Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding.

Area of Science:

• Molecular Ecology
• Bioinformatics
• Population Genetics

Background:

• Amplicon-based metabarcoding offers efficient species composition analysis.
• PCR amplification biases challenge accurate abundance estimation in metabarcoding.
• PCR-free methods increase workload and cost.

Purpose of the Study:

• To evaluate strategies for mitigating PCR amplification bias in metabarcoding.
• To assess the impact of primer design, PCR conditions, and locus selection on bias.
• To determine if bias can be corrected for reliable abundance estimates.

Main Methods:

• Analysis of multilocus datasets from diverse arthropod communities.
• Testing degenerate primers and conserved priming sites.
• Varying PCR template concentration and cycle numbers.
• Comparing locus-specific amplification with direct genomic DNA sequencing.

Main Results:

• Degenerate primers and conserved priming sites significantly reduced amplification bias.
• Reducing PCR cycles had minimal impact and decreased predictability.
• Excluding locus-specific amplification did not eliminate bias.
• Taxon-specific biases are predictable and can be corrected.

Conclusions:

• Amplicon-based metabarcoding can yield reliable abundance estimates.
• Strategic primer selection is key to minimizing bias.
• Predictable biases allow for the application of correction factors.
• Copy number variation may influence abundance estimates in both amplicon-based and PCR-free methods.