<i>KRAS</i> mutations observed in human NSCLC in order of decreasing frequency (http://www.sanger.co.uk/cosmic; accessed 14^th July 2015).

<p><i>KRAS</i> mutations observed in human NSCLC in order of decreasing frequency (<a href="http://www.sanger.co.uk/cosmic" target="_blank">http://www.sanger.co.uk/cosmic</a>; accessed 14^th July 2015).</p

<i>KRAS</i> mutant FFPE tissue DNA analysis using multiplex and duplex assays to detect <i>KRAS</i> mutant clones.

<p>All samples, except for S011, were analysed with multiplexes A, B and C (upper panels) and the <i>KRAS</i> 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 <i>KRAS</i> mutant DNA species not present in the multiplex are indicated by a yellow dashed square.</p

Correlation of NGS <i>KRAS</i> mutant allele frequency with digital PCR <i>KRAS</i> mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.

<p>Correlation of NGS <i>KRAS</i> mutant allele frequency with digital PCR <i>KRAS</i> mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.</p

<i>KRAS</i> multiplex digital PCR assays A-C and corresponding duplex assays.

<p>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.</p

<i>KRAS</i> duplex assays at optimal annealing temperature.

<p>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.</p

<i>KRAS</i> mutations observed in human cancer in order of decreasing frequency (http://www.sanger.co.uk/cosmic; accessed 14^th July 2015).

<p><i>KRAS</i> mutations observed in human cancer in order of decreasing frequency (<a href="http://www.sanger.co.uk/cosmic" target="_blank">http://www.sanger.co.uk/cosmic</a>; accessed 14^th July 2015).</p

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a <i>KRAS</i> fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics