HID Real-Time PCR Analysis Software v1
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1. 312 0 156 and 0 078 ng uL C values of each sample concentration and each calculated quantity were compared between the two software versions tested v1 0 and v1 1 Figure 2 Reproducibility and sensitivity plate layout for Quantifiler Duo samples 1 2 3 4 5 6 7 8 9 101112 Standard DNA dilution Sample DNA dilution HID Real Time PCR Analysis Software v1 1 User Bulletin aAOO0000000000 O Hrau D 10 ng L IODC CCOCCCEEE O 167 no 5 noL OOS SSCOOSSOO O 556 no 25 nail IDOOOOOOOOOOS O 125 noy 1 25 ng ul E OOOOOOOOO SD oe ra 0 625 ng L OODOODOODDD 02n 0312 ng F SSSSSSSOSSE osn 0 156 ng L ITIL ORIG 0 078 ng L 007 Validation and new features 3 Data collection Figure 3 Reproducibility and sensitivity plate layout for Quantifiler Human and Y Human Male samples 12 3 4 5 6 7 8 9 101112 Standard DNA dilution Sample DNA dilution NOOODDODOOCOODODD O ng 10 ng L BOOSSSSO0C0SSEES O 167 ng ut 5 ng uL cooggggooggg O 5 56 ng L 25 ng L DOOOOOOCOOOOOO O 1 85 ng L 1 25 ng uL EI OOOOOOOOOO 0 62 nit 0 625 ng uL FIPODODDOODODD tnt Q 0 312 ng L SOSSSSOOSSSS oon 0 156 ng ul H 0689 O8006 0 023 ng L 0 078 ng L Quantifiler Human Quantifiler Y Human Male 2 3 User interface The HID Real Time PCR Analysis Software v1 1 was tested to verify the accuracy of the evaluation H D specific quality flags as well as the plate setup and run monitoring The HID flag2. Bulletin 9 Validation and new features 6 Discussion 6 Discussion 6 1 Quantifiler Duo kit precision and accuracy 6 2 Quantifiler Duo Human and Male kits sensitivity 10 The data generated with the Quantifiler Duo kit did not demonstrate statistically significant differences between v1 0 and v1 1 for the human and the male targets The maximum mean Cy difference observed was 0 21 Cy for both the human 50 ng L standard and male 0 023 ng uL standard targets The mean difference in IPC Cy values was 0 14 Cy which is small but statistically significant A similar difference between the two 7500 Real Time PCR Systems used in the study suggests that the instruments might have introduced variability Statistical analysis of the standard curve metrics slope Y intercept and R2 did not demonstrate statistically significant differences between the two versions of the software Quantifiler Duo kit C values for the 007 dilution series showed a small but statistically significant difference for the human target only 0 05 C while the male and IPC targets showed no significant difference in these C values Calculated mean quantities showed more variability between versions of the software for both human 0 49 ng uL and male 0 54 ng uL targets We observed a similar difference between instruments suggesting that both the instruments and the individual standard curves introduced variability Quantifiler Human and Y
3. Manual PN 4344790 Applied Biosystems Quantifiler Duo DNA Quantification Kit User s Manual PN 4391294 Applied Biosystems HID Real Time PCR Analysis Software v1 0 Validation User Bulletin PN 4401643 HID Real Time PCR Analysis Software v1 1 User Bulletin 11 Validation and new features New features of v1 1 software New features of v1 1 software Software function New feature User interface Hybrid template access The Hybrid Quantifiler Human and Male plate option is available as a template on the home screen Adding a sample to a well Double clicking the well in the plate layout view opens the Add Sample dialog box so you can add a sample without returning to the Define Samples area See Figure 10 Start up preferences You can specify more start up preferences Default settings e Plate layout view e Show in wells screen Save settings as defaults in the plate layout view in addition to the well table view and also in the Show in Wells screen The new default settings and column arrangement in the well table view persist each time you open the software Setup and analysis settings can be different Analysis tools Omitting targets Omit either the human or male target from the standard curve during standard curve analysis in the Quantifiler Duo assay by selecting the well of interest then omitting the target See Figure 11 Selecting samples Select all
4. 2 replicates of the eight serial dilutions of standards ranging from 23 pg uL to 50 ng uL were prepared and run with each software version and instrument for the Quantifiler Duo kit only The reproducibility and sensitivity study used standard DNA sample 007 in these concentrations 10 5 2 5 1 25 0 625 0 312 0 156 and 0 078 ng uL Quantifiler Human and Male kits A sensitivity study compared results of the new Quantifiler Human and Y Human Male hybrid plate assay using both versions of the software The samples on the hybrid plate consisted of replicate standard curve dilution series for each kit as well as four replicate serial dilutions of standard control DNA 007 in these concentrations 10 5 2 5 1 25 0 625 0 312 0 156 and 0 078 ng pL Note For all validation studies identical sample dilutions were prepared and analyzed with each software version Two Applied Biosystems 7500 Real Time PCR System instruments with thermocycler firmware G2 10 were used for the validation Each 7500 instrument used for the validation was connected to two computers one with each version of the software v 1 0 and v1 1 The setup of the computer with v1 1 tested both the upgrade pathway and fresh installation for HID Real Time PCR Analysis Software v1 1 After software installation each instrument was calibrated using standard protocols to run the Regions of Interest ROI background optical and pure dye calibrations in addition to th
