The purpose of this user bulletin is to document validation studies using both theAmpFlSTR PCR Amplification Kits and the next generation AmpFlSTR PCR Amplification Kits to verify the successful amplification of samples on theVeriti thermal cycler with 0.2 mL block format.
This user bulletin documents two validation studies confirming the effectiveness of amplification using the Veriti 96-Well Thermal Cycler with 0.2 mL block format inconjunction with AmpFlSTR chemistry. These studies 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:
Note: For next generation kit validation information, see Appendix A, “Next generation AmpFlSTR PCR Amplification Kit validation on the Veriti 96-WellThermal Cycler with 0.2 mL sample block format” on page 16. This data includes information for the following five-dye AmpFlSTR PCR Amplification Kits:
In the study, we evaluated concordance, average peak heights, intra-color balance, peak height ratios, and dye artifacts for each thermal cycler/amplification kit combination. We performed statistical comparisons of the two thermal cyclers to ensure consistent results with the GeneAmp thermal cyclers and with the Veriti96-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.
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 kittested. The results were evaluated for concordance, average peak height, intra-colorbalance, and peak-height ratio. The positive controls provided in the AmpFlSTR kitswere 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
Amplification kits
used
The sensitivity study used a panel of seven male genomic DNA samples extracted from whole blood obtained from the Interstate Blood Bank. DNA samples werequantitated using the Quantifiler Human DNA Quantification Kit and diluted to 0.1ng/μL, 0.05 ng/μL, and 0.0125 ng/μL, using low TE buffer (10 mM Tris HCl, pH 8.0; 0.1mM EDTA, pH 8.0). Three input DNA amounts were used. For the AmpFlSTR kitsrequiring a total PCR reaction volume of 25 μL, amplification was performed usingDNA 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 reactionvolume of 50 μL, amplification was performed using DNA input amounts of 2 ng, 1 ngand 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 thermalcycler platform.
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 validationstudies to minimize variability. In addition, identical dilutions of samples were used fortesting on both of the thermal cycler platforms.
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 GeneAmpthermal 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 usedon 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),
Enter the information on the reaction volume as well as stages, cycles, times andtemperatures of the run
Select
Name and save the run method.Note: Run methods configured on one Veriti thermal cycler can be saved on aUSB drive and exported to other Veriti thermal cyclers to ensure uniformprotocols.
Fragment analysis To minimize variability, the study used one ABI PRISM 3100 Genetic Analyzer withone 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 withthe ABI PRISM 3100 Genetic Analyzer and AmpFlSTR PCR Amplification Kit user’s manuals. AmpFlSTR kit used the following reagent volumes per well:
• With dye set F: 8.5 μL Hi-Di™ Formamide, 0.5 μL GeneScan-500 ROX™ SizeStandard, 1.0 μL amplicon
• With dye set G5: 8.7 μL Hi-Di™ Formamide, 0.3 μL GeneScan-500 LIZSize Standard, 1.0 μL amplicon
The validation study used GeneMapper ID-X v1.0 software to analyze run filesgenerated by the Data Collection Software v1.1. Allele calls, peak heights, and basepair sizes were determined using the appropriate AmpFlSTR PCR AmplificationKit 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 usingeach AmpFlSTR kit. Profiles generated in the course of the studies were comparedto the control and male sample DNA profiles to determine whether all alleles werecalled identically. If an allele was not called identically in the control and sampleprofiles, 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 lowestnormalized peak height was divided by the highest normalized peak height and theresult was multiplied by 100.
Heterozygote peak height ratio
Within a heterozygous locus, the lower peak height of the two alleles was divided bythe 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 thepresence of artifacts and contamination throughout the validation study. Thisevaluation included the identification of any anomalous and reproducibly amplifiedproducts (one or more peaks of the same base pair size in two or more samples) or dyeartifacts. The evaluation included any peaks exceeding 50 relative fluorescent units (RFU) in the region greater than or equal to 100 base pairs.
Both the Veriti 96-Well Thermal Cycler with 0.2 mL block format and the GeneAmp PCR System 9700 thermal cycler 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 PCR System 9700 thermal cyclers. All AmpFlSTR kitsdemonstrated statistically significant results with the exception of the Identifiler Kit.
Figure 1
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 heights and standard deviations generated using both thermal cyclers with each of these kits are alsosimilar. 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.
