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2N Sample Identification forVeriDose CNV Analysis®

ABSTRACTOne of the main challenges in analyzing genes with copy numbervariations is finding a suitable “control” sample to be used for datanormalization. When running the Agena PGx Reporter, this 2N
sample is used to identify the 2N cluster in each assay and is what
all other samples in that run are compared to. Therefore, the control
cannot contain any gene deletions, duplications or hybrid alleles.
The sample can have SNPs in CYP2D6 that aren’t associated withCNVs or hybrids, but ideally the sample will be *1/*1.
The Agena VeriDose CNV panel contains 22 assays, targeting sevenregions of the CYP2D6 gene. Because the assays span the entire
gene, we are able to detect not only copy number variations, butalso hybrid alleles, where a portion of the CYPD6 gene is swapped
for the highly homologous CYP2D7 gene. These hybrid alleles areimportant since their presence confers the same functional outcome
as a gene deletion.

“Because each of these22 assays is unique andhas its own performance,the 2N control sample
needs to be carefully
chosen to be as“normal” as possible.”

Because each of these 22 assays is unique and has its own
performance, the 2N control sample needs to be carefully chosen
to be as “normal” as possible. Agena has identified a commerciallyavailable gDNA sample (Roche #11691112001) that clusters as closely
as possible with the 2N samples that were used during panel validation. If no other 2N samples are available in yourlab, this can be used as a control sample. However, we have seen some instances were DNA extraction kit can impactclustering and can cause the Roche gDNA sample to fall outside of the 2N group.
Therefore, it is Agena’s recommendation that every lab running clinical samples identify an in-house 2N control whose
performance most closely mimics that of the 2N samples in each assay. Following are two methods that can be used toidentify a suitable 2N control.

1The MassARRAY System is for research use only. Not for use in diagnostic procedures.

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STEPS
FORIDENTIFYINGSUITABLE2NSAMPLE
Stepsforidentifyinga AsuitablesampleStepsforidentifyinga suitable2N2N
sampleMethod 1

MethodMethod1 is1recommended for customers that have a large sample set (>75 samples) that allows for clearThis
methodThismethodis recommendedcustomers
thathavea largesample(>75samples)that
allowsThis
method
is1N,
recommended
forforcustomers
thathavea large
samplesetset(>75samples)that
allowsidentificationof
2N, and 3N+ clusters.
clear
identification
and3N+clusters.
forforclear
identification
of of
1N,1N,2N,2N,
and
3N+clusters.
1 Run samples on VeriDose CNV and view the Call Cluster Plots in Height View in Typer Analyzer. Identify the 2Nclusterin the sample
set(this will
generally
bewherethemajorityofthesamples
cluster
andfall close toRun
samples
VeriDose
CNV
andview
CallCluster
Plotsin HeightView
in Typer1. 1.Run
samplesonon
VeriDose
CNV
and
view
thetheCall
Cluster
Plots
in Height
View
in should
Typerthe45° angle inmost assays),see
Figure in
1. the sample set (this will generally be where the majority ofAnalyzer.
Identify
cluster
Analyzer.
Identify
thethe2N2Nclusterin thesample set (this will generally be where the majority ofsamples
clusterandshould
closeangle
in mostassays).
thethesamplesclusterandshouldfallfall
closeto tothethe45°45°
angle
in mostassays).
Figure 1: Samples needed to identify 2N cluster

CYP2D6_5Prime_Exon1-2

High Mass Height

High Mass HeightCYP2D6_Exon1-2

Low Mass Height

Sufficient
samples
to identify
identify
2N cluster
cluster(88samples)
Sufficient
samples
to identify
2N 2N
cluster(88 (88samples)
Sufficient
samples
tosamples)

Low Mass Height

Insufficient
samplesto identify
2N
clustersamples)
Insufficient
samples
to2N(28samples)
Insufficient
samplestoidentify
identify
2Nclustercluster(28(28samples)2 2.
Identify
aasample
that
falls
as
close
asclosepossible
tothe middle
ofmiddlethe2N ofcluster.

