Methyltransferase-assisted single duplex sequencing for detecting circulating tumor DNA

Abstract

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. This study presents MASD-seq, a duplex sequencing method for highly accurate circulating tumor DNA (ctDNA) detection. The approach is combined with cost-efficient tumor mutation profiling in tissue samples for minimal residual disease monitoring. Duplex error correction from a single read pair is enabled by MASD-seq through physical linkage of DNA strands, coupled with methylation conversion and M.SssI protection. Error rates of 5.4 × 10−7 for CpG>TpG and 4.1 × 10−8 for transversions are achieved by MASD-seq in cell lines, and age-associated mutational burdens are detected in blood. CpG>TpG mutations in colorectal cancer are enriched up to 4-fold by MspI-RRS, capturing a correctable mutation burden comparable to that of the whole-genome sequencing. Colorectal cancer patients are distinguished from healthy controls with high accuracy (AUC = 0.97) using this approach. Duplex error correction from a single read pair is enabled by MASD-seq through physical linkage of DNA strands, coupled with methylation conversion and M.SssI protection. Error rates of 5.4 × 10−7 for CpG>TpG and 4.1 × 10−8 for transversions are achieved by MASD-seq in cell lines, and age-associated mutational burdens are detected in blood. CpG>TpG mutations in colorectal cancer are enriched up to 4-fold by MspI-RRS, capturing a correctable mutation burden comparable to that of the whole-genome sequencing. Colorectal cancer patients are distinguished from healthy controls with high accuracy (AUC = 0.97) using this approach. This study presents MASD-seq, a duplex sequencing method for highly accurate circulating tumor DNA (ctDNA) detection. The approach is combined with cost-efficient tumor mutation profiling in tissue samples for minimal residual disease monitoring.