Pharmacogenomics

Validation of Nala PGx Core® on CFX Opus 96 Real-Time PCR (RUO)
and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems

Caroline Mahendra, Nathaniel Ryan Sanjaya, Michely Arista Tenardi, Shan Mi LooNalagenetics Pte LtdAbstract

The clinical utility of pharmacogenomics (PGx) has been gaining momentum, due to findings that up to 70% ofadverse drug reactions (ADRs) are associated with genetic variations. Nala PGx Core® is a comprehensive
multi-gene panel employing qPCR-based methodology. This panel encompasses 18 genetic variants and2 CYP2D6 Copy Number markers, spanning four pharmacogenes: CYP2C9, CYP2C19, CYP2D6, and SLCO1B1.
An evaluation conducted across diverse ethnic groups in Singapore and Indonesia, utilizing the Bio-Rad
CFX96 TouchTM Real-Time PCR System registered for IVD use, demonstrated robust and accurate genotyping
results. With the introduction of the next-generation real-time PCR technology by Bio-Rad, here we report resultsfrom a validation study performed to assess the suitability of the qPCR platform for running Nala PGxCore® on both the CFX 96 Real-Time PCR (RUO) and CFX 96 Dx Real-Time PCR Systems.
Introduction
A validation study was performed to expand the utility of NalaPGx Core® for use with specific qPCR platforms, as each qPCR
platform may differ in its Cq range and Last Cycle Relative
Fluorescence Unit (RFU) measurements. Therefore the purpose ofthis study is to determine the acceptable settings and thresholds
for genotyping specimens with Nala PGx Core® on Bio-Rad CFX
Opus 96 Real-Time PCR systems (RUO and Dx) and evaluate
whether these conditions are similar to Nala PGx Core®’s Minimum
Viable Product (MVP) platform, the Bio-Rad CFX96 Touch™
Real-Time PCR System registered for IVD use. The scope ofstudy includes a determination of the Positive Control acceptance
ranges and the genotyping thresholds specifically for CFX Opus 96
Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time
PCR Systems.

NA18971, NA19143, NA19152, NA19153, NA19393, NA21114,
HG00140, HG01094, HG01845, NA11892, and NA11931. 9
HapMap samples were used for genotyping threshold validation
on Bio-Rad CFX Opus 96 (RUO and Dx): HG00366, HG00463,
HG01097, HG01883, HG02029, HG03686, HG03854, HG04210,and NA11881.

Materials and Method

qPCR reactions in this study were carried out using reagents
provided in Nala PGx Core®. The preparation procedure, platelayout, and qPCR reaction conditions are all provided in theinstructions for use (IFU) of the kit for the Nala PGx Core®.

DNA Samples

This study utilized a total of 39 DNA samples acquired from the HapMap
Project at the Coriell Institute of Medical Research. 30 HapMap
samples were used for the verification runs and to generate thegenotyping threshold on the CFX Opus 96 Real-Time PCR (RUO)
and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems: HG00111,
HG00358, HG01085, NA11992, NA12003, NA18861, NA06989,
NA07357, NA12006, NA12155, NA12762, NA12872, NA18526,
NA18552, NA18564, NA18592, NA18608, NA18959, NA18961,

The positive controls used in this study, specifically the Single
Nucleotide Polymorphism Positive Control (SNP PC) and the Copy
Number Variant Positive Control (CNV PC), are available in the NalaPGx Core® kit. Nuclease-free water is used as the no-templatecontrol (NTC).
DNA samples were diluted with Tris-EDTA buffer pH 8.0 to 2 ng/uL
before use as template for qPCR reactions.
qPCR Reagents

Genotype calls and CNV calculations in this study were performedon Nala CDS Core®, a companion software used in tandem withNala PGx Core® which provides the genotyping thresholds andCNV calculation methods.

Validation of Nala PGx Core® on CFX Opus 96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems

Results and Discussion
CFX Opus 96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD)
Real-Time PCR Systems and the Bio-Rad CFX96 TouchTM
Real-Time PCR System registered for IVD use all employ the same
method of detection. Thus, the experiments of this study aimed
to validate that the settings and thresholds used on Bio-Rad
CFX96 TouchTM Real-Time PCR System registered for IVD use
would produce reliable results if implemented on the CFX Opus 96
Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time
PCR Systems. As such, the settings and thresholds of the Bio-Rad
CFX96 TouchTM Real-Time PCR System registered for IVD use
were analyzed for application to the CFX Opus 96 Real-Time PCR(RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems
to determine whether adjustments were needed. The FAM, HEX,and Cy5 detection channels are used for genotyping in Nala PGx
Core®, and each channel requires its own specific settings andthresholds. The FAM channel corresponds to wild-type genotype
detection, while the HEX and Cy5 channels correspond to mutant
genotype detection.

