Analysis of circulating tumor DNA (ctDNA) offers a non-invasive means to track tumor dynamics and treatment response. Reliable quantification approaches are essential to leverage ctDNA as a biomarker for cancer monitoring. While digital PCR (dPCR) provides high precision and sensitivity, it requires prior identification of tumor-specific mutations. Conversely, next-generation sequencing (NGS) offers broader genomic coverage but is semi-quantitative, relying on variant allelic fraction (VAF), which can be confounded by cell-free DNA of non-tumor origin. We established a new quantitative NGS (qNGS) platform that allows absolute measurement of nucleotide variants. The system integrates unique molecular identifiers (UMIs) and quantification standards (QSs) — short synthetic DNA molecules engineered with distinct mutations to be uniquely identifiable in sequencing reads. The method’s accuracy was tested using plasma samples spiked with mutant DNA and pooled plasma from cancer patients, and further validated on samples from four non-small cell lung cancer (NSCLC) participants in the ELUCID trial. The qNGS assay exhibited excellent linearity and strong correlation with dPCR results for both experimental and patient plasma samples. In the ELUCID trial samples, the approach successfully quantified multiple variants simultaneously within individual plasma specimens. ctDNA concentrations differed significantly between baseline and post-therapy samples collected three weeks after the start of treatment. This study introduces a qNGS technique enabling absolute ctDNA quantification, unaffected by fluctuations in non-tumor cell-free DNA. Its application to serial NSCLC samples demonstrated simultaneous tracking of multiple mutations, highlighting qNGS as a reliable and versatile tool for precision oncology.