The heterozygote peak height ratio, or intralocus balance, was determined across theprofile for each of the AmpFlSTR kit 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 COfilerKit. Figure 2 shows the average heterozygote peak height ratios of the instrumentreplicates for each of the AmpFlSTR kit tested.
Figure 2
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, thedifferences 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 andGeneAmp 9700 96-well thermal cyclers. Figure 3 details the intra-color balance of each of the AmpFlSTR kit tested.
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 thesample input amounts, instrument replicate, full concordance percentage, and percentage of total alleles that fell below the detection threshold. Full profilepercentage (the percentage of samples yielding full profiles) and undetected allele percentages (the percentage of expected alleles that were not detected) are shown foreach 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-to-injection 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 l
Average Instrument Replicate Intra-Color Balance
40
50
60
70
80
90
100
ProfilerPlus
COfiler SGM Plus Identifiler MiniFiler™ Yfiler
Intr
a-C
olor
Bal
ance
GeneAmpPCRSystem9700
Veriti™0.2mLThermalCycler
Kit Input amountThermal
cycler replicate
Full profile percentage Undetected allele percentage
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-to-run 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 boxescorrespond 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 inthe center of the box plots, represent the peak height mean. The black and red asterisks are the calculated outliers.
Table 3 shows the average instrument replicate peak heights for all DNA inputamounts for each AmpFlSTR kit tested.
Table 3 l
Heterozygote peak height ratio
Each of the AmpFlSTR kits tested on each thermal cycler platform producedaverage 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 9700 and Veriti0.2 mL thermal cycler platforms with a maximum of 1% difference observed betweenthe data generated with each thermal cycler platform using the Identifiler , SGM Plus , and Minifiler™ Kits. Figure 6 shows representative data from the IdentifilerKit.
AmpFlSTR PCR Amplification Kit Average Peak Heights
Thermal Cycler
Profiler Plus COfiler SGM Plus Identifiler Minifiler™ Yfiler
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 l
AmpFlSTR PCR Amplification Kit Average Peak Height Ratios
Thermal Cycler Profiler Plus COfiler SGM Plus Identifiler Minifiler™
The data generated on the Veriti 0.2 mL 96-well thermal cycler did not contain anynew reproducible PCR artifacts that are not already published in the User’s Manualsfor the AmpFlSTR PCR Amplification Kits tested.
The negative amplification controls produced no amplification of DNA with any of the AmpFlSTR PCR Amplification Kits tested.
Concordance
All of the alleles from the 9947A and 007 control DNAs generated using the Veriti96-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 thermalcycler 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 aremore sensitive to thermal profile differences, such as Yfiler , demonstrated a smaller peak height difference (≈8%) than was observed with the Profiler Plus and COfilerkits. 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.
Other AmpFlSTR kits tested generated differences of <15%, which is within thevariability 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 peakheight 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 AmpFlSTRkit tested.
Intra-color balance
For all of the AmpFlSTR kits tested and both thermal cycler types, the results allexceeded 40% for the control DNA. The difference between the GeneAmp 9700 andVeriti thermal cycler intra-color balances was <5%, indicating a minor difference within the run-to-run variability of the ABI PRISM 3100 Genetic Analyzer.
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 theGeneAmp 9700 thermal cycler 9700 to 14% to 100% for the data generated by theVeriti thermal cycler. For all samples and AmpFlSTR PCR Amplification Kits tested, all alleles detected were concordant with known genotypes. However, allelicdrop-out was prevalent at lower DNA input amounts. Generally, the Veriti 0.2 mLthermal 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 cyclerplatforms. 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 relativefluorescent unit (RFU) ranges. Most of the variability of the peak heights wasattributed to variation in sample quality and concentration.
Heterozygote peak height ratio
Similar to the average peak heights data, peak height data from the GeneAmp 9700and 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 PRISM3100 Genetic Analyzer. All of the 2 ng, 1 ng and 0.5 ng samples for all of theAmpFlSTR 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 AmpFlSTRkits tested. The percent difference between the intra-color balance for the GeneAmp9700 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 decreasein 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
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.
