Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks
| Autor: | Wessam Mohamed Ahmed Mohamed, Shohei Ogata, Elisha Chatanga, Nariaki Nonaka, Ryo Nakao, Yurie Taya, Keita Matsuno, Gohta Kinoshita, Kodai Kusakisako, Mohamed Abdallah Mohamed Moustafa, Yuma Ohari |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Microbiology (medical) QH301-705.5 030231 tropical medicine Computational biology Biology Tick Microbiology DNA sequencing 18S ribosomal RNA Article 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Virology parasitic diseases Microbiome Biology (General) Gene protists eukaryotic microbiome Amplicon biology.organism_classification artificial nucleic acid tick 030104 developmental biology chemistry Nucleic acid next-generation sequencing DNA |
| Zdroj: | Microorganisms Microorganisms, Vol 9, Iss 1051, p 1051 (2021) Volume 9 Issue 5 |
| ISSN: | 2076-2607 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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