Abstract
Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270–14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.
Highlights
Ecosystems harbor an immense abundance of viruses
We demonstrate the power and potential of droplet digital polymerase chain reaction (PCR) (ddPCR) to address the absolute abundances of two abundant viral genotypes in the marine viral realm
The uncultured cosmopolitan virus 37-F6 discovered by single-virus genomics (SVG) (Martinez-Hernandez et al, 2017), is likely the most abundant marine viral genotype, which together with the viral isolate HTVC010P represent two viruses that have a major contribution on the C marine cycle
Summary
Ecosystems harbor an immense abundance of viruses. they probably infect all marine forms of life, the vast majority of them are bacteriophages, i.e., viruses that infect bacteria. A novel methodology based on single-virus genomics have unveiled the genome of the single-virus vSAG 37-F6, that possibly represents the most abundant viral population in the marine surface virosphere (Martinez-Hernandez et al, 2017) This methodology, in contrast to viral metagenomics (i.e., collection of all viral genetic material), allows to obtain the genome from single uncultured viruses one at a time by combining fluorescence-activated virus sorting (FAVS), wholegenome amplification and sequencing of individual sorted viruses from marine viral samples. VSAG 37-F6 virus represents the putative most abundant and widespread uncultured pelagiphage population with a vast unrecognized genomic microdiversity (Martinez-Hernandez et al, 2017, 2018), and a high in situ viral activity in coastal marine samples (Alonso-Sáez et al, 2018)
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