VirStrain

VirStrain is an RNA virus strain-level identification tool for short-read sequencing data.

Overview

VirStrain supports:

• Strain identification from single-end and paired-end short reads
• Strain identification from assembled contigs
• Construction of custom VirStrain databases
• Use of pre-built public databases for common viral species

Contact

• Email: heruiliao2-c@my.cityu.edu.hk
• Recommended version: v1.18
• Legacy note: v1.14 fixed some bugs, but did not include virstrain_contig or virstrain_merge

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Changelog

2026 updates

2026-04-05

• v1.18: VirStrain supports Python >=3.8 now!

2024 updates

2024-05-28

• v1.17: Added the -v parameter to display version information
  Available in the GitHub version only

2024-03-11

• v1.17: Synced all changes to both GitHub and Conda

2024-02-27

• Tem_Vs files are now named randomly in the GitHub version
• Added links for downloading pre-built databases

2023 updates

2023-10-12

• v1.14: Fixed a bug in v1.13 related to handling gzipped FASTQ files

2023-09-05

• Added a new function for contig-based viral strain identification
• Supports comprehensive identification across 45,619 strains from 28 viral species

2022 updates

2022-12-20

• v1.13: Fixed a database generation bug present in v1.12 of the Bioconda release

2022-12-16

• The VirStrain web server extension, StrainDetect, is now online:
  https://strain.ee.cityu.edu.hk

2022-11-10

• Added parameter -s to sort the most likely strain by site matches

2022-03-23

• Fixed a Perl script bug related to header name handling

2022-02-08

• Added an alternative method for downloading databases from Figshare

2022-02-05

• v1.12: VirStrain can now accept gzipped FASTQ input files

2021 updates

2021-11

• Added downloadable databases for two DNA viruses used in the paper:
  • HBV
  • HCMV
• Added a larger SARS-CoV-2 database
  See Supplementary Section 1.1 of the paper

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Requirements

Dependencies

• Python >= 3.8
  • Recommended: 3.10.19
  • Should work on python >3.11 as well
• Perl
• Python packages:
  • biopython >=1.78
  • matplotlib-base >=3.1
  • networkx >=2.4
  • numpy >=1.17
  • pandas >=1.0
  • plotly >=5.0
• Bowtie2
  Required for VirStrain version >= v1.18

If you use Conda, you can install required packages automatically with:

sh install_package.sh

If you install VirStrain via Bioconda or pip, you can ignore manual dependency installation.

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Installation

Supported platform: Linux / Ubuntu

Option 1: Install with Bioconda

Once Bioconda is configured:

# Create new conda environment and install VirStrain
conda create -n virstrain_env -c conda-forge -c bioconda virstrain=1.18 -y
# or mamba create -n virstrain_env -c conda-forge -c bioconda virstrain=1.18 -y

# Activate the environment
conda activate virstrain_env

chmod 755 bin/jellyfish-linux

Optional: you can also install VirStrain in your conda env directly (may get issues due to dependencies)

conda install -c bioconda virstrain
#or: mamba install -c bioconda virstrain

Option 2: Install with pip

pip install virstrain==1.18
chmod 755 bin/jellyfish-linux

Option 3: Manual installation

Make sure all dependencies are installed first.

git clone https://github.com/liaoherui/VirStrain.git
cd VirStrain
chmod 755 bin/jellyfish-linux
rm VirStrain_DB.tar.gz

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Command mapping

If you installed VirStrain via Bioconda or pip, use the following command names:

Source install command	Bioconda / pip command
python VirStrain.py -h	virstrain -h
python VirStrain_build.py -h	virstrain_build -h
python VirStrain_contig.py -h	virstrain_contig -h
python VirStrain_contigDB_merge.py -h	virstrain_merge -h

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Databases

Download the default reference database

After cloning the repository:

cd VirStrain
sh download.sh

Alternative download method

cd VirStrain
wget -qO- "https://figshare.com/ndownloader/files/34002479" | tar -zx

You may also download the database manually from Google Drive or Figshare and extract it with:

tar -zxvf <downloaded_file>

If all download methods fail, please contact the author by email.

Additional downloadable databases

DNA virus databases

sh download_dna.sh

Includes databases for:

• HBV
• HCMV

Larger SARS-CoV-2 database

sh download_scov2_big.sh

Contig-level database

sh download_contig_db.sh

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Pre-built database downloads

If the download scripts fail, pre-built databases are also available via Google Drive.

