Intercalating fluorescent dyes enable real-time monitoring of DNA amplification, but their use in preparative NGS library PCR has raised questions about whether they may introduce sequencing errors. In this study, researchers evaluated the effects of SYBR Green I, EvaGreen, and EvaGreen Plus during high-fidelity library amplification on the iconPCR platform. Human genomic DNA libraries were prepared with each dye or a no-dye control, sequenced on an Element AVITI system, and assessed using variant calling and mutational-signature analysis. Dye-containing libraries showed comparable overall quality to the no-dye control, with single-base error rates increasing by less than 3.5% and only modest changes in indel profiles. A complementary 16S rRNA amplicon experiment found that errors increased substantially with additional PCR cycles, while the incremental effect of SYBR Green I remained small at matched cycle counts. No coherent mutational signature attributable to dye exposure was identified. Together, these results indicate that intercalating dyes used during iconPCR amplification have minimal impact on NGS library mutation rates under the conditions tested, while cycle number is the dominant contributor to accumulated amplification error.