Nanopore Sequencing Is a Credible Alternative to Recover Complete Genomes of Geminiviruses

Abstract

Next-generation sequencing (NGS), through the implementation of metagenomic protocols, has led to the discovery of thousands of new viruses in the last decade. Nevertheless, these protocols are still laborious and costly to implement, and the technique has not yet become routine for everyday virus characterization. Within the context of CRESS DNA virus studies, we implemented two alternative long-read NGS protocols, one that is agnostic to the sequence (without a priori knowledge of the viral genome) and the other that use specific primers to target a virus (with a priori). Agnostic and specific long read NGS-based assembled genomes of two capulavirus strains were compared to those obtained using the gold standard technique of Sanger sequencing. Both protocols allowed the detection and accurate full genome characterization of both strains. Globally, the assembled genomes were very similar (99.5–99.7% identity) to the Sanger sequences consensus, but differences in the homopolymeric tracks of these sequences indicated a specific lack of accuracy of the long reads NGS approach that has yet to be improved. Nevertheless, the use of the bench-top sequencer has proven to be a credible alternative in the context of CRESS DNA virus study and could offer a new range of applications not previously accessible.

CODEHOP-Mediated PCR Improves HIV-1 Genotyping and Detection of Variants by MinION Sequencing

“…Despite the detection of minority DRMs in treatment-experienced patients at read frequencies higher than 30% by MinION sequencing, they were not detected by Sanger-based sequencing. The minority variants described in this study were not located in homopolymer regions generally associated with false base calling on different NGS platforms, including the Oxford Nanopore MinION ( 22 ). In addition, they were supported by a depth of coverage at drug resistance sites of >40, per-base sequencing quality of >20, and read frequency of >30%.…”

Metagenomic Studies of Viruses in Weeds and Wild Plants: A Powerful Approach to Characterise Variable Virus Communities

“…Due to the small genome size of plant viruses, ranging from 4 to 30 kb [75], long read sequencing can capture entire genome segments in a single read. For plant viruses, long read sequencing has been used on geminiviruses [76], tombusviridae [77], and several potyviruses including Wheat streak mosaic virus [78], Plum pox virus [79] and Yam mild mosaic virus [80]. However, the host plants in these studies were all crop plants, with the exception of the geminiviruses found in Medicago arborea, a shrub in the Fabaceae family [76].…”

“…For plant viruses, long read sequencing has been used on geminiviruses [76], tombusviridae [77], and several potyviruses including Wheat streak mosaic virus [78], Plum pox virus [79] and Yam mild mosaic virus [80]. However, the host plants in these studies were all crop plants, with the exception of the geminiviruses found in Medicago arborea, a shrub in the Fabaceae family [76]. In these studies, a variety of DNA and RNA HTS library preparation protocols are used, and the choice of library preparation affects the virus detection sensitivity.…”

Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses

“…Using this advanced approach, by assessing the long overlaps among multiplex amplicons, the accurately assembled and complete viral genome can be obtained, which can facilitate the rapid genomic surveillance of PPRV for better understanding its pathogenicity, evolution and transmission. Although a protocol for Oxford nanopore sequencing of PPRV has been established, adoption of the technology has been limited due to concerns around accuracy and high error rates associated with homopolymer lengths [15]. The nanopore device exhibits lower read-level sequencing accuracy than its short-read platform counterparts [16].…”