Positive Control (PC) Settings and Acceptance Criteria on Opus

Six plates of PC, each containing 3 runs, were run on the CFX
Opus 96 Real-Time PCR (RUO) System using the Nala PGx Core®
following the instructions provided here and were then analyzed
with Baseline Threshold settings from the Bio-Rad CFX96 TouchTM
Real-Time PCR System registered for IVD use: Single Threshold= 300.00, Begin = 10, End = 20. Results showed that the SNP9
assay amplification values detected in the Cy5 channel were too
low to cross the baseline threshold and generate a Cq value.
Therefore, the baseline threshold for SNP9 on Cy5 channel wasadjusted so that Cq values can be generated for testing purposes.
The preliminary baseline threshold was calculated at 10 times ofthe standard deviation for the NTC RFU obtained from the SNP9
assay across all plates. The run data from the 6 plates of PC werethen reanalyzed using the adjusted settings and were able togenerate Cq values (Figure 1), confirming that the Cy5 baseline
threshold setting is suitable for use. The runs were then reanalyzed
again on all assays and channels with the newly adjusted
settings, and were able to generate Cq values that passed the PC
acceptance criteria (Figure 2).

Fig. 1. Amplification plots for the Cy5 channel of the 6 plates containing the PC run on the Bio-Rad CFX Opus 96 Real-Time PCR (RUO) system with theadjusted baseline threshold.
Resulting run files from the Cy5 channel from the 6 plates of PC run on CFX Opus 96 Real-Time PCR (RUO) were analyzed with the adjusted baseline thresholdsettings (A – F) and were able to generate Cq values. This finding shows that the adjusted setting is suitable for use on CFX Opus 96 Real-Time PCR (RUO).

Validation of Nala PGx Core® on CFX Opus 96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems

Fig. 2. Cq values of the 6 plates containing the Positive Control (PC) run on CFX Opus 96 Real-Time PCR (RUO) system on FAM, HEX, and Cy5 channels obtained
after analysis using the adjusted baseline threshold settings.
The 6 plates containing the PC that were run on CFX Opus 96 Real-Time PCR (RUO) system were reanalyzed for all assays and channels (A, FAM channel; B, HEX channel;
C, Cy5 channel) using the adjusted baseline threshold settings. All assays across all channels yielded Cq values that were within the ranges of the PC acceptance criteria (redline), showing that the adjusted baseline threshold setting is suitable for use with the CFX Opus 96 Real-Time PCR (RUO) system.

Validation of Nala PGx Core® on CFX Opus 96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems

One additional plate of PC was run on Bio-Rad CFX Opus 96
Dx (IVD) Real-Time PCR System and analyzed with the adjusted
settings, and was able to pass the acceptance criteria as well
(Figure 3).

Fig. 3. Cq values of the validation Positive Control (PC) plate run on CFX Opus 96 Dx (IVD) Real-Time PCR System for the FAM, HEX, and Cy5 channels following
analysis using the adjusted baseline threshold settings.
The plate of PC run on Bio-Rad CFX Opus 96 Dx (IVD) Real-Time PCR System was also analyzed for all assays and channels (A, FAM channel; B, HEX channel; C, Cy5channel) using the adjusted baseline threshold settings. All assays across all channels yielded Cq values that were within the ranges of the PC acceptance criteria (red line),showing that the adjusted baseline threshold setting is also suitable for use with Bio-Rad CFX Opus 96 Dx (IVD) Real-Time PCR System.

Validation of Nala PGx Core® on CFX Opus 96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems

These results showed that the adjusted settings are suitable foruse on both the CFX Opus 96 Real-Time PCR (RUO) and the CFX
Opus 96 Dx (IVD) Real-Time PCR Systems, as all PC runs wereable to generate Cq values that passed the acceptance criteria.
Thus, the same PC acceptance criteria validated for use of theNala PGx Core® on the Bio-Rad CFX96 TouchTM Real-Time PCR
System registered for IVD use is verified for use on CFX Opus 96
Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time
PCR Systems as well.
Nala PGx Core® Genotyping Thresholds on CFX Opus 96 (RUO and Dx)

30 HapMap samples of varying genotype calls in all Nala PGx
Core® assays were run on both Bio-Rad CFX Opus 96 RUO andDx. The runs were analyzed using baseline settings generated inthe previous section, and genotypes were called using the Bio-Rad
CFX96 TouchTM Real-Time PCR System registered for IVD use thatis available for use with Nala CDS Core®. Genotype call results
showed that all assays were called with concordance rate of >95%
except for SNP1 assay (Table 1). Additionally, CNV calculations
performed in Nala CDS Core® using the settings of the Bio-Rad
CFX96 TouchTM Real-Time PCR System registered for IVD use werealso fully concordant. Hence, only SNP1 genotyping thresholdrequired revision to be suitable for use with Bio-Rad CFX Opus 96(RUO and Dx); genotyping thresholds for all other assays remain
the same as Bio-Rad CFX96 TouchTM Real-Time PCR System
registered for IVD use.

