Jan2015 rm selection and design summary

1. Genome in a Bottle
Reference Material Selection and Design Working Group
January 29-30, 2015
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2. Presentations
• Horizon Diagnostics
– FFPE Blocks of GiaB Reference Cell Lines
• Discount for Consortium Members
– FFPE treatment increases mutation background
• 0.04-0.15%, 4 samples, increasing formalin intensity
• Horizon believes that FFPE compromized positions
up/downstream contribute to false positives
– Engineered multiple mutations in cell lines, including
translocation
– Haploid cell line to improve turn around and
frequencies of engineered cell lines
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3. Presentations
• Accrometrix
– 550 common cancer mutations as synthetic
molecules, diluted in GM24385
– Frequencies: 5-35%
– 30 site multi lab study showed utility in assessing
testing performance
– Dilution series tracks with expected frequency
change
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4. Presentations
• Jason Lih / Frederick National Lab for Cancer
– Plasmids perform with similar efficiency as
genomes in dilution series
• Compared serial dilutions of synthetic constructs with
serial dilution of V600E cell line
• Parallel slopes
– Detected 47 out of 51 mutations in multiplex
• InDels near homopolymer regions were weak spot of
detection
• Sensitivity of detection varies by pipeline (eg.
BWA/exome vs. TS3.2/targeted vs. TS4.0/targeted)
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5. Evaluation of Synthetic Controls
Goal Planning
• Hypothesis: Synthetic controls are good surrogates for DNA isolated from
a clinical sample
– Evidence to support/refute hypothesis
– Clear statement of scope
• If delta, where, how much
– Quantifiable difference
– What constitutes evidence
• Allele frequency
• Efficiency
– Tumor Type Scope
• Solid tumors
– Variant Classes
• Need List
• Need Priorities
– See MiSeq Dx review memo
• Scope sufficient for clinical application
– Verify synthetic material
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6. Workplan
Synthetic Reference Material Evaluation
Hypothesis
Synthetic controls are good surrogates for DNA isolated
from a clinical sample
Questions we like to address
In NGS pipelines, do synthetic mutant constructs mixed
with normal DNA perform similar to endogenous
mutations in human DNA or human FFPE DNA
Do allele frequencies obtained from synthetic constructs
mixed into wt human DNA approximate those from
mutant human samples
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Somatic Mutation Controls for Wet Lab and Bioinformatics Pipeline

7. Proof of Principle Study
DNA Mixing Study
Compare quantitative and qualitative mutation detection
between serial dilutions of human FFPE DNA, cell line
references, and synthetic DNAs (linear plasmids, oligos)
Mutations Selected For Hypothesis Testing
• BRAF V600E
• EGFR T790M
• EGFR DEL746-750
• others
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8. Analysis
• Compare expected vs. detected allele frequencies
– Do detected synthetic mutational frequencies match
expected frequencies
– Are frequency changes in dilution series of tumor FFPE
DNA , cell lines and synthetic DNAs similar
– Is mutation detection (yes/no) identical between tumor
FFPE DNA, cell lines and synthetic DNAs
– Are expected and detected frequencies of verified human
sample, cell line DNAs and synthetic samples similar
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9. Study Samples
• Establish NGS test panel baseline for diluent DNA sample (GM24385)
– Use GM24385 to make dilutions (Ashkenazi Son, GiaB Reference)
• Determine allele frequencies in serial dilutions of DNA from human FFPE
samples (25%, 12.5%, 6.25%)
– BRAF V600E
– EGFR del746-750
– EGFR T790M (pending availability?)
• Use NGS test panel to determine mutation frequencies of selected
synthetic mutant DNAs in dilution series (25%, 12.5%, 6.25%)
– Use multiplex of all synthetic mutations
• Determine allele frequencies in Horizon cell line DNA containing the
three mutations
– 16%, 12.5%, 6.25%
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Samples and serial dilutions (19 samples) are distributed from a common
stock to participating labs to control variation
1 Library
2? samples x 3 Libraries
3 samples x 3 Libraries
3 Libraries
Total: 19 Libraries

10. Analysis Results
Qualitative (Simulated)
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Simulated - Cartoon!
FFPE Synthetic Cell Line
6.25 12.5 25 6.25 12.5 25 6.25 12.5 16
BRAF V600E
EGFR Del746-750
EGFR T790M
Others 1
Other 2
….

11. Analysis Results (Simulated)
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0
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0 5 10 15 20 25 30
FFPE
Synthetic
Cell Line
Expected Frequency
MeasuredFrequency

12. Needs
• Participating Laboratories
• Funding Support
– Sequencing Reagents
• Illumina?
• PGM?
– Synthetic reference samples & dilutions
• SeraCare
• Acrometrix / Thermo Fisher
• Accugenomics
• Horizon Diagnostics
– Verified human DNA samples from FFPE tissue
• Mickey Williams / Frederick National Lab for Cancer
– GM24385 (diluent DNA)
• Data hosting of results
• Statistician(s)
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