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HORIZON DISCOVERY
Using Reference Standards to Analyse the Sensitivity and
Specificity of Sequencing Technologies and Pipelines
Hadas Amit, Vicky Spivey, Javier Armisen-Garrido, Jessie Bwanali, Jonathan Frampton, Paul Morrill, Karin Schmitt
Introduction
Next Generation Sequencing (NGS) offers significant advantages for mutation profiling, mainly the ability
to detect all mutation types, including Singly Nucleotide Polymorphisms (SNPs), Copy Number Variations
(CNVs), insertions/deletions and translocations, in many samples and many genes simultaneously. NGS
also provides a read-out of the mutation frequency. However, in order to utilize NGS in routine clinical
practice, operators are required to validate all aspects of the NGS workflow, to ensure that genetic
variations are correctly identified in diagnostic practice. A method for validation is required that goes
beyond the current use of CEPH sample cell lines. In response to the need for a better reference standard
for NGS, Horizon Diagnostics has created a range of multiplex HDx™ Reference Standards covering many
commonly assayed cancer mutations.
Development of a low frequency Multiplex Reference Standard
Building on our experience with the Multiplex Reference Standards we looked to address the need for
Reference Standards containing mutant frequencies below 1%. A multiplex was developed containing five
EGFR mutations at defined mutant allele frequencies ranging from 0.05% - 2%. Mutant allele frequencies
observed by digital PCR were all within 10% of predicted for 2%, 1% and 0.5% standards (Table 2).
Deviation is higher at the lower allele frequencies, which reflects assay specific background (e.g. EGFR
T790M) and increased variability when testing at these concentrations.
EGFR Multiplex
#1
#2
#3
#4
#5
#6
WT
Predicted mutant
allele frequency (%)
2.0%
1.0%
0.50%
0.20%
0.10%
0.05%
0%
Δ746-750
1.9
1.0
0.48
0.20
0.10
0.05
0.007
G719S
1.9
1.0
0.50
0.20
0.12
0.06
0.022
L858R
2.0
1.1
0.55
0.20
0.10
0.05
0.001
L861Q
2.0
1.0
0.52
0.21
0.12
0.06
0.003
T790M
2.0
1.0
0.53
0.24
0.15
0.09
0.032
Desired
500
500
500
5000
5000
5000
5000
Observed
555
536
525
5105
5088
5038
4970
Generation of Multiplex Reference Standards
Leveraging Horizon’s proprietary genome editing technology, we reconstituted clinically relevant cancer
genes in human cell lines. Critically, this approach enables us to define virtually every characteristic of our
Reference Standard, from the molecular constitution of the genome (desired mutational change), to the
DNA output associated with each product batch (different frequencies in the multiplex mixture) (Figure 1,
Table 1).
Observed mutant
allele frequency (%)
Total Copies
EGFR per µL
Table 2. Quantification of allele frequency in EGFR multiplexes by digital PCR. Frequency of all five EGFR mutations as well
total EGFR concentration was quantified using digital PCR.
Results confirm the suitability of this workflow for obtaining a Multiplex Reference Standard with multiple
mutant alleles at frequencies as low as 1 copy per 2000 wild type, equivalent to 1 mutant cell in 1000.
These standards offer a sustainable and highly defined source of reference material to laboratories,
proficiency schemes, and manufacturers relying on the use of NGS for clinical diagnostics.
Development of Formalin-Compromised HDx™ Reference Standards
Figure 1. Engineering cell lines using GENESIS™ technology. rAAV genomes are designed to include a mammalian selection
marker flanked by two regions of homology to the target gene. The desired mutation to be introduced is included in one of
these homology arms. When human somatic cells are infected with rAAV particles, the single stranded viral genomes direct
efficient homologous recombination leading to introduction of the mutation at the required endogenous locus. Abbreviations:
ITR, Inverted Terminal Repeat.
Verification Step
Current Assay
1. Confirm consistency of mixed ratios
Digital PCR
2. Confirm molecular weight of DNA
Gel Agarose Electrophoresis
3. Confirm absence of PCR inhibitors
qPCR
4. Confirm DNA concentration (DNA) or yield (FFPE)
Nanodrop ™ or QuantiFluor™ dsDNA System
Fixation using formalin is a critical step in the preparation of histological sections. It ensures the
preservation of tissue architecture and cell morphology by cross-linking biomolecules. If fixation is not
carried out under optimal conditions a tissue specimen can be irreversibly damaged. Methods of fixation
vary according to sample types. Longer fixation periods may cause a high degree of DNA fragmentation
and an increase in cross-links between biomolecules. This can result in reduced amplifiability of DNA thus
detrimentally affecting the patient’s ability to receive critical therapy.