5. 30 5 v1 1 Duo IPC CT 30 29 5 IPC Cr Value 29 28 5 28 0 62 1 85 5 56 16 7 50 Standard Concentration ng uL Overall the two versions of the software produced data that was highly similar or identical Statistical analysis showed no significant difference between the Duo Human and Duo Male targets data not shown Statistical analysis of the IPC Cy values demonstrated a statistically significant difference between data produced by the two versions of the software However the mean difference was 0 14 Cy between software versions only slightly more than the mean difference of 0 12 Cy between instruments HID Real Time PCR Analysis Software v1 1 User Bulletin Validation and new features 5 Results Standard curve metrics Figure 5 shows the average slope and Y intercept values for the Quantifiler Duo kit generated by each software version Differences in slope Y intercept and R2 standard curve metrics between the software versions were statistically insignificant R data not shown Figure 5 Mean slope and Y intercept values for the Quantifiler Duo kit generated by each software version Quantifiler Duo Slope and Y Intercept Values Duo Human 5 2 Reproducibility and sensitivity Slope m Duo Male Slope Duo Human Y Intercept J Duo Male Y Intercept Y Intercept v1 1 Sensitivity differed slightly between the two software versions in studies using the 007 DNA stand
6. Human Male kits Sensitivity differed more between software versions in studies using the Quantifiler Human and Y Human Male kits The HID Real Time PCR Analysis Software v1 1 reported values that were 0 7 Cy less for the Human kit and 0 4 Cy more for the Y Human Male kit than the HID Real Time PCR Analysis Software v1 0 As a result mean differences in quantity calculations were 0 33 ng uL for the Quantifiler Human kit and 0 16 ng uL for the Quantifiler Y Human Male kit Re analysis of raw data from each run with each version of the software produced identical Cy values demonstrating that there is no difference between the C7 algorithms used by the two versions of the software The differences in data generated by v1 0 and v1 1 may be attributable to small differences in plate preparation and sample run during different experiments HID Real Time PCR Analysis Software v1 1 User Bulletin 7 Conclusions 8 References Validation and new features 7 Conclusions e This validation study demonstrated the reliability and reproducibility of the new HID Real Time PCR Analysis Software v1 1 for performing quantitation using the Quantifiler Human Quantifiler Y Human Male and Quantifiler Duo DNA quantification kits and the 7500 Real Time PCR System e HID Real Time PCR Software v1 1 generated C values and standard curve metrics slope Y intercept and R2 values with the Quantifiler Duo kit that were nearly identical to data generate
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8. applied S8 biosystems by Life technologies HID Real Time PCR Analysis Software v1 1 USER BULLETIN NOVEMBER 2010 SUBJECT Validation and new features In this user bulletin Overview This user bulletin covers Valid atOMis saata senene iiaeia i de Goths 6 AB aa be eae Gee he Reese ae 2 1 Materials and methods 0 0 0 000 e eee eects 2 2 Experimental setup 2 0 0 c eects 3 3 Data collection 0 0 00 c ccc teens 4 4 D ta analysis eean 40 baa Shee A GEM TSM LAS eed OA a E Peers Fhe 4 PROS NS S cece sae ese a esse has teanty ine Gene Une Gee ee Gane geese ene a 5 6 DISCUSSION nananana annann 10 T CONCUSSIONS cnc cece vee ee bee oy deka oo ak CaS Gos ia aN e EE 11 8 Referenco cit doccicecd iet eR Seb Beans HOR Sanh Gon dan Bae dented Soe Pala aes 11 New features of v1 1 software 0 0 0 0 000 c eee eee 12 The HID Real Time PCR Analysis Software v1 1 was designed specifically for the 7500 Real Time PCR System and for the Quantifiler Human Quantifiler Y Human Male and Quantifiler Duo DNA Quantification Kits Quantifiler specific templates and quality flags as well as STR reaction and dilution calculation tools enable streamlined data and sample assessment within the software New features of the HID Real Time PCR Analysis Software v1 1 include streamlined sample entry enhanced reporting and export capability and improved data analysis tools Validation consisted of the experiments
