Aug2015 horizon diagnostics

1. HORIZON DIAGNOSTICS
What is the impact of formalin treatment on molecular
assays and how can we utilise the Genome in a Bottle
samples to answer this question?
Genome in a Bottle Reference Materials
Dr Jonathan Frampton
Associate Director, Products
Horizon Discovery, Cambridge, UK

2. 2
Disclaimer
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other commitment whatsoever in relation to, securities. Although reasonable care has been taken to ensure that the facts stated in this Presentation are
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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
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3. 3
What is the impact of assay failure
in your laboratory and how do you
monitor for it?

4. 4
NGS Workflow and Sources of Variability
Tumour sample
Analysis
Action
DNA extraction
DNA Quantification Library Preparation Sequencing Alignment/Mapping
Variant Calling/
Confidence Scoring
Reference Materials

5. 5
Horizon Diagnostics NGS Reference Material Roadmap
Q4 2014 Q1 2015 Q2 2015 Q3 2015 Q4 2015
FFPE Sections
based RM
DNA based RM
RNA?
Gene Editing…?
Asian Son
FFPE Sections
based RM
DNA based RM
RNA?
Gene Editing…?
Q-Seq NGS Reference Standards Range
Tru-Q Collection
Formalin Compromised
RM Format
Cell free DNA
RM Format
Structural Standards
RNA
RM Format
more….
Ashkenazim Trio (Father, Mother, Son)

6. 6
How are the reference standards manufactured and validated?
All products are currently RUO
Quality controlled building blocks
State of the Art QC Processes

7. 7
What are we doing?
y = 178.84x - 918.67
R² = 0.9876
0
100
200
300
400
500
600
700
800
900
1000
6 6.5 7 7.5 8 8.5 9 9.5 10
DNAYield(ng)
Core Diameter (mm)
Varying Core Diameter; Fixed Cell Density
Core diameter
Linear (Core diameter)
Understanding every aspect of the process so we can control it

8. 8
What factors are we looking at?
y = 3E-06x + 56.375
R² = 0.8924
0
100
200
300
400
500
600
700
800
900
0.00E+00 5.00E+07 1.00E+08 1.50E+08 2.00E+08 2.50E+08
DNAYield(ng)
Cell Density (Cells/ml)
Varying Cell Density; Fixed Core Diameter
SW48 Cell Density Plot
Linear (SW48 Cell Density Plot)
5e7 cells/ml 1e8 cells/ml 1.5e8 cells/ml 2e8 cells/ml
Understanding every aspect of the process so we can control it

9. 9
DNA Yield; Understanding the process from every angle
0µm
800µm
Analysing yield consistency across the FFPE Block

10. 10
Cell Count; Understanding the process from every angle
0µm
800µm

11. 11
What do the statistics look like?
Table Analyzed Section Depth
ANOVA summary
F 2.001
P value 0.0851
P value summary ns
Are differences among means statistically significant? (P < 0.05) No
R square 0.3722
Brown-Forsythe test
F (DFn, DFd) 0.6147 (8, 27)
P value 0.7577
P value summary ns
Significantly different standard deviations? (P < 0.05) No
Bartlett's test
Bartlett's statistic (corrected) 16.99
P value 0.0302
P value summary *
Significantly different standard deviations? (P < 0.05) Yes
ANOVA table SS DF MS F (DFn, DFd) P value
Treatment (between columns) 554111 8 69264 F (8, 27) = 2.001 P = 0.0851
Residual (within columns) 934442 27 34609
Total 1.489e+006 35
Data summary
Number of treatments (columns) 9
Number of values (total) 36
0µm
800µm

12. 12
Formalin Fixation; How does it impact a sample?
Formalin Compromised DNA Degradation

13. 13
Formalin Fixation; Controlling the process
Standard fixation:
High molecular weight
Extended fixation:
Medium degradation (peak 2kb)
Super fixation:
High degradation (peak 200bp)

14. 14
Impact of Formalin on DNA Quantification
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.

15. 15
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

16. 16
What next?
Do we initiate gene editing of the GIAB samples?
Do we develop formalin compromised samples?
How can you use these samples to understand the robustness of NGS workflows?

17. 17
Structural Multiplex Standard
Variant Type Mutation
Expected Fractional
Abundance (%) or CNV:
SNV High GC GNA11 Q209L 5.6
SNV High GC AKT1 E17K 5.6
SNV Low GC KRAS G13D 5.6
SNV Low GC Pi3Ka E545K 5.6
Long Insertion EGFR V769 ins 5.6
Long Deletion EGFR (delE746-A750) 5.3
Fusion ROS1 translocation 5.6
Fusion RET translocation 5.6
CNV MET amplification 4.5 copies
CNV MYC-N amplification 9.5 copies
SNP EGFR_G719S 5.3
Short Deletion MET_p.V237fs 4.8
SNV High GC NOTCH1_p.P668S 5
Short Deletion FLT3_p.S985fs 5.6
Short Deletion BRCA2_p.A1689fs 5.6
Short Deletion FBXW7_p.G667fs 5.6
How can we combine these or our technology with the GIAB samples?

18. 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
Jonathan Frampton, PhD
Associate Director, Products
j.frampton@horizondiscovery.com
+44 1223 655580