Methyltransferase-assisted single duplex sequencing for detecting circulating tumor DNA

Accurate circulating tumor DNA (ctDNA) detection is limited by high sequencing errors. To address this, we developed Methyltransferase-Assisted Single Duplex sequencing (MASD-seq), which physically links Watson and Crick strands to enable error correction using double-stranded information within a single read pair. MASD-seq employs enzymatic methyl-sequencing to disrupt strand pairing and uses CpG methyltransferase pre-methylation to enable detection of CpG>TpG and transversion mutations in DNA duplexes. In parallel, MspI digestion enriches tumor CpG>TpG mutations and reduces genome complexity. MASD-seq achieves error rates of 5.4 × 10^-7 for CpG>TpG and 4.1 × 10^-8 for transversions in cell lines, allowing detection of age-associated mutational burdens in blood. Tissue analyses show a 4-fold enrichment of CpG>TpG mutations and a 1.6-fold increase in total mutations in high microsatellite instability (MSI-H) colorectal cancer (CRC), and 3-fold and 1.2-fold enrichments in microsatellite stable (MSS) tumors, respectively, yielding a correctable mutation burden comparable to the whole genome. Clinical testing demonstrates an area under the curve (AUC) of 0.97 for ctDNA detection in CRC patients. MASD-seq is a highly accurate sequencing method, suggesting its potential for future clinical applications.