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AmpFℓSTR® Yfiler™ PCR Amplification Kit

1. Mark Sample for Deletion 80 120 160 200 240 280 320 eh 1200 800 400 0 A roe A m A Ee Mark Sample for Deletion 80 120 160 200 240 280 320 m 1200 800 400 0 f rae An 2 4 Mark Sample For Deletion 80 120 160 200 240 280 320 1600 1200 800 0 A La we anai A M 2 Mark Sample for Deletion 80 120 160 200 240 280 320 1600 4200 800 anl 0 AL mE UE E A 14 AmpFtSTR Yfiler PCR Amplification Kit User Guide Chapter 10verview 1 Workflow overview Workflow overview Perform PCR Perform electro phoresis Analyze data Extract DNA Quantify DNA Quantifiler Duo DNA Quantification Kit Prepare reactions AmpF STR Yfiler PCR Amplification Kit Perform PCR gt GeneAmp PCR System 9700 Cycler Veriti 96 Well Thermal Cycler a b sa gl ii mar 3100 3100 Avant 3130 3130xl 3500 3500xL 310 Genetic Genetic Analyzer Genetic Analyzer Genetic Analyzer Analyzer 2 GeneMapper 10 lt GeneMapper ID X or GeneMapper D Software AmpFtSTR Yfiler PCR Amplification Kit User Guide 15 1 Overview Instrument and software overview Instrument and software overview Data Collection and GeneMapper D or D X Software Instrument and software compatibility About multicomponent an
2. 00000 ccc cece ee RR KK KK KK KK 32 Import panels ak ch L ba d k et ta eb Iber uU Pared 33 Create an analysis method 00000 e kk kk eee 37 General tab settings nann ccc cece tte kk kk kk kk eee eee eee 38 Allele tab SettingS iis bert D Ged LOCA RR PET e ene 39 Peak Detector tab settings kK kk kk kk kk kk kk 40 Peak Quality tab setings E RINT EI UE DUNUA 41 Quality Filana tab Settings Sus Sa Wer saa ya eire RE kana Dey K RA n La Ay mead WAA 42 Create size standard conss kw xa ban nue e ah a ee a end kal V ku d k h 42 Analyze and edit sample files with GeneMapper ID Software 44 Examine and edit a project 2 kk kk kK kk kK kK kk sess kk kk kk kk kk kk 45 For more iniformatlon useless lle aee ame ne ame dd ew ed a Wan kad TREE m 46 Section 4 2 GeneMapper D X 48 Overview of GeneMapper D X Software 48 299 04 ce ose eee aim EAR Wan eq dud 48 Beloreyourstart Aata ae nia a a aea E tet pietre po ER De edes 48 Set up GeneMapper D X Software for data analysis 49 Panel bin and stutter file version 0 0 kk kk kk kk ce 49 Before using the software f
3. 4 Control Mark Sampie far AmpFtSTR Yfiler PCR Amplification Kit User Guide 95 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Sensitivity study DNA concentration and peak height Allelic dropout 96 The calculated slope and R values for each of the plotted curves were equivalent showing comparable relationships between peak height and DNA input amount for the Test and Control mixes Figure 25 Figure 25 Sensitivity study linear regression plot of combined mean referenced peak height for three genomic DNA samples 1 6 Lots Control 1 4 H Control B Testa z E A Test B 3 1 2 F F 1 0 0 8 9 9 0 6 0 4 0 2 0 0 0 200 400 600 800 1000 DNA Concentration pg Allelic dropout was observed only for amplifications of 125 pg where dropout of a single allele was observed for different replicates of Test A Sample 3 Figure 26 and Figure 27 These results can be explained by stochastic variation and sampling from dilute DNA solutions Allelic dropout results can therefore be considered equivalent between Test and Control mixes Figure 26 Sensitivity study electropherogram of 125 pg Sample 3 amplified with Test A mix One allele at the DYS635 locus in the NED dye yellow channel is below the analysis threshold of 50 RFU Y scale 300 RFU is er af
4. 75 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility Allele Mean Standard Deviation DY438 8 223 69 0 06 9 228 68 0 06 10 233 63 0 07 11 238 59 0 06 12 243 63 0 05 13 248 66 0 05 DYS448 17 280 49 0 04 18 286 58 0 03 19 292 70 0 05 20 298 92 0 05 21 305 51 0 04 22 312 25 0 06 23 318 60 0 10 24 324 88 0 08 AmpFtSTR Yfiler PCR Amplification Kit User Guide 75 g lt 3 o 22 2 E S 2 Chapter 5 Experiments and Results Extra Peaks in the electropherogram Extra Peaks in the electropherogram Causes of extra peaks Stutter products 76 Peaks other than the target alleles may be detected on the electropherogram displays Several causes for the appearance of extra peaks including the stutter product at the n 4 position incomplete 3 A nucleotide addition at the n 1 position artifacts and mixed DNA samples see SWGDAM Guideline 2 8 on page 86 A stutter is a well characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller or less frequently one repeat larger than the major STR product Butler 2001 Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the mai
5. C Kek Sangi for Mark Semple for Deletion r LLL ALL NR MES Merk Sangle for Mm m d Oe tm Ayen any n gt a ned ge a a EN M AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement 5 Sensitivity study Figure 27 Sensitivity study electropherogram of 125 pg Sample 3 amplified with Test A mix One allele at the DYS437 locus in the PET Red dye channel is below the analysis threshold of 50 RFU Y scale 500 RFU n w m m J Mark Sangle for Dalasa Merk Sample for Kurk Sample for Daaba U e zh 3 3 hy 2 le S S e 2 00 E 0 j m N lt 3 2 5 e 3 AmpFtSTR Yfiler PCR Amplification Kit User Guide 97 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Inhibition study Genotype Genotypes for Test and Control mixes were 100 concordant Table 7 concordance Table 7 Sensitivity study genotype concordance DNA Input Amount Reagent Mix Genotype Concordance 125 pg Test A 99 Test B 100 Test C 100 Control A 100 Control 100 250 pg Test A 100 Test B 100 Test C 100
6. Table 5 shows the Yfiler Kit gene diversity in three populations listed as percentages Table5 Yfiler9 Kit Gene Diversity values across three different U S populations TT African American U S Caucasian U S Hispanic n 333 n 254 n 175 DYS458 0 755 0 808 0 77 DYS19 0 748 0 541 0 645 DYS385a b 0 951 0 855 0 931 DYS393 0 619 0 412 0 507 DYS391 0 423 0 54 0 52 DYS439 0 629 0 663 0 665 DYS635 0 701 0 682 0 71 DYS392 0 419 0 615 0 671 Y GATA H4 0 599 0 604 0 575 DYS437 0 495 0 624 0 583 DYS438 0 528 0 622 0 712 DYS448 0 685 0 651 0 726 2 Gene diversity D aQ 2p where n sample size p allele frequency Johnson et al 2003 27 In addition to the alleles that were observed and recorded in the Life Technologies databases other known alleles have been published or reported to us by other laboratories Some of these alleles occur at a low frequency and include several microvariants Furedi et al 1999 Schoske et al 2004 89 g lt e 3 D e 5 ei E e 2 Mutation rate 90 Chapter 5 Experiments and Results Mutation rate Discriminatory capacity of haplotypes Table 6 shows the discriminatory capacity DC and the number of unique haplotypes UH for each Y STR marker combination listed The discriminatory capacity was determined by dividing the number of different haplotypes by the number of samples i
7. Representative electropherograms from the inhibition study are shown in Figure 31 and 32 Figure 31 Inhibition study representative electropherograms using uninhibited Control DNA 007 Y scale 4000 RFU Control A Mwk Semple for Delta Control B Yawk rgo for Delo ime Test A Chew Sar le for Dalat Test B 100 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement 5 Conclusions Figure 32 Inhibition study representative electropherograms using Control DNA 007 inhibited with12 ng uL Humic Acid Y scale 2500 RFU Control A Park Sangle for 1x5 185 25 25 ns Control B Mark sangle for Deletion ER e 3 D 5 a 2 e 3 Ww 2 m 2 N lt 3 2 o E o Allelic dropout No allelic dropout events were seen for any Test or Control mixes tested on uninhibited Control DNA 007 and Control DNA 007 inhibited with Humic Acid Conclusions Laboratories can expect to obtain equivalent quality profiles across a wide range of forensic samples when using the Yfiler Kit containing the AmpliTaq Gold enzyme and 10X PCR Buffer II manufactured by Life Technologies as compared to the original Yfiler Kit containin
8. 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Peak End Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of Yfiler 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 Size calling method The Yfiler Kit has been validated using the Local Southern sizing method Select alternative sizing methods only after performing the appropriate internal validation studies Normalization A Normalization checkbox is available on this tab in GeneMapper ID X Software v1 2 for use in conjunction with data run on the Applied Biosystems 3500 Series Genetic Analyzers Users of this version of software should perform laboratory evaluations to determine whether to use the Normalization feature for analysis of Yfiler Kit data AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Peak Quality tab ji Analysis Method Editor settings
9. 2 Select the Size Standards tab click New select the Basic or Advanced radio button then click OK 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 GS500 75 400 2007 08 09 13 23 gmidx Advanced CE F HID GS500 75 450 2007 08 09 13 24 gmidx Advanced CE G5 HID GS5500 2006 10 11 13 12 2 gmidx Advanced GS600 LIZ 2012 07 05 22 15 gmidx Advanced GS600 LIZ Normalization 80 400 2007 06 27 01 43 1 gmidx Advanced GS600 LIZ 80 400 2007 06 27 01 43 1 gmidx Advanced C lt o 19 xe P gt lt 02 S 3 Enter a name for example CE_G5_Yfiler_GS500 as shown below In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified in on page 60 The example below is for the GeneScan 500 LIZ Size Standard Size Standard Editor Edit Size Standard Description Name CE G5 Yfiler GS500 Security Group GeneMapper ID X Security Group v Description Size Standard Dye Size Standard Table Size in Basepairs 100 0 139 0 150 0 160 0 200 0 300 0 340 0 350 0 400 0 450 0 AmpFtSTR Yfiler PCR Amplification Kit Use
10. 46 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software For more information O D 5 lt 5 5 n E B AmpFtSTR Yfiler PCR Amplification Kit User Guide 47 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 file or a hid file Using GeneMapper ID X Software you can then analyze and interpret the data from fsa files GeneMapper ID X Software v1 0 1 or higher or hid files GeneMapper ID X Software v1 2 or higher Instruments Refer to Instrument and software overview on page 16 for a list of compatible instruments Before you start When using GeneMapper ID X Software v1 0 1 or higher to perform human identification HID analysis with AmpF STR kits be aware that 48 HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID X Software calculates allelic bin offsets by using an average of all ladders that use the
11. The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a PCR System 9700 with the silver 96 well block and were electrophoresed and detected using an Applied Biosystems 3100 Genetic Analyzer Table 4 Haplotypes of samples in Figure 19 Allele Sample A Sample B DYS19 14 15 DYS385a 11 13 DYS385b 14 15 DYS389 13 12 DYS389II 31 28 DYS390 24 23 DYS391 10 10 DYS392 13 11 DYS393 13 14 DYS437 15 16 DYS438 11 10 DYS439 12 13 AmpFtSTR Yfiler PCR Amplification Kit User Guide 87 Chapter 5 Experiments and Results Population data Allele Sample A Sample B DYS448 19 21 DYS456 17 15 DYS458 18 16 DYS635 23 22 Y GATA H4 12 12 The results of 1 ng total male male DNA mixture studies are shown in Figure 19 The limit of detection is when the minor component is present at approximately one tenth of the concentration of the major component and a threshold of 50 RFU The limit of detection for the minor component is influenced by the combination of genotypes in the mixture Figure 19 Mixtures of two male DNA samples and and A B ratios 1 ng input DNA The alleles attributable to the minor component even when the major component shares an allele are highlighted Population data SWGDAM Guid
12. 3 B 9 10 11 12 13 14 15 16 17 15 12 20 21 22 25 24 2 DYS458 DYS19 DYS385 AmpFtSTR Yfiler PCR Amplification Kit User Guide 77 Chapter 5 Experiments and Results Extra Peaks in the electropherogram Addition of 3 A 78 Figure 10 Stutter percentages for the DYS393 DYS391 DYS439 DYS635 DYS392 loci The DYS392 3 bp and 3 bp stutter percentages are shown in blue and grey respectively 5 10 E t 2 n 8 9 10 11 12 13 14 15 16 7 8 9 10 11 12 13 8 9 10 11 12 13 14 15 2 2 2 7 8 9 10 11 12 13 14 15 16 17 18 DYS393 DYS391 DYS439 DYS392 Figure 11 Stutter percentages for the Y GATA H4 DYS437 DYS438 and DYS448 loci 17 4 5 10 4 E 9 a HER 8 e i 3 t i i i s 14 e 0 8 9 10 11 12 13 13 14 15 16 17 8 9 10 1 2 3 17 18 19 20 21 22 23 24 Y GATA H4 DYS437 DYS438 DYS448 AmpliTaq Gold enzyme like many other DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 Magnuson et al 1996 This non template addition results in a PCR product that is one base pair longer than the actual target sequence and the PCR product with the extra nucleotide is referred to as the A form Figure 12 AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Section 5 1 Developmental Validation Extra Peaks i
13. AmpFtSTR Yfiler PCR Amplification Kit User Guide 25 e 9 ES T gt S 5 El m 5 a E 8 ER 5 Q ra E 5 3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument Prepare the samples for electrophoresis immediately before loading 1 26 O RO SOR ety BOs 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 8 7 jL 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 Pipette 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 e 1 uL of PCR product or allelic ladder Note For blank wells add 10
14. EN ERROR KR 66 Experiment conditions Wla yaba mh c od d 66 Developmental validation 000 ccc eee kk kk kk kk kk kk kk kk kk ka 67 SWGDAM Guideline 1 2 1 kK kk kK KK KK eee n 67 SWGDAM Guideline 2 1 0 1 Au kk kk kk cece eee EK KK e sn 67 PGR COMPONCMIS act csi eri is Ae pi CA ir e e rar T age Deer t ad 67 Thermal cycler parameters M M kk kk kk kk kk kK kk kk sss 67 PCR cycle number 2 vie Biss Ru Ag a MICE whe In eel OE 68 Accuracy precision and reproducibility kk kk kk kk kk kk KK KK KK KK KK ee 69 SWGDAM Guideline 2 9 2 d Ra dikk a K k E buk ele Sad a ks a 69 Precision and size windows 54 s04 EERE Eo G R EN 70 Extra Peaks in the electropherogram 4 We kk kk kk kk kK kK KK kk kK kK kk kK kk KK kk kK kk kk kk 76 Causesof extra D aks i d laa RR RR AREE EE Xua 76 Stutter products dd gie yer uer id sa mm prppmppmzzza 76 Addition Of uico shinee cc ugmmmmmmnmm m 78 Den 79 Characterization of ed ela 80 SWGDAM Guideline 2 1 _ KK KK KK 80 Nature of the polymorphisms 00000 cece eee kk kk kk kk kk kk kk kk 80 Inlieritanc zc iih ee haa n e in EA meh ein men nt 81 IM DING REKE eC tA Un OR M oe 81 Species specificity ELT IB ee al Rates i tM e rm 81 SWGDAM Guideline 2 2 i
15. 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 Prinz M Ishii A Coleman A Baum Shaler 2001 Validation and casework application of a Y chromosome specific STR multiplex Forensic Sci Int 120 177 88 Redd A J Agellon A B Kearney V A Contreras V A Karafet T Park H de Knijff P Butler J M Hammer 2002 Forensic value of 14 novel STRs on the human Y chromosome Forensic Sci Int 130 97 111 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Science Communications July 2004 Volume 6 3 Available at www fbi gov hq lab fsc current standards 2004 03 standards02 htm Schoske R Vallone P M Kline M C Redman J W Butler J M 2004 High throughput Y STR typing of U S populations with 27 regions of the Y chromosome using two multiplex PCR assays Forensic Sci Int 139 107 21 Smith R N 1995 Accurate size comparison of short tandem repeat alleles amplified by PCR Biotechniques 18 122 128 Sparkes R Kimpton C Watson S Oldroyd N Clayton T Barnett L Arnold J Thompson C Hale R Chapman J Urquhart A and Gill P 1996a The validation of a 7 locus multiplex STR test for use in forensic casework I Mixt
16. Three CEPH family DNA sets were examined 1 ng of DNA from each sample was amplified using the Yfiler Kit and the Identifiler Kit followed by analysis using a 3100 Genetic analyzer The families examined included 1333 9 offspring 7 males 1340 7 offspring 5 males and 1345 7 offspring 5 males representing 23 meiotic divisions The Identifiler Kit results confirmed that the loci are inherited according to Mendelian rules as reported in the literature 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 The Yfiler Kit results confirmed that the loci were inherited according to a Y linked father to son transmission In no case was the maternal grandfather s Y haplotype found in the offspring In family 1345 one son 1345 7356 had a DYS458 18 allele while the rest of his male relatives had a DYS458 17 allele In family 1340 one son 1340 7342 had a DYS458 16 allele while the rest of his male relatives had DYS458 17 Calculation of a mutation rate based on this small population size would be inaccurate due to the small sample size The samples were reamplified and reinjected to confirm the allele call The Yfiler Kit loci have been mapped and the chromosomal location on the Y chromosome is known based on the nucleotide sequence of the Y chromosome The Genbank accession numbers for representative sequences are DYS19 X77751 ACO017019
