Quantitative PCR-Based Method to Assess Cell-Free DNA Quality, Adjust Input Mass, and Improve Next-Generation Sequencing Assay Performance

Abstract

Cell-free (cf)DNA-based testing has undergone increasingly wide adoption, including assays for the detection of circulating tumor DNA. Due to nucleosome protection, cfDNA has a distinctive fragment size of 160 to 180 bp. However, cfDNA can be contaminated with high molecular weight genomic DNA from blood cells released in plasma during sample collection. Such contamination can lead to decreased sensitivity or inconsistent results in cfDNA next-generation sequencing assays. This article describes a technical advancement in which a quantitative PCR method is used for high molecular weight contamination assessment and input mass adjustment, and has been demonstrated to improve consistency of performance in a circulating tumor DNA next-generation sequencing workflow.