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AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide (Pub

1. African U S Native Allele American Caucasian 357 349 z n 191 11 3 0 14 t t t 12 29 13 32 81 39 66 32 72 13 5 32 7 31 6 38 4 71 14 0 98 1 43 0 86 0 79 15 t 0 29t t t 0251338 15 0 141 t t t 16 5 32 4 73 2 41 2 62 17 10 78 17 34 21 21 9 95 18 5 60 6 30 4 14 7 07 19 14 15 13 75 22 76 29 58 20 6 02 14 61 13 79 9 69 21 14 01 2 58 2 59 2 38 22 13 17 4 01 7 41 15 18 23 10 78 11 46 11 36 11 78 24 9 80 11 75 8 45 7 85 25 8 12 10 60 5 17 3 14 26 1 96 2 72 0 691 0 791 27 0 141 0 141 t t 28 t 1 1 0351358 lt 11 0 421 0 14 t t 11 t t t 0 26t 12 0 56t t 0 17t t 13 0 70t 0 29t 0 17t t 14 12 04 15 76 7 41 6 81 15 30 53 25 36 39 14 40 84 15 2 0 14 t t 16 28 57 22 78 26 72 32 98 17 19 47 18 19 16 03 9 95 18 6 72 16 48 8 97 8 38 19 0 84 1 00 1 03 0 791 20 t t 0 34 t AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 87 Chapter 5 Experiments and Results African U S Native Allele American Caucasian 357 349 191 D5S818 7 0 141 t 6 72 15 71 8 5 46 t 0 691 t 9 1 68 4 15 5 17 6 02 10 6 72 5 44 5 17 4 19 11 25 49 39 26 39 14 41 10 12 36 41 35 24 29 31 23 30 13 21 57 15 47 12 59 9 42 14 2 38 0 14 0 69 0 26 15 t 0 29 0 18 t 16 t
2. C 0 2 0 lt o 0 p e v e e 2 0 Inthe Bin Set field select the IdentifilerDirect GS500 Bins v1X bin set imported previously and configure the parameters as shown GeneMapper ID X Software allows you to specify 4 types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the stutter ratio apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the IdentifilerDirect GS500 Stutter v1X txt file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use AmpFtSTR9 Identifiler Direct PCR Amplification Kit User Guide 55 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis Peak Detector tab settings Analysis Method Editor Peak Detection Algorithm Advanced
3. 31 Set up the 3500 3500xL instruments for electrophoresis 31 Prepare samples for electrophoresis on the 3500 3500xL instruments 31 HW Section 3 3 3730 instrument sse nn 33 Set Up the 3730 instrument for 33 Prepare samples for electrophoresis on the 3730 instrument 33 AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 27 Chapter3 Perform Electrophoresis Allelic ladder requirements Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples Number of One din Number of samples per allelic Instrument allelic ladders injection ladder s to run equals 3100 Avant or 3130 1 per 4 injections 4 samples 15 samples 1 allelic ladder 3100 or 3130xl 1 per injection 16samples 15 samples 1 allelic ladder 3500 1 per 3 injections 8 samples 23 samples 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples 1 allelic ladder 3730 2 per injection 48 samples 46 samples 2 allelic ladders IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single and multiple capillary runs with larger size variations seen between samples injected in multiple capillary runs We recommend the above frequency of allelic lad
4. reference documents Genetic dala Operatin Collection 9 Run modules and conditions References Analyzer System Software Applied 1 0 Windows GeneScan36Avb DyeSetG5Module 3100 3100 Avant Genetic Analyzers Biosystems NT Injection condition 3 kV 5sec Protocols for Processing AmpFtSTR 3100 Avant e GS600v2 0Analvsis as PCR Amplification Kit PCR Products TOR EN User Bulletin Part no 4332345 Applied 2 0 Windows e HIDFragmentAnalysis36 POPA 1 3100 3100 Avant Genetic Analyzers Biosystems 9 2000 Injection condition 3kV 10 sec Using Data Collection Software v2 0 3100 Dye Set G5 Protocols for Processing AmpFtSTR PCR Amplification Kit PCR Products User Bulletin Part no 4350218 1 1 Windows GeneScan36vb DyeSetG5Module 3100 3100 Avant Genetic Analyzers NT Injection condition 3kV 10 sec Protocols for Processing AmpFtSTR PCR Amplification Kit PCR Products GS600v2 0Analysis anc EE User Bulletin Part no 4332345 Applied 3 0 Windows e HIDFragmentAnalysis36 POP4 1 Applied Biosystems 3130 3130xl Biosystems 9 XP Injection conditions Genetic Analyzers Using Data 3130 3130xl Collection Software v3 0 Protocols for 3130 3 kV 5 iS ser Processing AmpFISTRO PCR 3130x 3 kV 10 sec Amplification Kit PCR Products User Dye Set G5 Bulletin Part no 4363787 t We conducted validation studies for the Identifiler Direct Kit using this configuration AmpFtSTR Identifiler Direct PCR A
5. eee eee ees 61 FOr more information epe Es AS EAA UES PR ee KR N Q ek 47 Section 4 1 D Software Overview of D Software Instruments Before you start GeneMapper ID Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the Data Collection Software stores information for each sample in an fsa file Using GeneMapper ID Software v3 2 1 software you can then analyze and interpret the data from the fsa files Refer to Instrument and software overview on page 13 for a list of compatible instruments When using GeneMapper ID Software v3 2 1 to perform human identification HID analysis with AmpF STR kits be aware that HID analysis requires at least one allelic ladder sample per run folder Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis For multiple ladder samples the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 35 n Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis When the software imports multiple run folders into a project only the ladder
6. Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation 29 2 206 14 206 21 0 031 0 041 30 208 10 208 17 0 024 0 039 30 2 210 08 210 15 0 019 0 040 31 212 09 212 16 0 028 0 036 31 2 214 06 214 13 0 025 0 041 32 216 07 216 15 0 032 0 045 32 2 218 04 218 12 0 030 0 038 33 220 06 220 14 0 022 0 042 33 2 222 01 222 07 0 029 0 045 34 224 13 224 21 0 020 0 041 34 2 226 02 226 11 0 030 0 042 35 228 10 228 18 0 027 0 047 35 2 230 02 230 10 0 036 0 052 36 232 01 232 10 0 032 0 046 37 236 07 236 15 0 030 0 040 38 240 00 240 09 0 036 0 045 D2S1338 15 306 40 306 56 0 032 0 056 16 310 49 310 64 0 036 0 049 17 314 56 314 72 0 034 0 048 18 318 62 318 77 0 038 0 040 19 322 69 322 84 0 025 0 044 20 326 74 326 89 0 035 0 049 21 330 81 330 95 0 030 0 042 22 334 87 335 00 0 029 0 047 23 338 90 339 05 0 039 0 052 24 342 94 343 08 0 039 0 047 25 346 99 347 13 0 031 0 050 26 350 99 351 13 0 040 0 051 27 354 94 355 06 0 031 0 050 28 359 08 359 21 0 031 0 054 AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 69 Chapter 5 Experiments and Results Accuracy precision and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation D3S1358 12 111 35 111 49 0 034 0
7. Ranges Perform Analysis Sizing internal Full Range All Sizes validation studies to determine Smoothing and Baselining 7 settings Min Peak Half Width Smoothing None Light Polynomial Degree O Heavy Peak Window Size Baseline Window 51 pts Slope Threshold Peak Start 0 0 0 0 Size Calling Method Peak End 2nd Order Least Squares O 3rd Order Least Squares Normalization Cubic Spline Interpolation Local Southern Method Global Southern Method Use Normalization if applicable Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of Identifiler Direct Kit data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID X Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Sizecalling method This kit has been validated using the Local Southern sizing method Before using alternative sizing methods perform the appropriate internal validation studies Normalization v1 2 or higher For use with 3500 data Perform internal validation studies to determine whether to use the Normalization feature for analysis of I
8. a ale dul 107 Example electrophoresis plate layout WW kk kk kk kk kk kk kK kk KK kK KK kK KK kK kK sss 107 APPENDIX D PCR Work Areas 109 Work area setup and lab design WW kk kk kk kk kk kk kk kK kK kk KK KK KK KK kK sse 109 PGR setupworkatrea usce Ah a K la Kes EUM eae TA 109 Amplified DNA work area 2 2 kk kk kk kk kk kk kk KK kK kK kk kK kk kk kk ll 110 APPENDIX E 5 111 Chemicalsafety si gt Ax nike b a eR d l ey Ey E G 112 Specific chemical handling KK KK KK KK eens 112 Biologicalliazardisafety xalkani b n aa ly kik Lela bie g r dik Wilay eed pes 112 Bibliography 115 Documentation and Support 119 Related documentation eits ee a a E AE A 2 KERA E EEL 5L My 24 sh 119 Obtairi Support s s cie cc 120 Limited Product Warranty adasini ERR t We kal ale kh ark d n rak kk RAN RR RENATA A 120 NGO OX stern 121 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide About This Guide IMPORTANT Before using this product read and understand the information the Safety appendix in this document Revision history Revision Date Description A May 2
9. 11121314 18 1617 18 1920 567895101 8 9 101112131415 B 9 1011121314 151617 181920 2122232425262728 0351358 0138317 0165539 0281338 76 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 5 Experiments and Results Extra peaks in the electropherogram Figure 10 Untreated paper workflow Bode Buccal DNA Collector sample stutter percentages for 0195433 vWA and 018551 loci E Io o weran mn PUA i on aUe 9 101112131415 1617 18 111213141516 17 18 1920 212223 6 B 8 101112 10 1112 1314 15 16 17 18 1920 212223242826 0195433 018851 Figure 11 Untreated paper workflow Bode Buccal DNA Collector sample stutter percentages for 055818 and FGA loci blood samples blue buccal samples Percent Stutter B 8 10 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 42 43 44 45 46 47 48 49 60 5162 53 D58818 FGA AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 77 Chapter 5 Experiments and Results Extra peaks in the electropherogram Addition of 3 A nucleotide 78 Table 4 Treated paper workflow FTA9 card sample marker specific stutter filter percentages for Identifiler Direct Kit loci Locus Stuttert CSF1PO 8 48 0135317 9 39 0165539 9 42 D18S51 12 89 D19S433 11 15 D21S11 10 42 D2S1338 11 77 D3S1358 11 45 D5S818 9
10. a cs 3730 Genetic 3100 3100 Avant 3130 3130xl 3500 3500xL Genetic Analyzer Genetic Analyzer Genetic Analyzer Analyzer ET EEG GeneMapper D X Software GeneMapper D Software AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 1 Overview 1 Instrument and software overview Instrument and software overview Data collection and analysis software Instrument and software compatibility About multicomponent analysis How multicomponent analysis works This section provides information about the data collection and analysis software versions required to run the Identifiler Direct Kit on specific instruments The data collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time As the instrument measures sample fluorescence with its detection system the data collection software collects the data and stores it The data collection software stores information about each sample in a sample file fsa files for 31xx and 3730 instruments and hid files for 3500 instruments which is then analyzed by the analysis software Instrument Data collection software Analysis software 3100 3100 Avant 1 1 3100 GeneMapper ID 1 0 3100 Avant Software v3 2 1 e GeneMapper D X 20 Software v1 0 1 or later 3130 3130x 3 0 3730 3 1 3500 3500xL 3500 Series Data Collection Software v1 0
11. Create an analysis method as explained in Create an analysis method on page 40 Create a size standard as explained in Create a size standard on page 45 Define custom views of analysis tables Refer to the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 for more information Define custom views of plots Refer to the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 for more information To import the Identifiler Direct Kit panel and bin set from the Life Technologies web site into the GeneMapper ID Software v3 2 1 database 1 Download and open the file containing panels and bins a From the Support menu of www lifetechnologies com select Support Software Downloads Patches amp Updates gt GeneMapper ID Software v 3 2 Updates amp Patches and download the file Identifiler Direct Analysis Files GMID b Unzip the file AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis 2 Start the GeneMapper ID Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 3 Select Tools Panel Manager 4 Find then open the folder containing the pane
12. 6 e Goo SOTD Oc 0 g9 o o 9o ONE o soo ese o OPERE co 7 o PUNE o Percent Stutter vay 2 0 ERR SS Oooo PITA TUe So o o 6789101112 10 111213 14 15 16 17 18 1920 2122 23 24 2526 9 10 111213 14 15 16 17 18 1112 1314 15 16 17 18 1920 212223 TPOX D18S51 D19S433 Figure 7 Treated paper workflow FTA9 card sample stutter percentages for 055818 FGA loci red blood samples blue buccal samples o ROME BGs o oo 8 o Percent Stutter 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 42 43 44 45 46 47 48 49 50 51 52 53 10 1112 13 14 15 055818 AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 75 Chapter 5 Experiments and Results Extra peaks in the electropherogram Figure 8 Untreated paper workflow Bode Buccal DNA Collector sample stutter percentages for 0851179 021511 075820 and CSF1PO loci z t 2 o 8 9 10 1112 13 14 15 16 17 24 26 26 27 28 29 30 3132 33 34 36 36 6 7 B 9 10 1112 13 14 15 7 8 9 10 1112 13 14 15 0851179 021511 075820 CSF1PO Figure 9 Untreated paper workflow Bode Buccal DNA Collector sample stutter percentages for 0351358 THO1 0135317 0165539 and 0251338 loci z g o
13. Dispense 25 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells place a MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold plated silver 96 well block or a Veriti 96 well Thermal Cycler or a ProFlex PCR System as described in Perform PCR on page 26 IMPORTANT The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96 well block Use of this thermal cycling platform may adversely affect performance of the AmpF STR Identifiler Direct PCR Amplification Kit AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 2 Perform PCR Swab substrates prepare reactions Swab substrates prepare reactions Note Performance verification and optimization experiments for this protocol were conducted using 4N6FLOQSwabs OmniSwabs and Puritan swabs air dried and stored at r
14. bin sets and marker stutter will not be imported into the GeneMapper ID X Software database Create an analysis Use the following procedure to create an analysis method for the Identifiler Direct method Kit IMPORTANT Analysis methods are version specific so you must create an analysis method for each version of the software For example an analysis method created for GeneMapper ID X Software version 1 2 is not compatible with earlier versions of GeneMapper ID X Software or with GeneMapper ID Software version 3 2 1 1 Select Tools GeneMapper ID X Manager to open the GeneMapper ID X Manager GeneMapper ID X Manager Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings AmpFtSTR9 Identifiler Direct PCR Amplification Kit User Guide 53 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis 2 Select the Analysis Methods tab then click New to open the Analysis Method Editor with the General tab selected 3 Enter the settings shown in the figures on the following pages Note The Analysis Method Editor closes when you save your settings To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs 4 After you enter the settings on all tabs click Save General tab Analysis Method Editor setti
15. cell line DNA in 0 04 sodium azide and buffer t See Table 1 on page 10 for profile AmpF STR Identifiler9 Direct Allelic Ladder Contains amplified alleles See Table 1 on page 10 for a list of alleles included in the allelic ladder 1 tube 50 0 uL 1 tube 100 uL The Control DNA 9947A is included at a concentration appropriate to its intended use as an amplification control i e to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype The Control DNA 9947A is not designed to be used as a DNA quantitation control and laboratories may expect to see variation from the labelled concentration when quantitating aliquots of the Control DNA 9947A 14 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 1 Overview 1 Materials and equipment Standards for For the Identifiler Direct Kit the panel of standards needed for PCR amplification samples PCR product sizing and genotyping are e AmpE STR Identifiler Direct Control DNA 9947A A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR Identifiler Direct Allelic Ladder e GeneScan 500 LIZ Size Standard or GeneScan 600 LIZ Size Standard v2 0 Used for obtaining sizing results These standards which have been evaluated as internal size standards yield precise sizing results for Identifiler Direct Kit PCR
16. 67 Chapter 5 Experiments and Results Accuracy precision and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation 17 302 68 302 82 0 034 0 052 18 306 82 306 99 0 042 0 053 19 310 96 311 11 0 043 0 060 20 315 10 315 25 0 031 0 048 21 319 23 319 38 0 031 0 049 22 323 42 323 57 0 038 0 054 23 327 48 327 63 0 043 0 055 24 331 59 331 74 0 031 0 052 25 335 69 335 83 0 029 0 052 26 339 81 339 96 0 044 0 052 27 343 92 344 04 0 037 0 051 D19S433 9 101 38 101 46 0 032 0 039 10 105 28 105 36 0 030 0 036 11 109 20 109 28 0 027 0 042 12 113 14 113 22 0 028 0 038 12 2 115 15 115 21 0 032 0 038 13 117 11 117 17 0 030 0 045 13 2 119 11 119 17 0 028 0 038 14 121 07 121 14 0 022 0 045 14 2 123 10 123 17 0 035 0 047 15 125 09 125 13 0 031 0 048 15 2 127 12 127 16 0 026 0 045 16 129 11 129 16 0 034 0 044 16 2 131 17 131 20 0 028 0 044 17 133 17 133 22 0 033 0 044 17 2 135 24 135 27 0 022 0 043 021511 24 184 40 184 51 0 035 0 042 24 2 186 39 186 50 0 023 0 043 25 188 35 188 44 0 025 0 040 26 192 30 192 39 0 029 0 043 27 196 27 196 33 0 026 0 042 28 200 14 200 21 0 039 0 043 28 2 202 11 202 18 0 028 0 042 29 204 08 204 16 0 031 0 041 68 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 5 Experiments and Results Accuracy precision and reproducibility
17. Direct Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Identifiler Direct Control DNA 9947A are also listed in the table Table 1 AmpF STR Identifiler Direct PCR Amplification Kit loci and alleles Locus Chromosome Control designation location Alleles included in Allelic Ladder Dye label DNA 9947A 0851179 8 8 9 10 11 12 13 14 15 16 17 18 19 6 FAM 13 13 021511 21411 2 921 24 24 2 25 26 27 28 28 2 29 29 2 30 30 2 31 30 30 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 075820 7911 21 22 6 7 8 9 10 11 12 13 14 15 10 11 CSF1PO 5433 3 34 6 7 8 9 10 11 12 13 14 15 10 12 0351358 12 13 14 15 16 17 18 19 vic 14 15 THO1 11p15 5 4 5 6 7 8 9 9 3 10 11 13 3 8 9 3 0135317 13922 31 8 9 10 11 12 13 14 15 11 11 0165539 16q24 qter 5 8 9 10 11 12 13 14 15 11 12 0251338 2935 37 1 15 16 17 18 19 20 21 22 23 24 25 26 27 28 19 23 0195433 19q12 13 1 9 10 11 12 12 2 13 13 2 14 14 2 15 15 2 16 NED 14 15 16 2 17 17 2 vWA 12p12 pter 11 12 13 14 15 16 17 18 19 20 21 22 23 24 17 18 TPOX 2p23 2per 6 7 8 9 10 11 12 13 8 8 018551 1821 3 7 9 10 10 2 11 12 13 13 2 14 14 2 15 16 17 15 19 18 19 20 21 22 23 24 25 26 27 Amelogenin X p22 1 22 3 Y p11 2 X Y PET9 X D5S818 5421 31 7 8 9 10 11 12 13 14 15 16 11 11
18. Direct PCR Amplification Kit User Guide Overview Importance of validation Experiment conditions Experiments and Results B Overview ood ee RR Ren Mate eet ettet 63 Accuracy precision and reproducibility 8 64 Extra peaks in the 73 Characterization Of loci ose resse heb k etie ee kl kk k k RENE WEE Y 79 W Species Specificity ess ees en orte teme oer enses mei nie eee als 80 lI Sensit vity A Ke set eiue iat eb uM rte E tes 81 B Stability eerte RC mete eese a see a Ros 83 a Population data sce THERE ep EU 86 BH Mutationxdle z2 o wn wa aw K si n e ese emia betes Naber by 97 Probability of identity s ee nn 98 Probability of paternity exclusion 99 This chapter provides results of the developmental validation experiments we performed using the Identifiler Direct Kit for samples punched from FTA cards 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 Kimpton Watson et al 1996 Sparke
19. Figure 13 Figure 13 Representative electropherograms from a species specificity study including positive and non template controls NTC Control DNA 9947A 350 Microbial pool 80 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Sensitivity SWGDAM guideline 2 3 Blood on FTA9 cards Buccal cells on FTA or Indicating FTA cards and buccal cells on Bode DNA Collectors Chapter 5 Experiments and Results Sensitivity Figure 13 shows amplification for Control DNA 9947A 1 ng panel 1 chimpanzee 1 ng panel 2 pig 10 ng panel 3 cat 10 ng panel 4 microbial DNA pool equivalent to 105 copies of Candida albicans Enterococcus faecalis Escherichia coli Fusobacterium nucleatum Lactobacillus casei Staphylococcus aureus Streptococcus mitis Streptococcus mutans Streptococcus salivarius and Streptococcus viridans panel 5 and the non template control panel 6 The extracted DNA samples were amplified with the Identifiler Direct Kit and analyzed using the Applied Biosystems 3130x Genetic Analyzer Primates gorilla chimpanzee orangutan and macaque 1 ng each e Non primates mouse dog pig cat horse hamster rat chicken and cow 10 ng each e Microorganisms Candida albicans Enterococcus faecalis Escherichia coli Fusobacterium nucleatum Lactobacillus casei Staphylococcus aureus Streptococcus mitis Streptococcus mutans Streptococcus salivarius
20. J Legal Med 109 195 204 Straub R E Speer M C Luo Y Rojas K Overhauser J Ott J and Gilliam 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Suido H Nakamura M Mashimo P A Zambon J J and Genco R J 1986 Arylaminopeptidase activities of the oral bacteria J Dent Res 65 1335 1340 Waiyawuth W Zhang L Rittner C Schneider P M 1998 Genetic analysis of the short tandem repeat system D125391 in the German and three Asian populations Forensic Sci Int 94 25 31 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L Walsh P S 1998 SWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis J Forensic Sci 43 854 870 Wallin J M Holt C L Lazaruk K D Nguyen T H Walsh PS 2002 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci 47 52 65 Walsh P S Fildes N J Reynolds 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus Nucleic Acids Res 24 2807 2812 Weber J and Wong C 1993 Mutation of human short tandem repeats Hum Mol Genet 2 1123 1128 Weir B 1990 Genetic Data Analysis Sinauer Associates Sunderland MA Wiegand P and Kleiber M 2001 Less is more length reduction of STR amplicons using redesigned primers Int J Legal Med 114 285 287 AmpFtSTR Id
21. of PCR product or Allelic Ladder Note For blank wells add 10 uL of Hi Di Formamide 5 Sealthe reaction plate with appropriate septa then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom Heat the plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Place the sample tray on the autosampler Or Sor Start the electrophoresis run 32 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 3 3 3730 instrument Set Up the 3730 instrument for electrophoresis Section 3 3 3730 instrument Set Up the 3730 instrument for electrophoresis Reagents and parts Appendix B Ordering Information on page 103 lists the required materials not supplied with the Identifiler Direct Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Electrophoresis The following table lists data collection software and the run modules that you can use software setupand to analyze Identifiler Direct Kit PCR products For details on the procedures refer to the documents listed in the table reference documents Operatin Data P 9 collection Run module References system software Windows XP 3 01 GeneMapper 36 POP7 Applied Biosystems 3730 D
22. p A Markov chain unbiased exact test to estimate the P value of the Hardy Weinberg test with multiple alleles Hexp Expected heterozygosity H observed heterozygosity AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 5 Experiments and Results Population data African U S Native American Caucasian 357 349 E n 191 0251338 HW X 0 409878 0 537758 0 975972 0 722543 HW G2 p 0 962501 0 407932 0 973054 0 760953 HW Exact p 0 7838 0 3488 0 9794 0 5825 HExp 0 8936 0 8823 0 8529 0 8428 0 8768 0 8653 0 8379 0 801 0351358 HW X 0 947371 0 670787 0 681659 0 087223 HW G2 p 0 907905 0 654776 0 852278 0 175807 HW Exact p 0 2967 0 2814 0 4684 0 0614 HExp 0 7681 0 7986 0 7361 0 7028 0 7955 0 8166 0 7414 0 7382 055818 HW X 0 993751 0 859805 0 944725 0 073002 HW G2 p 0 989776 0 520417 0 979044 0 08025 HW Exact p 0 958 0 462 0 4662 0 0205 HExp 0 7476 0 6931 0 7351 0 7378 0 7479 0 7077 0 7586 0 6806 075820 HW X 0 987668 0 571989 0 336834 0 324754 HW G2 0 969887 0 44694 0 687948 0 289733 HW Exact p 0 9818 0 2286 0 4028 0 1276 HExp 0 7758 0 8117 0 7822 0 7858 0 7955 0 7908 0 7862 0 7487 0851179 HW X p 0 067164 0 545414 0 047783 0 446248 HW G2 p 0 568837 0 275218 0 302937 0 760077 HW Exact p 0 2176 0 3264 0 0304 0 1656 HExp 0 7925 0 8047 0 7853 0 7
23. punch in the center of the blood stain For buccal samples on treated paper punch in the center of the buccal transfer or punch in the optimal spot based on past experiences For buccal samples collected with the Bode Buccal DNA Collector punch from near the tip of the collector Insufficient lysis of the swab head Ensure swab heads are incubated for 20 minutes in 400 pL Prep N Go Buffer AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 101 Appendix A Troubleshooting Observation Possible causes Recommended actions More than two alleles present at a locus Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Amplification of stutter product n 4 nt position See Stutter products on page 73 Incomplete 3 A base addition n 1 nt position See Addition of A nucleotide on page 78 Be sure to include the final extension step of 60 C for 10 minutes in the PCR Signal exceeds dynamic range of instrument off scale data Ensure cycle number is optimized according to instructions on page 17 Repeat PCR amplification using fewer PCR cycles or use your laboratory s SOP to analyze off scale data Poor spectral separation bad matrix Follow the steps for creating a spectral file Confirm that Filter Set G5 modules are installed and used for analysis Contamination carr
24. 0 035 0 056 19 169 22 169 40 0 035 0 056 FGA 17 214 31 214 49 0 035 0 046 18 218 33 218 5 0 037 0 046 19 222 38 222 56 0 020 0 047 20 226 40 226 58 0 036 0 046 21 230 42 230 60 0 032 0 046 22 234 46 234 65 0 033 0 047 23 238 49 238 69 0 038 0 048 24 242 54 242 73 0 038 0 054 25 246 57 246 78 0 033 0 050 26 250 62 250 82 0 039 0 059 26 2 252 63 252 82 0 040 0 045 27 254 63 254 82 0 035 0 053 28 258 69 258 89 0 038 0 051 29 262 75 262 95 0 045 0 053 30 266 81 267 04 0 033 0 054 30 2 268 66 268 85 0 042 0 062 31 2 272 72 272 93 0 039 0 052 32 2 276 78 277 01 0 037 0 055 33 2 280 85 281 07 0 044 0 053 42 2 317 96 318 20 0 042 0 057 43 2 322 08 322 31 0 051 0 056 44 2 326 18 326 43 0 039 0 059 45 2 330 33 330 55 0 046 0 060 46 2 334 34 334 56 0 039 0 047 47 2 338 43 338 65 0 047 0 056 48 2 342 59 342 80 0 047 0 064 50 2 350 71 350 91 0 040 0 053 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 71 Chapter 5 Experiments and Results Accuracy precision and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation 51 2 354 67 354 87 0 048 0 058 THO1 4 162 79 162 92 0 034 0 054 5 166 84 166 97 0 034 0 048 6 170 88 171 00 0 030 0 047 7 174 88 175 01 0 028 0 046 8 178 89 179 01 0 031 0 045 9 182 87 182 98 0 031 0 042 9 3 185 90 186 02 0 025 0 049 10 186 83 186
25. 151 153 155 157 159 161 163 165 167 169 171 173175 177 179181183 g 5 B5 B B B B B B B B B B B B B 0851179 iil 173 36 Y 0 31 ad Reference Samples AmpFtSTR9 Identifiler Direct PCR Amplification Kit User Guide 51 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis 9 Import IdentifilerDirect GS500 Stutter v1X txt a Select the IdentifilerDirect GS500 v1 folder in the navigation panel File Edit Bins View Help g m Set IdentifilerDirect GS500 Bins v1X v 11 S gf Panel Manager IdentirlerDirect GS500 Panels v1x null b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the Identifiler Direct Analysis Files GMIDX folder d Select IdentifilerDirect GS500 Stutter v1X txt then click Import Note Importing this file associates the marker stutter ratio with the bin set in the IdentifilerDirect GS500 Bins v1X folder Import Marker Stutter 3 Identifiler Direct Analysis Files GMIDX paa IdentifilerDirect_GS500_Bins_v1X txt 1dentifilerDirect GS500 Panels vix txt IdentifilerDirect GS500 Stutter v1X txt E ReadMe IDDirect vix txt IdentifilerDirect GS500 Stutter viX txt All Files 10 View the imported marker stutters in the navigation pane a Select the Iden
26. 3 0 171 t 17 0 14 t 0 17t t 075820 6 T 0 14 0 17 t 7 0 421 1 29 1 72 0 521 8 18 77 16 48 11 72 13 09 9 13 73 17 62 6 21 8 12 10 34 45 27 22 27 41 21 99 11 19 89 18 05 28 79 28 80 12 10 78 14 76 20 17 24 08 13 1 54 3 72 3 45 3 40 14 0 42 0 72 0 34 t 15 t t t T D8S1179 8 0 421 2 29 0 341 0 521 9 0 421 1 15 0 341 0 261 10 2 38 9 74 8 45 4 71 11 3 92 6 02 5 86 3 40 12 13 31 14 04 12 07 11 52 13 23 25 32 52 32 93 37 43 14 30 11 21 35 26 21 30 63 15 20 17 9 89 10 86 9 42 16 4 62 2 72 2 41 1 57 17 1 12 0 291 0 521 0 521 18 0 281 t t t 19 t 1 t AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data African U S Native Allele American Caucasian 357 349 E n 191 0135317 8 3 08 12 18 9 66 4 97 9 2 52 7 74 21 72 17 80 10 3 78 4 44 9 14 13 61 11 24 51 29 80 23 10 24 35 12 46 22 30 80 20 86 23 04 13 15 41 11 17 10 17 7 85 14 4 34 3 72 5 34 8 12 15 0 14 0 14 0 261 D16S539 5 t t t 8 3 22 1 72 1 72 0 79 9 19 05 10 46 9 31 12 30 10 10 92 5 59 15 69 15 45 11 31 51 31 95 30 17 30 89 12 18 77 30 23 29 48 27 75 13 14 85 16 76 11 55 10 73 14 1 54 3 01 2 07 2 09 15 0 14 0 291 t t D18551 7 t 1 t t 9 0 14 t T t 10 0 281 0 86 0 521 0 791 10 2 0 141 t t Y 11 0 28 1 15 1 21 t 12 7 00 13 90 10
27. 34 14 92 13 4 34 12 18 14 48 9 16 13 2 0 421 t T t 14 6 86 16 76 15 52 26 96 14 2 0 28t t t 15 19 47 13 61 16 55 12 04 16 16 53 13 61 11 72 10 73 17 18 21 12 32 14 14 14 66 18 11 90 7 74 6 72 2 62 19 6 02 4 44 4 14 3 93 20 4 90 1 72 2 24 1 83 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 89 Chapter 5 Experiments and Results Population data African U S Native Allele American Caucasian S oou American n 357 n 349 li 191 21 2 10 1 00 1 03 1 31 22 0 701 0 431 0 521 0 791 23 0 421 0 141 0 521 0 261 24 T 0 14 0 17 t 25 t t 0 17t U 26 1 1 t t 27 t D19S433 9 t 0 14 0 17 10 1 54 t t t 11 7 14 0 72 0 52t 0 52t 11 2 0 14 t 0 17 T 12 10 78 7 74 6 21 3 14 12 2 6 30 0 571 1 90 t 13 29 83 28 94 16 03 17 80 14 21 01 34 10 31 72 24 87 14 2 4 20 0 86 5 00 3 66 15 4 76 15 76 13 45 13 35 15 2 3 36 2 72 8 79 10 73 16 2 38 4 15 4 31 3 93 16 2 2 38 1 72 2 93 1 83 17 t 0 29t 0 17t 0 79t 17 2 0 28t 0 291 d 2 88 18 2 0 14 0 29 1 05 021511 24 t t 24 2 0 14 0 431 0 171 t 24 3 t t t 25 t t t 25 2 t 0 141 0 171 t 26 0 14 0 14 0 171 t 27 5 04 4 58 1 21 0 52 28 22 97 16 76 9 14 6 28 28 2 t t t T 29 19 33 20 49 21 21 16 75 29 2 0 14 t 0 52 0 26 90 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide
28. 3500xL instruments 31 Section 3 3 3730 instrument kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK KK KK k 33 Set Up the 3730 instrument for electrophoresis 33 Reagents and parts xg ad sl phe ARE kcha RES edd Ke paid el ERR ed 33 Electrophoresis software setup and reference lt 33 Prepare samples for electrophoresis on the 3730 5 33 CHAPTER 4 Analyze Data 35 Section 4 1 GeneMapper ID Software 35 Overview of GeneMapper ID Software 35 tis tiat er S m nee Lesbo DPA oy ds ch bee QUAD EU ecd eua xr 35 Before Vou StarE cto eer estet et pc ese Sc C RN Tee Rc ed 35 Set up GeneMapper D Software for data 36 File Names sese lx ud RAP UI EIER NU ME ee bee ne Tue T 36 Before using the software for the first time 2 8 288 36 Import panels and bins kK kk kk rr 36 Create an analysis method n n kk kk kk kk kk kk kk kK kK kk kK kk kK kK kk kk kk kk kk kk kk kk kk k kk 40 Gerieral tab settings uj lt s xi ka kaleka ER b EET REED ML RERUM 40 Allele tab 5 5 e rl
29. 41 Peak Detector tab settings 2 kK kk kk kk kk kk kk kk kk k 42 Peak Quality tab settingS KELA 2N eh e NE 43 Quality Flags tab senings uk sun Ee ad RAN e ae SEW 44 Create a size standard WWW kk kk kk kk kk kk kk kk kk KK kK KK KK kK n 45 Analyze and edit sample files with GeneMapper ID Software 46 Examine andedita project ete DV Ib EDI I REM eer ERN ead rr ER 47 For more information cede ecd ee ERR RR de Elka ed Se Leah eee faa ded VE 47 Section 4 2 GeneMapper D X 48 Overview of GeneMapper D X Software 48 struments exce bi EA On ee ye ed IRE Eu 48 Before oes aS eec toe ee Ee 48 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Contents Set up GeneMapper D X Software for data analysis 49 iile 2 ade ES A IP Lg ee tiva oium 49 Before using the software for the first time kk kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK 49 Import panels bins and marker stutter kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK k 49 Create an analysis method MAW kk kk kk kk kk kk kk kK KK KK KK KK KK kk kK kK kk kk kK kk kk 53 General tab settings kk kk kk kK kK kk kk
30. 89 D7S820 8 60 0851179 9 54 FGA 11 62 TH01 4 76 TPOX 5 27 vWA 11 99 t These percentages are used as stutter filters in GeneMapper D Software IdentifilerDirect GS500 Panels v1 and GeneMapper D X Software IdentifilerDirect GS500 Stutter v1X txt 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 nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence The PCR product with the extra nucleotide is referred to as the A form The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The AmpF STR Identifiler Direct PCR Amplification Kit includes two main design features that promote maximum addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 25 min The final extension step gives the AmpliTaq Gold DNA polymerase additional time to complete A addition to all double stranded PCR products STR systems where each allele is represented by two peaks that are one nucleotide apart that have not been optimized for addition may have split peaks AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Characterization of loci Figur
31. 94 0 035 0 047 11 190 79 190 89 0 025 0 046 12 201 62 201 71 0 034 0 045 TPOX 6 221 96 222 07 0 030 0 043 7 225 93 226 04 0 035 0 044 8 229 90 230 01 0 027 0 043 9 233 86 233 98 0 032 0 039 10 237 86 237 98 0 023 0 049 11 241 83 241 96 0 028 0 037 12 245 84 245 95 0 032 0 043 13 249 83 249 93 0 027 0 044 vWA 11 154 16 154 27 0 025 0 044 12 158 30 158 44 0 029 0 054 13 162 40 162 54 0 034 0 045 14 166 62 166 78 0 029 0 048 15 170 56 170 70 0 028 0 046 16 174 57 174 71 0 028 0 045 17 178 56 178 71 0 028 0 045 18 182 51 182 66 0 032 0 044 19 186 48 186 60 0 031 0 045 20 190 41 190 53 0 026 0 043 21 194 29 194 43 0 032 0 044 22 198 21 198 33 0 025 0 043 23 202 06 202 18 0 034 0 040 24 206 38 206 48 0 031 0 040 72 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 5 Experiments and Results Extra peaks in the electropherogram Extra peaks in the electropherogram Causes of extra Peaks other than the target alleles may be detected on the electropherogram Causes peaks 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 2005 Mulero et al 2006 Sequence analy
32. Chapter 5 Experiments and Results Population data African U S Native Allele American Caucasian 357 349 z n 191 29 3 0 14 t ji t 30 17 23 25 21 29 31 34 29 30 2 1 40 3 30 2 93 1 83 31 7 98 7 16 6 72 5 76 31 2 7 98 9 46 8 62 18 85 32 1 12 1 43 1 55 0 791 32 2 5 88 7 16 12 93 9 69 33 0 561 t 0 52 33 2 3 78 3 30 4 14 3 66 34 1 26 t t 34 1 0 14 d t 34 2 0 14 0 29 0 86 0 79 35 2 94 0 34 T 35 1 0 14 t 35 2 t 0 141 t 36 0 84 t 37 0 281 t 38 0 14 t FGA 16 t 0 14 t 16 1 0 14 L t t 17 t 0 29t 0 17t t 17 2 0 14t i t 18 0 70 2 72 0 52 1 31 18 2 1 40 F t 19 6 72 6 16 7 07 10 21 19 2 0 28t t t 20 7 00 13 90 7 41 12 30 20 2 t 0 14 t 21 12 89 16 91 14 66 12 83 22 21 57 16 91 17 24 10 47 22 2 0 28 1 29 0 34 0 261 22 3 0 141 0 14 T t 23 14 99 15 19 11 90 15 97 23 2 0 14 0 14 0 861 0 26 24 17 51 13 75 15 34 15 71 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 91 Chapter 5 Experiments and Results Population data African U S Native Allele American Caucasian oou American n 357 n 349 li 191 24 2 t 0 14 0 17 t 25 7 98 8 60 14 14 14 14 26 3 50
33. Direct Kit loci are all tetranucleotide short tandem repeat STR loci The length differences among alleles of a particular locus result from differences in the number of 4 nt repeat units AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 79 Chapter 5 Experiments and Results Species specificity Mapping We have subjected to sequencing all the alleles in the AmpF STR Identifiler Direct Allelic Ladder In addition other groups in the scientific community have sequenced alleles at some of these loci Among the various sources of sequence data on the Identifiler Direct Kit loci there is consensus on the repeat patterns and structure of the STRs The Identifiler Direct Kit loci have been mapped and the chromosomal locations have been published Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Barber and Parkin 1996 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 Identifiler Direct Kit provides the required specificity for detecting primate alleles Other species do not amplify for the loci tested Nonhuman studies Nonhuman DNA may be present in forensic casework samples The data from Identifiler Direct Kit experiments on nonhuman DNA sources are shown in
34. FGA 4q28 17 18 19 20 21 22 23 24 25 26 26 2 27 28 23 24 29 30 30 2 31 2 32 2 33 2 42 2 43 2 44 2 45 2 46 2 47 2 48 2 50 2 51 2 10 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 1 Overview 1 Product overview Allelic ladder Figure 1 shows the allelic ladder for the Identifiler Direct Kit See Allelic ladder requirements on page 28 for information on ensuring accurate genotyping Figure 1 GeneMapper D X Software plot of the AmpF STR Identifiler Direct Allelic Ladder Is El 8 bl hell i ell sll l ls Is 2 lel 330 s Elka kl E L ji Jill be 21 22 f23 e bs ls 27 AmpFtSTR Identifiler9 Direct PCR Amplification Kit User Guide 11 1 Chapter 1 Overview Workflow Workflow Perform PCR Perform electro phoresis Analyze data 12 c 9 co Treated untreated paper Swab a E substrates substrates on ow ow SE Sz Lyse in 2 a TM o vE Prep n Go Buffer a kartis Manual CPA200 or CPA300 Punch instrument Prepare reactions Untreated paper only Prep n Go Buffer AmpF STR Identifiler Direct PCR Amplification Kit Prepare reactions AmpF STR Identifiler Direct PCR Amplification Kit Perform PCR 4 GeneAmp PCR System 9700 Cycler pror PCR System Veriti 96 Well Thermal Cycler
35. GeneMapper ID X Software v1 2 or later t We conducted validation studies for the Identifiler Direct Kit using these configurations Life Technologies fluorescent multi color dye technology allows the analysis of multiple loci including loci that have alleles with overlapping size ranges Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components The four dyes used in the Identifiler Direct Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ is used to label the GeneScan 500 LIZ Size Standard or the GeneScan 600 LIZ Size Standard v2 0 Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on Life Technologies instruments the fluorescence signals are separated by a diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern The 6 FAM dye emits at the shortest wavelength and is displayed as blue followed by the VIC dye green NED dye yellow PET dye red and LIZ dye orange Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 2 The goal of
36. In the Size Standard Table enter the peak sizes specified in on page 45 The example below is for the GeneScan 500 LIZ9 Size Standard xi Edit r Size Standard Description 1 Description Size Standard Dye yo gt l r Size Standard Table Size in Basepairs l 750 m 100 0 g 139 0 4 1500 s 1600 6 2000 7 lama la 350 0 ig 400 0 ja 4500 OK Cancel Analyze and edit sample files with GeneMapper D Software 1 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project The names of the settings shown are the names suggested in the sections above If you named the settings differently select the names you specified Parameter Settings Sample Type Select the sample type Analysis Method IdentifilerDirect AnalysisMethod v1 the name of the analysis method you created Panel IdentifilerDirect GS500 Panels v1 Size Standard cnet or the name of the size standard you created 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 Part no 4335617 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis Us
