Figure 5 – Coriander Genome annotation of the novel Potyvirus now named Coriander Potyvirus. Assays were then designed recognising the coat protein sequence for future use in field to test any future coriander.

Figure 1 - The MiSeq Bench top

NG Sequencer. Illumina

Plant viral disease is a constant threat to food security causing significant damage and currently with no treatment. Correct virus identification is crucial to deploy effective control strategies to protect crops. Next Generation Sequencing (NGS) can be used as a universal and unbiased method for virus identification. NGS improves significantly upon limitations of previous methods particularly in speed and generation of information. This technology also allows for assay development and further novel species discovery whilst aiding in food security.

1. Introduction Plant pathology begins by analysing

symptoms, these methods can

significant take time and bias results.

Next Generation Sequencing (NGS)

offers an alternative method to

diagnose plants without bias and

generate new information on the

infection. This poster highlights the use

of the MiSeq along with genomic and

bioinformatics analysis to determine

the cause of disease symptoms,

identify novel viruses, annotate the

genomes of viruses and develop assays

for future use.

Table 1. Highlights the diversity of samples and results from NGS.

Suraj Rai 1,2 Dr Ian Adams2

University of York, Department of Biology, York, YO105DD, UK The Food & Environment Agency , York, YO411LZ , UK

Next Generation Sequencing for Virus Discovery, Identification and Food Security

Overall this poster highlights the success of NGS deployed as virus diagnostic tool. Next Generation Sequencing is an highly useful diagnostic tool for

plant pathology & viral diagnostics5. Crucial aspects are generating a good library, the correct use of

bioinformatics and extra information on the sequenced data. Future analysis must be given in utilising the exome data for

resistance genes.

1- Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng

Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K,

Friedman N, Regev A. Full-length transcriptome assembly from RNA-seq data without a reference genome.

Nat Biotechnol. 2011 May 15;29(7):644-52

2-Huson, DH, Mitra, S, Weber, N, Ruscheweyh, H, and Schuster, SC (2011). Integrative analysis of

environmental sequences using MEGAN4. Genome Research, 21:1552-1560

3-MICHAEL J. ADAMS, JOHN F. ANTONIW AND FREDERIC BEAUDOIN. MOLECULAR PLANT PATHOLOGY (2005),

6, ( 4 ) , 471–487 Overview and analysis of the polyprotein cleavage sites in the family Potyviridae.

5- Virus Taxonomy, Classification and Nomenclature of Viruses, Ninth Report of the International Committee

on Taxonomy of Viruses Editors Andrew M.Q. King, Michael J. Adams, Eric B. Carstens, and Elliot J. Lefkowitz

6- K. Prabha, V. K. Baranwal, R. K. Jain. Indian Journal of Virology May 2013 Applications of Next Generation

High Throughput Sequencing Technologies in Characterization, Discovery and Molecular Interaction of Plant

Viruses

l

2. Methods Overview

Figure 3 – Full NGS

Diagnostics workflow.

Unknown Virus RNA Extraction

Virus Identification

3. Results

4. Conclusions & Future Directions

→ Extracting Virus RNA to gain a

quantifiable yield of RNA using

ng/µl, 260/280 ratios and

absorbance peaks.

→ Ligating synthesised cDNA with

adapters to be able to pool samples

and sequence allows us to

multiplex entire batches of

different samples.

→ Sequencing and monitoring to

maintain quality above >30,

alignment of the sequences and

BLAST and MEGAN Tree analysis to

determine viral identity.

A) RNA extraction and purification from diseased samples followed by quantification, ensuring quality.

B) cDNA synthesis, adapter ligations, library enrichment followed by validations, ensuring optimal concentrations.

C) Sample libraries pooling, validation and sequencing.

D) Sequence alignments and bioinformatic analysis for identification.

5. References

Overall the method is designed to

allow for swift processing of

samples to ensure rapid results

whilst also maintaining validity. Figure 2 – Virus Infected Coriander.

Our diseased samples showed a variety of vir infections. Examples in

the Phlox and Coriander are highlighted below*.

A

Figure 4 – Phlox Neighbour-joining tree using 500 boostrap replicates for coat proteins of Mosaic Viruses. Confirms the virus as SpiMV-3.

B

C

D

Sample Identification Result

Phlox Paniculata Spiranthes Mosaic Virus 3*

Coriander Novel Potyvirus CyoPV *

Tarenna D Fungal and Virus Infections

Maize Potential MCMV

Cabbage Potential Varicosavirus

Lantana Camara Potential Fungal Infections