Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA

Circulating tumor DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-Seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-Seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the parts-per-million range in patient samples. We profiled 678 specimens from 213 patients with B-cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B-cell lymphoma. In patients with undetectable ctDNA by CAPP-Seq after two cycles of therapy, an additional 25% have ctDNA detectable by PhasED-Seq and have worse outcomes. Finally, we demonstrate the application of PhasED-Seq to solid tumors.