Identifya samplethatfalls
possible
middleofcluster.
2.
Identify
sample
that
falls
as as
close
as as
possibleto tothethe
thethe2N2Ncluster.
Checkperformancethatsamplein otherassaysensuresamplefalls
in thegroup
3.3 3.Check
performanceof ofthatsamplein otherassays
to toensure
thethesamplefalls
in the2N2N
groupof that sample in other assays to ensure the sample falls in the 2N group in all assays. If a Check performance
inallassays.
Ifasample
hasa
hybridallele,
it
can
look
like
a2N
in
some
assays,
but
will
fall
insample
all assays.
If a sample
haslooka hybrid
allele,it canlookbut
like
2Ninineither
somewill infallotherhas a hybrid
allele, it canlike a 2N
in someassays,willa falltheassays,
1N or 3Nbutclusterin eitherorclusterin otherassays.
Below
is an
examplea samplethatlikeinassays.
eitherthethe1Norexample
3N3N
cluster
in otherassays.
Below
is an
exampleofa actually
sample
that
looks
Below
is 1N
anof a sample
that
looks
like
2N
in CYP2D6_Exon1-1
butof
contains
alooks*68likehybrid2N
in CYP2D6_Exon1-1
containsa(see*68
hybridalleleandfalls
in thecluster2Nin andCYP2D6_Exon1-1
butbutactuallycontainsa *68
hybridandso sofalls
in the1N1Nclusteralleleso falls in the 1N clusterinactually
CYP2D6_Intron4-1
Figure2).allele
in CYP2D6_Intron4-1.
in CYP2D6_Intron4-1.

The MassARRAY System is for research use only. Not for use in diagnostic procedures.

2

WHITE PAPER- Method 1 (continued)
Figure 2: Same sample showing different clustering in two assays
CYP2D6_Intron4-1

High Mass Height

High Mass HeightCYP2D6_Exon1-1

Low Mass Height

Low Mass Height

Same sample showing different clustering in two assays

Oncea suitablesampleis identified,PGxreportusing theselected
44. Oncea suitablesampleis identified,
run therun
PGxthereportusing
the selected
sampleas thesample
CYP2D6as
2N thecontrol. It


isCYP2D6
recommended
that a previously
characterizedthat
sampleset be usedcharacterized
during the identificationa control
sample2N control.
It is recommended
a previously
sample ofset
be usedsothat theof the Agena
reportsample
can be checked
the known
copy numbersof yourcan
samples.
duringtheresults
identificationof aPGxcontrol
so that withthe resultsof CNV
the Agena
PGx reportbeThiswillensure
the2N
thathasbeen
selected
issuitableasacontrolsample.
checked with the known CNV copy numbers of your samples. This will ensure the 2N that has

been selected is suitable as a control sample.
5 Compare the PGx Report to the expected outcomes for each sample. If all samples give the correct CNV call, this5. isCompare
PGx Reportto theshow
expectedoutcomes
fora different
each sample.
If allre-runsamples
give thea suitable the2N control.
If the results
discrepancies,
selectsample andthe report.
RepeatcorrectCNVcall, thisisisachieveda suitable
If the results show discrepancies, select a
until
100%
concordancewith 2N
the control.
known dataset.
different sample and re-run the report. Repeat until 100% concordance is achieved with theknown dataset.

Method 2

This method is recommended for customers that have a small data set that does not allow for
identification of the 2N cluster in the Call Cluster Plot. This method requires that the Roche gDNA,mentioned above, be run on the same plate as your samples.
1. Run samples and Roche DNA on VeriDose CNV.
3
2. Run the PGx Report with Roche DNA selected as the CYP2D6 2N control.
The MassARRAY System is for research use only. Not for use in diagnostic procedures.
3. Open the “AssaysPerSample.pdf” file that is generated in the PGx Report folder.
4. One diagram will be generated per sample, showing that samples’ performance on all assays.

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Method 2
This method is recommended for customers that have a small data set that does not allow for identification of the 2Ncluster in the Call Cluster Plot. This method requires that the Roche gDNA, mentioned above, be run on the same plate
as your samples.
1 Run samples and Roche DNA on VeriDose CNV.

2 Run the PGx Report with Roche DNA selected as the CYP2D6 2N control.
3 Open the “AssaysPerSample.pdf” file that is generated in the PGx Report folder.
4 One diagram will be generated per sample, showing that samples’ performance on all assays. Scroll through tofind a sample that falls as close to the 2N line as possible, for all assays.

