Genome in a Bottle Working GroupReference Material (RM) Selection and Design

NIST WorkshopAugust 14 + 15, 2014

Andrew Grupe

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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RM Selection & Design SummaryMona 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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RM Selection & Design SummaryJonathan 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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RM Selection & Design SummaryOther 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

RM Selection & Design SummaryOther 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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RM Selection & Design SummaryAdditional 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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Evaluation of Synthetic ControlsGoal 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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