5. References

3. Sequence Analysis & Diagnosis (Example Using Coriander)

Next Generation Sequencing offers a high throughput and un-

biased technology to detect and diagnose plants symptomatic

with unknown diseases. These sequenced plants can also

generate complete genomes and characterise new viruses. We

have proved its potential and now we are researching on methods

to improve diagnostics and deliver these results worldwide,

making an impact rapidly for response to diseases.

2. Methods & Materials

RNA Extraction

• Lyse Samples Using Liquid Nitrogen

• RNA extracted using RNeasy kits®

• Calculate RNA yields using Spectrophotometer

Library

Prep

• cDNA Synthesis from RNA

• Ligation of adapters onto synthesised cDNA

• Loading into MiSeq Cartridge Sequencing Kit

Sequence

& Analysis

• MiSeq run – Quality > 30

• Alignment of reads using Trinity1

• Further analysis utilising BLAST and MEGAN2

1. Introduction

Plant pathology begins by analysing symptoms, often

these methods can take time and potentially bias results

due to the focus on symptomatic plants. Next Generation

Sequencing (NGS) offers an alternative method to

diagnose plants without bias by directly sequencing

symptomatic and asymptomatic plants. We are utilising

the MiSeq to sequence and analyse the RNA present in

samples to determine rapidly the cause of disease

symptoms which are mainly plant viruses.

Sample Species Sequencing & Diagnosis Results Future Directions

Pepper (UK) Genome of Pepper mild mottle virus PMMov Forward onto Plant Virology

Phlox (UK) Detected Spiranthes Mosaic Virus 3 SpiMV3 New disease in the UK reported

Parsley (UK) Novel Rhobidoviridae detected Koch's postulates testing

Coriander (UK) Possible novel Potyvirus Genome Annotation

Maize (Kenya) Maize Lethal Necrosis viruses Reported back to Kenyan partners

Lily (UK) No Conclusive Viruses Detected Virology now reanalysing symptoms

Carrot (UK) Novel combinations of mottle viruses Forward into disease management

Tomato (Sweden) Novel Tomato virus detected Reported to Virology for analysis

Table 1. Highlights the diversity of samples that NGS can be utilised for, the international sources and the

findings of diagnostics. Importantly there is always a direction for the future utilising these results.

MSc Bioscience Technology Department of Biology SURAJ RAI

DR. IAN ADAMS

NEXT GENERATION SEQUENCING AS A PLANT DISEASE DIAGNOSTIC TOOL

Figure 2. Methods to Sequence.

→ Preserving the RNA and trying to

gain a good yield of RNA using

ng/µl, 260/280 ratios and

absorbance peaks are crucial.

→ 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 to determine

disease source.

Figure 1. The MiSeq Bench top Sequencer

The analysis of the Coriander (UK) sample diagnosed with a possible novel

Potyvirus utilised a complementary poly protein sequence generated from

the sequenced bases, alongside further research into the Potyviridae

species and known cleavage sites to annotate the genome3. Overall this

allowed us to create a genome organization map for the unknown Potyvirus

Figure 4. This organization map allows us to then BLAST the individual

mature proteins to then classify our new Potyvirus and generate a tree.

Figure 3. Coriander sample with symptoms Figure 4. The Genome Organization Map of Novel Potyvirus.

Shows the ten mature proteins produced by the Novel Portyvirus. This map is further used to separate and BLAST individual proteins for classification. Information on each protein can be found in Adams et al3.

4. Conclusions Within our example for Coriander (UK) we concluded that the cause of yellowing was caused by the VDMV and are now writing a genome annotation paper to share the sequence. Overall this poster highlights the potential of NGS as a plant disease diagnostic tool, how extra resources are required and how important bioinformatics to confirm any data. Next Generation Sequencing is now becoming an accepted diagnostic tool for plant pathology5. Crucial aspects in its use are generating a good library and the correct use of bioinformatics on the sequenced data. Particular care must be given in analysis by utilising extra information to confirm any results.

Figure 5. Novel Potyvirus Coat Protein Tree. The key criteria for species demarcation within the Potyviridae family is a sequence identity of less than 76% in the Coat protein4. The novel Potyvirus matches significantly with the coat protein of the Vanilla Distortion Mosaic Virus (VDMV). Following the taxonomic guidelines and as due to no sequence available for the VDMV we suggest that our Potyvirus is either a new strain of or simply VDMV.

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