Visualization of the Evolution and Transmission of Circulating Vaccine-Derived Poliovirus (cVDPV) Outbreaks in the African Region

Equipment

We used shell commands on a Linux-based operating system with superuser privileges.

Computational requirements: We used a workstation or a server with a 64-bit Linux-based operating system, possessing 12 GB RAM and sufficient hard disk space (at least 500 GB) to store the files used and produced in the analysis. The commands given in this analysis protocol were validated on Ubuntu (24.04 LTS) Linux Distribution running on a Microsoft Azure cloud server (https://azure.microsoft.com/en-gb) and on a personal laptop running the same system.

Software and datasets

1. Required software

This protocol uses the following tools and Nextstrain software to perform the phylogeographic analysis:

a. Anaconda (https://github.com/conda/conda)

b. Nextstrain https://github.com/nextstrain [7]

c. Augur https://github.com/nextstrain/augur [7]

d. Auspice https://github.com/nextstrain/augur [7]

e. MAFFT (https://github.com/GSLBiotech/mafft [8]

f. IQTREE http://www.iqtree.org [9]

All requisite tools and their dependents were installed (personal computer or workstation) before proceeding with the analysis.

2. Datasets

The protocol uses the poliovirus VP1 gene sequence datasets made publicly available by the National Center for Biotechnology Information (NCBI): https://www.ncbi.nlm.nih.gov/nucleotide/ for development. Similar datasets are routinely generated in AFRO-supported laboratories in WHO AFRO under the supervision of the global network of poliovirus laboratories (Global Poliovirus Laboratory Network, GPLN).

The installation steps for all tools used in this protocol and the instructions for downloading the requisite datasets are given in the following section.

Procedure

The individual steps involved in this protocol and the Augur modules used in each step are summarized in Figure 1.

Figure 1. Different steps described in this protocol and the Augur modules used in each of the analysis steps

A. Install Anaconda

Anaconda is an open-source distribution of Python that simplifies the management of Python packages and environments. To install Anaconda, use the following commands:

$ wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh

$ echo 'exportPATH="~/miniconda3/bin:$PATH"' >> ~/.bashrc

Follow the on-screen instructions to continue the installation. The anaconda3 folder can be found in the directory shown in the installer script (Miniconda3-latest-Linux-x86_64.sh). The installation is then activated and tested by running the following commands:

$ source ~/.bashrc

$ conda list

B. Install Nextstrain command-line interface (Nextstrain-CLI)

Use the commands below to install Nextstrain directly in a Conda environment.

$ conda create -n nextstrain

$ conda activate nextstrain

$ curl -fsSL --proto '=https' https://nextstrain.org/cli/installer/linux | bash

$ nextstrain setup --set-default conda

Enter the interactive Nextstrain shell in the current directory (.).

$ nextstrain shell .

To check whether Nextstrain was successfully installed, use the following command:

$ nextstrain version

The version number shown in the output should be 8.5.3 or higher. Augur is a bioinformatics toolkit for phylogenetic analysis, whereas Auspice is an interactive visualization tool for phylogenomic data [7].

Test Augur and associated Auspice for visualization:

$ augur --help

$ auspice --help

C. Test MAFFT and IQ-TREE installations

MAFFT (multiple alignment using fast Fourier transform) is required by Augur to perform multiple-sequence alignments [8]. IQ-TREE is an open-source tool for constructing maximum-likelihood trees using phylogenetic data [9]. IQ-TREE is required by Augur for constructing a phylogenetic tree from sequence data. To test these two installations, use the following command:

$ mafft - - help

$ iqtree - - help

D. Download poliovirus Sabin 2 vaccine reference

Before proceeding with the analysis, download the Sabin 2 vaccine reference strain from NCBI in GenBank format (https://www.ncbi.nlm.nih.gov/genbank/). For this analysis, the strain with the accession number AY082679 is downloaded.

E. Download publicly available VP1 gene sequence dataset

Sequence datasets are downloaded from NCBI (https://www.ncbi.nlm.nih.gov/nucleotide/) in FASTA format using the accession numbers provided in File S1.

