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140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
140127 rm selection wg summary
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140127 rm selection wg summary

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  • 1. Genome in a Bottle Working Group Reference Material (RM) Selection and Design NIST Workshop January 27 & 28, 2014
  • 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. 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. 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. 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. 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. 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. 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. 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. 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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