37. Let the lysate stand at room temperature for at least 15 minutes to cool the lysate for accurate pipetting 6 Transfer the sample lysate out of the sample plate into tubes or plates for storage then discard the deep well plate containing the swab heads 7 Proceed to the next section to prepare the reactions or see Store the sample lysate on page 25 Prepare the 1 Calculate the volume of each component needed to prepare the reactions using reactions the table below Reaction component Volume per reaction Master Mix 12 5 uL Primer Set 12 5 uL AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 23 24 Chapter 2 Perform PCR Swab substrates prepare reactions Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT This kit has been optimized for a 25 uL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples Using a lower PCR reaction volume may reduce the ability of Identifiler Direct Kit chemistry to generate full STR profiles Prepare reagents Thaw the Master Mix and the Primer Set then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 C and therefore do not require subsequent thawing Do not refreeze the rea
38. RFU 3500 Series 3000 12 000 RFU 18 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 2 Perform PCR Treated paper substrates prepare reactions Treated paper substrates prepare reactions Sample prep Do not add water to the wells on the reaction plate before adding the punches If guidelines your laboratory is experiencing static issues with the paper discs you may prepare and dispense the 25 uL reaction mix into the wells of the reaction plate before adding the punches Make the punch as close as possible to the center of the sample to ensure optimum peak intensity Increasing the size of the punch may cause inhibition during PCR amplification For manual punching Place the tip of a 1 2 mm Harris Micro Punch on the card hold the barrel of the Harris Micro Punch do not touch the plunger gently press and twist 1 4 turn then eject the punch in to the appropriate well on the reaction plate For automated punching Please refer to the User Guide of your automated or semi automated disc punch instrument for proper guidance Prepare the 1 Add samples to the reaction plate reactions Wells Add the following to wells of a MicroAmp Optical 96 Well Reaction Plate Negative control 1 2 mm blank disc Test samples 12mm sample disc Positive control For 25 cycles m 3 uL of Control DNA9947A IMPORTANT Do not For 26 and 27 cycles 2 uL of Control DNA 9947A addab
39. Sample Quality per project based on sample SOS SSPK OMR SQ Total of Samples 8 Al thresholds met __ One or more thresholds not met Samples 6 0 6 60 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 2 ID X Software Examine and edit a project Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Analysis Summary tab of the Project window assuming the analysis is complete For more information For more information about any of these tasks refer to GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 e GeneMapper ID X Software Version 1 0 Quick Reference Guide Part no 4375670 GeneMapper ID X Software Version 1 0 Reference Guide Part no 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide Part no 4396773 e GeneMapper ID X Software Version 1 2 Reference Guide Part no 4426481 e GeneMapper ID X Software Version 1 2 Quick Reference Guide Part no 4426482 2 0 lt D Ne 0 p x 00 g oO AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 61 For more information Chapter 4 GeneMapper ID X Software 62 AmpFtSTR Identifiler
40. for PCR based genotyping and cloning Biotechniques 21 700 709 Mansfield E S Robertson J M Vainer M Isenberg A R Frazier R R Ferguson K Chow S Harris D W Barker D L Gill P D Budowle B McCord 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 Mills K A Even D and Murray J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 Momhinweg E Luckenbach C Fimmers R and Ritter 1998 D351358 sequence analysis and gene frequency in a German population Forensic Sci Int 95 173 178 Moretti T Baumstark A Defenbaugh D Keys K Smerick J and Budowle B 2001 Validation of short tandem repeats STRs for forensic usage Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples J Forensic Sci 46 3 647 660 Mulero J J Chang C W and Hennessy L K 2006 Characterization of N 3 stutter product in the trinucleotide repeat locus DYS392 J Forensic Sci 51 826 830 Nakahori Y Takenaka O and Nakagome Y 1991 A human X Y homologous region encodes amelogenin Genomics 9 264 269 Nei M 1973 Analysis of gene diversity in subdivided populations Proc Natl Acad Sci LISA 70 3321 3323 Nei M 1978 Estimation of average heterozygosity and genetic distance from a sma
41. indicator of precision within a run Use the following procedure to create the size standard for the Identifiler Direct Kit 1 Select Tools GeneMapper ID X Manager to open the GeneMapper ID X Manager 58 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 2 Select the Size Standards tab then click New GeneMapper ID X Manager Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Type Description CE F HID G5500 75 400 2007 08 09 13 23 gmidx Advanced CE F HID G5500 75 450 2007 08 09 13 24 C gmidx Advanced CE G5 HID GS500 2011 04 18 13 15 gmidx Advanced Open Save As Export 3 Complete the Name field as shown below or with a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the drop down list In the Size Standard Dye field select Orange In the Size Standard Table enter the peak sizes specified on page 58 The example below is for the GeneScan 500 LIZ Size Standard O 2 0 lt Rv o S X n 9 m 2 0 Size Standard Editor Edit Size Standard Description Name CE G5 IdentifilerDirect 65500 Security Group GeneMa
42. kK kk KK kk m n 54 Allel tab settings ci teme reco Utopia Mum hr 55 Peak Detector tab settings kk kk kk kk kk kK kK kk KK KK KK kk kk sse 56 Peak Quality tab settings 57 SQ amp GQ tab settings 02 s xuy k RR ERE I l k k any le bates 58 Create a size standard M A kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK kk kK kk KK kK kk kk kk ka 58 Analyze and edit sample files with GeneMapper D X Software 60 Examine and edit a project lk kk kk kk kk kk kk kk kK kk KK kK kK kK kk kk kk kk kk kk kk kk kk kk kk kk kk ka 61 For more iriformatlon ses a ke ra 61 m CHAPTER 5 Experiments and 63 ida se Nite ee nA iad VENE KARA DEN Kal Key KA O 63 lmportance of Validation 23 5 Q el PUR EE 63 Experiment conditions s a gk kenek wi kaya OAS al ka DARIN ERR 63 Accuracy precision and reproducibility kk kk kk kk kk KK KK KK KK KK KK KK kK KK KK kK k b4 SWGDAM guideline 1 251 SP ehh tea DE bi V PRIMIS 64 SWGDAM guideline 2 9 kk kk kk kk 64 Accuracy A YEKA dues eu ncn sre viria ccr 64 Precision and size 65 Extra peaks in the
43. kk kk kk kk k 13 How multicomponent analysis works 13 Materials and equipment kk kk kk kk kk kk kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk 14 Kitcontentsand storage 225 sis ees khe RU EA LAXE EE 14 Standards for samples n nsan kk kk kk kk kk kk kk kk kK kk kk kK kk kK kk kk kk kk e 15 CHAPTER 2 17 Optimize PCR cycle ra 17 Select samples and prepare plates 17 Determine optimum conditions WWW kk kk kk kk k kk kk kK KK kk kK kk kk kk kk kk kk kk kk kk kk 18 Treated paper substrates prepare reactions 19 Sample prep guidelines n kk kk kk kk kk kk kk kk kK kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk k 19 Prepare the 5 kk kk kk kk kk kk kk kk 19 Untreated paper substrates prepare reactions 21 Sample prep guidelines M M WWW kk kk kk kk kk kk kk kK kK kk kk kk kk kk kk kk kk kk kk kk kk kk k 21 Prepare the 5 kk kk kk kk kk kk kk kk 21 Swab substrates prepare reactions kk kk kk kk kk kk kK kK KK kK kk kk kk kK kK kk ka 23 Sample prep guidelines M M WWW kk kk kk kk kk kk kk
44. kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk k 23 Prepare the sample lysate kk kk kk kK KK KK KK KK kk kK kk kk kk kk kk kk kk kk kk kk 23 Prepare the reactions dan k lA RAUM diwan Weg dina k l ada 23 Store the sample lysate WW kk kk kk kk kk kk kk kk kk KK KK kK KK kk kk en 25 PR DD DD DD e r e ow 26 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 3 Contents Mm CHAPTERS Perform Electrophoresis 27 Allelic ladder requirements n nannu nunnurnar erann renren kk kk kk I kk kk k 28 Section 3 1 3100 3100 Avant and 3130 3130xl instruments 29 Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis 29 Reagents and parts ciis idei daa ER d p ADA ark ni ELM RIED eM 29 Electrophoresis software setup and reference lt 29 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instruments 30 Section 3 2 3500 3500xL instruments 31 Set up the 3500 3500xL instruments for 31 Reagents and parts kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk rr 31 Electrophoresis software setup and reference lt 31 Prepare samples for electrophoresis on the 3500
45. multicomponent analysis is to correct for spectral overlap AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 13 Chapter 1 Overview Materials and equipment Figure 2 Emission spectra of the five dyes used in the Identifiler Direct Kit 6 FAM A Dyes VIC NED PET LIZ Boo o 2 o 2 E o AN T E 9 2 o Materials and equipment Kit contents and storage 650 Wavelength nm The Identifiler Direct Kit contains sufficient quantities of the following reagents for 200 reactions Part no 4467831 or 1000 reactions Part no 4408580 at 25 uL reaction IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Table 2 Kit Contents and Storage Component Description 200 reaction 1000 reaction Storage AmpF STR Contains enzyme salts dNTPs 2 tubes 1 bottle 12 5 mL 15 to 25 C upon Identifiler Direct carrier protein and 0 04 1 25 mL each receipt 2 to 8 C after Master Mix sodium azide initial use AmpF STR Contains forward and reverse 2 tubes 1 bottle 12 5 mL Identifiler9 Direct primers to amplify human DNA 1 25 mL each Primer Set targets AmpF STR9 Contains 2 ng uL human female 1 tube 50 0 uL 1 tube 50 0 uL Identifiler Direct Control DNA 9947A
46. n gs General allele Peak Detector Peak Quality SQ amp GQ Settings r Analysis Method Description Mame IdentifilerDirect AnalysisMethod v1x Security Group GeneMapper ID X Security Group Description Instrument Analysis Type In the Name field either type the name as shown or enter a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the drop down list The Description and Instrument fields are optional 54 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 2 ID X Software 4 Set up GeneMapper ID X Software for data analysis Allele tab settings Analysis Method Editor General Allele Peak Detector Peak Quality 5Q amp GQ Settings Bin Set IdentifilerDirect GS500 Bins v1X Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off Value 0 0 0 2 0 0 MinusA Ratio 0 0 0 0 0 0 MinusA Distance 0 0 0 0 0 0 0 0 0 0 0 0 Global Minus Stutter Ratio 0 0 0 0 0 0 Global Minus Stutter Distance From 0 0 3 25 0 0 To 00 4 75 0 0 Global Plus Stutter Ratio 0 0 0 0 0 0 Global Plus Stutter Distance From 00 0 0 0 0 To 00 0 0 0 0 Amelogenin Cutoff 0 0 Range Filter Factory Defaults
47. number of tests with p value 0 05 were 0 in the African American and U S Caucasian populations 1 in the U S Hispanic population D851179 p 0 0304 and 2 in the Native Americans 021511 p 0 0118 D55818 p 0 0205 These are no more than would be expected by chance No more alleles were observed to be in linkage disequilibrium than would be expected by chance alone The average observed heterozygosity across the 15 STR loci was 0 804 in the African American population 0 792 in the U S Caucasian sample population 0 793 in the Hispanic sample population and 0 757 in the Native Americans The most heterozygous locus was FGA mean observed heterozygosity across all populations of 0 875 and the least heterozygous STR locus was TPOX mean observed heterozygosity across all populations of 0 677 Table 6 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler STR loci in four U S populations African U S Native U S Hispanic American Caucasian 290 American n 357 n 349 191 CSF1P0 HW X 0 13649 0 926431 0 951476 0 839278 HW G2 p 0 08902 0 894972 0 918038 0 728023 HW Exact p 0 0762 0 2688 0 5456 0 6148 HExp 0 7829 0 7267 0 7051 0 7398 Hy 0 7703 0 7421 0 7138 0 7958 t HW X p probability value of X test for Hardy Weinberg equilibrium HW G p probability value of the G statistic of the Likelihood Ratio test for multinomial proportions HW Exact
48. of allele sizes obtained on the Applied AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 65 66 Chapter 5 Experiments and Results Accuracy precision and reproducibility Biosystems 3130 1 Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 nt The instance of a sample allele sizing outside the 0 5 nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 nt or less Smith 1995 For sample alleles that do not size within a 0 5 nt window the PCR product must be rerun to distinguish between a true off ladder allele versus measurement error of a sample allele that corresponds with an allele in the allelic ladder Repeat analysis when necessary provides an added level of confidence to the final allele assignment GeneMapper ID Software and GeneMapper ID X Software automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling the allele as OL Off ladder Maximum precision is obtained with a set of capillary injections on each of the supported platforms however the determined allele sizes will vary between the different platforms Cross platform sizing differences occur from a number of factors including type and concentration of polymer run temperature and electrophoresis conditions Variations in sizing can also occur between runs on the same instrument and betwe
49. of paternity exclusion Table 8 shows the Probability of Paternity Exclusion Pg values of the Identifiler Chapter 5 Experiments and Results Probability of paternity exclusion Direct Kit STR loci individually and combined Table 8 Probability of Paternity Exclusion values for the Identifiler Direct Kit loci Locus African ME U S Hispanic Native American Caucasian American CSF1PO 0 545 0 496 0 450 0 409 D2S1338 0 748 0 725 0 671 0 399 D3S1358 0 591 0 630 0 495 0 510 D5S818 0 506 0 440 0 525 0 601 D7S820 0 591 0 582 0 574 0 492 D8S1179 0 580 0 680 0 599 0 601 D13S317 0 383 0 487 0 638 0 370 D16S539 0 649 0 566 0 567 0 428 D18S51 0 760 0 731 0 767 0 329 D19S433 0 601 0 531 0 678 0 360 D21S11 0 737 0 708 0 586 0 399 FGA 0 760 0 766 0 739 0 309 THO1 0 492 0 566 0 618 0 646 TPOX 0 521 0 329 0 392 0 687 vWA 0 709 0 625 0 555 0 528 Combined 0 9999996 0 9999992 0 9999990 0 9999527 The value is the probability averaged over all possible mother child pairs that a random alleged father will be excluded from paternity after DNA typing of the Identifiler Direct Kit STR loci Chakraborty and Stivers 1996 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 99 Chapter 5 Experiments and Results Probability of paternity exclusion 100 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Troubleshooting Follow the actions recomme
50. products Order the GeneScan 500 LIZ Size Standard Part no 4322682 or the GeneScan 600 LIZ Size Standard v2 0 Part no 4408399 separately AmpE STR Identifiler Direct Allelic Ladder Developed for accurate characterization of the alleles amplified by the Identifiler Direct Kit The Allelic Ladder contains most of the alleles reported for the 15 autosomal loci Refer to page 10 for a list of the alleles included in the Allelic Ladder AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 15 1 Chapter 1 Overview Materials and equipment 16 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Optimize PCR cycle 8 17 Treated paper substrates prepare 19 m Untreated paper substrates prepare reactions 21 Swab substrates prepare reactions 23 W Perform PCR zc eg estet mult eu og auld ete eats ete mates edge 26 Optimize PCR cycle number Before using the Identifiler Direct Kit for the first time perform a single initial sensitivity experiment to determine the appropriate cycle number to use during internal validation studies and operational use of the Identifiler Direct Kit This experiment accounts for instrument to instrument and sample to sample variations If you are processing multiple sam
51. the Identifiler kit Estimation of spontaneous or induced germline mutation at genetic loci can be achieved by comparing the genotypes of offspring to those of their parents From such comparisons the number of observed mutations are counted directly In previous studies genotypes of ten STR loci that were amplified by the SGM Plus PCR Amplification Kit were determined for a total of 146 parent offspring allelic transfers meioses at the Forensic Science Service Birmingham England One length based STR mutation was observed at the 018511 locus mutations were not detected at any of the other nine STR The 018511 mutation was represented by an increase of one 4 nt repeat unit allele 17 was inherited as allele 18 single step mutation The maternal paternal source of this mutation could not be distinguished AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 97 5 Chapter 5 Experiments and Results Probability of identity Additional Additional studies Edwards et al 1991 Edwards et al 1992 Weber and Wong 1993 mutation studies Hammond et al 1994 Brinkmann et al 1995 Chakraborty et al 1996 Chakraborty et al 1997 Brinkmann et al 1998 Momhinweg et al 1998 Szibor et al 1998 of direct mutation rate counts produced Larger sample sizes for some of the Identifiler Direct Kit loci Methods for modifications of these mutation rates to infer mutation rates indirectly for thos
52. to open the GeneMapper Manager GeneMapper Manager Lest ____ owner________nstrmert _____Analya s Type mese jmmmemje le og 2 Select the Analysis Methods tab then click New to open the New Analysis Method dialog box 3 Select HID and click OK to open the Analysis Method Editor with the General tab selected 4 Enter the settings shown in the figures on the following pages Note The Analysis Method Editor closes when you save your settings To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs 5 After you enter settings in all tabs click Save General tab Analysis Method Editor HID settings 40 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis In the Name field either type the name as shown for consistency with files supplied with other AmpF STR kits or enter a name of your choosing The Description and Instrument fields are optional Allele tab settings xi Peak Detector Peak Quality Quality Flags Bin Set identifilerDirect_GS500_Bins_v1 Y Q v Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Penta Hexa Cut off Value o2 MinusA Ratio Minus Distance From foo o n o n To f
53. 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 e Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipette tips sterile disposable hydrophobic filter plugged Pipettors AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 109 Appendix PCR Work Areas Amplified DNA work area Tube decapper autoclavable e Vortex Amplified DNA work area IMPORTANT Place the thermal cyclers in the Amplified DNA Work Area You can use the following systems GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block IMPORTANT The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the Aluminium 96 Well Block Use of this thermal cycling platform may adversely affect performance of the Identifiler Direct Kit Veriti 96 Well Thermal Cycler 110 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for labo
54. 0 minutes Copan 4N6 FLOQSwabs Mark Sample for Mak Sample for Delebor i M u lu 1 Puritan swabs Figure 18 Amplification of buccal cells on fresh Copan 4N6FLOQSwabs and lysed with Prep n Go Buffer at room temperature 115 155 195 23 275 51 AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 85 Population data Chapter 5 Experiments and Results Population data SWGDAM guideline 2 7 Overview Population samples used in these studies Identifiler Direct Kit allele frequencies 86 The distribution of genetic markers in populations should be determined in relevant population groups SWGDAM July 2003 To interpret the significance of a match between genetically typed samples you must know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of a suspects reference sample then the suspect is excluded as the donor of the biological evidence that was tested An exclusion is independent of the frequency of the two genotypes in the population 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 relevan
