Data Sheet: DNA Analysis
VeraCode® ADME Core Panel
Investigate pharmacogenetic variations associated with drug metabolism faster than ever before.
Highlights
• Standardized Content
184 biomarkers in 34 genes, covering >95% of the
PharmaADME Core list
• Rapid Genotyping
Assay 32 samples per run within 8 hours with only
2.5 hours hands-on time
• Highest Quality Data
Specific assay chemistry and integrated quality controls
enable ≥99.5% concordance and ≥99.5% reproducibility
Introduction
Understanding genetic variability associated with drug response
and disposition is a key step towards the realization of personalized
medicine. Biomarkers that indicate levels of absorption, distribution,
metabolism, and excretion (ADME) need to be analyzed and studied
for each newly developed potential pharmaceutical compound. The
quicker this analysis is completed, the sooner a compound can be
categorized as a possible drug candidate or discarded, enabling
faster, less expensive product development. To help researchers
succeed in their ADME pursuits, Illumina introduces the VeraCode
ADME Core Panel.
The VeraCode ADME Core Panel offers the most complete representation of the ADME content that matters most. Focused on the
standardized PharmADME Core list1 and leveraging the innovative
VeraCode technology2, this panel streamlines drug metabolism
biomarker analysis, allowing these studies to proceed faster than ever
before. With the VeraCode ADME Core Panel, researchers can analyze
key ADME content in as little as one day. In addition, the VeraCode
ADME Panel provides a number of internal controls to ensure precise,
verifiable results.
High-Value Content
The PharmaADME Working Group, consisting of industry and academic
experts, has developed a list of genetic content considered standard
for pharmacogenetic drug studies, including genes considered valid according to FDA guidance3. Focusing on this standardized PharmaADME
Core list enables more comprehensive coverage of the most biologically
relevant biomarkers spanning complex regions of the genome.
The optimized VeraCode ADME Core Panel covers >95% of these
carefully selected markers, including validated DNA-based biomarkers
of drug response3, genes with demonstrated association to model
drugs, and experimental biomarkers (Table 1). This core content
provides the essentials for investigating pharmacogenetic associations
to therapeutic substances. Researchers will be able to identify candi-
date genes associated with drug ADME for follow-on safety studies,
improve their understanding of genotype-specific differential drug
metabolism to analyze effects on dosing, and learn more about which
alleles may be indicative of drug response.
Precisely Assay Complex Genomic Regions
ADME-related genes are often found in complex regions of the
genome that are difficult for most genotyping assay technologies to
target precisely. To overcome this challenge, the VeraCode ADME
Core Panel is processed using a highly specific version of the
GoldenGate® assay9. To extract a particular region of interest from
the rest of the genome, a single primer extension step is conducted
with a primer optimized to avoid potentially conflicting regions of the
genome. This makes it possible to assay homologous genomic
regions that otherwise would suffer from cross reactivity with close
relatives (Figure 1). For further specificity, this is followed by an allelespecific primer extension and ligation step. The result is an assay that
provides the highest quality data (Table 2).
Quick Assay Turnaround
Within the VeraCode ADME Core Panel, complex regions are isolated
into three highly optimized reaction pools. In combination with the
initial primer extension step, this makes it possible to quickly and
precisely genotype several complex regions of the genome, including
tri-allelic SNPs, SNPs in close proximity, indels, homologous regions,
and copy number variants. One technician can easily assay up to
32 samples per run within a single work shift with less than 2.5 hours
of hands-on time (Figure 2). Up to two plates (64 samples), can be
processed simultaneously.
