test sample contains PCR inhibitor e g
153. rophoresis 1 In the Tree pane of the Data Collection software click GA Instruments gt ga 3100 or ga 3100 Avant gt Plate Manager DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 22 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup 2 Complete the New Plate dialog box a Enter a name for the plate b Optional Enter a description for the plate record c In the Application drop down list select GeneMapper Generic for manual analysis or GeneMapper lt Instrument Name gt for autoanalysis d In the Plate Type drop down list select 96 Well e Enter a name for the owner f Enter a name for the operator The following figure shows an example of a completed New Plate dialog box g Click OK The GeneMapper Software Plate Editor opens To set up data collection software for electrophoresis continued DRAFT May 25 2005 9 53 am 3100_part2 fm Setting Up the Electrophoresis Run AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 23 3100 3100 Avant Electrophoresis Setup 3 Complete the plate record 4 If you want to perform more than one run a Select Edit gt Add Sample Run Additional Results Group Instrument Protocol and Analysis Method columns are added to the right end of the plate record b Complete the columns for the additional runs To set up data collection software for electr
154. rophoresis Analyze data GeneMapper ID Software v3 2 GeneScan Software Automated Data Review and Summary Baseline Multicomponenting Manually Review and QC Manually Edit and Confirm Sizing Allele Calling Data Summary Genotyper Software 3100 3100 Avant 310 Data Collection Software stores unanalyzed data in sample files fsa Data Collection Software stores unanalyzed data in sample files fsa Compare results against haplotype database Multicomponenting Baselining Sizing Allele Calling Section 5 2 Using GeneMapper ID Software v3 2 to Analyze AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 5 Analyzing Data GeneMapper ID Software Section 5 2 Using GeneMapper ID Software v3 2 to Analyze AmpFlSTR Yfiler Kit Data This section covers Overview 5 6 Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data 5 7 Analyzing Sample Files With GeneMapper ID Software 5 21 Examining and Editing GeneMapper ID Software Results 5 23 Chapter 5 Analyzing Data 5 6 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software Overview What Does GeneMapper ID Software Do GeneMapper ID Software is
155. rs We strongly recommend that the allele sizes obtained be compared to the sizes obtained for known alleles in the AmpFlSTR Yfiler Kit Allelic Ladder from the same run and then converted to genotypes as described in the Automated Genotyping section Refer to Table 6 1 DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 10 AmpFlSTR Yfiler PCR Amplification Kit User s Manual for the results of injections of the AmpFlSTR Yfiler Allelic Ladder For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 Table 6 1 Precision results of nine injections of the AmpFlSTR Yfiler Allelic Ladder ABI PRISM 310 Genetic Analyzer Allele Mean Standard Deviation DYS456 13 104 51 0 05 14 108 31 0 05 15 112 16 0 04 16 116 04 0 04 17 119 90 0 05 18 123 82 0 05 DYS389I 10 142 87 0 04 11 147 28 0 04 12 151 80 0 06 13 156 43 0 07 14 160 66 0 05 15 164 81 0 07 DYS390 18 192 26 0 05 19 195 99 0 04 20 199 93 0 05 21 203 85 0 06 DRAFT August 7 2006 8 20 am ExperimentsResults fm Accuracy Precision and Reproducibility AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 11 22 207 83 0 05 23 211 90 0 04 24 215 90 0 05 25 219 88 0 06 26 223 84 0 06 27 227 80 0 07 DYS389II 24 253 05 0 05 25 257 17 0 06 26 261 19 0 07 27 265 38 0 08
156. rt Chapter 2 Extracting and Quantifying DNA 2 6 AmpFlSTR Yfiler PCR Amplification Kit User s Manual DRAFT May 25 2005 9 53 am PCRAmpTitle fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual Chapter 3 PCR Amplification I DRAFT May 25 2005 9 53 am PCRAmpTitle fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3 1 3 PCR Amplification 3 This chapter covers PCR Work Areas 3 2 Required User Supplied Materials and Reagents 3 4 Preparing the Reactions 3 6 Performing PCR 3 8 Amplification Using Bloodstained FTA Cards 3 9 Chapter 3 PCR Amplification 3 2 AmpFlSTR Yfiler PCR Amplification Kit User s Manual PCR Work Areas Work Area Setup and Lab Design Many resources are available for the appropriate design of a PCR laboratory If you are using the Yfiler kit for forensic DNA testing refer to Forensic Laboratories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 http www ojp usdoj gov nij scidocs htm If you are using the Yfiler kit for parentage DNA testing refer to the Guidance for Standards for Parentage Testing Laboratories American Association