55. 009 New document B August 2009 Add Experiments and Results chapter C October 2009 Update screen shots for Panel Manager D September 2010 Change copyright page information E July 2011 Add 200 reaction kit Bode Buccal DNA Collector Prep n Go Buffer and 3100 Avant 3130 and 3500 3500xL Genetic Analyzer information October 2011 Add information for Prep n Go Buffer to Experiments and Results chapter March 2012 Change copyright page information May 2012 Add heat protocol for buccal swab lysate preparation Add results for additional swab types February 2015 Add information for the ProFlex PCR System Purpose The AmpFtSTR Identifile Direct PCR Amplification Kit User Guide provides information about our instruments chemistries and software associated with the AmpF STR Identifiler Direct PCR Amplification Kit AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide About This Guide Purpose 8 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Product overview Purpose Substrate examples Product description Overview Product Overview da h n eb k V ete erke ak 9 Workflow eiue Dee REDE AR Da W ay bb a Mar bn a kh 12 Instrument and software overview 13 Materials and equipment W Wk kK kK KK KK KK KK KK KK KK KK ee 14 The AmpF STR Identifiler Direct PCR Amplificati
56. 052 13 115 45 115 58 0 034 0 046 14 119 44 119 58 0 034 0 047 15 123 37 123 49 0 035 0 053 16 127 55 127 67 0 033 0 051 17 131 74 131 86 0 029 0 048 18 135 85 135 96 0 035 0 050 19 139 96 140 07 0 036 0 056 D5S818 7 133 85 133 95 0 037 0 048 8 137 96 138 06 0 040 0 046 9 142 31 142 42 0 032 0 045 10 146 78 146 89 0 033 0 044 11 151 13 151 26 0 032 0 043 12 155 36 155 50 0 027 0 042 13 159 51 159 67 0 020 0 045 14 163 57 163 73 0 032 0 044 15 167 60 167 76 0 030 0 055 16 171 63 171 77 0 036 0 049 D7S820 6 255 09 255 23 0 031 0 047 7 259 11 259 25 0 038 0 048 8 263 13 263 27 0 036 0 049 9 267 16 267 29 0 029 0 041 10 271 20 271 32 0 041 0 048 11 275 23 275 37 0 032 0 051 12 279 26 279 40 0 037 0 047 13 283 28 283 43 0 035 0 049 14 287 32 287 45 0 043 0 052 15 291 35 291 49 0 037 0 053 D8S1179 8 122 84 122 95 0 030 0 046 9 126 91 127 01 0 027 0 053 10 131 01 131 10 0 031 0 052 11 135 14 135 24 0 037 0 051 70 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy precision and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation 12 139 33 139 43 0 029 0 059 13 143 90 144 02 0 027 0 045 14 148 36 148 48 0 034 0 045 15 152 70 152 82 0 022 0 044 16 156 93 157 09 0 026 0 041 17 161 08 161 24 0 026 0 046 18 165 14 165 33
57. 125391 STR locus in an Austrian population sample sequencing data and allele distribution Forensic Sci Int 90 197 203 Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S Iovannisci D M Woo S Winn Deen E S 1994 High density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 4527 4534 Grubwieser P Muhlmann R Berger B Niederstatter H Palvic M Parson W 2006 A new mini STR multiplex displaying reduced amplicon lengths for the analysis of degraded DNA Int J Legal Med 120 115 120 Guo S W and Thompson E A 1992 Performing the exact test of Hardy Weinberg proportion for multiple alleles Biometrics 48 361 372 Guthmiller J M Vargas K G Srikantha R Schomberg L L Weistroffer P L McCray and Tack 2001 Susceptibilities of oral bacteria and yeast to mammalian cathelicidins Antimicrob Agents Chemother 45 3216 3219 Hammond Jin L Zhong Y Caskey C and Chakraborty 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 189 Holt C Stauffer C Wallin J et al 2000 Practical applications of genotypic Surveys for forensic STR testing Forensic Sci Int 112 91 109 Kalinowski S T 2006 HW QuickCheck an easy to use computer program for checking genotypes for agreement with Hardy Wei
58. 2 72 6 90 4 45 26 2 t A a 0 52 29 0 561 i t 30 t t 30 2 0 14 t 31 2 t t 32 2 1 t 33 2 t a 34 2 0 14 t 42 2 t i 43 2 t t 44 2 0 28 t 45 2 t 1 0 261 46 2 0 14 4 J t 47 2 t t 48 2 0 14 t 50 2 t t 51 2 t t THO1 4 t Ti 5 0 281 0 431 0 171 t 6 11 06 20 49 22 76 20 68 7 42 86 21 78 33 62 43 98 8 20 73 11 46 8 45 5 24 8 3 t t t t 9 t t t 6 28 9 3 11 62 29 08 20 34 23 56 10 0 98 0 431 0 521 0 261 11 t t t qd 13 3 0 14 t t t TPOX 6 6 72 0 141 0 34 T 7 2 24 t 0 34t 0 261 8 36 13 53 30 49 66 37 96 92 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results 5 Population data African U S Native Allele American Caucasian 357 349 E n 191 9 21 15 11 60 7 24 4 19 10 9 24 4 30 4 66 3 40 11 21 43 25 93 27 24 39 27 12 3 08 4 73 10 52 14 92 13 t t t t vWA 11 0 28t t 0 17t t 12 u t t 0 26t 13 1 26 0 43t t 0 26t 14 7 14 8 31 6 90 4 45 15 20 03 11 32 10 00 7 07 16 26 75 23 35 34 31 32 98 17 20 59 24 50 21 55 33 51 18 14 71 22 49 18 45 15 45 19 6 72 8 31 7 07 4 71 20 1 96 1 15 1 38 1 051 21 0 281 t 0 171 0 261 22 0 281 t t t 23 t t t t 24 t 0 14t t t minimum allele frequency 0 796 for the African American database 0 7 for the U S Caucasian database 0 996 for the U S Hispanic database and 1 3 for the Native American databa
59. 320 24 320 42 0 027 0 051 11 324 30 324 45 0 033 0 055 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy precision and reproducibility Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation 12 328 34 328 49 0 036 0 053 13 332 37 332 52 0 033 0 047 14 336 42 336 57 0 038 0 052 15 340 46 340 60 0 036 0 045 0135317 8 216 56 216 75 0 033 0 050 9 220 55 220 72 0 020 0 051 10 224 53 224 70 0 035 0 043 11 228 52 228 70 0 037 0 048 12 232 58 232 76 0 037 0 049 13 236 48 236 66 0 031 0 051 14 240 40 240 60 0 037 0 044 15 244 40 244 59 0 038 0 048 D16S539 5 252 22 252 42 0 040 0 050 8 264 17 264 35 0 030 0 052 9 268 18 268 35 0 040 0 051 10 272 15 272 33 0 031 0 048 11 276 16 276 33 0 034 0 047 12 280 15 280 34 0 039 0 050 13 284 16 284 33 0 032 0 052 14 288 17 288 33 0 029 0 058 15 292 17 292 36 0 037 0 055 D18S51 7 261 88 261 98 0 028 0 045 9 269 99 270 12 0 039 0 058 10 274 08 274 20 0 031 0 045 10 2 276 08 276 20 0 029 0 054 11 278 15 278 28 0 040 0 047 12 282 22 282 35 0 036 0 049 13 286 27 286 40 0 038 0 053 13 2 288 28 288 42 0 040 0 050 14 290 37 290 50 0 033 0 049 14 2 292 39 292 50 0 037 0 053 15 294 47 294 60 0 038 0 050 16 298 55 298 70 0 041 0 053 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide
60. 403 0 7899 0 8424 0 8 0 6806 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 95 Chapter 5 Experiments and Results Population data African U S Native American Caucasian n do An American 357 349 191 0135317 HW X 0 014379 0 711127 0 353995 0 813948 HW G2 p 0 609389 0 871173 0 190736 0 814681 HW Exact p 0 3818 0 667 0 2415 0 6851 HExp 0 6977 0 7797 0 8251 0 8222 0 6695 0 7364 0 8207 0 8168 D16S539 HW X 0 433216 0 67702 0 058631 0 996396 HW G2 p 0 482435 0 594871 0 37601 0 981384 HW Exact p 0 3753 0 4328 0 3068 0 9986 HExp 0 7939 0 7632 0 7747 0 7766 0 8203 0 7822 0 7828 0 7853 018551 HW X 0 999844 0 628334 0 999203 0 343027 HW G2 p 1 0 872113 0 999492 0 798859 HW Exact p 0 978 0 0982 0 9152 0 2265 HExp 0 8694 0 8769 0 8761 0 8463 0 8824 0 8682 0 8862 0 8377 0195433 HW X 0 91703 0 806717 0 731222 0 810711 HW G2 p 0 83419 0 999765 0 975476 0 898389 HW Exact p 0 4517 0 69 0 3475 0 4301 HExp 0 8364 0 7659 0 8310 0 8430 0 8011 0 7622 0 8414 0 822 021511 HW X p 0 985687 0 936146 0 0 HW G2 p 1 0 999757 0 999794 0 712937 HW Exact p 0 7627 0 7861 0 6476 0 0118 HExp 0 8585 0 8427 0 8290 0 8003 0 8711 0 8567 0 7931 0 801 FGA HW X p 0 0 904953 0 263223 0 999686 HW G2 p 1 0 999812 0 960137 0 999946 HW Exact p 0 9761 0 4459 0 0
61. 55818 1 01851 yellow 26449 3500 15 19 FGA 14 AMEL red 1080 1140 x 15 055818 red 1280 1800 11 amp JiReference Samples 16 FGA red 208 25 360 0 2324 38 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis 8 Select D8S1179 to display the Bin view for the marker in the right pane Panel Manager File Edit Bins View BH x 8111 Bin Set aentitierbirect 65500 Bins mEnE ji IdentifilerDirect GS500 v1 EE IdentifilerDirect GS500 Panels v 7 8 9 10 11 12 13 10 021911 o D75820 5 1 03 351358 THO1 3 p135317 08 0165539 E 0251338 07 0195433 L VW 2 TPOX 0 6 5 D18551 a AMEL D55818 0 5 L FGA 04 E Reference Samples 03 02 9 Click Apply then OK to add the Identifiler Direct Kit panel and bin set to the GeneMapper ID Software database IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper ID Software database AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 39 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Create an analysis To create an HID analysis method for the Identifiler Direct Kit method 1 Select Tools GeneMapper Manager
62. 891 0 9161 HExp 0 8659 0 8686 0 8751 0 8746 0 8824 0 8854 0 8724 0 8482 96 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Concordance studies Mutation rate Chapter 5 Experiments and Results 5 Mutation rate African U S Native American Caucasian 357 349 E n 191 THO1 HW X 0 961911 0 997905 0 649467 0 329461 HW G2 p 0 940414 0 99169 0 617212 0 318591 HW Exact p 0 8286 0 9716 0 4495 0 1377 HExp 0 7323 0 7866 0 7666 0 7016 0 7395 0 7822 0 8103 0 6492 HW X 0 765163 0 801518 0 875348 0 333914 HW G2 0 611014 0 757735 0 913091 0 229017 HW Exact p 0 7247 0 5775 0 8356 0 0647 HExp 0 7643 0 6311 0 6607 0 6765 0 7563 0 6304 0 6759 0 6178 HW X 0 925176 0 005048 0 641684 0 994248 HW G2 p 0 964308 0 218817 0 934427 0 997184 HW Exact p 0 7033 0 0564 0 7066 0 8845 HExp 0 8141 0 8081 0 7818 0 7457 0 8571 0 8138 0 7759 0 7277 We compared allele calls between the Identifiler and Identifiler Direct Kits The genotype data from the 200 analyzed treated paper workflow samples showed 100 concordance between the Identifiler and Identifiler Direct Kits The genotype data from 84 buccal samples processed using Prep n Go Buffer and the Identifiler Direct Kit showed 100 concordance to allele calls generated for purified DNA samples analyzed with
63. Amplification Kit User Guide 9 Product overview About the primers Chapter 1 Overview The Identifiler Direct Kit employs the same primer sequences as used in the AmpF STR Identifiler PCR Amplification Kit Degenerate primers for the loci D8S1179 vWA 0165539 are included in the AmpF STR Identifiler Direct Primer Set to address mutations in the primer binding sites The addition of the degenerate primers allows for the amplification of those alleles in samples containing the mutations without altering the overall performance of the Identifiler Direct Kit Non nucleotide linkers are used in primer synthesis for the following loci CSF1PO 0135317 0165539 0251338 For these primers non nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis Loci amplified by Butler 2005 Grossman et al 1994 and Baron et al 1996 Non nucleotide linkers enable reproducible positioning of the alleles to facilitate inter locus spacing The combination of a five dye fluorescent system and the inclusion of non nucleotide linkers allows for simultaneous amplification and efficient separation of the 15 STR loci and Amelogenin during automated DNA fragment analysis Table 1 shows the loci amplified their chromosomal locations and the corresponding the kit fluorescent marker dyes The AmpF STR Identifiler
64. Biological hazard safety A WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best pract
65. Genetic Analyzers 4410231 8 Strip Septa for 3500 3500xL Genetic Analyzers 4410701 96 Well Septa for 3500 3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container 3500 series 4410715 For a complete list of parts and accessories for the 3500 3500xL instrument refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide PN 4401661 3730 Analyzer materials 3730 DNA Analyzer Capillary Array 36 cm 4331247 GeneScan 500 LIZ Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Hi Di Formamide 4311320 Running Buffer 10X 4335613 DS 33 Matrix Standard Kit Dye Set G5 4345833 96 Well Plate Septa 4315933 MicroAmp Optical 96 Well Reaction Plate N8010560 POP 7 Polymer for the 3730 Genetic Analyzer 4332241 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 105 Appendix B Ordering Information Equipment and materials not included Source For a complete list of parts and accessories for the 3730 instrument refer to Appendix of the Applied Biosystems 3730 3730xl DNA Analyzer Getting Started Guide Part no 4359476 PCR Amplification MicroAmp 96 Well Tray N8010541 MicroAmp Reaction Tube with Cap 0 2 mL N8010540 MicroAmp 8 Tube Strip 0 2 mL N8010580 MicroAmp 8 Cap Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96
66. ID Software for data analysis Peak Detector tab settings 42 Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced I rRanges Analysis Sizing Full Range m All Sizes Start Pt fo Start Si Stop fi 0000 5 and Baselining Smoothing C None Light C Heavy Baseline Window 5 pts r Size Calling Method C 2nd Order Least Squares C 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method C Global Southern Method r Peak Detection Peak Amplitude Thresholds R G Y Min Peak Half Width Polynomial Degree Peak Window Size Slope Threshold 34 Peak Start Peak End mm p m s ms E x Factory Defaults Perform internal validation studies to determine settings IMPORTANT Perform the appropriate internal validation studies to determine the peak amplitude thresholds for interpretation of Identifiler Direct Kit data Fields include e Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these p
67. NA Analyzer Dye Set G5 RCT Human Identification Validation Report t We conducted concordance studies for the Identifiler Direct Kit using this configuration Contact your sales or support representative to obtain a copy of the 3730 DNA Analyzer Human Identification Validation Report Prepare samples for electrophoresis on the 3730 instrument Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Reagent reaction reaction GeneScan 500 0 3 uL OR GeneScan 600 0 5 uL LIZ Size Standard LIZ Size Standard v2 0 Hi Di Formamide 8 7 uL Hi Di Formamide 8 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your experiments and results AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 33 wo A wo e 5 e x is 3 0 5 Le 3 Chapter 3 3730 Instrument Prepare samples for electrophoresis on the 3730 instrument 2 Pipet the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each well of a MicroAmp Op
68. OP 49 Polymer for 3130 3130xl Genetic Analyzers 4352755 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 104 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Appendix B Ordering Information Equipment and materials not included Item Source GeneScan 500 LIZ9 Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10X 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 Hi Di Formamide 4311320 For a complete list of parts and accessories for the 3130xl instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide Part no 4352716 3500 3500xL Analyzer materials Anode buffer container 4393927 Cathode buffer container CBC 4408256 POP 4 polymer 960 samples for 3500 3500xL Genetic Analyzers 4393710 POP 4 polymer 384 samples for 3500 3500xL Genetic Analyzers 4393715 DS 33 Matrix Standard Kit Dye Set G5 4345833 GeneScan 600 LIZ Size Standard v2 0 4408399 Conditioning reagent 4393718 8 Capillary array 36 cm for 3500 Genetic Analyzers 4404683 24 Capillary array 36 cm for 3500xL Genetic Analyzers 4404687 96 well retainer amp base set Standard 3500 3500xL Genetic Analyzers 4410228 8 Tube retainer amp base set Standard for 3500 3500xL
69. PCR 26 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide prepare reactions 23 sample preparation guidelines 23 samples 9 types 9 validation 85 I technical support 120 Terms and Conditions 120 thermal cyclers for use with kit 110 programming 26 training information 120 treated paper 19 PCR 26 prepare reactions 19 sample preparation guidelines 19 U untreated paper 21 PCR 26 prepare reactions 21 sample preparation guidelines 21 V validation characterization of loci 79 developmental 64 effect of DNA quantity 82 experiments to evaluate 63 importance of 63 mutation rate 97 population data 86 probability of identity 98 probability of paternity exclusion 99 sensitivity 81 size deviation sample and ladder alleles 64 species specificity 80 swabs 85 W warranty 120 work area amplified DNA 107 110 PCRsetup 109 setup and lab design 109 workflow overview 12 Index 123 Index 124 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support technologies www lifetechnologies com A Thermo Fisher Scientific Brand 5 February 2015
70. Perform Homozygous min peak height internal Heterozygous min peak height validation studies to Heterozygote balance determine settings Min peak height ratio morphology Max peak width basepairs j 2 rr Pull up peak Pull up ratio Joos Allele number 2 Max expected alleles Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of Identifiler Direct Kit data AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 43 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Quality Fla gs tab Analysis Method Editor HID settings IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform the appropriate internal validation studies to determine the appropriate values to use in your laboratory 44 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Create a size The size standards for the Identifiler Direct Kit use the following size standard peaks standard in their definitions GeneScan 500 LIZ Size Standard peak GeneScan 600 LIZ Size Standard v2 0
71. Software for data analysis d Select IdentifilerDirect GS500 Bins vl1 txt then click Import Note Importing this file associates the bin set with the panels in the IdentifilerDirect GS500 Panels v1 folder C Import Bin Set Look in a Identifiler Direct Analysis Files GMID AEE identitilerDirect_Gss in idertifierDirect GS500 Panels v1 bd 2 ReadMe IDDirect v1 txt File name Jaentitierbirect 65500 Bins v1 tt Import Fies ot type an ries Cancel 7 View the imported panels in the navigation pane a Double click the IdentifilerDirect GS500 v1 folder to view the IdentifilerDirect GS500 Panels v1 folder b Double click the IdentifilerDirect GS500 Panels v1 folder to display the panel information in the right pane 2 Panel Manager File Edit Bins View Maker Nene Dye Color Min Size Max Size Control Alleles 1 eue nso fess fis Foes 2 pas bue 1845 2475 30 gen D7S820 pue 2910 2985 10 11 _ 1 4 CSF1PO bue 30242 34863 1012 351358 5 351358 green 580 1480 145 a green 1590 2050 833 0135317 green 20565 25016 t 0251338 D16s539 gren 2553 30181 1142 0195433 0251338 green 3048 37031 1923 pe 10 0195433 yelow 1010 1480 1445 Ee 11 wa velow 151 0 2135 firas AMEL 12 yellow 216 99 260 99 8 0
72. USER GUIDE applied biosystems Fe technologies AmpF STR Identifiler Direct PCR Amplification Kit for use with 200 reaction kit Part no 4467831 1000 reaction kit Part no 4408580 Publication Part Number 4415125 Rev J technologies For Forensic or Paternity Use Only Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Important Licensing Information These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses TRADEMARKS 2015 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified AmpliTaq Gold is a registered trademark of Roche Molecular Systems Inc Windows and Windows Vista are
73. Well Base N8010531 MicroAmp Clear Adhesive Film 4306311 MicroAmp Optical Adhesive Film 4311971 MicroAmp Optical 96 Well Reaction Plate N8010560 Other user supplied materials Hi Di Formamide 25 mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Vortex MLS t For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions 106 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide C Plate Layouts Example PCR plate layout The following layout is recommended for use with the sensitivity experiment on page 17 Create 3 identical plates for amplification at 3 different cycle numbers ELE tee tet te te pepo te samp 1 samp 8 Samp 15 Samp 15 Samp 22 samp 22 do i f Ees pee e a JL 5 A EEE EEE NI Seet mar pL oL HETI Pp EE EEE i 1 1 ol Example electrophoresis plate layout The following layout is recommended for use with the sensitivity experiment on page 17 Cycle 1 Cycle 2 Cyde 3 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 107 Appendix C Plate Layouts Example
