Validation of COI metabarcoding primers for terrestrial arthropods

Area of Science:

• Ecology and Evolutionary Biology
• Genetics and Genomics
• Molecular Biology

Background:

• Metabarcoding is vital for biodiversity and ecosystem assessment by analyzing species composition in bulk samples.
• Primer set selection significantly impacts species recovery in metabarcoding, yet terrestrial arthropod primers are under-evaluated.
• Amplification bias among taxa must be minimized for reliable metabarcoding results.

Purpose of the Study:

• To evaluate the performance of 36 primer sets for terrestrial arthropod metabarcoding.
• To identify primer sets that maximize species recovery and minimize amplification bias.
• To recommend optimal primer sets for arthropod biodiversity studies.

Main Methods:

• Tested 36 primer sets on a mock community of 374 insect species.
• Assessed amplification success using gradient PCRs and selected 21 primer sets for metabarcoding.
• Validated top primer sets on a Malaise trap sample and evaluated performance at varying annealing temperatures.

Main Results:

• Eight primer sets, particularly those with inosine or high degeneracy, recovered over 95% of species in the mock community.
• Malaise trap results aligned with mock community data; short amplicons from some primers potentially caused false positives.
• Primer set performance varied slightly with annealing temperature, but some sets consistently outperformed others.

Conclusions:

• Certain primer sets, like BF3 + BR2, are highly effective for arthropod metabarcoding, offering maximal taxonomic resolution.
• The fwhF2 + fwhR2n primer set is suitable for degraded DNA due to its shorter amplicon.
• This research provides essential guidance for selecting optimal primers, improving the accuracy of biodiversity assessments.