HORIZON DIAGNOSTICS
What is the impact of formalin treatment on molecular
assays and how can we utilise the Genome in a Bo...
2
Disclaimer
This Presentation does not constitute or form any part of an offer to sell, or invitation to purchase or appl...
3
What is the impact of assay failure
in your laboratory and how do you
monitor for it?
4
NGS Workflow and Sources of Variability
Tumour sample
Analysis
Action
DNA extraction
DNA Quantification Library Preparat...
5
Horizon Diagnostics NGS Reference Material Roadmap
Q4 2014 Q1 2015 Q2 2015 Q3 2015 Q4 2015
FFPE Sections
based RM
DNA ba...
6
How are the reference standards manufactured and validated?
All products are currently RUO
Quality controlled building b...
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 1...
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 ...
9
DNA Yield; Understanding the process from every angle
0µm
800µm
Analysing yield consistency across the FFPE Block
10
Cell Count; Understanding the process from every angle
0µm
800µm
11
What do the statistics look like?
Table Analyzed Section Depth
ANOVA summary
F 2.001
P value 0.0851
P value summary ns
...
12
Formalin Fixation; How does it impact a sample?
Formalin Compromised DNA Degradation
13
Formalin Fixation; Controlling the process
Standard fixation:
High molecular weight
Extended fixation:
Medium degradati...
14
Impact of Formalin on DNA Quantification
Observations:
1. There is variation in the
concentration of DNA from
matched p...
15
Formalin induced mutation detection
Formalin Intensity
1. Utilised clonal wild type cell line
2. Treated cell pellets w...
16
What next?
Do we initiate gene editing of the GIAB samples?
Do we develop formalin compromised samples?
How can you use...
17
Structural Multiplex Standard
Variant Type Mutation
Expected Fractional
Abundance (%) or CNV:
SNV High GC GNA11 Q209L 5...
Your Horizon Contact:
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e info@horizondiscovery.com
w www.horizondiscovery.com
H...
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Aug2015 horizon diagnostics

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Aug2015 horizon diagnostics

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  • Pleasure to be here to today to tell you more about Horizon and our suite of technologies based around a core expertise in human genome editing and how we are applying this to better understand the human genome, find new validated targets and support targeted drug discovery with predictive, genetically-defined, in vitro models that accurately represent target patient groups.
  • Aug2015 horizon diagnostics

    1. 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. 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. 3 What is the impact of assay failure in your laboratory and how do you monitor for it?
    4. 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. 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. 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. 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. 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. 9 DNA Yield; Understanding the process from every angle 0µm 800µm Analysing yield consistency across the FFPE Block
    10. 10. 10 Cell Count; Understanding the process from every angle 0µm 800µm
    11. 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. 12 Formalin Fixation; How does it impact a sample? Formalin Compromised DNA Degradation
    13. 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. 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. 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. 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. 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. 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

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