F. Preparation of input files

To use Nextstrain for phylogenetic analysis and visualization, prepare the following input files (Table 1):

Table 1. List of input files required to run the phylogeographic analysis pipeline.

1. polio_sequences.fasta

This is the single FASTA file containing a collection of poliovirus sequences to be analyzed. For this analysis, use the sequence dataset downloaded from NCBI. Each sequence in the FASTA file has the unique strain identity of the virus as the sequence header. A sample sequence record for the FASTA file is shown in Figure 2.

Figure 2. Sample record for poliovirus accession number PQ497685 strain in the sequences.fasta format

2. polio_metadata.tsv

A tab-delimited metadata file that describes the sequences given in the FASTA file. The various fields included in the metadata file are as follows:

a. Required fields: strain, virus, date.

For each strain ID in the polio_sequences.fasta file, there should be an entry under the strain column in the metadata file.

b. Additional fields (if using published data): Accession, Authors, URL, Title, Journal, Paper_URL.

c. To infer ancestral traits, additional information fields such as continent, region, country, province, state, and city should be included in the metadata file if available.

3. auspice_config.json

This file is needed to set various display options for visualization. A sample config file is available as File S2.

4. lat_longs.tsv

A tab-separated file containing latitudes and longitudes for all regions, countries, states, and cities in the dataset (Figure 3). This file is used to display geographic traits during visualization.

Figure 3. Summary screenshot of the lat_longs.tsv file required by Nextstrain for visualizing geographic traits

Data analysis

A. Alignment to the reference genome

Augur uses MAFFT to perform multiple-sequence alignments. To create an alignment file using Augur, use the following command:

$ augur align --sequences --reference-sequence --output --fill-gaps –remove reference --nthreads <8>

B. Constructing the phylogenetic tree

Augur uses IQTREE as the default software to construct a phylogenetic tree from the multiple-sequence alignment file. The branch lengths in the tree are a measure of nucleotide divergence.

The following command will generate a phylogenetic tree in Newick format (.nwk):

$ augur tree --alignment --output --nthreads <8>

C. Refining the phylogenetic tree

The raw tree constructed in the previous step is further processed by Augur using TreeTime to adjust the branch lengths according to the sampling dates of the sequences. In the analysis, the root of the tree is specified by giving the sequence name AY082679.1 explicitly with the --root parameter of the refine command. The --clock-rate parameter is used to run the analysis using a fixed evolutionary rate to produce a robust time-resolved phylogeny, and the --clock-filter-iqd parameter filters out genes that do not follow the evolutionary rate or molecular clock. For poliovirus genomes, this rate is fixed at 0.01 or 1 × 10^-2 substitutions per site per year. To produce a time-resolved tree, use the following command:

$ augur refine --tree --alignment --metadata --output-tree --output-node-data --timetree --coalescent opt --clock-rate 0.01 --clock-std-dev 0.04 --date-inference marginal --divergence-unit mutations --date-confidence --clock-filter-iqd 4 –-stochastic-resolve

D. Annotating ancestral traits

Augur can use the time tree to infer the region and country of all internal nodes. The ancestral traits for all nodes can be annotated using the following command:

$ augur traits --tree --metadata --output --columns continent country --confidence --sampling-bias-correction 2.5

E. Inferring ancestral sequences and nucleotide mutations

The following command identifies the nucleotide mutations of the branches of the tree and infers the ancestral strain of each node:

$ augur ancestral --tree --alignment --output-node-data --inference joint --infer-ambiguous

F. Inferring amino acid mutations

The following command identifies the amino acid mutations using the reference genome and ancestral sequences:

$ augur translate --tree --ancestral-sequences --reference-sequence < AY082679.gb> --output

G. Exporting output files for visualization

The following command exports all output files generated in the previous steps of the analysis as a single JSON file to visualize the data using Nextstrain:

$ augur export v2 --tree --metadata --node-data --auspice-config auspice_config.json --lat-longs lat_longs.tsv --colors colors.tsv --output auspice/polio_global.json

H. Viewing the data

To visualize the output, use the following command:

$ nextstrain view auspice/ --allow-remote-access

This command starts the Auspice server on port 4000. The output is visualized through a browser by navigating to http://127.0.0.1:4000/ or using the IP address of the machine on which the Auspice service is running and navigating to http://IP_ADDRESS_OF_MACHINE:4000/ to display the interactive Auspice page (Figure 4). For the links, follow the steps given at https://docs.nextstrain.org/en/latest/learn/augur-to-auspice.html. The hyperlinks correspond to a locally operated server through Auspice (installation and instructions are detailed in the protocol), which helps the user to view the phylogeny on their own system through a browser.

