jan2016 horizon giab
TRANSCRIPT
HORIZON DIAGNOSTICS
Designing Reference MaterialsNatalie LaFranzo, PhD
Product Manager, NGS Products
Genome in a Bottle Consortium – Spring Meeting 2016
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Disclaimer
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Initial Questions
• What variant types/formats are needed by the community? – Is a matched wild type always required (LOD)?
– What level of multiplexing/frequencies are desired?
– More variant types vs. multiples of single type?
• How do we provide reference materials to meet those needs?
• Do these materials fit with existing/future regulatory guidelines?
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Manufactured under
Engineering HDx Reference Standards
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Horizon Diagnostics
HDx Reference Standards offer a sustainable source of reference material to laboratories,
proficiency schemes and manufacturers, providing an unprecedented level of control.
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Formalin Fixation; How does it impact a sample?
Formalin Compromised DNA Degradation
Not all formalin treatments are created equal, but how close to clinical archives can
we get?
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Evaluating the Effects of Formalin
Important parameters to evaluate: • How does formalin affect the pre-analytical workflow?
• Can we make a sequencing library out of this material?
• How does formalin affect the analytical workflow? • Does this require a matched wild-type standard?
VariantExpected
AFStandard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6
KIT D816V 10% 11.5% 11.5% 10.5% 21.9% 20.1% 18.0%
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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%
Mu
tati
on
Fre
qu
ency
Sample preparation may interfere with assay sensitivity and specificity
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How to test the robustness and sensitivity of your workflow and assay
Sensitivity of your Assay
Formalin Intensity
Robustness and Sensitivity of your Workflow
FFPE
DNA
Robustness of your Assay
QMRS Formalin Compromised (fcDNA) QMRS Tru-Q
QMRS FFPE GIAB FFPE
Most verified mutations are oncology-relevant, however, other disease-relevant mutations are often
present and available for analysis.
QMRS = Quantitative Multiplex Reference Standard
GIAB = Genome in a Bottle Reference Standards
Structural Multiplex
3 levels of fcDNA coming soon!
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cfDNA Reference Standards – now available
Example trace showing the fragment sizes collected by D1000 DNA ScreenTape assay, comparing cfDNA HDxReference Standards (Red and Green traces) to cfDNA
extracted from human plasma (Blue trace). cfDNA from human plasma was provided by CareDx,Inc. Leftmost peaks-internal marker for the assay. Rightmost peaks-fragmented
materials.
Summarized in Application Note available for download.
1% Multiplex I cfDNA Reference Standard assessed using Droplet Digital PCR (blue), Ion Torrent (orange) and MiSeq (grey).
All assays utilized amplicon-based enrichment.
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What next?
Do we initiate gene editing of the GIAB samples?
What is the ideal representation of a formalin-compromised standard?
Are BRCA standards required for germline, somatic or both?
Universal Reference Standard – DNA/RNA from same sample?
Internal vs. Externally-verified Reference Standard – what is more suitable?
What are the top variants/variant types needed?• High GC, Low GC, difficult to sequence regions,
large rearrangements, drug-resistance markers
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Developing a Universal Reference Standard
Genetically Defined Mutant Cell Lines
EML4-ALK
FISH IHCgDNA RNA
EML4-ALK FFPE curl
FISH
IHC
gDNA
RNA
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
Natalie LaFranzo, PhD
Product Manager, NGS Products
(314) 400-6636