This document summarizes a presentation about reference standards for next generation sequencing (NGS). Horizon Diagnostics has developed genomic DNA and formalin-fixed, paraffin-embedded (FFPE) reference standards containing defined mutations at known allelic frequencies to validate NGS workflows and monitor assay performance. Multiplex reference standards contain up to 40 mutations at low allelic frequencies down to 1.3% that can be quantified using digital PCR. Several laboratories demonstrated they could accurately detect the mutations in Horizon's reference standards using different NGS platforms. The standards help evaluate sensitivity, specificity, and limits of detection on NGS assays.

1. Sponsored by Horizon Diagnostics:
The Use of Standards in NGS: Managing Variability in NGS?
Dr Sarah Leigh Brown, Cambridge Institute Genomics Core,
CRUK-CI.
HDx™ Reference Standards and Reference Materials for Next
Generation Sequencing somatic variant pipelines
Dr Louisa Ludbrook, Horizon Discovery, Cambridge, UK
NGS Sheffield, Nov 11th 2014

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3. 33
Genome Translation
precision genome editing
(GENESIS™)
Modelling patient genetic variations
that define disease biology, drug
response & resistance
Precision functional genomics;
targeted gene knock-outs & knock-ins
Capacity to make 200 models / year
Custom generation services
Human Cellular
Disease Models
Gene-X, Mutant And
Normal isogenic cell lines
X-MAN™
Genetically defined disease
models & endogenous pathway
reporters to study target biology
Rational screening tools for
novel biomarker & target-ID &
validation
Drug screening
>500 off-the-shelf disease
models
Tumour
Microenvironment
Assay development &
screening CRO (Horizon
Discovery Services)
Suite of specialist assays including
hypoxia, 3D, senescence, autophagy
& pathway analysis
Endogenous mutations placed
under selective pressure to illicit
true disease phenotypes & drug
responses
Portfolio of 50+ 2D and 3D Assays
Right Drug
Right Patient
X-MAN™ surrogate
patient profiling
Predicting responsive or resistant
patients before entering clinical
trials using wide panels of X-MAN
disease models
Drug profiling service
COLTHERES, PREDICT, INSIGHT EU
FP6 Translational Consortia
HDx™ Reference
Standards
Genetically defined DNA or
paraffin embedded cells in
specific wild type vs. mutant
ratios
Portfolio of genomic reference
standards for multiple
platforms; Sanger, qPCR, NGS,
IHC & FISH
What we do

4. NGS Labs
• Support workflow
analysis and setup
• Provision of positive
reference standard for
everyday run control
Molecular Labs
• Support bridging studies
and setup of NGS
• Provision reference
standards for singleplex
assays
OEM Partnerships
• Support assay/platform
development
• Provision of reference
material for validation
studies
• Supply of internal kit
reference standards
HDx™ Reference Standards
Every Assay, Every Run, Every Confidence

5. NGS Workflow and sources of variability
Tumour sample
Action
Analysis
DNA extraction
Variant Calling/
Confidence Scoring
Reference Materials
DNA Quantification Library Preparation Sequencing Alignment/Mapping

6. Overview of HDx™ Reference Standards
Product Product Detail Features Application
Genomic DNA Singleplex Defined allelic frequency
(e.g. KRAS 1% G12A)
6
Routinely monitor the performance
of your molecular assay
(Sanger Sequencing, Therascreen,…)
Multiplex Multiple mutations in one tube
(e.g. EGFR 5% G719S; 5% L858R, 5%
T790M,…)
Monitor the performance of your
NGS platform
FFPE Sections Singleplex
or
Multiplex
Defined standards in FFPE format Monitor the workflow from DNA
extraction to genotyping
FFPE Blocks Standard
Genotype
A complete block
(~800sections@15μm)
Consistent material for assay
validation studies
Multiplex
Genotype
Select your genotypes
Select your allelic frequencies
Consistent and customised material
for assay validation studies
Cell Slides FISH Slides Defined chromosomal abnormalities
(EML/ALK)
Validate your FISH probes and
workflow
IHC Slides
COMING SOON
Defined protein expression levels
(ROS1, c-MET, EGFR, HER2,
EML4/ALK)
Routinely monitor the performance
of IHC workflow

7. Creating cell lines- Horizon Discovery’s Gene Editing Platform
7
“Wild type cell line”
Single Cell Dilute
Clonal wild type cell line Cell Line Validation
Gene Engineering Technology (GENESIS™)
Clonal mutant cell line Cell Line Validation

