Dye shift: a neglected source of genotyping error in molecular ecology

Area of Science:

• Molecular Ecology
• Genetics
• Bioinformatics

Background:

• Genotyping error is a critical concern in molecular ecology, with dye-induced mobility shift (dye shift) often overlooked in automated genotyping using 3-primer systems.
• Uncorrected dye shift can lead to allele mis-scoring and the false identification of new alleles, particularly when different fluorescent dyes are used for the same locus across reactions.

Purpose of the Study:

• To investigate and quantify the impact of dye shift on genotyping accuracy using a standard set of fluorophore labels.
• To assess the prevalence of dye shift correction in published 3-primer studies and evaluate existing correction factors.

Main Methods:

• Genotyped identical loci using four distinct fluorophore labels from a standard dye set.
• Measured size estimates for individual alleles across different dye applications to determine dye shift magnitude.
• Reviewed scientific literature for evidence of dye shift accounting in 3-primer studies and examined cited correction factors.

Main Results:

• Allele size estimates varied significantly (2.07–3.68 bp) depending on the fluorophore used, indicating substantial dye shift.
• The PET fluorophore exhibited the strongest dye shift effects, and the magnitude of the shift was inversely proportional to locus size.
• Limited evidence suggests dye shift is routinely addressed in 3-primer studies, and some correction factors found in literature were potentially erroneous.

Conclusions:

• Dye shift represents a significant, often neglected, source of genotyping error in molecular ecology.
• Researchers must implement stringent quality control measures, especially when using different dyes for the same locus.
• Generic correction patterns for dye shift are unreliable; locus-specific or dye-specific corrections are strongly advised.