Assessing the effects of primer specificity on eliminating numt contamination in DNA barcoding

The identification of species using DNA barcodes is sometimes hindered by coamplification of nuclear mitochondrial pseudogenes (numts) with orthologous mitochondrial COI gene. Researchers have suggested that utilizing more specific primers will reduce the chance of numt coamplification, but no quantification has been performed to date. In this study, we assess the effect of primer specificity in reducing numt coamplification using four taxa from the order Orthoptera representing 2 highly divergent ensiferan families and 2 divergent lineages within the family Acrididae. We present the analyses of data obtained from cloned sequences generated from PCR reactions using three sets of primers of varying specificity (Folmer primers, Orthoptera-specific primers, and species-specific primers) and estimate the number of numts present in the four taxa. Our findings suggest that even the most specific primers can coamplify numts, which implies that primer specificity is not the best solution to avoid numts.