User Bulletin. Veriti 96-Well Thermal Cycler AmpFlSTR Kit Validation. Overview

User Bulletin Veriti 96-Well Thermal Cycler AmpFlSTR Kit Validation June 2009 SUBJECT: AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format In this user bulletin This user bulletin documents a study verifying the successful amplification of samples on the Veriti 96-Well thermal cycler with 0.2 ml block format: Overview...................................................... 1 Validation studies................................................ 3 Materials and Methods........................................... 4 Data analysis................................................... 5 Results........................................................ 6 Discussion.................................................... 13 Conclusions................................................... 15 References.................................................... 16 Overview This user bulletin documents a validation study confirming the effectiveness of amplification using the Veriti 96-Well Thermal Cycler with 0.2 ml block format in conjunction with AmpFlSTR chemistry. The study compared data generated on the Veriti thermal cycler with data generated on the GeneAmp PCR System 9700 thermal cycler. The data verified reproducibility, reliability, and accuracy of amplification on each thermal cycler with the following representative four-dye and five-dye AmpFlSTR PCR amplification kits: AmpFlSTR COfiler PCR Amplification Kit AmpFlSTR Identifiler PCR Amplification Kit AmpFlSTR MiniFiler PCR Amplification Kit AmpFlSTR Profiler Plus PCR Amplification Kit AmpFlSTR SGM Plus PCR Amplification Kit AmpFlSTR Yfiler PCR Amplification Kit

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format In the study, Applied Biosystems evaluated concordance, average peak heights, intracolor balance, peak height ratios, and dye artifacts for each thermal cycler/amplification kit combination. Applied Biosystems performed statistical comparisons of the two thermal cyclers to ensure consistent results with the GeneAmp thermal cyclers and with the Veriti 96-Well Thermal Cycler with 0.2 ml block format. Validation studies focused on repeatability and sensitivity to ensure data quality. Repeatability and sensitivity studies confirmed that the AmpFlSTR amplification chemistry produces high quality data when run on the Veriti 96-Well Thermal Cycler with 0.2 ml block format. 2 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

Validation studies Validation studies Repeatability In the repeatability study, 42 positive control samples and one negative control were amplified on three Veriti and three 9700 thermal cyclers for each AmpFlSTR kit tested. The results were evaluated for concordance, average peak height, intra-color balance, and peak-height ratio. The positive controls provided in the AmpFlSTR kits were used for all of the repeatability testing: 9947A DNA for the Identifiler, Profiler Plus, and COfiler kits 007 DNA, for the MiniFiler, SGM Plus, and Yfiler kits Sensitivity The sensitivity study used a panel of seven male genomic DNA samples extracted from whole blood obtained from the Interstate Blood Bank. DNA samples were quantitated using the Quantifiler Human DNA Quantification Kit and diluted to 0.1 ng/µl, 0.05 ng/µl, and 0.0125 ng/µl, using low TE buffer (10 mm Tris HCl, ph 8.0; 0.1 mm EDTA, ph 8.0). Three input DNA amounts were used. For the AmpFlSTR kits requiring a total PCR reaction volume of 25 µl, amplification was performed using DNA input amounts of 1 ng, 0.5 ng and 0.125 ng from each of the seven male genomic DNA samples. For the AmpFlSTR kits requiring a total PCR reaction volume of 50 µl, amplification was performed using DNA input amounts of 2 ng, 1 ng and 0.250 ng from each of the seven male genomic DNA samples. Data was generated from four replicate amplifications of each DNA input amount for each of the six AmpFlSTR kits. The study evaluated and compared concordances, average peak heights, intra-color balances, and heterozygote peak height ratios for each thermal cycler platform. Amplification kits used Table 1 AmpFlSTR PCR Amplification Kit lot and part numbers AmpFlSTR Kit Part Number Lot Number Repeatability Study Sensitivity Study Profiler Plus 4303326 0708120/0706115 0801125 COfiler 4305246 0710091/0705086 0710091 SGM Plus 4307133 0712131 0711130 Identifiler 4322288 0801092 0801092 Minifiler 4373872 0806012 0712008 Yfiler 4359513 0801035 0708030 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 3

