GenEpi-BioTrain - Virtual training 19 - Genomic Tools in Outbreak Investigations - Case Study Cholera Outbreak

Topic outline

• Welcome to the virtual training on Genomic Tools in Outbreak Investigation!

  Welcome to the virtual training on Genomic Tools in Outbreak Investigation!

  ℹ️ Please Note

  You are welcome to explore the available resources and video recordings. Please note that the resources in this course come from the Virtual Training 19 held in August–September 2025, and certificates are only issued to participants who attended the live webinar sessions.

  
  Course Description

  This two-half-day training course explores the application of genomic tools in outbreak investigations using the 2010 Haitian cholera outbreak - "Population Genetics of Vibrio cholerae from Nepal in 2010: Evidence on the Origin of the Haitian Outbreak" (DOI: 10.1128/mBio.00157-11) - as a case study. Through guided analysis of molecular and genomic data, participants will compare traditional typing methods (e.g., Pulsed-field gel electrophoresis-PFGE) with modern approaches like Whole-Genome Sequencing-Based Typing (WGST), and develop skills in interpreting phylogenetic relationships, antimicrobial resistance (AMR), and virulence profiles, and genomic epidemiology in a real-world context. The course includes a simulation exercise to reinforce practical understanding.

  Target audience

  Bioinformaticians, Microbiologists, Epidemiologists, and Clinicians with basic Bioinformatics skills.

  Pre-requisites

  This training, specifically the simulated exercise, has some Prerequisites. In anticipation of the first session, we invite you to:

  1. Read carefully the 'Software requirements' and prepare your tools. 

  Format

  This virtual training consists of two sessions held on Wednesday, August 27, and Wednesday, September 3, from 9:00 AM to 12:30 PM (CEST).

  

  
  

  The practical exercises assigned in Session 1 should be completed before the next session. The discussion of the simulated exercise will take place during Session 2.
  

  

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  Aim and Objectives

  Aim and Objectives

  Course Description

  This two-half-day training course explores the application of genomic tools in outbreak investigations using the 2010 Haitian cholera outbreak - "Population Genetics of Vibrio cholerae from Nepal in 2010: Evidence on the Origin of the Haitian Outbreak" (DOI: 10.1128/mBio.00157-11) - as a case study. Through guided analysis of molecular and genomic data, participants will compare traditional typing methods (e.g., Pulsed-field gel electrophoresis-PFGE) with modern approaches like Whole-Genome Sequencing-Based Typing (WGST), and develop skills in interpreting phylogenetic relationships, antimicrobial resistance (AMR), and virulence profiles, and genomic epidemiology in a real-world context. The course includes a simulation exercise to reinforce practical understanding.

  Intended Learning Outcomes (ILOs)

  Upon completion of this workshop, participants will be able to:

  1. Describe the epidemiological context of the 2010 Haitian cholera outbreak.
  2. Explain the differences in resolution and applicability between PFGE and WGST.
  3. Use simple bioinformatics tools to infer phylogenies from genomic data.
  4. Analyze and interpret Single Nucleotide Polymorphism (SNP) matrices and maximum parsimony trees.
  5. Correlate antimicrobial resistance, and virulence profiles with epidemiological data.
  6. Assess evidence to infer outbreak sources using genomic data.
  7. Communicate findings from genomic investigations in a structured report.
  8. Critically evaluate the role of genomics in global health surveillance.
• Software Requirements

  Software Requirements

  Equipment and Tools

  To participate effectively in the simulated exercise, ensure your machine meets the following requirements and has the appropriate tools installed.

  Equipment:

  • A laptop with internet access.
  • Sample datasets (provided latter). 
  • For command-line usage: macOS or Linux recommended; Windows users should use WSL (Windows Subsystem for Linux) or Docker. 
  • Mamba or Conda (installed through, for example,       Miniforge — https://github.com/conda-forge/miniforge) installation is mandatory for managing software environments and packages.

  Tool Setup:

  • Participants may choose to use web-based (basic) tools or command-line (advanced) tools depending on their comfort and experience.
  • Pre-install the command-line tools before Session 1 for a smooth workflow.
  • Web tools require no installation — access via provided links.

