Development and optimization of a diagnostic system based on Illumina sequencing of genus wide PCR amplicons for the detection of viruses of grapevines
In this study we present a poly-specific PCR-based, high-throughput sequencing (HTS) diagnostic system together with an appropriate data analysis pipeline for the diagnosis of grapevine viruses. Poly-specific and virus-specific primers were established to be capable of detecting and identifying 3...
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| Language: | en |
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University of Pretoria
2017
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| Online Access: | http://hdl.handle.net/2263/63362 Wayland, J 2017, Development and optimization of a diagnostic system based on Illumina sequencing of genus wide PCR amplicons for the detection of viruses of grapevines, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/63362> |
| Summary: | In this study we present a poly-specific PCR-based, high-throughput sequencing
(HTS) diagnostic system together with an appropriate data analysis pipeline for the
diagnosis of grapevine viruses. Poly-specific and virus-specific primers were
established to be capable of detecting and identifying 37 grapevine infecting viruses
from 11 genera. An analysis pipeline using CLC Genomics workbench was
developed by utilising various defined artificial samples which were assembled and
sequenced on the Illumina MiSeq platform. A threshold for percentage mapped
reads of 0.4% during reference mapping was established to discriminate between
presence or absence of viruses associated with reads. Various criteria for the
evaluation of de novo assembled contigs and BLAST results were identified based
on virus hits, E-value, percentage query overlap and percentage amplicon overlap.
Various RT-PCR systems were used to screen 62 grapevine samples (field collected
and candidate nuclear vines) for their virus populations. Seven samples were
selected for Illumina MiSeq sequencing, and the data was analysed as per the
optimized pipeline. The threshold established for reference mapping and the criteria
for BLAST analysis was successfully implemented, proving the applicability of this
PCR and HTS-based system in grapevine diagnostics. This system was compared
to the standard ELISA system routinely utilised during certification. In our study,
when samples evaluated by RT-PCR were tested using ELISA for the presence of
GLRaV-1, -2 and -3, a false-negative rate in ELISA of 14.3% was observed,
confirming that RT-PCR is the more sensitive test of the two. The capability of RTPCR
to readily detect viruses present in low concentrations in woody plants, the
availability of primers for virus identification, the ease and rapidity of the technique,
together with constant improvement of HTS platforms especially in the area of cost
makes this an extremely useful method for grapevine virus diagnostics. === Dissertation (MSc)--University of Pretoria, 2017. === Winetech === Microbiology and Plant Pathology === MSc === Unrestricted |
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