MEDUSA: Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy

Highly accurate DNA sequencing is essential for many applications. Duplex sequencing-based methods achieve unparalleled accuracy by requiring reads from both strands of the original DNA duplex to match. Yet, methods to prepare 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. 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 and cell-free DNA and found a residual SNV frequency within a median 1.23-fold (range 0.92-1.88; p View larger version (12K): org.highwire.dtl.DTLVardef@1ddbc77org.highwire.dtl.DTLVardef@802ca9org.highwire.dtl.DTLVardef@f443ecorg.highwire.dtl.DTLVardef@974bdb_HPS_FORMAT_FIGEXP M_FIG C_FIG