Jan2016 seracare giab update d yuzuki
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This document describes reference materials developed by SeraCare Precision Medicine for next generation sequencing applications. It discusses Seraseq tools that provide mutations, aneuploidies, fusions and cell-free DNA references to validate assays. Specifically, it details the Seraseq Solid Tumor Mutation Mixes which contain cancer-related single nucleotide variants and indels down to 1% allele frequency to test assay sensitivity. A prototype cell-free DNA reference material is also described that contains 9 mutations across a 2-log allele frequency range from 5% to 0.1% to validate plasma DNA sequencing.
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Jan2016 seracare giab update d yuzuki
- 1. SeraCare Precision Medicine Reference Materials GIAB Dale Yuzuki M.A., M.Ed. Market Development Director dyuzuki@seracare.com @daleyuzuki January 28 2016
- 2. Solid Tumor Cell-free fetal DNA Cell-free tumor DNA Seraseq Solid Tumor Mutation Mix-I • AF20 • AF10 • AF8 AF5 AF3 AF1 Seraseq FFPE Multiplex RNA Fusions • 12 plex Seraseq™ Tools for NGS Seraseq Aneuploidy Reference Materials • T21 • T18 • T13 • Triplex (T21,18,13) • 4%ff-12%ff Infectious Disease Seraseq ctDNA Reference Materials • 9 Mutations • Allele Frequency from 5% down to 0.1% • Full Process Seraseq HIV-1 DR Reference Materials Inherited Disease Cardio- myopathy Panel
- 3. BRAF V600E KRAS G12D FLT3 D834Y TP53 R273H Quantitate (via dPCR) and Pool Multiple Constructs Formulate into reference genomic DNA GM24385 at defined ratio 10% 20% AF Seraseq™ Solid Tumor Mutation Mix–I 26 Individual Mutation Biosynthetics PIK3CA H1047R PIK3CA N1068fs*4 EFGR E764_A750del MPL W515L RET M918T ATM C353fs*5 ~1000bp Mutation Of Interest (MOI) Vector backbone Vector backbone
- 4. COSMIC ID Gene Mutation Type CDS Amino Acid Change COSM6224 EGFR Homopolymer c.2573T>G p.L858R COSM715 FGFR3 Homopolymer c.746C>G p.S249C COSM28758 GNAQ Homopolymer c.626A>C p.Q209P COSM33765 AKT1 Homopolymer c .49G>A p.E17K COSM21924 ATM Small Deletion c.1058_1059delGT p.C353fs*5 COSM14105 SMAD4 Small Insertion c.1394_1395insT p.A466fs*28 COSM17559 NPM1 Large Insertion c.863_864insTCTG p.W288fs*12 COSM6225 EGFR Large Deletion c.2236_2250del15 p.E746_A750delELREA COSM476 BRAF SNV c.1799T>A p.V600E COSM521 KRAS SNV c.35G>A p.G12D COSM775 PIK3CA SNV c.3140A>G p.H1047R COSM763 PIK3CA SNV c.1633G>A p.E545K COSM584 NRAS SNV c.182A>G p.Q61R COSM10648 TP53 SNV c.524G>A p.R175H COSM5664 CTNNB1 SNV c.121A>G p.T41A COSM28747 IDH1 SNV c.394C>T p.R132C COSM6240 EGFR SNV c.2369C>T p.T790M COSM18918 MPL SNV c.1544G>T p.W515L COSM13127 APC SNV c.4348C>T p.R1450* COSM783 FLT3 SNV c.2503G>T p.D835Y COSM736 PDGFRA SNV c.2525A>T p.D842V COSM965 RET SNV c.2753T>C p.M918T COSM27887 GNAS SNV c.601C>T p.R201C COSM10662 TP53 SNV c.743G>A p.R248Q COSM1314 KIT SNV c.2447A>T p.D816V COSM12600 SNV c.1849G>T p.V617F JAK2 4 SNVs (HP) 4 INDELs 18 SNVs Seraseq™ Mutation Targets
- 5. Seraseq AF20 Tested Across 3 Runs 5 0 5 10 15 20 25 30 35 VariantAlleleFrequency One Lot of Seraseq AF20 Tested Across 3 Runs (Ion Ampliseq) 102226 Run 1 Avg 102226 Run 2 Avg 102226 Run 3
