USER BULLETIN
Veriti® 96-Well Thermal Cycler
AmpFlSTR® Kit Validation
Pub. No. 4440754 Rev. B
SUBJECT:
AmpFlSTR® PCR Amplification Kit validation on the
Veriti® 96-Well Thermal Cycler with 0.2 mL sample block format
■
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
■
Validation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
■
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
■
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
■
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
■
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
■
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
■
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Appendix A, Next generation AmpFlSTR® PCR Amplification Kit validation on the Veriti® 96-Well Thermal Cycler with 0.2 mL sample block format
■
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■
Validation Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
■
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
■
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
■
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
■
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
■
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Purpose of this user bulletin
The purpose of this user bulletin is to document validation studies using both the
AmpFlSTR® PCR Amplification Kits and the next generation AmpFlSTR® PCR
Amplification Kits to verify the successful amplification of samples on the Veriti®
thermal cycler with 0.2 mL block format.
For Forensic or Paternity Use Only.
Overview
Overview
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 in
conjunction 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:
• 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
Note: For next generation kit validation information, see Appendix A, “Next
generation AmpFlSTR® PCR Amplification Kit validation on the Veriti® 96-Well
Thermal Cycler with 0.2 mL sample block format” on page 16. This data includes
information for the following five-dye AmpFlSTR® PCR Amplification Kits:
• AmpFlSTR® Identifiler® Direct PCR Amplification Kit
• AmpFlSTR® Identifiler® Plus PCR Amplification Kit
• AmpFlSTR® NGM™ PCR Amplification Kit
• AmpFlSTR® NGM SElect™ PCR Amplification Kit
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 Veriti® 96Well 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
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
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
Plus®
4307133
0712131
0711130
Identifiler®
4322288
0801092
0801092
Minifiler™
4373872
0806012
0712008
Yfiler®
4359513
0801035
0708030
SGM
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
3
Materials and methods
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 MenuConvert a Method9600 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® kit used the following reagent volumes per well:
• With dye set F: 8.5 µL Hi-Di™ Formamide, 0.5 µL 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
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Data analysis
Sizing and
genotyping
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.
Statistical analysis
and calculations
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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
5
Results
Results
Repeatability
Concordance
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® kits
demonstrated statistically significant results with the exception of the Identifiler® Kit.
Figure 1 Average peak height values of thermal cycler instrument replicates. Error bars
indicate the average standard deviation observed for the instrument replicates.
Instrument Replicate Average Peak Height Values
3500
3000
GeneAmp®
PCR System
9700
RFU
2500
2000
Veriti™
0.2mL
Thermal
Cycler
1500
1000
500
0
Profiler
Plus®
COfiler®
SGM
Plus®
Identifiler® MiniFiler™
Yfiler®
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 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.
6
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Heterozygote peak height ratio
The heterozygote peak height ratio, or intralocus balance, was determined across the
profile 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 COfiler® Kit. Figure 2 shows
the average heterozygote peak height ratios of the instrument replicates for each of the
AmpFlSTR® kit tested.
Figure 2 Average heterozygote peak height ratios of the three instrument replicates. Error bars
indicate the average instrument replicate standard deviations.
Average Peak Height Ratios
105
Peak Height Ratio
100
GeneAmp®
PCR System
9700
95
90
85
80
Veriti™
0.2mL
Thermal
Cycler
75
70
65
Profiler
Plus®
COfiler®
SGM Plus®
Identifiler®
MiniFiler™
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® kit tested.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
7
Results
Figure 3 Intra-color balance of AmpFlSTR® PCR Amplification Kits. Error bars indicate the
average instrument replicate standard deviations.
