From Sequencing to Awareness

Bioinformatic Open Days 2020

C I Mendes • Mário Ramirez Lab

Twitter: @ines_cim

The dengue virus

| Transmission & Disease

The dengue virus

doi:10.1038/nrmicro1690

| Transmission & Disease

Sequential infection increases the risk of a severe form of the infection - dengue hemorrhagic fever.

The dengue virus

Geographical distribution of dengue cases reported worldwide, July to September 2019

 17 September 2019 - laboratory-confirmed autochthonous case of dengue in Barcelona, Spain

| Transmission & Disease

The dengue virus

| The virus

Dengue hemorrhagic fever:

  • The virus presents 4 different serotypes, sub-classified in many genotypes
  • After "primary" infection with one serotype, "secondary" infections by one or more of the other serotypes can precipitate ‘antibody dependant enhancement’ (ADE)
  • Infection with one type gives little immune protection against the other types

The dengue virus

| The virus

  • DENV-1  - genotypes I-V​​
  • DENV-2 - genotypes Asian I, Asian II, Cosmopolitan, American, Asian/American & Sylvatic​
  • DENV-3 - genotypes I-V​
  • DENV-4 - genotypes I - III & Sylvatic​

https://doi.org/10.1371/journal.pntd.0001876.g002

https://doi.org/10.1371/journal.pntd.0000757

Thailand

Viet Nam

The dengue virus

DENV: (+)ssRNA (~11Kb; 1 ORF)​

The single polyprotein encodes:​

  • Structural Proteins:​

    • C – capsid​

    • prM – pre-membrane​

    • M - membrane

    • E - envelope​

  • Non-Structural Proteins: ​

    • NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5​​

| The virus

The dengue virus

| The virus

Empower the use of HTS to monitor the dissemination of the disease

RNA Extraction

 PCR
Amplification

HTS Sequencing

➔

➔

How do the different genotypes model transmission and infection?

| The Workflow

  • Ready to use but customizable
  • Scalable
  • Reproducible
  • Stand-alone (confidential data)
  • Easy to explore and share results

Requirements

A solution

A ready-to-use, reproducible, scalable workflow for the identification, serotyping, genotyping and phylogenetic analysis of DENV from HTS data

| The Workflow

DENV Identification

  • 3830 complete DENV genomes from the NIAID Virus Pathogen Database and Analysis Resource (ViPR)​
  • complete genome sequence ​
  • human host (exception of DENV-1 III, monkey)​
  • collection year (1950-2018)​

In Silico Typing:​

  • 161 representative sequences of all sero and genotypes.​

| The Workflow

| Test datasets

Shotgun Metagenomics dataset:

  • 9 plasma samples​
  • 13 serum samples​
  • 1 spiked sample with the 4 serotypes​
  • Positive and Negative controls​
nextflow run DEN-IM.nf -profile slurm_shifter --fastq="fastq/*_{1,2}.*"
  • HPC with 300 Cores/Processing Power and 3 TB RAM
  • On average, each sample took 7 minutes to analyse. A total of 75 CPU hours were used to analyse the 25 samples, with a total of 17Gb in size. This analysis resulted in 69Gb of data.

| Test datasets

| Where to find it?

 https://doi.org/10.1101/628073

Git, Nextflow (java) and a container engine (Docker, singularity, shifter...).​

apt-get install git
curl -s https://get.nextflow.io | bash 

apt-install docker-ce

Clone (or run remotely)

git clone https://github.com/B-UMMI/DEN-IM.git

This work was funded by: FCT - "Fundação para a Ciência e a Tecnologia" (SFRH/BD/129483/2017) and the Abel Tasman Talent Program grant from the UMCG, University of Groningen, Groningen, The Netherland​

Special thanks to E Lizarazo, M P Machado, D N Silva, A Tami, M Ramirez, N Couto, J W A Rossen, J A Carriço

Copy of DEN-IM

By Pedro Cerqueira

Source: Inês Mendes

Copy of DEN-IM

Slide presentation for BOD'2020

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