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format Materials and Methods Reagents One lot of each of the AmpFlSTR PCR Amplification Kits listed in Table 1 on page 3, except the Profiler Plus kit and the COfiler kit, was utilized for each of the validation studies to minimize variability. In addition, identical dilutions of samples were used for testing on both of the thermal cycler platforms. Instruments Amplification Three Veriti 0.2 ml and GeneAmp 9700 96-well thermal cyclers were used for the repeatability study. The sensitivity study used two each of the Veriti and GeneAmp thermal cyclers due to a sensor error on one of the Veriti thermal cyclers. All samples were amplified using MicroAmp Optical 96-well Reaction Plates (0.2 ml) and clear adhesive covers. To ensure correct temperature ramping, 9600 emulation mode was used on both thermal cycler platforms for both studies. The following method was used to run the Veriti thermal cycler in 9600 emulation mode: 1. Select Tools Menu Convert a Method 9600 Emulation Mode (right arrow), 2. Enter the information on the reaction volume as well as stages, cycles, times and temperatures of the run 3. Select 4. Name and save the run method. Note: Run methods configured on one Veriti thermal cycler can be saved on a USB drive and exported to other Veriti thermal cyclers to ensure uniform protocols. Fragment analysis To minimize variability, the study used one ABI PRISM 3100 Genetic Analyzer with one 16-capillary array and Data Collection Software v1.1 to analyze all samples. Run modules specific to each of the dye sets (F and G5) were used in accordance with the ABI PRISM 3100 Genetic Analyzer and AmpFlSTR PCR Amplification Kit user s manuals. AmpFlSTR kits used the following reagent volumes per well: With dye set F: 8.5 µl Hi-Di Formamide, 0.5µL GeneScan-500 ROX Size Standard, 1.0 µl amplicon With dye set G5: 8.7 µl Hi-Di Formamide, 0.3 µl GeneScan-500 LIZ Size Standard, 1.0 µl amplicon 4 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

Data analysis Data analysis Sizing and genotyping Statistical analysis and calculations The validation study used GeneMapper ID-X v1.0 software to analyze run files generated by the Data Collection Software v1.1. Allele calls, peak heights, and base pair sizes were determined using the appropriate AmpFlSTR PCR Amplification Kit panel, bin set, and stutter file. Data generated by each instrument was compared using the parameters detailed below. Statistical analysis consisted of t-tests and paired t-tests using Microsoft Excel Analysis Toolpak with a 95% confidence interval. Concordance Prior to the validation study, profiles of the male samples were determined using each AmpFlSTR kit. Profiles generated in the course of the studies were compared to the control and male sample DNA profiles to determine whether all alleles were called identically. If an allele was not called identically in the control and sample profiles, further investigation was performed to determine whether the allele was not present or was called incorrectly. Average peak heights The average peak height was calculated from peak heights generated with GeneMapper ID-X software. The average peak heights from each thermal cycler were compared. Intra-color balance Normalized peak heights were used to calculate the intra-color balance. For a heterozygous locus, the two allele peak heights were averaged. For a homozygous locus, the single allele peak height was divided by two. For each color, the lowest normalized peak height was divided by the highest normalized peak height and the result was multiplied by 100. Heterozygote peak height ratio Within a heterozygous locus, the lower peak height of the two alleles was divided by the larger allele peak height and the result was multiplied by 100. Dye artifacts and negative controls data All samples tested in the reproducibility and sensitivity studies were evaluated for the presence of artifacts and contamination throughout the validation study. This evaluation included the identification of any anomalous and reproducibly amplified products (one or more peaks of the same base pair size in two or more samples) or dye artifacts. The evaluation included any peaks exceeding 50 relative fluorescent units (RFU) in the region greater than or equal to 100 base pairs. AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 5

