Abstract 3621: High efficiency detection of low frequency alleles in cell-free DNA

Abstract

Abstract Cancer is a genetic disease, caused by a variety of alterations to a cell's genome. One specific type of alteration associated with cancer is point mutations, and Next-generation sequencing (NGS) is a key tool to identify those mutations. Unlike inherited mutations, cancer mutations can be found at very low frequencies, mainly because of tumor heterogeneity and their dilution in the germline background. Cell-free DNA (cfDNA) derived from blood plasma, is a promising material for cancer research and diagnosis, and can be readily obtained via non-invasive procedures. However, the amount of tumor DNA and, hence, the amount of rare allele in the cfDNA are typically very low. Sensitive methods are therefore needed for the detection of somatic, cancer-associated mutations in cfDNA isolated from liquid biopsies to support both discovery and clinical decision making in oncology. To overcome these challenges, we developed at Rubicon Genomics an NGS library preparation kit, namely ThruPLEX® Plasma-seq, optimized specifically for cfDNA at input levels from less than 1 ng to 30 ng. We designed experiments to determine the lower limit of SNV detection under these assay conditions. Six individual ThruPLEX® Plasma-seq libraries generated from 10 ng cfDNA were enriched with the Agilent SureSelect XT2 reagents and ClearSeq Comprehensive Cancer panel (151 genes). After sequencing to >500X coverage, SNP calling was performed using Agilent SureCall software to identify the SNPs specific to each sample. In a second step, the cfDNA isolated from two of these plasma samples with known SNP differences were mixed to generate various spike-in frequencies (10%, 5%, 2% and 0.5%) prior to preparing libraries and enrichment. This is to simulate low allele frequency, and to determine the sensitivity of ThruPLEX® Plasma-seq under the outlined assay conditions. Our results showed that it was possible to detect specific alleles down to an actual 0.6% with the theoretical 0.5% spike-in ratio. The percent of on­target bases was over 45%. The sensitivity (SNPs detected/total SNPs analyzed) of our test was above 97% for 10%, 5%, and 2% frequencies and 70% for 0.5%. This is a demonstration of ThruPLEX® Plasma-seq to produce libraries with high enough complexity to detect low frequency alleles from limited amounts of cfDNA from plasma. Citation Format: Kamran Shazand, Jing Ning, Anthony Popkie, Egon Ranghini, John Paul Jerome. High efficiency detection of low frequency alleles in cell-free DNA. [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 3621.