Figure 4. Nextstrain visualization output showing phylogeny and inferred transmissions

For the example of the visualization of the evolution and transmission of cVDPV outbreaks provided in this protocol, a visual representation of five continents is provided, color-coded by country of sequence collection. The phylogenetic tree (Phylogeny) is shown alongside the geographical view (Geography). The time-resolved phylogenetic tree shows the inferred dates on the X-axis and inferred poliovirus evolution on the Y-axis. This inferred phylogeographic representation may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective supplementary immunization activities and improved outbreak response and surveillance when focused on specific geographical locations of interest at regional, national, or sub-national levels of investigation, for example, the inference of regional patterns of poliovirus transmission within a continent. Additionally, it may provide improved surveillance, timely and effective outbreak response when combined with other epidemiological tools (e.g., vaccination coverage) and climatic and population movement datasets.

Validation of protocol

This protocol or parts of it has been used and validated in the following research article(s):

• Hadfield et al. [7]. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 34(23): 4121–4123. https://doi.org/10.1093/bioinformatics/bty407 (Figure 1).

General notes and troubleshooting

The protocol is based on a validated pipeline developed by Nextstrain https://nextstrain.org/. The Data analysis section provides sufficient information regarding validation.

Possible software, installation, and command-line issues may arise due to software and hardware incompatibility and software versions. Additional issues may include input file configuration, which should follow the format described in the protocol. Possible troubleshooting questions and answers are addressed in the Nextstrain documentation available at https://docs.nextstrain.org/en/latest/reference/faq.html.

Supplementary information

The following supporting information can be downloaded here:

1. File S1. Poliovirus tab-delimited text file describing all sequences in the “polio_sequences.fasta” file (polio_metadata.tsv).

2. File S2. Text file in JSON format specifying visualization settings (auspice_config.json).

Acknowledgments

This protocol is adapted from the Nextstrain project [7]. The authors would like to thank Marta Villa Monge for the high-resolution images used in this article. This work is supported by a Gates Foundation Grant (grant number INV-07033: Polio Genomics Expansion in Africa).

Authors contribution: Conceptualization: D.C.O., K.T., H.A., J.E.C., A.K.W.K., Data Curation: D.C.O., A.Y.D.W., Formal Analysis: D.C.O., A.Y.D.W., J.K., J.E.C., Funding Acquisition: H.A., K.R., O.M.D., J.E.C., N.M., J.A.A., A.K.W.K., Investigation: D.C.O., S.K., C.O.I., M.E.F.G.E.D., P.E.M.B.S., I.I.I., A.J., I.N., H.A., Methodology: D.C.O., S.K., C.O.I., M.E.F.G.E.D., P.E.M.B.S., I.I.I., A.J., I.N., D.O.O., C.K., C.G., J.K., K.T., H.A., J.E.C., Resources: K.T., H.A., K.R., O.M.D., N.M., J.A.A., A.K.W.K., Software: D.C.O., J.K., D.O.O., C.K., C.G., J.K., K.T., K.R., N.M., J.A.A., A.K.W.K., Supervision: H.A., J.E.C., A.K.W.K., Validation: D.C.O., J.K., D.O.O., C.K., C.G., H.A., O.M.D., J.E.C., Visualization: D.C.O., B.T., A.Y.D.W., J.K., Writing - Original Draft Preparation: D.C.O., B.T., H.A., K.R., O.M.D., J.E.C., N.M., A.K.W.K., Writing - Review Editing: D.C.O., J.E.C., A.K.W.K.

Competing interests

No competing interests to be declared.

References

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