Course outside EMBL-EBI
CABANA Workshop: NGS analysis applied to virome sequencing in agricultural systems
Virome sequencing is a high throughput method to identify and classify communities of viruses, not only in plants but also animals and humans. We will focus on the sequencing of small-interfering RNAs (siRNAs) to discover the virus population in plant samples. The resulting information analysis relies on viral databases that are constantly evolving, and software tools constantly being updated. Therefore, we will explore all of these aspects in this workshop.
The workshop will consist of two modules: i) a wet bench workshop for training around the experimental design and DNA library preparation, and ii) a bioinformatics module overviewing multiple applications of Virome Sequencing, and its application in agricultural systems. People interested in this workshop may apply for both modules (limited to 10 participants) or only for the bioinformatics module (limited to 25 participants). Selection of participants will be based on application form.
The bioinformatics module includes a pipeline for detailed computational analysis of virome data; including genomic analysis tools such as Virus-DetectW, phylogenetic and recombination analysis with MegaX and RDP and network analysis using the statistical package R. Also, publicly available resources for virus analysis and their application will be implemented during the workshop.
The detection of pathogens is an essential task for people working on agricultural systems. In recent years the use of sequencing technologies has enabled the discovery and description of new viruses, even communities of pathogenic entities within crops, for example potato and sweet potato. This is relevant for surveillance and to develop strategies to control these pathogens across borders.
Who is this course for?
This course is aimed at wet-lab researchers at any career stage within Latin America undertaking pathogen population genetics and surveillance, interested in applying next generation sequencing to virus identification. Those working in plant protection organizations and/or academia are welcome to apply, and priority will be given to those who have not yet attended a CABANA workshop.
Please note this course will be taught in English, however the trainers are fluent in Spanish, and can offer language support where feasible.
A number of travel fellowships are available for this course - early-stage researchers and researchers from underrepresented groups are especially encouraged to apply for CABANA travel fellowships. You can apply for travel fellowships via the course application form. Priority will be given to those who have not previously been awarded a CABANA fellowship.
Knowledge in R and NGS technologies is required. Therefore, these free resources should be studied beforehand:
What will I learn?
After this course you will be able to:
- Use the miRNA kit to extract nucleic acids
- Implement the workflow to prepare small RNA libraries
- Recall and explain the applications of this protocol
After the computational training course you will be able to:
- Plan and design experiments for virome sequencing in your organism of interest
- Assess applications of virome sequencing to detection of agricultural diseases, discovery of biological control agents, biodiversity studies and plant phytobiome
- Implement pipelines for analyzing viromes and interpretation of results
- Use public databases for viral genomics
During this course you will learn about:
- Extraction of viral nucleic acids from plant tissue with the miRNA kit
- siRNA quality parameters
- Library preparation for small RNAs using custom primer
- DNA library quality check
- Sequencing of libraries on the NextSeq instrument
During the computational training course you will learn about:
- Databases including Genbank & ENADiverse software to analyze virus populations such as: VirusDetect-W, Geneious, UGene, Mega, RDP
- R for analysis of sequencing experiments
- Download RStudio: https://rstudio.com/products/rstudio/download/#download
- R libraries required (for network analysis):
International Potato Center (CIP), Peru
International Potato Center (CIP), Peru
CIBCM - University of Costa Rica, Costa Rica
CITA-UCR, Costa Rica
DCLab - UCR, Costa Rica
University of Florida
EEAFBM-UCR, Costa Rica
Day 1 – Monday 24th February 2020
|09:00-09:15||Welcome and Instructions||Rebeca Campos|