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format Results Repeatability Concordance Both the Veriti 96-Well Thermal Cycler with 0.2 ml block format and the GeneAmp PCR System 9700 thermal cycler produced correct genotypes of all amplified positive control samples when used with each of the AmpFlSTR PCR Amplification Kits tested. All results were 100% concordant with the previously established genotyping results. Average peak heights Figure 1 shows the average peak heights for the instrument replicates run on both the Veriti and GeneAmp 9700 thermal cyclers. All AmpFlSTR kits demonstrated statistically significant results with the exception of the Identifiler kit. Instrument Replicate Average Peak Height Values 3500 3000 2500 GeneAmp PCR System 9700 RFU 2000 1500 1000 Veriti 0.2mL Thermal Cycler 500 0 Profiler Plus COfiler SGM Plus Identifiler MiniFiler Yfiler Figure 1 Average peak height values of thermal cycler instrument replicates. Error bars indicate the average standard deviation observed for the instrument replicates. The average peak height values generated using the SGM Plus, Identifiler, MiniFiler, and Yfiler kits demonstrated <15% difference between the thermal cyclers. This variation is consistent with the observed run-to-run variability of the ABI PRISM 3100 Genetic Analyzer. The average peak 6 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

Results heights and standard deviations generated using both thermal cyclers with each of these kits are also similar. Larger differences ( 30%) were observed with the Profiler Plus and COfiler kits. These differences may have been due to amplification preparation by different operators and use of different kit lots. Heterozygote peak height ratio The heterozygote peak height ratio, or intralocus balance, was determined across the profile for each of the AmpFlSTR kits tested. These ratios were >70% for the Identifiler, SGM Plus, Profiler Plus, and COfiler kits and 65% for the MiniFiler kit. A minor but statistically significant difference of about 1% was observed between data generated by the thermal cycler platforms using the COfiler kit. Figure 2 shows the average heterozygote peak height ratios of the instrument replicates for each of the AmpFlSTR kits tested. Average Peak Height Ratios 105 100 Peak Height Ratio 95 90 85 80 75 70 65 Profiler Plus COfiler SGM Plus Identifiler MiniFiler GeneAmp PCR System 9700 Veriti 0.2mL Thermal Cycler Figure 2 Average heterozygote peak height ratios of the three instrument replicates. Error bars indicate the average instrument replicate standard deviations. Intra-color balance The intra-color balance for all kits except the MiniFiler and Yfiler kits showed statistically significant differences between thermal cycler platforms. However, the differences between the thermal cyclers were minimal, with the Identifiler kit producing the largest difference observed ( 5%). In addition, all kits met and exceeded 40% intra-color balance when amplified with both the Veriti 0.2 ml and GeneAmp 9700 96-well thermal cyclers. Figure 3 details the intra-color balance of each of the AmpFlSTR kits tested. AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 7

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format Average Instrument Replicate Intra-Color Balance 100 Intra-Color Balance 90 80 70 60 50 GeneAmp PCR System 9700 Veriti 0.2mL Thermal Cycler 40 Profiler Plus COfiler SGM Plus Identifiler MiniFiler Yfiler Figure 3 Intra-color balance of AmpFlSTR PCR Amplification Kits. Error bars indicate the average instrument replicate standard deviations. Sensitivity Concordance Profiles of the seven male genomic DNA samples were generated by each thermal cycler and compared, to identify any discordant samples. Table 2 on page 8 shows the sample input amounts, instrument replicate, full concordance percentage, and percentage of total alleles that fell below the detection threshold. Full profile percentage (the percentage of samples yielding full profiles) and undetected allele percentages (the percentage of expected alleles that were not detected) are shown for each thermal cycler format. Discordant alleles were not observed because non-full profile samples were the result of allelic drop-out. With 0.125 ng and 0.250 ng DNA input amounts, the full profile percentage is highly variant for both thermal cycler instruments, across the six kits that were tested. The full profile percentage ranged from 14% to 100%. Quantitation, stochastic variation, sample quantity and injection-toinjection variability are all factors that affect the likelihood of obtaining a full profile from a sample with relatively little DNA. Generally, the Veriti thermal cycler had fewer allele drop-outs and a higher percentage of full profiles. Table 2 Full profile percentage and undetected allele percentage by thermal cycler, AmpFlSTR PCR Amplification Kit, and DNA input amount KIT Input amount Thermal cycler replicate Full profile percentage Undetected allele percentage GeneAmp Veriti 0.2 ml GeneAmp Veriti 0.2 ml COfiler 0.250 ng 1 62.5% 75.0% 7.3% 2.6% 8 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