  1. Snippy (Command-line – Advanced)

  • Objective: Call SNPs from assembled genomes and generate a core SNP alignment.
  • URL with detailed instructions: https://github.com/tseemann/snippy.
  • Installation (using Bioconda):

  conda config --add channels conda-forge conda config --add channels bioconda conda config --set channel_priority strict # Installation using mamba mamba install snippy   # Verify installation snippy --version snippy --check

  2. CSI Phylogeny (Web – Basic)

  • Objective: Upload FASTA files and a reference genome to call SNPs, filter them, and automatically infer a phylogenetic tree.
  • Access: Use the online interface (no installation needed).
  • URL: https://cge.food.dtu.dk/services/CSIPhylogeny/.

  3. MINTyper (Web + CLI – Basic)

  • Objective: Automatically select a reference, call and mask SNPs, and build a phylogenetic tree (Neighbor-Joining-NJ or Maximum Likelihood-ML).
  • Access:
  • Web interface: https://cge.food.dtu.dk/services/MINTyper/.
  • CLI (optional install): pip install mintyper.

  4. IQ‑TREE (Command-line – Advanced)

  • Objective: Perform maximum-likelihood phylogenetic inference from SNP alignments.
  • URL with detailed instructions: https://github.com/iqtree/iqtree3.
  • Installation (Bioconda):

  # Installation using mamba mamba install iqtree # Verify installation iqtree --version iqtree --help

  5. iTOL (Visualization – Basic)

  • Objective: Visualize and annotate phylogenetic trees (Neighbor-Joining-NJ or Maximum Likelihood-ML).
  • Access:
  • iTOL: Web-based (sign-up link https://itol.embl.de/).
  • MEGA: Downloadable desktop GUI (Windows, macOS, Linux — installation file provided in https://www.megasoftware.net/).

  6. AMRFinderPlus (Command-line – Advanced)

  • Objective: Detect AMR and biocide tolerance genes, virulence factors, and other stress-resistance elements.
  • URL with detailed instructions: https://github.com/ncbi/amr. 
  • Installation (Bioconda):

  # Installation using mamba mamba install ncbi-amrfinderplus   # Verify installation amrfinder --version amrfinder --help

  7. ResFinder (Web – Basic)

  • Objective: Upload genomes to detect AMR genes or associated mutations via a user-friendly interface.
  • URL: http://genepi.food.dtu.dk/resfinder.

  8. CholeraFinder (Web – Basic)

  • Objective: Detect Vibrio cholerae-specific virulence genes.
  • URL: https://cge.food.dtu.dk/services/CholeraeFinder/.

  9. IslandViewer 4 (Web – Basic)

  • Objective: Identify genomic islands using an online interface.
  • URL: https://www.pathogenomics.sfu.ca/islandviewer.

  10. VCGIDB (Web – Basic)

  • Objective: Explore V. cholerae-specific genomic island predictions.
  • URL: http://leb.snu.ac.kr/vcgidb/index.

  
  

  
  
• Session 1: Introduction to Molecular Epidemiology and WGS in Outbreak Investigation

  Session 1: Introduction to Molecular Epidemiology and WGS in Outbreak Investigation

  This session will cover the following:

  1. Introduction to Vibrio cholerae and cholera. Setting the scene: Case Study-Haiti
  2. Virulence Factors and Genomic Islands in V. cholerae
  3. Typing Tools: From PFGE to WGS
  4. Phylogenetic Tree Interpretation and WGS Tools
  5. Exercise Introduction: Outbreak Analysis

  • Recording of session 1- Opening

  • Recording of session 1- PART 1: Introduction to Vibrio cholerae and cholera. Setting the scene: Case Study-Haiti

  • FileEpidemiological background of cholera and the 2010 Haiti outbreak
  • Recording of session 1- PART 2: Virulence Factors and Genomic Islands in V. cholerae

  • FileV cholerae virulence ceg
  • Recording of session 1- PART 3: Typing Tools: From PFGE to WGS

  • FileTyping Tools PFGE to WGS JOAM
  • Recording of session 1- PART 4: Phylogenetic Tree Interpretation and WGS Tools

  • FileSNPs cgMLST epidemiology presentation
  • Recording of session 1- PART 5: Exercise Introduction: Outbreak Analysis

  • FileAnnotation AMR Virulence MGE JOAM
• Simulated Exercise between sessions

  Simulated Exercise between sessions

  You will receive a small set of assembled Vibrio cholerae genomes (FASTA files) derived from Haitian and Nepalese isolates. Additional isolates can also be provided for further comparative genomic analysis.

  Your task is to replicate the phylogenetic analysis done in the Hendriksen et al. (2011) study, using freely available or the recommended tools during Session 1 (you can use different tools).