74. ale w dad eee Rx da 85 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 5 Contents Popula tion datain doc E npe cava 86 SWGDAM guideline 27 e 86 OVERVIEWS zoe er nee san xa Patel DUE RE eU REL mr 86 Population samples used in these 5 86 Identifiler Direct Kit allele frequencies 86 Evaluation of Hardy Weinberg equilibrium WWW kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK 94 Conceordance st dles lt i ence ADDE LhTeep Sed ASOYA Y ban eid 97 Mutation rate SEEVD DDB B gt P gt P gt gt P gt P gt 0 gt DDDL pD XXBD gt ZDI AMIN NNVN M MZMI I N MJ J MMDMMIM NNN NN MM 97 Additional mutation studies k kk kk kk kk KK kK KK KK KK KK KK KK KK KK KK kk kk kk kk kk kk 98 Probability of ide niya Ai ka ettet nk e w EI Cer RE tU 98 Probability of paternity exclusion WWW kk kk kk kk kk kk kk KK KK KK KK KK KK KK kK KK kk kk kk kK kk kk ka 99 APPENDIX 101 APPENDIX B Ordering Information 103 Equipment and materials not included 020c cece eee eee eee eens 103 APPENDIX Plate Layouts 107 Example PCR plate layout u me eee te ie tava Mia oi RT
75. alysis as a completion bar extending to the right with the percentage completed indicated The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Analysis Summary tab is displayed upon completion of the analysis The figure below shows the analysis summary window after analysis ID X Identifiler Direct Example gmidx Is Logged In Database GBOLDROYNJO9E File Edit Analysis View Tools Admin Help DEB mu B Table setting 31x Data Analysis gB Project amp C3dentifiler Direct Example Analysis Completed Samples Analysis Summary Genotypes Analysis Summary Select run folder to display Sample Status Total of Samples lr Unanalyzed 0 Analyzed 8 p Analysis Setting Changed __ Mm D Click a link below to display a filtered Samples Table containing only the samples selected Allelic Ladder Quality per run folder based on SQ and CGQ only Run Folder JL of Analyzed Ladders Le a Identifiler Direct Example Data 2 2 o0 0 Control Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Control Total of Samples 8 Al thresholds met __ One or more thresholds not met Positive Control 0 0 0 Custom Control 0 Negative Control 0 Total 0
76. and Streptococcus viridans equivalent to 105 copies These microorganisms are commonly found in the oral cavity Suido et al 1986 Guthmiller et al 2001 All the primate DNA samples amplified producing fragments within the 100 to 350 base pair region Lazaruk et al 2001 Wallin et al 1998 The microorganisms chicken cat hamster rat rabbit and mouse samples did not yield detectable product Horse cow dog and pig samples produced a 104 bp fragment near the Amelogenin locus in PET dye When appropriate the range of DNA quantities able to produce reliable typing results should be determined SWGDAM July 2003 The Identifiler Direct Kit has been optimized at 25 uL PCR reaction volume to overcome the PCR inhibition expected when amplifying blood samples directly from unpurified 1 2 mm FTA discs Depending on the volume of blood spotted onto the card DNA quantities present on the 1 2 mm disc may vary from laboratory to laboratory It is essential for your laboratory to optimize the PCR conditions based on the types of blood samples received or based on your standard operating protocol used in the spotting of blood onto FTA cards Refer to page 17 for instructions on PCR optimization The Identifiler Direct Kit has been optimized at 25 uL PCR reaction volume to overcome the PCR inhibition expected when amplifying buccal cells directly from unpurified 1 2 mm FTA discs or Indicating FTA discs Depending o
77. ative for information This product is not available for sale in the US 96 well deep well plate 4392904 Table 11 User supplied materials Item Source AmpF STR Identifiler Direct PCR Amplification Kit 200 reaction 4467831 AmpF amp STR Identifiler Direct PCR Amplification Kit 1000 reaction 4408580 Prep n Go Buffer untreated paper substrate 4467079 Prep n Go Buffer buccal swab 4471406 3100 Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130xl Genetic Analyzer Capillary Array 36 cm 4315931 POP 4 Polymer for 3100 3100 Avant Genetic Analyzers 4316355 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10X 402824 Hi Di Formamide 4311320 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 250 uL Glass Syringe array fill syringe 4304470 5 0 mL Glass Syringe polymer reserve syringe 628 3731 For a complete list of parts and accessories for the 3100 instrument refer to Appendix B of the 3700 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide Part no 4335393 3130xl Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130xl Genetic Analyzer Capillary Array 36 cm 4315931 P
78. available from major laboratory suppliers MLS Table 10 Equipment Equipment Source 3100 3100 Avant Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3730 Genetic Analyzer Contact your local Life Technologies sales representative GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block 4314878 Silver 96 Well Sample Block N8050251 Gold plated Silver 96 Well Sample Block 4314443 Veriti 96 Well Thermal Cycler 4375786 ProFlex 96 Well PCR System 4484075 Tabletop centrifuge with 96 Well Plate Adapters optional MLS Harris Manual Punch 1 2 mm MLS CPA200 Semi Automated Punch Instrument with a 1 2 mm punch head CPA300 Fully Automated Punch Instrument with a 1 2 mm punch head Contact your local Life Technologies support representative for information Bode Buccal DNA Collector 4467893 This part number is not available for sale in the US Copan FLOQSwabs9 Contact your local Life Technologies support representative for information AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 103 Appendix B Ordering Information Equipment and materials not included Equipment Source NUCLEIC CARD system Contact your local Life Technologies support represent
79. cale may be unusually high Off scale peaks were not included in the evaluation of stutter characterized here Figure 4 Treated paper workflow FTA9 card sample stutter percentages for D8S1179 021511 075820 and CSF1PO loci red blood samples blue buccal samples o o 2 a t 9 o a o DORPER gem eS o hd o o o ONTENTS co 9 Om 400 O e e qomman oo e oog cO PENES 6 oo wap gee 8 9 10 1112 13 14 15 16 17 24 25 26 27 28 29 30 3132 33 34 35 36 6 7 8 9 10 1112 13 14 15 7 8 9 10 1112 1314 15 D8S1179 D21S11 D7S820 CSF1PO Figure 5 Treated paper workflow FTA card sample stutter percentages for 0351358 TH01 0135317 0165539 and 0251338 loci red blood samples blue buccal samples Percent Stutter e H La H 4 L 8 E oo 11121314 1516 17 18 1920 567891011 8 9 101112131415 8 9 1011121314 1516 17 18 1920 21222324 25 26 27 28 0351358 1 D13S317 0165539 0251338 74 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 5 Experiments and Results Extra peaks in the electropherogram Figure 6 Treated paper workflow FTA9 card sample stutter percentages for 0195433 vWA TPOX and D18S51 loci red blood samples blue buccal samples e nh ET e o b omo QJ ONE Gem 8999699
80. ction mix for 3 seconds then centrifuge briefly Dispense 25 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells place a MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold plated silver 96 well block or a Veriti 96 well Thermal Cycler or a ProFlex PCR System as described in Perform PCR on page 26 IMPORTANT The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96 well block Use of this thermal cycling platform may adversely affect performance of the Identifiler Direct Kit AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 2 Perform PCR Untreated paper substrates prepare reactions Untreated paper substrates prepare reactions Sample prep guidelines Prepare the reactions Make the punch as close as possible to the center of the sample to ensure optimum peak in
81. dentifiler Direct Kit data 56 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Peak Quality tab settings Analysis Method Editor Section 4 2 ID X Software Set up GeneMapper ID X Software for data analysis General Allele Peak Detector Peak Quality 5Q amp GQ Settings Min Max Peak Height LPH MPH Homozygous min peak height Perform Heterozygous min peak height ntern al E efi validation Max Peak Height MPH studies to determine Peak Height Ratio PHR s etti n g s Min peak height ratio Broad Peak BD Max peak width basepairs Allele Number AN Max expected alleles Allelic Ladder Spike C 0 2 lt o 0 e v gt n e e 2 0 Spike Detection Enable v Cut off Value 0 2 IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds maximum peak height threshold and the minimum peak height ratio threshold for interpretation of Identifiler Direct Kit data AmpFtSTR9 Identifiler Direct PCR Amplification Kit User Guide 57 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis SQ amp GQ tab Analysis Method Editor setti n gs General Allele Peak Detector Peak Quality 5Q amp GQ Settings Quality weig
82. 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 AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 83 Chapter 5 Experiments and Results Stability DNA on FTA cards Aged blood on FTA cards and aged buccal cells on Indicating FTA cards were prepared to examine the sample on substrate stability Finger prick blood spotted onto FTA card and buccal samples swabbed and transferred using the EasiCollect devices were collected on three individuals over the course of 30 weeks The Identifiler Direct Kit was used to amplify the aged FTA samples in a GeneAmp PCR System 9700 with the gold plated silver 96 well block and were electrophoresed and detected using an Applied Biosystems 3130xl Genetic Analyzer The results of the aged blood on FTA card are shown in Figure 15 and the results of the aged buccal cells on Indicating FTA card are shown in Figure 16 The analysis revealed that the age of the FTA samples did not impact the performance of the AmpF STRO Identifiler Direct Kit Figure 15 Amplification o
83. der injections which should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment It is critical to genotype using an allelic ladder run under the same conditions as the samples because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions 28 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis Appendix B Ordering Information on page 103 lists the required materials not Reagents and parts supplied with the Identifiler Direct Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Electrophoresis software setup and the documents listed in the table The following table lists data collection software and the run modules that can be used to analyze Identifiler Direct Kit PCR products For details on the procedures refer to
84. e 12 Omitting the final extension step results in split peaks due to incomplete A nucleotide addition Data are from an Applied Biosystems 3130xl Genetic Analyzer using the AmpF STR Identifiler Direct Kit 130 No Extension Artifacts Artifacts and anomalies are seen in all molecular biological systems Artifacts are typically reproducible while anomalies are non reproducible intermittent occurrences that are not observed consistently in a system for example spikes and baseline noise Reproducible artifacts have not been seen in data produced on the genetic analyzers used during developmental validation of the Identifiler Direct Kit Characterization of loci SWGDAM guideline 2 1 Nature of the polymorphisms The basic characteristics of a genetic marker must be determined and documented SWGDAM July 2003 This section describes basic characteristics of the 15 loci and the sex determining marker Amelogenin that are amplified with the Identifiler Direct Kit These loci have been extensively characterized by other laboratories The primers for the Amelogenin locus flank a 6 nucleotide deletion within intron 1 of the X homologue Amplification results in 107 nt and 113 nt products from the X and Y chromosomes respectively Sizes are the actual nucleotide size according to sequencing results including 3 A nucleotide addition The remaining Identifiler
85. e loci where the rates are not large enough to be measured directly and or to account for those events undetectable as Mendelian errors Probability of identity Table 7 shows the Probability of Identity Pj values of the Identifiler Direct Kit loci individually and combined Table 7 Probability of Identity values for the Identifiler9 Direct Kit STR loci Locus Brian p U S Hispanic Native American Caucasian American CSF1PO 0 079 0 132 0 141 0 123 D2S1338 0 023 0 027 0 038 0 043 D3S1358 0 097 0 076 0 112 0 158 D5S818 0 104 0 147 0 115 0 110 D7S820 0 085 0 063 0 083 0 081 D8S1179 0 074 0 064 0 089 0 104 D13S317 0 132 0 079 0 056 0 056 D16S539 0 077 0 097 0 090 0 082 D18S51 0 033 0 031 0 031 0 046 D19S433 0 042 0 087 0 049 0 044 D21S11 0 037 0 044 0 047 0 074 FGA 0 034 0 035 0 032 0 031 THO1 0 109 0 079 0 097 0 134 TPOX 0 089 0 188 0 168 0 159 vWA 0 066 0 066 0 080 0 103 Combined 1 31 X 10 18 5 01 x 10 18 7 65 X 10 18 3 62 X 10 17 The Pr value is the probability that two individuals selected at random will have an identical Identifiler Direct Kit genotype Sensabaugh 1982 The P values for the populations described in this section are then approximately 1 7 64 X 1017 African American 1 2 00 X 1017 U S Caucasian 1 1 31 X 1017 U S Hispanic and 1 2 76 X 1016 Native American 98 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Probability
86. e typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems 3130xl Genetic Analyzer with POP 4 polymer The x axis in Figure 3 represents the nominal nucleotide sizes for the AmpF STR Identifiler Direct Allelic Ladder The dashed lines parallel to the x axis represent 0 25 nt windows The y axis represents the deviation of each sample allele size from the corresponding allelic ladder allele size All sample alleles are within 0 5 nt from a corresponding allele in the allelic ladder AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy precision and reproducibility Figure3 Size deviation of 200 blood samples on card analyzed on the Applied Biosystems 3130xl Genetic Analyzer CSFIPO 0251338 0351358 055818 075820 0851179 0135317 0165539 018551 0195433 021511 FGA THO TPOX vWA AMEL 0 n 9 O O0 O 2 c S 0 00 UTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 100 120 140 160 180 200 220 240 260 280 300 320 340 360 Allele Size nt Precision and size Sizing precision allows for determining accurate and reliable genotypes Sizing windows precision was measured on the Applied Biosystems 3130 1 Genetic Analyzer The recommended method for genotyping is to employ a 0 5 nt window around the size ob
87. eaks e Size calling method The Identifiler Direct Kit has been validated using the Local Southern sizing method Before using alternative sizing methods perform the appropriate internal validation studies AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis 3500 3500xL and 3730 data 3500 3500xL and 3730 data Overall peak heights for the data are approximately 3 times higher than peak heights obtained for samples run on the 31xx series instruments Evaluate validation data carefully to determine the appropriate Peak Amplitude Thresholds for reliable analysis 3730 data only Due to differences in the resolution of peaks using POP 7 polymer versus POP 49 polymer reduce the Peak Window Size setting in GeneMapper ID Software from 15 pts to 11 pts to obtain accurate genotyping results For more information Refer to User Bulletin Applied Biosystems 3500 3500xL Genetic Analyzer Protocols for Analysis of AmpFtSTR PCR Amplification Kit PCR Products and Validation Summary Part no 4469192 Contact your sales or support representative to obtain a copy of the 3730 DNA Analyzer Human Identification Validation Report D lt 97 o 9 5 90 E E Peak Quality tah x settings 9 General Allele Peak Detector Peak Quality quality Flags Signal level
88. efly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Prepare the plate assembly on the autosampler Start the electrophoresis run AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 3 2 3500 3500xL instruments 3 Set up the 3500 3500xL instruments for electrophoresis Section 3 2 3500 3500xL instruments Set up the 3500 3500xL instruments for electrophoresis Reagents and parts Appendix B Ordering Information on page 103 lists the required materials not supplied with the Identifiler Direct Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum e a 55 eb a e x E 3 Q e 3 5 Z Electrophoresis The following table lists data collection software and the run modules that you can use software setupand to analyze Identifiler Direct Kit PCR products For details on the procedures refer to the documents listed in the table reference documents Genetic pata Operatin Collection g Run modules and conditions References Analyzer System Software Applied 3500 Data Windows HID36_POP4 Applied Biosystems 3500 Bi
89. electropherogram ssssssssslslllllllllssl kK kk kK kk kk kk k 73 alt s s of xtra p aKS ac ana h ke kh le WER Yek Meee bene ER CA kih dark 73 St tter ProdUCtS E sks Saas bac os nade une ERU ee 73 Addition of 3 Anucleotide Eka xal s 78 unsere nte x m a ee d eel ih send 79 Characterization G locl i cieli Walk Ka Wind eph Pelee eine s 79 SWGDAM guideline 2T vi PURIS y PU epe rem way kr edt erii 79 Nature of the polymorphisms nn nassaan kk kk kk kk kk kk kk kk kk kk kk kk kk kk 79 MAP DING ooi Eh o t E ee ebrei ote pons iet 80 Species Specificity unir eic RA RR ENSE A MJ 80 SWGDAM Guideline 2 2 kk kk kk kk kk kk kk kk kk 80 SID Na EE 81 SWGDAM guideline 2 3 a n nu nek kK Ka kk k K kk k lek kk k l kk e 81 Bi6 d n FTA Cards ees e he REEL Rae ean tus 81 Buccal cells on FTA or Indicating FTA cards and buccal cells on Bode DNA Collectors 81 Effect of DNA quantity on results kk kk kk kk kk kk kk kk KK kK KK kk KK KK KK KK kk kk kk kk kk lk 82 Stabilityz s ss ee DD tactu uS 83 SWGDAM guideline 24 occie ree aie eaa kk eye ee kela i ekl hr 83 DNA on cinta Seah enr qe cT Opine 84 DNA on buccal Swabs Re e eR a
90. electrophoresis plate layout 108 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide PCR Work Areas Work area setup and lab design Lek KK KK KK KK KK KK KK KK KK KK KK KK 109 PCR Sel piWOklK alf ya 502y k n mik s Wale eee enn eens 109 Amplified DNA work area KK KK KK KK KK KK KK KK 110 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory If you are using the AmpF STR Identifiler Direct PCR Amplification Kit for Forensic DNA testing refer to Forensic Laboratories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 Parentage DNA testing refer to the Guidance for Standards for Parentage Relationship Testing Laboratories American Association of Blood Banks 7th edition 2004 The sensitivity of the Identifiler Direct 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 contamination by human DNA Wear gloves at all times and change them frequently Close sample tubes when not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note We do not intend these references for laboratory design to constitute all precautions and care necessary for
91. en runs on different instruments of the same platform type because of these factors We recommend strongly that the allele sizes obtained should be compared to the sizes obtained for known alleles in the AmpF STR Identifiler Direct Allelic Ladder from the same run and then converted to genotypes as described in Before you start on page 35 and 48 Refer to Table 3 for the results of five runs of the AmpF STR Identifiler Direct Allelic Ladder For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 In Table 3 the mean sizes for all the alleles in each run 16 capillaries were calculated The mean range shown in the table represents the lowest and highest mean size values obtained across all five runs Similarly the standard deviation for the allele sizing was calculated for all the alleles in each run The standard deviation range shown in Table 3 represents the lowest and highest standard deviation values obtained across all five runs Table 3 Precision results of five runs 16 of the AmpF STR Identifiler Direct Allelic Ladder Applied Biosystems 3130xl Genetic Analyzer Allele Mean Standard deviation Amelogenin X 106 26 106 43 0 033 0 044 Y 111 92 112 06 0 032 0 046 CSF1PO 6 304 04 304 20 0 038 0 053 7 308 09 308 26 0 033 0 052 8 312 15 312 32 0 038 0 047 9 316 20 316 37 0 033 0 048 10
