Degenerate PCR Primers to Reveal the Diversity of Giant Viruses in Coastal Waters

Abstract

“Megaviridae” is a proposed family of giant viruses infecting unicellular eukaryotes. These viruses are ubiquitous in the sea and have impact on marine microbial community structure and dynamics through their lytic infection cycle. However, their diversity and biogeography have been poorly characterized due to the scarce detection of Megaviridae sequences in metagenomes, as well as the limitation of reference sequences used to design specific primers for this viral group. Here, we propose a set of 82 degenerated primers (referred to as MEGAPRIMER), targeting DNA polymerase genes (polBs) of Megaviridae. MEGAPRIMER was designed based on 921 Megaviridae polBs from sequenced genomes and metagenomes. By applying this primer set to environmental DNA meta-barcoding of a coastal seawater sample, we report 5595 non-singleton operational taxonomic units (OTUs) of Megaviridae at 97% nucleotide sequence identity. The majority of the OTUs were found to form diverse clades, which were phylogenetically distantly related to known viruses such as Mimivirus. The Megaviridae OTUs detected in this study outnumber the giant virus OTUs identified in previous individual studies by more than an order of magnitude. Hence, MEGAPRIMER represents a useful tool to study the diversity of Megaviridae at the population level in natural environments.

An Optimized Metabarcoding Method for Mimiviridae

“…Effective methods for the characterization of the community structures of Mimiviridae and related viruses have been the amplification and sequencing (i.e., "meta-barcoding") of highly conserved genes such as the major capsid protein genes, DNA polymerase family B (polB) and DNA mismatch repair protein genes [10,[18][19][20][21]. Recently, Li et al proposed a novel meta-barcoding method (i.e., the MEGAPRIMER method) to investigate Mimiviridae diversity and revealed a hitherto unknown level of Mimiviridae richness in environmental samples [22,23]. Their approach used a set of 82 degenerate primers, which were designed to cover diverse Mimiviridae polB genes identified in the Tara Oceans metagenomic dataset [22,23].…”

“…Recently, Li et al proposed a novel meta-barcoding method (i.e., the MEGAPRIMER method) to investigate Mimiviridae diversity and revealed a hitherto unknown level of Mimiviridae richness in environmental samples [22,23]. Their approach used a set of 82 degenerate primers, which were designed to cover diverse Mimiviridae polB genes identified in the Tara Oceans metagenomic dataset [22,23]. Analysis of a coastal water sample with the MEGAPRIMER method resulted in the identification of 5,595 non-singleton operational taxonomy units (OTUs) at 97% nucleotide identity [23].…”

“…Their approach used a set of 82 degenerate primers, which were designed to cover diverse Mimiviridae polB genes identified in the Tara Oceans metagenomic dataset [22,23]. Analysis of a coastal water sample with the MEGAPRIMER method resulted in the identification of 5,595 non-singleton operational taxonomy units (OTUs) at 97% nucleotide identity [23]. The same approach has also revealed the Mimiviridae community structures in samples from hot spring freshwater, brackish water of the mangroves, and the Sea of Japan [22].…”

“…The same approach has also revealed the Mimiviridae community structures in samples from hot spring freshwater, brackish water of the mangroves, and the Sea of Japan [22]. Although the development of the MEGAPRIMER method helped to characterize Mimiviridae communities more effectively and deeply than other methods such as shotgun metagenomics or metatranscriptomics, the original MEGAPRIMER method was time consuming due to the requirement of 82 individual PCR amplifications per sample [23]. In the first study [23], the authors sequenced only the amplicons that were visible in gel electrophoresis.…”

“…Although the development of the MEGAPRIMER method helped to characterize Mimiviridae communities more effectively and deeply than other methods such as shotgun metagenomics or metatranscriptomics, the original MEGAPRIMER method was time consuming due to the requirement of 82 individual PCR amplifications per sample [23]. In the first study [23], the authors sequenced only the amplicons that were visible in gel electrophoresis. However, this approach was subjective in deciding whether an amplification was successful or not.…”