Genome in a Bottle Working Group 
Reference Material (RM) Selection and Design 
NIST Workshop 
August 14 + 15, 2014 
Andre...
RM Selection & Design Workgroup 
• Derivative products based on NIST RMs 
– Acrometrix/Thermo Fisher 
• RMs for cancer and...
RM Selection & Design Summary 
Mona Shahbazian, Thermo Fisher 
• Synthetic constructs with clinical cancer 
mutations 
– D...
RM Selection & Design Summary 
Jonathan Frampton, Horizon Diagnostics 
• Engineered 40 cancer relevant mutations in 4 
rec...
RM Selection & Design Summary 
Other Cancer Relevant RMs 
• Mickey Williams, NCI 
– Uses set of engineered 13 plasmids wit...
RM Selection & Design Summary 
Other Cancer Relevant RMs 
• To use existing RMs for Cancer relevant 
applications 
– We ne...
RM Selection & Design Summary 
Additional Family Sample RMs 
• Prefer to select one additional large family 
– Ethnically ...
Evaluation of Synthetic Controls 
Goal Planning 
• Hypothesis: Synthetic controls are good surrogates for DNA isolated fro...
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Aug2014 working group report rm selection and design

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Aug2014 working group report rm selection and design

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Aug2014 working group report rm selection and design

  1. 1. Genome in a Bottle Working Group Reference Material (RM) Selection and Design NIST Workshop August 14 + 15, 2014 Andrew Grupe
  2. 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 | 2
  3. 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 | 3
  4. 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 | 4
  5. 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 | 5 Need interlab study to assess the qualifications of synthetic materials vs. full genomic DNA to inform utility of synthetic RMs
  6. 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? | 6
  7. 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 | 7
  8. 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 | 8

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