Detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a low level, is challenging. Single Molecule, Real-Time (SMRT®) Sequencing is ideal for minor variant detection because of its ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach.

Here, we characterize the Sequel System for the detection of low-frequency somatic variants using constructs containing mutations in coding regions in EGFR, NPM1, AKT1 and JAK2 representing deletion, insertion, substitution and homopolymer variants. Wild type and mutant amplicons, provided by SeraCare, were mixed and serially diluted from 10% down to 0.1% allelic frequency. Independent SMRTbell libraries were constructed for each dilution point, sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants. We will demonstrate sensitivity of the PacBio Systems to detect mutations down to 0.1%.

Citation Format: Primo Baybayan, Laura Nolden. Detection of low-frequency somatic variants using single-molecule, real-time sequencing [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 5366. doi:10.1158/1538-7445.AM2017-5366