Why Plasmids Addition of alien barcode 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 ID: 784838
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Slide1
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
Slide2*
MOI
Alien Barcode (6bp)
~1000bp
*
MOI at highest mutation frequency per gene
Control Plasmids for Reference
Standards
*
Cloning
Verify by Sanger
Precise quantification
Blend
with normal at pre-defined
ratios
2
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, …
Slide3Control 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
Slide4Assay Detects
M
utations at 5% Frequency4
Miseq/TSCAPGM/Ampliseq
Observed Frequency
20%CPSC10%CPSC5%CPSC2.5%CPSC1.25%CPSC20%CPSC10%CPSC5%CPSC2.5%CPSC
1.25%CPSCCEPHCEPH
Slide5List of MOIs in Control Plasmids
AKT1
_p.E49KAKT1_p.E17KAKT2_p.V90LAKT3_p.Q124LAPC_p.R1450*ARHGAP5_p.E622K ATM_p.D1853NATR_p.I1264fs*24BRAF_p.V600ECTNNB1_p.T41AERCC1_p.A96EFBXW7_p.R465HFGFR3_p.S249CGABRA6_p.V255LGABRG2_p.Y452CGNAQ_p.Q209LGNAS_p.R201CKRAS_p.G12VMET_p.Y1253DMLH1_p.V384DMSH2_p.G751fs*12MTOR_p.L888FNF1_p.R1362*NRAS_p.Q61RPARP1
_p.G913fs*4PARP2_p.D133APDGFRA_p.D842VPIK3CA_p.E545KPIK3CA_p.H1047RPTEN_p.R130*PTPN11_p.E76KRAD51_p.Q145*RB1_p.R320*NBN_p.P748SDNMT3A_R882CEGFR_E746_A750delEGFR_L858REGFR_T790MEZH2_Y646FFLT3_D835YIDH1_R132CERBB2_M774_A775insAYVM
IDH2_R140QIDH2_R172KJAK2_V617FKIT_D816VMPL_W515LMYD88_L265PNPM1_W288fs*12RET_M918TTP53_R273HTP53_R248QTP53_R175H……
5
Challenge MOIs (in homopolymer region)APC_T1556fs*, (insA in AAAAAACT)PTEN_ K267fs*9 , (delA in AAAAAAGG)NF1_M799fs*22 , (delA in CCAAAA)