157. s Manual 5 41 Analyzing Data Genotyper Software These functions are described in greater detail in the following sections The Calculate locus Offsets macros use offset categories also described below to perform these functions Categories In the Genotyper software each allele is defined by a category Each category contains information about the allele size size range and dye color To view the list of categories in the AmpFlSTR Yfiler Template select View gt Show Categories Categories for each locus are listed under the locus name Note that the software calls a locus a group In the Categories window each locus has two sets of categories Allele categories Designated by lt locus name gt for example DYS392 The Genotyper software uses the categories in this group for allele assignment Offset categories Designated by lt locus name os gt for example DYS392 os The Calculate locus Offsets macros use the categories in this group to find the allele peaks in the allelic ladder and to determine the correct offset values for each allele category Identifying the First Allele Peak in Each Allelic Ladder The macros use values specified in the first offset category each allelic ladder has its own group of offset categories to identify the first leftmost peak in each allelic ladder For example the first offset category for the DYS392 allele 7 os instructs the Genotyper software to find all
158. s believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity To examine the effects of hematin on the amplification results obtained by the AmpFlSTR Yfiler kit DNA samples were amplified using the AmpFlSTR Yfiler kit reagents including the BSA containing PCR reaction mix in the presence of increasing concentrations of purified hematin The concentrations of hematin used were 0 M 10 M 12 M 16 M 20 M and 24 M No preferential amplification was observed in the presence of increasing amounts of hematin 0 min 1 min 2 min 4 min 8 min 12 min DRAFT August 7 2006 8 20 am ExperimentsResults fm Stability AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 33 Figure 6 14 DNA amplified with the AmpFlSTR Yfiler kit in the presence of varying concentrations of hematin 0 M 10 M 12 M 16 M 20 M and 24 M analyzed on the ABI PRISM 3100 Genetic Analyzer 0 M 10 M 12 M 16 M 20 M 24 M DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 34 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Mixture Studies SWGDAM Guideline 2 8 The ability to obtain reliable results from mixed source samples should be determined SWGDAM July 2003 Evidence samples may contain DNA from more than one individual The possibility of multiple contri
159. sis parameters 3 Defining a size standard 4 Performing data analysis Creating a Project A project is a file containing references to a set of sample files that you want to analyze and display together You can create a new project and add any combination of sample files allowing you to analyze and display samples from different runs Adding a sample file to the project sets up a link between the project and the sample file The file itself is not imported into the project If you enabled autoanalysis the Data Collection Software automatically launches the GeneScan application creates a project and adds sample files to the project If you did not enable autoanalysis you must create the project manually and add the sample files to the project as explained in the following procedure To create a new project 1 Select File gt New The Create New dialog box opens 2 Click the Project icon An untitled Analysis Control window opens 3 Add sample files to the project by Selecting Project gt Add Sample Files to add one or more sample files from the hard drive Selecting Project gt Add file name to add open sample files Analyzing Sample Files Using GeneScan Software AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 27 Analyzing Data GeneScan Software Note The GeneScan Analysis Software version 3 7 CD ROM contains two applications Mac to Win and Win to Mac which you can use