17. dye label 12 lack of amplification effect of DNA quantity on results 83 population data allele frequencies 89 population data samples used in studies 89 locus See loci low TE buffer 19 M materials and equipment included in kit 18 not included with kit 105 mixed samples 86 multicomponent analysis 16 N negative control sample preparation 21 AmpF amp TR Yfiler PCR Amplification Kit User Guide 0 operating systems 16 25 27 29 panels check version 49 50 import 33 50 PCR amplification of tetranucleotide STR loci stutter peak 76 inhibitor causing lack of amplification 84 performing 22 setup 109 thermal cycling conditions programming 22 PCR work areas 105 109 population genetics allele frequencies 89 populations and samples used in the studies 89 positive control sample preparation 21 primers volume per reaction 21 Q quantification DNA 19 R reaction mix volume per reaction 21 reactions preparing for PCR 21 reagents not included with kit 105 user supplied 19 references 119 run module electrophoresis 25 27 29 S safety biohazard 112 chemical 112 Safety Data Sheets SDSs obtaining 116 sample preparation 21 DNA negative control 21 DNA positive control 21 standards 18 software instrument compatibility 16 stutter check version 49 50 AmpFSTR Yfiler PCR Amplification Kit User Guide Index import 50 stutter peak or product 76 support obtaining 116 T techni
18. 49 Analyze and edit sample files with GeneMapper ID X Software 62 Examine and edita project sise suk een een 63 Section 4 1 GeneMapper ID Software Overview of GeneMapper D Software GeneMapper ID Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the data collection software stores information for each sample in a fsa file Using GeneMapper ID Software v3 2 1 software you can then analyze and interpret the data from the fsa files Instruments Refer to Instrument and software overview on page 16 for a list of compatible instruments AmpFtSTR Yfiler PCR Amplification Kit User Guide 31 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Before you start 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 Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run fol
19. Control A 100 Control B 100 500 pg Test A 100 Test B 100 Test C 100 Control A 100 Control B 100 1 ng Test A 100 Test B 100 Test C 100 Control A 100 Control B 100 Inhibition study An inhibition series of 1 ng control DNA 007 consisting of uninhibited control and Humic Acid at a final concentration of 12 ng uL in replicates of five was amplified using each of Test and Control mixes The amount of each inhibitor tested was titrated to cause an approximate 50 reduction in overall peak height of the samples Results were evaluated for mean referenced peak height minimum referenced peak height intracolor balance and levels of allelic dropout Mean referenced Uninhibited Control DNA 007 Test and Control mixes displayed no significant peak height difference in mean referenced peak height minimum referenced peak height and minimum displayed the same changes in intracolor balance as described in the reproducibility study For the Humic Acid inhibited DNA the Test Mixes containing both in house refe renced peak components showed slightly better results for mean referenced peak height minimum height and referenced peak height and intracolor balance for the FAM PET and NED dye intracolor balance 98 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement Inhibition study channels compared to the Control Mixes or the Test Mix
20. DYS385 AC022486 293950 DYS389 AC011289 AF140635 DYS390 AC011289 DYS391 G09613 AC011302 DYS392 G09867 06152 DYS393 G09601 AC06152 DYS437 002992 DYS438 AC002531 DYS439 AC002992 DYS448 AC025227 6 DYS456 010106 2 DYS458 AC010902 4 DYS635 G42676 011751 and Y GATA C4 G42673 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 Yfiler Kit provides the required degree of specificity such that it is specific to primates Other species do not amplify for the loci tested Nonhuman Studies Nonhuman DNA may be present in forensic casework samples The Yfiler Kit provides the required degree of specificity for the species tested Figure 14 AmpFtSTR Yfiler PCR Amplification Kit User Guide 81 g lt e 3 D e 5 E s 2 82 Chapter 5 Experiments and Results Species specificity Figure 14 Representative electropherograms from a species specificity study including positive and negative control Male Microbial pool Negative control 3200 2400 1600 00 o 3200 2400 1e00 wo 0 200 150 100 50 0 200 150 100 60 0 200 160 100 60 0 200 160 100 0 Li The following experiments were conducted to investigate interpretation
21. General Allele Peak Detector ji Peak Quality 50 amp GQ Settings Min Max Peak Height LPH MPH Homozygous min peak height H Perform Heterozygous min peak height internal validation Max Peak Height MPH studies to determine settings Peak Height Ratio PHR Min peak height ratio Broad Peak BD Max peak width basepairs C 0 2 0 o 0 p e v e e 2 0 Allele Number AN Max expected alleles Allelic Ladder Spike Cut off Value Factory Defaults Save Cancel 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 Yfiler Kit data AmpFtSTR Yfiler PCR Amplification Kit User Guide 59 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis SQ amp GQ tab settings Analysis Method Editor Quality weights are between and 1 General Allele Peak Detector Peak Quality 50 amp GQ Settings Sample and Control GQ Weighting Broad Peak BD Allele Number AN Out of Bin Allele BIN Low Peak Height LPH Overlap OVL i Max Peak Height MPH Marker Spike SP 0 3 Off scale OS Peak Height Ratio PHR C
22. KK KK KK KK KK kk 105 APPENDIX PCR Work Areas 109 Work area setup and lab design WW kk kk kk kk kk kk kk kK kK eee kK kK KK KK kK kK kK kk kk e 109 PCR i i b xake kad n dn k kk Ses kak alan dinek l Ka ran kl l Lee 109 Amplified DNA work area n kk kk kk kk kk kk kk kK kk KK kK KK kk kK kk kk kk n rl 110 APPENDIX D 5 111 Chemical salei scite oe ees ittis es pend PII den 112 Biological hazard safety kk kk cece KK KK KK KK kk KK n 112 AmpFtSTR Yfiler PCR Amplification Kit User Guide Contents Documentation and Support 115 Relatedidocumentationig inns efie vnnd KE dan A dau At Bats 115 Obtain SDSS ssi ee b ER Ile IS VeL VE a RV E EU RII 116 Obtaln SUppOrl eurer wee RA UR RR EU gales E EH HJ II H N E 116 Limited Product Warranty WW kk kk kk kk kk kk kK KK KK kK KK n 117 Bibliography qud iri aree dua os eue 119 Did Xs co suce eres rae E ees 123 AmpFtSTR Yfiler PCR Amplification Kit User Guide 7 Contents 8 AmpFtSTR Yfiler 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 Dat
23. User Guide Documentation and Support Related documentation Document title M AmpFtSTR Yfiler PCR Amplification Kit Human Identification Application Note 040302 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 AmpFtSTR PCR Amplification Kit PCR Products 4332345 User Bulletin 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 issues related to software data 4445098 hardware and consumables Note Additional user bulletins may be available at www lifetec
24. and the GeneMapper ID X Software Peaks in the stutter position that are above the highest observed stutter percent will not be filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated For evaluation of mixed samples see Mixture studies on page 86 The measurement of percent stutter may be unusually high for main peaks that are off scale AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Extra Peaks in the electropherogram Figure 8 Stutter percentages for the DYS456 DYS3891 DYS390 and DYS3891I 17 16 4 15 144 i1 13 i i 12 i 11 i 1 iw t j g 10 o 8 9 t 1 i IS Plo 3 a n 5 ij i 6 s 2 9 2 5 1 0 T T T T T T T 13 14 15 16 17 18 o 1 12 13 14 15 18 19 20 21 22 23 24 25 26 27 24 25 26 27 28 29 30 31 32 33 DYS456 DYS389I DYS390 DYS389II Figure 9 Stutter percentages for the DYS458 DYS19 and DYS385 loci The 4 bp and 2 bp stutter percentages for DYS19 are shown in blue and green respectively v 16 15 t 63 5 t Y i H 1 10 4 1 1 x i i 4 t 2 5 i 8 oue i a H i 1 t 6 i 3 1 14 15 s 17 15 19 2 fo 12 13 14 15 1 17 18
25. dtt ead all ed 7 heb tar Btn n aliat nl ply M eger ime nd ETT MERERI TUER RORIS ORE AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement 5 Sensitivity study Sensitivity study For the sensitivity study dilution series of three genomic DNA samples were amplified 1 ng three replicates each 0 5 ng 0 25 ng and 0 125 ng four replicates each The results were evaluated for mean referenced peak height degree of linearity between input DNA concentration and peak height level of allelic dropout at 125 pg and genotype concordance Mean referenced Mean referenced peak height observations were consistent between all Test and peak height Control mixes Figure 23 demonstrating equivalent performance Figure 24 Figure 23 Sensitivity study mean referenced peak heights three genomic DNA samples 125 250 500 1000 Lot Control U ED 3 D 5 Q 9 Ww E 2 d E Control B 3 D 2 O 2 Mean Referenced Peak Height 125 250 500 1000 125 250 500 1000 DNA Concentration pg Figure 24 Sensitivity study representative electropherograms for Sample 2 amplified using 125 pg input DNA Y scale 550 RFU ns i j l Control A 7 Mark for
26. failed for the sample The Genotypes tab becomes available after analysis see the figure on the next page Project window after analysis 2 lt GeneMapper ID v3 2 1 Untitled gmid Is Logged In DEAR File Edt Analysis View Tools Help FS UH DE e AmpFLSTR Table 58 a Project If Samples Z Yfiler Example Status Sample File Sample Type Analysis Method Panel Size Standard Run Name ji l Allelic Ladder fsa Allelic Ladder Yfiler__AnalysisMethod_ Yfiler_v2 CE_G5 Y filer Example l2 M Sample 1 fsa Positive Control filer AnalysisMethod Yfiler v2 Y filer_G v filer Example l Sample 10 fsa Negative Control Yfiler _AnalysisMethod_ Yfiler v2 2 4 ix Sample 11 fsa Sample Yfiler AnalysisMethod Yfiler v2 e er_G te V filer Example 5 Sample 12 fsa Sample Yfiler_AnalysisMethod_ Yfiler_v2 V filer Example 6 Sample 13 fsa Sample Yfiler AnalysisMethod Yfiler v2 Yfiler Yfiler Example 7 Sample 2fsa Sample Yfiler AnalysisMethod Yfiler v2 G Y filer Example 8 Sample 3 tsa Sample Yfiler _AnalysisMethod_ Yfiler_v2 E Y filer Example I Sample 4 fsa Sample Yfiler AnalysisMethod filer v2 Yfiler G Y filer Example 10 Sample 5 fsa Sample Yfiler AnalysisMethod Yfiler v2 Yfiler v filer Example n Sample 6 fsa Sample Yfiler AnalysisMetho
27. fluorescent dyes 16 G gels 85 gene diversity values 89 GeneMapper ID Software analyze project 44 create analysis method 37 create size standard 42 60 examine and edit project 45 import panels and bins 33 overview 16 31 setup 32 GeneMapper ID X Software analyze project 62 check version of panels bins and stutter 49 50 create analysis method 55 examine and edit project 63 overview 16 48 setup 49 GeneScan size standard about 18 dye label 16 volume per reaction 26 27 29 genetics allele frequencies 89 populations and samples used in studies 89 genotype determining 69 exclusion of suspects 88 H hematin effecton DNA samples 84 Hi Di formamide volume per reaction 26 27 29 import HID size standard 42 60 panels and bins 33 panels bins and stutter 50 instrumentation 310 genetic analyzer 16 29 3100 3100 Avant genetic analyzer 16 25 3130 3130xl genetic analyzer 16 25 3500 3500xL genetic analyzer 16 25 27 124 software compatibility 16 kit allelic ladder 18 amplification 11 contents 18 control DNA 18 description 11 DNA polymerase 18 21 fluorescent dyes 16 loci amplification 12 PCR reaction mix 18 21 primers 11 18 20 reagents 18 supported instruments 11 thermal cyclers for use with 110 L limited product warranty 117 LIZ size standard about 18 volume per reaction 26 27 29 loci allele frequencies in the population databases 89 chromosomal location 12 differential amplification 85
28. for 310 377 systems 4318159 MicroAmp 8 tube strip 0 2 mL N8010580 MicroAmp 96 well base holds 0 2 mL reaction tubes N8010531 MicroAmp 96 well full plate cover N8010550 MicroAmp 96 well tray retainer set 403081 POP 4 polymer for the 310 Genetic Analyzer 402838 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the 370 Genetic Analyzer User Guide Pub no 4317588 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 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide 107 Ordering Information Equipment and materials not included Item Source Tris HCL pH 8 0 MLS EDTA 0 5 M MLS Vortex MLS t For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handl
29. for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpF STR Kit Validation Part no 4440754 Initial Denature Anneal Extend Final Final hold incubation step extension HOLD CYCLE 30 HOLD 95 94 61 72 60 4 C 11 min 1 min 1min 1min 80 min 2 Load the plate into the thermal cycler and close the heated cover 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 be sure to 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 3 Start the run 4 On completion of the run store the amplified DNA and protect from light If you are storing the DNA Then place at lt 2 weeks 2 to 8 C 15 to 25 C 2 weeks IMPORTANT Store the amplified products so that they are protected from light 22 AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Perform Electrophoresis Allelic ladder requirements SA KK KK KK KK KK KK eee KK KK KK KK KK KK 23 Section 3 1 3100 3100 Avant and 3130 3130xl instruments 25 Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis 25 Prepare samples for e
30. 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 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 Yfiler PCR Amplification Kit User Guide Appendix DSafety 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
31. of Yfiler Kit results from nonhuman DNA sources The extracted DNA samples were amplified in Yfiler Kit reactions and analyzed using the 3100 Genetic Analyzer e Primates Gorilla chimpanzee orangutan and macaque 1 0 ng each Non primates Mouse dog pig cat horse chicken and cow 10 ng each Microorganisms Candida albicans Neisseria gonorrhoeae Escherichia coli 0157 H7 Bacillus subtilis Staphylococcus aureus and Lactobacillus rhamnosus 5 ng each The chimpanzee and gorilla DNA samples produced partial profiles within the 100 330 base pair region The remaining species tested did not yield reproducible detectable products AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Section 5 1 Developmental Validation Sensitivity Sensitivity SWGDAM Guideline When appropriate the range of DNA quantities able to produce reliable typing results should 23 be determined S WGDAM July 2003 Effect of DNA The amount of input DNA added to the Yfiler Kit should be between 0 5 and 1 0 ng quantity on results Figure 15 on page 84 The DNA sample should be quantitated prior to amplification and importance of using a system such as the Quantifiler Human DNA Quantitation Kit ABC Part no 4343895 or the Quantifiler Y Human Male DNA Quantification Kit quantitation Part no 4343906 The final DNA concentration should be in the range of 0 05 0 10 ng uL so that 0 05 0 10 ng of DNA will be added to the PC
32. of the alleles included in the AmpF STR Yfiler Allelic Ladder 18 AmpFtSTR Yfiler PCR Amplification Kit User Guide Perform PCR Required user supplied reagents eh 19 a DNA quantification K RHAR D R a RW NS Ye RI RU He k 19 Prepare the amplification kit 21 Perform o PU Up eR pe b 22 Required user supplied reagents In addition to the Yfiler Kit reagents the use of low TE buffer 10 mM Tris 0 1 mM EDTA pH 8 0 is recommended You can prepare the buffer as described in the procedure below or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together e 10mLof 1 M Tris HCl pH 8 0 02 mL of 0 5 M EDTA pH 8 0 990 mL glass distilled or deionized water Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature DNA quantification Importance of Quantifying the amount of DNA in a sample before amplification allows you to quantification determine whether or not sufficient DNA is present to permit amplification and to calculate the optimum amount of DNA to add to the reaction The optimum amount of DNA for the Yfiler Kit is 1 0 ng in a maximum input volume of 10 uL for 30 PCR cycles AmpFtSTR Yfiler PCR Amplification Kit User Guide 19 Perform PCR DNA quantification If too much DNA is added to the PCR reaction th
33. the 310 instrument 2 Pipette the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each 0 2 mL sample tube add e 25 uL of the formamide size standard mixture 1 5 uL of PCR product or allelic ladder Note For blank wells add 25 uL of Hi Di Formamide 5 Sealthe tubes with the appropriate septa then briefly centrifuge to ensure that the contents of each tube are mixed and collected at the bottom Heat the tubes in a thermal cycler for 3 minutes at 95 C Immediately place the tubes on ice for 3 minutes Place the sample tray on the autosampler Ensure that an injection list is prepared St Ber ZA Start the electrophoresis run 30 AmpFtSTR Yfiler PCR Amplification Kit User Guide Analyze Data Section 4 1 GeneMapper ID Software 31 Overview of GeneMapper ID Software 31 Set GeneMapper ID Software for data 32 Analyze and edit sample files with GeneMapper ID Software 44 Examine and edita project 6 eee KK KK KK 45 For more information 0 66 ee k hh hh hen 46 Section 4 2 GeneMapper ID X Software 48 Overview of GeneMapper ID X Software 48 Set up GeneMapper ID X Software for data 1
