PO-317 Improved performance and workflow using sanger sequencing for low-level somatic mutation detection from low amount of formalin-fixed paraffin-embedded (FFPE) DNA

Abstract

IntroductionSanger sequencing is often used in oncology research applications for molecular profiling of cancers. Applied Biosystems Minor Variant Finder (MVF) Software now enables low-level variant detection in Sanger sequencing traces.RAS mutational testing is frequently performed by clinical researchers due to the strong correlation between RAS mutational profiles of colorectal cancers and their anti–epidermal growth factor receptor (EGFR) response.Material and methodsWe have improved and simplified the workflow for the extended RAS research assay. The ready-to-use plates are preloaded with primers for all the eight frequently studied hotspot regions of KRAS and NRAS genes thus only the PCR mix and templates need to be added. Minimised hands-on time, extra convenience and flexibility gained by using the SeqStudio Genetic Analyzer which utilises an all-in-one cartridge and provides simple plate setup with easy-to-use touch-screen interface and real-time/remote data monitoring.Results and discussionsThe performance of the assay on the SeqStudio instrument was tested in the laboratory of Dr. Quagliata (University of Basel, Switzerland) on twenty-two FFPE DNA samples derived from colon cancer biopsies. Variants (ranging from 5.25% to 91.1%) were successfully detected even from research samples where less than 1 ng of input DNA per reaction was used. Only limited amount of microdissected FFPE sections were available for 6 research samples where DNA concentrations were below 1 ng (down to 0.094 ng).ConclusionRAS mutations down to 5% level can be detected even from less than 1 ng FFPE DNA with simplified workflow, fast turnaround time and low cost per sample.