Frederick National Laboratory for Cancer Research

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

Frederick National Laboratory for Cancer Research

*MOI Alien Barcode (6bp)

~1000bp

* MOI at highest mutation frequency per gene

Control Plasmids for Reference Standards

*Cloning

Verify by SangerPrecise quantification Blend with normal at pre-defined ratios

2

1. Access Specificity, Sensitivity, and Limit of detection during assay development2. Internal controls to monitor assay performance run by run 3. Reference materials for comparing results of assays performed by different

platforms, operators, times, and sites4. Applicable for all kinds of assays - amplicon targeted seq, exome capture,

Haloplex, HRMC (PCR), Pyroseq, Sanger seq, …

Frederick National Laboratory for Cancer Research

Control Plasmid Spike-in CEPH (CPSC)

*BRAF V600E

*KRAS G12V

PIK3CA H1047R

* AKT1 F35L

* METY1253D

*

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

Frederick National Laboratory for Cancer Research

Assay Detects Mutations at 5% Frequency

4

0 5 10 15 20 250

5

10

15

20

25

30

PIK3CA_ITLinear (PIK3CA_IT)MET_ITLinear (MET_IT)KRAS_ITLinear (KRAS_IT)BRAF_IT

0 5 10 15 20 250

5

10

15

20

25

PIK3CA_MiseqLinear (PIK3CA_Miseq)MET_MiseqKRAS_MiseqLinear (KRAS_Miseq)

Miseq/TSCAPGM/Ampliseq

Ob

served F

requ

ency

20

%C

PS

C

10

%C

PS

C

5%

CP

SC

2.5

%C

PS

C

1.2

5%

CP

SC

20

%C

PS

C

10

%C

PS

C

5%

CP

SC

2.5

%C

PS

C

1.2

5%

CP

SC

CE

PH

CE

PH

Frederick National Laboratory for Cancer Research

List 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.V255L

GABRG2_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.E545K

PIK3CA_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)