Liquid biopsies harness cell-free DNA (cfDNA) in the bloodstream, which can detect cancer in its early stages. However, the precision required to discern between malignant and benign genetic markers, and the sensitivity needed for disease monitoring, make their development challenging. The formulation and utilization of specific reference materials are crucial for the successful development of these assays.

Twist Bioscience initially introduced the Twist Pan-Cancer cfDNA Reference Standards to serve as a cfDNA analog reference material. This material featured a broad range of ctDNA variants and closely emulated the size and distribution of cfDNA fragments. It included synthetically-printed variants like single nucleotide variants (SNVs), insertion-deletions (INDELs), and structural variants (SVs). The reference standard covers over 400 variant sites across 84 genes, including literature-curated, clinically-relevant variant sites. Despite its comprehensive features, the first version had its drawbacks. The method used to derive the wild type (WT) background from donor derived cfDNA led to the introduction of artifacts, which limited the ability to test for low VAFs (<0.1%) at certain sites.

We have developed an enhanced version of the Twist Pan-Cancer Reference Standards, which offers a stable background genotype and a reduced background error rate. Evaluating this new reference standard with the Twist Standard V2 target enrichment system and custom capture panels targeting clinically-relevant variants, there is a clear separation between measured VAF of the lowest variant-positive standard and WT background. In addition, a side-by-side analysis showed that the error rate of this standard (V2) is markedly lower than both its predecessor (V1) and other market competitors. Importantly, its accuracy aligns closely with that of native cfDNA, and we demonstrate that it is an appropriate substrate for 'limit of blank' (LoB) studies of a given variant detection assay. The updated reference materials were created at multiple variant allele frequency (VAF) dilutions, and characterized using droplet digital PCR (ddPCR) and Next-Generation Sequencing (NGS).

In summary, the Twist Pan-Cancer cfDNA Reference Standards V2 emerges as a valuable asset for assay developers and clinical labs using highly multiplexed NGS-based assays and qPCR/dPCR methods. Such a reference standard paves the way for more effective liquid biopsies in clinical research.