Training Workshop for the Implementation of MinION Sequencing for Influenza Viruses 

February 6-8, 2020

Irotama Resort

Santa Marta, Colombia

Workshop Objective

This three-day workshop, held February 6-8th, 2020 in Santa Marta, Colombia, introduced new technologies and advanced informatics approaches that can be used for infectious disease genomic characterization in resource-limited settings. Using the portable, low-cost Oxford Nanopore MinION sequencing platform, we will introduce the essential laboratory and computational skills required to perform extensive genetic characterization of viral pathogens, using influenza A virus as a primary demonstration. Topics will include sample preparation, bioinformatics, and phylogenetic approaches to characterize newly sequenced sampled against publicly available data.

By the completion of the workshop, attendees were provided access and training to all essential bioinformatics tools required to establish real-time genetic characterization capabilities in their primary laboratories or at field sites using the low-cost Oxford Nanopore MinION sequencing platform. Best practices for implementing these new technologies in research, clinical, and public health laboratories were emphasized.

 

Instructors/staff:

Johns Hopkins University: Tom Mehoke, Peter Thielen

Fogarty International Center: Martha Nelson, Nidia Trovao

 

Workshop Agenda

Day 1 (Thursday Feb 6) 

Introduction to Genomics Technologies and Oxford Nanopore Sequencing

 

Time	Event	Presenter
9:00am	Welcome and Introductions Pre-session surveys	Peter Thielen
9:30am	Low-Resource Genome Sequencing with Oxford Nanopore: Outbreak response and Influenza Applications	Peter Thielen
10:00am	Data Analysis Objectives: Rapid Characterization and Consensus Sequence Generation	Peter Thielen
11:00am	BREAK
11:15am	Rapid Talks: Single Slide Presentations from Attendees	Attendees
12:00pm	Lunch / Surveillance Meeting Field Trips

 

Day 2 (Friday Feb 7):

Laboratory Considerations and Basic Bioinformatics

 

Time	Event	Presenter
9:00am	Practical Considerations: Establishing a Genomics Wet Laboratory Efficient isolation of nucleic acids (DNA/RNA) Targeted and untargeted sequencing approaches Accurate quantification of DNA Minimizing contamination	Peter Thielen
9:30am	Concepts: Optimized Influenza A Virus Sample Preparation with Ligation reagents Time- and reagent-optimizing considerations Sequencing of influenza multi-segment PCR amplicons	Peter Thielen
10:00am	BREAK
10:15am	Hands on: Sequencing with the MinION	Peter Thielen
11:45pm	Data Manipulation and Exploratory Data Analysis Complete consensus generation on generated data	Tom Mehoke
12:15pm	Lunch
1:15pm	Introduction to Phylogenetics Concepts 101 talks on principles of phylogenetics Common mistakes	Martha Nelson
2:15pm	Using data produced by the MinION Outline of general analysis workflow Initial processing vs. in depth analysis	Tom Mehoke
2:45pm	BREAK
3:00pm	Hands-on: Processing data produced so far Visualization of processed data Introduction to folder hierarchy Consensus sequence generation	Tom Mehoke
5:00pm	Day 2 Review Revisit data from the earlier sequencing run Recap of topics Post-session evaluations

 

Day 3 (Saturday Feb 8):

Phylogenetics

 

Time	Event	Presenter
9:00am	Identifying Genomes and Building a Genetic Sequence Dataset Genbank	Nídia Trovão
10:00am	Multiple Sequence Alignment and Building a Phylogenetic Tree AliView MEGA	Nídia Trovão
11:00am	BREAK
11:15am	Post-processing of Phylogenetic Trees FigTree	Martha Nelson
12:15pm	Lunch
1:15pm	How To: Interpret a Phylogenetic Tree	Martha Nelson
2:15pm	Final Recap Open Discussion: Moving forward with resource development and applied research Post-session evaluations
3:00pm	BREAK
3:15pm	Time to Work With Your Own Data	All instructors available for questions

 

 

 

 

POSTPONED

New date TBD

 

FIC MISMS workshop 

Influenza epidemiology, digital surveillance and evolutionary dynamics

March 12-13, 2020

ANISE meeting, Livingstone, Zambia

 

Link for Registration

This training workshop is held in conjunction with the March 2020 meeting of the ANISE network in Zambia and follows scientific research sessions and training modules led by the GISAID and WHO teams earlier in the week.

Information on influenza epidemiology and evolutionary dynamics is particularly useful to guide intervention strategies, optimize surveillance, and vaccine design, but such studies remain scare in low-income settings. Digital surveillance uses novel data streams (Google searches, Twitter, data from participatory surveillance systems) to help monitor and forecast influenza activity in settings where traditional surveillance data may be lacking, sparse, or lag by several weeks. Phylogenetic analyses of influenza sequence data are important to shed light on the global, regional and local migration of influenza viruses, their evolution, and the match of circulating strains with available vaccines.

The objective of this two-day MISMS workshop is to train epidemiologists and virologists from Africa (particularly from countries with available data) on the use of the quantitative epidemiological methods for digital surveillance and forecasting, and on phylogenetic analyses for phylodynamics and phylogeography using the BEAST package.

This workshop will include two separate modules devoted to (i) epidemiology and (ii) phylogenetics. Both modules will include theory and practice sessions. Participants will be invited to work with their own data and with publicly available sample datasets.

Participants in the epidemiological module will be taught how to use different R packages for statistical analyses of influenza time series (lab-confirmed cases, ILI or SARI) and develop models using digital surveillance signals (Google, Twitter). They will learn how to use and analyse various Google tools, such as Google searches, Google correlates, and Google Trends. Short-term influenza forecasting models will also be presented. Participants will be walked through examples using sample datasets, and if time allows, a few hours will be devoted to the analysis of the participants’ own data. There will be a live demo of InfluenzaNet, an on-line platform for influenza participatory surveillance.

Participants in the phylogenetic module will learn how to prepare a sequence dataset, explored it using maximum likelihood phylogenies and use the BEAST platform to perform advanced phylogenetic analyses to understand the spatial and evolutionary dynamics of influenza viruses. BEAST is a cross-platform program for Bayesian analysis of molecular sequences using Markov chain Monte Carlo (MCMC) algorithms. Outputs include rooted, time-measured phylogenies inferred using strict or relaxed molecular clock models. It can be used as a method of reconstructing phylogenies but is also a framework for testing evolutionary hypotheses without conditioning on a single tree topology. BEAST uses MCMC to average over tree space, so that each tree is weighted proportional to its posterior probability.

Participants will be provided a sample FASTA influenza datasets and walked through an example of BEAST analysis. An overview of the possible uses of BEAST will be discussed. If time allows, a few hours will be devoted to the analysis of the participants’ own data.

 

Faculty:

Trevor Bedford, Fred Hutchinson Cancer Institute, WA, USA (TBC)

Daniela Paolotti, Institute for Scientific Interchange, Italy

Mauricio Santillana, Boston Children Hospital, MA, USA (TBC)

Kaiyuan Sun, NIH-Fogarty, Bethesda, MD, USA

Nídia Sequeira Trovão, NIH-Fogarty, Bethesda, MD, USA

Cécile Viboud, NIH-Fogarty, Bethesda, MD, USA