IMPACT OF FLUORESCENT DYES ON MUTATIONS IN NEXT GENERATION SEQUENCING LIRBARY PREPARATION

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Fluorescent DNA-binding dyes (e.g., ethidium bromide) were historically considered mutagenic.
• The potential for these dyes to induce mutations during DNA replication persists, despite recent research.
• Real-time PCR instruments use fluorescent dyes for amplification monitoring, raising concerns about mutations in downstream sequencing.

Purpose of the Study:

• To evaluate the mutagenic impact of SYBR and EvaGreen dyes during iconPCR amplification.
• To assess the effect of these dyes on mutation calling and mutational signatures in sequencing data.
• To determine the significance of dye-induced mutations in fluorescence-guided amplification for downstream analyses.

Main Methods:

• Samples were amplified using the iconPCR instrument with SYBR or EvaGreen.
• Routine mutation calling was performed on amplified samples.
• Mutational signature analysis was conducted to identify dye-specific mutation patterns.
• Sequence analysis focused on substitutions and indel domains.

Main Results:

• Sequence analysis revealed minimal impact of fluorescent dyes on PCR reactions.
• Observed changes in mutation rates were subtle and largely within the experimental noise.
• Mutational signature analysis did not identify significant signatures attributable to the dyes.
• No key signatures were found for either substitutions or indels related to dye use.

Conclusions:

• Intercalating fluorescent dyes have a minimal mutational impact during fluorescence-guided amplification.
• The mutagenic effects of SYBR and EvaGreen are negligible for most sequencing applications.
• Dye-induced mutations can be disregarded except in highly sensitive next-generation sequencing (NGS) studies.