Long-read viral metagenomics captures abundant and microdiverse viral populations and their niche-defining genomic islands

Abstract

Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and ‘universal’ gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The ‘VirION’ (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

Genome‐resolved viral and cellular metagenomes revealed potential key virus‐host interactions in a deep freshwater lake

“…In light of this hypothesis, the high abundance and diversity of actinoviruses may result from an arms race with their hosts, leading to genomic diversification of the hosts to avoid mortality induced by a high viral load. It should be noted that such microdiversification of viral genomes poses challenges for metagenomic assembly (Martinez‐Hernandez et al ., ; Roux et al ., ; Warwick‐Dugdale et al ., ); indeed, 109 LBVCs encoded the actinoviral hallmark whiB gene (Supporting Information Dataset S1), suggesting that the majority of actinoviral genomes were not recovered as LBVGs (i.e., circularly assembled). Although further investigation is beyond the scope of the present study, our results revealed that, analogous to their hosts, actinoviruses are one of the most diverse, abundant, ubiquitous, and active viral groups in freshwater systems.…”

“…Alignments between individual genomes can be found in Supporting Information Alignment S1. [Color figure can be viewed at wileyonlinelibrary.com] Roux et al, 2017b;Warwick-Dugdale et al, 2019); indeed, 109 LBVCs encoded the actinoviral hallmark whiB gene (Supporting Information Dataset S1), suggesting that the majority of actinoviral genomes were not recovered as LBVGs (i.e., circularly assembled). Although further investigation is beyond the scope of the present study, our results revealed that, analogous to their hosts, actinoviruses are one of the most diverse, abundant, ubiquitous, and active viral groups in freshwater systems.…”

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

“…Recently, nanopore sequencing has been used for metagenomic forays into the virosphere (Warwick-Dugdale et al 2019) and studies focusing on transmission routes Faria et al 2017). Furthermore, viral transcriptomes have been investigated using nanopore sequencing of cDNA (Moldován et al , 2018aTombácz et al 2017), being subject to bias from reverse transcription and amplification.…”

Identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge, Guyana

“…In addition, the system is simple to use, fast, high portability, thus, allows it to be used on real-time analysis of sequencing. MinION has been increasingly applied in the fields of virus detection, viral whole-genome sequencing, and viral evolution [19][20][21][22]. NGS was also successfully applied in response to the event described in this paper.…”