Average Instrument Replicate Intra-Color Balance
100
GeneAmp®
PCR
System
9700
Intra-Color Balance
90
80
70
Veriti™
0.2mL
Thermal
Cycler
60
50
40
Profiler
Plus®
Sensitivity
COfiler®
SGM Plus® Identifiler® MiniFiler™
Yfiler®
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-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 Full profile percentage and undetected allele percentage by thermal cycler, AmpFlSTR® PCR Amplification Kit, and
DNA input amount
Kit
8
Input amount
Full profile percentage
Undetected allele percentage
Thermal
cycler
replicate
GeneAmp®
Veriti® 0.2 mL
GeneAmp®
Veriti®
0.2 mL
COfiler®
0.250 ng
1
62.5%
75.0%
7.3%
2.6%
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%
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Kit
Input amount
Full profile percentage
Undetected allele percentage
Thermal
cycler
replicate
GeneAmp®
Veriti® 0.2 mL
GeneAmp®
Veriti® 0.2 mL
(continued)
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%
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™
Profiler
0.5 ng
2
100.0%
100.0%
0.0%
0.0%
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%
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%
Profiler
SGM
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
9
Results
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-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 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
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
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® 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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
11
Results
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
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
12
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
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® 96Well 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.
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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
13
Discussion
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 9700 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.
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-torun variability of the ABI PRISM® 3100 Genetic Analyzer.
Artifacts and
negative controls
14
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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
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 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 Guide,
(PN 4323291).
Applied Biosystems AmpFlSTR® Minifiler™ PCR Amplification Kit User Guide,
(PN 4374618).
Applied Biosystems AmpFlSTR® Profiler Plus® PCR Amplification Kit User Guide,
(PN 4303501).
Applied Biosystems AmpFlSTR® SGM Plus® PCR Amplification Kit User Guide,
(PN 4309589).
Applied Biosystems Veriti® Thermal Cycler User Guide, (PN 4375799).
Applied Biosystems AmpFlSTR® Yfiler® PCR Amplification Kit User Guide, (PN 4358101).
ABI PRISM® 3100 Genetic Analyzer User's Manual (PN 4315834).
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
15
Overview
Appendix A Next generation AmpFlSTR® PCR Amplification Kit
validation on the Veriti® 96-Well Thermal Cycler with 0.2 mL
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 in
conjunction with new 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, sensitivity, accuracy, and inhibition
resistance of amplification on each thermal cycler with the following next generation
AmpFlSTR® PCR amplification kits:
• AmpFlSTR® Identifiler® Plus PCR Amplification Kit
•
AmpFlSTR® Identifiler® Direct PCR Amplification Kit
• AmpFlSTR® NGM™ PCR Amplification Kit
• AmpFlSTR® NGM SElect™ PCR Amplification Kit
In the study, we evaluated genotype concordance, average peak heights, intracolor and
intralocus 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 GeneAmp® thermal
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
0.2 mL 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 NGM
SElect™ - 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
16
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Sensitivity
The sensitivity study included three next generation AmpFlSTR® kits; Identifiler®
Plus, NGM™, and NGM SElect™. Each kit was tested on two Veriti® thermal cyclers
and 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 fragment
analysis.
Accuracy
The accuracy study with the Identifiler® Direct Kit was performed on two Veriti®
thermal 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) per
sample per thermal cycler. The PCR reactions were performed with the standard
protocol 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 with
25 cycles, and one with 26 cycles. The PCR products were injected once on a 3130xl
instrument for fragment analysis.
Inhibition
The inhibition study included three next generation AmpFlSTR® kits: Identifiler® Plus,
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 and cycle number according
to the kit user guide. The PCR products were injected once on a 3130xl instrument for
fragment analysis.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
17
Materials and methods
Materials and methods
Amplification kits
and samples
One lot of each of the AmpFlSTR® PCR Amplification Kits listed in Table 6 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.
