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Abstract Extraction of cell free DNA (cfDNA) from a patient’s blood sample is a crucial preanalytic step of the liquid biopsy process. Whether testing is being performed for early disease diagnosis, therapy selection, or disease and treatment surveillance, the input material must be of sufficient quantity as well as high quality to return informative data. In addition, accurate capture of the full cfDNA content from a sample is necessary to evaluate tumor growth or recurrence. Fragmentomics is an emerging field that can indicate tumor origin among other information, and again, accurate and precise cfDNA content is critical. The cfDNA content in patient plasma is highly variable, and sample mass is quite limited compared to that of resected tumors. Healthy donor plasma, though abundant, varies in cfDNA levels batch to batch and should not contain any somatic variants relevant to liquid biopsy assay development or validation. Despite the challenges associated with cfDNA isolation and ctDNA analysis there are few reference materials available to optimize laboratory process for these preanalytic challenges. Here we evaluate various commercially available extraction kits using novel Seraseq® cfDNA Extraction Reference Materials. Genomic DNA from the GM24385 cell line was blended with 4 biosynthetic DNA EGFR variants targeted at 1% allele frequency and verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The mixed DNA was fragmented and size selected to create ctDNA-like size profiles. The ctDNA was mixed into a synthetic plasma matrix to simulate native cfDNA samples and diluted to three distinct concentrations (20, 50, and 80 ng/mL). Verification of concentration was performed using the QIAGEN QIAamp Circulating Nucleic Acid Kit for extraction. The final materials were also extracted with other kits: the Maxwell RSC ccfDNA Plasma Kit, the MagMAX Cell-Free DNA Isolation kit, the Zymo MAGicBead™ cfDNA Isolation Kit, and the Takara NucleoSnap cfDNA Kit. Yields were measured with Qubit dsDNA BR assay and EGFR variants were measured using Bio-Rad ddPCR. Other sites evaluated the materials as well, using various kits. The kits showed variable yields, but there was a clear difference between the cfDNA extracted from each reference material correlating with the intended increasing concentration. The EGFR variants were all present at ~1% regardless of the cfDNA concentration and the kit used for extraction. Seraseq ctDNA Plasma Reference Materials serve to evaluate extraction of cfDNA in liquid biopsy testing workflows without having to source donor plasma, which is limited and variable in cfDNA content. The consistency and availability of these biosynthetic materials allow for optimization without risking patient samples. Citation Format: Dana Ruminski Lowe, Sanchita Jamindar, Andrew Anfora, David Merriam, Catherine Huang, Russell Garlick, Bharathi Anekella. Evaluation of commercially available cell free DNA extraction kits using biosynthetic reference materials abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5040.
Lowe et al. (Fri,) studied this question.