160. so applied to two other categories in the DYS392 group OL Allele and allele 6 The OL Allele category is specified to span the range of known DYS392 alleles to catch off ladder alleles that do not size within one of the allele categories Allele 6 in this case is a virtual allele category meaning that this allele is not present in the allelic ladder The virtual category exists to assign an allele designation to allele 6 which is a known allele not included in the allelic ladder Because allele 6 is specified to have the same offset value as allele 7 the allele category sizes for these two alleles differ by exactly 3 bp the difference in their reference sizes Specifying a size for allele 6 that is 3 bp shorter than allele 7 is generally expected to be a reasonable estimate since alleles 6 and 7 differ by a single repeat unit 3 bp Another example of virtual allele categories can be seen in the DYS385 categories The DYS385 group contains virtual allele categories such as 14 2 and 17 2 The offset value for allele 14 2 is the same as for allele 14 In this case since reference sizes for these two alleles differ by 2 bp the category size used for allele 14 2 will be 2 bp longer than for allele 14 Likewise the offset for allele 17 2 is the same as for allele 17 so the allele category size for allele 17 2 will be 2 bp longer than for allele 17 Kazam 20 Filter Macro The Kazam 20 Filter macro applies a
161. ssion Wavelength nm Dyes 6 FAM PET NED VIC LIZ 0 20 40 60 80 100 500 550 600 650 700 Materials and Equipment AmpFlSTR Yfiler PCR Amplification Kit User s Manual 1 7 Materials and Equipment Kit Contents and Storage Each Yfiler kit contains materials sufficient to perform 100 reactions at a 25 L reaction volume IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpFlSTR Yfiler Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum Table 1 2 Yfiler kit contents Reagent Contents Quantity Storage AmpFlSTR Yfiler Primer Set Forward and reverse primers to amplify human male DNA target 1 tube 0 55 mL 2 to 8 C AmpFlSTR Yfiler PCR Reaction Mix MgCl2 dNTPs and bovine serum albumin in buffer with 0 05 sodium azide 1 tube 1 1 mL tube 2 to 8 C AmpFlSTR Yfiler Allelic Ladder Allelic ladder containing amplified alleles refer to Loci Amplified by the Kit on page 1 2 for a list of alleles included in the ladder 1 tube 50 L 2 to 8 C AmpFlSTR Control DNA 007 0 10 ng L human male genomic DNA in 0 05 sodium azide and buffer refer to Loci Amplified by the Kit on page 1 2 for profile 1 tube 0 3 mL 2 to 8 C AmpliTaq Gold DNA Polymerase DNA polymerase 5 U L 2 tubes
162. stutter ratio filters specific to the Yfiler panel defined by Applied Biosystems Note For more information about allele filters refer to Chapter 3 of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 and User Bulletin New Features and Installation Procedures for GeneMapper ID Software Version 3 2 PN 4352543 Setting Up GeneMapper ID Software v3 2 for Analyzing AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 19 Analyzing Data GeneMapper ID Software Peak Detector The software uses the peak detection parameters to specify the minimum peak height to limit the number of peaks detected Although GeneMapper ID software displays peaks that fall below the specified height in electropherograms the software does not label or determine the genotype of these peaks The analysis range is set by the user based on location of the primer peak and size standard peaks Note For more information on peak detection algorithms refer to Appendix A of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 and User Bulletin New Features and Installation Procedures for GeneMapper ID Software Version 3 2 PN 4352543 Table 5 2 HID_Advanced analysis method settings continued Tab Settings 400 Chapter 5 Analyzing Data 5 20 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyz
163. t Consumable Reservoir Septa 4315932 Array fill syringe 250 L glass syringe 4304470 Polymer reserve syringe 5 0 mL glass syringe 628 3731 For a complete list of parts and accessories for the 3100 3100 Avant instrument refer to Appendix B of the ABI PRISM 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide PN 4335393 310 Analyzer materials Table 1 3 Equipment Equipment Source Chapter 1 Overview 1 10 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Capillaries 47 cm 50 m i d internally uncoated green 402839 0 5 mL Sample Tray 5572 96 Well Tray Adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 10 Genetic Analyzer Buffer with EDTA 402824 Genetic Analyzer Retainer Clips 96 Tube Tray Septa Clips 402866 Genetic Analysis Sample Tubes 0 5 mL 401957 Genetic Analysis Septa for 0 5 mL Sample Tubes 401956 Matrix Standard Set DS 33 6FAM VIC NED PET and LIZ dyes for 310 377 systems 4318159 MicroAmp 8 strip Reaction Tubes N801 0580 MicroAmp 96 Well Support Base holds 0 2 mL reaction tubes N801 0531 MicroAmp 96 Well Full Plate Cover N801 0550 MicroAmp 96 Well Tray Retainer Sets 403081 POP 4 Performance Optimized Polymer 402838 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the ABI PRISM 310 Genetic Analyzer User Guide
