Natalie LaFranzo discusses Horizon Diagnostics' work designing reference materials for next generation sequencing. She addresses questions about what variant types and formats are needed, how to provide materials to meet those needs, and whether the materials fit regulatory guidelines. LaFranzo then summarizes Horizon's approach to evaluating the effects of formalin fixation on DNA and developing reference standards for formalin-compromised and cell-free DNA samples. She concludes by asking what next steps the community recommends, such as editing GIAB samples or developing universal DNA/RNA standards.

1. HORIZON DIAGNOSTICS
Designing Reference Materials
Natalie LaFranzo, PhD
Product Manager, NGS Products
Genome in a Bottle Consortium – Spring Meeting 2016

2. 2
Disclaimer
This Presentation does not constitute or form any part of an offer to sell, or invitation to purchase or apply for or enter into any contract or make any
other commitment whatsoever in relation to, securities. Although reasonable care has been taken to ensure that the facts stated in this Presentation are
accurate and that the opinions expressed are fair and reasonable, the contents of this Presentation have not been formally verified by Horizon Discovery
plc (the “Company”) or any other person. Accordingly, no representation or warranty, expressed or implied, is made as to the fairness, accuracy,
completeness or correctness of the information and opinions contained in this Presentation and no reliance should be placed on such information or
opinions. Further, the information in this Presentation is not complete and is subject to updating, revision, further verification and amendment. Neither
the Company, nor any of its subsidiaries, nor any of its respective members, directors, officers or employees nor any other person accepts any liability
whatsoever for any loss howsoever arising from any use of such information or opinions or otherwise arising in connection with this Presentation.
Accordingly, information contained in the Presentation is being supplied to you solely for your information and may not be copied, reproduced or further
distributed to any person or published in whole or in part, for any purpose. In particular, the distribution of this Presentation in certain jurisdictions may
be restricted by law, and persons into whose possession this Presentation comes should inform themselves about, and observe, any such restrictions. Any
failure to comply with these restrictions may constitute a violation of laws of any such jurisdiction.
This Presentation includes certain forward-looking statements, estimates and projections with respect to the anticipated future performance of Horizon
Discovery plc, its products and the markets in which it operates. Forward-looking statements involve risks and uncertainties. Actual events could differ
materially from those projected herein and such statements, estimates and projections reflect the various assumptions made by the Company which
assumptions may or may not prove to be correct. These forward-looking statements speak only as at the date of this Presentation. The Company
expressly disclaims any obligation or undertaking to disseminate any updates or revisions to any forward-looking statements contained in the
Presentation to reflect any change in the Company’s expectations with regard thereto or any change in events, conditions or circumstances on which any
such statements are based.
No part of this Presentation, or the fact of its distribution, should form the basis of or be relied upon in connection with any contract or commitment or
investment decision whatsoever. This Presentation does not constitute a recommendation regarding the securities of the Company.
By participating in and/or accepting delivery of this Presentation you agree to be bound by the foregoing restrictions and the other terms of this
disclaimer.

3. 3
Initial Questions
• What variant types/formats are needed by the
community?
– Is a matched wild type always required (LOD)?
– What level of multiplexing/frequencies are desired?
– More variant types vs. multiples of single type?
• How do we provide reference materials to meet those
needs?
• Do these materials fit with existing/future regulatory
guidelines?

4. 4
Manufactured under
Engineering HDx Reference Standards

5. 5
Horizon Diagnostics
HDx Reference Standards offer a sustainable
source of reference material to laboratories,
proficiency schemes and manufacturers,
providing an unprecedented level of control.

6. 6
Formalin Fixation; How does it impact a sample?
Formalin Compromised DNA Degradation
Not all formalin treatments
are created equal, but how
close to clinical archives can
we get?

7. 7
Evaluating the Effects of Formalin
Important parameters to evaluate:
• How does formalin affect the pre-analytical workflow?
• Can we make a sequencing library out of this material?
• How does formalin affect the analytical workflow?
• Does this require a matched wild-type standard?
Variant
Expected
AF
Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6
KIT D816V 10% 11.5% 11.5% 10.5% 21.9% 20.1% 18.0%

8. 8
Formalin induced mutation detection
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%
MutationFrequency
Sample preparation may interfere with assay sensitivity and specificity

9. 9
How to test the robustness and sensitivity of your workflow and assay
Sensitivity of your Assay
Formalin Intensity
Robustness and Sensitivity
of your Workflow
FFPE
DNA
Robustness of your Assay
QMRS Formalin Compromised (fcDNA) QMRS Tru-Q
QMRS FFPE GIAB FFPE
Most verified mutations are oncology-relevant,
however, other disease-relevant mutations are often
present and available for analysis.
QMRS = Quantitative Multiplex Reference Standard
GIAB = Genome in a Bottle Reference Standards
Structural
Multiplex
3 levels of fcDNA coming soon!

10. 10
cfDNA Reference Standards – now available
Example trace showing the fragment sizes collected by
D1000 DNA ScreenTape assay, comparing cfDNA HDx
Reference Standards (Red and Green traces) to cfDNA
extracted from human plasma (Blue trace). cfDNA from
human plasma was provided by CareDx,Inc. Leftmost peaks-
internal marker for the assay. Rightmost peaks-fragmented
materials.
Summarized in Application Note available for download.
1% Multiplex I cfDNA Reference Standard assessed using Droplet
Digital PCR (blue), Ion Torrent (orange) and MiSeq (grey).
All assays utilized amplicon-based enrichment.

11. 11
What next?
Do we initiate gene editing of the GIAB samples?
What is the ideal representation of a formalin-compromised standard?
Are BRCA standards required for germline, somatic or both?
Universal Reference Standard – DNA/RNA from same sample?
Internal vs. Externally-verified Reference Standard – what is more suitable?
What are the top variants/variant types needed?
• High GC, Low GC, difficult to sequence regions,
large rearrangements, drug-resistance markers

12. 12
Developing a Universal Reference Standard
Genetically Defined Mutant Cell Lines
EML4-ALK
FISH IHCgDNA RNA
EML4-ALK FFPE curl
FISH 
IHC 
gDNA 
RNA 

13. Your Horizon Contact:
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e info@horizondiscovery.com
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Your Horizon Contact:
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e info@horizondiscovery.com
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Natalie LaFranzo, PhD
Product Manager, NGS Products
n.lafranzo@horizondiscovery.com
(314) 400-6636