92. entifiler Direct PCR Amplification Kit User Guide Documentation and Support Related documentation Document title A 3100 3100 Avant Data Collection v2 0 User Guide 4347102 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin 4350218 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpFtSTR9 PCR Amplification Kit PCR Products 4332345 User Bulletin Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation User Bulletin 4440754 ProFlex PCR System Kit Validation User Bulletin 100031595 Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection Software v3 0 User Bulletin 4363787 Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide 4352716 Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 Applied Biosystems 3130 3100xl DNA Analyzers User Guide 4331468 Applied Biosystems 3500 3500xL Genetic Analyzer Quick Reference Card 4401662 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Data Collection v1 0 4401661 Applied Biosystems 3500 3500xL Genetic Analyzer User Bulletin Solutions to i
93. er Guide Part no 4338775 46 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software Examine and edit a project 3 Click gt Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis During a run The status bar displays the progress of analysis as both A completion bar extending to the right with the percentage completed 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 The Genotypes tab becomes available after analysis fGeneMapper ID v3 2 1 IdentifilerDirect Example gmid Is Logged In File Edit Analysis View Tools Help I Project identifier Di ei 2 d E Ii FI Ej rave seting Ho r m Bi Samples Genotypes Status Sample File Sample Name Sample Type Analysis Method Panel Size Standard 1 lx Identifiler FT amp 01 A01 fsa Identifiler 01 Sample IdentifilerDirect amp nalysisMethod v1 IdentifilerDirect GS500 Panels v1 CE GS ldentifiler 2 lx Identifiler FT amp 02 B01 fsa Identifiler 02 Sample IdentifilerDirect AnalysisMethod v1 IdentifilerDirect GS500 Panels v1 CE G5 Identifier 3 Identifiler Q3 C01 fsa Identifiler 03 Sample IdentifilerDirect AnalysisMethod v1 Identif
94. f blood on FTA card stored for various amounts of time at room temperature 84 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide DNA on buccal swabs Chapter 5 Experiments and Results 5 Stability Aged buccal cell samples on 4N6FLOQSwabs Whatman OmniSwabs and Puritan swabs were also prepared to verify their respective sample on substrate stability Buccal swabs were collected from 40 individuals on each swab type over the course of three months The aged swab samples were processed with Prep n Go Buffer amplified using the Identifiler Direct Kit in a GeneAmp PCR System 9700 with the gold plated silver 96 well block and were electrophoresed and detected using an Applied Biosystems 3130xI Genetic Analyzer Figure 17 shows the results of the aged buccal samples collected on each swab type and lysed at 90 C for 20 minutes For comparison Figure 18 shows the results of fresh buccal samples collected on Copan 4N6FLOQSwabs and lysed at room temperature The analysis revealed that buccal samples on the swab types tested air dried immediately after collection and aged up to three months at room temperature produce acceptable profiles when amplified with the Identifiler Direct Kit Figure 17 Amplification of buccal cells on aged Copan 4N6FLOQSwabs OmniSwabs and Puritan swabs stored for 3 months at room temperature and lysed with Prep n Go Buffer at 99 C for 2
95. frequency in population 93 mapping 80 lysate prepare 23 M marker stutter import 49 master mix volume per reaction 19 21 23 materials and equipment 14 materials not included with kit 103 multicomponent analysis 13 mutation studies 98 mutation STR 97 N normalization 56 0 off ladder alleles 65 operating systems 29 31 33 P panels import 36 49 PCR optimize cycle number 17 perform 26 setup 109 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide PCR work areas 103 109 percent stutter off scale peaks 74 relation to allele length 73 precision and size windows 65 precision sizing 65 Prep n Go Buffer 9 17 21 primers about 10 Amelogenin 79 volume per reaction 19 21 23 probability of identity definition 98 values 98 project examination and editing 61 punches size 19 21 R reaction mix for PCR 20 22 24 reactions prepare for PCR 19 21 23 reagents not included with kit 103 run module electrophoresis 29 31 33 S safety biohazard 112 chemical 112 sample discs size 19 21 sample files fsa 36 49 size deviation sample alleles and ladder alleles 64 size standard create 45 58 GS 50 and GS 600 15 size standard create 45 58 sizing precision 65 species specificity 80 split peaks nucleotide addition 78 standards for samples 15 STRBase 86 stutter products 73 substrates swab 23 treated paper 19 untreated paper 21 support obtaining 120 swab
96. g in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 Select Tools Panel Manager Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane Panel Manager File Edit Bins View Help NEN Panel Manager b Select File Import Panels to open the Import Panels dialog box c Navigate to then open the Identifiler Direct Analysis Files GMIDX folder that you unzipped in step 1 AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 49 C 0 2 0 lt RV o 0 p e v gt ee e p 2 0 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis 5 Select IdentifilerDirect GS500 v1X then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager IdentifilerDirect GS500 Panels v1X This folder contains the panel and associated markers Import Panels Identifiler Direct Analysis Files GMIDX e EE E 1dent filerDirect GS500 Bins vix txt IdentifilerDirect GS500 Panels v1X txt E IdentifilerDirect_GSS00_Stutter_v1X txt E ReadMe IDDirect vix txt IdentifilerDirect GS500 Panels viX txt All Files 6 Import IdentifilerD
97. gents Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the reaction mix for 3 seconds then centrifuge briefly Dispense 25 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate Add samples to the reaction plate Add the following to wells of a MicroAmp Optical Wells 96 Well Reaction Plate Negative control 3 uL of Prep n Go Buffer Test samples 3 uL of lysate Positive control For 25 cycles 3 uL of Control 9947A For 26 and 27 cycles 2 uL of Control DNA 9947A For 28 cycles 1 uL of Control DNA 9947A Note The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells place a MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad Vortex the reaction mix at medium speed for 3 seconds Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide C
98. ggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number Centrifuge the plate to ensure the punches are immersed in the Prep n Go Buffer Calculate the volume of each component needed to prepare the reactions using the table below Reaction component Volume per reaction Master Mix 12 5 uL Primer Set 12 5 uL Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 21 22 Chapter 2 Perform PCR Untreated paper substrates prepare reactions IMPORTANT The Identifiler Direct Kit has been optimized for a 25 uL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples Using a lower PCR reaction volume may reduce the ability of Identifiler Direct Kit chemistry to generate full STR profiles Prepare reagents Thaw the Master Mix and the Primer Set then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 C and therefore do not require subsequent thawing Do not refreeze the reagents Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the reaction mix for 3 seconds then centrifuge briefly
99. hapter 2 Perform PCR Swab substrates prepare reactions 10 Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold plated silver 96 well block or a Veriti 96 well Thermal Cycler or a ProFlex PCR System as described in Perform PCR on page 26 IMPORTANT The Identifiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96 well block Use of this thermal cycling platform may adversely affect performance of the Identifiler Direct Kit Store the sample Cap the sample lysate storage tubes or seal the sample lysate storage plate with lysate MicroAmp Clear Adhesive Film Store the sample lysate as needed If you are storing the sample lysate Then place at lt 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C These storage recommendations are preliminary pending the results of ongoing stability studies The effects of multiple freeze thaw cycles on the lysate have not been fully evaluated Therefore multiple freeze thaw cycles are not recommended AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 25 Perform PCR 26 Chapter 2 Perform PCR Program the thermal cycling conditions Whenusing the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode Whenusing the Veriti 96 Well Thermal Cycler refer to the following document for instructions on how to configure the Veriti instru
100. hts are between and 1 Sample and Control GQ Weighting Broad Peak BD Out of Bin Allele BIN Overlap OVL Marker Spike SPK Control Concordance CC Weight 1 0 SQ Weighting Broad Peak BD fos Allelic Ladder GQ Weighting Spike SSPK SPK 50 amp GQ Ranges Sizing Quality From 0 75 Genotype Quality From 0 75 Allele Number AN Low Peak Height LPH Max Peak Height MPH Off scale OS Peak Height Ratio PHR Only applicable to controls Off scale OS to 1 0 From 0 0 to 0 25 to 1 0 From 0 0 to 0 25 Reset Defaults IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform appropriate internal validation studies to determine the appropriate values to use Create a size standard The size standards for the Identifiler Direct Kit uses the following size standard peaks in their definitions GeneScan 500 LIZ Size Standard peak sizes GeneScan 600 LIZ Size Standard v2 0 peak sizes 75 100 139 150 160 200 300 350 400 and 450 80 100 114 120 140 160 180 200 214 220 240 250 260 280 300 314 320 340 360 380 400 414 420 440 and 460 Note The 250 nt and the 340 nt peaks in the GeneScan 500 LIZ Size Standard not included in the size standard definition These peaks can be used as an
101. ices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 113 Appendix Safety Biological hazard safety 114 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Bibliography Akane A Matsubara K Nakamura H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 Bonferroni C E 1936 Teoria statistica delle classi e calcolo Belle probabilita Publicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8 3 62 Barber M D and Parkin B H 1996 Sequence analysis and allelic designation of the two short tandem repeat 018551 and 0851179 Intl J Legal Med 109 62 65 Baron H Fung S Aydin A Bahrig S Luft F C Schuster H 1996 Oligonucleotide ligation assay OLA for the diagnosis of familial hypercholesterolemia Nat Biotechnol 14 1279 1282 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 Bender K Farfan M J Schneide
102. ied over from the disc punching tool Clean the disc punching tool thoroughly If necessary include a blank punch step in between the sample punches Incomplete denaturation of double stranded DNA Use recommended amount of Hi Di Formamide and perform heat denaturation step according to the instructions in Chapter 3 Perform Electrophoresis Some but not all loci visible on electropherogram of DNA Test Samples Disc size used in the amplification reaction was greater than 1 2 mm Repeat amplification using a use 1 2 mm punch size Insufficient volume of swab lysate added to the reaction Repeat amplification using the recommended lysate input volume Less than 25 uL of PCR reaction volume was used Repeat amplification using the recommended PCR reaction volume of 25 pL STR profiles contain many off scale alleles PCR cycle number was too high Perform sensitivity experiment page 17 to determine the optimal PCR cycle number based on the sample type For blood samples Too much liquid blood was spotted onto paper substrate Spot 100 uL of liquid blood per sample area 102 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Equipment and materials not included Ordering Information Table 10 and Table 11 list required and optional equipment and materials not supplied with the Identifiler Direct Kit Unless otherwise noted many of the items are
103. ilerDirect GS500 Panels v1 CE G5 Identifier 4 Lg Identifiler FT amp 04 D01 fsa Identifiler 04 Sample IdentifilerDirect A amp nalysisMethod v1 IdentifilerDirect GS500 Panels v1 CE G5 Identifier 5 w Identifiler FT amp 05 E01 fsa Identifiler FT amp 05 Sample IdentifilerDirect AnalysisMethod v1 IdentifilerDirect GS500 Panels v1 CE G5 Identifier 5 ln Identifiler FT amp Q6 F01 fsa Identifiler FT amp 06 Sample IdentifilerDirect AnalysisMethod v1 IdentifilerDirect GS500 Panels v1 CE G5 Identifier 7 lx Ladder G01 fsa Ladder Allelic Ladder IdentifilerDirect amp nalysisMethod v1 IdentifilerDirect GS500 Panels v1 CE G5 Identifiler 8 w Ladder G02 fsa Ladder Allelic Ladder identifilerDirect_AnalysisMethod_v1 IdentifilerDirect GS500 Panels v1 CE G5 Idertifiler Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Samples tab of the Project window assuming the analysis is complete For more information For details about GeneMapper ID Software features allele filters peak detection algorithms and project editing refer to GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Part no 4335523 e GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Part no 4338775 Installation Procedures and New Feature
104. irect GS500 Bins v1X txt a Select the IdentifilerDirect GS500 Panels v1X folder in the navigation pane File Edit Bins View Help a XX Bin Set IdentifilerDirect G5500 Bins IX v Ilii S f Panel Manager K AmpFLSTR Panels v1x i IdentifilerDirect IdentirilerDirect G5500 Panels v1x null b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the Identifiler Direct Analysis Files GMIDX folder d Select IdentifilerDirect GS500 Bins v1X txt then click Import Note Importing this file associates the bin set with the panels in the IdentifilerDirect GS500 v1X folder Import Bin Set amp Identffier Direct Analysis Files GMIDX 3 IdentifilerDirect GS500 Bins viX txt E 1dentifilerDirect GS500 Panels vix txt 1dentifilerDirect GS500 Stutter viX txt E ReadMe IDDirect vix txt IdentifilerDirect GS500 Bins viX txt All Files 50 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 7 View the imported panels in the navigation pane a Double click the IdentifilerDirect GS500 v1X folder b Double click the IdentifilerDirect GS500 Panels v1X folder to display the panel information in the right pane and the markers below it File Edit Bi
105. ition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 115 Bibliography 116 Coble M D and Butler J M 2005 Characterization of new miniSTR loci to aid analysis of degraded DNA J Forensic Sci 50 43 53 DeFranchis R Cross N C P Foulkes N S and Cox 1988 A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Res 16 10355 DNA Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 Quality assurance standards for forensic DNA testing laboratories Drabek J Chung D T Butler J M McCord B R 2004 Concordance study between Miniplex assays and a commercial STR typing kit J Forensic Sci 49 859 860 Edwards A Civitello A Hammond H and Caskey C 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats J Hum Genet 49 746 756 Edwards A Hammond H A Lin J Caskey C T and Chakraborty 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Frank W Llewellyn B Fish P et al 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework J Forensic Sci 46 642 646 Glock B Dauber E M Schwartz D W Mayr W R 1997 Additional variability at the D
106. lankdisctothe For 28 cycles 1 uL of Control DNA 9947A positive control well Note The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number 2 Calculate the volume of each component needed to prepare the reactions using the table below Reaction component Volume per reaction Master Mix 12 5 uL Primer Set 12 5 uL Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The Identifiler Direct Kit has been optimized for a 25 uL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples Using a lower PCR reaction volume may reduce the ability of Identifiler Direct Kit chemistry to generate full STR profiles AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 19 20 Chapter 2 Perform PCR Treated paper substrates prepare reactions 3 Prepare reagents Thaw the Master Mix and the Primer Set then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 and therefore do not require subsequent thawing Do not refreeze the reagents Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the rea
107. le number to determine the optimum conditions for use in your laboratory Suggested cycle numbers for different sample type and substrate combinations are listed below AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 17 2 Chapter 2 Perform PCR Optimize PCR cycle number Sample Substrate type Treated paper Untreated paper Swab Blood 25 26 27 cycles 25 26 27 cycles N A Buccal 26 27 28 cycles 26 27 28 cycles 26 27 28 cycles Note Our testing has not included blood samples on swab substrates This sample type is not frequently used for the collection of database or casework reference samples Note To minimize the effect of instrument to instrument variation use the same thermal cycler to amplify all three plates To maximize result quality prepare and amplify Plate 1 then repeat for Plates 2 and 3 Do not prepare all three plates simultaneously Determine 1 Run the PCR products on the appropriate CE platform using the recommended optimum protocol see Chapter 3 Perform Electrophoresis on page 27 conditions 2 Based on the results of the sensitivity study select the appropriate PCR cycle number for future experiments Our studies indicate the optimum PCR cycle number should generate profiles with the following heterozygote peak heights with no instances of allelic dropout and minimal occurrence of off scale allele peaks Instrument Heterozygous peak height 31xx 1000 3000
108. ll number of individuals Genetics 89 583 590 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Science Communications July 2004 Volume 6 3 Available at www fbi gov hq lab fsc current standards 2004 03 standards02 htm Sensabaugh G F 1982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handbook Prentice Hall Inc New York pp 338 415 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the D21511 locus Hum Mol Genet 1 67 Shin C H Jang P Hong K M Paik M K 2004 Allele frequencies of 10 STR loci in Koreans Forensic Sci Int 140 133 135 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 117 Bibliography 118 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 7 locus multiplex STR test for use in forensic casework I Mixtures ageing degradation and species studies Int J Legal Med 109 186 194 Sparkes R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 1996b The validation of a 7 locus multiplex STR test for use in forensic casework II Artifacts casework studies and success rates Int
109. ls and bins a Select Panel Manager in the navigation pane Panel b Select File Import Panels to open the File Edit Bins View Emm m mimi E Panel Manager Import Panels dialog box c Navigate to then open the Identifiler Direct Analysis Files GMID folder that you unzipped in step 1 on page 36 D lt 5 9 ES 90 E 5 Select IdentifilerDirect GS500 Panels v1 txt then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager IdentifilerDirect GS500 v1 This folder contains the panel and associated markers x Lookin Cd identifier Direct Analysis Files GMD amp e IdentifilerDirect_GS500_Bins_v1 txt d identifilerDirect GS500 Panels v1 tt ReadMe_lDDirect_v1 txt Desktop File name dertifilerDirect_65500_Panels_v1 txt Import Files of type Files v Cancel 6 Import IdentifilerDirect GS500 Bins vl1 txt a Select the IdentifilerDirect GS500 v1 folder in the navigation pane File Edit Bins View m m BIJI rset amp iPanel Manager Wil IdentifilerDirect GS b Select File gt Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the Identifiler Direct Analysis Files GMID folder AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 37 n Chapter 4 Analyze Data Set up GeneMapper ID
