A universal duplex sequencing approach for accurate detection of somatic mutations

Somatic mutations arise from endogenous and exogenous mutagenic processes, accumulating over time and contributing to aging and disease. Detecting these rare mutations in non-clonal tissues remains a significant challenge due to the high error rates, limited genome coverage, and substantial DNA input requirements of existing sequencing approaches. Here, we introduce UDSeq, a high-accuracy, cost-effective, single-molecule duplex sequencing protocol designed to overcome these limitations. We place UDSeq in the context of existing duplex sequencing approaches, demonstrating that it achieves an exceptionally low error rate of ~2.5 × 10 -9 per base pair, supports whole-genome and targeted capture sequencing from as little as 100 picograms of DNA, and delivers up to four times more usable duplex molecules than current state-of-the-art methods from the same input. We demonstrated the broad applicability of UDSeq through a series of in vitro and in vivo mutagenesis experiments, accurately capturing known mutational signatures induced by environmental carcinogens in human cell lines, rodents, and non-model organisms. We further applied UDSeq to normal tissues from a 70-year-old individual, revealing organ-specific mutational burdens and the activity of distinct mutational processes. With its high accuracy, low input requirements, and wide applicability, UDSeq provides a powerful and scalable tool for studying mutational processes across diverse biological contexts. Its versatility supports applications in cancer research, aging, and environmental exposure, expanding our capacity to characterize somatic mutations in both healthy and diseased tissues.