34. the items are available from major laboratory suppliers MLS Table 9 Equipment Equipment Source 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer for Human Identification Applied Biosystems 310 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 Veriti 96 Well Thermal Cycler 4375786 Silver 96 well sample block N8050251 Gold plated silver 96 well sample block 4314443 Tabletop centrifuge with 96 well plate adapters optional MLS Table 10 User supplied materials Source AmpFSTR Yfiler9 PCR Amplification Kit 4427368 3100 Analyzer materials 96 well plate septa 4315933 Reservoir septa 4315932 3100 3130x 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide 105 Ordering Information Equipment and materials not included Item Source DS 33 Matrix
35. 19 90 0 05 18 123 82 0 05 0 53891 10 142 87 0 04 11 147 28 0 04 12 151 80 0 06 13 156 43 0 07 14 160 66 0 05 15 164 81 0 07 DYS390 18 192 26 0 05 19 195 99 0 04 20 199 93 0 05 21 203 85 0 06 22 207 83 0 05 23 211 90 0 04 24 215 90 0 05 25 219 88 0 06 AmpFtSTR Yfiler PCR Amplification Kit User Guide 71 Chapter 5 Experiments and Results Accuracy precision and reproducibility Allele Mean Standard Deviation 26 223 84 0 06 27 227 80 0 07 DYS389Il 24 253 05 0 05 25 257 17 0 06 26 261 19 0 07 27 265 38 0 08 28 269 42 0 08 29 273 36 0 06 30 277 63 0 07 31 281 76 0 09 32 285 78 0 07 33 289 93 0 05 34 293 94 0 06 DYS458 14 130 98 0 05 15 134 87 0 06 16 138 81 0 03 17 142 95 0 05 18 147 31 0 05 19 151 72 0 05 20 155 94 0 04 DYS19 10 176 06 0 07 11 179 98 0 05 12 183 84 0 05 13 187 76 0 03 14 191 64 0 05 15 195 49 0 05 16 199 32 0 05 17 203 20 0 06 18 207 09 0 07 19 211 02 0 06 DYS385 a b 7 242 79 0 05 8 246 89 0 07 9 250 94 0 04 10 254 98 0 07 72 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility Allele Mean Standard Deviation 11 259 04 0 08 12 263 08 0 06 13 267 2
36. 4 0 05 14 271 38 0 06 15 275 47 0 10 16 279 56 0 08 17 283 70 0 07 18 287 79 0 05 19 292 06 0 06 20 296 19 0 07 21 300 42 0 06 22 305 06 0 12 23 309 50 0 07 24 313 99 0 10 25 318 39 0 05 DYS393 8 100 26 0 05 9 104 19 0 04 10 108 05 0 04 11 112 04 0 04 12 115 98 0 04 13 119 89 0 04 14 123 89 0 04 15 127 80 0 05 16 131 95 0 04 DYS391 7 150 88 0 08 8 155 27 0 06 9 159 67 0 06 10 163 83 0 05 11 167 94 0 07 12 172 00 0 07 13 176 03 0 06 DYS439 8 197 84 0 05 9 201 70 0 03 10 205 68 0 05 11 209 46 0 04 AmpFtSTR Yfiler PCR Amplification Kit User Guide 73 g lt e 3 D 2 EX S Qo 2 e Chapter 5 Experiments and Results Accuracy precision and reproducibility Allele Mean Standard Deviation 12 213 47 0 03 13 217 41 0 03 14 221 42 0 05 15 225 17 0 04 DYS635 GATA C4 20 246 43 0 07 21 250 49 0 06 22 254 45 0 06 23 258 49 0 03 24 262 45 0 06 25 266 56 0 06 26 270 56 0 03 DYS392 7 291 38 0 05 8 294 39 0 07 9 297 44 0 06 10 300 30 0 06 11 303 91 0 07 12 307 44 0 07 13 310 64 0 08 14 313 74 0 07 15 317 12 0 11 16 320 45 0 08 17 323 54 0 09 18 326 79 0 10 Y GATA H4 8 122 01 0 06 9 125 98 0 06 10 129 97 0 07 11 134 01 0 04 12 138 09 0 03 13 142 37 0 05 DYS437 13 182 53 0 05 14 186 45 0 07 15 190 40 0 04 16 194 25 0 04 17 198 07 0 03
37. 7 Y STR loci during automated DNA fragment analysis AmpFtSTR Yfiler PCR Amplification Kit User Guide 11 1 Overview Product overview Loci amplified by The following table shows the loci amplified their chromosomal locations and the the kit corresponding fluorescent marker dyes The AmpF STR Yfiler Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Control DNA 007 are also listed in the table Table 1 Yfiler Kit loci and alleles Locus Alleles included in AmpF STR Yfiler Dye Control DNA designation Allelic Ladder label 007 DYS456 13 14 15 16 17 18 6 FAM 15 DYS389 10 11 12 13 14 15 DYS390 18 19 20 21 22 23 24 25 26 27 24 DYS389 I 24 25 26 27 28 29 30 31 32 33 34 29 DYS458 14 15 16 17 18 19 20 VIC 17 DYS19 10 11 12 13 14 15 16 17 18 19 15 DYS385 a b 7 8 9 10 11 12 13 14 15 16 17 18 19 20 11 14 21 22 23 24 25 DYS393 8 9 10 11 12 13 14 15 16 NED 13 DYS391 7 8 9 10 11 12 13 11 DYS439 8 9 10 11 12 13 14 15 a DYS635 20 21 22 23 24 25 26 24 DYS392 7 8 9 10 11 12 13 14 15 16 17 18 13 Y GATA H4 8 9 10 11 12 13 PET9 13 DYS437 13 14 15 16 17 P15 DYS438 8 9 10 11 12 13 12 DYS448 17 18 19 20 21 22 23 24 19 Allelic ladder Figure 1 shows the all
38. Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 Quality assurance standards for forensic DNA testing laboratories 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 Furedi S Woller J Padar Z Angyal M 1999 Y STR haploty ping in two Hungarian populations Int J Legal Med 113 38 42 Gonzalez Neira A Elmoznino M Lareu M V Sanchez Diz P Gusmao L Prinz M Carracedo A 2001 Sequence structure of 12 novel Y chromosome microsatellites and PCR amplification strategies Forensic Sci Int 122 19 26 Gusmao L Gonzalez Neira A Pestoni C Brion M Lareu M V Carracedo A 1999 Robustness of the Y STRs DYS19 DYS389 I and II DYS390 and DYS393 optimization of a PCR pentaplex Forensic Sci Int 106 163 72 Hall A and Ballantyne J 2003 The development of an 18 locus Y STR system for forensic casework Anal Bioanal Chem 376 1234 46 Holt C Stauffer C Wallin J Lazaruk L Nguyen T Budowle B and Walsh P 2000 Practical applications of genotypic Surveys for forensic STR testing Forensic Sci Int 112 91 109 Johnson C L Warren J H Giles R C Staub R W 2003 Validation and uses of a Y chromosome STR 10 plex for forensic and paternity laboratories J Forensic Sci 2003 48 6 1260 8 AmpFtSTR Yfiler PCR Amplification Kit
39. AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement 5 Reproducibility study Reproducibility study For the reproducibility study 12 replicates of control DNA 007 at 1 ng input and three negative control replicates were amplified The results were evaluated for intracolor balance stutter percentage and the presence signal intensity and location of artifacts Intracolor balance The Test mixes delivered lower intracolor balance results for the FAM dye channel blue but improved intracolor balance results for NED dye channel yellow compared to the Control mixes The levels of intracolor balance obtained for the Test and Control mixes all fall within the expected range of performance for the Yfiler Kit Figure 20 Figure 20 Reproducibility study intracolor balance Lot Control E Control Test A U e 9 E QO 5 Q D 9 00 os m N 5 2 A Tet B di o e 2 Intracolor Balance Percentage Stutter Stutter percentage results for each marker were comparable across all Test and Control percentages mixes Figure 21 AmpFtSTR Yfiler PCR Amplification Kit User Guide 93 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Reproducibility study Figure 21 Reproducibility study mean stu
40. E Buffer with 0 1 mM EDTA More than one allele present at a locus except for DYS385a b Some but not all loci visible on electropherogram of DNA test samples Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Amplification of stutter product Mixed sample Test sample DNA is severely degraded See Stutter products on page 76 If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Test sample contains high concentrations of a PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test Poor peak height Incorrect thermal cycler parameters Check the protocol for correct thermal cycler balance parameters GeneAmp PCR System 9700 with Use GeneAmp PCR System 9700 with silver or Aluminum 96 Well block or third gold plated silver blocks only or the Veriti 96 Well party thermal cyclers Thermal Cycler 104 AmpFtSTR Yfiler PCR Amplification Kit User Guide Equipment and materials not included Ordering Information Table 9 and Table 10 list required and optional equipment and materials not supplied with the Yfiler Kit Unless otherwise noted many of
41. O Sj ao on a ow O identi Identifiler v1 1X null Identifiler_v1 1 zs i amp EZ MiniFiler_v1 1X MiniFiler_v1 1 nul HE NGM_SElect_v2 1X NGM SElect v2 1X null m z NGM_v3 1X NGM v3 1X null in Se 10 Profiler_Plus CODIS_v1 1 null 8 Profiler Plus v1 1X iE Profil amp CProfiler v1 1x 11 Profiler Plus v1 1X null 12 Profiler v1 1X null H O SEfiler Plus v1 1X DS ewl ani AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 8 Select and expand Yfiler_v1 1X in the navigation pane then select B_DYS389II to display the Bin view for the marker in the right pane Panel Manager File Edit Bins View Help aP gt lt ig ND OE UU Bin Set ampFLSTR_Bins_v2x FLERE n u o sf Panel Manager m C AmpFLSTR Panels v1x 10 S E AmpFLSTR Panels v2X tC COfiler CODIS v1 1X 03 S O filler v1 1x a Y DYS391 a Y DYS439 07 a Y DYS635 Y DYS392 0 R_Y_GATA_H4 R_DYS437 R_DYS438 04 R_DYS448 B_DYS456 0 3 B_DYS3891 B_DYS390 24 26 26 27 28 29 30 31 32 33 34 08 05 C gt D o 15 o P e g 0 2 9 0 1 n wcACO 0 0 241243 245 247 249 251 253 255 257 259 261 263 265 267 269 271 273 275 277 273 281283 285 287 289 291293 295 237 299 301303 305 307 ass Refere
42. Panel Manager a Select Panel Manager in the navigation pane Ele Edit Bins View Help b Expand the Panel Manager folder and any sub folders to identify the analysis file version already installed for your kit choice i 4 Check the version of files available for import into the Panel Manager a Select Panel Manager then select File Import Panels to open the Import Panels dialog box b Navigate to then open the Panels folder and check the version of panel bin and stutter files installed 5 If newer versions are available on the website download and import as described below Impo rt pa nels To import the Yfiler Kit panel bin set and marker stutter from our web site into the bins and marker GeneMapper ID X Software database stutter 1 50 a b Download and open the file containing panels bins and marker stutter Go to www lifetechnologies com support Software Patches amp Updates GeneMapper ID X Software Download the file AmpFLSTR Analysis Files GMIDX Unzip the file Start the GeneMapper ID X Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 Select Tools Panel Manager Find then open the folder containing the panels bins and marker stutter a Select File Import Panels to open the Import Select Panel Manager in the n
43. Plus v1 E ldertifilerDirect GS500 L AmpFLSTR_Yfiler_Pan 8 amp mpFLSTR NGM v2 b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the x V Applied Biosystems GeneMapper Panels folder d Select AmpFLSTR Yfiler Bins v2 txt then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR Yfiler Panel v2 folder Import Bin Set Look in Panels E AmpFLSTR_Bins_v1 tt K3 E AmpFLSTR Bins v2 bxt My Recent E AmpFLSTR Panels v1 txt Documents E AmpFLSTR Panels v2 txt AmpFISTR_Yfiler_Bin_v2txt E AmpFISTR_Y filer_Panel_v2 tot Desktop E 2 My Documents File name AmpFISTR Yfiler Bin v2ibd Ws Files of type Files x 34 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis 6 View the imported panels in the navigation pane a Double click the AmpFLSTR_Yfiler_Panel_v2 folder b Double click the Yfiler_v2 folder to display the panel information in the right pane and the markers below it P Panel Manager File Edit Bins View Binset Binset v2 ii 5 Bi OOOO E amp 3Panel Manager Marker Name Dye Color Min Size Size Control Alleles amp LAmpFLSTR Panels v1 B DYS456 blue 100 0 127 0 15 8 amp mpFLSTR_MiniFi
44. R reaction in a volume of 10 uL If the sample contains degraded DNA amplification of additional DNA may be beneficial If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in the following g lt e 3 D e 5 ei E e 2 Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem for two reasons Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate which results in poor spectral separation pull up Incomplete A nucleotide addition The sample can be re amplified using less DNA 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide 83 Chapter 5 Experiments and Results Stability Stability SWGDAM Guideline 2 4 Lack of amplification of some loci Effect of inhibitors 84 Figure 15 Effect of amplifying 1 ng 500 pg 250 pg 125 pg and 62 pg of male control DNA 007 Data analyzed using the 3100 Geneti
45. STR_Panels_v1X NGM_SElect_v2 1 nui AmpFLSTR Panels v2X Identifiler Plus v1 1X nul MAM es ut b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR Yfiler PCR Amplification Kit User Guide 51 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR_Bins_v2X txt then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR_Panels_v2X folder Import Bin Set Lookin G AmpFLSTR Analysis Files GMIDX My Recent B ampFistR_Stutter_v2x Documents El ReadMe AmpFLSTR v2X My Documents 48 File name AmpFLSTR Bins v2X txt My Computer Files of type all Files 7 View the imported panels in the navigation pane a Double click the AmpFLSTR Panels v2X folder b Double click the Yfiler v1 1X folder to display the panel information in the right pane Panel Manager File Edit Bins View Help ux mE MN Bin Set AmpFLSTR Bins v2X l lii W EE 8 R Panel Manager Panel Name Comment Ofiler_CODIS_v1 1X null YFiler_v1 1 null Identifiler CODIS v1 1X null Identifiler Direct v1 1X null cH Yfiler_v1 1x HE Identifiler_CODIS_v1 1 m n Identifiler_Direct_v1 1X m 2 Identifiler_Plus_v1 1 Identifiler_Plus_v1 1X null OP
46. Software for data analysis c Select B_DYS389II to display the Bin view for the marker in the right pane Panel Manager File Edit Bins View mE x gg m m JI Bin set 5 amp iPanel Manager 8 L amp mpFLSTR Panels v1 8 L amp mpFLSTR MiniFiler GS500 Panels v1 8 C amp mpFLSTR SEfiler Plus Panels v1 amp LAmpFLSTR Panels v2 8 C AmpFLSTR Identifiler Plus v1 8 L7 ldertifilerDirect GS500 v1 5 A AmpFLSTR Yfiler Panel v2 E E Yfiler_v2 B_DYS456 B_DYS3891 G_DYS458 G_DYS19 G_DYS385 Y_DYS393 Y_DYS391 Y_DYS439 Y_DYSB35 Y_DYS392 R_Y_GATA_H4 R_DYS437 R_DYS438 R_DYS448 Reference Samples ler Binset v2 B 9 n al 247 253 259 265 271 277 283 288 285 301 B_DYS389II 8 Click Apply then OK to add the AmpF STR 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 36 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Create an analysis The HID Advanced analysis method for the Yfiler Kit uses the method AmpFLSTR_Yfiler_Bins_v2 file described in step 5 on page 34 Use the following procedure to create a HID analysis method for the Yfiler Kit 1 Select Tools GeneMapper Manager to open the GeneMapp
47. 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 3100 Avant instrument refer to Appendix B of the 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide Pub no 4335393 3130xl Analyzer materials 96 well plate septa 4315933 Reservoir septa 4315932 3100 3130xl Genetic Analyzer capillary array 36 cm 4315931 POP 49 polymer for 3130 3130xl Genetic Analyzers 4352755 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ9 Size Standard 4322682 OR OR GeneScan 600 LIZ9 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 3130 3130xl instrument refer to Appendix A of the Applied Biosystems 9 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide Pub no 4352716 3500 3500xL Analyzer materials Anode buffer container ABC 4393927 Cathode buffer container CBC 4408256 POP 49 polymer 960 samples for 3500 3500xL Genetic Analyzers 4393710 POP 49 polymer 384 samples for 3500 3500xL Genetic Analyze
48. USER GUIDE applied biosystems by Life technologies AmpF STR Yfiler PCR Amplification Kit for use with 100 reaction kit Part no 4359513 Publication Number 4358101 Revision 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 TRADEMARKS 2014 Thermo Fisher Scientific Inc All rights reserved All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Life Technologies is a Thermo Fisher Scientific Brand TaqMan and AmpliTaq Gold are registered trademarks of Roche Molecular Systems Inc TaqMan is used under permission and license Microsoft Windows and Windows Vista are registered trademarks of Microsoft Corporation Minitab is a registered trademark of Minitab Inc Mac OS is a registered tr
49. User Guide 119 Bibliography 120 Kayser M Kittler R Erler A Hedman M Lee A C Mohyuddin A Mehdi S Q Rosser Z Stoneking M Jobling M A Sajantila A Tyler Smith C 2004 A comprehensive survey of human Y chromosomal microsatellites Am J Hum Genet 2004 74 1183 97 Kayser M Sajantila A Mutations at Y STR loci implications for paternity testing and forensic analysis 2001 Forensic Sci Int 118 116 21 Kwok S and Higuchi 1989 Avoiding false positives with PCR Nature 339 237 238 Lazaruk K Walsh P S Oaks F Gilbert D Rosenblum B B Menchen S Scheibler D Wenz H M Holt C Wallin J 1998 Genotyping of forensic short tandem repeat STR systems based on sizing precision in a capillary electrophoresis instrument Electrophoresis 19 86 93 Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J I Lowe A L Ghosh S and Collins F S 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Biotechniques 21 700 709 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 and McCord B R 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 Moretti T Baumstark A Defenbaugh D
