Novel, single-tube, multiplex rhPCR technology for detection of low-frequency variants in BRCA1 and BRCA2

Targeted next-generation sequencing (NGS) technologies serve as critical tools for cancer diagnostics. For example, individuals with pathological BRCA1 and BRCA2 (BRCA1/2) mutations have a dramatically increased risk for certain cancers, including breast and ovarian cancer. Those mutations are either germline or somatically acquired, and play an important role in cancer risk assessment, prevention of malignancy, and management of disease. A variety of methodologies are currently available to screen for BRCA1/2 mutations. Amplicon-based sequencing technologies are prevalent due to their easy workflow but have specific challenges due to inherent limitations of PCR, such as difficulties with amplification of overlapping amplicons for contiguous coverage in a single-tube or with detection of somatic low-frequency variants due to high polymerase error rates. We developed a robust, highly multiplex rhPCR workflow to screen for BRCA1/2 mutations across their entire coding regions in a single-tube. This method allows for detection of low-frequency variants through use of unique molecular identifiers (UMIs), which correct for PCR and sequencing errors and dramatically increase specificity. This method supports a wide DNA input range (1 to 50 ng) from cell-free, formalin-fixed paraffin-embedded (FFPE), or small biopsy samples. Our rhAmpSeq™ chemistry uses a two-enzyme system coupled with uniquely designed RNA-DNA hybrid primers, which enable the amplification of overlapping, multiplexed amplicons. Thus, we eliminate the need to split precious DNA samples into multiple tubes and prevent UMI hopping. Here, we demonstrate the performance of our novel, highly specific amplicon-based technology for detection of low-frequency variants of BRCA1/2 and compare our technology to current industry standards. Citation Format: Nicole Sponer, Yu Wang, Junzhou Wang, Shengyao Chen, Kevin Lai, Scott Rose, Patrick Lau. Novel, single-tube, multiplex rhPCR technology for detection of low-frequency variants in BRCA1 and BRCA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5310.