rsv_epidemiology_2025

A collection of code and data used for "Molecular Epidemiology of Respiratory Syncytial Virus in Switzerland from 2020 to 2024" publication.

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src/genome_assembly

This directory contains input data and metadata used for genome assembly and downstream analysis of RSV samples.

data/ Folder Structure

data/
├── annotation.tsv
├── primers/
├── ref_genomes/
└── samples/

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annotation.tsv

Sample metadata file.
Columns:

• NGS_ID: Full sample ID (matches FASTQ filenames)
• Virus Type: Either HRSVA or HRSVB
• amplicon_types: Comma-separated amplicon types (500bp, 1000bp, 2000bp in the original analysis)
• Pools: Pool 1, Pool 2 in the original analysis, different for separate even/odd amplicon pool experiments

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primers/

CSV files specifying primer schemes used for different virus types and amplicon sizes.

Naming: rsv_{a|b}_primers_{500bp|1000bp|2000bp|revised}{|_even|_odd}.csv

• RSV A or B;
• Amplicon sizes; revised for pooled together from the original analysis;
• empty for combined odd & even amplicons, different otherwise.

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ref_genomes/

Reference genome FASTA files:

• rsva_ref.fasta
• rsvb_ref.fasta

(Additional BWA index files may be created here, can be cleaned with cleanup)

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samples/

One folder per sample (named by NGS_ID in annotation.tsv). Each contains:

<NGS_ID>/
├── <NGS_ID>_R1.fastq.gz
└── <NGS_ID>_R2.fastq.gz

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Configuration (config.yaml)

The config.yaml file specifies various parameters for running the pipeline. Below is a breakdown of the options in the current configuration:

Parameter	Description	Example
base_dir	The base directory where the project is located.	"." (current directory)
res_dir_name	The name of the directory where results will be stored.	"results"
ann_filename	The name of the annotation file in src/genome_assembly/data. This file containis metadata for the samples.	"annotation.tsv"
read_length	TrimGalore min length.	80
min_cov	The minimum coverage threshold for variants.	100
freq_threshold	The frequency threshold: bases with the main allele of lower values are treated as undefined	0.9
allow_cleanup	A flag that determines if intermediate files should be cleaned up after execution. Set to True to enable cleanup.	True

Example Commands

Here are some example commands run from src/genome_assembly:

1. Run the genome assembly pipeline

snakemake --configfile config.yaml --cores 1 --use-conda -p

2. Cleanup intermediate files

snakemake cleanup --cores 1

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Phylogeny Analysis

This is the Nextstrain build for respiratory syncytial virus (RSV).

Input Data

Input metadata and sequences for RSV-A and RSV-B are available via https://data.nextstrain.org

• RSV-A sequences

• RSV-A metadata

• RSV-B sequences

• RSV-B metadata

These data are generously shared by labs around the world and deposited in NCBI genbank by the authors. Please contact these labs first if you plan to publish using these data. RSV sequences and metadata can be downloaded in the /ingest folder using nextstrain build --cpus 1 ingest or nextstrain build --cpus 1 . if running directly from the /ingest directory.

The ingest pipeline is based on the Nextstrain mpox ingest workflow (https://github.com/nextstrain/mpox/tree/master/ingest). Running the ingest pipeline produces ingest/data/{a,b}/metadata.tsv and ingest/data/{a,b}/sequences.fasta.

ingest/vendored

This repository uses git subrepo to manage copies of ingest scripts in ingest/vendored, from nextstrain/ingest. To pull new changes from the central ingest repository, first install git subrepo, then run:

See ingest/vendored/README.md for instructions on how to update the vendored scripts.

Run Analysis Pipeline

The workflow produces whole genome and G gene trees for RSV-A and RSV-B. To run the workflow, use snakemake -j4 -p --configfile config/configfile.yaml and nextstrain view auspice to visualise results.

Installation

Follow the standard installation instructions for Nextstrain's suite of software tools.

Data use

We gratefully acknowledge the authors, originating and submitting laboratories of the genetic sequences and metadata for sharing their work. Please note that although data generators have generously shared data in an open fashion, that does not mean there should be free license to publish on this data. Data generators should be cited where possible and collaborations should be sought in some circumstances.

Update example data

Example data is used by CI. It can also be used as a small subset of real-world data.

Example data should be updated every time metadata schema is changed. To update, run:

nextstrain build --docker . update_example_data -F

Sending data to the nextclade_data repo

From within the destination directory, run

rsync -a <path-to>/rsv/nextclade/datasets/ .

Updates for lab samples analysis:

To construct a tree with an addition of lab samples, the annotation and the sequences have to be located in input_lab_samples/{a_or_b}/ with the following names:

• Lab Annotation (comb_ann.tsv): The lab annotation TSV file includes sample details, including patient number (Patient_Nr), year of sample collection (Year), month of sample collection (Months), and unique identifiers (NGS_ID). The NGS_ID field is used as the index for merging the metadata.
• Nextclade Results (nextclade_results.tsv): The TSV file is generated by the Nextclade tool from the same sequences, it contains columns related to the quality of the sequence and the clade assignment of lab samples, such as clade, qc.overallScore, qc.overallStatus, alignmentScore, alignmentStart, alignmentEnd, coverage
• sequences.fasta: FASTA file with the same indices, as NGS_ID values.

The pipeline then concatenates these sequences and annotation with the opensource ones, constructs a tree for all lab samples + a selected number of background open-source ones. Otherwise, the pipeline works as the master one. One can add filters by country/region, modifying rsv_epidemiology_2025/src/phylogenetic_analysis/workflow/snakemake_rules/core.smk, --query in the filter_os rule. For example:

        --query "({params.min_coverage}) & (source == 'NCBI') & (region == 'Europe')"

Results: Phylogenetic Tree Analysis

The following phylogenetic tree is an example of RSV A tree on lab and open-source RSV samples, colored by origin: NCBI or USB (University Hospital Basel).

Example Commands

Here are some example commands run from src/phylogenetic_analysis:

1. Run the phylogeny reconstruction pipeline

snakemake --cores 1 --use-conda -p

2. Cleanup

snakemake clobber --cores 1

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