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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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

200

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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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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 info@horizondiscovery.comw 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 info@horizondiscovery.comw www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

Jonathan Frampton, PhDAssociate Director, Productsj.frampton@horizondiscovery.com +44 1223 655580