8. Cell Validation Test Assay
Confirm identity of parental cell line STR and/or SNP6.0
Confirm integration in the correct
locus
8
Cell Line Validation
Clonal heterozygous
mutant cell line
gDNA locus specific PCR & Sanger Sequencing
Confirm expression of modified allele cDNA-PCR & Sanger Sequencing
Confirm clonality
Droplet digital PCR, gDNA PCR & Sanger
Sequencing
Confirm gene copy number Droplet digital PCR
Clonal
wild type cell line
Fully validated genetically defined isogenic cell line pair

9. Genomic DNA Standards verified using Digital™ PCR
Gene Mutation Allelic Frequency
BRAF V600K 50% 10% 5% 1% 0.5% 0.1% 0.05%
KRAS G12D 50% 10% 5% 1% 0.5% 0.1% 0.05%
Stoichiometric dilutions are accurate down to 0.1% and 0.05%

10. Validation of NGS somatic variant pipelines
Parameters that may be assessed:
• Sensitivity do you detect the targets if they are present?
• Specificity can you distinguish between false/true positives?
• Limit of Detection what AF is your limit, does it vary mutation to mutation?
• Repeatability run-to-run, same and different variants
• Reproducibility inter-lab, same result elsewhere?
• Robustness what is the effect of sample quality on performance?
DNA and FFPE HDx™ Reference Standards are manufactured within a
Quality Management System which complies with the requirements
of ISO 9001:2008 and ISO 13485:2003.

11. Multiplex Reference Standards for NGS workflow analysis
Genetically Defined Mutant Cell Lines
Precise DNA dilutions
Digital PCR Analysis
Quantitative Multiplex
Reference Standards
 Quantitative Multiplex Reference Standard
 A ‘ladder’ of 11 engineered fixed allelic
frequency variants (1% to 24.5% AF)
 Validated endogenous variants (20)
 Predicted endogenous variants (>200)
 Genomic DNA Format:
1ug @ 50ng/μl - £160
 FFPE Format: Vial containing 1 FFPE section
that will provide >400ng (5 sections - £195)
 Formalin Compromised (Paraffin-Free) DNA
Grade I and Grade II formalin intensity (1ug
- £160)

12. Case Study Data for Q-Seq FFPE HDx™ Reference Standards
Source:
Horizon
Discovery
Partner A Partner B Partner C Partner D
Platform:
QX100™ Droplet
Digital™ PCR
System
AmpliSeq Cancer
Panel
Ampliseq Cancer
Hotspot Panel v2
Ampliseq Cancer
Hotspot Panel v2
(Average of 8
runs)
TruSeq Panel
Sequencing Depth N/A 3000-4000x Average 5000x 2000X 150k+
Gene Mutation Observed mutant ratio
BRAF V600E 10.2 9.9 9.1 10.3 9
KIT D816V 10.4 10.0 11.0 10.1 Not Tested
EGFR
ΔE746 -
A750
2.0 2.3 Not detected Not detected Not Tested
EGFR L858R 2.7 2.7 2.1 2.4 Not Tested
EGFR T790M 0.9 0.8 Not detected Not detected Not Tested
EGFR G719S 24.4 23.7 23.1 24.8 Not Tested
KRAS G13D 16.1 16.3 12.35 15.5 15.4
KRAS G12D 5.0 5.2 Not detected 5.1 5.5
NRAS Q61K 12.8 9.0 12.7 12.6 13.5
PIK3CA H1047R 18.6 16.7 16.8 17.9 17.0
PIK3CA E545K 8.9 3.2 8.4 8.8 10.0
Multiplex Reference Standard can be used to identify false negative NGS reads

13. Q-Seq HDx™ Reference Standards for Next Generation Sequencing
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

14. Case Study Data for Tru-Q HDx™ Reference Standards
Source:
Horizon
Diagnostics
Predicted %
5% blend 2.5% blend 1.3% blend
Horizon
Diagnostics
Observed %
Partner
Platform: N/A
QX100™
Droplet
Digital™
PCR System
Ion Torrent
Gene
Mutatio
n
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

15.  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 BRAF cKIT EGFR FGFR2
FLT3 GNAQ GNA11 IDH1 IDH2 JAK2 KRAS
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 NRAS MLL PI3K
Y1253D MLL/ENL
PTEN
ΔEX6/EX7
ROS1
ROS1
RUNX1
RUNX1/RUNX1T1
Q61H
A146T
Mutation Coverage

16. What’s coming next?
Low Copy Number /
cfDNA Reference Standards
Translocation / RNA
Reference Standards
…and more

17. Your Horizon Contact:
Louisa Ludbrook
Head of Sales
l.ludbrook@horizondiscovery.com
+44 1223 655589
www.horizondx.com
Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Tel: +44 (0) 1223 655 580 (Reception / Front desk) Fax: +44 (0) 1223 655 581 Email: info@horizondx.com Web: www.horizondx.com