Results KIT Input amount Thermal cycler replicate Full profile percentage Undetected allele percentage GeneAmp Veriti 0.2 ml GeneAmp Veriti 0.2 ml COfiler 0.250 ng 2 82.1% 78.6% 2.0% 2.9% Identifiler 0.125 ng 1 18.8% 21.9% 11.0% 8.3% Identifiler 0.125 ng 2 21.7% 14.3% 10.3% 8.7% MiniFiler 0.125 ng 1 100.0% 96.4% 0.0% 0.2% MiniFiler 0.125 ng 2 82.1% 100.0% 1.1% 0.0% Profiler Plus 0.250 ng 1 78.1% 85.7% 1.0% 0.8% Profiler Plus 0.250 ng 2 92.9% 100.0% 0.4% 0.0% SGM Plus 0.250 ng 1 42.9% 53.6% 1.6% 1.4% SGM Plus 0.250 ng 2 78.6% 42.9% 1.4% 1.7% Yfiler 0.125 ng 1 82.1% 89.3% 1.0% 0.6% Yfiler 0.125 ng 2 85.7% 100.0% 0.8% 0.0% COfiler 1.0 ng 1 100.0% 100.0% 0.0% 0.0% COfiler 1.0 ng 2 100.0% 100.0% 0.0% 0.0% Identifiler 0.5 ng 1 100.0% 100.0% 0.0% 0.0% Identifiler 0.5 ng 2 100.0% 100.0% 0.0% 0.0% MiniFiler 0.5 ng 1 100.0% 100.0% 0.0% 0.0% MiniFiler 0.5 ng 2 100.0% 100.0% 0.0% 0.0% Profiler Plus 1.0 ng 1 100.0% 100.0% 0.0% 0.0% Profiler Plus 1.0 ng 2 100.0% 100.0% 0.0% 0.0% SGM Plus 1.0 ng 1 100.0% 100.0% 0.0% 0.0% SGM Plus 1.0 ng 2 100.0% 100.0% 0.0% 0.0% Yfiler 0.5 ng 1 100.0% 100.0% 0.0% 0.0% Yfiler 0.5 ng 2 100.0% 100.0% 0.0% 0.0% COfiler 2.0 ng 1 100.0% 100.0% 0.0% 0.0% COfiler 2.0 ng 2 100.0% 100.0% 0.0% 0.0% Identifiler 1.0 ng 1 100.0% 100.0% 0.0% 0.0% Identifiler 1.0 ng 2 100.0% 100.0% 0.0% 0.0% MiniFiler 1.0 ng 1 100.0% 100.0% 0.0% 0.0% MiniFiler 1.0 ng 2 100.0% 100.0% 0.0% 0.0% Profiler Plus 2.0 ng 1 100.0% 100.0% 0.0% 0.0% Profiler Plus 2.0 ng 2 100.0% 100.0% 0.0% 0.0% SGM Plus 2.0 ng 1 100.0% 100.0% 0.0% 0.0% SGM Plus 2.0 ng 2 100.0% 100.0% 0.0% 0.0% Yfiler 1.0 ng 1 100.0% 100.0% 0.0% 0.0% Yfiler 1.0 ng 2 100.0% 100.0% 0.0% 0.0% AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 9

