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The Pathologist / App Notes / 2019 / Comparison of cfDNA Reference Material Prepared using Enzymatic Fragmentation or Sonication for the Validation of Liquid Biopsy Assays

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

05/28/2019

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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

Introduction

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.

Methods

DNA extracted from engineered cancer cell lines, representing the Multiplex 1 blend at 5% or 0.1%, was fragmented by mechanical or enzymatic shearing. In addition, a size selection step was included to obtain a fragment size distribution profile that closely mimics real cfDNA samples. The allele frequency of specific variants was confirmed by ddPCR. The eight-sample cfDNA material experimental set was externally tested on the Illumina TruSight Tumor15 (TST-15) panel and the Oncomine Breast cfDNA Assay v2 (OBA v2) to assess library preparation and variant calling performance.

NGS: Sequencing was performed on the MiSeqDx system in RUO mode and the Ion S5 for the TST-15 and OBA v2 assays respectively. MiSeqDx system filter settings for analysis with Variant Studio (and automatic analysis) were: Read depth >500 and MAF >2%.

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