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

NIST WorkshopJanuary 27 & 28, 2014

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

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Preferred Reference Standard CriteriaReference 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

AgendaRM 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 (TGen-

Illumina)• 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

AgendaRM 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

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DNA Samples at NIST

• NA12878• Eastern European Ashkenazi Jewish father-mother-

son 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

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SummaryPrimary 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 father-

mother-son trio • huAA53E0

– Son of Chinese trio• hu91BD69

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SummaryOther 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

• Low level mutations not observed in NGS verification

• 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

• 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

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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 dashboardOpportunity for independently supplied reference materials

SummaryLarge 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

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Assess In vitro fertilized eggs (embryos) and parents as potential reference material source => needs legal reviewCould use parents as reference material and embryo sequence to support parental reference

SummaryTumor-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

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• 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)