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format Average peak heights The average peak heights were calculated for each AmpFlSTR kit tested and each DNA input amount. Generally, the Veriti 0.2 ml and GeneAmp 9700 thermal cyclers generated comparable data for each DNA input amount with each amplification kit. The average peak height differences between the thermal cycler platforms across all input amounts were within normal run-torun variability of the ABI PRISM 3100 Genetic Analyzer for all kits tested, with a maximum 13% difference observed with the COfiler kit. Figures 4 and 5 show example data from the Profiler Plus and MiniFiler AmpFlSTR kits at each DNA input amount. The orange and green boxes correspond to the two GeneAmp 9700 thermal cycler and Veriti 0.2 ml thermal cycler peak height averages, respectively. The black and red dots, generally located in the center of the box plots, represent the peak height mean. The black and red asterisks are the calculated outliers. Figure 4 Average peak heights for the Profiler Plus AmpFlSTR PCR Amplification Kit. 10 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

Results Figure 5 Average peak heights for the AmpFlSTR MiniFiler PCR Amplification Kit. Table 3 shows the average instrument replicate peak heights for all DNA input amounts for each AmpFlSTR kit tested. Table 3 Average instrument replicate peak heights for AmpFlSTR PCR Amplification Kits AmpFlSTR PCR Amplification Kit Average Peak Heights Thermal Cycler Profiler Plus COfiler SGM Plus Identifiler MiniFiler Yfiler GeneAmp 9700 619.05 550.60 606.45 453.67 1837.90 1261.40 Veriti 0.2 ml 611.95 625.85 551.45 490.98 1799.15 1263.45 Heterozygote peak height ratio Each of the AmpFlSTR kits tested on each thermal cycler platform produced average heterozygote peak height ratios of >70% at the recommended DNA input amount amounts of 1.0 ng (25 µl PCR reaction volume) and 2.0 ng (50 µl PCR reaction volume). Values were comparable between the GeneAmp AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 11

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format 9700 and Veriti 0.2 ml thermal cycler platforms with a maximum of 1% difference observed between the data generated with each thermal cycler platform using the Identifiler, SGM Plus, and MiniFiler kits. Figure 6 shows representative data from the Identifiler kit. Figure 6 Heterozygote peak height ratios observed with the Identifiler kit data generated by the Veriti and GeneAmp 9700 thermal cyclers. Table 4 Average instrument replicate peak height ratios for AmpFlSTR PCR Amplification Kits AmpFlSTR PCR Amplification Kit Average Peak Height Ratios Thermal Cycler Profiler Plus COfiler SGM Plus Identifiler MiniFiler GeneAmp 9700 87.51 88.48 87.57 85.22 82.05 Veriti 0.2 ml 87.22 88.65 88.34 84.76 81.56 12 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

