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

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Title: Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks
Authors: Taya, Yurie Browse this author
Kinoshita, Gohta Browse this author
Mohamed, Wessam Mohamed Ahmed Browse this author
Moustafa, Mohamed Abdallah Mohamed Browse this author
Ogata, Shohei Browse this author
Chatanga, Elisha Browse this author
Ohari, Yuma Browse this author →KAKEN DB
Kusakisako, Kodai Browse this author →KAKEN DB
Matsuno, Keita Browse this author
Nonaka, Nariaki Browse this author →KAKEN DB
Nakao, Ryo Browse this author →KAKEN DB
Keywords: artificial nucleic acid
eukaryotic microbiome
next-generation sequencing
Issue Date: 13-May-2021
Publisher: MDPI
Journal Title: Microorganisms
Volume: 9
Issue: 5
Start Page: 1051
Publisher DOI: 10.3390/microorganisms9051051
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.
Type: article
Appears in Collections:獣医学院・獣医学研究院 (Graduate School of Veterinary Medicine / Faculty of Veterinary Medicine) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

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