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Poster De Conférence Année : 2018

Third Generation Sequencing Technologies to Decipher Genomic Structures of Recombinant-prone Viruses

Résumé

INTRODUCTION/CONTEXT The development of the “third-generation sequencing” platforms, such as the Pacific Biosciences PacBio sequencing system and more recently the Oxford Nanopore MinION device, have yet to be exploited, and have generated particular interest within the scientific community. These methodologies open up new possibilities, such as providing the capability for minimal library preparation and long reads (up to 10 kilobases), thus enabling true linkage to be established between variants within single genomes, and resolving assembly issues that often give incorrect genomic organization. These long-read sequencing platforms especially facilitate the analysis of viral genome structure, including recombination events, which are generally difficult to ascertain using second-generation platforms such as Illumina and Ion Torrent. In fact, current second-generation sequencing technologies have played a driving role to address questions relating to viral genome organization, epidemiology, and investigations of outbreaks by characterizing both partial- (such structural proteins) and whole-genome sequencing (WGS). In the specific case of recombinant-prone viruses, e.g. members of Picornaviridae family, second-generation sequencing technologies have often unveiled the limits of the approach, notably when determining precise viral genomic reconstruction and recombination hotspots. Foot-and-mouth disease (FMD) is considered one of the most contagious diseases of livestock, which can lead to huge economic losses. This disease, present in Africa, Asia and South America, is caused by a virus from the Picornaviridae family, genus Aphthovirus, referred to as FMD virus (FMDV). Seven different FMDV serotypes have been described (A, O, C, SAT1, SAT2, SAT3 and Asia1). The genome of FMDV comprises a positive-sense single-stranded RNA approximately 8300 nucleotides in length. The viral genome contains a single long ORF, encoding a large polyprotein, further processed into 13 viral mature proteins, whose 4 structural proteins (VP1-VP4). The extensive genetic diversity in FMDV is attributed to the poor proof-reading ability of the viral RNA dependent RNA polymerase, with large viral population size and high replication rates. Then, FMDV evolves through genetic drift, where positive selection contributes to fixation of mutations in the capsid coding regions. Although the VP1 coding region of FMDV is useful for isolate characterization, it is relatively short (only ~8% of the genome length) and, consequently, phylogenetic trees generated from closely related FMDV sequences recovered within outbreak clusters are typically flat, with poor resolution. For this reason, the use of WGS to discriminate between closely related viruses has become commonplace and has subsequently been applied to both human and animal pathogens. However, incongruences between phylogenies from individual sub-genomic regions suggest that recombination also plays a role in FMDV evolution. Recombination events have indeed been demonstrated within the FMDV genome and have highlighted the fact that particular regions of the FMDV genome appear to be more prone to intertypic recombination than others. The number of exchanges of genome sequences encoding for nonstructural proteins seems to be much more important and numerous, than the events involving the sequences encoding parts of the capsid-coding region. It is therefore important to identify the set of recombination events in FMDV full genome sequences, and to determine the distribution of these events across the FMDV genome. Recombination events are of particular interest as a source for driving FMDV diversity giving rise to FMDV outbreaks. Third-generation sequencing technologies could thus allow to bridge the gap in resolving genome structure uncertainties for such virus. METHODS Four isolates of FMDV were sequenced using MiSeq Illumina platform (second-generation) and MinION Oxford Nanopore Technologies (third-generation). Two of these samples were collected from cattle in 2011 from Balochistan Province in Pakistan (PAK-6; PAK-9) and the others originated from Benin (BEN-017, BEN-036) in 2010. The whole genome sequencing (WGS) with Illumina technology were performed using Nextera XT kit in order to produce paired-end reads of approximately 150pb each. The MinION libraries were prepared using 1D2 Sequencing chemistry and Flow cell MIN-10 to obtain one unique long read covering the entire genome of the virus (8Kb). For second-generation data analysis, the four FMDV genomes were reconstructed using a dedicated pipeline with classic state-of-the-art bioinformatics tools. Third-generation long reads were analyzed using a long reads analysis workflow (including Albacore and Canu Minimap softwares). In both approaches, phylogenetic trees were established using the Mafft tool, allowing to consolidate the geographical origin and the serotype of all isolates and to help solve the recombination events. A global genomics analysis approach for mapping recombination hotspots appeared to be necessary, particularly for such datasets where the identities of the parental sequences involved in recombination are unknown. More specifically, within the current data study-set, it is generally unknown which FMDV sequence is the recombinant and which is no recombinant. Mapping of the positions of recombination is done by a phylogenetic-compatibility analysis using phylogeny tree scanning, applied to both publicly available full genomes and newly sequenced isolates. Phylogenetic tree scanning is based on recording the order of each variant in an alignment, giving a successive serie of phylogenetic trees (rooted neighbor-joining trees, 100 bootstrap replicates, and where all branches with <70% support are collapsed, moving windows of 300nt and intervals of 100nt), and hence examining the positions in the alignment where phylogenetic relationships change. To investigate the extent of recombination within the data set, the aligned sequences were examined using the Recombination Detection Program in RDP4, in order to infer breakpoint positions and recombinant sequences for every detected potential recombination event. The results of this analysis are in agreement with the phylogenetic-compatibility analysis in that the distribution of observed breakpoints appears to be non-random. CONCLUSION. Incongruent tree topologies between the structural and non-structural coding regions of FMDV isolates suggest that the VP1 phylogeny may not be appropriately reflecting the evolutionary histories of different FMDV isolates. We therefore analyzed the existence of differences in the frequency of recombination between species by an extended comparison of sequences that included all available complete genome sequences available from public databases. Using exhaustive comparisons of fragment sets generated from alignments or the complete genome sequence of the species, it is possible to map regions of phylogenetic incongruity and infer sites of favored recombination using a phylogenetic compatibility matrix (PCM). The results of these FMDV breakpoint distribution and phylogenetic-compatibility analyses reflect a clear partitioning of structural and non-structural genes in the organization of the genome. This organization facilitates component swapping or recombination that frequently occurs among such viruses. Confident construction of transmission trees from phylogenetic data, through spatio-temporal epidemiological data, using MinION nanopore sequencing, offers an exciting potential to FMDV diagnostics, and more specifically for resolving recombination scenarios when comparing different field isolates. Such approaches, integrating both novel technological sequencing instruments, together with phylogenetic and epidemiological data, will help understand mechanisms involving the recombination patterns observed in FMDV and other picornaviruses, and will eventually lead to novel insights into epidemiological and phylogeographics issues in FMDV outbreaks.
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anses-04440933 , version 1 (06-02-2024)

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  • HAL Id : anses-04440933 , version 1

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Mathias Vandenbogaert, Charlotte Balière, Aurélia Kwasiborski, Véronique Hourdel, Laure Diancourt, et al.. Third Generation Sequencing Technologies to Decipher Genomic Structures of Recombinant-prone Viruses. JOBIM (Journées Ouvertes Biologie, Informatique et Mathématiques), Jul 2018, Marseille, France. ⟨anses-04440933⟩
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