# A PCR primer design method for identifying spider mite species using k-mer counting

## TL;DR

This paper introduces a new method using bioinformatics and k-mer counting to design PCR primers that can distinguish between closely related spider mite species.

## Contribution

The novel contribution is a bioinformatics method for designing species-specific PCR primers using RNA-Seq data and k-mer counting.

## Key findings

- The method generated species-specific primer candidates for 19 spider mite species.
- Species-specific primer pairs were successfully obtained for 17 out of 19 species.
- The method is expected to be applicable to other taxa beyond spider mites.

## Abstract

Using PCR to distinguish closely related species can be difficult because they may have very similar genomes. Advances in bioinformatics make it possible to design PCR primers that are species-specific. In this study, we developed a bioinformatics method for extracting species-specific primer candidate sequences (i.e., unpaired primers that were specific to a single species) from RNA-Seq data sets of 19 species of spider mites (Acari, Tetranychidae). Using k-mer counting, we obtained between 257 and 48,621 species-specific unpaired primer candidates for the 19 species. We then manually obtained a second primer that was also species-specific. The primer pairs were then confirmed to work in the target species and not to work in the non-target species. Finally, species-specific primer pairs were obtained for 17 of the 19 species tested. Such species-specific primers may be used for practical species discrimination by optimizing multiplex PCR. Our primer design method is expected to be applicable to other taxa.