110. mat PDF versions of this guide and the documents listed above are available at www lifetechnologies com Note To open the user documentation available from the our web site use the Adobe Acrobat Reader software available from www adobe com For HID support In North America Send an email to HIDTechSupport lifetech com or call 888 821 4443 option 1 Outside North America Contact your local support office For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Limited Product Warranty 120 Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Sy
111. mbols fsa sample files 36 49 hid sample file 48 nucleotide addition defined 78 efficiency of 78 Numerics 3100 3100 Avant instruments 29 3500 3500xL instruments 31 3730 instrument 31 A accuracy and reproducibility 64 alleles low frequency 93 off ladder 65 allelic ladder figure 11 requirements for accurate genotyping 28 volume per reaction 30 32 34 analysis method 40 53 analysis method create 40 analyze a project 46 60 artifacts in data 79 B bins import 49 bins import 36 49 biohazard safety 112 blood samples 9 17 19 Bode Buccal DNA Collector 9 17 21 buccal samples 9 17 19 21 buccal swabs 23 C characterization of loci validation 79 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Index chemical safety 112 concordance studies 97 contents of kit 14 control DNA about 15 Copan FLOQSwabs 85 treated cards 9 D data collection software 13 data accuracy precision and reproducibility of 64 data artifacts 79 data for different populations 86 developmental validation 64 DNA effect of quantity figure 83 negative control sample preparation 19 21 24 positive control sample preparation 19 21 24 sensitivity 81 your sample preparation 19 21 24 documentation related 119 E electropherogram causes of extra peaks 73 extra peaks 73 species specificity 80 84 85 electrophoresis Data Collection Software 29 31 data collection software 33 prepare sample
112. ment to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation PN 4440754 When using the ProFlex PCR System refer to the ProFlex PCR System Kit Validation User Bulletin Pub no 100031595 for more information gt t Optimum cycle number Final Final step Denature Anneal Extend extension hold HOLD CYCLE HOLD HOLD 95 C 94 C 59 C 72 C 60 C 4 C 11 min 20 sec 2 min 1 min 25 min t Determine the optimum cycle number for your laboratory according to the instructions on page 17 Load the plate into the thermal cycler and close the heated cover Start the run On completion of the run store the amplified DNA If you are storing the DNA Then place at 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C IMPORTANT Protect the amplified products from light AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Perform Electrophoresis Allelic ladder requirements W KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK K 28 Section 3 1 3100 3100 Avant 3130 3130xl 29 Set up the 3100 3100 Avant and 3130 3130xl instruments for electrophoresis 29 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl MOUNES vere uro eR urbe ee up debt pe eee quete sums 30 Section 3 2 3500 3500xL instruments
113. mplification Kit User Guide 29 o D 3 E e 3 5 Z 3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instruments Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instruments Prepare the samples for electrophoresis immediately before loading 1 0 pesce ON 30 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per Reagent Ber reaction reaction GeneScan 500 0 3 uL OR GeneScan 600 05 LIZ9 Size Standard LIZ Size Standard v2 0 Hi Di Formamide 87 Hi Di Formamide 8 5 pL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your experiments and results Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate add 9uL of the formamide size standard mixture 1uL of PCR product or Allelic Ladder Note For blank wells add 10 uL of Hi Di Formamide Seal the reaction plate with appropriate septa then bri
114. n the collecting devices used the collection methods applied and the swab to FTA transfer protocol employed DNA quantities present on the 1 2 mm disc may vary from sample to sample and from laboratory to laboratory It is essential for your laboratory to optimize the PCR conditions based on the types of buccal samples received or based on your standard operating protocol used in transferring saliva from a buccal swab onto an FTA card or Indicating FTA cards Refer to page 17 for instructions on PCR optimization AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 81 5 Experiments and Results Sensitivity Effect of DNA If too much DNA is added to the PCR reaction the increased amount of PCR product quantity on results that is generated can result in Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem because Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate This inaccuracy results in poor spectral separation pull up Incomplete A nucleotide addition To ensure minimal occurrence of offscale data when using the Identifiler Direct Kit optimize PCR c
115. nberg expectations Molecular Ecology Notes 6 974 979 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Mol Genet 1 287 Kong X Murphy K Raj T He C White P S Matise T C 2004 A combined linkage physical map of the human genome Am J Hum Genet 75 1143 1148 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Bibliography Kwok S and Higuchi 1989 Avoiding false positives with PCR Nature 339 237 238 Lareu M V Pestoni M C Barros F Salas A Carracedo A 1996 Sequence variation of a hypervariable short tandem repeat at the 0125391 locus Gene 182 151 153 Lazaruk K Walsh P S Oaks F Gilbert D Rosenblum B B Menchen S Scheibler D Wenz H M Holt C Wallin J 1998 Genotyping of forensic short tandem repeat STR systems based on sizing precision in a capillary electrophoresis instrument Electrophoresis 19 86 93 Levene H 1949 On a matching problem in genetics Ann Math Stat 20 91 94 Li H Schmidt L Wei M H Hustad T Leman M L Zbar B and Tory 1993 Three tetranucleotide polymorphisms for loci D351352 0351358 0351359 Hum Mol Genet 2 1327 Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J I Lowe A L Ghosh S Collins F S 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications
116. nded in this appendix to troubleshoot problems that occur during analysis Table 9 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the AmpF amp STR Identifiler9 Direct Control DNA 9947A and the DNA test samples at all loci Incorrect volume or absence of Identifiler Direct Master Mix or Identifiler9 Direct Primer Set Repeat amplification No activation of AmpliTaq Gold DNA Polymerase Repeat amplification making sure to hold reactions initially at 95 C for 11 minutes Master Mix not vortexed thoroughly before aliquoting Identifiler Direct Primer Set exposed to too much light Vortex the Master Mix thoroughly Store the Primer Set protected from light PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Use of incorrect thermal cycling parameters Check the protocol for correct thermal cycling parameters MicroAmp Base used with tray retainer set and tubes in GeneAmp 9700 Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected Prepare PCR product as described in Chapter 3 Perform Electrophoresis on page 27 Degraded formamide Sample punch location was not optimal Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide For blood samples on treated paper
117. ns View Help ux EE Ss Bin Set IdentiflerDirect GSS00 Bins v1 7 E oO S sf Panel Manager Maker Name Dye Color Min Size Size Control Alleles AmpFLSTR Panels v1X n pss Blue 118 0 183 5 13 E IdentifilerDirect 5500_ 1 D21511 Blue 184 5 247 5 30 1 0851179 D75820 Blue 251 0 2985 10 11 5 D21511 5 1 Blue 302 12 348 63 10 12 U 075820 D351358 Green 98 0 148 0 14 15 GH CSFIPO THO1 Green 159 0 2050 8 9 3 zi green 7 D135317 Green 205 65 250 16 D THO1 3 g D135317 8 10165539 Green 2553 301 81 0165539 9 D251338 Green 3048 370 31 E 0251338 10 D195433 Yellow 1010 148 0 tH 0195433 i VWA 11 vwa Yellow 1510 2135 12 TPOX Yellow 216 99 260 99 718551 13 1014551 Yellow 264 49 1850 0 15 19 8 Select D8S1179 to display the Bin view for the marker in the right pane Panel Manager File Edit Bins View Help mx umm S gy Panel Manager E AmpFLSTR Panels v1x S O IdentifilerDirect GS500 vix S IdentifilerDirect G5500 Panels v1x LU Bin Set IdentifilerDirect G5500 Bins v1x v ELERE BEBNHBBB o 1 0 0 9 0 8 075820 CSFIPO D351358 THO1 0135317 0165539 0251338 0195433 VWA TPOX D18551 AMEL D55818 FGA 07 0 6 05 04 0 3 0 2 0 1 0 0 113 115 117 119 121 123 125 127 129 131 133 135 137 139 141 143 145 147 149
118. o 0 o 0 5 Minus Stutter Ratio n o 0 o Minus Stutter Distance From j 25 ja 75 o n Plus Stutter Ratio o 0 o 0 Plus Stutter Distance From 0 o 0 o 0 o n Amelogenin Cutoff Range Filter Factory Defaults In the Bin Set field select the IdentifilerDirect GS500 Bins v1 bin set imported previously and configure the stutter distance parameters as shown GeneMapper ID Software v3 2 1 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the stutter ratio Toapply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data Toapply the stutter ratios contained in the IdentifilerDirect GS500 Panels v1 file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 41 n Chapter 4 Analyze Data Set up GeneMapper
119. on Kit is a short tandem repeat STR multiplex assay optimized to allow direct amplification of single source Blood and buccal samples on treated paper substrates without the need for sample purification Blood and buccal samples collected on untreated paper substrates and treated with Applied Biosystems Prep n Go Buffer Buccal samples collected on swab substrates and treated with Applied Biosystems Prep n Go Buffer The Identifiler Direct Kit amplifies 15 autosomal STR loci 0851179 021511 075820 CSF1PO 0351358 THOI D13S317 0165539 0251338 0195433 018551 055818 and the sex determining marker Amelogenin a single PCR reaction The Treated paper Copan NUCLEIC CARD system or Whatman FTA cards Untreated paper Bode Buccal DNA Collector or 903 paper Swab Copan 4N6FLOQSwabs Identifiler Direct Kit contains all the necessary reagents for the amplification of human genomic DNA The reagents are designed for use with the following Applied Biosystems instruments Applied Biosystems 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x1 Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer Applied Biosystems 3730 Genetic Analyzer GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block Veriti 96 Well Thermal Cycler ProFlex PCR System AmpFtSTR Identifiler Direct PCR
120. oom temperature for up to three months Sample prep Detach buccal swab heads from the swab shaft for lysis guidelines e Lysis is performed under heated conditions using Prep n Go Buffer Part no 4471406 for buccal swabs in either of the following formats 1 5 mL tubes with a heat block VWR Scientific Select dry heat b lock or similar 96 well deep well plate Part no 4392904 with an oven and a metal plate adaptor Robbins Scientific Model 400 Hybridization Incubator or similar Agilent Benchtop Rack for 200 ul Tubes V Bottom Plates metal Part no 410094 or similar IMPORTANT Do not use a plastic plate adaptor For optimum performance lysis of a whole swab is recommended To preserve the sample evaluate lysis of a half swab Prepare the 1 Preheat the heat block to 90 or the oven with metal plate adaptor to 99 sample lysate 2 Add 400 uL Prep n Go Buffer for buccal swabs Part no 4471406 to 1 5 mL tubes or the appropriate wells of a 96 well deep well plate Part no 4392904 3 Into each tube or well put the entire head of each swab If you are using tubes cap the tubes Let the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample 4 After 20 minutes remove the tubes or the deep well plate from the heat block or oven Note To minimize the risk of contamination do not remove the swab heads from the sample lysate plate before transferring the lysate 5
121. osystems Collection XP Injection conditions 1 2kV 15 sec 3500xL Genetic Analyzer User 3500 u on Dye Set 05 Guide Part no 4401661 Applied Windows HID36_POP4 PN eae ec AA o Analyzers Quick Reference Card Biosystems Vista Injection conditions 1 2kV 24 sec Part no 4401662 3500xL Dye Set 65 Prepare samples for electrophoresis on the 3500 3500xL instruments Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and GeneScan 600 LIZ Size Standard v2 0 needed to prepare the samples Reagent Volume per reaction GeneScan 600 LIZ Size Standard v2 0 0 5 uL Hi Di Formamide 8 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your experiments and results AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 31 Chapter 3 3500 3500xL Instruments Prepare samples for electrophoresis on the 3500 3500xL instruments 2 Pipet the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each well of a MicroAmp Optical 96 Well Reaction Plate add 9uL of the formamide size standard mixture 1
122. p determining the correct files to use contact your local Life Technologies Human Identification representative or go to www appliedbiosystems com Before you use GeneMapper ID X Software to analyze data files GeneMapper ID X Software v1 0 1 or higher for fsa files GeneMapper ID X Software v1 2 or higher for hid files Import panels bins and marker stutter into the Panel Manager as explained in Import panels bins and marker stutter on page 49 Create an analysis method as explained in Create an analysis method on page 53 Create a size standard as explained in Create size standard on page 58 Define custom views of analysis tables Refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 for more information Define custom views of plots Refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Part no 4375574 for more information To import the Identifiler Direct Kit panel bin set and marker stutter from the Life Technologies web site into the GeneMapper ID X Software database 1 Download and open the file containing panels bins and marker stutter a From the Support menu of www lifetechnologies com select Support Software Downloads Patches amp Updates GeneMapper ID X Software gt Updates amp Patches and download the file Identifiler Direct Analysis Files GMIDX b Unzip the file Start the GeneMapper ID X Software then lo
123. ple type and substrate combinations for example buccal samples on treated paper and buccal samples on swabs perform separate sensitivity experiments for each sample type and substrate to be used for testing The Identifiler Direct Kit is optimized to amplify unpurified Single source blood samples on treated paper or untreated paper Buccal samples on treated paper untreated paper or swabs When amplifying single source unpurified samples using the Identifiler Direct Kit you should expect to see greater variation in peak height from sample to sample than is expected with purified samples Careful optimization of the cycle number will help to minimize this variation Select samples and 1 Select 26 of each sample substrate type Ensure the selected samples represent a prepare plates typical range of samples analyzed in your laboratory IMPORTANT The number of samples recommended for this study has been chosen to allow you to complete electrophoresis using a single 96 well plate thus minimizing the impact of run to run variation on the results Examples of PCR and electrophoresis plate layouts are provided on page 107 2 Prepare the samples and the reactions as described in the protocols later in this chapter Prepare sufficient PCR reagents to complete amplification of three replicate plates 3 Create three identical PCR plates see page 107 for a suggested plate layout 4 Amplify each plate using a different cyc
124. pper ID X Security Group Description Size Standard Dye M r Size Standard Table 1 75 0 2 100 0 3 139 0 4 150 0 5 160 0 6 2000 7 300 0 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 59 n Chapter 4 GeneMapper ID X Software Analyze and edit sample files with GeneMapper ID X Software Analyze and edit sample files with GeneMapper D X Software 1 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project The names of the settings shown are the names suggested in the sections above If you named the settings differently select the names you specified Parameter Settings Sample Type Select the sample type Analysis Method IdentifilerDirect AnalysisMethod v1X or the name of the analysis method you created Panel IdentifilerDirect GS500 Panels v1X Size Standard CE 65 IdentifilerDirect 65500 the name of the size standard you created For more information about how the Size Caller works or about size standards refer to the GeneMapper ID X Software v1 2 Reference Guide Part no 4426481 3 Click P Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis During a run The status bar displays the progress of an
125. r P M 2004 Preparation of degraded human DNA under controlled conditions Forensic Sci Int 139 134 140 Brinkman B Klintschar M Neuhuber F Huhne J and Rolf B 1998 Mutation rate in human microsatellites Influence of the structure and length of the tandem repeat Am J Hum Genet 62 1408 1415 Brinkman B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Intl J Legal Med 107 201 203 Butler J M 2005 Forensic DNA Typing Burlington MA Elsevier Academic Press Butler J M Shen Y McCord B R 2003 The development of reduced size STR amplicons as tools for analysis of degraded DNA J Forensic Sci 48 1054 1064 Chakraborty R Kimmel M Stivers D Davison L and Deka R 1997 Relative mutation rates at di tri and tetranucleotide microsatellite loci Proc Natl Acad Sci USA 94 1041 1046 Chakraborty R Stivers D and Zhong Y 1996 Estimation of mutation rates from parentage exclusion data applications to STR and VNTR loci Mutat Res 354 41 48 Chakraborty R and Stivers D N 1996 Paternity exclusion by DNA markers effects of paternal mutations J Forensic Sci 41 671 677 Chung D T Drabek J Opel K L Butler J M and McCord 2004 A study of the effects of degradation and template concentration on the amplification efficiency of the Miniplex primer sets J Forensic Sci 49 733 740 Clark J M 1988 Novel non templated nucleotide add
126. r chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Specific chemical Chemical Phrase handling 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides Biological hazard safety Q WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards 112 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Appendix E Safety E
127. ratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 111 Appendix E Safety Chemical safety Chemical safety Aq WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly fo
128. s Validation studies included testing on the following sample substrate combinations Unpurified single source blood or buccal samples on FTA paper treated paper substrate TM Buccal samples on a Bode Buccal DNA Collector untreated paper substrate Additional performance verification studies included testing on Copan 4N6FLOQSwabs swab substrate We did not perform mixture or inhibition studies during the developmental validation of the Identifiler Direct Kit because these tests are not relevant for the intended use of this chemistry Accuracy precision and reproducibility SWGDAM guideline 1 2 1 SWGDAM guideline 2 9 Accuracy 64 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 The extent to which a given set of measurements of the same sample agree with their mean and the extent to which these measurements match the actual values being measured should be determined SWGDAM July 2003 Laser induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2000 and Wallin et al 2002 However accuracy and reproducibility of Identifiler Direct Kit profiles have been determined from various sample types Figure 3 illustrates the size differences that ar
129. s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele verify the sample result according to your laboratory s protocol Set up GeneMapper D Software for data analysis File names Before using the software for the first time Import panels and bins 36 The file names shown in this section may differ from the file names you see when you download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com To analyze sample files fsa using GeneMapper ID Software v3 2 1 for the first time Import panels and bins into the Panel Manager as explained in Import panels and bins on page 36
