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 MaterialsDr Jonathan FramptonAssociate Director, Products
Horizon Discovery, Cambridge, UK
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What is the impact of assay failure in your laboratory and how do you monitor for it?
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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
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Horizon Diagnostics NGS Reference Material Roadmap
Q4 2014 Q1 2015 Q2 2015 Q3 2015 Q4 2015
FFPE Sectionsbased RM DNA based RM RNA?
Gene Editing…?
Asian Son
FFPE Sectionsbased RM DNA based RM RNA?
Gene Editing…?
Q-Seq NGS Reference Standards Range
Tru-Q Collection Formalin CompromisedRM Format
Cell free DNA RM Format
Structural Standards
RNA RM Format
more….
Ashkenazim Trio (Father, Mother, Son)
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How are the reference standards manufactured and validated?
All products are currently RUO
Quality controlled building blocks
State of the Art QC Processes
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What are we doing?
6 6.5 7 7.5 8 8.5 9 9.5 100
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200
300
400
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800
900
1000
f(x) = 178.84375 x − 918.666666666666R² = 0.987642788683483
Varying Core Diameter; Fixed Cell Density
Core diameterLinear (Core diameter)
Core Diameter (mm)
DNA
Yiel
d (n
g)
Understanding every aspect of the process so we can control it
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What factors are we looking at?
4.00E+07 6.00E+07 8.00E+07 1.00E+08 1.20E+08 1.40E+08 1.60E+08 1.80E+08 2.00E+08 2.20E+080
100
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f(x) = 0.0000032025 x + 56.375R² = 0.892362144717498
Varying Cell Density; Fixed Core Diameter
SW48 Cell Density Plot
Linear (SW48 Cell Density Plot)
Cell Density (Cells/ml)
DNA
Yiel
d (n
g)
5e7 cells/ml 1e8 cells/ml 1.5e8 cells/ml 2e8 cells/ml
Understanding every aspect of the process so we can control it
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DNA Yield; Understanding the process from every angle
0µm
800µm
Analysing yield consistency across the FFPE Block
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Cell Count; Understanding the process from every angle
0µm
800µm
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What do the statistics look like?
Table Analyzed Section Depth
ANOVA summaryF 2.001P value 0.0851P value summary ns
Are differences among means statistically significant? (P < 0.05) NoR square 0.3722
Brown-Forsythe testF (DFn, DFd) 0.6147 (8, 27)P value 0.7577P value summary nsSignificantly different standard deviations? (P < 0.05) No
Bartlett's testBartlett's statistic (corrected) 16.99P value 0.0302P value summary *Significantly different standard deviations? (P < 0.05) Yes
ANOVA table SS DF MS F (DFn, DFd) P valueTreatment (between columns) 554111 8 69264 F (8, 27) = 2.001 P = 0.0851Residual (within columns) 934442 27 34609Total 1.489e+006 35
Data summaryNumber of treatments (columns) 9Number of values (total) 36
0µm
800µm
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Ladd
er (b
p)Su
per fi
xatio
nEx
tend
ed Fi
xatio
nSt
anda
rd Fi
xatio
nSu
per F
ixatio
n
Formalin Fixation; How does it impact a sample?
Formalin Compromised DNA Degradation
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Formalin Fixation; Controlling the process
Standard fixation: High molecular weight
Extended fixation:Medium degradation (peak 2kb)
Super fixation:High degradation (peak 200bp)
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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.
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Formalin induced mutation detection
Formalin Intensity
1. Utilised clonal wild type cell line2. Treated cell pellets with four different
formalin conditions3. 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%
Mut
ation
Fre
quen
cy
Sample preparation may interfere with assay sensitivity and specificity
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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?
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Structural Multiplex StandardVariant 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 5.6Long 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?
Your Horizon Contact:
t + 44 (0)1223 655580f + 44 (0)1223 655581e [email protected] www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Your Horizon Contact:
t + 44 (0)1223 655580f + 44 (0)1223 655581e [email protected] www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Jonathan Frampton, PhDAssociate Director, [email protected] +44 1223 655580