An Optimized Metabarcoding Method for Mimiviridae.

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

doi:10.3390/microorganisms8040506

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.

Highlights

Mimiviridae is a family of nucleocytoplasmic large DNA viruses (NCLDVs), or the proposed order “Megavirales” [1]
Mimiviridae Community Profiles were Coherent across Different Primer Cocktails
We generated 15 datasets of Mimiviridae polymerase family B (polB) amplicon sequences from four samples collected at four distinct locations (Osaka Bay and Uranouchi Inlet station F, J, and M) through either deep or shallow sequencing (Table 1)

Summary

Introduction

Mimiviridae is a family of nucleocytoplasmic large DNA viruses (NCLDVs), or the proposed order “Megavirales” [1]. The first member of the Mimiviridae family is the amoeba-infecting giant mimivirus (Acanthamoeba polyphaga mimivirus) with a particle diameter of 750 nm and a large genome encoding over 1,000 genes [2,3]. While this first-discovered member of Mimiviridae infects amoeba, other members infect unicellular algae or heterotrophic protists other than amoeba [4,5,6]. The genome sizes of alga-infecting Mimiviridae are mostly from 370 kb to 560 kb, but larger genomes up to 668 kb have been reported [5,7] Their virion sizes range from 140 nm to 310 nm [4,7]. The genome size of heterotrophic protist-infecting Mimiviridae ranges from 600 kb to 1,500 kb, and their icosahedral heads measure from 300 nm to 750 nm, though the tupanvirus virion has a long tail (~0.5 μm) in addition to its capsid [2,8,9]

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Conclusion