Table 6 AmpFlSTR® PCR Amplification Kit part numbers and lot numbers
AmpFlSTR® kit
Part number
Lot number
Identifiler® Plus
4427368
1006006
Identifiler®
4408580
1005006
NGM™
Direct
4415021
1004003
NGM SElect™
4457889
1010001
Table 7 DNA samples used in each test
Test
Reproducibility
Samples
Sources
0.1 ng/µL and 2 ng/uL 9947A
Kit positive control
0.1 ng/µL 007
Sensitivity
3 Male genomic DNA samples in 0.1 ng/µL,
0.05 ng/µL. 0.0125 ng/µL, 0.00625 ng/µL, and
0.0031 ng/µL
SeraCare collection
Accuracy
3 Male blood samples on FTA card
IBB collection
3 Male buccal samples on FTA card
Internal collection
0.1 ng/µL 007 + Hematin at 25, 160, 250 µM
Internal
Inhibition
0.1 ng/µL 007 + Hematin at 30, 260, 310 µM
0.1 ng/µL 007 Pristine
Instruments
Table 8 Instruments used in all studies
Instruments†‡
Part number
Serial number
Veriti® 96-Well Thermal Cycler
4375786
299020040
Veriti® 96-Well Thermal Cycler
4375786
299020002
Gold Plated 96-Well GeneAmp® PCR System 9700
4314878
805S8100303
Gold Plated 96-Well GeneAmp® PCR System 9700
4314878
805S9100817
3130xl
1401-003
3130xl Genetic Analyzer
† To ensure correct temperature ramping, 9600 emulation mode was used on both thermal cycler platforms for
all studies.
‡ To minimize variability, the study used one ABI PRISM® 3130xl Genetic Analyzer according to the AmpFlSTR®
PCR Amplification Kit user guides.
18
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Capillary
electrophoresis
consumables
Table 9 Electrophoresis consumables used in all studies
Consumables†
Part number
Lot number
DS-33 Matrix Standard Kit (G5)
4345833
1007064
GS600 LIZ® Size Standard
4408399
1008006
HiDi™
4311320
1008015
POP-4 Polymer™ for 3130/3130xl
4352757
1008085
36 cm Capillary Array for 3130/3130xl
4315930
30E09045
Formamide
† AmpFlSTR® kits used the following reagents per well; 8.5 µL Hi-Di™ formamide, 0.5 µL GS600 LIZ Size
Standard, and 1.0 µL amplicon.
Data analysis
Sizing and
genotyping
The validation study used GeneMapper® ID-X v1.2 software to analyze run files
generated by the Data Collection Software v3.0. All the data were analyzed with the
same Analysis Method settings using peak amplitude threshold at 50 RFU. Allele calls,
peak heights, and base pair sizes were determined using the appropriate AmpFlSTR®
PCR Amplification Kit panel, bin set, and stutter file.
Statistical analysis
and calculations
Data generated by each instrument was compared using the parameters detailed
below. Statistical analysis consisted of t-tests and paired t-tests using Minitab®
statistical analysis software v15 with 95% confidence interval.
Genotype concordance and allele dropout
Prior to the validation study, profiles of the DNA samples were determined using each
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 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. The percent of the allele drop out was calculated using 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.
Intra-locus balance (heterozygote peak height ratio)
Within a heterozygous locus, the lower peak height of the two alleles was divided by
the 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 highest
average peak height, then multiplied by 100.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
19
Results
Stutter ratio
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 from
GeneMapper® ID-X v1.2 and run through a Microsoft Excel® Macro program
developed internally. The identified stutter ratios were compared with kit stutter ratio
setting in GeneMapper® ID-X v1.2 software. A few sample plots were selected for
manually screening for stutter ratios.
Dye artifacts and negative control data
All samples tested in the reproducibility 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
marker reading region of each kit.
Results
Reproducibility
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® PCR Amplification 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 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 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 replicates
run on both the Veriti® and GeneAmp® 9700 thermal cyclers. All AmpFlSTR® kits
demonstrated no statistically significant difference between the two platforms
The average peak height values generated using the Identifiler® Plus, Identifiler®
Direct, NGM™, and NGM SElect™ Kits demonstrated <10% difference between the
thermal cyclers. This variation is consistent with the observed reaction-to-reaction
20
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
variability of the GeneAmp® 9700 thermal cyclers. The injection-to-injection variation
on the ABI PRISM® 3130xl Genetic Analyzer might also contribute to the observed
differences. The performance of the two thermal cycler platforms were consistently
similar with the kit positive control DNA.
Figure 7 Average peak height values of kit positive control DNA on the thermal cycler
instruments. Error bars indicate the standard deviation observed among instrument and sample
replicates.
Intra-locus balance (heterozygote peak height ratio)
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-locus balance
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% and were not
statistically significant.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
21
Results
Figure 8 Average intra-locus balance of kit positive control DNA on the thermal cycler
instruments. Error bars indicate the standard deviation observed among instrument and sample
replicates.
Intra-color balance
The intra-color balance was determined across the positive control DNA profiles for
each of the AmpFlSTR® kits tested. Figure 9 compares the average intra-color balance
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.
22
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Figure 9 Average intra-color balance of kit positive control DNA on the thermal cycler
instruments. Error bars indicate the standard deviation observed among instrument and sample
replicates.
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 three male DNA
samples with different concentrations when used with Identifiler® Plus, NGM™, and
NGM SElect™ Kits. All results were 100% concordant with the previously 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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
23
Results
Table 10 Allele dropout percentages and overall average peak heights by thermal cycler,
AmpFlSTR® PCR Amplification Kit, and DNA input amount
Kit
Identifiler® Plus
NGM SElect™
NGM™
DNA input
(ng)
Allele drop-out
percentage
Average peak height
(RFU)
GeneAmp®
9700
Veriti®
0.2 mL
GeneAmp®
9700
Veriti®
0.2 mL
0.031
26.9
27.1
102
105
0.062
4.1
5.1
145
149
0.125
0.0
0.0
353
357
0.5
0.0
0.0
1,377
1,367
1.0
0.0
0.0
2,175
2,230
0.031
48.5
43.4
57
61
0.062
13.4
9.3
108
117
0.125
0.3
0.3
214
239
0.5
0.0
0.0
911
1,000
1.0
0.0
0.0
1,721
1,814
0.031
27.4
24.0
102
101
0.062
2.6
3.6
162
152
0.125
0.2
0.0
370
357
0.5
0.0
0.0
1,420
1,311
1.0
0.0
0.0
2,325
2,254
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 generally less than 10% with a maximum 19% difference observed
with the NGM SElect™ Kit and 0.5 ng DNA input. These differences 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 at each DNA input amount. The
orange and green boxes correspond to the two GeneAmp® 9700 thermal cyclers and
Veriti® 0.2 mL thermal cyclers respectively. The black dots, generally located in the
center of the box plots, represent the peak height mean. The black asterisks are the
calculated outliers.
24
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Figure 10 Average peak heights for the AmpFlSTR® Identifiler® Plus PCR Amplification Kit.
Figure 11 Average peak heights for the AmpFlSTR® NGM™ PCR Amplification Kit
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
25
Results
Figure 12 Average peak heights for the AmpFlSTR® NGM SElect™ PCR Amplification Kit.
Intra-Locus Balance (Heterozygote peak height ratio)
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 between the
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.
26
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Table 11 Comparison of average intra-locus balance between Veriti® and 9700 for male DNA
samples with different input amounts
% Intra-locus balance
Kit
DNA input (ng)
Identifiler® Plus
NGM™
NGM
SElect™
GeneAmp® 9700
Veriti® 0.2 mL
0.031
69.2
71.8
0.062
70.4
71.1
0.125
74.7
75.6
0.5
85.3
86.1
1.0
89.5
89.6
0.031
69.8
70.4
0.062
69.4
69.0
0.125
74.6
76.5
0.5
86.5
86.0
1.0
89.2
89.2
0.031
73.1
72.8
0.062
70.6
69.4
0.125
73.8
71.5
0.5
85.6
85.5
1.0
88.4
88.5
Intra-color balance
The intra-color balance was determined across all male DNA samples for each of the
AmpFlSTR® kits tested. Table 12 compares the average intra-color balance between the
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 were not
calculated. For all kits, the average peak height ratios were >50%. The performance
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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
27
Results
Table 12 Comparison of average intra-color balance between Veriti® and 9700 for male DNA
samples with different input amounts
% Intra-color balance
Kit
Identifiler® Plus
Dye
B
G
R
Y
NGM™
B
G
R
Y
28
DNA input
(ng)
GeneAmp®
9700
Veriti® 0.2 mL
0.125
61.5
60.9
0.5
73.6
74.0
1.0
72.3
73.7
0.125
58.1
61.6
0.5
69.7
70.9
1.0
74.4
72.2
0.125
68.7
70.4
0.5
76.0
79.9
1.0
82.3
83.2
0.125
62.2
64.5
0.5
67.7
75.8
1.0
75.2
74.0
0.125
66.4
59.3
0.5
79.2
81.7
1.0
86.4
86.1
0.125
56.6
63.7
0.5
65.9
65.7
1.0
