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Aug2013 horizon dx engineered cell line reference materials
1. Horizon Diagnostics
Addressing the Variability of Molecular Assays: The Need for Reference Standards
Jonathan Frampton
Diagnostics Product Manager
2. 2
Undefined Cell Line Reference Materials
Working with a laboratory that were able to detect EGFR ΔE746-A750 using
Sanger Sequencing down to LOD of 2%
EGFR ΔE746-A750
Sample Description
Actual Allelic
Frequency
1% 7%
2% 14%
5% 28%
7.5% 42%
10% 41%
15% 51%
100% 94%
Digital PCR highlighted that a “2% mutant” was actually a “14% mutant”
3. 3
Undefined Cell Line Reference Materials
HCC827 cell line containing EGFR ΔE746-A750
Cell Line EGFR Copy Number
HeLa Source 1 2.6
HeLa Source 2 2.7
HCC827 Source 1 15
HCC827 Source 2 38
Non-integer copy number suggests a heterogeneous cell line population
Variable EGFR copy number depending on the source material
4. What is a Reference Standard
4
Independently/Externally validated reference material
Allows you to understand the sensitivity and limit of detection of your assay
Allows you to monitor the reproducibility of your molecular assay
Quantitative Reference standards are a critical part of the development and
QC of molecular assays
Reference Standards will tell you if your assay is working today.
5. 5
Using Horizon Discovery’s Gene Editing Platform
“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
6. Functional Genomics
Disease modelling
Reference Standards
6
> 40 different parental cell lines now targeted; 500 projects, covering 16 tissue types
Examples of what you can accomplish with GENESIS
7. 7
Cell Line Validation
Clonal heterozygous
mutant cell line
Cell Validation Test Assay
Confirm identity of parental cell line STR and/or SNP6.0
Confirm integration in the correct locus 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 X-MAN isogenic cell line pair
9. Quantitative Molecular Reference Standards
9
Genomic DNA
Stoichiometric Dilutions
Mutant Wild type
FFPE Processing
FFPE Mixed Ratio
Sections
Mutant Wild type
10. Genomic DNA Standards verified using Digital™ PCR
Stoichiometric dilutions are accurate down to 0.1% and 0.05%
Gene Mutation Allelic Frequency
B-Raf V600K 50% 10% 5% 1% 0.5% 0.1% 0.05%
K-Ras G12D 50% 10% 5% 1% 0.5% 0.1%
11. 11
FFPE Block Production
B-Raf 50% V600K
Prepare block
and sections
B-Raf 100% wild type B-Raf 5% V600K
Mix cells
9:1
Droplet Digital PCR
BioRad QX100
Our proprietary FFPE technology allows us to accurately control:
• Number of cells and cores per block
• Homogenous cell distribution throughout the block
• Allelic frequency
• Section thickness & DNA content
12. 12
H+E
DNA
H+E Staining & DNA Extraction demonstrates the homogeneity and
consistency of our FFPE technology
FFPE Block Consistency
13. FFPE Block Allelic Frequency
13
Gene Mutation
B-Raf V600E 25% 5% 3.5% 1%
B-Raf V600K 50% 5% 1% ---
Consistent allelic frequency throughout the FFPE Blocks
14. 14
Quantitative Multiplex DNA Reference Standard
Precise DNA dilutions
Digital PCR Analysis
Quantitative Multiplex DNA Reference Standard
X-Man Genetically Defined Mutant Cell Lines
15. 15
Quantitative Multiplex DNA Reference Standard
Permits validation of a wide range of multiplex platforms including Next
Generation Sequencing (NGS)
Chromosome Gene Variant %
7q34 BRAF V600E 10.5
4q11-q12 cKIT D816V 10.0
7p12 EGFR ΔE746 - A750 2.0
7p12 EGFR L858R 3.0
7p12 EGFR T790M 1.0
7p12 EGFR G719S 24.5
12p12.1 KRAS G13D 15.0
12p12.1 KRAS G12D 6.0
1p13.2 NRAS Q61K 12.5
3q26.3 PI3KCA H1047R 17.5
3q26.3 PI3KCA E545K 9.0
Initial offering covers 11 mutations – Available Now
16. 16
External Data – Ion Torrent
External partners using independent assays confirm accurate allelic frequencies
Source: Horizon Discovery Partner A Partner B Partner C
Platform:
QX100™ Droplet
Digital™ PCR System
AmpliSeq Cancer
Panel
Ampliseq Cancer
Hotspot Panel v2
Ampliseq Cancer
Hotspot Panel v2
(Average of 8 runs)
Sequencing Depth N/A 3000-4000x Average 5000x 2000X
Gene Mutation Observed mutant ratio
BRAF V600E 10.2 9.9 9.1 10.3
KIT D816V 10.4 10.0 11.0 10.1
EGFR ΔE746 - A750 2.0 2.3 Not detected Not detected
EGFR L858R 2.7 2.7 2.1 2.4
EGFR T790M 0.9 0.8 Not detected Not detected
EGFR G719S 24.4 23.7 23.1 24.8
KRAS G13D 16.1 16.3 12.35 15.5
KRAS G12D 5.0 5.2 Not detected 5.1
NRAS Q61K 12.8 9.0 12.7 12.6
PIK3CA H1047R 18.6 16.7 16.8 17.9
PIK3CA E545K 8.9 3.2 8.4 8.8
20. 20
Custom Multiplex Options
Cover a range of mutations including:
• Single nucleotide polymorphisms
• insertion-deletions
• 5’UTRs
• Frameshifts
DNA or FFPE format available (Xenograft an option)
22. Your Horizon Contact:
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
Dr Jonathan Frampton
Product Manager
j.frampton@horizondiscovery.com
+44 (0) 1223 815299
Hinweis der Redaktion
Who am I?
What is the purpose of the presentation?
Heterogenous samples (mixed clones)
Are you using internal kit controls?
Have you made controls yourself from a cell line?
Can perform standard KI and KO approaches with enhanced precision over any technique.
Can uniquely spatial, inducible and (soon) conditional genomic alterations.
Examples of this are current projects modelling amplifications and translocation (EML4-Alk / Crizotinib Dx Standard)
Are you using internal kit controls?
Have you made controls yourself from a cell line?