164. t Window Chapter 5 Analyzing Data 5 48 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software The Kazam macro which removes labels from specific peaks might have removed labels from peaks that should be labeled If so edit the macro to change the filter percentage for the locus Table 5 5 on page 5 39 lists the filter percentages for Yfiler kit loci The peak filtering included in the Kazam macro is intended only as a tool and guideline You should base final conclusions on careful examination of the STR profiles You can manually filter stutter peaks as explained in the following procedure To filter stutter peaks 1 In the Step Window for the Kazam macro scroll down to the line that reads Select category DYS390 2 Five rows below select the line that reads Remove labels from peaks followed by an 861 higher labeled peak within 3 25 to 4 75 bp 3 Select Macro gt Edit Step to open the Filter Labels window In the Filter Labels window there are four options check boxes for filtering In this example the filtering option for DYS390 is denoted in the last check box This filtering option includes another check box that reads higher by at least 861 Using the AmpFlSTR Yfiler Kit Template for Automatic Genotyping AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 49 Analyzing Data Genotyper Software 3 contin
165. ta Collection Software v2 0 click GA Instruments gt ga3100 or ga3100 Avant gt Protocol Manager 2 In the Instrument Protocol pane click New to open the Protocol Editor dialog box 3 Complete the Protocol Editor dialog box a Enter a name for the protocol b Optional Enter a description for the protocol c In the Type drop down list select REGULAR d In the Run Module drop down list select HIDFragmentAnalysis36_POP4_1 e In the Dye Set drop down list select G5 4 Click OK To create a results group 1 In the Tree pane of Data Collection Software v2 0 click GA Instruments gt Results Group 2 Click New The Results Group Editor displays 3 Complete the General tab a Type a Results Group Name The name can be used in naming and sorting sample files It must be unique b Optional Type a Results Group Owner c Optional Type a Results Group Comment DRAFT May 25 2005 9 53 am 3100_part2 fm Chapter 4 Performing Electrophoresis 4 18 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 3100 3100 Avant Electrophoresis Setup 4 Select the Analysis tab then a Select an Analysis Type Note Step b below applies only to GeneMapper lt Instrument Name gt b Select an Analysis Action c Type the GeneMapper ID software Login ID d Type the GeneMapper ID software login password The login ID and password are created in the GeneMapper ID software Options Users
166. tab To create a results group continued If you select Then lt None gt Only unanalyzed sample files are generated GeneMapper Generic Autoanalysis is not enabled and only fsa files are generated GeneMapper lt Instrument Name gt Autoanalysis of completed runs is enabled If you select Then Use with Setting from Automated Processing tab page 4 21 Do Autoanalysis Samples are analyzed after each run of 16 or 4 samples When every run completes Do Autoanalysis and Results Entry Group Complete Samples are analyzed after all samples using the same results group have been run Only when the result group is complete DRAFT May 25 2005 9 53 am 3100_part2 fm Setting Up the Electrophoresis Run AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 19 3100 3100 Avant Electrophoresis Setup 5 Select the Destination tab then use the default destination or define a new location for data storage a Click Use Custom Location then click Browse to navigate to a different location for saving b Click Test to test the Location path name connection If it passes a message box displays Path Name test successful If it fails a message box displays Could not make the connection Please check that the Path Name is correct click and try to establish a connection c Click OK To create a results group continued To use a Then default location ski
167. the capillary is placed carefully in the laser detection window Once a satisfactory matrix file has been made this matrix file can be applied to subsequent runs It is not necessary to run matrix standard samples for each new capillary To verify the accuracy of the matrix file 1 Apply the new matrix file to the Matrix Standard Sample Files as follows a In the Analysis Control window highlight the Sample File column by clicking in the Sample File title row b Select Sample gt Install New Matrix c Select the new matrix file located in the ABI folder in the System folder and click Open 2 Analyze the matrix standard samples as follows a Select Settings gt Analysis Parameters and verify that the settings are correct b In the Analysis Control window select all five colors in each sample row for all of the matrix standard samples c Click the Analyze button 3 In the Results Control window examine the results for all five colors for each of the matrix standard samples For example the 6 FAM dye matrix standard results should have peaks for blue Evaluate the baseline A pattern of pronounced peaks or dips in any of the other four colors indicates that the color separation may not be optimal DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 38 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup Setting Up the Electrophoresis Run Co
