Assessing the effects of primer specificity on eliminating numt co-amplification in DNA barcoding

DNA barcoding is a method of species identification based on sequencing a short mitochondrial DNA fragment of Cytochrome Oxidase I (COI), but its ability to correctly diagnose be limited by the presence of nuclear mitochondrial pseudogenes (numts). Numts can be co-amplified with the mitochondrial ortholog when using universal primers, which can lead to incorrect species identification and an overestimation of the number of species. Some researchers have proposed that using more specific primers may help eliminate numt co-amplification, but the efficacy of this method has not been thoroughly tested. In this study, we investigate the effect of primer specificity on eliminating numt co-amplification by comparing cloned COI sequences in four orthopteran lineages. We find that numts can be co-amplified in three of the four taxa using standard barcoding primers. Increased primer specificity can help reduce, but does not eliminate numt co-amplification in all species tested. We also find that a number numts do not have stop codons or indels, making it difficult to distinguish them from mitochondrial orthologs, thus putting the efficacy of barcoding quality control measures under question. Our findings suggest that the taxonomic impact of numt co-amplification is large and more care is necessary to identify and eliminate numts when using DNA barcoding for species diagnosis. We also characterize numts of COII and ND5.