The validation of the Veriti 96-Well Thermal Cycler with 0.2 mL block format wasdesigned to evaluate the amplification performance of AmpFlSTR PCRAmplification 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 generated on theGeneAmp PCR System 9700 thermal cycler. Analysis metrics compared weregenotype concordance, average peak height, heterozygote peak height ratio, intra-color balance, and artifact identification. The validation study results demonstrate thegeneration of high quality AmpFlSTR kit data using the Veriti thermal cycler with0.2 mL block format. In addition, the data generated by each of the thermal cycler platforms was either statistically similar or relatively comparable to the datagenerated by the other. Thus, the Veriti thermal cycler with 0.2 mL block formatexhibits reliable performance for human identification applications.
Applied Biosystems AmpFlSTR COfiler PCR Amplification Kit User Bulletin,(PN 4306116).
Applied Biosystems AmpFlSTR Identifiler PCR Amplification Kit User Guide,(PN 4323291).
Appendix A Next generation AmpFlSTR PCR Amplification
Kit validation on the Veriti 96-Well Thermal Cycler with 0.2mL sample block format
Overview
Purpose 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 inconjunction with new AmpFlSTR chemistry. The study compared data generatedon the Veriti thermal cycler with data generated on the GeneAmp PCR System 9700 thermal cycler. The data verified reproducibility, sensitivity, accuracy, andinhibition resistance of amplification on each thermal cycler with the following next generation AmpFlSTR PCR amplification kits:
In the study, we evaluated genotype concordance, average peak heights, intracolor andintralocus balance, allele drop out rate with inhibitor, and dye artifacts for each thermal cycler/amplification kit combination. We performed statistical comparisons of the two thermal cyclers to ensure consistent results between the GeneAmpthermal cyclers and the Veriti 96-Well Thermal Cycler with 0.2 mL block format.
This validation studies confirmed that the AmpFlSTR amplification chemistry produces high quality data when run on the Veriti 96-Well Thermal Cycler with 0.2mL block format.
Validation Studies
Reproducibility All four next generation AmpFlSTR kits were tested in the reproducibility study.Each kit was tested on two Veriti thermal cyclers and two 9700 thermal cyclers with 12 replicates of the kit positive control DNA, and 3 replicates of NTC in one PCR plate. All kits were run with standard protocol and cycle number according to the kit user guide (Identifiler Plus - 28 cycles; Identifiler Direct - 27 cycles; NGM™ and NGMSElect™ - 29 cycles). The PCR products were injected once on a 3130xl instrument for fragment analysis:
• 9947A DNA for the Identifiler Plus and Identifiler Direct Kits• 007 DNA for the NGM™ and NGM SElect™ Kits
The sensitivity study included three next generation AmpFlSTR kits; IdentifilerPlus, NGM™, and NGM SElect™. Each kit was tested on two Veriti thermal cyclersand two 9700 thermal cyclers with 3 genomic DNA samples at 5 input amounts each (1 ng, 0.5 ng. 0.125 ng, 0.0625 ng, and 0.031 ng), and 3 replicates for each input. All samples of each kit were amplified on one PCR plate for each thermal cycler. All kits were run with standard protocol and cycle number according to the kit user guide(Identifiler Plus - 28 cycles; Identifiler Direct - 27 cycles; NGM™ and NGM SElect™
- 29 cycles). The PCR products were injected once on a 3130xl instrument for fragmentanalysis.
The accuracy study with the Identifiler Direct Kit was performed on two Veritithermal cyclers and two 9700 thermal cyclers. Three male buccal swaps on FTA cards and 3 male blood stains on FTA cards were tested with 12 punches (replicates) persample per thermal cycler. The PCR reactions were performed with the standardprotocol according to the kit user guide. Two buccal swab samples were amplified with 26 cycles, and one with 27 cycles. Two blood samples were amplified with25 cycles, and one with 26 cycles. The PCR products were injected once on a 3130xlinstrument for fragment analysis.
The inhibition study included three next generation AmpFlSTR kits: IdentifilerPlus, NGM™, and NGM SElect™. Each kit was tested on two Veriti and two 9700 thermal cyclers using 1 ng 007 DNA spiked with three concentrations of hematin targeted at 100%, 50%, and 0% peak height reduction. The Identifiler Plus Kit was tested with 30, 260, and 310 μM of hematin spiked into the pristine DNA. The NGM™
and NGM SElect™ Kits were tested with 25, 160, 250 uM of hematin spiked into the pristine sample. Four replicates of each inhibited sample at each concentration of hematin were tested against three replicates of 1 ng 007 pristine DNA and three replicates of the kit positive control. All kits were run with standard protocol andcycle number according to the kit user guide. The PCR products were injected onceon a 3130xl instrument for fragment analysis.
