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

Abstract

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.