This document summarizes Horizon Diagnostics' work to develop reference standards for next-generation sequencing assays. They have created cell line mixtures containing defined mutations at various frequencies to assess assay performance. Testing showed the assays could reliably detect mutations down to an allelic frequency of 2.5% and sometimes 1.3%, but not consistently at 1%. Formalin treatment of DNA was found to affect quantification and variant calling. Horizon Diagnostics offers a comprehensive suite of reference standards containing mutations relevant to cancer therapeutics.

1. Redefining Reference Standards
Dr Jonathan Frampton
Product Manager

2. 2
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3. Impact of formalin treatment on template and assay performance
Goal:
To assess the effect of formalin on genomic DNA and the on assay performance for
somatic variant detection.
1. Created matched mutant and wild
type cell lines
2. Generated precisely defined HDx™
mixed cell pellets
3. Formalin treat one pellet from each
pair
4. Extract genomic DNA from both
pellets
5. Quantitate gDNA
6. Assess allelic frequency by dPCR
Defined HDx™ cell line mixture
No Formalin
Treatment
Formalin
Treatment
DNA Extraction DNA Extraction
DNA
Quantification
DNA
Quantification

4. DNA quantification of formalin-compromised gDNA
Three methodologies employed to
perform quantitation:
• Quantifluor Assay
• Agilent Tapestation
• Nanodrop
Observations:
1. There is variation in the
concentration of DNA from
matched pairs (overestimation in
formalin vs no formalin).
2. The Nanodrop data shows a
greater overestimation of
concentration in formalin vs no
formalin samples from matched
pairs.

5. Mutant detection on formalin-compromised DNA by digital PCR
Non-compromised
DNA
Formalin-
[bp] compromised DNA
Observations:
Begin to see the subtle variation in variant
calling Sample between Expected formalin Genotype vs Formalin
no formalin
Treatment
matched pairs.
Mutant Allelic
Frequency
Measured
1 5.0% B-Raf V600E - 5.2
2 5.0% B-Raf V600E + 5.5
3 2.5% B-Raf V600E - 2.7
4 2.5% B-Raf V600E + 3.7
5 1.0% B-Raf V600E - 1.0
6 1.0% B-Raf V600E + 1.3
7 0.5% B-Raf V600E - 0.6
8 0.5% B-Raf V600E + 0.6
9 0.2% B-Raf V600E - 0.2
10 0.2% B-Raf V600E + 0.4
TapeStation analysis (above) and digital PCR
genotyping (below) of matched pairs.
Expected and measured allelic frequencies
Implications:
Artifacts – eg effects of formalin on DNA by
deamination affect variant calling, potentially
by increasing the mutant to wild type ratio.
Sample Expected Genotype Formalin
Treatment
Mutant Allelic
Frequency
Measured
1 5.0% Mutant - 5.2
2 5.0% Mutant + 5.5
3 2.5% Mutant - 2.7
4 2.5% Mutant + 3.7
5 1.0% Mutant - 1
6 1.0% Mutant + 1.3
7 0.5% Mutant - 0.6
8 0.5% Mutant + 0.6
9 0.2% Mutant - 0.2
10 0.2% Mutant + 0.4
Mutation Frequency

6. Impact of Formalin treatment on wild type samples
6
Formalin Intensity
1. Utilised clonal wild type cell line
2. Treated cell pellets with four different
formalin conditions
3. Analyzed allelic frequency by digital PCR
Sample Expected Genotype Mutant Allelic
Frequency
Measured
1 0% Mutant 0.04%
2 0% Mutant 0.04%
3 0% Mutant 0.07%
4 0% Mutant 0.15%
Sample preparation may interfere with assay sensitivity and specificity
Mutation Frequency

7. Case Study 2. Tru-Q NGS Reference Standards
EGFR
mutants
K-Ras
mutants
B-Raf
mutants
N-Ras
mutants
PIKCA
mutants
Quantification by Droplet Digital PCR
C Blend 1
10 mutations
at 5%
C Blend 2
10 mutations
at 5%
C Blend 3
10 mutations
at 5%
Quantification by Droplet Digital PCR
Quantification by Droplet Digital PCR
A Blend
40 Mutations
@ 1.3%
B Blend 1
20 Mutations
at 2.5%
B Blend 2
20 Mutations
at 2.5%
C Blend 4
10 mutations
at 5%
14 Additonal
Biomarkers
1.3%
20 copies per μl
Quantification by Droplet Digital PCR
Dilution
Series with
wild type

8. Case Study 2. Data for Tru-Q NGS Reference Standards
5% blend 2.5% blend 1.3% blend
8
Source:
Horizon
Diagnostics
Predicted %
Horizon
Diagnostics
Observed %
Partner
Platform: N/A
QX100™
Droplet
Digital™ PCR
System
Ion Torrent
Gene Mutation
BRAF V600M 4.0 4.4 3.5
EGFR T790M 4.2 3.9 4.3
EGFR L858R 4.2 4.2 3.5
EGFR L861Q 4.2 4.1 3.6
KIT D816V 5.0 5.4 6.4
KRAS G12A 5.0 5.7 4.9
KRAS G12R 5.0 5.2 4.6
NRAS Q61K 5.0 4.9 3.3
Specific and Sensitive down
to 5% allelic frequency
Horizon
Diagnostics
Predicted %
Horizon
Diagnostics
Observed %
Partner
N/A
QX100™
Droplet
Digital™ PCR
System
Ion Torrent
2.0 2.2 2.1
2.1 2.0 2.1
2.1 2.0 2.3
2.1 2.1 1.8
2.5 2.6 3.2
2.5 3.0 2.5
2.5 2.9 2.6
2.5 2.5 2.5
Horizon
Diagnostics
Predicted %
Horizon
Diagnostics
Observed %
Partner
N/A
QX100™
Droplet
Digital™ PCR
System
Ion Torrent
1.0 1.0 1.9
1.0 1.1 missing
1.0 1.1 missing
1.0 1.0 missing
1.3 1.3 1.5
1.3 1.4 missing
1.3 1.3 missing
1.3 1.2 missing
Specific and Sensitive down
to 2.5% allelic frequency
Not sensitive to detect down
to 1% for all variants

9.  Horizon Diagnostics’ suite of reference material includes standards for the increasing number of ‘rare’
mutations being targeted for cancer therapeutics, which by definition are hard to find in clinical samples.
ABL1 AKT1 ALK B-Raf cKIT EGFR FGFR2
FLT3 GNAQ GNA11 IDH1 IDH2 JAK2 K-Ras
9
G12V
E17K
Q209L
V600E
V600K
V600R
R132C
R132H
G719S
T790M
L858R
L861Q
ΔE746-A750
V617F
S252W
G12A
G12C
G12D
G12R
G12S
G12V
G13D
T315I
D835Y
L1601P
F1174L
R1275Q
F1245V
Q209L
Q61H
Q61K
Q61L
Q61R
D816V
R140Q
R172K
E542K
E545K
H1047R
EML4/ALK V600M
V600G
Δ1836
NOTCH1 MET N-Ras MLL PI3K
Y1253D MLL/ENL
PTEN
ΔEX6/EX7
ROS1
ROS1
RUNX1
RUNX1/RUNX1T1
Q61H
A146T
Mutation Coverage