An Optimized Metabarcoding Method for Mimiviridae
| Autor: | Keizo Nagasaki, Tetsuya Hayashi, Takashi Yoshida, Florian Prodinger, Hiroyuki Ogata, Yoshihito Takano, Romain Blanc-Mathieu, Kento Tominaga, Hisashi Endo, Yanze Li, Yasuhiro Gotoh, Daichi Morimoto, Kimiho Omae |
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| Přispěvatelé: | Kyoto University [Kyoto], Kyushu University [Fukuoka], Régulateurs du développement de la fleur (Flo_RE ), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Kochi University, The Canon Foundation (No. 203143100025), JSPS/KAKENHI (Nos. 26430184, 17H03850, 18H02279, 16H06279 (PAGS)), Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (Nos. 16H06429, 16K21723, 16H06437), The Kyoto University Foundation, Collaborative Research Program of the Institute for Chemical Research, Kyoto University (Nos. 2019-33, 2018-31, 2017-25, 2016-28), Kyoto University, Kyushu University, Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA) |
| Rok vydání: | 2020 |
| Předmět: |
Microbiology (medical)
DNA polymerase Computational biology Microbiology Genome ‘Megaviridae’ 03 medical and health sciences chemistry.chemical_compound Virology MEGAPRIMER Megaviridae Mimiviridae Environmental DNA lcsh:QH301-705.5 Gene 030304 developmental biology 0303 health sciences biology 030306 microbiology primer cocktail biology.organism_classification lcsh:Biology (General) chemistry Metagenomics [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology biology.protein DNA |
| Zdroj: | Microorganisms Microorganisms, MDPI, 2020, 8 (4), pp.506. ⟨10.3390/microorganisms8040506⟩ Microorganisms, Vol 8, Iss 506, p 506 (2020) Microorganisms, 2020, 8 (4), pp.506. ⟨10.3390/microorganisms8040506⟩ Volume 8 Issue 4 |
| ISSN: | 2076-2607 |
| DOI: | 10.3390/microorganisms8040506 |
| Popis: | Mimiviridae is a group of viruses with large genomes and virions. Ecological relevance of Mimiviridae in marine environments has been increasingly recognized through the discoveries of novel isolates and metagenomic studies. To facilitate ecological profiling of Mimiviridae, we previously proposed a meta-barcoding approach based on 82 degenerate primer pairs (i.e., MEGAPRIMER) targeting the DNA polymerase gene of Mimiviridae. The method detected a larger number of operational taxonomic units (OTUs) in environmental samples than previous methods. However, it required large quantities of DNA and was laborious due to the use of individual primer pairs. Here, we examined coastal seawater samples using varying PCR conditions and purification protocols to streamline the MEGAPRIMER method. Mixing primer pairs in &ldquo cocktails&rdquo reduced the required amount of environmental DNA by 90%, while reproducing the results obtained by the original protocol. We compared the results obtained by the meta-barcoding approach with quantifications using qPCR for selected OTUs. This revealed possible amplification biases among different OTUs, but the frequency profiles for individual OTUs across multiple samples were similar to those obtained by qPCR. We anticipate that the newly developed MEGAPRIMER protocols will be useful for ecological investigation of Mimiviridae in a larger set of environmental samples. |
| Databáze: | OpenAIRE |
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