9. ard with each of the Quantifiler kits Quantifiler Duo kit Cy values for the human target in the 007 dilution series indicated a small though statistically significant mean difference of 0 05 C between the software versions in studies with the Quantifiler Duo kit data not shown Mean Cy values for the male target did not differ significantly between the software versions As shown in Figure 6 and Figure 7 human and male target quantities calculated with HID Real Time PCR Analysis Software v1 1 differed slightly 0 49 ng L for the human target 0 54 ng L for the male target Because the mean Cy values were nearly identical we attribute the difference in target quantities to the differences in the standard curves No statistical difference was observed in the IPC C values of the 007 dilution series HID Real Time PCR Analysis Software v1 1 User Bulletin Validation and new features 5 Results Quantifiler Human and Male kits We observed more variable sensitivity between the software versions in studies with the Quantifiler Human and Y Human Male kit As shown in Figure 8 and Figure 9 the HID Real Time PCR Analysis Software v1 1 reported lower mean Cy values and higher calculated quantities with the Quantifiler Human kit and higher mean Cy values and lower calculated quantities with the Quantifiler Y Human Male kit In addition v1 1 of the software reported a higher IPC C value than v1 0 Figure 6 Mean quantities of huma
10. d or otherwise controlled by Life Technologies either expressly or by estoppel 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners TaqMan is a registered trademark of Roche Molecular Systems Inc A Part Number 4460964 Rev A 11 2010 446 0964 Mj applied Headquarters Technical Resources and Support biosystems 5791 Van Allen Way Carlsbad CA 92008 USA For the latest technical resources and support information s Phone 760 603 7200 for all locations please refer to our Web site at by Lefe technologies www lifetechnologies com www appliedbiosystems com
11. d with HID Real Time PCR Analysis Software v1 0 Quantity values demonstrate a statistically significant difference but that difference is attributable to differences in the individual standard curves e Quantifiler Human and Y Human Male kit assay results demonstrated some variability between the software versions v1 0 and v1 1 Further re analysis of the raw data with each software version produced identical results Thus the variability could be a result of factors such as instrument assay setup or time between assay runs e All workflows run methods HID quality flags and dilution calculations were defined and utilized identically for each version of the software Federal Bureau of Investigation 2000 Quality assurance standards for forensic DNA testing laboratories Forensic Science Communications Online July 2000 Available at http www fbi gov hq lab fsc backissu july2000 codis2a htm Scientific Working Group on DNA Analysis Methods SWGDAM Revised validation guidelines approved July 2003 Available at http www fbi gov hq lab fsc backissu july2000 codis2a htm Applied Biosystems HID Real Time PCR Analysis Software v1 1 Getting Started Guide PN 4401644 Applied Biosystems Quantifiler Kits User Bulletin Validation Using SDS Software Version 1 2 3 on the Applied Biosystems 7500 Real Time PCR System and the ABI PRISM 7000 Sequence Detection System PN 4374659 Applied Biosystems Quantifiler Kits User s
12. e TaqMan RNase P Instrument Verification Plate with both HID Real Time PCR Analysis Software v1 0 and v1 1 HID Real Time PCR Analysis Software v1 1 User Bulletin Validation and new features 2 Experimental setup 2 Experimental setup 2 1 Quantifiler Duo kit precision and accuracy study Eight serially diluted samples prepared from the Quantifiler Duo kit stock standard were analyzed Figure 1 shows the plate setup for the Quantifiler Duo kit Each of the eight standard dilutions was run 12 times on each plate then Cy values were compared In addition six standard curves were generated per plate for slope Y intercept and R2 comparisons Figure 1 Precision and accuracy plate layout for Quantifiler Duo kit 1 2 3 4 5 6 7 8 9 101112 Standard DNA dilution NOOQOQOOO0OOO00OOO O Pangi F OOSOOOOOOOD 1677n 7OOOOOOOOOOOO 556 noL A ILLLALLALALAA JE Beun ESOS OOOOOOOD Om FiISOOOOOOOOOOOD Om FOOOOOOOOOOOS 0 068 rH 119 OOOO OOOOOOS Sr 2 2 Quantifiler Duo Human and Y Human Male kit reproducibility and sensitivity study Control DNA sample 007 was diluted and tested using the Quantifiler Duo Human and Y Human Male kits and each software version A single standard curve dilution series was prepared and used to test each software version with the diluted control DNA samples As Figure 2 and Figure 3 show eight dilutions of sample DNA 007 were prepared in these concentrations 10 5 2 5 1 25 0 625 0
