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. 2022 Nov;8(11):mgen000895.
doi: 10.1099/mgen.0.000895.

Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly

Aurélie Dotto-Maurel  1 , Camille Pelletier  1 , Benjamin Morga  1 , Maude Jacquot  1 , Nicole Faury  1 , Lionel Dégremont  1 , Maëlis Bereszczynki  2 , Jean Delmotte  2 , Jean-Michel Escoubas  2 , Germain Chevignon  1
Affiliations

Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly

Aurélie Dotto-Maurel et al. Microb Genom. 2022 Nov.

Abstract

Whole-genome sequencing is widely used to better understand the transmission dynamics, the evolution and the emergence of new variants of viral pathogens. This can bring crucial information to stakeholders for disease management. Unfortunately, aquatic virus genomes are usually difficult to characterize because most of these viruses cannot be easily propagated in vitro. Developing methodologies for routine genome sequencing of aquatic viruses is timely given the ongoing threat of disease emergence. This is particularly true for pathogenic viruses infecting species of commercial interest that are widely exchanged between production basins or countries. For example, the ostreid herpesvirus type 1 (OsHV-1) is a Herpesvirus widely associated with mass mortality events of juvenile Pacific oyster Crassostrea gigas. Genomes of Herpesviruses are large and complex with long direct and inverted terminal repeats. In addition, OsHV-1 is unculturable. It therefore accumulates several features that make its genome sequencing and assembly challenging. To overcome these difficulties, we developed a tangential flow filtration (TFF) method to enrich OsHV-1 infective particles from infected host tissues. This virus purification allowed us to extract high molecular weight and high-quality viral DNA that was subjected to Illumina short-read and Nanopore long-read sequencing. Dedicated bioinformatic pipelines were developed to assemble complete OsHV-1 genomes with reads from both sequencing technologies. Nanopore sequencing allowed characterization of new structural variations and major viral isomers while having 99,98 % of nucleotide identity with the Illumina assembled genome. Our study shows that TFF-based purification method, coupled with Nanopore sequencing, is a promising approach to enable in field sequencing of unculturable aquatic DNA virus.

Keywords: Crassostrea gigas; Illumina; ostreid herpesvirus type 1; oxford nanopore technologies; tangential flow filtration; virus.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
(a) Workflow for the processing of OsHV-1 inoculum on tangential flow filtration (TFF) devices with three membrane pore sizes (750, 500 and 300 kDa). After filtration on each TFF-device, the volumes of retentates (R) and permeates (P) were supplemented to 10 ml with filtered seawater (FSW, 0,02 µm). (b) qPCR monitoring of OsHV-1 loads (in genomic units per µl) at the different steps of TFF. (c) Visualization and quantification of virus-like particles (VLP) by epifluorescence microscopy in retentates of TFF devices of 750 kDa (R-750) and 500 kDa (R-500). An eyepiece with a 10×10 grid reticle was used for counting VLP (note in our setup the grid reticle is 100×100 µm). (d) Kaplan-Meier survival curves of oysters injected with 100 µl of OsHV-1 inoculum, or retentates of TFF-devices of 750, 500, 300 kDa (R-750, R-500 and R-300, respectively), or permeate of TFF device 300 kDa (P-300) or FSW as negative control. ****P <0.0001; ns not significant (Mantel–Cox Log-rank test).
Fig. 2.
Fig. 2.
Flow diagram to illustrate the bioinformatic pipelines used to assemble OsHV-1 genome from (a) Illumina short-read or (b) Nanopore long-read.
Fig. 3.
Fig. 3.
Global sequencing statistics. (a) Histogram representing percentage of sequenced bases (green) and reads (blue) that aligned to OsHV-1 (dark) or to C. gigas (regular) genomes or that did not align to these genomes (light). (b) Raincloud plot representing the size distribution of the Nanopore reads that aligned to OsHV-1 (dark blue) or to C. gigas (blue) genomes or that did not align to these genomes (light blue). Each panel is made of a density plot, a dot plot where each dot corresponds to one Nanopore read and a box plot showing average read size (vertical coloured bar) and N50 (coloured circle).
Fig. 4.
Fig. 4.
Schematic representation of OsHV-1 whole-genome assembly outputs for (a) Illumina short-read sequencing and (b) Nanopore long-read sequencing.
Fig. 5.
Fig. 5.
OsHV-1 whole-genome assemblies' evaluation by read mapping. Upper panel represent Illumina short-read coverage on the Illumina OsHV-1 NR genome. Black circles indicate bottom line of the two losses of coverage identified in the Illumina OsHV-1 NR genome. Middle panel represent Illumina de novo assembled contigs (green), OsHV-1 genomic region annotation with the pink bar representing the stem-loop and the ONT de novo assembled contig (green). Bottom panel represent ONT long-read coverage on the ONT OsHV-1 genome. C: Contig.
Fig. 6.
Fig. 6.
OsHV-1 major isomer validation. Each panel corresponds to a 42 kb long OsHV-1 genomic region made of UL fragment plus IRL, X, IRS, US and TRS. The different panels correspond to the different OsHV-1 isomers made by permutation of UL and US regions. Each panel is made of a diagram of reads coverage, a diagram of OsHV-1 region annotation and a diagram representing long-read alignment that span at least one fragment of the UL and one fragment of the US. Read bases are color-coded from conserved (dark blue), to less conserved (light blue) with gaps in sequence colored in red.
Fig. 7.
Fig. 7.
ONT and Illumina OsHV-1 whole-genome comparison. A total of 187 bp differences are identified between both genomes. Overall, 7 bp correspond to SNPs (black bar), 24 bp correspond to SNPs in small homopolymer (red bar), 41 bp correspond to a homopolymer size variation (green bar), and 115 bp correspond to a copy number variation (purple bar). Numbers next to the bars correspond to the number of variable bases at that position.
Fig. 8.
Fig. 8.
Phylogenetic tree of the 11 OsHV-1 NR version of the genomes available in public database, plus the Nanopore and Illumina NR genomes generated in the present study (red). The tree was mid-point rooted. Bootstrap values are shown at nodes (100 replicates).
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