50. ademark of Apple Inc Contents About This Guide J zoo Ex kk kk kk kK KK kK kk kk kk kk kok kk kok kk kk ere 9 Revisi n HIStory Naa mm X Ya SREP eee I DICH E EY 9 PURPOSE ML 9 M CHAPTER 11 Produc overview xu o bo baleen a hoe OD en daa H He 11 Aortic UR he io AA e e AT AE E 11 Product description oido ee pe lr 11 Aboutthe primers ee cee hooks lee HH DOR ROUEN S 11 Loci amplified by the kit kk A kk kk kk kk kk kk kK KK KK KK KK eese 12 Allelic ladder profile s bern ore t tete er te bees 12 Control DNA 007 profile 0000 eect n 14 Workflow OVervIew lt ks sd ced a ue merae erre oe Re ARR ka i E Re did 15 Instrument and software overview 16 Data Collection and GeneMapper ID or ID X Software 16 Instrument and software compatibility lk kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KIR 16 About multicomponent analysis MM kk kk kk kk kk kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk k 16 How multicomponent analysis works 4 W kk kk kk kk kk kk kk kk kK kk eee ee kk kk kk kk kk k 16 Materials and equipment MM 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 18 Kit contents and storage WW kk kk
51. allele size from the corresponding allelic ladder allele size All sample alleles are within 0 5 bp of a corresponding allele in an allelic ladder AmpFtSTR Yfiler PCR Amplification Kit User Guide 69 Chapter 5 Experiments and Results Accuracy precision and reproducibility Precision and size windows 70 Figure 7 Size deviation of 78 samples analyzed on the 3100 Genetic Analyzer Precision ER A DYS3889II x DYS390 A DYS331 DYS392 o DYS393 DYS437 2 8 a o a 7 o 0 5438 nDvs438 0 5448 o 075456 x 05458 x 0 5635 225 Allele Size bp x YGATAH4 Sizing precision allows for determination of accurate and reliable genotypes Sizing precision was measured on the 3100 Genetic Analyzer The recommended method for genotyping is to use a 0 5 bp window around the size obtained for each allele in the AmpF STR Yfiler Allelic Ladder A 0 5 bp window allows for the detection and correct assignment of alleles Any sample allele that sizes outside a window could be either of the following e An off ladder allele for example an allele of a size that is not represented in the AmpF STR Yfiler Allelic Ladder An allele that does correspond to an allelic ladder allele but whose size is just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele m
52. 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 flags sample alleles that do not size within the prescribed window around an allelic ladder allele It is important to note that while the precision within a set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences arise from a number of parameters including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can also be found between runs on the same instrument and between runs on different instruments because of these parameters We strongly recommend that the allele sizes obtained be compared to the sizes obtained for known alleles in the AmpF STR9 Yfiler Allelic Ladder from the same run and then converted to genotypes For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 g lt o e 3 e S E s 2 Table 3 Example of precision results of nine injections of the AmpF STR Yfiler Allelic Ladder run on the 3100 Genetic Analyzer Allele Mean Standard Deviation DYS456 13 104 51 0 05 14 108 31 0 05 15 112 16 0 04 16 116 04 0 04 17 1
53. alysis How multicomponent analysis works 16 This section provides information about the Data Collection Software versions required to run the AmpF STR Yfiler PCR Amplification 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 which is then analyzed by the GeneMapper ID or ID X Software Table 2 Software specific to each instrument Instrument Operating Data Colection Analysis software system Software 3500 3500xL Windows XP 3500 Series GeneMapper D X Software Windows Data Collection v1 2 or higher Vista Software v1 0 3130 3130xl Windows XP 3 0 GeneMapper D Software v3 2 1 31001 3100 Windows9 NT 1 1 3100 id Avant 1 0 3100 Avant amp GeneMapper ID X Windows 2000 20 Software v1 0 1 or higher 310 Windows XP 3 1 Windows NT 3 0 Windows 2000 We conducted validation studies 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
54. alyzed using the 3100 Genetic Analyzer No preferential amplification was observed in the denaturation temperature experiments Of the tested annealing temperatures 59 60 and 61 C produced robust profiles At 63 C the yield of the majority of loci was significantly reduced This should pose no problem with routine thermal cycler calibration and when following the recommended amplification protocol Preferential amplification was not observed at standard annealing temperature of 61 C Figure 5 An amplification of 1 ng of genomic DNA amplified while varying the annealing temperature analyzed on the 3100 Genetic Analyzer AJ 120 150 180 20 340 17 200 0 59 C 60 C 61 C 62 C Ll tala f The Yfiler Kit reactions were amplified for 28 29 30 31 and 32 cycles on the GeneAmp PCR System 9700 using 1 0 ng of three DNA samples As expected PCR product increased with the number of cycles Figure 6 A full profile was generated at 28 cycles off scale data were collected for several allele peaks at 32 cycles While none of the cycle numbers tested produced nonspecific peaks 30 cycles was found to give optimal sensitivity when the amplified products were examined on 3100 Genetic Analyzers At 30 cycles high ratios of female to male DNA amplify reliably and specifically following the conditions outlined in this user manual Figure 18 on page 87 AmpFtSTR Yfiler PCR Ampl
55. avigation pane s Panel Manager File Edit Bins View Help Panels dialog box Navigate to then open the AmpFLSTR Analysis Files GMIDX folder that you unzipped in step 1 on page 50 4 LJ X a 4 Em Panel Manager AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 5 Select AmpFLSTR_Panels_v2X or the version you installed then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR_Panels_v2X This folder contains the panels and associated markers Import Panels Lookin G AmpFLSTR Analysis Files GMIDX i c E AmpFLSTR_Bins_v2X My Recent gl AmpFLSTR Stutter v2X Documents ReadMe_AmpFLSTR_v2x My Documents ys File name AmpFLSTR Panels v2X bxt My Computer of type All Files C 0 2 o 0 p e v gt e p 2 0 6 Import AmpFLSTR Bins v2X txt a Select the AmpFLSTR Panels v2X folder in the navigation pane Panel Manager File Edit Bins Yiew Help X ke Bin Set i sf Panel Manager AmpFLSTR Bins jw jlji amp I E i BSE Comment Panel Name AmpFLSTR_NGMSElect_v2x Ofiler v1 1X null SGM_Plus_v1 1 C AmpFLSTR_P mpFL
56. bin set GeneMapper ID X Software v1 0 1 or higher 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 To apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the AmpFLSTR Panels v2X 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide 57 Peak Detector tab settings 58 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis Analysis Method Editor Peak Detection Algorithm Advanced Ranges Perform EITE dedi validation studies to determine settings Smoothing and Baselining Min Peak Half Width Smoothing None j Light Polynomial Degree O Heavy Peak Window Size Baseline Window 51 pts Peak Start Size Calling Method
57. bserved Additional studies need to be performed for other loci in order to estimate their average mutation rate AmpFtSTR Yfiler PCR Amplification Kit User Guide Mutation rate Section 5 1 Developmental Validation g lt e 3 o 5 E S 2 e 2 AmpFtSTR Yfiler PCR Amplification Kit User Guide 91 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Overview Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement Overview Experiments 92 As part of an ongoing program to exercise greater control over raw materials used in the AmpF amp STR PCR Amplification Kits manufacturing of the AmpliTaq Gold enzyme and 10X PCR Buffer II Tris KCl buffer components is transitioning from Roche Molecular Systems to Life Technologies Manufacturing of both components by Life Technologies will be conducted according to the same specifications used previously by Roche The in house components are established raw materials in our next generation kits for example the NGM NGM SElect and Identifiler Plus Kits We performed studies to compare the performance of the Yfiler Kit containing the in house components updated kit with the performance of the original kit focusing on studies most relevant to forensic DNA testing see SWGDAM Guidelines effective January 1 2011 These studies while not exhaustive are in our opinion appropriate for a ma
58. c Analyzer fi DEN TEN 4 d diy il i db 11 3 uan li u tt Lb Lu ti yl i lii il i Note The y axis scale is magnified for the lower amounts of DNA The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults has been extensively documented In most instances assessment of the effects of these factors on new forensic DNA procedures is not required However if substrates and or environmental and or chemical insults could potentially affect the analytical process then the process should be evaluated using known samples to determine the effects of such factors SWGDAM July 2003 As with any multi locus system the possibility exists that not every locus will amplify This is most often observed when the DNA sample contains PCR inhibitors or when the DNA sample has been severely degraded Valuable information may be obtained from partial profiles Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis et al 1988 Akane et al 1994 It is believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity To examine the effects of hematin on the amplification results obtained by the Yfiler Kit DNA samples were amplified using the Yfiler Ki
59. cal support 116 thermal cyclers for use with kit 110 programming conditions 22 training information on 116 troubleshooting 103 U user supplied reagents 19 V validation accuracy precision reproducibility 69 characterization of loci 80 developmental 66 experiments to evaluate 66 importance of 66 mixture studies 86 mutation rate 90 population data 88 sensitivity 83 species specificity 81 stability 84 thermal cycler parameters 67 W warranty 117 work area amplified DNA 110 PCRsetup 109 setup and lab design 109 workflow overview 15 125 Index 126 AmpF amp TR Yfiler 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 03 April 2014
60. containing the in house buffer and the enzyme purchased from Roche Molecular Systems Results for the VIC9 dye channel were more comparable across all Test and Control mixes Figure 28 29 and 30 Figure 28 Inhibition study mean referenced peak height Inhibitors HA Humic Acid PRI Pristine or Uninhibited DNA Eu at Q 9 amp 2 25 amp 20 15 io 0 5 gt gt ye C SP PO Pye SG SPF X KERA RR ees 9 7 CS ARD d RN 47 Dye Color and Locus Lot Control H Control B p Figure 29 Inhibition study minimum referenced peak height Inhibitors Humic Acid PRI Pristine or Uninhibited DNA Minimum Referenced Peak Height Lot 9 Contro A E Control B Test A Test B AmpFtSTR Yfiler PCR Amplification Kit User Guide 99 e 3 o 5 a 2 e 3 Ww 2 m 2 N lt 3 2 o E o 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Inhibition study Figure 30 Inhibition study intracolor balance Y axis intracolor balance percentage versus X axis dye color Inhibitors HA Humic Acid PRI Pristine or Uninhibited DNA B G R HA_007 PRI_007 Lot Control E Control B Test A Test 8 gt Test Intracolor Balance Percentage
61. ct File Import Panels to open the Import Panels dialog box c Navigate to then open the x Applied Biosystems GeneMapper Panels folder where x is the drive on which the GeneMapper ID Software is installed 4 Select AmpFLSTR_Yfiler_Panel_v2 txt then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR_Yfiler_Panel_v2 This folder contains the panels and associated markers 2 Import Panels C3 Panels E AmpFLSTR_Bins_v1 txt E AmpFLSTR_Bins_v2 bt My Recent E AmpFLSTR Panels v1 bt Documents E AmpFLSTR Panels v2 E AmpFISTR_Y filer_Bin_v2 txt AmpFISTR_Yfiler_Panel_v2txt My Documents File name AmpFISTR_Yiler_Panel_v2 txt My Computer Files of type Files AmpFtSTR Yfiler PCR Amplification Kit User Guide 33 D lt 97 o 9 5 90 E 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis 5 Import AmpFLSTR_Yfiler_Bins_v2 txt a Select the AmpFLSTR_Yfiler_Panel_v2 folder in the navigation pane 2 Panel Manager File Edit Bins View BE H L Bin Set ampristr_vfiler_Binset_v2 5 amp iPanel Manager Panel Hame Con Z AmpFLSTR_Panels_v1 Pies J non 7 AmpFLSTR MiniFiler GS500 Panels v1 7 AmpFLSTR SEfiler Plus Panels v1 amp LAmpFLSTR Panels v2 E LAmpFLSTR Identifiler
62. d Yfiler v2 V tiler Example 12 7 Sample Yfiler_AnalysisMethod_ Yfiler_v2 Y filer Example 13 Sample 8 fsa Sample Yfiler AnalysisMethod Yfiler v2 Y filer te V filer Example 14 l n Sample 9 fsa Sampie Yfiler _AnalysisMethod_ Yfiler_v2 Y file Te Y filer Example Kil n __ WMWMRM J SXMZvZ a Progress Status 0 Stor For more information about any of these tasks refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide 45 CD D ied o 19 o e 2 2 4 Chapter 4 Analyze Data For more information For more information For details about GeneMapper ID Software features allele filters peak detection algorithms and project editing refer to e GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 e Installation Procedures and New Features for GeneMapper ID Software Software Version v3 2 User Bulletin Pub no 4352543
63. d 95 bp and red 80 bp dye Low level artifacts in the calling region may or may not be reproducible Figure 13 demonstrates reproducible artifacts while using the Yfiler Kit Consider these artifacts when interpreting data AmpFtSTR Yfiler PCR Amplification Kit User Guide 79 Chapter 5 Experiments and Results Characterization of loci Figure 13 Examples of baseline noise and reproducible artifacts in data produced on the 3100 Genetic Analyzer Peak Amplitude Threshold set to 25 RFU to illustrate artifacts C_DYS458 pvsis 170 20 22 80 s0 100 no 120 130 140 130 160 D D D B 180 190 Genotyping may result in the detection of these artifacts as off ladder alleles or OL Alleles Note A high degree of magnification y axis is used in this figure to illustrate these artifacts data produced on capillary electrophoresis instrument platforms Characterization of loci SWGDAM Guideline 2 1 Nature of the polymorphisms 80 The basic characteristics of a genetic marker must be determined and documented SWGDAM July 2003 This section describes basic characteristics of the 17 loci that are amplified with the Yfiler Kit These loci have been extensively characterized by other laboratories Gusmao et al 1999 Butler et al 2002 Gonzalez Neira et al 2001 Hall and Ballantyne 2003 Redd et al 2002 Schoske et a
64. ders 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 the sample result needs to be verified according to the laboratory s protocol Set up GeneMapper D Software for data analysis File names Before using the software for the first time 32 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 support gt Software Patches amp Updates GeneMapper ID Software Before you can analyze sample fsa files using GeneMapper ID Software v3 2 1 for the firs
65. ditor General Allele Peak Detector Peak Quality 5Q amp GQ Settings Analysi De ion Yfiler_AnalysisMbthod_v2Xx 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 dropdown list The Description and Instrument fields are optional 56 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper 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 SQ amp GQ Settings Bin Set AmpFLSTR_Bins_v 2X Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off Value 0 0 0 0 0 0 Ratio 0 0 0 0 0 0 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 0 0 4 75 0 0 C 0 2 0 o 0 p e v gt e p 2 0 Global Plus Stutter Ratio 0 0 0 0 0 0 Global Plus Stutter Distance From 00 0 0 0 0 To 0 0 0 0 0 0 Amelogenin Cutoff 0 0 Range Filter Factory Defaults Save Cancel e In the Bin Set field select the AmpFLSTR Bins v2X
66. e Description A November 2004 New document B June 2005 _ C August 2006 _ D September 2010 E April 2011 Change to limited licensing information March 2012 Change to limited licensing information G August 2012 e Remove Mac 0S procedures Add 3100 3100 Avant 3130 3130xl 3500 3500xL Genetic Analyzer information Added GeneMapper D Software and GeneMapper ID X Software information e Add validation experiments and results for buffer and enzyme kit component changes H September 2012 Correct PCR final extension time error in rev G J April 2014 Change the number of PCR cycles from 28 to 30 on page 19 Purpose The AmpFtSTR Yfile PCR Amplification Kit User Guide provides information about the Life Technologies instruments chemistries and software associated with the AmpF STR Yfiler PCR Amplification Kit AmpFtSTR Yfiler PCR Amplification Kit User Guide About This Guide Purpose 10 AmpFtSTR Yfiler PCR Amplification Kit User Guide Overview B Product overview dk cen eee hm hh 11 Workflow overview 15 B Instrument and software overview 16 Materials and equipment 0 nnn 18 Product overview Purpose The AmpF STR Yfiler PCR Amplification Kit is a short tandem repeat STR multiplex assay that amplifies 17 Y STR loci in a single PCR amplificatio