One lot of each of the AmpFlSTR PCR Amplification Kits listed in Table 6 wasutilized 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.
The validation study used GeneMapper ID-X v1.2 software to analyze run filesgenerated by the Data Collection Software v3.0. All the data were analyzed with thesame Analysis Method settings using peak amplitude threshold at 50 RFU. Allelecalls, 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 Minitabstatistical analysis software v15 with 95% confidence interval.
Genotype concordance and allele dropout
Prior to the validation study, profiles of the DNA samples were determined usingeach AmpFlSTR kits. Profiles generated in the course of the studies were compared to the control and male sample DNA profiles to determine whether all alleles werecalled identically. If an allele was not called identically in the control and sampleprofiles, further investigation was performed to determine whether the allele was not present or was called incorrectly. The percent of the allele drop out was calculatedusing the number of allele drop out (<50 RFU) divided by the total number of expected alleles, then multiply by 100%.
Average peak heights
The average peak height was calculated by taking an average of the heterozygous peak heights in each marker, and dividing each homozygous peak height by 2. The average peak heights from each thermal cycler were compared.
Within a heterozygous locus, the lower peak height of the two alleles was divided bythe higher peak height, then multiplied by 100.
Intra-color balance
The average peak heights were used to calculate the intra-color balance. Between multiple loci in a color, the lowest average peak height was divided by the highestaverage peak height, then multiplied by 100.
Shutter ratio was the ratio of the stutter divided by the +4 bp adjacent allele peak. The sample plot sizing table for FAM™, VIC , NED™, and PET™ dyes was exported fromGeneMapper ID-X v1.2 and run through a Microsoft Excel Macro program developed internally. The identified stutter ratios were compared with kit stutter ratiosetting in GeneMapper ID-X v1.2 software. A few sample plots were selected formanually screening for stutter ratios.
Dye artifacts and negative control data
All samples tested in the reproducibility studies were evaluated for the presence ofartifacts and contamination throughout the validation study. This evaluation included the identification of any anomalous and reproducibly amplified products (one or morepeaks of the same base pair size in two or more samples) or dye artifacts. Theevaluation included any peaks exceeding 50 relative fluorescent units (RFU) in the marker reading region of each kit.
Genotype 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 PCRAmplification Kits tested. All results were 100% concordant with the previously established genotyping results.
Stutter ratio
The percent stutter ratio was examined for both the 9700 and Veriti thermal cyclers. No stutter ratio identified in this study exceeded the stutter ratio settings in GeneMapper ID-X v1.2 software for all AmpFlSTR PCR Amplification Kits tested.The Veriti thermal cyclers generated similar stutter ratios as the 9700 platform.
Artifacts and negative control samples
The data generated on the Veriti 0.2 mL 96-well thermal cycler did not contain anynew reproducible PCR artifacts that are not already published in the User’s Manualsfor the AmpFlSTR PCR Amplification Kits tested.
The negative amplification controls generated similar baseline profiles on both Veriti and 9700 thermal cyclers, and produced no amplification of DNA with any of the AmpFlSTR PCR Amplification Kits tested.
Average peak heights
Figure 7 compares the average peak heights for the positive control DNA replicatesrun on both the Veriti and GeneAmp 9700 thermal cyclers. All AmpFlSTR kitsdemonstrated no statistically significant difference between the two platforms
The average peak height values generated using the Identifiler Plus, IdentifilerDirect, NGM™, and NGM SElect™ Kits demonstrated <10% difference between the thermal cyclers. This variation is consistent with the observed reaction-to-reaction
variability of the GeneAmp 9700 thermal cyclers. The injection-to-injection variation on the ABI PRISM 3130xl Genetic Analyzer might also contribute to the observeddifferences. The performance of the two thermal cycler platforms were consistently similar with the kit positive control DNA.
The intra-locus balance was determined across the positive control DNA profiles for each of the AmpFlSTR kits tested. Figure 8 compares the average intra-locusbalance between the Veriti and GeneAmp 9700 thermal cyclers. These ratios were >85% for all kits. The differences between the two platforms were less than 6% andwere not statistically significant.