Table 1: ADME core genes (34 genes)
ABCB1
CYP2C19
DPYD
SLC22A1
SLC22A2
TPMT
ABCC2
CYP2C8
GSTM1
ABCG2
CYP2C9
GSTP1
SLC22A6
UGT2B15
UGT1A1
CYP1A1
CYP2D6
GSTT1
SLCO1B1
UGT2B17
CYP1A2
CYP2E1
NAT1
SLCO1B3
UGT2B7
CYP2A6
CYP3A4
NAT2
SLCO2B1
VKORC1
CYP2B6
CYP3A5
SLC15A2
SULT1A1
The essential genes that populate the VeraCode ADME Core Panel
include several with well-known associations to model drugs. For example,
CYP2D6 has been associated with the metabolism of certain antidepressants, antopsychotics, beta blockers like metoprolol, opioids like morphine,
cancer drugs like Tamoxifen and many others4–8,CYP2C19 has been
associated with the metabolism of the anti-platelet Plavix, and CYP2C9 and
VKORC1 are most well known for their association to warfarin sensitivity.
For a detailed VeraCode ADME Core SNP list please contact your account
manager or visit www.illumina.com/VeraCodeADME.
Data Sheet: DNA Analysis
Figure 1: Highly Specific Genotyping Assay
100%
97%
2C19
2C18
100%
100%
2C9
2C8
SNP
Region -specific primer
Extended product
GoldenGate oligos
GoldenGate
Genotyping Assay
CYP2C9*2
(Conventional)
CYP2C9*2
(Conventional )
CYP2C9*2
CYP2C9*2
(VeraCode
ADME Core)
(VeraCode ADME Core )
Verified
Verified
*2HETs
HETs
Intensity (B)
Intensity (B)
Verified
Verified
*2HETs
HETs
*1
Intensity (A)
Intensity (A)
The CYP450 gene family includes a tetrad of closely related genes: CYP2C18, CYP2C19, CYP2C9 and CYP2C8. Using an upfront targeting approach, a specific
gene of interest can be pulled out from the genome, avoiding other homologous regions, and can each be analyzed precisely with excellent discrimination.
Figure 2: Fast, Simplified Workflow
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32 DNA
Samples
NaOH
Targeting
Reaction
NaOH
Capture
by PMP
Oligo
Annealing
P2
Biotin
|
WashSNP locus
T
P3
Wash
-
Biotinylate genomic DNA
Pre-PCR (2 hr 15 min)
Ligation
Multiplexed
Universal PCR
P2
Hybridization
T
A
Ad
dre
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P3
Scan
Wash
Extend, ligate, elute
P1
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P2
P3
Amplify by universal PCR
Post-PCR (5 hr 15 min)
Using the VeraCode ADME Core Panel, 32 samples can be processed per run. An initial round of primer extension is used to target regions of interest into
three optimized assay pools using biotinylated primer mixes. Products are isolated onto paramagnetic particles for subsequent allele-specific extension and
ligation followed by universal PCR amplification. A single plate transfer applies amplified material to VeraCode beads for hybridization and scanning on the
BeadXpress® Reader. The total assay time is <8 hours with 2.5 hours of technical hands-on time. One technician can process up to two plates simultaneously for
64 samples.
Internal Controls
Carefully designed positive and negative internal control assays are
included in each reaction within the VeraCode ADME Core Panel
to monitor sample processing and quality control (Figure 3). These
controls aid in the identification of low DNA quality or quantity, reagent
failures, operator failures, and run performance issues.
A panel of 13 high minor allele frequency SNPs are included as a positive control in each assay pool to create a sample barcode and enable
detection of sample pipetting errors. Negative controls are included in
each reaction to ensure proper pairing and position of assay subpools.
Data Sheet: DNA Analysis
Table 2: ADME Core Panel Performance Specifications
Parameter
Performance
Specification
Locus Success
Illumina Validation
(465 samples,
3 users, 3 systems)
≥95%
≥95%
Assay Time, 32 samples
DNA to data
Hands-on time
≤8 hr
7 hr 38 min
≤2.5 hr
1 hr 37 min
Sample Throughput
1 user, 1 run, 1 plate
32
32
Integrated Software
1 user, 1 run, 2 plates
64
64
VeraScan offers integrated system software with a convenient user
interface that manages user authentication, enforces instrument
maintenance, and produces detailed sample reports. Genotype data
generated from a run is displayed in concise plots on the screen and is
automatically translated into star allele calls in .CSV and .PDF reports.
