2. 2
Disclaimer
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3. 3
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?
5. 5
Horizon Diagnostics
HDx Reference Standards offer a sustainable
source of reference material to laboratories,
proficiency schemes and manufacturers,
providing an unprecedented level of control.
6. 6
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?
7. 7
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?
Variant
Expected
AF
Standard 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%
8. 8
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
9. 9
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!
10. 10
cfDNA Reference Standards – now available
Example trace showing the fragment sizes collected by
D1000 DNA ScreenTape assay, comparing cfDNA HDx
Reference 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.
11. 11
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
12. 12
Developing a Universal Reference Standard
Genetically Defined Mutant Cell Lines
EML4-ALK
FISH IHCgDNA RNA
EML4-ALK FFPE curl
FISH
IHC
gDNA
RNA
13. 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
Natalie LaFranzo, PhD
Product Manager, NGS Products
n.lafranzo@horizondiscovery.com
(314) 400-6636
Editor's Notes
HDx Reference Standards are offered in multiple formats, which allows for the each portion of the workflow to be evaluated.
gDNA (top right quadrant): Highly-characterized, high molecular weight genomic DNA is provided ready for direct input into NGS library prep. Includes Quantitative Multiplex (11 variants from 1%-24.5%); Tru-Q Series (40 mutations blended at 5%, 2.5% and 1.25% blends); Structural Multiplex (complex variants including CNVs and gene fusions), as well as individual mutants (Base-Seq products)
Formalin-Compromised (top left): Degraded, genomic DNA is extracted from formalin-treated cells (early-access product, more info coming soon), and allows you to evaluate the effect of formalin treatment on your workflow. Available as Quantiative Multiplex in different formalin intensities
FFPE (bottom left): Highly-characterized cells are blended, mildly fixed with formalin and then embedded in paraffin wax. This product is ready for extraction alongside patient samples to evaluate the pre-analytical portion of the workflow. Available as Quantitative Multiplex blend and Genome in a Bottle samples (Multi-platform Personal Genome Project data available)