168. these tasks refer to ABI PRISM 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin PN 4350218 For information about setting up the 3100 3100 Avant instrument for use with other Data Collection Software versions refer to the appropriate user manuals Related Documentation on page xiii provides a list of related documentation To set up the 3100 3100 Avant instrument 1 Power on the computer The OrbixWeb Daemon software automatically launches 2 Power on the 3100 3100 Avant instrument wait for solid green light 3 Select Start gt Programs gt Applied Biosystems gt Data Collection gt Run Data Collection 3100 v2 0 or Run Data Collection 3100 Avant v2 0 The Service Console opens when all the applications are running the Data Collection Viewer window opens 4 Fill the reservoirs and place them on the autosampler 5 Install or replace the capillary array if necessary 6 Install the polymer blocks if necessary 7 Add or change the polymer if necessary DRAFT May 25 2005 9 53 am 3100_part2 fm Performing a Spectral Calibration AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 9 3100 3100 Avant Electrophoresis Setup Performing a Spectral Calibration A spectral calibration creates a matrix or spectral to correct for the overlapping of fluorescence emission spectra of the dyes Data collection software applies this spectral to raw data durin
169. to bottom 500 pg of male DNA 500 pg male DNA with 500 ng female DNA 250 pg male DNA with 500 ng female DNA 500 ng female DNA Male Male Mixture Studies Forensic samples may contain body fluids or tissues originating from more than one male Mixtures of two male DNA samples were examined at various ratios 1 1 to 1 10 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a Silver 96 Well GeneAmp PCR System 9700 and were electrophoresed and detected using an ABI PRISM 3100 Genetic Analyzer 0 5 ng Male 1 1000 1 2000 500 ng Female DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 36 AmpFlSTR Yfiler PCR Amplification Kit User s Manual The haplotypes of the samples in Figure 6 16 are the following The results of the mixed DNA samples are shown in Figure 6 16 where sample A and sample B were mixed according to the ratios provided For these 1 ng total DNA mixture studies the limit of detection is when the minor component is present at approximately one tenth of the concentration of the major component and a threshold of 50 RFU The limit of detection for the minor component is influenced by the combination of genotypes in the mixture Allele Sample A Sample B DYS19 14 15 DYS385a 11 13 DYS385b 14 15 DYS389I 13 12 DYS389II 31 28 DYS390 24 23 DYS391 10 10 DYS392 13 11 DYS393 13 14
170. ts must be greater than the GeneScan Software PAT in order to be recognized labeled by Genotyper software Understanding the AmpFlSTR Yfiler Kit Template About the Yfiler Template Table 5 4 lists the macros contained in the Yfiler Kit Template and summarizes their functions Detailed descriptions of each macro are provided in subsequent sections Chapter 5 Analyzing Data 5 38 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data Genotyper Software Table 5 4 Macros contained in the Yfiler Template Macro Name Function Reference Check GS500 Automatically labels the size standard peaks and examines the 250 bp peak Kazam Automatically determines genotypes based on the allelic ladder applying different stutter filters for each locus Kazam Macro on page 5 39 Kazam 20 Filter Automatically determines genotypes based on the allelic ladder applying a 20 stutter filter to all loci Kazam 20 Filter Macro on page 5 43 Make Allele Table Stores genotyping information in a table Can be used for data generated by the ABI PRISM 310 3100 and 3100 Avant instruments For more information about the Make Table macros refer to Chapter 10 of the AmpFlSTR Profiler Plus PCR Amplification Kit User s Manual PN 4303501 310 Make Table Stores genotyping information in a table Can be used for data generated by the 310 instrument Understan
