Table 1.
KRAS mutations observed in human cancer in order of decreasing frequency (http://www.sanger.co.uk/cosmic; accessed 14th July 2015).
Table 2.
KRAS mutations observed in human NSCLC in order of decreasing frequency (http://www.sanger.co.uk/cosmic; accessed 14th July 2015).
Fig 1.
KRAS duplex assays at optimal annealing temperature.
Droplet populations observed for each duplex assay tested with wild-type and relevant mutant cell line gDNA or oligonucleotide at the optimal annealing temperature e.g. G12V panel top left shows droplet populations seen with WT for G12V assay, G12V assay, NCI-H727 gDNA and NCI-H1975 gDNA present. HEX amplitude is up to 6000 on the x axis and FAM amplitude up to 11000 on the y-axis of each panel. Key: Black drops- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets.
Fig 2.
Four different KRAS multiplex digital PCR assays combining G12C, G12V and G12D mutant assays and corresponding duplex assays.
KRAS G12C mutant droplet populations are indicated by a red dashed square, KRAS G12D mutant populations by a blue dashed square and KRAS G12V mutant populations by a yellow dashed square. Each multiplex assay, combining all relevant FAM and HEX assays, KRAS WT gDNA and G12V, D and C mutant gDNA, is shown in the top panel. The corresponding duplex assay for each mutation, using the same FAM and HEX assay concentration with KRAS WT DNA and the appropriate KRAS mutant DNA present, is shown in the panels below each multiplex assay. Multiplex 1 (top left panel) is an assay combination of 900 nM primers and 500 nM G12C probe, 562.5 nM primers and 312.5 nM G12D probe and 225 nM primers and 125 nM G12V probe with 450 nM primers and 250 nM WT for G12C probe. Multiplex 2 uses the same concentration of G12V and WT for G12C assay as Multiplex 1 but also contains 450 nM primers and 250 nM G12C probe and 675 nM primers and 375 nM G12D probe. Multiplex 3 uses the same concentration of KRAS mutant assays as in Multiplex 2 but with the WT for G12V probe assay present, used at the same concentration as the WT for G12C assay in Multiplex 1. Multiplex 4 is an assay combination of the G12C assay as in Multiplex 2 with 225 nM primers and 125 nM G12D probe and 675 nM primers and 375 nM G12V probe with the WT for G12D assay at the same concentration as the WT for G12C assay in Multiplex 1. Key: black- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets. The same scale is used for all panels (HEX amplitude up to 7000 and FAM amplitude up to 22000).
Table 3.
Detection of KRAS mutant clones in FFPE tissue DNA using next generation sequencing (NGS) and KRAS multiplex digital PCR assays.
Fig 3.
KRAS multiplex digital PCR assays A-C and corresponding duplex assays.
Multiplex A (top left panel) is an assay combination of 900 nM primers and 500 nM G13C probe (red dashed square), 450 nM primers and 250 nM G12C probe (blue dashed square) and 225 nM primers and 125 nM G12V probe (yellow dashed square). Multiplex B (top middle panel) is an assay combination of 675 nM primers and 375 nM G12S probe (red dashed square), 450 nM primers and 250 nM G12D probe (blue dashed square) and 225 nM primers and 125 nM G13D probe (yellow dashed square). Multiplex C (top right panel) is an assay combination of 675 nM primers and 375 nM G12R probe (red dashed square), 450 nM primers and 250 nM G12A probe (blue dashed square) and 900 nM primers and 500 nM Q61H probe (yellow dashed square). Multiplex C has 900 nM primers and 500 nM Q61H wild-type probe in addition to a G12C wild-type assay. All other wild-type droplet populations shown, except in the Q61H duplex assay, are 450 nM primers and 250 nM G12C wild-type probe. All panels in the left and centre columns show a FAM amplitude up to 18000 and an HEX amplitude up to 6000. Panels in the right column have a FAM amplitude up to 18000 and a HEX amplitude up to 11000.
Fig 4.
Correlation of mutant DNA fraction detected in multiplex and duplex assays using cell line gDNA or oligonucleotides (left) and in FFPE tissue DNA (right).
Fig 5.
KRAS mutant FFPE tissue DNA analysis using multiplex and duplex assays to detect KRAS mutant clones.
All samples, except for S011, were analysed with multiplexes A, B and C (upper panels) and the KRAS mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a KRAS mutant DNA species not present in the multiplex are indicated by a yellow dashed square.
Fig 6.
KRAS mutant biclonal gDNA analysis using multiplex and duplex assays to detect KRAS mutant clones.
All samples were analysed with multiplexes A, B and C (upper panels) and the KRAS mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a KRAS mutant DNA species not present in the multiplex are indicated by a yellow dashed square.
Table 4.
Detection of KRAS mutant clones in biclonal gDNA samples using next generation sequencing (NGS) and KRAS multiplex digital PCR assays.
Two individual library preparations were analysed for each sample.
Fig 7.
Correlation of NGS KRAS mutant allele frequency with digital PCR KRAS mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.