5 Example suitable samples:
The MassARRAY System is for research use only. Not for use in diagnostic procedures.

4

WHITE PAPER- Method 2 (continued)Note that
not allassayswillassays
fall exactly
2N line.
assaysthe VeriDose
CNVwill consistently
Notethat
not allwillon
falltheexactly
onSomethe 2Nline.in Someassaysin panelthe VeriDose
CNV skew
towardspanel
the 3Nwill
cutoff(ex.
Intron6andIntron7
assays).
consistently skew towards the 3N cutoff (ex. Intron6 and Intron7 assays).

6. Once a suitable sample is identified, run the PGx report again, this time with the selected
sample used as the CYP2D6 2N control. It is recommended that a previously characterized
sample set be used during the identification of a control sample so that the results of theAgena PGx report can be checked with the known CNV copy numbers of your samples. Thiswill Systemensure
2N usethathasbeen
is suitable as a control sample.
The MassARRAY
is forthe
researchonly.
Not foruse inselected
diagnostic procedures.
7. Compare the PGx Report to the expected outcomes for each sample. If all samples give thecorrect CNV call, this is a suitable 2N control. If the results show discrepancies, select a5

WHITE PAPER- Method 2 (continued)
6 Once a suitable sample is identified, run the PGx report again, this time with the selected sample used as

the CYP2D6 2N control. It is recommended that a previously characterized sample set be used during theidentification of a control sample so that the results of the Agena PGx report can be checked with the known
CNV copy numbers of your samples. This will ensure the 2N that has been selected is suitable as a controlsample.

7 Compare the PGx Report to the expected outcomes for each sample. If all samples give the correct CNV call, thisis a suitable 2N control. If the results show discrepancies, select a different sample and re-run the report. Repeat
until 100% concordance is achieved with the known dataset.

OTHER CONSIDERATIONS
Unusually high number of 1N and 3N samples
If the number of samples with deletions or duplications is unexpectedly high and/or there are a large number of samples
with hybrid calls, it’s possible the 2N control that was used during reporting is not suitable. If it appears rare haplotypes
are being reported too frequently, we recommend reviewing the 2N control sample and overall data quality using thefollowing checks:Ensure that all samples show similar extension rates and peak heights in the cluster plots. For guidance, refer tothe ‘PCR inhibitors’ section.
Confirm that samples of similar concentrations have comparable peak heights. Large deviations from thisexpected pattern, due to low extension and yield, suggest a sample issue.
Inspect the calculated copy number values to ensure that they are close to integer values, rather than between
two copy number values (ex. 2.06 rather than 2.42).
Review the confidence and coefficient of variation values for the predicted copy number calls. If a sample is listed
as discordant, check cluster plots.
Confirm that samples cluster into well-defined, well-separated copy number groups in the Typer Cluster Plot.

If comparing the data generated by Agena’s VeriDose CNV panel with the Thermo Fisher Exon 9 TaqMan assay, be awarethat TaqMan is not able to detect Exon 9 hybrid alleles (*4N and *36). This results in TaqMan underestimating copy
number for samples containing hybrids.

DNA NormalizationFor optimal results, normalize DNA to the recommended 5-10 ng/uL concentration and use 2uL input for thePCR reaction.
The DNA amount in each sample, and in each technical replicate on a single plate, should be similar(i.e. 10-20 ng).

Sufficient genomic DNA input may be verified by alternate technologies (ex. Nanodrop).

Check OD260/280 and OD260/230 for ratios over 1.7. Lower ratios may indicate the presence of PCR inhibitors.

The MassARRAY System is for research use only. Not for use in diagnostic procedures.

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WHITE PAPERSample Types

Different sample types and samples prepared by different extraction methods can amplify with different
efficiencies.
Samples analyzed together on the same plate should be of the same sample type and extraction method.
If using an in house 2N control, it should be of the same sample type and extraction method as your clinicalsamples.

Note: a small number of control samples (e.g. a few Coriell samples on a 384-well plate) should not strongly impact
analysis of buccal or blood derived samples)

PCR inhibitors

If running samples extracted from blood, residual hemoglobin may cause samples to skew, resulting in copy
number being reporter as either higher or lower than a 2N control that has no hemoglobin present.
Other inhibitors may cause similar skewing.

The MassARRAY System is for research use only. Not for use in diagnostic procedures.

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Phone: +1.858.882.2800
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