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

The National Institute of Standards and Technology (NIST) 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 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, perhaps due to differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multi-laboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and ensuring their quality assurance.

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