13. listed below comparing Quantifiler Human Y Human Male and Duo kit data collected and analyzed with the HID Real Time PCR Analysis Software v1 1 to data collected and analyzed using the HID Real Time PCR Analysis Software v1 0 The data generated by the validation experiments demonstrates that the HID Real Time PCR Analysis Software v1 1 is reliable and reproducible in performing HID Quantifiler kit assays Our experiments with the kit demonstrated some difference between software versions Cy values and quantity values for the in Quantifiler Duo kit and also in Cy values for the Quantifiler Human and Y Human Male kit However this variability could be a result of factors such as instrument assay setup or time between assay runs Validation studies performed e Precision and Accuracy e Reproducibility and Sensitivity Validation and new features Validation Validation 1 Materials and methods 1 1 Reagents 1 2 Instruments To minimize variation due to hand pipetting and lot to lot differences the following setup procedures were employed throughout the study One lot of the Quantifiler Human Y Human Male and Duo kits was used for all validation studies A master mix consisting of the primer set and PCR reaction buffer was made daily for all of the plates run on that day A master mix of each sample dilution was prepared before pipetting into the plate wells Quantifiler Duo kit For the precision and accuracy study 1
14. n target DNA determined with the Quantifiler Duo kit by each version of the software Error bars indicate standard deviations Quantifiler Duo Human Target Sensitivity Quantities t N Calculated Concentration ng uL a 10 5 2 5 1 25 0 625 0 312 0 156 0 078 Input ng uL HID Real Time PCR Analysis Software v1 1 User Bulletin 7 Validation and new features 5 Results Figure 7 Mean quantities of male target DNA determined with the Quantifiler Duo kit by each version of the software Error bars indicate standard deviations Quantifiler Duo Male Target Sensitivity Quantities Calculated Concentration ng uL o a 2 5 1 25 0 625 0 312 0 156 0 078 Input ng uL Figure 8 Mean Cy of human target DNA determined with the Quantifiler Human kit by each version of the software Error bars indicate standard deviations Quantifiler Human Sensitivity C Values 36 34 32 30 28 26 24 22 20 CT 10 5 2 5 1 25 0 625 0 312 0 156 0 078 Input 8 HID Real Time PCR Analysis Software v1 1 User Bulletin Validation and new features 5 Results Figure 9 Mean Cy of human male target DNA determined with the Quantifiler Y Human Male kit by each version of the software Error bars indicate standard deviations Quantifiler Y Human Male Sensitivity C Values 40 38 36 34 32 5 30 28 26 24 22 20 10 5 2 5 1 25 0 625 0 312 0 156 0 078 Input HID Real Time PCR Analysis Software v1 1 User
15. s were analyzed to determine if samples were flagged correctly given the thresholds set Plate setup and performance were evaluated during the course of data collection and setup 3 Data collection Run methods for each Quantifiler kit were configured as outlined in the respective Quantifiler kit user manuals Although there is no 9600 Emulation mode checkbox in HID Real Time PCR Analysis Software v1 1 or v1 0 a calculated ramp up of 64 and ramp down of 100 was used in all validation experiments to mimic the 9600 Emulation mode in SDS v1 2 3 4 Data analysis All runs were analyzed using the manual analysis mode in each software version with the baseline set to start at cycle 3 and end at cycle 15 The manual Cy threshold was set to 0 2 4 HID Real Time PCR Analysis Software v1 1 User Bulletin 5 Results Validation and new features 5 Results 5 1 Precision and For the precision and accuracy studies the following values were determined accuracy e Average C7 IPC Cy values Slope Y Intercept R2 Cy Results Figure 4 Mean Cr values for the Quantifiler Duo kit with human and Internal PCR Control IPC targets generated by each software version at each DNA standard concentration Error bars indicate standard deviations v1 0 Duo Human Quantifiler Duo Precision and Accuracy Human and IPC Targets Target E v1 1 Duo Human 38 Cr Value 0 023 0 068 0 21 31 Target v1 0 Duo IPC CT
16. unknown samples for STR kit selection and dilution calculations with one click Sorting results You can sort data by column or row in the Results table The exported data file shows data sorted the way you chose for the Results table Exporting and reporting options Exporting jpg of standard curve Includes slope Y intercept and R2 Standard curve The standard curve includes data points for standard samples only Well selection You can export and print results for specific wells or for all wells Results table Printed Results Table contains all report data in v1 0 reports contained a Report Table and Report Summary each with some of the data Long sample names A sample name in a report can be up to 100 characters long 12 HID Real Time PCR Analysis Software v1 1 User Bulletin Validation and new features New features of v1 1 software Figure 10 One step sample addition and well assignment Duo Human 6 27 Duo IPC Duo Male 6 52 Well Duo Human Duo Standard 4 HID Real Time PCR Analysis Software v1 1 User Bulletin 13 For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice Life Technologies Corporation assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIE
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