Abstract
Abstract FFPE tumor biopsies are being increasingly used as a source of DNA for translational research in pursuit of personalized medicine. In addition, circulating cell-free DNA in liquid biopsy fractions shows promise as a means of tracking tumor burden, tumor regression, and/or cancer relapse. However, both sources come with significant DNA quality risks related to sample preparation that can result in downstream sequencing failures or false negatives. For FFPE samples, chemical fixation of the tissue crosslinks DNA, resulting in DNA fragmentation and damage that cannot be completely reversed. For liquid biopsy samples, contamination with genomic DNA can reduce sensitivity for detecting the tumor-specific fraction of ccfDNA. The ProNex® DNA QC Assay is a multiplex qPCR assay that provides both quantification and quality control for small or fragmented DNA samples. ProNex® DNA QC Assay includes a 75bp, 150bp, and 300bp target as well as an internal positive control. Unlike absorption, fluorescent DNA binding dyes, and electrophoresis-based methods, qPCR can be used to quantify specifically amplifiable DNA able to participate in downstream PCR or sequencing applications. The ratio of amplification at 75bp and 300bp sizes can be used to assess fragmentation of FFPE samples, which is correlated with improved coverage uniformity in NGS applications. With ccfDNA, the ratio can also be used to determine the degree of genomic DNA contamination and indicate when further clean-up is necessary prior to sequencing. Citation Format: Trista L. Schagat, Jennifer Mook, Leta Steffen, Doug Wieczorek, Curtis Knox. Measuring DNA quantity, FFPE degradation, and genomic DNA contamination of ccfDNA in a single assay [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 2526.
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