67. e procedures start with the Analysis Summary tab of the Project window assuming the analysis is complete AmpFtSTR Yfiler PCR Amplification Kit User Guide 63 Chapter 4 GeneMapper ID X Software For more information For more information For more information refer to GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 e GeneMapper ID X Software Version 1 0 Quick Reference Guide Pub no 4375670 GeneMapper ID X Software Version 1 0 Reference Guide Pub no 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide Pub no 4396773 GeneMapper ID X Software Version 1 2 Reference Guide Pub no 4426481 GeneMapper ID X Software Version 1 2 Quick Reference Guide Pub no 4426482 GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 64 AmpFtSTR Yfiler PCR Amplification Kit User Guide Experiments and Results Section 5 1 Developmental Validation 66 Overview soe et ea ere eem A pU Ed d 66 Developmental validation LL WAK KK KK eee eee KK KK KK 67 Accuracy precision and reproducibility LSS KK KK KK KK 69 Extra Peaks in the electropherogram 66 6 KK KK KK KK KK KK KK KK 76 Characterization Of JOCL cesa ce ne v e ae e ke kin saga 80 Species specificitys Js veu ie e
68. elic ladder for the Yfiler Kit See Allelic ladder requirements profile on page 23 for information on ensuring accurate genotyping 12 AmpFtSTR Yfiler PCR Amplification Kit User Guide Chapter 10verview Product overview Figure 1 GeneMapper D X Software plot of the AmpF STR Yfiler Allelic Ladder _ Mark Sample For Deletion 95 125 165 205 245 EJ 325 D y 00 40 20 e Mark Sample for Deletion 95 125 165 05 245 205 325 500 400 200 100 on A A hi ARE A 14 5 le 17 Bs s Bo 19 L ja 2 G 15 Ge z s s E e amp Be ey ka G2 G Bs Bs 7 s Be l Ea Bs Be E Mark Sample for Deletion 85 125 165 205 245 295 325 40 300 200 100 ola A a eee NS X LE e E bol od la s ps ks bs b EJ bl bo lJ ea 2 Gs Ds Bo E3 bs 2 0 242 MM op b do 7 Mark Sample fer Deletion 85 125 165 205 245 225 325 AmpFtSTR Yfiler PCR Amplification Kit User Guide 13 Overview Product overview Control DNA 007 Figure 2 shows amplification of Control DNA 007 using the Yfiler Kit profile Figure 2 1 ng of Control DNA 007 amplified with the Yfiler9 Kit and analyzed on the Applied Biosystems 3130xl Genetic Analyzer
69. eline 2 7 Overview 88 The distribution of genetic markers in populations should be determined in relevant population groups GWGDAM July 2003 To interpret the significance of a match between genetically typed samples it is necessary to know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of the suspect s reference sample then the suspect is excluded as the donor of the biological evidence tested An exclusion is independent of the frequency of the two genotypes in the population AmpFtSTR Yfiler PCR Amplification Kit User Guide Population samples used in these studies Gene diversity values Analyzing the population data AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Population data If the suspect and evidence samples have the same genotype then the suspect is included as a possible source of the evidence sample The probability that another unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant population s The Yfiler Kit was used to generate the population data provided in this section Samples were collected from individuals throughout the United States with no geographical preference Population Number of samples African American 333 U S Caucasian 254 U S Hispanic 175
70. en the increased amount of PCR product that is generated can result in e Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data are problematic 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 and it results in poor spectral separation pull up Incomplete A nucleotide addition When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile Methods of quantifying DNA Life Technologies provides several kits for quantifying DNA in samples See the references cited in the following table for details about these kits Product Description Quantifiler Human DNA Quantification Kit Part no 4343895 and Quantifiler9 Y Human Male DNA Quantification Kit Part no 4343906 For more information see Quantifiler Human DNA Quantification Kits User s Manual Pub no 4344790 Properties The Quantifiler Human and Quantifiler Y Human Male Kits are highly specific for human DNA and they individually detect total human or male DNA respectively The kits detect single stranded and degraded DNA How t
71. er Manager Q GeneMapper Manager Projects Table Settings Plot Settings Matrices Size Standards Last Saved Owner Instrument Analysis Type IdentifilerDirect _HID_v1 2010 05 05 10 24 1 gmid HID Identifiler Plus amp nalysisMet 2011 05 19 14 41 1 amid HID Default analysis Microsatellite Default 2010 01 27 14 58 0 gmid Microsatellite Factory Provided 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 The figures below show the settings for each tab of the Analysis Method Editor Configure settings as shown unless the instructions state otherwise 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide 37 D lt 97 9 5 90 E 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis General tab settings Analysis Method Editor HID In the Name field either type the name as shown or enter a name of your choosing The Description and Instrument fields are optional 38 AmpFtSTR Yfiler PCR Amplification Kit User Guide Sec
72. f 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 Safety Data Sheets SDSs are available from www lifetechnologies com sds Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide Documentation and Support Limited Product Warranty Limited Product Warranty Life Technologies and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sa
73. g AmpliTaq Gold enzyme and 10X PCR Buffer II manufactured by Roche Molecular Systems AmpFtSTR Yfiler PCR Amplification Kit User Guide 101 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Conclusions 102 AmpFtSTR Yfiler PCR Amplification Kit User Guide Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis Table 8 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the AmpF STRO Control DNA 007 and the DNA test samples at all loci Incorrect volume or absence of PCR Master Mix or Yfiler Primer Set Repeat amplification No activation of AmpliTaq Gold 9 DNA Polymerase Repeat amplification making sure to hold reactions initially at 95 C for 11 minutes Master Mix not vortexed thoroughly before aliquoting Vortex the Master Mix thoroughly Yfiler9 Primer Set exposed to too much light Store the Primer Set protected from light GeneAmp 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 Tubes not seated tightly in the thermal cycler during amplification Push reaction tubes firmly into contact with block after first cycle Repeat test Wrong PCR reaction tube Use M
74. genotyping 23 volume per reaction 26 28 30 amplification differential amplification of loci 85 loci 12 AmpliTaq Gold DNA Polymerase catalyzing the ad dition of a 3 A nucleotide 78 B bins check version 50 import 33 50 biohazard safety 112 buffer new 92 C chemical safety 112 contents of kit 18 control DNA 007 14 18 control DNA 9947A 18 D Data Collection Software overview 16 data analysis 89 AmpFSTR Yfiler PCR Amplification Kit User Guide Index degraded DNA 85 developmental validation 66 differential amplification of loci 85 discriminatory capacity 90 DNA control about 18 effect of DNA quantity on results 83 how degraded DNA affects which loci amplify 85 negative control reaction 21 positive control reaction 21 quantification 19 quantification methods 20 sample preparation 21 test sample 21 using agarose gel analysis to examine the DNA 85 documentation related 115 E effect of inhibitors 84 electropherogram addition of a3 A nucleotide 78 stutter peak 76 electrophoresis Data Collection Software 25 27 29 preparing samples on the 310 instrument 29 preparing samples on the 3100 3100 Avant or 3130 3130xl instrument 26 preparing samples on the 3500 3500xL instrument 27 reagents and parts 25 27 29 references 25 27 29 run module 25 27 29 setup 25 27 29 emission spectra 17 enzyme new 92 equipment not included with kit 105 evidence exclusion of suspects 88 123 Index F
75. hey work The Quantifiler DNA Quantification Kits consist of target specific and internal control 5 nuclease assays The Quantifiler Human and Quantifiler Y Human Male Kits contain different target specific assays human DNA or human male DNA respectively that each consist of two locus specific PCR primers and one TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence The kits each contain a separate internal PCR control IPC assay which consists of an IPC template DNA a synthetic sequence not found in nature two primers for amplifying the IPC template and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC Quantifiler Duo DNA Quantification Kit Part no 4387746 For more information see Quantifiler9 Duo DNA Quantification Kit User s Manual Part no 4391294 Properties The Quantifiler Duo Kit is highly specific for human DNA This kit combines the detection of both total human and male DNA in one PCR reaction The kit detects single stranded and degraded DNA How it works The Quantifiler Duo DNA Quantification Kit consists of target specific and internal control 5 nuclease assays The Quantifiler Duo kit combines two human specific assays in one PCR reaction for total human DNA and human male DNAJ The two human DNA specific assays each consist of two PCR primers and a TagMan probe The TagMan probes for the human DNA and human male DNA assays are
76. hnologies com Applied Biosystems 3730 3730xl Genetic Analyzer Getting Started Guide 4359476 GeneAmp PCR System 9700 Base Module User s Manual N805 0200 Veriti 96 Well Thermal Cycler AmpFtSTR9Kit Validation User Bulletin 4440754 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit Users Manual PrepFiler9 Forensic DNA Extraction Kit User Guide 4390932 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 ID X Software Version 1 1 Mixture Analysis Getting Started Guide 4396773 AmpFtSTR Yfiler PCR Amplification Kit User Guide 115 Documentation and Support Obtain SDSs Document title eL MN GeneMapper ID X Software Version 1 1 Mixture Analysis Quick Reference Guide 4402094 GeneMapper ID X Software Version 1 2 Reference Guide 4426481 GeneMapper ID X Software Version 1 2 Quick Reference Guide 4426482 Obtain SDSs Obtain support 116 Portable document format PDF versions o
77. hresholds for interpretation of Yfiler 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 Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks e Size calling method The Yfiler Kit has been validated using the Local Southern sizing method Select alternative sizing methods only after performing the appropriate internal validation studies 40 AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Peak Quality tab Analysis Method Editor HID settings General Allele Peak Detector Peak Quality Quality Flags T Signal level Perform Homozygous min peak height internal validation Heterozygous min peak height s E studies to Heterozygote balance determine settings Min peak height ratio r Peak morphology Max peak width basepairs Pull up peak Pull up ratio D lt 97 o 9 5 90 E S r Allele number Max expected alleles Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and h
78. icroAmp Reaction Tubes with Caps for the GeneAmp PCR System 9700 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 23 Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide AmpFtSTR Yfiler PCR Amplification Kit User Guide 103 Troubleshooting Observation Possible causes Recommended actions Positive signal from AmpF STR Control DNA 007 but partial or no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantitate DNA and add 1 0 ng of DNA Repeat test Test sample contains high concentration of PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpFZSTR MiniFiler Kit Dilution of test sample DNA in water or wrong buffer for example TE formula with incorrect EDTA concentration Redilute DNA using low T
79. ies 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 AmpFLSTR Yfiler Panel v2 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide 39 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Peak Detector tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced mI Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds FulRange v all Sizes 50 R 50 G 50 o v 5 Smoothing and Baselining Min Peak Half Width 2 Smoothing O None Polynomial Degree 3 Light O Heavy Peak Window Size 15 r Slope Threshold Baseline Window 51 pts Peak Start Size Calling Method Peak End 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Factory Defaults Perform internal validation studies to determine settings IMPORTANT Perform the appropriate internal validation studies to determine the peak amplitude t
80. ification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility Figure 6 An amplification of 1 ng of genomic DNA amplified while varying the cycle number analyzed on the 3100 Genetic Analyzer EX m zi ms Pe 28 29 30 db Li di d d d g lt e 3 D e S E e 2 i LLL LA Lil Lb 32 BB888BB 2882238 888828 288888 Accuracy precision and reproducibility SWGDAM Guideline The extent to which a given set of measurements of the same sample agree with their mean and 29 the extent to which these measurements match the actual values being measured should be determined SWGDAM July 2003 Laser induced fluorescence detection systems of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2001 and Wallin et al 2001 However accuracy and reproducibility of Yfiler Kit profiles have been determined from various sample types Figure 7 illustrates the size differences that are typically observed between sample alleles and allelic ladder alleles on the 3100 Genetic Analyzer with POP 4 polymer The x axis in Figure 7 represents the nominal base pair sizes for the AmpF STR Yfiler Allelic Ladder and the dashed lines parallel to the x axis represent the 0 5 bp windows The y axis is the deviation of each sample
81. ile 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 Parameter Settings Sample Type Select the sample type Analysis Method Yfiler_AnalysisMethod_v2 or the name of the analysis method you created Panel Yfiler_v2 Size Standard CE_G5_Yfiler_GS500 or the name of the size standard you created t The Yfiler9 Kit was originally validated using the GeneScan 500 LIZ9 Size Standard If you use the GeneScan 600 LIZ Size Standard v2 0 as an alternative perform the appropriate internal validation studies to support the use of this size standard with the Yfiler Kit Note For more information about how the Size Caller works refer to the GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 3 Click B Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis As a completion bar extending to the right with the percentage indicated With text messages on the left 44 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software Examine and edit a project The table displays the row of the sample currently being analyzed in green or red if analysis
82. iles to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software The instructions and examples in this section refer to the latest version of panel bin and stutter file available at the time of publication Before using the Before you use GeneMapper ID X Software v1 0 1 or higher for fsa files v1 2 or software for the higher for hid files to analyze data for the first time you must do the following first time 1 Check the version of panel bin and stutter files installed with the GeneMapper ID X Software as explained in Check panel bin and stutter file version below 2 Check www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software to determine if newer files are available 3 If updated files are available download and import the files into the GeneMapper ID X Software as explained in Import panels bins and marker stutter on page 50 Note When downloading new versions of analysis files refer to the associated Read Me file for details of changes between software file versions If you have validated previous file versions for data analysis conduct the appropriate internal verification studies before using new file versions for operational analysis 4 Create an analysis method as explained in Create an analysis method on page 55 5 C
83. includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 AmpFtSTR Yfiler PCR Amplification Kit User Guide 113 Safety Biological hazard safety 114 AmpFtSTR Yfiler PCR Amplification Kit
84. ing any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions 108 AmpFtSTR Yfiler PCR Amplification Kit User Guide PCR Work Areas Work area setup and lab design 0 6 6 ccc KK KK KK KK 109 PCR setup work area 0 6 6 eee enn HA 109 Amplified DNA work area 6 eens 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 Yfiler 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 Yfiler 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 using PCR technology PCR