Reports can be configured to include specified genes and/or alleles
within the ADME Core Panel for more focused analysis.
Average Call Rate
≥98%
99.5%
Concordance*
≥99.5%
99.8%
Reproducibility
≥99.5%
99.8%
To ensure data integrity, the software only reports assay results
when the controls perform as expected. Data from each assay pool
is bioinformatically combined to create pharmacogenetic profiles for
each sample. Sample identity is verified by the software using the
13 positive control SNPs present in each assay. Additionally, panel
results are translated into a barcode that uniquely identifies each sample.
This barcode is included in the analysis report for added traceability.
For added convenience, VeraReport, an off-line reporting tool that
enables review of past runs and off-site analysis in other areas of the
lab, is also included.
Conclusion
* Concordance with reference data from the Infinium® assay, Genome Analyzer,
and Sanger sequencing.
In a study with 465 DNA samples, the VeraCode ADME Core Panel
produced data with a high average call rate (99.49%), excellent
reproducibility (99.77% on average), and high concordance
(99.84% overall) with whole-genome genotyping and sequencing data
for genotyping assays, and with quantitative PCR10 for CNV assays.
Figure 3: Fully Integrated Quality Control
Internally “barcoded” samples
10101100...0011
10101100...0011
10101100...0011
The VeraCode ADME Core Panel provides researchers with the most
coverage of biologically relevant pharmacogenetic information. With a
streamlined assay chemistry and precise, integrated internal controls,
researchers can quickly and reliably assess pharmacogenetic profiles
for their drug metabolism studies.
AOP4C
MTR4C
AOP4B
MTR4B
AOP4A
MTR4A
Correct pairing of oligo pools is internally controlled
MTR = Subpool specific Targeting Mix
AOP = Subpool specific Assay Oligo Pool
Each VeraCode ADME Core Panel includes internal controls to monitor
assay performance and detect potential error. Internally barcoded samples
control for correct sample position and DNA quality and quantity while
providing added traceability through a run. Negative controls monitor the
correct pairing and position of the targeting mix and assay oligos within
assay subpools.
Data Sheet: DNA Analysis
References
Ordering Information
1. www.pharmaadme.org
2. http://www.illumina.com/downloads/VeraCodeTechSpot.pdf
Product
3. H
uang SM, Goodsaid F, Rahman A, Frueh F, Lesko LJ (2006) Application of
pharmacogenomics in clinical pharmacology. Toxicol Mech Methods.16: 89–99.
VeraCode ADME Core Panel
4. http://medicine.iupui.edu/clinpharm/ddis/table.asp
Quantity
Catalog No.
32 samples
VC-901-0201
160 samples
VC-901-0200
5. G
oldstein JA (2001) Clinical relevance of genetic
polymorphisms in the human CYP2C subfamily.
Br J Clin Pharmacol. 52: 349–355.
6. B
adyal DK and Dadhich AP (2001) Cytochrome P450 and drug interactions.
Indian J Pharmacol. 33: 248–259.
7. B
ristol-Myers Squibb/Sanofi Pharmaceuticals Partnership (2009) Package
Insert, PLAVIX (clopidogrel bisulfate) tablet, film coated.
8. B
ristol-Myers Squibb (2009) Package Insert, Anticoagulant Coumadin®
Tablets (Warfarin Sodium Tablets, USP) Crystalline.
9. http://www.illumina.com/downloads/GoldenGateVeraCodeWhitePaper.pdf
10. McCarroll SA, Hadnott TN, Perry GH, Sabeti PC, Zody MC, et al. (2006)
Common deletion polymorphisms in the human genome. Nat Genet. 38: 86–92.
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Pub. No. 170-2009-009 Current as of 03 November 2010