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

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posted on 2016-12-09, 00:00 authored by Alexandra S. Whale, Alison S. Devonshire, George Karlin-Neumann, Jack Regan, Leanne Javier, Simon Cowen, Ana Fernandez-Gonzalez, Gerwyn M. Jones, Nicholas Redshaw, Julia Beck, Andreas W. Berger, Valérie Combaret, Nina Dahl Kjersgaard, Lisa Davis, Frederic Fina, Tim Forshew, Rikke Fredslund Andersen, Silvia Galbiati, Álvaro González Hernández, Charles A. Haynes, Filip Janku, Roger Lacave, Justin Lee, Vilas Mistry, Alexandra Pender, Anne Pradines, Charlotte Proudhon, Lao H. Saal, Elliot Stieglitz, Bryan Ulrich, Carole A. Foy, Helen Parkes, Svilen Tzonev, Jim F. Huggett
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 KRAS 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.

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