The last cycle RFU values of each sample in SNP1 assay were thengraphed on a scatterplot to confirm that the candidate thresholds
in both FAM and HEX channels were lower than the lowest HET
datapoint, and were able to correctly call all the corresponding
samples, while remaining higher than the highest suppressed
genotype datapoints in order to avoid mistaken calls of those
suppressed genotypes (Figure 4). The thresholds set for both FAM
and HEX channels fulfill these requirements and were then usedas the final genotyping threshold for SNP1 assay on Bio-Rad CFXOpus 96 (RUO and Dx).
After adjusting the genotyping threshold for SNP 1 assay, theconcordance rate became 100% as all samples were called
correctly to their actual genotype. All other assays will use thesame genotyping threshold as Bio-Rad CFX96 Touch IVD as all 30
HapMap samples were called with >95% concordance in thoseassays (Table 1).

Table 1. Genotype Concordance Rate for Calls Determined Using theGenotyping Threshold and Settings of the Bio-Rad CFX96 TouchTM
Real-Time PCR System registered for IVD use. HapMap samples were runon Bio-Rad CFX Opus 96 (RUO and Dx) and their genotypes were called withthe genotyping threshold of the Bio-Rad CFX96 TouchTM Real-Time PCR System
registered for IVD use. The percentage in red represents assays that are called withconcordance rate of <95%.

To adjust the genotyping threshold for SNP1, the last cycle RFU values
for each genotype, homozygous wild-type (WT), heterozygous (HET),and homozygous mutant (MT), in the SNP1 assay across all 30
HapMap samples were compiled and analyzed using our proprietarystatistical method. The results provide a candidate threshold todetermine amplification detected in each channel, and thus can beused to call genotypes.

Fig. 4. Scatterplots of the last cycle Relative Fluorescent Unit (RFU) ofHapMap samples using in the SNP1 assay.
Last cycle RFU values of each sample in the SNP1 assay were plotted as ascatterplot (WT samples in blue, HET in orange, MT in gray) and were comparedto the adjusted SNP1 genotyping threshold (yellow line) to confirm the thresholdsetting provided correct calls of the samples. A, scatterplot confirms that the SNP1
genotyping threshold on FAM channel was able to correctly call samples, since it
is lower than the lowest HET datapoint and higher than the highest MT datapoint;B, similarly, the SNP1 genotyping threshold on HEX channel was also able tocorrectly call samples, since it is lower than the lowest HET datapoint and higher
than the highest WT datapoint.
WT - wild type; HET - heterozygous; MT - homozygous mutant.

Validation of Nala PGx Core® on CFX Opus 96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems

Validating Genotyping Threshold for CFX Opus 96 Real-Time PCR (RUO) andthe CFX Opus 96 Dx (IVD) Real-Time PCR Systems

A total of 9 HapMap samples were run, analyzed, and genotyped
using the final baseline settings and genotyping thresholds
discussed in sections above. The resulting analysis showed thatall 9 samples were called correctly across all assays, and CNV
calculations were concordant (Table 2).
Table 2. Validation of the genotyping concordance rate for samples calledusing the genotyping threshold of the CFX Opus 96 Real-Time PCR (RUO)
and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems. Nine HapMap
samples were run, analyzed and genotyped using the CFX Opus 96 Real-Time PCR(RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems with newly adjusted
thresholds, resulting in correct calls for all 9 samples across all assays.

with their true genotype calls and CNV. This finding validates thatNala PGx Core® is suitable for use on CFX Opus 96 Real-Time PCR(RUO) and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems.
References
Kothary AS et al. (2021). Validation of a multi-gene qPCR-based pharmacogenomics
panel across major ethnic groups in Singapore and Indonesia. Future Medicine
Pharmacogenetics Vol. 22, No. 16. https://www.futuremedicine.com/doi/10.2217/pgs-2021-0071, assessed September 8, 2023.

Visit bio-rad.com/CFXOpus for more information on Real-TimePCR Instruments and www.nalagenetics.com for more informationon Nala PGx Core®.
BIO-RAD and CFX96 Touch are the trademarks of Bio-Rad Laboratories, Inc. in certain
jurisdictions. All trademarks used herein are the property of their respective owner.
© 2023 Bio-Rad Laboratories, Inc.

Conclusion
Nala PGx Core® demonstrated similar performance on CFX Opus
96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD) Real-Time
PCR Systems as the Bio-Rad CFX96 TouchTM Real-Time PCR
System registered for IVD use. Nala PGx Core® exhibited similar
performance on both the CFX Opus 96 Real-Time PCR (RUO)
and the CFX Opus 96 Dx (IVD) Real-Time PCR Systems whencompared to the Bio-Rad CFX96 TouchTM Real-Time PCR System
registered for IVD use. Only 2 adjustments were made to the initial
settings and thresholds for the CFX96 TouchTM Real-Time PCR
System registered for IVD use: the Cy5 baseline settings for running
the PC and the SNP1 threshold for genotype call samples. The restof the settings and thresholds in CFX96 TouchTM Real-Time PCR
System registered for IVD use were already suitable and applicableto CFX Opus 96 Real-Time PCR (RUO) and the CFX Opus 96 Dx (IVD)
Real-Time PCR Systems. With the new settings and thresholds,
reference samples with known genotype calls across all assays andCYP2D6 CNV values were analyzed, resulting in 100% concordanceBio-RadLaboratories, Inc.
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