85. kK kk kk kk kK kk kk 89 MUitation rates cos 90 Section 5 2 Performance Validation After Buffer and Enzyme Component Replacement 000 kk kk kk kk kk kk kK tee kk kk eee kk ka 92 OVERVIEW ica Deme enced Aes faa nei ona cu xb LM ERR detiene ted 92 Experiments RNMNMMME DMDMNNNMNDNNnNnNnNnRnEEHnEnDnDMDIRnRnRHEDEDnDEBDDEHHnHHnEHnHEHnHnNn n EHHEHHREHHMMP LNEA 92 Reproducibility Study os HH HH EH E HRIH re Aber d 93 Intracolorbalance An e etc yt deka fecal dts 93 Stutter percentages MM WW kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk n 93 Rr 94 Sensitivity StUGY LA ME Mo tero Ia 95 Mean referenced peak height WW kk kk kk kk kk kk kk kK kK kK kK kk kK KK kk kk kk kK kk kk kk kk kk 95 DNA concentration and peak height n nannan nanan kK kK KK KK KK KK KK KK KK KK KK KK KK kk 96 Allelic dropout 4 5 cence pr ue DAR WAP ade pe dae peed es 96 Genotype concordance 02 a a A A 98 set oh ee oats oot cp 98 Mean referenced peak height minimum referenced peak height and intracolor balance 98 AllelicdEODOUb cm ob ttm Mae da Ansa et lt d roli 101 CONCLUSIONS c 101 APPENDIX A Troubleshooting 103 APPENDIX Ordering Information 105 Equipment and materials not included 0 cece cece KK
86. ke k GR baka ad qe ehe 81 SOT SI UV L Y eos xod LU NE Opi Rc Lec onde yek ed t doo tpi Ar te 83 jc T 84 Mixture st dies 5 E pe gt RU RUE ER RU Ee Op M 86 Population datane eee eue e eS 88 BNN eee xr m de we hea Re dose gable baat EUR 90 Section 5 2 Performance Validation After Buffer and Enzyme Component Replacements debite a 92 OVET VIEW DnE ud e de i Sie ls Re Die Reg etel Bow eta 92 EXPOriMen S s 2 ss tenet ME rpm 92 Reproducibility study 93 Sensitivity study ore bere AG REN e EE eeu SEE eene 95 Inhibitioristudy lt lt xuy nnn bes de ab ea dee n ka du ee S qure 98 Conclusions e 101 AmpFtSTR Yfiler PCR Amplification Kit User Guide 65 Chapter 5 Experiments and Results Overview Section 5 1 Developmental Validation Overview Importance of validation Experiment conditions 66 This chapter provides results of the developmental validation experiments we performed using the AmpF STR Yfiler PCR Amplification Kit 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
87. kk kk kk kk kk kk kk kk kK KK kk kk kk kk kk kk kk kk kk kk kk kk 18 Standards for samples escis tee aA nim EN Tp ER eR L Ai EZA AA 18 M CHAPTER 2 19 Required user supplied reagents WWW kk kk kk kk kk kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk kk kk 19 DNA quarntificatln gt a xa S la kab e ca ah n xala ko euet De Rohe we rae 19 Importance of quantification s kk kk kk kk kK kK KK KK KK KK KK kK KK KK KK kK kK KK KK kk 19 Methods of quantifying D NA a a n Kenan eh nk a Edla NA d A aA n a Eek rn 20 Prepare the amplification kit lt 8 8 21 Perform PCR yana na a WEN Aw e kar n k wad dei due riae pe ae kn WE ka k n ned 22 m CHAPTER Perform Electrophoresis 23 Allelic ladder requirements le 23 Section 3 1 3100 3100 Avant and 3130 3130xl instruments 25 Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis 25 Reagents and parts ao uds eR ee R ja a SEQUI pii ES 25 Electrophoresis software setup and reference documents 25 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument 26 AmpFtSTR Yfiler PCR Amplification Kit User Guide 3 C
88. l 2004 DYS392 is a trinucleotide repeat DYS438 is a pentanucleotide repeat and DYS448 is a hexanucleotide repeat Their allele differences result from differences in the number of repeat units 3 bp 5 bp and 6 bp respectively The remaining Yfiler Kit loci are tetranucleotide short tandem repeat STR loci The length differences among alleles of these particular loci result from differences in the number of 4 bp repeat units We have sequenced all the alleles in the AmpF STR Yfiler Allelic Ladder In addition other groups in the scientific community have sequenced alleles at some of these loci Redd et al 2002 www cstl nist gov biotech strbase y strs htm Among the various sources of sequence data on the Yfiler Kit loci there is consensus on the repeat patterns and structure of the STRs AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Inheritance Mapping Section 5 1 Developmental Validation Species specificity The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from 39 families of Utah Mormon French Venezuelan and Amish descent These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring Consequently the CEPH family DNA sets are ideal for studying inheritance patterns Begovich et al 1992
89. labeled with VIC and FAM dyes respectively In addition the kit contains an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye 20 AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Chapter 2Perform PCR 2 Prepare the amplification kit reactions Prepare the amplification kit reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below DNA sample Volume per reaction AmpF STR PCR Reaction Mix 9 2 uL AmpF STR Yfiler Primer Set 5 0 uL AmpliTaq Gold DNA Polymerase 0 8 uL 2 Prepare reagents Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers Thaw the PCR Reaction Mix and the Primer Set then vortex all reagent tubes including the enzyme for 3 seconds and centrifuge briefly before opening the tubes IMPORTANT Thawing is required only during first use of the Primer Set and PCR Reaction Mix After first use these reagents are stored at 2 to 8 C and therefore they do not require subsequent thawing Do not refreeze these reagents 3 Prepare the master mix Pipette the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the master mix for 3 seconds then centrifuge briefly 5 Dispense 15 uL of the master mix into each reaction well of a MicroAmp Optical 96 We
90. 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 Yfiler Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ dye 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 the Life Technologies instruments the fluorescence signals are separated by 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 it is displayed as blue followed by the VIC dye green NED dye yellow PET dye red and LIZ dye orange AmpFtSTR Yfiler PCR Amplification Kit User Guide Chapter 10verview 1 Instrument and software overview 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 3 The goal of multicomponent analysis is to correct for spectral overlap Figure 3 Emission spectra of the five dyes used in the AmpF STR Yfiler Kit Dyes VIC NED PET LIZ c o 2 E N E o 2 Wavelength AmpFtSTR Yfi
91. le 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 Yfiler PCR Amplification Kit User Guide 117 Documentation and Support Limited Product Warranty 118 AmpFtSTR Yfiler 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 Begovich A B McClure G R Suraj V C Helmuth R C Fildes N Bugawan T L Erlich H A and Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region J Immunol 148 249 58 Butler J M Schoske R Vallone P M Kline M C Redd A J Hammer M F 2002 A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers Forensic Sci Int 129 10 24 Butler J M 2001 Forensic DNA Typing San Diego CA Academic Press Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 DeFranchis R Cross N C P Foulkes N S and Cox T M 1988 A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Res 16 10355 DNA
92. lectrophoresis on the 3100 3100 Avant or 3130 3130xl NOT testes pecie 26 Section 3 2 3500 3500xL Series 27 Set up the 3500 3500xL instrument for electrophoresis 27 Prepare samples for electrophoresis on the 3500 3500xL instrument 27 Section 3 3 310 Instrument 0 22 43 2250 enhn 29 Set up the 310 instrument for electrophoresis 29 Prepare samples for electrophoresis on the 310 instrument 29 Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples Number of allelic One Number of samples per Instrument injection ladders to allelic ladder s equals 3100 Avant or 3130 1 per 4 injections 4 samples 15 samples 1 allelic ladder 3100 or 3130xl 1 per injection 16 samples 15 samples 1 allelic ladder 3500 1 per 3 injections 8 samples 23 samples 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples 1 allelic ladder 310 1 per 10 injections 1sample 9 samples 1 allelic ladder 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 reco
93. ler PCR Amplification Kit User Guide 17 Overview Materials and equipment Materials and equipment Kit contents and storage The AmpF STR Yfiler PCR Amplification Kit Part no 4427368 contains materials sufficient to perform 100 amplifications at 25 uL amplification IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Component Description 100X Volume Storage AmpF amp STR Yfiler Contains MgCl dNTPs and bovine serum 1 tube 1 1 mL 15 to 25 C on receipt PCR Reaction Mix albumin in buffer with 0 0596 sodium azide 2to 8 C after initial use AmpF STR Yfiler Contains forward and reverse primers to 1 tube 0 55 mL Primer Set amplify human male DNA target AmpFSTR Yfiler Allelic Ladder Contains amplified alleles 1 tube 0 05 mL See Table 1 on page 12 for a list of alleles included in the allelic ladder AmpF STR Control DNA 007 Contains 0 10 ng L human male DNA in 1 tube 0 3 mL 0 05 sodium azide and buffert See Table 1 on page 12 for profile AmpF STR Control DNA 9947A Contains 10 ng uL human female DNA in 1 tube 0 025 mL 0 0596 sodium azide and buffer See Table 1 on page 12 for profile AmpliTaq Gold DNA Polymerase Contains enzyme with an activity of 5 U uL 2tubes 0 05 mL 15
94. ler_GSS500_Panels_v1 B DYS368l blue 1340 1780 13 amp amp mpFLSTR SEfiler Plus Panels v1 B DYS390 1850 2450 24 8 15 _ _ 2 B_DYS388II blue 2460 3020 29 AmpFLSTR_Identifiler_Plus_v1 8 L7 ldentifilerDirect GS500 v1 G DYS458 green 133 0 165 0 17 Eb FA AmpFLSTR filer Panel v2 G DY518 green 1670 2190 15 G DYS385 green 2350 3230 1114 E B DYS456 B DYs3asl Y DYS393 yellow 104 0 144 0 13 B_DYS390 a Y DYS39 yelow 1460 181 0 11 _ 53831 holv pvs4s9 yelow 1920 2360 12 41 Y DYS635 yelow 2410 2740 24 G DYS385 12 Y DYS382 yelow 2860 3350 13 Y DYS3893 13 R Y GATA Hd red 1140 1500 h3 YDYS 14 R DYS437 1740 2100 115 YS4 15 RDYS438 red 2155 2565 12 Y DYS392 16 RDYS448 red 2730 3320 hs R_Y_GATA_H4 E m R_DYS437 R_DYS438 R_DYS448 als olal ae oln D lt 5 90 E B Olan amp 1 ol sl amp 1 el amp 1 el amp 1 el el ale amp iReference Samples 7 View the markers and display the Bin view in the navigation pane a Select the AmpFLSTR Yfiler Panel v2 folder to display its list of kits in the right pane b Double click the Yfiler v2 folder to display its list of markers below it AmpFtSTR Yfiler PCR Amplification Kit User Guide 35 4 Chapter 4 Analyze Data Set up GeneMapper ID
95. les and conditions References Analyzer System Software Applied 3500 Data Windows e HID36 POP4 Applied Biosystems 3500 3500xL Biosystems Collection XP Injection conditions 1 2kV 15 sec Genetic Analyzer User Guide 3500 Software Pub no 4401661 10 Dye Set G5 ra Applied Windows HID36 POP4 Applied Biosystems codon Biosystems Vista 9 Injection conditions 1 2kV 24 sec kn yar dui S e nen 35001 J m Reference Card Pub no 4401662 Dye Set 65 Prepare samples for electrophoresis on the 3500 3500xL instrument 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 LIZ9 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 results and experiments AmpFtSTR Yfiler PCR Amplification Kit User Guide 27 28 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3500 3500xL instrument 9 9 Pipette the required volumes of components into an appropriately sized polypropylene t
96. limitations which are critical for sound data interpretation in casework Sparkes Kimpton Watson et al 1996 Sparkes Kimpton Gilbard et al 1996 Wallin et al 1998 We performed experiments to evaluate the performance of the Yfiler 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 We performed additional studies according to the revised guidelines from the Scientific Working Group on DNA Analysis Methods SWGDAM July 10 2003 This DNA methodology is not novel Moretti et al 2001 Frank et al 2001 Wallin et al 2002 and Holt et al 2001 Based on these standards we conducted experiments which comply with Standards 1 0 and 2 0 and its associated subsections Whereas this DNA methodology is not novel Standard 8 1 2 and its related subsections have been addressed Holt et al 2001 and Wallin et al 2001 This chapter will discuss many of the experiments we performed and examples of the results we obtained We used conditions that produced maximum PCR product yield and a window in which reproducible performance characteristics were met These experiments while not exhaustive are appropriate for a manufacturer in our opinion Each laboratory using the Yfiler Kit should perfor
97. ll Reaction Plate or each MicroAmp tube 6 Prepare the DNA samples DNA sample To prepare Negative control Add 10 uL of low TE buffer 10mM Tris 0 1mM EDTA pH 8 0 Test sample Dilute a portion of the test DNA sample with low TE buffer so that 1 0 ng of total DNA is in a final volume of 10 uL Add 10 uL of the diluted sample to the reaction mix Positive control Add 10 uL of 007 control DNA 0 1 ng L The final reaction volume sample or control plus master mix is 25 uL 7 Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes 8 Centrifuge the tubes at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders if using 96 well plates 9 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 Note The Yfiler 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 Yfiler Kit AmpFtSTR Yfiler PCR Amplification Kit User Guide 21 2 Perform PCR Perform PCR Perform PCR 1 Program the thermal cycling conditions When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode When using the Veriti 96 Well Thermal Cycler refer to the following document
98. lt k sik k la ak cee ss nnn 81 MD UT eid wie bb HH HF mnn 83 SWGDAM Guideline 259 4 54 55 2 peed ee ied bete pe ep ure ute 83 Effect of DNA quantity on results and importance of quantitation 83 Stability en des ioe 84 SWGDAM Guideline 2 4 _ KK as wh ge eel ae 84 Lack of amplification of Some loci WW kk kk kk kk kk kk KK 84 El lec toli DI QE ea ocd het ead eed Da N ee A A nea See ER ips 84 Degraded DNA oes Wan uM ee 85 AmpFtSTR Yfiler PCR Amplification Kit User Guide 5 Contents M Studies cs deuten far Toe dr hee eh ba ees EE pops ee ee 86 SWGDAM Guideline 2 8 4 lt ag cee eee Se Chad pe REEL 86 Male female mixture studies 86 Male male mixture studies 0 0 0 0 ccc ccc cence en 87 e tts aeu oer ou tbt Mcr eM dou ota ett 88 SWGDAM Guideline 2 7 AW kk kk kk kk kk kk kk kk kK kk kk kk kk kk k kk kk he 88 IN eMe Deed pts tet ette teet td 88 Population samples used in these studies 0000 0c cee eee KK KK KK KK KK KK 89 Gene diversity values kk kk kk kk cece kk kK kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk eee 89 Analyzing the population data kK KK kk KK kk
99. lysis s GeneMapper ID X Yfiler Example gmidx Is Logged In Database GBOLDROYNJOSE File Edit Analysis View Tools Admin Help e H BB 5 P Table Setting 31xx Data Analysis E 2 amp l ne S Project Samples Analysis Summary Genotypes m EZ Yfiler Example C 0 2 0 o 0 e v gt e p E 0 Analysis Summary Select run folder to display ier Example Sample Status Total of Samples 0 Unanalyzed Analyzed 14 Analysis Setting Changed 0 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 Total of Analyzed Ladders m A 3 3 o o Yfiler Example Control Quality per project based on sample SOS SSPK MIX OMR SQ CGQ Control Type if Total 3t of Samples Positive Control 0 0 0 Custom Control 0 0 0 Negative Control 0 0 0 Total 0 0 0 Sample Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Total of Samples 8 All thresholds met One or more thresholds not m Samples j 0 l Analysis Completed Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data Thes
100. m appropriate validation studies AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Section 5 1 Developmental Validation Developmental validation Developmental validation SWGDAM Guideline Developmental validation is the demonstration of the accuracy precision and reproducibility 1 2 1 of a procedure by the manufacturer technical organization academic institution government laboratory or other party SWGDAM July 2003 Critical reagent concentrations and reaction conditions such as thermal cycling parameters AmpliTaq Gold DNA polymerase activation cycle number to produce reliable locus specific amplification and appropriate sensitivity have been determined SWGDAM Guideline The reaction conditions needed to provide the required degree of specificity and robustness 2 1 0 1 must be determined These include thermocycling parameters the concentration of primers magnesium chloride DNA polymerase and other critical reagents SWGDAM July 2003 g lt e 3 D e 5 E e 2 PCR components The concentration of each component of the Yfiler Kit was examined The PCR components are Tris HCl pH 8 3 KCl dNTPs primers AmpliTaq Gold DNA Polymerase MgCl bovine serum albumin and sodium azide The concentration for a particular component was established to be in the window that meets the reproducible performance characteristics of specificity and sensitivity Figure 4 The pe
101. ment 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 Yfiler PCR Amplification Kit User Guide 111 Safety Chemical safety Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended
