Scaling liquid biopsy biomarker discovery: Multi-center, analytical validation of high resolution ctDNA profiling using TSO500 v2 on NovaSeq X Plus

Illumina's TruSight™ Oncology 500 (TSO500) ctDNA v2 assay, enables comprehensive genomic profiling from plasma, allowing non-invasive, repeatable monitoring of tumor evolution during clinical studies. The assay covers a broad panel of 523 clinically relevant cancer genes and provides sensitive detection of key biomarkers such as SNVs, indels, CNVs, gene fusions, MSI, and TMB, all from a simple blood draw. Therefore, this RUO assay is well-suited for exploratory biomarker discovery, response monitoring, and mechanistic insights during immunotherapy and targeted therapy development without the need for fresh tissue biopsies. A validation study was performed to characterize the analytical performance of TSO500 ctDNA v2 assay on the NovaSeq™ X Plus platform for comprehensive genomic profiling of 523 cancer-related genes in ctDNA. The assay was evaluated at two CellCarta laboratories (Antwerp, Belgium and Naperville, USA). Accuracy was assessed using reference materials with a variant allele frequency (VAF) threshold of 0.5% for SNVs and indels. Across both sites, the assay demonstrated >95% overall percent agreement for SNV, indel, CNV, MSI, and TMB results, with positive percent agreement >93.5% and negative percent agreement >95% for SNV, indel, and CNV (no negative reference materials were available for MSI and TMB). Precision assessments showed >97% concordance for intra-run, inter-run, and inter-operator comparisons across all variant classes. Limit of detection verification confirmed 100% detection of SNVs down to 0.25% VAF, indels down to 0.5% VAF, and CNVs at >1.0 fold change. The optimal input for the assay was determined to be 20 ng ctDNA; however, evaluation of lower inputs (10 ng and 5 ng) demonstrated that while these samples did not consistently meet QC thresholds, SNV and indel detection remained robust, with >98% concordance for SNVs and >95% for indels. Lower input material may therefore be used for exploratory analyses, with the understanding that reduced coverage may limit detection sensitivity (potential false negatives) but does not introduce false-positive variant calls. These results confirm that the TSO500 ctDNA v2 assay delivers reproducible, high-resolution genomic profiling across sites, supporting its applicability for exploratory biomarker analyses and longitudinal response monitoring in clinical studies.