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Multi-Laboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements



Hua-Jun He, Erica V. Stein, Yves Konigshofer, Thomas Forbes, Farol L. Tomson, Russell Garlick, Emiko Yamada, Tony Godfrey, Toshiya Abe, Koji Tamura, Michael Borges, Michael Goggins, Sandra Elmore, Margaret L. Gulley, Jessica L. Larson, Lando Ringel, Brian C. Haynes, Corinne Camalier, Chris Karlovich, Biswajit Das, P. M. Williams, Aaron Garnett, Anders Stahlberg, Stefan Filges, Lynn Sorbara, Mathew R. Young, Sudhir Srivastava, Kenneth D. Cole


We conducted a multi-laboratory assessment to determine the suitability of a new commercially- available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele fractions (VAF): 2%, 0.5%, 0.125%, and 0 %. The variants include single nucleotides, insertions, deletions, and two structural variations selected both for their clinical importance, and to challenge the performance of next generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild- type and tumor DNA (ctDNA) in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, and automated methods resulted in lower yields. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods (including targeted amplicon or hybrid capture with and without molecular barcodes). This multi-laboratory assessment demonstrates that the new reference material is an appropriate tool for comparing the performance of different approaches and ensuring the reproducibility of ctDNA measurements including pre-analytical, analytical, and post-analytical data analysis steps.
Journal of Molecular Diagnostics


cancer, reference material, digital PCR, next generation sequencing, biomarkers, variants, allele fraction


He, H. , Stein, E. , Konigshofer, Y. , Forbes, T. , Tomson, F. , Garlick, R. , Yamada, E. , Godfrey, T. , Abe, T. , Tamura, K. , Borges, M. , Goggins, M. , Elmore, S. , Gulley, M. , Larson, J. , Ringel, L. , Haynes, B. , Camalier, C. , Karlovich, C. , Das, B. , Williams, P. , Garnett, A. , Stahlberg, A. , Filges, S. , Sorbara, L. , Young, M. , Srivastava, S. and Cole, K. (2019), Multi-Laboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements, Journal of Molecular Diagnostics, [online],, (Accessed April 14, 2024)
Created May 1, 2019, Updated October 12, 2021