MEDUSA: Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• High-accuracy DNA sequencing is crucial for various applications.
• Duplex sequencing methods rely on matching reads from both DNA strands for accuracy.
• Current dsDNA preparation methods can introduce errors by resynthesizing DNA strands.

Purpose of the Study:

• To develop a method that minimizes dsDNA resynthesis during preparation for duplex sequencing.
• To enhance the accuracy and yield of duplex sequencing.
• To introduce MEDUSA (Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy) for improved sequencing fidelity.

Main Methods:

• MEDUSA repairs and blunts fragmented dsDNA.
• Apyrase is used to digest residual deoxynucleotide triphosphates (dNTPs).
• Restricted dA-tailing is employed to prevent DNA resynthesis.

Main Results:

• MEDUSA achieved full genome coverage with duplex yields comparable to traditional methods.
• Residual single nucleotide variant (SNV) frequency was minimally increased compared to a theoretical ideal.
• The method demonstrated broad compatibility with standard dsDNA fragmentation and library preparation kits.

Conclusions:

• MEDUSA significantly enhances duplex sequencing accuracy by minimizing resynthesis.
• The protocol is simplified and broadly applicable to various DNA samples, including sheared genomic and cell-free DNA.
• MEDUSA enables high-breadth or high-depth duplex sequencing with reduced false discoveries.