Broad range virus indexing through NGS: the SafePGR case study. Michel GRISONI CIRAD -UMR PVBMT

Transcription

1 Broad range virus indexing through NGS: the SafePGR case study Michel GRISONI CIRAD -UMR PVBMT

2 Rationale of the SafePGR project The distribution of healthy plants is a key factor for reducing crop loss and economic damages due to virus The capacity to detect and identify virus in PGR is crucial for Quarantines and Biological Resources Centers The advent of New Generation Sequencing (NGS) opens the way for considerable progress in virus diagnosis (ELISA, PCR, Microarray) No need to have prior sequence information Wide range detection (across Families) Deep knowledge of virus population in a plants

3 UMR BGPI UMR AGAP UMR PVBMT UR ASTRO UMR BFP CBA ISOplexis Guadeloupe Montpellier Réunion Guadeloupe Bordeaux Azores Madeira

4 Objectives of the SafePGR project Develop Metagenomics-based tools : Increase knowledge on molecular diversity of virus Improve plant virus diagnosis Secure plant tranfers EXEMPLE D IMAGE

5 Six plant models Five collections Garlic La Réunion & Azores Sugarcane Montpellier, Guadeloupe & Azores Sweet potato Azores & La Réunion Banana Guadeloupe, Azores & Madeira Yam Montpellier & La Réunion Vanilla La Réunion

6 SafePGR workflow Increase knowledge on virus in BRCs material Conventional PCR based methods / generic primers Improve diagnosis tools Set up NGS protocols for virus detection in PGR 16 accessions - 6 plant species Data mining for plant virus diversity (EST) Develop bio-informatics pipeline for the analysis of NGS data EXEMPLE D IMAGE Validation step: Large scale NGS of PGR present in BRCs (Guadeloupe, La Réunion, Azores, Madeira, Montpellier)

7 Organization of the project EXAMPLE OF PICTURE

8 Virus diversity (conventional detection tools approach) (RT) PCR- seq [Sanger] of virus present in PGR using degenerate primers (17 virus genus / 10 families) Setup of improved PCR-based detection protocols At Cirad Réunion : Garlic : potyvirus (2), carlavirus (2), allexivirus (5) Yam : potyvirus (1), badnavirus (1): No Potex, Macluravirus, Secoviridae EXAMPLE OF PICTURE Sweet potato : No virus found (poty, crinivirus, carlavirus, begomovirus) Vanilla : potexvirus (1), potyvirus (1), tobamovirus (1)

9 Metagenomics approach 1 - Methodological step Compare several sequencing methods (16 accessions of known virological status / plant species) Set up bioinformatics tools to retrieve viral sequences EXAMPLE OF PICTURE Mining ESTs Databases for viral sequences Analysis of first NGS datasets

10 Three viral nucleic acids targets ADN / ARN Viral particle Semi purification rpcr1 rpcr2 dsrna CF11 INRA Bordeaux ML Roossinck Kit INtRON Small RNAs Trizol Phenol:Chlo Kit mirvana UMR BGPI

11 NGS datasets SafePGR1 / 454 Roche: 1 st 1/8: CF11BDX-96 2 nd 1/8: CF11BDX th 1/8: CF11BDX th 1/8: VIRMPL-96 5 th 1/8: VIRMPL th 1/8: VIRMPL bp 352 bp UMR BGPI

12 Three viral nucleic acids targets ADN / ARN Viral particle Semi purification rpcr1 rpcr2 dsrna CF11 INRA Bordeaux ML Roossinck Kit INtRON Small RNAs Trizol Phenol:Chlo Kit mirvana UMR BGPI

13 Viral particle semipurification From Victoria/Blinkova (Victoria, 2009 / Blinkova,2010) modified by Emmanuel Fernandez (UMR BGPI) Sample Grinding step Filtration 0,45 µm Ultracentrifugation rpm and resuspension V A N A Virion Associated Nucleic Acids DNA and RNA extraction (96-wells plate) Macherey- Nagel Digestion RNase et DNase

14 C F 11 I N R A B o r d e a u x Sample Extraction Buffer Phenol:Chloroforme Grinding step RNA précipitation Absolute Ethanol Elution Elution Washing steps dsarn Ethanol + Sodium Acetate RNA precipitation RNase DNAse +Proteinase Column filled with CF11 cellulose

15 Metagenomics approach 1 First results Comparison of several sequencing methods (16 accessions of known virological status / plant species) Set up bioinformatics tools to retrieve viral sequences EXAMPLE OF PICTURE Mining ESTs Databases for viral sequences Analysis of first NGS datasets

16 virus identified in ESTs Sugarcane Sugarcane mosaic virus (Potyvirus) Sorghum mosaic virus (Potyvirus) Sugarcane yellow leaf virus (Polerrovirus) Sugarcane streak mosaic virus (unassigned) ESTs accessible via Genbank (2013) Sugarcane: ~ EST Vanilla: 31 EST Yam: ~ EST sweet potato: ~ EST Garlic: ~ EST Banana: ~ EST Sweet potato Sweet potato chlorotic stunt virus (Crinivirus) Sweet potato virus G (Ipomovirus) Sweet potato feathery mottle virus (Potyvirus) Garlic Shallot latent virus (Carlavirus) Allexvirus : Garlic virus B, Garlic virus E (>1000 ESTs) novel Foveavirus (?) (2 EST) Banana novel Tobamovirus(?) (1 EST) Yam Novel Secoviridae (ARN1: 6 EST; ARN2: 59 EST) UMR BFP

17 New putative Foveavirus(es) in Garlic CRA0011 CRA0016 Clichés Anne Saison ANSES-LSV / SCIAM Angers Foveavirus Scafold of Fovea-like virus 454 Roche & Sanger direct sequencing

18 18 putative new virus identified in Deep-seq data (16 accessions / plant species - CF11/siRNA/dsRNA data) Banana Garlic Not many viral reads (BSV, BanMMV) 1 Foveavirus (~8kb), 1 Luteovirus (~4kb), 2 Umbravirus (~2kb) Sugarcane SwPotato 1 Mastrevirus (~full g), 1 Closterovirus (~8kb) 2 Mitovirus?(~1kb), 1 Soymovirus?(~4kb), 1 Mastrevirus (contigs) Yam 1 Potexvirus (~6kb), 1 Macluravirus (~4kb), 2/3 Closteroviridae, 1 Flexiviridae (~full g) Vanilla 1 Potexvirus (~6kb), 1 Flexiviridae (~full g), 1 Caulimovirus? UMR BFP

19 Vanilla virus X a new Potexvirus? Virus particles in vanilla leaves Clichés Anne Saison ANSES-LSV / SCIAM Angers TubeCapture RT-PCR test Lad. 100pb ORSV CR0064 CymMV CR688 VVX CR2148 VVX CR2150 VVX CR2169 VVX Pos. C Grind. Buffer 743 pb Full genome >35% mismatches / all potexvirus

20 Where we are Demonstrated the power of Deep sequencing (454 Roche) for indexing plant genetic resources Increased knowledge on plant virus biodiversity (21 novel putative virus species, new sequences of known virus) Development and optimization of 10 PCR-based diagnostic tools Plant virus specific metagenomics protocols and bioinformatics tools available Increased skills in metagenomics at BRC VATEL

21 Where we go. Validation of the methods (~ 1200 samples / 4 BRCs) Processing accessions the core collection of BRC VATEL (273 samples) Set up appropriate NGS-based diagnostic tools according to specific purpose (indexing, certification, controls ) Adapt NGS-based diagnostic tools to evolving sequencing technologies. Transfer the methods to other crops such as.. CASSAVA!

22 Thank you