  • Practical

    Here you will find the practical exercises assigned in Session 1 that should be completed before the next session. The discussion of the simulated exercise will take place during Session 2.
    

    Task: Reproduce the phylogenetic analysis from Hendriksen et al. (2011) using tools of your choice.

    Some suggestions (see more suggestions for tools above):

    •Online: CSI Phylogeny, MINTyper, iTOL, MEGA, Microreact
    •Command-line: Snippy, IQ-TREE / RAxML

    Aim:
    

    •Identify clusters and closely related isolates
    •Determine how Haitian and Nepalese isolates group
    •Estimate the likely source of the outbreak strain based on tree topology & SNP distances

    Data for the SimEx:
    

    Available isolates:

    •24 Nepalese V. cholerae
    •3 Haitian V. cholerae
    •5 additional V. cholerae isolates linked to the 2010 Haitian outbreak (optional to include in your analysis)

    Data format:
    

    •Illumina sequences
    •Provided as zipped FASTA files

    Where to download the data?:

    •All files are available on ScienceData via this link: https://sciencedata.dk/shared/7ba608b7d30942995cc1e94baa475102

  • An announcement will be made once the datasets and materials for the exercise are available.

    
    
    

  • FileAdditional Isolates Metadata

    Excel file containing metadata (e.g., location, source, year) for the additional isolates.
    

  • FileCode in PDF format
  • FileCode in R Markdown

    Important: the commands aren’t “one-click copy and paste”. You’ll need to replace placeholders with your own files/paths before running.

    Edit before you run:

    REF → set to your reference FASTA (put it in your working folder).

    sra_list.txt → add the SRR accessions (or skip if you already have assemblies).

    samples_reads.tsv → map your sample IDs to the downloaded *_1.fastq.gz / *_2.fastq.gz.

    If using existing assemblies, ensure they’re at assemblies/<ID>/contigs.fa (or adjust the paths).

    THREADS → match your CPU; on Apple Silicon keep --platform linux/amd64.

    How to use it:

    The code chunks are Bash and meant to be copied to your terminal (Docker required).

    The .Rmd is set to show code but not execute it (eval = FALSE), so knitting won’t run anything.

    
    

• Session 2: Interpretation of Results and Discussion on the Application of WGS in Outbreak Scenarios
  This topic

  Session 2: Interpretation of Results and Discussion on the Application of WGS in Outbreak Scenarios

  This session will cover the following:

  1. Guided Discussion using Slido
  2. Comparative and Critical Analysis of the Results
  3. Case Study-Haiti Reflections
  4. Troubleshooting and Q&A (distributed among the other topics)

  • Recording of session 2- Opening

  • Recording of session 2- Guided Discussion using Slido

  • FileSNPs cgMLST epidemiology presentation results session
  • Recording of session 2- Comparative and Critical Analysis of the Results

  • FileExercise AMR Virulence MGE JOAM
  • Recording of session 2- Case Study-Haiti Reflections

  • FileEpidemiological background of cholera and the 2010 Haiti outbreak v1 part 2
• Contacts

  Contacts

  For any questions regarding the this webinar series, please use the discussion forum.

  If you have questions regarding other training initiatives please contact: courses@ecdc.europa.eu.

  If you have technical issues please contact the ECDC Service Desk at servicedesk@ecdc.europa.eu.
• Delivery

  Delivery
  To help you get the most out of the discussion in Session 2, we ask you to prepare a short written report on your findings from the simulated exercise, which replicates a SNP-based phylogenetic analysis of Vibrio cholerae isolates published in "Population Genetics of Vibrio cholerae from Nepal in 2010: Evidence on the Origin of the Haitian Outbreak" (DOI: 10.1128/mBio.00157-11).

  This short report will make it easier for you to answer the questions we will pose during the Slido discussion in Session 2. It does not need to be long or formal — just a concise summary of your main observations and interpretations.

  Specifically, your report should include:

  • A screenshot of your SNP-based phylogenetic tree, with the key clades labelled (e.g., Haitian outbreak isolates, Nepalese isolates, reference isolates).

  • A brief interpretation of your findings:

    • Which isolates form a clade?

    • What does the tree suggest about the possible geographic origin of the Haitian outbreak?

  • A short summary of detected AMR genes, virulence factors, or notable genomic elements identified in the isolates.

  • Any notes on limitations or uncertainties in your analysis (e.g., sample representativeness, potential biases, phylogenetic resolution).

  Having this document ready will help you actively participate in the discussion and support your answers with evidence.