Abstract 2404: Quality assessment of circulating cell-free DNA using multiplexed droplet-digital PCR

Abstract

Abstract Recent proof-of-principle studies have demonstrated potential utility of sequencing cell-free DNA in cancer diagnostics. However, little is understood about the effect of fragment size distributions in plasma DNA on the performance of sequencing-based assays. We developed a multiplexed assay to perform one-step analysis of DNA quantity and integrity from minute amounts of cell-free DNA using picoliter droplet digital PCR. Primers were designed to amplify 5 short PCR products (67-71 bp) and 4 long PCR products (439-522 bp) from independent quiescent regions of the human genome. Oligonucleotide probes targeting each of the short and long products were labeled with fluorescent dyes FAM and TET respectively. We evaluated the performance of our assay using control human genomic DNA sheared using sonication to an average of 150 bp, 300 bp, 500 bp and 1000 bp fragment sizes. We observed that relative quantities of short and long PCR products reflect integrity of sheared input DNA. We further observed that accurate quantification and assessment of integrity could be achieved using picogram amounts of cell-free DNA (as few as 200 pg in current assessments). Finally, our results were predictive of diversity and obtainable depth-of-coverage in next-generation sequencing libraries made from cell-free DNA samples. Citation Format: Tania Contente-Cuomo, Muhammed Murtaza. Quality assessment of circulating cell-free DNA using multiplexed droplet-digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2404. doi:10.1158/1538-7445.AM2015-2404