Discussion Intra-color balance Data from both thermal cyclers demonstrated similar intra-color balance for each of the AmpFlSTR kits tested. The average intra-color balance exceeded 40% for all kits tested. Table 5 shows the average intra-color balance of each kit tested. Table 5 Average instrument replicate intra-color balance for AmpFlSTR PCR Amplification Kits AmpFlSTR PCR Amplification Kit Intra-color Balance Thermal Cycler Profiler Plus COfiler SGM Plus Identifiler MiniFiler Yfiler GeneAmp 9700 73.27 81.50 66.69 64.38 76.15 58.88 Veriti 0.2 ml 74.17 81.29 65.43 65.65 75.83 59.54 Artifacts and Negative Control Samples The data generated on the Veriti 0.2 ml 96-well thermal cycler did not contain any new reproducible PCR artifacts that are not already published in the User s Manuals for the AmpFlSTR PCR Amplification Kits tested. The negative amplification controls produced no amplification of DNA with any of the AmpFlSTR PCR Amplification Kits tested. Discussion Repeatability Concordance All of the alleles from the 9947A and 007 control DNAs generated using the Veriti 96-Well Thermal Cycler with each of the AmpFlSTR PCR Amplification Kits tested were correctly genotyped by the GeneMapper ID-X software and concordant with the data generated using the GeneAmp PCR System 9700 thermal cycler. Average peak height Average peak heights of the positive control DNA were similar across the thermal cycler platforms. Profiler Plus and COfiler data exhibited about a 30% difference between the two thermal cycler platforms, potentially due to sample processing at different times by different operators and using different kit lots. Other kits that are more sensitive to thermal profile differences, such as Yfiler, demonstrated a smaller peak height difference ( 8%) than was observed with the Profiler Plus and COfiler kits. In addition, data generated on the Veriti thermal cycler showed less peak height variability than the data generated on the GeneAmp 9700 thermal cycler for most kits tested. AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 13

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format Other AmpFlSTR kits tested generated differences of <15%, which is within the variability of the ABI PRISM 3100 Genetic Analyzer. Heterozygote peak height ratio For all of the kits and both thermal cycler platforms, all of the heterozygote peak height ratio results demonstrated an intralocus balance of >70% for the control DNA of each AmpFlSTR kit tested. The largest difference observed between the GeneAmp 9700 and the Veriti thermal cyclers was 1%. The average intralocus balance range for all of the kits was between 83% and 95%, with similar intralocus balance observed between the thermal cycler platforms and with each AmpFlSTR kit tested. Intra-color balance For all of the AmpFlSTR kits tested and both thermal cycler types, the results all exceeded 40% for the control DNA. The difference between the GeneAmp 9700 and Veriti thermal cycler intra-color balances was <5%, indicating a minor difference within the run-to-run variability of the ABI PRISM 3100 Genetic Analyzer. Sensitivity Concordance The DNA input amounts of 2.0 ng, 1.0 ng and 0.50 ng produced full profiles and 100% concordance with the known male profiles. The DNA input amounts of 0.125 ng and 0.25 ng produced 100% concordance, but these results also demonstrated wide variability of detected alleles, ranging from 18% to 100% for the data generated by the GeneAmp 9700 thermal cycler to 14% to 100% for the data generated by the Veriti thermal cycler. For all samples and AmpFlSTR PCR Amplification Kits tested, all alleles detected were concordant with known genotypes. However, allelic drop-out was prevalent at lower DNA input amounts. Generally, the Veriti 0.2 ml thermal cycler produced more full profile samples than the GeneAmp 9700 thermal cycler, with a lower percentage of allele drop-outs. Average peak height For all of the kits, average peak height data from the GeneAmp 9700 thermal cycler showed a strong correlation with the data from the Veriti thermal cycler. At most, there was a 13% difference in average peak height between the two thermal cycler platforms. In general, the differences in peak heights did not trend toward either the GeneAmp 9700 thermal cycler or the Veriti thermal cycler. The peak heights obtained for different DNA input amounts were within the expected relative fluorescent unit (RFU) ranges. Most of the variability of the peak heights was attributed to variation in sample quality and concentration. 14 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