171. ts using low amounts of input DNA DRAFT August 7 2006 8 20 am ExperimentsResults fm Chapter 6 Experiments and Results 6 30 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Figure 6 12 Effect of amplifying 1 ng 500 pg 250 pg 125 pg and 62 pg of male control DNA 007 Note that the y axis scale is magnified for the lower amounts of DNA analyzed using the ABI PRISM 3100 Genetic Analyzer 1 ng 500 pg 250 pg 125 pg 62 pg DRAFT August 7 2006 8 20 am ExperimentsResults fm Stability AmpFlSTR Yfiler PCR Amplification Kit User s Manual 6 31 Stability SWGDAM Guideline 2 4 The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults has been extensively documented In most instances assessment of the effects of these factors on new forensic DNA procedures is not required However if substrates and or environmental and or chemical insults could potentially affect the analytical process then the process should be evaluated using known samples to determine the effects of such factors SWGDAM July 2003 Lack of Amplification of Some Loci As with any multi locus system the possibility exists that not every locus will amplify This situation is most often observed when the DNA sample contains PCR inhibitors or when the DNA sample has been severely degraded Valuable information may be obtained from
172. ued For each labeled peak e g peak A in the locus size range this filtering option examines the very next i e greater in bp size labeled peak peak B The label will be removed from peak A if peak B meets both of the specified criteria Peak B is higher by at least 861 Peak B is within 3 25 to 4 75 bp The percentage value in this filtering option is calculated as follows peak B peak A peak A x 100 percentage value For example if peak A 291 RFU and peak B 2797 RFU then the percentage value is calculated as follows 2797 291 291 x 100 861 In this example the label will be removed from peak A provided that the filter option specifies a threshold of 861 and that peak B is within 3 25 to 4 75 bp of peak A Conventionally percent stutter is calculated peak A peak B x 100 percent stutter The percentage value that is used in the Genotyper software filtering option F can be derived from the conventional percent stutter expression S F 10 000 S 100 For example if the desired stutter percent threshold for DYS390 is 10 4 then the percentage value that should be used in the Genotyper software filtering option is F 10 000 10 40 100 861 4 To use a filter value different than 861 for DYS390 enter another value then click Replace To filter stutter peaks continued Chapter 5 Analyzing Data 5 50 AmpFlSTR Yfiler PCR Amplification Kit Use
173. ure 1 5 L allelic ladder or sample Heat 95 C for 3 then place on ice for 3 Perform electrophoresis ABI PRISM 310 Analyzer ABI PRISM 3100 3100 Avant Extract and quantify data PCR amplify data Analyze data DRAFT May 25 2005 9 53 am 310_part2 fm Overview AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 29 310 Analyzer Setup 310 Analyzer Quick Reference Tables The following tables provide information that users familiar with the 310 instrument can use to get started analyzing samples from Yfiler Kit experiments Condition Setting Dye Set DS 33 Matrix Standards PN 4312131 Filter Set G5v2 Size Standard GeneScan 500 LIZ Size Standard PN 4322682 Run Module GS STR POP4 1mL G5v2 Analysis Method If using GeneMapper ID software create an analysis method as explained in Creating HID Analysis Methods on page 5 12 If using GeneScan software AnalyzeGSsample bat Polymer 3100 Performance Optimized Polymer 4 POP4 7 mL PN 4316355 Capillary 310 Capillaries 47cm x 50 m i d internally uncoated PN 402839 Running Buffer 10 Genetic Analyzer Buffer with EDTA 25 mL PN 402824 DRAFT May 25 2005 9 53 am 310_part2 fm Chapter 4 Performing Electrophoresis 4 30 AmpFlSTR Yfiler PCR Amplification Kit User s Manual 310 Analyzer Setup Before You Begin Before using the instrument use the following checklist to determine
174. urified and amplified without transferring the evidence Our studies have indicated that a 1 2 mm bloodstained punch contains approximately 5 20 ng DNA Accordingly an appropriate cycle number for this high quantity of DNA is 27 cycles It is recommended that each laboratory determine the cycle number based upon individual validation studies In the example shown in Figure 3 1 a 1 2 mm punch of a bloodstained FTA card was purified using three washes with FTA Purification Reagent and two washes with 1X TE buffer After drying at room temperature overnight the punch was then amplified directly in the MicroAmp tube for 27 cycles 5 Store the amplified DNA IMPORTANT Protect the amplified products from light To run PCR continued If you are storing the DNA Then place at lt 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C Chapter 3 PCR Amplification 3 10 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Figure 3 1 AmpFlSTR Yfiler kit results from a 1 2 mm FTA bloodstain punch 27 cycle amplification analyzed on the ABI PRISM 3100 Genetic Analyzer 08 2006 Part Number 4358101 Rev C DRAFT August 7 2006 8 15 am ElectrophoresisTitle fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual Chapter 4 Electrophoresis I DRAFT May 25 2005 9 53 am ElectrophoresisTitle fm AmpFlSTR Yfiler PCR Amplification Kit User s Manual 4 1 4 Performing Electrophoresis 4 This chapter covers