102. mmend the above frequency of allelic ladder injections which should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment AmpFtSTR Yfiler PCR Amplification Kit User Guide 23 24 Chapter 3 Perform Electrophoresis Allelic ladder requirements 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 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis Reagents and parts Electrophoresis software setup and AmpF STR Yfiler PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect Ordering Information on page 105 lists the required materials not supplied with the the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum documents listed in the table The following table lists Data C
103. n e European minimal haplotype DYS19 DYS385a b DYS3809I IL DYS390 DYS391 DYS392 DYS393 e Scientific Working Group DNA Analysis Methods S WGDAM recommended Y STR panel European minimal haplotype plus DYS438 and DYS439 Additional highly polymorphic loci DYS437 DYS448 DYS456 DYS458 DYS635 Y GATA C4 and Y GATA H4 Product The Yfiler Kit contains all the necessary reagents for the amplification of human description male specific genomic DNA The reagents are designed for use with the following Life Technologies instruments Applied Biosystems 3100 3100 Avant Genetic Analyzer e Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer 310 Genetic Analyzer e GeneAmp PCR System 9700 with the Silver 96 Well Block e GeneAmp PCR System 9700 with Gold plated Silver 96 Well Block Veriti 96 Well Thermal Cycler About the primers Non nucleotide linkers are used in primer synthesis for the DYS438 and DYS456 loci For these primers non nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis Butler 2005 Grossman et al 1994 and Baron et al 1996 Non nucleotide linkers enable reproducible positioning of the alleles to facilitate interlocus 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 1
104. n Software and the run modules that can be software setup and used to analyze Yfiler Kit PCR products For details on the procedures refer to the documents listed in the table reference documents Pala Operatin Collection 3 Run modules and conditions References System Software 3 1 Windows e GS STR POP4 1mL G5 v2 md5 370 Genetic Analyzer User s Manual Windows or XP Injection condition Pub no 4317588 3 0 Br 15 kV 5 sec 310 Protocols for Processing AmpFtSTR PCR Windows Amplification Kit Products with Microsoft Windows NT and NT Operating System User Bulletin Windows Pub no 4341742 2000 Prepare samples for electrophoresis on the 310 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 Volume per reaction GeneScan 500 LIZ9 Size Standard or 0 75 uL GeneScan 600 LIZ Size Standard v2 0 Hi Di Formamide 24 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 results and experiments AmpFtSTR Yfiler PCR Amplification Kit User Guide 29 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on
105. n allele Walsh et al 1996 It has been reported that the DYS19 tetranucleotide repeat locus displays the typical 4 bp stutter but also a 2 bp stutter Prinz et al 2001 Gusmao et al 1999 The DYS392 trinucleotide repeat locus displays the typical 3 bp stutter but also a smaller 3 bp stutter Sequence analysis of this 3 bp stutter revealed that the product contains an additional repeat unit relative to the true allele peak The proportion of the stutter product relative to the main allele stutter percent is measured by dividing the height of the stutter peak by the height of the main allele peak Such measurements have been made for amplified samples at the loci used in the Yfiler Kit All data were generated on the 3100 Genetic Analyzer Some of the general conclusions from these measurements and observations are as follows For each Yfiler Kit locus the stutter percent generally increases with allele length as shown in Figure 8 through Figure 11 on the following pages Smaller alleles display a lower level of stutter relative to the longer alleles within each locus This is reflected in Figure 8 through Figure 11 where minimal data points are plotted for some smaller alleles as stutter could not be detected for many of these samples Each allele within a locus displays percent stutter that is reproducible The highest observed stutter percent for each locus is included as the filter in the GeneMapper ID Software
106. n that population Schoske et al 2004 A unique haplotype is defined as one that occurs only once in a given population The number of unique haplotypes is usually less than the number of different haplotypes in any given population Table 6 Discriminatory capacity and number of unique haplotypes for three U S populations African American U S Caucasian U S Hispanics Y STR marker N 333 N 254 N 175 combination DC UH DC UH DC UH Minimal 84 6 249 74 8 162 85 1 136 haplotype t U S 91 3 286 83 8 196 90 3 146 haplotype t U S haplotype 91 9 286 85 8 202 91 4 148 DYS437 Yfiler 99 1 327 98 8 248 98 3 169 haplotype t The minimal haplotype includes the markers DYS19 DYS385 a b DYS389 1 11 DYS390 DYS391 DYS392 DYS393 t The U S haplotype includes the minimal haplotype loci plus DYS438 and DYS439 Estimation of spontaneous or induced germline mutation at genetic loci may be achieved through comparison of the genotypes of offspring to those of their parents From such comparisons the number of observed mutations are counted directly In previous studies haplotypes of eight loci amplified by the Yfiler Kit were determined for a total of 4999 parent son Kayser and Sajantila 2001 Fourteen mutations were identified and an overall average mutation rate was estimated at 2 80 103 In two confirmed father son pairs mutation at two Y STRs were o
107. n the electropherogram The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The Yfiler Kit includes two main design features that promote maximum A addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 80 minutes This final extension step gives the AmpliTaq Gold DNA Polymerase extra time to complete A addition to all double stranded PCR product STR systems that have not been optimized for maximum A addition may have split peaks where each allele is represented by two peaks one base pair apart Figure 12 Split peaks resulting from incomplete A nucleotide addition due to omission of the 80 minute extension step g lt e 3 D e 5 E e 2 Final Extension Lack of full A nucleotide addition may be observed in Yfiler Kit results when the amount of input DNA is greater than recommended protocols This is because more time is needed for AmpliTaq Gold DNA Polymerase to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA will also result in off scale data Artifacts Artifacts or anomalies have been seen in data produced on genetic analyzers when using the Yfiler Kit In amplified samples artifacts in the non calling region may appear in the green 88 bp black 80 an
108. nce Samples B DYsss8ll 9 Import AmpFLSTR Stutter v2X a Select the AmpFLSTR Panels v2X folder in the navigation panel Panel Manager File Edit Bins Yiew Help B E E S Bin Set AmpFLSTR_Bins_ NE RE BEBE S Panel Manager Panel Name AmpFLSTR_NGMSElect_v2x Ofiler v1 1X null e 2 AmpFLSTR_NGM_v3x Plus v1 1X null C AmpFLSTR Panels v1X MEME NGM_SElect_v2 1 null 1 ese eet AMpPFLSTR_Panel Identifiler Plus v1 1x null b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR Yfiler PCR Amplification Kit User Guide 53 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR Stutter v2X then click Import Note Importing this file associates the marker stutter ratio with the bin set in the AmpFLSTR v2X folder Import Marker Stutter Lookin G AmpFLSTR Analysis Files GMIDX ampFLsTR_Bins_v2x 4 AmpFLSTR_Panels_v2 My Recent B AmpFLSTR Stutter v2X Documents El ReadMe AmpFLSTR v2X My Documents AmpFLSTR_Stutter_v2x txt My Computer Files of type all Files 10 View the imported marker stutters in the navigation pane a Double click the AmpFLSTR Panels v2X folder
109. ng the Yfiler Kit As the DNA became increasingly degraded the loci became undetectable according to size Preferential amplification was not observed The loci failed to robustly amplify in the order of decreasing size as the extent of degradation progressed Figure 17 AmpFtSTR Yfiler PCR Amplification Kit User Guide 85 Chapter 5 Experiments and Results Mixture studies Figure 17 Multiplex amplification of DNA samples incubated with DNAse top panel 1 ng of DNA with no DNAse added remaining panels 2 ng of DNA incubated with DNAse 300 2400 1600 00 n 3200 2400 1 00 wo L 3200 240 1600 wo 0 00 wo 40 200 D 800 s00 40 200 o Mixture studies SWGDAM Guideline 2 8 Male female mixture studies 86 The ability to obtain reliable results from mixed source samples should be determined SWGDAM July 2003 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting the results We recommend that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures Evidence samples that contain body fluids and or tissues originating from more than one individual are an integral component of forensic casew
110. nt peak in the GeneScan 500 LIZ Size Standard is not included in the size standard definition This peak can be used as an indicator of precision within a run Use the following procedure to create the appropriate size standard 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 42 AmpFtSTR Yfiler9 PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis 2 Select the Size Standards tab click New select the Basic or Advanced radio button then click OK GeneMapper Manager Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Last Saved Owner Type Description CE G5 HID GS500 2010 09 08 15 04 3 gmid BasiciAdvanced 3 Enter a name for example CE G5 Yfiler GS500 as shown below In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified in on page 42 The example below is for the GeneScan 500 LIZ Size Standard D lt 5 9 ES 5 90 E 2 Size Standard Editor CE GS Yfiler GS500 AmpFtSTR Yfiler PCR Amplification Kit User Guide 43 4 Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper ID Software Analyze and edit sample files with GeneMapper ID Software 1 In the Project window select F
111. nufacturer Each laboratory using the Yfiler Kit should assess their own requirements for evaluation of kits Our studies compared the performance of two Roche manufactured enzyme and buffer lots Control mixes with three new lots of buffer and two new lots of enzyme manufactured by Life Technologies Test mixes Studies were performed using Test mixes containing both the enzyme and buffer manufactured by Life Technologies Test Control A mix Control B mix Test A mix Test B mix Test C mix Material Buffer Control Buffer Control Buffer Test Buffer Test Buffer Test Buffer Lot 1 Lot 2 Lot 1 Lot 2 Lot 3 Enzyme Control Control TestEnzyme Control Test Enzyme Enzyme Enzyme Lot 1 Enzyme Lot 2 Lot 1 Lot 2 Lot 2 Each of the five mixes listed above were used to conduct reproducibility sensitivity and inhibition studies All amplifications were performed using a GeneAmp PCR System 9700 with either silver or gold plated silver block using the recommended amplification conditions and cycle number for the Yfiler Kit All data was run on an Applied Biosystems 3130xl Genetic Analyzer running Data Collection Software v3 0 and analyzed using GeneMapper ID X Software Subsequent data analysis was performed using Minitab Statistical Software To minimize the effect of injection to injection variation on result interpretation peaks heights for all studies were normalized using an in house multicolor reference standard
112. ollection Software and the run modules that can be used to analyze Yfiler Kit PCR products For details on the procedures refer to the reference documents Genetic Data Operatin Collection 9 Run modules and conditions References Analyzer System Software Applied 3 0 Windows HIDFragmentAnalysis36_POP4_1 Applied Biosystems 3130 3130xl Biosystems XP Injection conditions Genetic Analyzers Using Data 3130 3130xl 3130 3 kV 5 sec Collection Software v3 0 Protocols for Processing AmpFtSTR PCR 3130x 3 kV 10 sec Amplification Kit PCR Products User Dye Set G5 Bulletin Pub no 4363787 3100 2 0 Windows HIDFragmentAnalysis36_POP4_1 3100 3100 Avant Genetic Analyzers 2000 Injection condition 3kV 10 sec Using Data Collection Software v2 0 Protocols for Processing AmpFtSTR Dye Set 65 Ua PCR Amplification Kit PCR Products User Bulletin Pub 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 M User Bulletin Pub no 4332345 3100 Avant 1 0 Windows GeneScan36Avb DyeSetG5Module 3700 3100 Avant Genetic Analyzers NT Injection condition 3 kV 5sec Protocols for Processing AmpFtSTR PCR Amplification Kit PCR Products GS600v2 0Analysis LC User Bulletin Pub no 4332345 We conducted validation studies using these configurations
113. omozygous minimum peak height thresholds and the minimum peak height ratio threshold for interpretation of Yfiler Kit data AmpFtSTR Yfiler PCR Amplification Kit User Guide 41 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Quality Flags tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Quality weights are between 0 and 1 Quality Flag Settings Overlap 08 PQY Thresholds Sizing Quality From 0 75 to 1 0 Genotype Quality From 0 75 to 1 0 Spectral Pull up Control Concordance Broad Peak 0 8 Low Peak Height Out of Bin Allele os Off scale Peak Height Ratio From 0 0to 0 25 From 0 0 to 028 Factory Defaults 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 for interpretation of Yfiler Kit data Create size The size standards for the Yfiler Kit use the following size standard peaks in their standard definitions GeneScan 500 LIZ9 Size Standard peak sizes GeneScan 600 LIZ Size Standard v2 0 peak sizes 75 100 139 150 160 200 300 340 350 400 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
114. ontents Section 3 2 3500 3500xL Series instruments 27 Set up the 3500 3500xL instrument for electrophoresis 27 Reagents and parts esc bre o RE et Le dut e Oe SE eld 27 Electrophoresis software setup and reference lt 27 Prepare samples for electrophoresis on the 3500 3500xL instrument 27 Section 3 3 310 Instrument kk kk kk kk kk kk cette KK KK KK x 29 Set up the 310 instrument for electrophoresis WW kk kk kk kk kk KK KK KK KK KK KK kk 29 Reagents and parts isse edn E eed nay Ee ena ahd ka mene Yek e nde 29 Electrophoresis software setup and reference lt 29 Prepare samples for electrophoresis on the 310 29 CHAPTER 4 Analyze Data E dnin A araya E hen 31 Section 4 1 GeneMapper ID Software 31 Overview of GeneMapper ID Software 31 elits rro Re ca 31 Befoneyou Start sana k eB xak n ale eae eg A 32 Set up GeneMapper ID Software for data analysis 32 Fil inames he ahs Rem dealer eR eerta r escenas 32 Before using the software for the first time
115. ontrol Concordance CC Weight 1 0 Only applicable to controls r5Q Weighting Broad Peak BD 05 Allelic Ladder GQ Weighting 50 amp GQ Ranges Spike SSPKJSPK Off scale OS Sizing Quality From 0 75 to 1 0 From 0to 0 25 Genotype Quality From 0 75 to 1 0 From 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 size The size standards for the Yfiler Kit use the following size standard peaks in their standard definitions GeneScan 500 LIZ Size Standard peak sizes GeneScan 600 LIZ Size Standard v2 0 peak sizes 75 100 139 150 160 200 300 340 350 400 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 peak in the GeneScan 500 LIZ Size Standard is not included in the size standard definition This peak can be used as an indicator of precision within a run Use the following procedure to create the appropriate size standard 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 60 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis
116. or the Yfiler Kit method IMPORTANT Analysis methods are version specific so you must create an analysis method for each version of the software For example an analysis method created for GeneMapper ID X 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 C 0 2 0 i o 0 p e v gt ee e p 2 0 mM Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings 2 Select the Analysis Methods tab then click New to open the Analysis Method Editor with the General tab selected 3 The figures below show the settings for each tab of the Analysis Method Editor Configure the Analysis Method Editor tab settings as shown in the figures below unless the instructions state otherwise 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 settings in all tabs click Save AmpFtSTR Yfiler PCR Amplification Kit User Guide 55 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis General tab settings Analysis Method E
117. or the first time MM kk kk kk kk kk KK KK KK KK KK RR KK KK KK KK 49 Forlmore TIT Ob OT Olya ek en hn Re R 245000 seg Nose Wa te 49 Check panel bin and stutter file version 02 0 cece KK KK KK KK KK KK KK KK 50 Import panels bins and marker stutter kk kk kk kk kk 000 cece KK KK KK KK KK KK KK KK kK kk 50 Create an analysis method kk kk kk e eect kK kK kk kk kk kK kk kk kk kk kk kk kk 55 General tab settings kate Ak ala SES bey CA Skee sta ei dite ERRARE 56 AmpFtSTR Yfiler PCR Amplification Kit User Guide Contents Allele tab settings HH HH m 57 Peak Detector tab settings 00 0c eee eee kk kk kK kk kk kk 58 Peak Quality ta3b Setlirng gt sk xu aku kulak A eed de abe Chee eee eed ee 59 50 amp GQ tab Settings eeiam a rm ed le wa al Kawan d das 60 Create size staridard ossai sonna Sa an lal Sa da lak a ee ela e Boa 60 Analyze and edit sample files with GeneMapper ID X Software 62 Examine and edit a project kk kk kk kk kK kK KK cent n 63 Eormore information ice desi eee RETI eem Gale eU Rui ee RAP e DOR 64 m CHAPTER5 Experiments and Results 65 Section 5 1 Developmental Validation 66 OVERVIEW DD Ec T 66 Importance of validation 3 uim ea Reged KARE VEX