- 6. Lot-to-Lot Consistency: Three lots of Seraseq AF20 tested on Ion Ampliseq 6 0 5 10 15 20 25 30 35 VariantAlleleFrequency Lot 102226 Lot 10119135 Lot 10119136
- 7. Freeze Thaw Stability: Single lot 5-cycle stress test 7 0 5 10 15 20 25 30 35 40 45 50 VariantAlleleFrequency 5 FT Replicate 1 5 FT Replicate 2 Non- stressed Replicate 1 Non- stressed Replicate 2
- 8. Testing Across 3 Different NGS Assays 8 0 5 10 15 20 25 30 VariantAlleleFrequency SeraSeq Solid Tumor Mutation Mix -I (AF20) Ion AmpliSeq Hotspot Panel v2 Custom Ion AmpliSeq Assay TruSeq Amplicon Cancer Panel
- 9. Seraseq AF1-10 Sensitivity Panel
- 10. Seraseq AF1-10 Panel 0 2 4 6 8 10 12 14 16 18 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 AF10 AF8 AF5 AF3 AF1
- 11. Seraseq Mutation Mix-II • 20 SNVs (from 18) • 5 SNVs as part of HP’s (from 4) • 13 InDels (from 4) • March availability 11
- 12. SeraCare Oncology Fusion Prototype • Prototype expresses 12 fusion RNAs simultaneously • Fusion RNAs are in FFPE cells as full process controls • Technology scalable and fully customizable Engineered fusion RNA in human cell line Formalin Fix and FFPE embed cell pellets
- 13. Fusion Partners included RNA Fusion Primary Cancer Tissue 1 EML4-ALK Lung 2 NPM1-ALK Lymphoid 3 KIF5B-RET Lung 4 NCOA4-RET Thyroid 5 CD74-ROS1 Lung 6 SLC34A2-ROS1 Lung, Stomach 7 TPM3-NTRK1 Lung, Large Intestine 8 TFG-NTRK1 Thyroid (rare) 9 FGFR3-BAIAP2L1 Urinary tract (rare) 10 FGFR3-TACC3 Urinary tract, CNS 11 PAX8-PPARG Thyroid 12 ETV6-NTRK3 Kidney, Breast, Soft Tissue
- 14. Seraseq™ Oncology Fusion Prototype and the Archer™ FusionPlex Lung Thyroid Panel 14 Fusion Prototype Negative Control Fusion Spanning Reads EML4-ALK 90 NPM-ALK 136 KIF5B-RET 199 NCOA4-RET 341 CD74-ROS1 54 SLC34A2-ROS1 38 TPM3-NTRK1 127 TFG-NTRK1 124 FGFR3- BAIAP2L1 361 FGFR3-TACC3 396 Pax8-PPARG 152 ETV6-NTRK3 195
- 16. • Technology – Biomimetic – Full process controls • Products – Format any DNA mutation Circulating Tumor DNA
- 17. Towards a ctDNA Process control Building on the recent project success with biomimetic reference materials for non-invasive prenatal screening (NIPS), we are now working to extend our technology platform to ctDNA applications
- 18. Fragmented ctDNA dilution series, 10% to 0.1% AF -Detection of the BRAF V600E mutation use Bio-Rad PrimePCR mutation assay -Demonstrates linearity of the fragmented material across a 2 log series 7-Mutation Dilution series
- 19. NGS data using Swift Accel-Amplicon 56G Oncology Panel • All mutation detected at consistent allele frequencies • Swift reports confidence in their assay at >1% AF, however, CV doubles at <5% AF 19 LOG2scale Linearscale Boxes indicate mutations on same plasmid
- 20. Summary • Seraseq Mutation Mix-I (AF20) and (AF1-10) • Aneuploidy and Cardiomyopathy materials available • Prototype Seraseq ctDNA Reference: 9 SVs/indels from 5% down to 0.125%; scalable on both mutations and AF’s • Prototype Seraseq FFPE Fusion RNA Reference: 12 fusions with 10-fold dynamic range, scalable platform