Estimating genetic variability in non-model taxa: a general procedure for discriminating sequence errors from actual...

Area of Science:

• Evolutionary Biology
• Molecular Biology
• Genetics

Background:

• Genetic variation is fundamental to evolution, but errors in its measurement can skew biological estimates.
• Multi-template PCR cloning methods are prone to misincorporation errors, which are hard to detect in multi-gene families.
• Previous studies often lack detailed methods for detecting and correcting PCR-induced errors.

Purpose of the Study:

• To empirically estimate the base misincorporation rate of a common PCR-cloning method.
• To assess the impact of these errors on genetic variation studies.
• To provide a framework for accounting for PCR errors in gene discovery and diversity assessments.

Main Methods:

• A single-copy mitochondrial gene from one individual was used to minimize template DNA variation.
• The rate of base misincorporation was calculated per site and per duplication.
• The empirically determined error rate was applied to data from the Phospholipase A(2) toxin family in Ovophis monticola.

Main Results:

• The base misincorporation rate was estimated at 1.62×10(-3) errors per site.
• The distribution of errors followed a binomial distribution.
• The observed differences in the pitviper toxin family data closely matched the expected error distribution.

Conclusions:

• The PCR-cloning method exhibits a significant base misincorporation rate.
• Empirically determining this error rate is essential for accurate genetic variation analysis.
• This approach is vital for reliable gene discovery and diversity assessments using error-prone PCR methods.