Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks

Abstract

Ticks serve as 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 interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.

Tick-Borne Zoonotic Diseases and Its Control

“…Using artificial nucleic acids, mainly peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). Additionally, the PNA-or LNA-based methods were suitable for pancreatic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi [104]. For detection of TBE virus and Bluetongue virus in ticks and realtime RT-PCR is also done [105] Indirect immunofluorescence is applied for detection of Borrelia spirochetes [106].…”

LNA blockers for improved amplification selectivity

“…The higher melting temperatures (T m s) of oligonucleotides that include LNA bases provide greater specificity and new functions. They have been used successfully as PCR primers/probes 4-6 , as antisense reagents 7-9 , as selective binders for distinguishing single-nucleotide variants [10][11][12][13][14][15][16][17][18] , as agents for selective capture/ degradation 19,20 , and as polymerization/splicing blockers [21][22][23][24][25][26][27][28] . Each of these roles requires that the LNA bind tighter than the corresponding pure DNA, but some functions may also require additional attributes that could be affected by the number and location of LNA bases within the oligonucleotide.…”

“…Furthermore, proteins may interact with LNAs differently than with standard DNAs, dictating whether LNA or DNA should occupy a particular position.While LNA blockers have been used in multiple situations, those studies have little commonality to provide insight into preferred designs. Some use chimeric [11][12][13]15,21,23,26 or pure 10,17,27 LNAs of 16 nt or shorter, while others use chimeric LNAs of 20 nt or longer 16,22,24 For 20-mers, there are more than one million possible LNA-DNA configurations for each of the more than 1 trillion possible sequences. In addition, functional predictions of how different LNAs will perform as primers, blockers, or in other roles are even less well characterized because additional factors beyond the well-studied T m 29-33 may play a critical role in how different LNAs perform [34][35][36] .…”