The intra-color balance was determined across the positive control DNA profiles foreach of the AmpFlSTR kits tested. Figure 9 compares the average intra-colorbalance between the Veriti and GeneAmp 9700 thermal cyclers. These ratios were >50% for all kits. There was no statistically significant difference between the two platforms except for Identifiler Direct Kit, which had a maximum difference of 15%. The difference was well within the observed reaction-to-reaction variability of the GeneAmp 9700 thermal cyclers.
Sensitivity Genotype concordance and allele dropout
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 threemale DNA samples with different concentrations when used with Identifiler Plus,NGM™, and NGM SElect™ Kits. All results were 100% concordant with thepreviously established genotyping results.
Allele drop outs were observed in the samples with low DNA input. Table 10 compares the allele drop out rates and the overall average peak heights between the two thermal cycler platforms for each AmpFlSTR kit and DNA input amount. There was no significant difference in the performances between the Veriti and the 9700 instruments.
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 thermalcyclers generated comparable data for each DNA input amount with eachamplification kit. The average peak height differences between the thermal cycler platforms across all input amounts were generally less than 10% with a maximum 19% difference observed with the NGM SElect™ Kit and 0.5 ng DNA input. Thesedifferences were within normal run-to-run variability of 9700 thermal cycler and ABI PRISM 3130xl Genetic Analyzer for all kits tested. Figure 10, 11, and 12 illustrate the average peak height data with Identifiler Plus, NGM™, and NGM SElect™ Kits ateach DNA input amount. The orange and green boxes correspond to the twoGeneAmp 9700 thermal cyclers and Veriti 0.2 mL thermal cyclers respectively. Theblack dots, generally located in the center of the box plots, represent the peak heightmean. The black asterisks are the calculated outliers.
The intra-locus balance was determined across all male DNA samples for each of the AmpFlSTR kits tested. Table 11 compares the average intra-locus balance betweenthe Veriti and GeneAmp 9700 thermal cyclers for each kit and each DNA input.For all kits, the average peak height ratios were >85% with 0.5 ng and 1.0 ng DNA inputs, and were slightly lower for the low DNA inputs. The differences between the two platforms were less than 4% and were not statistically significant.
The intra-color balance was determined across all male DNA samples for each of theAmpFlSTR kits tested. Table 12 compares the average intra-color balance betweenthe Veriti and GeneAmp 9700 thermal cyclers for each dye color in each kit. Due to significant allele drop outs, the data for 0.031 ng and 0.062 ng DNA inputs werenot calculated. For all kits, the average peak height ratios were >50%. Theperformance from the two platforms were comparable in most cases. The maximum difference was 17% in the NGM™-NED™ dye channel with 0.125 ng input.
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 blood and buccal samples when used with the AmpFlSTR Identifiler Direct PCR Amplification Kit. All results were 100% concordant with the previously establishedgenotyping results.
Average peak heights
The average peak heights were calculated for blood and buccal samples withIdentifiler Direct Kit. The results are illustrated in Figure 13 on page 30. The orange and green boxes correspond to the two GeneAmp 9700 thermal cyclers and Veriti0.2 mL thermal cyclers respectively. The black dots located in the center of the boxplots represent the peak height mean. The black asterisks are the calculated outliers.
Generally, the Veriti thermal cycler had equivalent or better performance compared to 9700. The Veriti thermal cycler consistently gave slightly better peak height thanthe 9700 across all markers with similar amounts of variability. The maximum percentdifference between Veriti and 9700 thermal cyclers in peak height was found in marker D135S317 at 20.7% for blood samples and 23.0% for buccal samples. Thesedifferences were within normal run-to-run variability of 9700 thermal cycler and ABI PRISM 3130xl Genetic Analyzer.
The Intra-locus balance were calculated for blood and buccal samples withIdentifiler Direct Kit. The results are illustrated in Figure 14 on page 31. The orange and green boxes correspond to the GeneAmp 9700 thermal cyclers and Veriti 0.2mL thermal cyclers respectively. The black dots in the center of the box plots,represent the mean peak height ratio. The black asterisks are the calculated outliers.
The intra-locus balance for the 9700 and Veriti thermal cyclers showed no differencein mean value and amounts of variability. The differences between the 9700 and Veriti thermal cycler in average intra-locus balance were less than 3%.
