Aug2014 working group report rm selection and design

1. Genome in a Bottle Working Group
Reference Material (RM) Selection and Design
NIST Workshop
August 14 + 15, 2014
Andrew Grupe

2. RM Selection & Design Workgroup
• Derivative products based on NIST RMs
– Acrometrix/Thermo Fisher
• RMs for cancer and somatic variant calling
– Horizon Dx
• Do we need another large family and/or more
diversity
• What is the relative priority of transcriptome RMs
• Oncology – is high read depth on existing RMs needed
• Oncology - synthetics
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3. RM Selection & Design Summary
Mona Shahbazian, Thermo Fisher
• Synthetic constructs with clinical cancer
mutations
– Designed and manufactured by Thermo Fisher under
design control
– Cover 504 SNVs, 2 MNVs, 29 Del, 19 Ins
– Mixed with GM24385 (Ashk. Son)
– Frequencies given as: 5-15%, 15-35%
– Constructs have  100bp to each side of mutation
• Multiple mutations per construct
• Sequence proprietary
– Available: 8/15/2014
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4. RM Selection & Design Summary
Jonathan Frampton, Horizon Diagnostics
• Engineered 40 cancer relevant mutations in 4
receiver cell lines
– Available as ddPCR verified mixture, 1.3% each
mutant
– Mixes with fewer mutations at 5% and 2.5%
– MCF10A wt cell line used for dilution
• EML4/ALK engineered translocation
– Present at DNA & RNA level
• Experiments show that formalin treatment
increases allele frequencies of engineered
Horizon mutations
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5. RM Selection & Design Summary
Other Cancer Relevant RMs
• Mickey Williams, NCI
– Uses set of engineered 13 plasmids with cancer relevant
mutations routinely to evaluate assay performance
– NA12878 is routinely used as negative control
• Translocation RMs
– Synthetic samples for short term access more likely
• Have to understand suitability of synthetics
– For long term prefer genome-engineered samples
• Accommodate new technologies, eg. longer read
– Translocation RMs have to be available as DNA and RNA
– Artificial chromosome another option to make RM?
– Conclusion: Postpone translocations until we better understand
utility of RMs after assessing synthetic SNVs & Indels
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Need interlab study to assess the qualifications of synthetic materials
vs. full genomic DNA to inform utility of synthetic RMs

6. RM Selection & Design Summary
Other Cancer Relevant RMs
• To use existing RMs for Cancer relevant
applications
– We need higher read depth (targeted regions?) to
assess presence of lower frequency mutations in
RMs
• FFPE tissue: usually ≥ 5%
• Future - Circulating cell free DNA: 0.01 – 0.1%
– Are there sufficient NA12878 higher read depth
data sets for prototype analysis and tool
assessment for ‘somatic’ variant analyses?
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7. RM Selection & Design Summary
Additional Family Sample RMs
• Prefer to select one additional large family
– Ethnically diverse from existing RMs
• Admixture preferred for phasing
• African family an alternative
– IVF samples plus parents is least favorite option
• Rationale
– Avoid over-fitting of analysis pipelines
– Additional variety, including for mixing
experiments
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8. 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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