Effect of DNA input on analytical and clinical parameters of a circulating tumor DNA assay for comprehensive genomic profiling

Abstract Objectives Circulating tumor DNA (ctDNA) assays play an increasingly important role in cancer management. A new version of TruSight Oncology 500 (TSO500) ctDNA, v2, was recently released, and the recommended DNA input was decreased to 20 ng from 30 ng in the prior version. This study aimed to assess the analytical and clinical performance of TSO500 ctDNA v2 using varying input DNA. Methods Four replicates of two reference materials were prepared with varying DNA input. Nine residual clinical samples that were tested using TSO500 ctDNA v1 were also prepared with reduced DNA input compared to the initial tests. A total of 17 samples were subjected to library preparation, sequencing, and bioinformatics analysis according to the manufacturer’s protocol. The effects of varying DNA input on quality measures and variant detection accuracy were analyzed. Results The depth of coverage and variant detection sensitivity of the reference material gradually decreased with reduced DNA input. The total number of reads was unaffected, while the mean family size increased with the reduction in DNA input, attributing the decreased performance to reduced library complexity. Among the 61 single nucleotide variants and 17 indel variants previously detected using TSO500 ctDNA v1 with 30 ng of input DNA, only 45 (73.8 %) and 7 (41.2 %) were detected using v2 with 20 ng of input DNA, respectively. Conclusions The DNA input is a critical factor that determines assay performance and an input higher than recommended by the manufacturer should preferably be used for TSO500 ctDNA v2 for its optimal performance.