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A Plasmid based Reference/Control Strategy for Targeted NGS in Oncology

A Plasmid based Reference/Control Strategy for Targeted NGS in Oncology. Why Plasmids?: Addition of alien bar-code permits identification in a mixture

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A Plasmid based Reference/Control Strategy for Targeted NGS in Oncology

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  1. A Plasmid based Reference/Control Strategy for Targeted NGS in Oncology • Why Plasmids?: • Addition of alien bar-code permits identification in a mixture • Multiple plasmids can be mixed into the background of a human reference genome providing controls for multiple targets and sequence variants (missense, in/dels, homopolymers etc.) • Rapid design, manufacture and sequence verification • Possible disadvantage, does not represent a genome (i.e. large DNA fragments), therefore does it perform similarly to a human genomic DNA sample in the targeted assay? • Uses: • Reference Standards • Spike in assay positive controls

  2. Control Plasmids for Reference Standards * MOI at highest mutation frequency per gene MOI Alien Barcode (6bp) * ~1000bp Cloning * Verify by Sanger Precise quantification Blend with normal at pre-defined ratios Access Specificity, Sensitivity, and Limit of detection during assay development Internal controls to monitor assay performance run by run Reference materials for comparing results of assays performed by different platforms, operators, times, and sites Applicable for all kinds of assays - amplicon targeted seq, exome capture, Haloplex, HRMC (PCR), Pyroseq, Sanger seq, … 2

  3. Control Plasmid Spike-in CEPH (CPSC) * * * * * BRAF V600E KRAS G12V PIK3CA H1047R AKT1 F35L MET Y1253D Mix with normal genomic DNA (mutant vs normal) at various copy number ratios 1:5 1:10 1:20 1:40 1:80 20% 10% 5% 2.5% 1.25% 3

  4. Assay Detects Mutations at 5% Frequency PGM/Ampliseq Miseq/TSCA Observed Frequency CEPH CEPH 5%CPSC 5%CPSC 10%CPSC 10%CPSC 2.5%CPSC 2.5%CPSC 20%CPSC 20%CPSC 1.25%CPSC 1.25%CPSC 4

  5. List of MOIs in Control Plasmids PIK3CA_p.H1047R PTEN_p.R130* PTPN11_p.E76K RAD51_p.Q145* RB1_p.R320* NBN_p.P748S DNMT3A_R882C EGFR_E746_A750del EGFR_L858R EGFR_T790M EZH2_Y646F FLT3_D835Y IDH1_R132C ERBB2_M774_A775insAYVM IDH2_R140Q IDH2_R172K JAK2_V617F KIT_D816V MPL_W515L MYD88_L265P NPM1_W288fs*12 RET_M918T TP53_R273H TP53_R248Q TP53_R175H …… AKT1_p.E49K AKT1_p.E17K AKT2_p.V90L AKT3_p.Q124L APC_p.R1450* ARHGAP5_p.E622K ATM_p.D1853N ATR_p.I1264fs*24 BRAF_p.V600E CTNNB1_p.T41A ERCC1_p.A96E FBXW7_p.R465H FGFR3_p.S249C GABRA6_p.V255L GABRG2_p.Y452C GNAQ_p.Q209L GNAS_p.R201C KRAS_p.G12V MET_p.Y1253D MLH1_p.V384D MSH2_p.G751fs*12 MTOR_p.L888F NF1_p.R1362* NRAS_p.Q61R PARP1_p.G913fs*4 PARP2_p.D133A PDGFRA_p.D842V PIK3CA_p.E545K Challenge MOIs (in homopolymer region) APC_T1556fs*, (insA in AAAAAACT) PTEN_ K267fs*9 , (delA in AAAAAAGG) NF1_M799fs*22 , (delA in CCAAAA) 5

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