|09:15-10:00||Introduction to deep sequencing / small RNA preparation||Segundo Fuentes|
|10:00-12:30||Processing of samples (RNA extraction with miRNA kit)||Segundo Fuentes / Ana Pérez|
|13:30-15:30||Processing of samples (RNA extraction with miRNA kit) - continued||Segundo Fuentes / Ana Pérez|
|16:00-17:30||siRNA library preparation: Adenylated 3’ adapter ligation (optional: 3’ adapter adenylation)||Segundo Fuentes / Ana Pérez|
|17:30||End of day 1|
Day 2 – Tuesday 25th February 2020
|09:00-12:00||siRNA library preparation: 5’ adapter ligation (3’ adapter adenylation - continued)||Segundo Fuentes / Ana Pérez|
|13:00-15:30||siRNA library preparation: Reverse transcription (RT), Agarose gel preparation, PCR test, Gel electrophoresis and Qiaxcel run||Segundo Fuentes / Ana Pérez / Ivannia Atmetlla|
|16:00-18:30||siRNA library preparation continued||Segundo Fuentes / Ana Pérez / Ivannia Atmetlla|
|18:30||End of day 2|
Day 3 – Wednesday 26th February 2020
|09:00-12:00||siRNA library preparation: PCR enrichment, Agarose gel preparation, Gel electrophoresis||Segundo Fuentes / Ana Pérez|
|13:00-15:00||siRNA library preparation: Gel electrophoresis and Qiaxcel run||Segundo Fuentes / Ana Pérez / Ivannia Atmetlla|
|15:30-16:30||siRNA library preparation: siRNA library purification||Segundo Fuentes / Ana Pérez|
|16:30||End of day 3|
Day 4 - Thursday 27th February 2020
|09:00-12:30||siRNA library preparation: siRNA library quantification (Qubit), Preparation of pooled siRNA libraries||Segundo Fuentes/ Ana Pérez / Ivannia Atmetlla|
|13:30-17:30||Q&A session of extraction protocols, library prep||All trainers|
|17:30||End of day 4|
Day 5 – Friday 28th February 2020
|09:00-12:00||Sequencing the libraries in the NextSeq - Illumina platform||Ricardo Chinchilla|
|13:00-14:00||Wrap-up and Feedback||Rebeca Campos|
|14:00||End of day 5|
Computational training programme
Day 1 – Monday 2nd March 2020
|08:00-8:30||Welcome and Instructions||Rebeca Campos|
|08:30-09:00||Introduction and CABANA presentation||Andrés Gatica|
|09:00-10:00||Short presentation of the participants about their research (2 min each) - Flash talks||Mauricio Montero|
|10:30-11:30||Theory of NGS for virus discovery||Jan Kreuze|
|11:30-12:30||Plan and design experiment||Segundo Fuentes|
|13:30-15:30||Example and practice of virome sequencing||Jan Kreuze / Ricardo I. Alcalȧ|
|16:00-17:30||Pipeline and tools for sequencing data||Jan Kreuze|
|17:30||End of day 1|
Day 2 – Tuesday 3rd March 2020
|08:00-08:30||Q&A of previous day||All trainers|
|08:30-10:00||Applying VirusDetect-W to previous datasets, interpretation of results||Jan Kreuze|
|10:30-12:00||Assembling complete genomes, quality checking||Segundo Fuentes|
|13:00-15:30||Network analysis of plant viromes and practical||Ricardo I. Alcalá|
|16:00-17:00||Practice with VirusDetect-W||Jan Kreuze|
|17:00||End of day 2|
Day 3 – Wednesday 4th March 2020
|08:30-09:00||Q&A of previous day||All trainers|
|09:00-10:30||Phylogenies with viral data (comparative genomics)||Jan Kreuze|
|11:00-12:30||Other tools for viral genomics: RDP (Recombination Detection Program), viral evolution, phylogenetics||Jan Kreuze|
|13:30-15:00||Practice on phylogenies & interpretation||Jan Kreuze|
|15:30-16:15||Project presentation: "Molecular marker assisted selection in tomato cultivars with resistance genes to Tomato yellow leaf curl virus (TYLCV)"||Natalia Barboza / Walter Barrantes|
|17:00-17:30||Wrap-up and feedback||Rebeca Campos|
|17:30||End of course|
Please note that the registration fee of $75000 CRC (wet-lab and computational training) or $30000 CRC (computational training only) includes:
- Lunch and refreshment breaks
- Course materials
- Use of a computer in the Training Room at Educación Continua throughout the course
- Course dinner on Tuesday 3rd March 2020
- Course certificate
There will be a maximum of 10 participants for the wet-lab workshop (mandatory to stay for the computational workshop) and 25 participants for the computational workshop. Registration is dependent on selection after successful completion of the application process.
Applications will close on January 24th 2020 at 23:30 noon (GMT)
To apply for this event, please visit and complete the application form. Incomplete applications will not be processed. You will need to pay by credit card once you are informed you are selected.
If you have any questions please email Prof. Rebeca Campos-Sanchez.