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Comparison of cfDNA Reference Material Prepared using Enzymatic Fragmentation or Sonication for the Validation of Liquid Biopsy Assays

Aldo Mele 1, Hannah Child 1, Katarzyna Wilczynska 1, Christian Paar 2 Simi Chacko 3 and Julie Wickenden 1

1 Horizon Discovery, United Kingdom 2 Kepler University Hospital, Austria 3 Atlantic Cancer Research Institute, Canada


Cell-free DNA (cfDNA) can be extracted from a routine patient blood sample and used to determine the genetic profile of a solid tumor located elsewhere within the body. This facilitates more informative disease management for the clinician, without the need for invasive surgery for the patient. With new cfDNA NGS assays being able to detect variants from as little as 2-10ng DNA, assay validation to ensure sufficient accuracy has never been so critical. Reference materials that closely mimic real cfDNA samples are essential to support this effort. Here we present results from a comparative study of DNA fragmentation methods applied during the production of cfDNA reference standards. We show a comparison between enzymatic fragmentation and mechanical shearing (sonication), and the benefit of including a size selection step for data accuracy and performance of NGS gene panel workflow.

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