MEDUSA: Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy

BACKGROUND: Detection of low-level genetic variants is crucial for many applications in clinical care and research, yet can be hampered by inaccuracies in DNA sequencing. Duplex sequencing-based methods achieve unparalleled accuracy by requiring reads from both strands of the original DNA duplex to match. Yet, methods to prepare double-stranded DNA (dsDNA) for sequencing may resynthesize portions of each DNA duplex and cause base damage errors on one strand to become indistinguishable from true mutations on both strands. METHODS: Here, we report MEDUSA (Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy), which minimizes dsDNA resynthesis to maximize duplex sequencing accuracy and yield. MEDUSA carefully repairs and blunts fragmented dsDNA, then employs apyrase to digest residual dNTPs, followed by restricted dA-tailing to prevent resynthesis. MEDUSA affords full genome coverage in a simplified protocol that is broadly compatible with dsDNA fragmentation and library preparation kits. We benchmarked MEDUSA on sheared genomic DNA derived from formalin-fixed paraffin-embedded tumor tissue or blood cells, and cell-free DNA. RESULTS: We found that MEDUSA measured a residual single-nucleotide variant frequency within a median 1.23-fold (range 0.92-1.88; P < 0.001) of what was expected if resynthesis was almost completely blocked, but with full genome coverage and duplex yields within a median 1.02-fold (range 0.33-1.46; P = 0.258) of traditional methods that do not limit resynthesis. CONCLUSIONS: MEDUSA could enable high breadth or depth of duplex sequencing while limiting false mutation discovery.