140127 rm selection wg summary

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140127 rm selection wg summary

  1. 1. Genome in a Bottle Working Group Reference Material (RM) Selection and Design NIST Workshop January 27 & 28, 2014
  2. 2. Reference Material – Intended Uses • Characterize Platforms & Methods – DNA sequencing – Existing & upcoming NGS technologies – Research applications – Clinical diagnostics applications • Not intended as reference material for – Validation of specific mutations in a panel | 2
  3. 3. Preferred Reference Standard Criteria Reference Lab Perspective • Commercially available (renewable and constant) • Cost-effective • Similar in performance to anticipated test samples • Contain representative type and number of mutations tested • Ability to adjust mutation frequency to monitor assay sensitivity • Recognized by agencies responsible for recommending standards
  4. 4. Agenda RM Selection and Design Working Group • Select one or two genomes as primary genomes? • NCI Cancer spike-ins (Mickey Williams, Jason Lih) • Tumor-normal pair & synthetic fusion constructs (TGenIllumina) • FFPE embedded reference material • Commercial Reference Materials, including Acrometrix and Horizon Diagnotics • Organize a potential interlaboratory study on these RMs – What is the necessary framework for sample access (prior and post interlab study) – How will we assess the interlab-study outcome – How will we communicate the study outcome – Define a testing protocol
  5. 5. Agenda RM Selection and Design Working Group • Future Reference Material Genomes – Priorities for selecting future genomes • • • • • Ancestry Larger families Consent for research & commercial use Trios, child only, … Should we select tumor normal pair(s) – How can they be recruited | 5
  6. 6. DNA Samples at NIST • NA12878 • Eastern European Ashkenazi Jewish father-motherson trio – PGP IDs: huAA53E0/hu8E87A9/hu6E4515 – Coriell IDs: GM24143/GM24149/GM24385 • Son of Chinese trio – PGP IDs: hu91BD69/hu38168C/huCA017E – Coriell IDs: GM24631/GM24695/GM24694 | 6
  7. 7. Summary Primary Genomes • Purpose for prioritization – Achieve highest accuracy of reference • High depth of characterization of one or two genomes • Sequence primaries on platforms with limited access • Lower level characterization of the other reference genomes • Prioritized genomes – Son of Eastern European Ashkenazi Jewish fathermother-son trio • huAA53E0 – Son of Chinese trio • hu91BD69 | 7
  8. 8. Summary Other types of Reference Materials • 53 plasmids with engineered cancer mutations (Mickey Williams, NCI) – – – – 1kb fragments Mutation in center Alien barcode in vicinity for differentiation from human DNA Sequence is Sanger confirmed • • Large set of engineered cancer mutations in 3 cancer cell lines (Brian Burke, Horizon Diagnostics) – – – – • Proprietary engineered cell lines Confirmed identities of parental cell lines Digital PCR confirmed mutational frequencies FFPE embedded cells / DNA available Engineered cell line controls (Kara Norman, Acrometrix/ Life Technologies) – – 8 different multi mix controls 12-26 variants per control • – – – • Low level mutations not observed in NGS verification 1-7 COSMIC variants / control Standards produced under cGMP / Quality System Proprietary cell lines FFPE embedded cells / DNA available 9 synthetic RNA fusion constructs (Han-Yu Chuang, Illumina/Tgen) – Spiked into COLO829 DNA ABRF: Assess performance and resemblance to ‘normal’ DNA in mixing study/spike in comparison (cell line spike ins, possibly also 53 plasmids) Prototype ‘User Repository; develop, evaluate & test performance dashboard Opportunity for independently supplied reference materials | 8
  9. 9. Summary Large Families • Can produce high accuracy sequence with inheritance check • Needs high coverage for one sample, lower coverage for remaining samples – See presentations by Francisco De La Vega and Michael Eberle Assess In vitro fertilized eggs (embryos) and parents as potential reference material source => needs legal review Could use parents as reference material and embryo sequence to support parental reference | 9
  10. 10. Summary Tumor-Normal Pairs • To resemble somatic mutation analysis workflow • Mixing of normal cell lines can mimic some aspects of workflow, but not completely • Advantages of matched cells are – – – – • • • • • Small number of changes Copy number changes in tumor sample Potential rearrangements in tumor sample => higher confidence in establishing tumor reference calls • Presence/absence of mutation at wide frequency spectrum Consider Ashkenazi father (hu6E4515), had colon tumor removed – assess tissue availability to generate cell line Sarcoma – large tumors that could serve as reference w/o cell line need Haematological Cancer & Solid Tumor Liaise with TCGA and others for sample access and selection ATCC: Tumor normal cell lines: HCC1187 , HCC2218 and –BL (normal) | 10

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