A novel NGS-compatible Enzymatic Strategy Enables Carryover Contamination Removal and Enhances Sequencing Performance.

Carryover contamination during DNA amplification can often lead to false positives. Traditional mitigation strategies often include physical separation and/or enzymatic decontamination. However, these methods have limitations, including logistical constraints and polymerase compatibility issues especially for Next Generation Sequencing (NGS). Here, we introduce a novel approach using 7-deaza-dGTP and Fpg for carryover amplicon degradation. When incorporated into library preparation, Fpg degrades 7-deaza-deoxyguanosine amplicons providing carryover protection comparable to the established dUTP/UDG strategy with over 95% carryover contamination removal. Unlike currently available carryover enzyme/dNTP solutions, 7-deaza-deoxyguanosine is compatible with many polymerases and does not impact substitution frequencies during sequencing. Additionally, due to its chemical properties, incorporating 7-deaza-dGTP during amplification improves GC bias during sequencing. In turn, this method is compatible with NGS, supports broader polymerase compatibility, and improves sequencing performance, particularly in AT- and GC-rich regions.