68.5
66.5
0.125
66.5
66.9
0.5
76.5
79.6
1.0
76.5
78.0
0.125
59.8
70.1
0.5
69.6
66.2
1.0
73.2
73.2
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
% Intra-color balance
Kit
NGM SElect™
Dye
B
G
R
Y
Accuracy
DNA input
(ng)
GeneAmp®
9700
Veriti® 0.2 mL
0.125
62.0
54.9
0.5
80.4
71.8
1.0
85.6
82.0
0.125
68.3
63.5
0.5
79.1
84.1
1.0
86.6
84.5
0.125
60.5
56.9
0.5
73.9
73.5
1.0
72.6
72.5
0.125
63.1
59.1
0.5
74.6
75.0
1.0
76.8
76.4
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 blood and buccal
samples when used with the AmpFlSTR® Identifiler® Direct PCR Amplification Kit.
All results were 100% concordant with the previously established genotyping results.
Average peak heights
The average peak heights were calculated for blood and buccal samples with
Identifiler® 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 Veriti®
0.2 mL thermal cyclers respectively. The black dots located in the center of the box
plots 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 than
the 9700 across all markers with similar amounts of variability. The maximum percent
difference 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. These
differences were within normal run-to-run variability of 9700 thermal cycler and ABI
PRISM® 3130xl Genetic Analyzer.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
29
Results
Figure 13 Average peak height values of blood and buccal samples on the thermal cycler
instruments in each dye channel.
Intra-Locus Balance (Heterozygote peak height ratio)
The Intra-locus balance were calculated for blood and buccal samples with Identifiler®
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® 0.2 mL 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 difference
in mean value and amounts of variability. The differences between the 9700 and Veriti®
thermal cycler in average intra-locus balance were less than 3%.
30
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Figure 14 The intra-locus balance of blood and buccal samples on the thermal cycler
instruments in each marker.
Intra-color balance
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 Veriti® 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 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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
31
Results
Figure 15 The intra-color balance of blood and buccal samples on the thermal cycler
instruments in each dye channel.
Inhibition
Allele dropout
Allele drop outs were observed in the 007 samples with high and medium
concentrations 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 9700
instruments
Table 13 Allele drop out percentage by thermal cycler, AmpFlSTR® PCR Amplification Kit, and
hematin concentrations
Kit
Thermal cycler
% High
hematin
% Medium
hematin
% Low
hematin
9700
61
1
0
Veriti®
57
1
0
9700
20
0
0
Veriti®
6
0
0
9700
0
0
0
Veriti®
0
0
0
Identifiler® Plus†
NGM™‡
NGM SElect™§
† Out of 232 total allele calls
‡ Out of 256 total allele calls
§ Out of 272 total allele calls
32
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Average remain peak heights
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 peak height of each loci was calculated as the percent ratio of the peak height
between the 007 DNA 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 were illustrated in different rows. Data from 9700 and Veriti®
platforms were presented in black and red colors respectively. There was no significant
difference between the two platforms.
Figure 16 Average remain peak heights of 007 DNA with different concentration of hematin on
the Veriti® and 9700 thermal cycler instruments for each AmpFlSTR kit tested
Intra-locus balance (Heterozygote peak height ratio)
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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
33
Results
Figure 17 Average intra-locus balance of 007 DNA with different concentration of hematin on
the Veriti® and 9700 thermal cycler instruments for each AmpFlSTR kit tested
Intra-color balance
The intra-color balance was determined for pristine 007 and 007 with different
concentrations of hematin for each of the AmpFlSTR® kits tested. Figure 18 compares
the average intra-color balance between the Veriti® and GeneAmp® 9700 thermal
cyclers for each kit (in columns) and each inhibitor concentration (in rows). The data
from Identifiler® Plus samples with high hematin concentration were not plotted due
to the significant allele drop out. There was no significant difference between the two
platforms.
