Abstract 3646: Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing

Abstract

Abstract Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust but also capable of accurately detecting low-frequency somatic mutations. Single Molecule, Real-Time (SMRT®) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays for studying cancer variants. Here we characterize SMRT Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standards from Horizon Diagnostics, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay. First, we sequenced a reference standard containing precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. This reference material recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot MASTR Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. We also sequenced a second sample containing a series of mixes, each with known mutations, at levels below 10% and down to 0.01%. PCR-amplified targets were 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. Citation Format: Steve Kujawa, Anand Sethuraman, Kevin Eng, Primo Baybayan, Lien Heyrman, Jurgen Del Favero. Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3646.