Comprehensive benchmarking of methods for mutation calling in circulating tumor DNA

Detection of somatic mutations in cell-free DNA (cfDNA) is challenging due to low variant allele frequencies and extensive DNA degradation. Here we develop a benchmarking strategy using longitudinal patient-matched cfDNA samples from individuals with colorectal and breast cancer. Samples with high and ultra-low levels of tumor-derived DNA are combined into controlled dilution series that preserve the properties of authentic cell-free DNA, including each patient's germline and blood-cell mutation backgrounds. Using deep whole-genome (150x) and exome (2,000x) sequencing, we define a reference set of ~37,000 single nucleotide variants and ~58,000 indels to benchmark nine somatic variant callers across varying ctDNA levels and sequencing depths. We also explore machine learning-based tuning of individual callers and identify features that improve accuracy in cfDNA. This benchmarking resource clarifies the detection limits of current approaches and provides practical guidance for selecting somatic variant calling methods in liquid biopsy applications.