175. utorials 4309961 Quantifiler Human DNA Quantification Kits User s Manual 4344790 AmpFlSTR Profiler Plus PCR Amplification Kit User s Manual 4303501 DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm How to Obtain Support AmpFlSTR Yfiler PCR Amplification Kit User s Manual xv How to Obtain Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm Preface xvi AmpFlSTR Yfiler PCR Amplification Kit User s Manual AmpFlSTR Yfiler PCR Amplification Kit User s Manual 1 1 1 Overview 1 This chapter covers Product Overview 1 2 Procedural Overview 1 4 Instrument and Software Overview
176. ve eyewear clothing and gloves Read and follow the guidelines in these publications U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 http bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Additional information about biohazard guidelines is available at http www cdc gov DRAFT May 25 2005 9 53 am 7x9_Preface_Protocol fm How to Obtain More Information AmpFlSTR Yfiler PCR Amplification Kit User s Manual xiii How to Obtain More Information Related Documentation The following documents are available on the HID Validation and Verification Atlas CD PN 402800 and on the Applied Biosystems Web site http docs appliedbiosystems com search taf ABI PRISM 3100 3100 Avant Data Collection v2 0 User Guide 4347102 ABI PRISM 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin 4350218 ABI PRISM 3100 Genetic Analyzer User Manual DC v1 1 4315834 ABI PRISM 3100 Avant Genetic Analyzer User Guide DC v1 0 4333549 ABI PRISM 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin 4332345 ABI PRISM 310 Genetic Analyzer User s Manual Macintosh 903565 ABI PRISM 310 Genetic A
177. wle 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 Prinz M Ishii A Coleman A Baum H J Shaler R C 2001 Validation and casework application of a Y chromosome specific STR multiplex Forensic Sci Int 120 177 88 Redd A J Agellon A B Kearney V A Contreras V A Karafet T Park H de Knijff P Butler J M Hammer M F 2002 Forensic value of 14 novel STRs on the human Y chromosome Forensic Sci Int 130 97 111 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Science Communications July 2004 Volume 6 3 Available at http www fbi gov hq lab fsc current standards 2004_03_standards0 2 htm Schoske R Vallone P M Kline M C Redman J W Butler J M 2004 High throughput Y STR typing of U S populations with 27 regions of the Y chromosome using two multiplex PCR assays Forensic Sci Int 139 107 21 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
178. zing AmpFlSTR Yfiler Kit Data AmpFlSTR Yfiler PCR Amplification Kit User s Manual 5 17 Analyzing Data GeneMapper ID Software To create an analysis method for the HID Advanced Mode 1 Select Tools gt GeneMapper Manager to open the GeneMapper Manager 2 Create an analysis method for HID_Advanced a Select the Analysis Methods tab and click New to open the New Analysis Method dialog box b Select HID and click OK to open the Analysis Method Editor with the General tab selected c In the General tab enter the name for the analysis method such as AmpFlSTR_Yfiler_AdvancedMode 3 Select the settings shown in Table 5 2 HID_Advanced analysis method settings IMPORTANT You must select your settings on all the tabs before you Click OK to save the analysis method and return to GeneMapper Manager Chapter 5 Analyzing Data 5 18 AmpFlSTR Yfiler PCR Amplification Kit User s Manual Data Analysis Overview Analyzing Data GeneMapper ID Software HID_Advanced Settings Table 5 2 HID_Advanced analysis method settings Tab Settings General Name AmpFlSTR_Yfiler_AdvancedMode Allele w Note GeneMapper ID Software v3 2 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameters for each type of repeat in the appropriate column Note Select Use marker specific stutter ratio if available If the box is selected the software applies the

AmpFLSTR Yfiler PCR Amplification Kit User`s Manual (PN 4358101C)

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