The Intra-color balance were calculated for blood and buccal samples with Identifiler Direct Kit. The results are illustrated in Figure 15 on page 32. The orange and green boxes correspond to the GeneAmp 9700 thermal cyclers and Veritithermal cyclers respectively. The black dots in the center of the box plots representthe mean peak height ratio. The black asterisks are the calculated outliers
The Veriti thermal cyclers performed slightly better in intra-color balance than 9700s in all dye channels, but the variability was similar on both platforms. The maximum differences were observed in the PET™ channel at 11.9% for blood samples and 11.1% for buccal samples. These differences were within normal run-to-run variability of 9700 thermal cycler and ABI PRISM 3130xl Genetic Analyzer.
Allele drop outs were observed in the 007 samples with high and mediumconcentrations of hematin. Table 13 compares the allele drop out rates between the two thermal cycler platforms for each AmpFlSTR kit and hematin concentrations. The Veriti thermal cyclers generated slightly less allele drop out than the 9700instruments
Figure 16 compares the average remain peak heights of the 007 DNA with the existence of hematin between the Veriti and GeneAmp 9700 thermal cyclers. The remain peakheight of each loci was calculated as the percent ratio of the peak height between the 007DNA with the inhibitor and the pristine DNA. Data from each AmpFlSTR kit were illustrated in different columns of the panel. Data from each inhibitor concentration wereillustrated in different rows. Data from 9700 and Veriti platforms were presented in black and red colors respectively. There was no significant difference between the twoplatforms.
The intra-locus balance was determined for pristine 007 and 007 with different concentrations of hematin for each of the AmpFlSTR kits tested. Figure 17 compares the average intra-locus balance between the Veriti and GeneAmp 9700 thermal cyclers for each kit (in columns) and each inhibitor concentration (in rows). There was no significant difference between the two platforms.
The intra-color balance was determined for pristine 007 and 007 with differentconcentrations of hematin for each of the AmpFlSTR kits tested. Figure 18compares the average intra-color balance between the Veriti and GeneAmp 9700thermal cyclers for each kit (in columns) and each inhibitor concentration (in rows). The data from Identifiler Plus samples with high hematin concentration were notplotted due to the significant allele drop out. There was no significant difference between the two platforms.
The genotype profiles of the DNA samples used in the reproducibility, sensitivity,accuracy, and inhibition tests were 100% concordant between Veriti and 9700 thermal cyclers with all the AmpFlSTR kits tested. They were the same as previously established genotyping results.
Allele drop outs were observed on both Veriti and 9700 thermal cyclers in the 0.031ng and 0.062 ng input DNA samples with all the AmpFlSTR kits tested. Minor dropouts were observed in the 0.125 ng input with NGM™ and NGM SElect™ Kits, but not with Identifiler Plus Kit.
The allele drop outs were also observed in the samples with hematin as PCRinhibitor. The Identifiler Plus Kit demonstrated the most allele drop out rate withboth high and medium level of inhibitor. The NGM™ Kit only demonstrated allele drop outs with high level of inhibitor. The NGM SElect™ Kit showed no allele drop out with any level of inhibitor.
The Veriti thermal cyclers generated slightly lower allele drop out rates than the 9700 instruments, indicating equivalent or better performance to amplify the DNA with low input or with PCR inhibition.
The average peak heights generated with all the AmpFlSTR kits tested were comparablebetween the Veriti and 9700 thermal cyclers. In average, the peak height differencebetween the two platforms was 5% to 10% with the reproducibility and sensitivitysamples, and 10% to 15% with the direct amplification samples. The actual differencebetween individual data points varied depending on the kit and sample used. For the Identifiler Direct samples, the Veriti thermal cyclers intended to generate strongersignals in all markers. For the Identifiler Plus, NGM™, and NGM SElect™ samples, theresults were mixed. The Veriti instruments provided higher signals in some markers butlower in others.
When inhibitor presented in the samples, the average remain peak heights was calculatedas the ratio to the pristine DNA. The results were also comparable between the Veritiand 9700 thermal cyclers with Identifiler Plus, NGM™, and NGM SElect™ kits. Theaverage difference was 5% to 15% depending on the kit. On both platforms, theIdentifiler Plus Kit was least resistant to the inhibition. With high concentration ofinhibitor, Identifiler Plus Kit remained no signals in most of the locus, while NGM™
and NGM SElect™ Kits still generated above-threshold peak heights.
