Abstract
We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.
Highlights
Metagenomic approaches have enabled the discovery of hundreds previously unknown virus species[1,2,3,4]
DBV is reportedly the most prevalent virus in yam[56] and transcriptionally active endogenous geminiviral (EGV) sequences have been identified integrated within the genomes of many yam species[57]
While our overarching goal was to assess whether the MinION should be used in plant virology to enhance research on, and the monitoring of, plant viruses, we challenged the MinION technology with the well-established Illumina sequencing technology and compared the results obtained using these two sequencing approaches
Summary
Metagenomic approaches have enabled the discovery of hundreds previously unknown virus species[1,2,3,4] These discoveries have strengthened our understanding of the ecological roles and impacts of viral communities, indicating that viruses are likely essential components of ecosystems as diverse as the human gut[5,6] and the oceans[7,8]. Failure to build large enough scaffolds or contigs can reduce the proportions of reads that can be reliably identified as being related to known virus species using alignment-based approaches such as BLAST24. This technical problem is compounded by the dearth of viral taxa that are represented in public nucleotide sequence databases such as GenBank. A consequence of this is that, up to 70% or more of the reads that are generated by some environmental viral metagenomic studies, end up being labeled as “dark matter” because they have no detectable homology to sequences within the public nucleotide sequence databases[19,25]
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