DRP007211
Analysis of eukaryotic microbiome in ticks Using blocker artificial nucleic acids and non-metazoan PCR
Ticks are important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interaction with pathogens. However, eukaryotic communities of ticks have not yet been extensively explored owing to the lack of suitable methods. Here, we report new methods developed to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using blockers based on artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, PCR using non-metazoan primers, called UNonMet-PCR, was performed. We performed each PCR using tick DNA, and the amplicons were sequenced on an Illumina MiSeq platform. The results showed that almost all sequences obtained using the conventional PCR method were derived from ticks, whereas the proportion of the microeukaryotic reads and their alpha diversity increased with all the methods newly employed in this study.