We have developed a Formalin-Compromised HDx Reference Standard that will serve as a control to
address features associated with extensive exposure to formalin of DNA extracted from formalin
compromised tissues. In order to demonstrate how different levels of fragmentation can be achieved,
genomic DNA extracted from a complex cell blend having been exposed to different levels of formalin was
analysed by the Screen Tape assay and Gel electrophoresis. A High Molecular Weight (HMW) Quantitative
Multiplex HDx Reference Standards containing the same set of mutants was used as a control (Figure 3).
We are able to reproducibly generate different and specific levels of DNA fragmentation.
Figure 3. Fragmentation of Formalin Compromised DNA HDx Reference
Standard. DNA fragmentation of DNA
extracted from cell blends fixed for
different periods was examined by agarose
gel electrophoresis and Genomic DNA
Screen Tape assay.
Table 1. Reference Standard Verification Steps. Both genomic DNA and FFPE HDx™ Reference Standards are extensively
characterised. Analysis of multiple FFPE sections across a block also confirms homogeneity of the cell core.
Using these cell lines, we have created several Multiplex Reference Standards containing many commonly
assayed cancer mutations. These are generated either by blending cell line derived genomic DNA that has
been precisely quantified, or by mixing the cells themselves to produce a cell pellet that is subsequently
Formalin-Fixed, Paraffin-Embedded (FFPE). Both are extensively quality tested with the allelic frequencies
of key oncogenic mutations validated using digital PCR.
The Tru-Q HDx Reference Standard portfolio covers 40 in allelic frequencies ranging from 5% -1%. Tru-Q
HDx Reference standards at the 5% Tier cover 10 mutations, at the 2.5% Tier cover 20 mutations and the
1% Tier cover 40 mutations. These results were compared with frequencies observed by an independent
laboratory using the Ion Torrent™ PGMs with various Ampliseq™ panels (Table 2).
Allele frequencies were analyzed by digital PCR and NGS (Ion Torrent). There is concordance of mutant
calling between the digital PCR and the Ion Torrent. This data demonstrates that our FormalinCompromised DNA Reference Standard can be used to validate assay and platform performance on very
challenging samples such as those derived from DNA extracted from formalin compromised tissues.
Observed frequencies at 5% and 2.5% using the IonTorrent are consistent with digital PCR. Inability to
detect several mutations at the 1% level by the Ion Torrent highlights the platforms limit of detection.
Source
Horizon
Diagnostics
Predicted %
Partner
Horizon
Diagnostics
Observed %
Platform
N/A
Ion Torrent
QX100™ Digital
PCR System
HMW Control
DNA
Partner
Horizon
Diagnostics
Observed %
Partner
Ion Torrent
QX100™ Digital
PCR System
Ion Torrent
Medium intensity Medium intensity High intensity
Formalin
Formalin
Formalin
Exposure
Exposure
Exposure
High intensity
Formalin
Exposure
Gene
Mutation
HMW Control
DNA
BRAF
V600E
10.5
10.0
10.7
13.0
11.7
13.0
EGFR
ΔE746 - A750
2.0
No call
2.2
2.0
2.2
4.0
EGFR
L858R
3.0
2.0
2.4
3.0
3.2
5.0
EGFR
G719S
24.5
24.0
24.3
27.0
23.7
24.0
KRAS
G13D
15.0
13.0
12.0
12.0
12.1
18.0
KRAS
G12D
6.0
8.0
3.0
6.0
6.4
7.0
NRAS
Q61K
12.5
11.0
10.6
10.0
12.4
13.0
PI3KCA
H1047R
17.5
22.0
18.1
22.0
18.6
25.0
PI3KCA
E545K
9.0
8.0
5.2
5.0
8.0
9.0
Table 3. Verification of the Formalin-Compromised DNA HDx Reference standard on an Ion Torrent NGS platform and QX100
DDPCR. Formalin-Compromised DNA HDx™ Reference Standards were analysed by the QX100 and by another laboratory using
an Ion Torrent.
Summary and Future Directions
• We have shown the importance of using a Reference Standards to validate the limit of detection of assays and platforms
including dealing with the effects for formalin-compromised material. Verification of these standards on the Ion Torrent
supports the suitability of these products on NGS platforms and data agrees broadly with digital PCR. These results validate
our standards as a useful new tool with clear utility as a standard for mutation profiling on NGS or equivalent multiplex
platforms.
• Horizon Diagnostics are pleased to offer the generation of Multiplex Reference Standards on a custom basis. Researchers
will be able to define the following features of their projects:
Figure 2. Comparison of Tru-Q HDx Reference Standards predicted allelic frequencies with those observed by QX100 digital
PCR and Ion Torrent.
Genomic DNA Tru-Q HDx Reference Standard was analysed by the QX100 and by another laboratory using an Ion Torrent.
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1. Target genotypes to be included (subject to cell line availability).
2. Allelic frequency of chosen genotypes.
3. Manufacturing of a custom standard either as genomic DNA or as an FFPE block.