The observed peak height difference between the Veriti and 9700 platforms were withinthe normal instrument-to-instrument and run-to-run variability of 9700 thermal cycler andABI PRISM 3130xl Genetic Analyzer.
Intra-locus balance The intra-locus peak balance generated with all the AmpFlSTR kits tested werecomparable between the Veriti and 9700 thermal cyclers. These heterozygote peak height ratios were >85% for all kits with the reproducibility samples and sensitivitysamples with 0.5 ng and 1.0 ng input amounts. The heterozygote peak height ratios were >70% for all kit Identifiler Direct Kit with the accuracy samples. The averagedifferences between the two platforms were less than 6% with reproducibility samples, less than 4% with the sensitivity samples, less than 3% with the accuracy samples, andwere not statistically significant.
When inhibitor presented in the samples, the intra-locus balance for the 9700 and Veriti thermal cyclers showed no significant difference in mean value and amounts of variability. The heterozygote peak height ratios were above 70% for all the kitstested.
Intra-color balance The intra-color peak balance generated with all the AmpFlSTR kits tested were
Stutter ratio
comparable between the Veriti and 9700 thermal cyclers. These peak height ratios within each dye channel were >50% for all kits with the reproducibility samples and sensitivity samples, and were >40% for Identifiler Direct Kit with the accuracy samples. In general, there was no statistically significant difference between the two platforms, but random differences did exist. In the reproducibility test, the maximum difference of 15% was observed in the VIC (green) dye in Identifiler Direct Kit. In the sensitivity test, the maximum difference of 17% was observed in the NED™(yellow) dye in NGM™ Kit. In the accuracy test, the maximum difference of 12% was observed in the PET™ (red) dye in Identifiler Direct Kit.
When inhibitor presented in the samples, the intra-color balance for the 9700 andVeriti thermal cyclers showed no significant difference in mean value and amounts of variability.
The Veriti thermal cyclers generated similar stutter ratios as the 9700 platform. No stutter ratio identified in this study exceeded the stutter ratio settings in GeneMapperID-X v1.2 software for all AmpFlSTR PCR Amplification Kits tested.
No reproducible artifacts other than those published in the AmpFlSTR kit user manuals were produced by either the GeneAmp 9700 or the Veriti thermal cyclerswith the amplified control DNA and male samples. Both platforms generated similar peak heights for these known artifacts as the published ranges. The artifact peak heights from the Veriti thermal cyclers were equivalent to those produced by the GeneAmp 9700 thermal cyclers.
No DNA contamination was observed in any negative control amplified throughoutthe validation studies. The Veriti thermal cyclers generated the baseline profiles as clean as the 9700 instruments. There was no amplification artifacts detected that was not among the published artifacts.
The results of this validation study demonstrated that the Veriti thermal cycler wascomparable to the validated 9700 thermal cycler in assay performance of AmpFlSTRNGM™, Identifiler Plus, Identifiler Direct, and NGM SElect™ kits. There was no significant difference in the peak height, intralocus balance, and intracolor balance between the two platforms except for a few cases where the Veriti thermal cycler was marginally better.
The genotype profiles were 100% concordant between the Veriti and 9700 thermal cyclers in all of the tests. The stutter ratios were within stutter ratio setting inGeneMapper ID-X v1.2 for each kit. No new artifact peaks, uneven or fluctuatebaseline were observed.
Allele dropout was observed at 0.062 ng or lower DNA input for each kit across both Veriti and 9700 thermal cyclers. The Veriti thermal cycler had equivalent or lowerallele dropout rate compared to the 9700.
The Veriti thermal cycler performed better than the 9700 thermal cycler in the hematin inhibition study, with fewer allele drop outs and less signal loss observed.
The validation study results demonstrate that the Veriti thermal cycler with 0.2 mLblock format could generate high quality AmpFlSTR kit data. The data generated byVeriti thermal cycler were either statistically similar or relatively comparable to thedata generated by the 9700 platform. Thus, the Veriti thermal cycler with 0.2 mLblock format exhibits reliable performance for human identification applications.
AmpFlSTR Identifiler Plus PCR Amplification Kit User Guide (Pub. no. 4440211).
AmpFlSTR Identifiler Direct PCR Amplification Kit User Guide (Pub. no. 4475401).
AmpFlSTR NGM™ PCR Amplification Kit User Guide (Pub. no. 4425511).
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