130. s Kimpton Gilbard et al 1996 Wallin et al 1998 We performed experiments to evaluate the performance of the Identifiler Direct Kit 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 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 we conducted experiments that comply with guidelines 1 0 and 2 0 and its associated subsections This DNA methodology is not novel Moretti et al 2001 Frank et al 2001 Wallin et al 2002 and Holt et al 2000 AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 63 Chapter 5 Experiments and Results Accuracy precision and reproducibility This chapter discusses many of the experiments we performed and provides examples of results obtained We chose conditions that produced optimum PCR product yield and that met reproducible performance standards It is our opinion that while these experiments are not exhaustive they are appropriate for a manufacturer of STR kits intended for forensic and or parentage testing use Each laboratory using the Identifiler Direct Kit should perform their own internal validation studie
131. s 30 31 33 references 29 31 33 run module 29 31 33 set up of 3100 3100 Avant instruments 29 set up of 3500 3500xL instruments 31 set up of 3730 instrument 31 33 emission spectra 14 equipment not included with kit 103 extra peaks causes 73 121 Index F five dye fluorescent system 10 fluorescent dyes 13 FSA sample files 36 49 FTA cards 9 17 19 G GeneMapper ID Software analyze a project 46 analyze files 46 data analysis 36 edit a project 47 examine a project 47 import panels and bins 36 size standard create 45 GeneMapper ID X Software analyze a project 60 analyze files 60 data analysis 49 marker stutter 49 panels bins and stutter import 49 size standard create 58 GeneScan size standard about 15 dye label 13 volume per reaction 30 31 33 GS 500 15 GS 600 15 H Hi Di formamide volume per reaction 30 31 33 l import bins 36 panels 36 panels bins stutter 49 size standard 45 58 Instrument and software compatibility 13 Instrument and special software compatibility 13 K kit contents 14 description 9 fluorescent dyes 13 122 instruments for use with 9 loci amplified 10 master mix 14 primers 10 14 purpose 9 reagents 14 storage 14 thermal cyclers for use with 110 L Limited Product Warranty 120 LIZ size standard about 15 volume per reaction 30 31 33 loci characterization 79 chromosomal location 10 combined genotype frequency 93 dye label 10 genotype
132. s for GeneMapper ID Software Software Version v3 2 User Bulletin Part no 4352543 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 47 D lt 97 o 9 ES 90 E g n Chapter 4 GeneMapper ID X Software Overview of GeneMapper ID X Software Section 4 2 GeneMapper D X Software Overview of GeneMapper D X Software GeneMapper ID X Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the data collection software stores information for each sample in a fsa or hid file Using GeneMapper ID X Software you can then analyze and interpret the data from the fsa files GeneMapper ID X Software v1 0 1 or higher or hid files GeneMapper ID X Software v1 2 or higher Instruments Refer to Instrument and software overview on page 13 for a list of compatible instruments Before you start When using GeneMapper ID X Software v1 0 1 or higher to perform human identification HID analysis with AmpF STR kits be aware that 48 HID analysis requires at least one allelic ladder sample per run folder Perform the appropriate internal validation studies if you want to use multiple ladder samples in an analysis For multiple ladder samples the GeneMapper ID X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder
133. samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele verify the sample result according to your laboratory protocol AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 2 ID X Software 4 Set up GeneMapper ID X Software for data analysis Set up GeneMapper D X Software for data analysis File names Before using the software for the first time Import panels bins and marker stutter The file names shown in this section may differ from the file names you see when you download or import files If you need hel
134. se is suggested by the National Research Council in forensic calculations Low frequency alleles Some alleles of the Identifiler Direct Kit loci occur at a low frequency For these alleles a minimum frequency 5 divided by 2n where n equals the number of individuals in the database was assigned for the Identifiler Direct Kit African American Asian U S Caucasian and U S Hispanic databases as suggested in the 1996 report of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Table 5 on page 86 The minimum reportable genotype frequency at each locus is 1 19 X 10 4 for the African American database 1 19 X 10 4 for the U S Caucasian database 1 70 X 10 4 for the U S Hispanic database and 2 97 X 10 4 for the Native American database p2 p 1 p where 0 0 01 Hence the minimum combined multilocus genotype frequency at 15 loci is 1 36 X 10 59 for the African American database 1 36 X 10 59 for the U S Caucasian database 2 86 X 10 57 for the U S Hispanic database and 1 23 x 10753 for the Native American database AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 93 Chapter 5 Experiments and Results Population data Evaluation of Hardy Weinberg equilibrium 94 Estimates of expected heterozygosity HExp were computed as described by Nei M 1973 using the program PopGene 1 32 Possible divergence from Hardy Weinberg expectations HWE was tes
135. sis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 The proportion of the stutter product relative to the main allele percent stutter is measured by dividing the height of the stutter peak by the height of the main allele peak Peak heights were measured for amplified samples at the loci used in the Identifiler Direct Kit Treated paper workflow 370 blood samples on FTA card and 299 buccal samples on Indicating FTA cards Untreated paper workflow 370 buccal samples on Bode Buccal DNA Collectors All data were generated on the Applied Biosystems 3130x Genetic Analyzer Some conclusions from these measurements and observations are For each Identifiler Direct Kit locus the percent stutter generally increases with allele length as shown in Treated paper workflow Figure 4 through Figure 7 on page 74 through page 75 Untreated paper workflow Figure 8 through Figure 11 on page 76 through page 77 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 The stutter value for each locus shown for the treated paper workflow in Table 4 on page 78 was determined by taking the mean plus three times the standard deviation These values are the stutter filter percen
136. sizes peak sizes 75 100 139 150 160 200 300 350 400 and 80 100 114 120 140 160 180 200 214 220 450 240 250 260 280 300 314 320 340 360 380 400 414 420 440 and 460 Note The 250 nt and the 340 nt peak in the GeneScan 500 LIZ Size Standard are not included in the size standard definition These peaks can be used as an indicator of precision within a run To create the size standard for the Identifiler Direct Kit 1 Select Tools GeneMapper Manager to open the GeneMapper Manager D lt fe o 9 5 90 E g xi Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Name Last Saved Owner Description 377 G5 HID GS500 2004 05 28 11 34 3 gmid Basic Advanced Provided 377 F HID GS500 2004 05 28 11 34 3 gmid Basic Advanced Factory Provided CE_G5_HID_GS500 2004 05 28 11 34 3 gmid Basic Advanced Factory Provided CE F HID GS500 2004 05 28 11 34 3 gmid Basic Advanced Factory Provided New Open Save AS Import Export 2 Select the Size Standards tab then click New AmpFtSTR9 Identifiler Direct PCR Amplification Kit User Guide 45 n Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper ID Software 3 Enter a name as shown below or enter a name of your choosing In the Size Standard Dye field select Orange
137. ssues related to software data 4445098 hardware and consumables Note Additional user bulletins may be available at www lifetechnologies com Applied Biosystems 3730 3730xl Genetic Analyzer Getting Started Guide 4359476 GeneAmp PCR System 9700 Base Module User s Manual N805 0200 Quantifiler9 Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit Users Manual AmpFtSTR Identifiler PCR Amplification Kit Users Manual 4323291 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin 4352543 GeneMapper ID X Software Version 1 0 Getting Started Guide 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide 4375670 GeneMapper ID X Software Version 1 0 Reference Guide 4375671 GeneMapper 10 Software Version 1 1 Mixture Analysis Getting Started Guide 4396773 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide 119 Documentation and Support Obtain support Document title GeneMapper 10 Software Version 1 1 Mixture Analysis Quick Reference Guide 4402094 GeneMapper ID X Software Version 1 2 Reference Guide 4426481 GeneMapper ID X Software Version 1 2 Quick Reference Guide 4426482 Obtain support Portable document for
138. t population s The Identifiler Kit prior to the addition of the D8S1179 degenerate primer was used to generate the population data provided in this section Samples were collected from individuals throughout the United States with no geographical preference Number Population of Samples provided by samples African American 357 Kentucky State Police and the Federal Bureau of Investigation U S Caucasian 349 U S Hispanic 290 Minnesota Bureau of Criminal Apprehension Memorial Blood Center of Minneapolis Native American 191 In addition to the alleles that were observed and recorded in the Life Technologies databases other alleles have been published or reported to Life Technologies by other laboratories see the STRBase at www cstl nist gov div831 strbase Table 5 shows the Identifiler Direct Kit allele frequencies in four populations listed as percentages Table 5 Identifiler Direct Kit allele frequencies African U S Native U S Hispanic i Allele American Caucasian 290 American n 357 n 349 191 CSF1PO 6 t t t t 7 4 62 0 14 0 341 8 7 56 0 291 0 177 0 52 9 3 78 1 72 0 86 8 38 10 27 87 24 21 23 10 30 89 11 20 59 31 91 28 28 21 99 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data
139. tages in the Identifiler Direct stutter file and will be used during the filtering step in GeneMapper ID Software or GeneMapper ID X Software Peaks in the stutter position that are above the stutter filter percentage will not be filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated Stutter percentages generated using the untreated paper workflow were calculated on a different smaller data set than was used for the original stutter calculations We used the stutter values of the most common alleles at each locus to compare the data sets There was no significant difference in the stutter values mean plus three times the standard deviation for the individual loci with the exception of 0351358 0 7 075820 0 4 0165539 0 2 FGA 1 2 For 0351358 075820 and FGA the stutter values were slightly lower than the original stutter values calculated for punches from FTA cards processed with the AmpFtSTR9 Identifiler9 Direct PCR Amplification Kit User Guide 73 Chapter 5 Experiments and Results Extra peaks in the electropherogram Identifiler Direct Kit The 0165539 stutter percentage mean plus three times the standard deviation was slightly higher than the original stutter value You should evaluate the impact of sample type on stutter percentages when implementing a direct amplification system The measurement of percent stutter for allele peaks that are off s
140. tained for each allele in the AmpF STR Identifiler Direct Allelic Ladder 0 5 nt window allows for the detection and correct assignment of alleles Any sample allele that sizes outside the specified window could be An off ladder allele that is an allele of a size that is not represented in the AmpF STR Identifiler Direct Allelic Ladder or An allele that corresponds to an allelic ladder allele but whose size falls just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument Table 3 on page 66 shows typical precision results obtained from five runs 16 capillaries run of the AmpF STR Identifiler Direct Allelic Ladder on the Applied Biosystems 3130xl Genetic Analyzer 36 cm capillary and POP 4 polymer sized using the GeneScan 500 LIZ Size Standard The results were obtained within a consecutive set of injections on a single capillary array Sample alleles may occasionally size outside of the 0 5 nt window for a respective allelic ladder allele because of measurement error The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Figure 3 illustrates the tight clustering
141. ted using various methods by calculating the unbiased estimate of the expected homozygote heterozygote frequencies Levene H Nei M 1978 and using chi square HW X p and likelihood ratio HW G p tests as implemented in the program PopGene 1 32 and with an exact test HW Exact p which is a Markov chain method based on 1000 shuffling experiments to estimate without bias the exact P value of the Hardy Weinberg test with multiple alleles Guo S W 1992 as implemented in the program GenePop 3 4 An inter class correlation test analysis Burrows composite measure of linkage disequilibria between pairs of loci and X tests for significance Weir B 1990 was performed separately in each population to detect any correlations between alleles at any of the pair wise comparisons of the 15 loci using the program PopGene 1 32 Observed heterozygosity expected heterozygosity information content and tests for detecting departures from Hardy Weinberg equilibrium are shown for each population in Table 6 While a number of the chi square tests gave seemingly significant p values putatively indicating departures from Hardy Weinberg equilibrium chi squared tests are very sensitive to small expected values as in the case of multiple rare alleles where the expected number of certain genotypes is 1 or fewer such as with some of these markers and can greatly inflate the test statistic in this situation Weir B 1990 With the exact test the
142. tensity Increasing the size of the punch may cause inhibition during PCR amplification If you are using a Bode Buccal DNA Collector Bode Take punch make the punch as close as possible to the tip of the DNA as close to TM DNA collector to ensure optimum peak intensity Collector the tip as Increasing the size of the punch may cause possible inhibition during PCR amplification For manual punching Place the tip of a 1 2 mm Harris Micro Punch on the card hold the barrel of the Harris Micro Punch do not touch the plunger gently press and twist 1 4 turn then eject the punch in to the appropriate well on the reaction plate For automated punching Please refer to the User Guide of your automated or semi automated disc punch instrument for proper guidance Add 2 uL of Prep n Go Buffer Part no 4467079 to the sample and negative control wells in a 96 well plate Do not add Prep n Go Buffer to the positive control wells Add samples to the reaction plate Add the following to wells of a MicroAmp Optical 96 Well Reaction Plate Negative control 1 2 mm blank disc Test samples 1 2 mm sample disc Positive control For 25 cycles 3 uL of Control DNA 9947A IMPORTANT Do not For 26 and 27 cycles 2 uL of Control DNA 9947A add a blank disc tothe For 28 cycles 1 uL of Control DNA 9947A positive control well Note The volumes of positive control are su
143. tical 96 Well Reaction Plate add 9uL of the formamide size standard mixture 1 of PCR product or Allelic Ladder Note For blank wells add 10 uL of Hi Di Formamide 5 Sealthe reaction plate with appropriate septa then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom Heat the plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Place the sample tray on the autosampler Or Sor Start the electrophoresis run 34 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Analyze Data Section 4 1 GeneMapper ID 35 Overview of GeneMapper ID Software 35 Set up GeneMapper ID Software for data 36 Analyze and edit sample files with GeneMapper ID Software 46 Examine and edita project KK KK KK nen 47 For more information ssis sss rado raan 334 444 414 Sl a14 dd r eee 47 Section 4 2 GeneMapper ID X 48 Overview of GeneMapper ID X Software 48 Set up GeneMapper ID X Software for data 49 Analyze and edit sample files with GeneMapper ID X Software 60 Examine and edita project
144. tifilerDirect GS500 v1X folder to display its list of markers in the right pane b Double click the IdentifilerDirect GS500 v1X folder to display its list of markers below it 52 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Section 4 2 ID X Software 4 Set up GeneMapper ID X Software for data analysis c Double click 0165539 to display the Stutter Ratio amp Distance view for the marker in the right pane Panel Manager File Edit Bins View Help IdentifilerDirect GSS00 Bins vix EKLERE E E HB Bi Please enter the stutter filter s for D165539 marker here If left blank the global stutter filt ax B Bin Set S Panel Manager AmpFLSTR_Panels_v1x S IdentifilerDirect_G5500_v1X S E IdentifilerDirect_GS500_Pane 0851179 D21511 D75820 CSF1PO D351358 THO1 D135317 D165539 Stutter Ratio amp Distance D251338 miaca22 Minus Stutter Plus Stutter Ratio From Distance To Distance Ratio From Distance 1 10942 3 25 4 75 1 ae gt _y 3 4 gt wl n B B BR B B B B C D 3 lt o 0 p e v ee p E 0 P E 11 Click Apply then OK to add the Identifiler Direct Kit panel bin set and marker stutter to the GeneMapper ID X Software database IMPORTANT If you close the Panel Manager without clicking Apply the panels
145. trademarks of Microsoft Corporation FTA and Whatman are trademarks of Whatman International Ltd Buccal DNA Collector is a trademark of Bode Technology Group Inc NUCLEIC CARD and FLOQSwabs are trademarks of Copan Italia S P A Contents About This GUIDE esci Reda warte es tiet arde ele ais Ros e 7 Revision history cesa Ro ERaI aHE HH H HHH HH HH HHHH a HHH e 7 Purposes eec env nU ILU Wee Rer ID He we ee dee RR as 7 M CHAPTER 9 Product oveEVIe Wesce tni i ADAE ES dak br n C LA 5 T eie i ee 9 ecce rtt eoe cette terga melee er pal isch ciet esci 9 Substrate examples 4 MA Wk kk kk kk kk kk kk kk kk kk kk kK kk kk kk kk kk kk kk kk n 9 Productdescriptionrs eley ftt s tt nA O 9 sella tn EB eA AT AER Ta ih s nn 10 Loci amplified by the kit lk kk kk kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK kK KK 10 Allelic ladder ssec o bes o N EH DE tele neta aad els 11 ela dilS WED eee a oe DD Hare recti ced rcr ud eA E E m 12 Instrument and software overview 2 00e cece eer 13 Data collection and analysis software 13 Instrument and software compatibility kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KIR 13 About multicomponent analysis WW kk kk kk kk kk kk kK eee kk kk kk kk kk kk kk
146. ycle number according to instructions on page 17 When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the alleles may occur because of stochastic fluctuation Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA 82 AmpFtSTR Identifiler Direct PCR Amplification Kit User Guide Chapter 5 Experiments and Results 5 Stability Figure 14 Effect of amplifying varying amounts of white blood cells WBCs spotted onto Indicating FTA discs 900 WBCs 5 3 ng DNA 450 WBCs 2 7 ng DNA 220 WBCs 1 3 ng DNA 110 WBCs 0 65 ng DNA 55 WBCs 0 33 ng DNA Stability SWGDAM guideline 2 4 Negative control Note that the y axis scale is magnified for the lower amounts of DNA analyzed using the Applied Biosystems 3130x Genetic Analyzer The amount of DNA on the Indicating FTA cards were calculated based on the assumptions of 100 cell lysis efficiency and that each cell contain 6 pg of DNA The results from white blood cells spotted onto Bode DNA Collectors were comparable to the results shown here obtained using the Identifiler Direct Kit with white blood cells spotted onto FTA Indicating Cards data not shown The ability to obtain results from DNA recovered from biological samples

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