34
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Figure 18 Average intra-color balance of 007 DNA with different concentration of hematin on
the Veriti® and 9700 thermal cycler instruments for each AmpFlSTR kit tested
Discussion
Genotype
concordance
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 out
Allele drop outs were observed on both Veriti® and 9700 thermal cyclers in the
0.031 ng and 0.062 ng input DNA samples with all the AmpFlSTR® kits tested. Minor
drop outs 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 PCR inhibitor.
The Identifiler® Plus Kit demonstrated the most allele drop out rate with both 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.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
35
Discussion
Average peak
height
The average peak heights generated with all the AmpFlSTR® kits tested were
comparable between the Veriti® and 9700 thermal cyclers. In average, the peak height
difference between the two platforms was 5% to 10% with the reproducibility and
sensitivity samples, and 10% to 15% with the direct amplification samples. The actual
difference between individual data points varied depending on the kit and sample
used. For the Identifiler® Direct samples, the Veriti® thermal cyclers intended to
generate stronger signals in all markers. For the Identifiler® Plus, NGM™, and NGM
SElect™ samples, the results were mixed. The Veriti® instruments provided higher
signals in some markers but lower in others.
When inhibitor presented in the samples, the average remain peak heights was
calculated as the ratio to the pristine DNA. The results were also comparable between
the Veriti® and 9700 thermal cyclers with Identifiler® Plus, NGM™, and NGM SElect™
kits. The average difference was 5% to 15% depending on the kit. On both platforms,
the Identifiler® Plus Kit was least resistant to the inhibition. With high concentration of
inhibitor, 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
within the normal instrument-to-instrument and run-to-run variability of 9700 thermal
cycler and ABI PRISM® 3130xl Genetic Analyzer.
Intra-locus balance
The intra-locus peak balance generated with all the AmpFlSTR® kits tested were
comparable between the Veriti® and 9700 thermal cyclers. These heterozygote peak
height ratios were >85% for all kits with the reproducibility samples and sensitivity
samples 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 average
differences 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, and
were 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 kits tested.
Intra-color balance
The intra-color peak balance generated with all the AmpFlSTR® kits tested were
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 and
Veriti® thermal cyclers showed no significant difference in mean value and amounts of
variability.
Stutter ratio
36
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 GeneMapper®
ID-X v1.2 software for all AmpFlSTR® PCR Amplification Kits tested.
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
Dye artifact
No reproducible artifacts other than those published in the AmpFlSTR kit user
manuals were produced by either the GeneAmp® 9700 or the Veriti® thermal cyclers
with 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.
Negative control
baseline
No DNA contamination was observed in any negative control amplified throughout
the 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.
Conclusions
The results of this validation study demonstrated that the Veriti® thermal cycler was
comparable to the validated 9700 thermal cycler in assay performance of AmpFlSTR
NGM™, 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 in
GeneMapper® ID-X v1.2 for each kit. No new artifact peaks, uneven or fluctuate
baseline 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 lower
allele 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 mL
block format could generate high quality AmpFlSTR kit data. The data generated by
Veriti® thermal cycler were either statistically similar or relatively comparable to the
data generated by the 9700 platform. Thus, the Veriti® thermal cycler with 0.2 mL
block format exhibits reliable performance for human identification applications.
References
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).
AmpFlSTR® NGM SElect™ PCR Amplification Kit User Guide (Pub. no. 4458841).
Applied Biosystems® Veriti® Thermal Cycler User Guide (Pub. no. 4375799).
Veriti® 96-Well Thermal Cycler AmpFlSTR® Kit Validation
37
The information in this guide is subject to change without notice.
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5 February 2015