Benchmarking of single‐virus genomics: a new tool for uncovering the virosphere

Abstract

Metagenomics and single-cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single-virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high-quality genomic data. We report a sequencing dataset of viral single-amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole-genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase-polymerase features, and WGA-X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology.

Into the Dark: Exploring the Deep Ocean with Single-Virus Genomics

“…The best WGA successful rates were obtained from a fresh sample (Mediterranean Sea; WGA success rate = 48.80%) while lower values were obtained in cryopreserved samples with the cryoprotectant glycerol Tris-EDTA (GlyTE) (WGA success rate = 15.56%), commonly used in single-cell genomics. This variation in the WGA success rate is in accordance with a previous SVG benchmarking study, in which the efficiency of the SVGs methodology was tested using different preservation methods [ 29 ]. Nevertheless, results of this work demonstrate the ability of SVGs to efficiently recover the genome from long-preserved samples (sorting and amplification performed ~4 years apart from sampling).…”

“…The whole-genome amplification (WGA) success rate was, as a mean, ~24%, slightly higher than those values obtained for surface seawater in a previous SVGs survey (17.22%) [ 18 ]. Variation in this rate was observed between the different samples, probably generated by the different preservation methods ( Table 1 ) as previously discussed [ 29 ]. The best WGA successful rates were obtained from a fresh sample (Mediterranean Sea; WGA success rate = 48.80%) while lower values were obtained in cryopreserved samples with the cryoprotectant glycerol Tris-EDTA (GlyTE) (WGA success rate = 15.56%), commonly used in single-cell genomics.…”

“…Nevertheless, results of this work demonstrate the ability of SVGs to efficiently recover the genome from long-preserved samples (sorting and amplification performed ~4 years apart from sampling). Likely, the use of fresh unfixed samples, along with short periods of time from sampling to sorting, guarantee a better genome recovery in SVGs [ 29 ].…”

The landscape of lysogeny across microbial community density, diversity and energetics

“…Building these coinfections networks is a current challenge because the hosts of the majority of phages identified from metagenomic analyses are unknown (Edwards et al ., 2016; Gregory et al ., 2019; Silveira et al ., 2020). New methods to assign phages to hosts, both in silico and in vitro , are beginning to shed light on these networks, including single‐cell and single‐virus sequencing (Labonté et al ., 2015; Garcia‐Heredia et al ., 2020; Martínez et al ., 2020) and proximity‐ligation DNA sequencing (Marbouty et al ., 2017; Yaffe and Relman, 2020).…”

Comparison of de novo assembly using long-read shotgun metagenomic sequencing of viruses in fecal and serum samples from marine mammals

“…Future studies could incorporate the use of biotinylated oligonucleotide probes (targeted viral enrichment) to capture viruses from complex matrices after sequencing library preparation to avoid upstream enzymatic treatments ( Briese et al, 2015 ; Martínez-Puchol et al, 2020 , 2022 ; Bonny et al, 2021 ). Although several studies use filtration ( Allen et al, 2011 ; Fontenele et al, 2019 ; Garcia-Heredia et al, 2021 ; Patterson et al, 2021 ; Crum et al, 2023 ; LaRocca et al, 2023 ) to reduce background host and bacterial nucleic acids, it too can cause losses of viruses in filtration.…”