Abstract 5360: DNA repair increases sequencing accuracy without altering actual mutation frequency in clinical samples

Abstract

Abstract Targeted cancer therapy based on genomic alterations can be remarkably effective. Currently, cancer genome profiling using next generation sequencing (NGS) is routinely applied in cancer care to guide personalized treatment. The accuracy of this profiling directly impacts therapeutic choices and the outcomes of patient care. We previously showed that false positive variants are abundant and can account for a major fraction of identified somatic variations in publicly available datasets (doi: http://dx.doi.org/10.1101/070334). These false positive variants show signs of mutagenic DNA damage. We further demonstrated that enzymatic DNA repair increases sequencing quality by lowering damage-induced background noise. Therefore, enzymatic DNA repair has the potential to improve sequencing accuracy, avoiding incorrect somatic variant calls and consequently reducing incorrect diagnostic conclusions. In this study, we investigated whether enzymatic DNA repair introduces any bias to NGS libraries using analysis by droplet digital PCR (ddPCR) and deep sequencing. DNA Reference Standards containing multiple common cancer mutations (Horizon Discovery, Inc.) were spiked into formalin-fixed paraffin-embedded (FFPE) DNA isolated from tumor samples from different tissue types at defined frequencies (0.5-10% quantified by ddPCR). Genotyping of the FFPE DNA ensured that they were free of any of the spiked-in mutations. After DNA repair and library preparation, mutation frequencies were quantified by ddPCR, and compared to the mutation levels in input DNA and control libraries without repair. Deep sequencing of 151 cancer genes including these spike-ins showed no difference in mutation frequency for the spiked-in mutations between the control and repair groups. However, the number of false positive variant calls was reduced in the repair group. Our data demonstrates that DNA repair significantly increases sequencing accuracy without altering the frequency of actual mutations in tumor samples. Citation Format: Pingfang Liu, Lixin Chen, Laurence Ettwiller, Eileen Dimalanta, Theodore B. Davis, Thomas C. Evans. DNA repair increases sequencing accuracy without altering actual mutation frequency in clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2017-5360