118. ork Therefore it is essential to ensure that the DNA typing system is able to detect DNA mixtures In the case of Y STRs the female DNA component is not amplified by the Y chromosome specific primers Male female mixture studies were performed up to a ratio of 1 2000 using three different female DNAs The amount of female DNA was kept constant at 500 ng and the amount of male control DNA was changed The female DNA did not cause any interference with the interpretation of the male Y STR profile as shown in Figure 18 Low level artifacts with female DNA have been occasionally observed in the black 136 bp and red 291 bp dye In general these artifacts peaks will not affect interpretation due to their intensity AmpFtSTR Yfiler PCR Amplification Kit User Guide Mixture studies Section 5 1 Developmental Validation Figure 18 Amplification of male Control DNA 007 in the presence of female DNA 9947A Profiles shown in the panels from top to bottom 500 pg of male DNA 500 pg male DNA with 500 ng female DNA 250 pg male DNA with 500 ng female DNA 500 ng female DNA 0 5 ng tt til ILE Ld LIT udi ILE Lib la 1 2000 al gad __ li ii i a g lt e 3 D m S E 2 500 ng female Male male mixture Forensic samples may contain body fluids or tissues originating from more than one studies male Mixtures of two male DNA samples were examined at various ratios 1 1 to 1 10
119. r Guide 61 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 Parameter Settings Sample Type Select the sample type Analysis Method Yfiler AnalysisMethod v2X or the name of the analysis method you created Panel Yfiler_v1 1X Size Standard CE 65 Yfiler GS500 or the name of the size standard you created For more information about how the Size Caller works or about size standards refer to the GeneMapper ID X Software v1 2 Reference Guide Pub no 4426481 62 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software Examine and edit a project 3 Click Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis as a completion bar extending to the right with the percentage 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 and the Genotypes tab becomes available upon completion of the analysis Analysis summary window after ana
120. reate a size standard as explained in Create size standard on page 60 6 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information 7 Define custom views of plots Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information For more For quick set up instructions refer to the GeneMapper ID X Software Version 1 0 Getting information Started Guide Pub no 4375574 For details about GeneMapper ID X Software features refer to e GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide Pub no 4375670 e GeneMapper ID X Software Version 1 0 Reference Guide Pub no 4375671 AmpFtSTR Yfiler PCR Amplification Kit User Guide 49 C 0 2 0 i o 0 e v gt e p 2 0 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis Check panel bin 1 and stutter file Start the GeneMapper ID X Software then log in with the appropriate user name and password version IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 2 Select Tools Panel Manager 3 Check the version of files imported into the Panel Manager s
121. rformance of the multiplex is most robust within a 20 window of magnesium chloride Figure 4 A 1 ng amplification of male genomic DNA varying the MgCl concentration analyzed on the 3100 Genetic Analyzer 30 20 10 2 li bid Ut a Laub 11 LAW hit l a Lh id lu j A AL 20 z 11 ta Al d li ji d4 d BN Thermal cycler Thermal cycling parameters were established for amplification of the Yfiler Kit in the parameters GeneAmp PCR Systems 9600 and 9700 Thermal cycling times and temperatures of GeneAmp PCR systems were verified Annealing and denaturation temperature windows were tested around each stipend to verify that a 1 0 C window produced a specific PCR product with the desired sensitivity of at least 1 ng of AmpF STR Control DNA 007 AmpFtSTR Yfiler PCR Amplification Kit User Guide 67 Chapter 5 Experiments and Results Developmental validation PCR cycle number 68 The effects of denaturation and annealing temperatures on the amplification of Yfiler Kit loci were examined using AmpF STR Control DNA 007 and two DNA samples The denaturation temperatures tested were 92 5 94 and 95 5 C all for 1 minute hold times on the GeneAmp PCR System 9700 The annealing temperatures tested were 59 60 61 62 and 63 C Figure 5 also for 1 minute hold times in the GeneAmp PCR System 9700 with the silver 96 well block The PCR products were an
122. rs 4393715 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 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 GeneScan 600 LIZ Size Standard v2 0 4408399 DS 33 Matrix Standard Kit Dye Set G5 4345833 For a complete list of parts and accessories for the 3500 3500xL instrument refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Pub no 4401661 106 AmpFtSTR Yfiler PCR Amplification Kit User Guide Appendix BOrdering Information Equipment and materials not included Itemt Source 310 Analyzer materials 310 DNA Analyzer capillary array 47 cm 402839 0 5 mL sample tray 5972 96 well tray adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10x 4335643 Genetic analyzer septa retainer clips for 96 tube sample tray 402866 Genetic analysis sample tubes 0 5 mL 401957 Septa for 0 5 mL sample tubes 401956 05 33 Matrix Standard Set 6 FAM VIC9 NED PET and LIZ9 dyes
123. same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the 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 the sample result needs to be verified according to the laboratory s protocol AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Set up GeneMapper D X Software for data analysis Panel bin and The file names shown in this section may differ from the file names you see when you stutter file version download or import files If you need help determining the correct f
124. 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 Yfiler PCR Amplification Kit User Guide 109 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 Yfiler 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 Yfiler Kit Veriti 96 Well Thermal Cycler 110 AmpFtSTR Yfiler 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 laboratories and the safety information provided in this document Before using an instru
125. t reagents including the BSA containing PCR reaction mix in the presence of varying concentrations of purified hematin The concentrations of hematin used were 0 uM 10 uM 12 uM 16 uM 20 uM and 24 uM No preferential amplification was observed in the presence of increasing amounts of hematin AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation 5 Stability Figure 16 DNA amplified with the Yfiler Kit in the presence of varying concentrations of hematin 0 10 uM 12 uM 16 uM 18 uM 20 uM and 24 uM analyzed the 3100 Genetic Analyzer g lt e 3 D m S E e 2 2200 2400 1600 800 0 2200 2400 1600 900 0 3200 2400 1600 00 2200 2400 1000 800 2200 2400 1600 800 0 2200 2400 1600 800 Degraded DNA As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This is due to the reduced number of intact templates in the size range necessary for amplification Degraded DNA was prepared to examine the potential for differential amplification of loci High molecular weight DNA was incubated with the enzyme DNase I for varying amounts of time The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each time point 2 ng of degraded DNA or 1 ng undegraded DNA was amplified usi
126. t time you need to Import panels and bins into the Panel Manager as explained in Import panels and bins on page 33 Create an analysis method as explained in Create an analysis method on page 37 Create a size standard as explained in Create size standard on page 42 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 for more information Define custom views of plots Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 for more information AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Import panels and To import the Yfiler panel and bin set into the GeneMapper ID Software v3 2 1 bins database 1 Start the GeneMapper ID Software then log in with the appropriate user name and password IMPORTANT If you need logon instructions refer to page 2 7 of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 2 Select Tools Panel Manager 3 Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane 2 Panel Manager File Edit Bins View E HB m m WW Ba set Highlight this ES Panel Manager b Sele
127. tion 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Allele tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags AmpFLSTR Yfiler Binset v2 Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Hexa Cut off Value 0 0 00 j 0 0 MinusA Ratio 0 0 0 0 j 0 0 MinusA Distance 0 0 00 J 0 0 To 00 0 0 0 0 Minus Stutter Ratio 0 0 0 0 J 0 0 Minus Stutter Distance From 0 0 3 25 0 0 To 00 475 0 0 Plus Stutter Ratio 0 0 0 0 i 0 0 Plus Stutter Distance From 0 0 0 0 0 0 00 0 0 0 0 D lt 97 o 9 5 90 E Amelogenin Cutoff Range Filter Factory Defaults In the Bin Set field select the AmpFLSTR Yfiler Bins v2 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 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 stud
128. to 25 C each t The AmpF STR Control DNA 007 is included at a concentration appropriate to its intended use as an amplification control i e to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype The AmpF STR Control DNA 007 is not designed to be used as a DNA quantitation control and you may see variation from the labelled concentration when quantitating aliquots of the AmpF STR Control DNA 007 Standards for For the Yfiler Kit the panel of standards needed for PCR amplification PCR product samples sizing and genotyping are e AmpFE STR Control DNA 007 A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR Yfiler 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 Yfiler Kit PCR products Order the GeneScan 500 LIZ9 Size Standard Part no 4322682 or the GeneScan 600 LIZ Size Standard v2 0 Part no 4408399 separately e AmpE STR Yfiler Allelic Ladder Allelic ladder developed by Life Technologies for accurate characterization of the alleles amplified by the Yfiler Kit The AmpF STR Yfiler Allelic Ladder contains most of the alleles reported for the 17 loci Refer to Table 1 on page 12 for a list
129. to display its list of kits in the right pane b Double click the Yfiler v1 1X folder to display its list of markers below it c Double click Y DYS392 to display the Stutter Ratio amp Distance view for the marker in the right pane Panel Manager File Edit Bins Yiew Help B g Bin Set AmpFLSTR Bins v2X v NN ug m BEBE B Panel Manager E E AmpFLSTR Panels v1X Please enter the stutter filter s for Y DYS392 marker here If left blank the global stutt S E AmpFLSTR Panels v2X applied H E COfiler CODIS v1 1x filer v1 1x Minus Stutter Plus Stutter t _DYS439 Ratio From Distance To Distance Ratio From Distance Y_DY5635 1622 2 25 3 75 0 0790 2 25 TAS qu O Stutter Ratio amp Distance H R_Y_GATA_H4 E R_DYS437 R_DYS438 11 Click Apply then OK to add the Yfiler Kit panel bin set and marker stutter to the GeneMapper ID X Software database I wl wl N IMPORTANT If you close the Panel Manager without clicking Apply the panels bin sets and marker stutter will not be imported into the GeneMapper ID X Software database 54 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Create an analysis Use the following procedure to create an analysis method f
130. tter percentage 0 13 mn Control W Control B 4 Test e 9H 2Z v gt Test C amp 0 10 S 0 09 5 g 0 08 0 07 2 0 06 0 05 0 04 e gt B ug Wo 2 SES Pe PLES 7 7 e oF 249 lt AZ A7 XIZ XIZ Dye Color and Locus Artifacts Known artifacts observed showed the same morphology signal intensity and location in all Test and Control mixes and did not exceed 50 RFU Figure 22 No new artifacts were observed in the Test mixes Figure 22 Reproducibility study known artifacts Y scale 50 RFU v c dye labeled artifacts at 75 and 115 bp a X LAL ar ett ETIN Ea em aeraalete rrr OY OOD aa ae ON ii OWEN dak FAM dye labeled artifact at 110 bp nat dence tin da ata Deja dabit atn Atta af E ONE ML LL Awa TEE ot T at 4 1 I ji j li 7 ji li RESM j a 94 50 RFU 75 350 bp Control A Control B Test A Test B Test C Control A Control B Test A Test B Test C neta liat Ae EA saldra lead l lsa NED dye labeled artifact at 88 bp PET dye labeled artifacts at 210 bp Bam y pi aea
131. uL of Hi Di Formamide Seal the reaction plate with appropriate septa then centrifuge the plate to ensure that the contents of each well are 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 then place on the autosampler Ensure that a plate record is completed and link the plate record to the plate Start the electrophoresis run AmpFtSTR Yfiler PCR Amplification Kit User Guide Chapter Perform Electrophoresis 3 Set up the 3500 3500xL instrument for electrophoresis Section 3 2 3500 3500xL Series instruments Set up the 3500 3500xL instrument for electrophoresis Reagents and parts Ordering Information on page 105 lists the required materials not supplied with the AmpF STR Yfiler PCR Amplification 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 can be software setup and used to analyze Yfiler Kit PCR products For details on the procedures refer to the documents listed in the table a 2 E 5 a o x a 0 e 5 reference documents Genetic Pata Operatin Collection 9 Run modu
132. ube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp optical strip tube add 9uL of the formamide size standard mixture e 1uL of PCR product or allelic ladder Note For blank wells add 10 uL of Hi Di Formamide Seal the reaction plate or strip tubes with the appropriate septa then centrifuge to ensure that the contents of each well are collected at the bottom Heat the reaction plate or strip tubes in a thermal cycler for 3 minutes at 95 C Immediately put the plate or strip tubes on ice for 3 minutes Prepare the plate assembly then place on the autosampler Ensure that a plate record is completed and link the plate record to the plate Start the electrophoresis run AmpFtSTR Yfiler PCR Amplification Kit User Guide Chapter 3 Perform Electrophoresis Set up the 310 instrument for electrophoresis Section 3 3 310 Instrument Set up the 310 instrument for electrophoresis Reagents and parts Ordering Information on page 105 lists the required materials not supplied with the AmpF STR Yfiler PCR Amplification 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 zi o 3 Z Electrophoresis The following table lists Data Collectio
133. ultiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections in one capillary run Table 3 on page 71 indicates typical precision results obtained from the seven injections of the AmpFSTR Yfiler Allelic Ladder analyzed on the 3100 Genetic Analyzer 47 cm capillary and POP 4 polymer The size standard used was GeneScan 500 LIZ Size Standard These results were obtained within a set of injections on a single capillary As indicated above sample alleles may occasionally size outside of the 0 5 bp window for a respective allelic ladder allele because of measurement error The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Figure 7 illustrates the tight clustering of allele sizes obtained on the 3100 Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 bp The instance of a sample allele sizing outside of the 0 5 bp window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 bp or less Smith 1995 AmpFtSTR Yfiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility For sample alleles that do not size within a 0 5 bp window the PCR product must be rerun to distinguish between a true off ladder allele vs measurement error of a sample
134. ures ageing degradation and species studies Int J Legal Med 109 186 194 Sparkes R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 1996b The validation of a 7 locus multiplex STR test for use in forensic casework II Artifacts casework studies and success rates Int J Legal Med 109 195 204 AmpFtSTR Yfiler PCR Amplification Kit User Guide Bibliography 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 and Walsh P S 2002 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci 47 52 65 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 2812 AmpFtSTR Yfiler PCR Amplification Kit User Guide 121 Bibliography 122 AmpFtSTR Yfiler PCR Amplification Kit User Guide Numerics 310 instrument 29 3130 3130xl instrument 25 3500 3500xL instrument 27 A A nucleotide addition by AmpliTaq Gold to 3 end of amplicon 78 agarose gel using to examine DNA 85 allele frequencies in the population databases 89 allelic ladder about 18 precision 69 profile 12 requirements for accurate

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