An optimized metabarcoding method for mimiviridae

Florian Prodinger, Hisashi Endo, Yasuhiro Gotoh, Yanze Li, Daichi Morimoto, Kimiho Omae, Kento Tominaga, Romain Blanc-Mathieu, Yoshihito Takano, Tetsuya Hayashi, Keizo Nagasaki, Takashi Yoshida, Hiroyuki Ogata

Research output: Contribution to journalArticle

Abstract

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 “cocktails” 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.

Original languageEnglish
Article number506
JournalMicroorganisms
Volume8
Issue number4
DOIs
Publication statusPublished - Apr 2020

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Virology
  • Microbiology (medical)

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    Prodinger, F., Endo, H., Gotoh, Y., Li, Y., Morimoto, D., Omae, K., Tominaga, K., Blanc-Mathieu, R., Takano, Y., Hayashi, T., Nagasaki, K., Yoshida, T., & Ogata, H. (2020). An optimized metabarcoding method for mimiviridae. Microorganisms, 8(4), [506]. https://doi.org/10.3390/microorganisms8040506