Conclusions Heterozygote peak height ratio Similar to the average peak heights data, peak height data from the GeneAmp 9700 and the Veriti thermal cyclers demonstrated a strong correlation with respect to heterozygote peak height ratio. The percent differences in peak height ratio, or intralocus balance, were 1%, which is well within the variability of the ABI PRISM 3100 Genetic Analyzer. All of the 2 ng, 1 ng and 0.5 ng samples for all of the AmpFlSTR kits tested yielded average peak height ratios of >70%. Intra-color balance Intra-color balances exceeded 40% for the male DNA panel for all of the AmpFlSTR kits tested. The percent difference between the intra-color balance for the GeneAmp 9700 and Veriti thermal cyclers was <2%. For some of the AmpFlSTR kits, such as the MiniFiler kit, the intra-color balance for certain dyes showed a marked decrease in percent balance when compared to the other colors. However, this effect was equivalent for both of the thermal cyclers, indicating that the variation was due to the amplification chemistry. Although the average intra-color balance was similar, the Yfiler kit produced larger differences between the thermal cycler platforms for the blue (13%) and green (10%) dye colors. These differences, however, are within the run-to-run variability of the ABI PRISM 3100 Genetic Analyzer. Artifacts and negative controls Artifacts The amplified control DNA and male samples generated by the GeneAmp 9700 and Veriti thermal cycler platforms were examined for artifacts not published in the AmpFlSTR kit user s manuals. No additional reproducible artifacts were observed. The published artifacts were also analyzed for significant changes in peak height. There were no significant changes in the artifact peak height. In addition, the artifact peak heights from the Veriti thermal cycler were equivalent to the artifact peak heights produced by the GeneAmp 9700 thermal cycler. Negative controls Negative controls amplified throughout the validation studies were examined for contamination and for extra PCR artifacts. No contamination was observed and there were no amplification artifacts detected that were not among the published artifacts. Conclusions The validation of the Veriti 96-Well Thermal Cycler with 0.2 ml block format was designed to evaluate the amplification performance of AmpFlSTR PCR Amplification Kit chemistry. The data generated by the AmpFlSTR Profiler Plus, COfiler, SGM Plus, Identifiler, MiniFiler, and Yfiler PCR Amplification Kits on the Veriti thermal cycler was also compared with data AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 15

AmpFlSTR PCR Amplification Kit validation on the Veriti 96-Well Thermal Cycler with 0.2 ml sample block format generated on the GeneAmp PCR System 9700 thermal cycler. Analysis metrics compared were genotype concordance, average peak height, heterozygote peak height ratio, intra-color balance, and artifact identification. The validation study results demonstrate the generation of high quality AmpFlSTR kit data using the Veriti thermal cycler with 0.2 ml block format. In addition, the data generated by each of the thermal cycler platforms was either statistically similar or relatively comparable to the data generated by the other. Thus, the Veriti thermal cycler with 0.2 ml block format exhibits reliable performance for human identification applications. References Applied Biosystems AmpFlSTR COfiler PCR Amplification Kit User Bulletin, (PN 4306116). Applied Biosystems AmpFlSTR Identifiler PCR Amplification Kit User s Manual, (PN 4323291). Applied Biosystems AmpFlSTR MiniFiler PCR Amplification Kit User Guide, (PN 4374618). Applied Biosystems AmpFlSTR Profiler Plus PCR Amplification Kit User s Manual, (PN 4303501). Applied Biosystems AmpFlSTR SGM Plus PCR Amplification Kit User s Manual, (PN 4309589). Applied Biosystems Veriti Thermal Cycler User Guide, (PN 4375799). Applied Biosystems AmpFlSTR Yfiler PCR Amplification Kit User s Manual, (PN 4358101). ABI PRISM 3100 Genetic Analyzer User's Manual (PN 4315834) 16 AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin

References AmpFlSTR PCR Amplification Kit Validation on the Veriti 96-Well Thermal Cycler User Bulletin 17

Information in this document is subject to change without notice. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS 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, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. TRADEMARKS: The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. All other trademarks are the sole property of their respective owners. For Research Use Only. Not for use in diagnostic procedures. Copyright 6/2009, Life Technologies Corporation. All rights reserved. Part Number 4440754 Rev. A 06/2009 Stock Number 112UB25-01 Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone 650.638.5800 Toll Free 800.345.5224 www.appliedbiosystems.com International Sales For our office locations please call the division headquarters or refer to our Web site at www.appliedbiosystems.com/about/offices.cfm