Name	Description	Download
VirStrain_DB.tar.gz	Databases containing SCOV2, H1N1, and HIV strains used in the paper	Google Drive
SCOV2_newBig.tar.gz	Expanded database containing additional SCOV2 strains	Google Drive
VirStrain_DNA_DB.tar.gz	Databases containing HBV and HCMV strains	Google Drive
VirStrain_contig_DB.tar.gz	Contig-level database	Google Drive

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Usage

If you installed VirStrain via Bioconda or pip, replace script-based commands with the corresponding installed commands shown above.

1) Identify RNA virus strains from short reads

Single-end reads

python VirStrain.py -i Test_Data/MT451123_1.fq -d VirStrain_DB/SCOV2 -o MT451123_SE_Test

Paired-end reads

python VirStrain.py -i Test_Data/MT451123_1.fq -p Test_Data/MT451123_2.fq -d VirStrain_DB/SCOV2 -o MT451123_PE_Test

High-mutation viruses such as HIV

Use the -m option.

Single-end:

python VirStrain.py -i <Read1> -d VirStrain_DB/HIV -o <Output_dir> -m

Paired-end:

python VirStrain.py -i <Read1> -p <Read2> -d VirStrain_DB/HIV -o <Output_dir> -m

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2) Identify viral strains from assembled contigs

python VirStrain_contig.py -i <Input_Contig_fasta> -d VirStrain_contig_DB -o VirStrain_Contig_Res

Convert read-based databases into a contig database

python VirStrain_contigDB_merge.py -i VirStrain_DB/SCOV2,VirStrain_DB/H1N1 -o VirStrain_contig_DB_merge

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3) Build a custom VirStrain database

Basic usage:

python VirStrain_build.py -i <Input_MSA> -d <Database_Dir>

Important header naming rule

Characters , and | are not allowed in sequence headers in <Input_MSA>.

Examples:

• Not allowed: >Strain_A, 2022
• Not allowed: >Strain_A|2022
• Allowed: >Strain_A_2022

Manual covering for small datasets or large viral genomes

For small strain collections (<1000 strains) or viruses with large genomes such as HCMV, you can use the manual covering function with -s to retain more useful sites.

Example:

python VirStrain_build.py -i <Input_MSA> -d <Database_Dir> -s 0.4

General guidance:

• 0.2–0.6 is usually a reasonable range for -s
• With very few strains (for example, 3 strains), a larger value such as -s 0.8 may also work

Restrict SNV site range

If you only want to use SNV sites from position x to y, use -r.

Example:

python VirStrain_build.py -i <Input_MSA> -d <Database_Dir> -s 0.4 -r 500-1000

Input format note

The input MSA must have the same format as an alignment generated by MAFFT:

https://mafft.cbrc.jp/alignment/software/

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Full command-line options

VirStrain.py — short-read strain identification

Default k-mer size: 25

VirStrain - An RNA virus strain-level identification tool for short reads.

Example:
python VirStrain.py -i Test_Data/MT451123_1.fq -p Test_Data/MT451123_2.fq -d VirStrain_DB/SCOV2 -o MT451123_PE_Test

required arguments:
    -i, --input_reads             Input FASTQ data
    -d, --database_dir            Path to VirStrain database

optional arguments:
    -h, --help                    Show help message and exit
    -o, --output_dir              Output directory (default: ./VirStrain_Out)
    -p, --input_reads2            Input FASTQ data for paired-end reads
    -c, --site_filter_cutoff      Site filtering cutoff used when calculating Vscore (default: 0.05)
    -s, --rank_by_sites           If set to 1, sort the most likely strain by site matches (default: 0)
    -f, --turn_off_figures        If set to 1, do not generate figures (default: 0)
    -m, --high_mutation_virus     Use for high mutation rate viruses such as HIV

VirStrain_build.py — custom database construction

Default k-mer size: 25

VirStrain - An RNA virus strain-level identification tool for short reads.

Example:
python VirStrain_build.py -i <Input_MSA> -d <Database_Dir>

required arguments:
    -i, --input_msa               Input MSA file (must match MAFFT output format)

optional arguments:
    -d, --database_dir            Output directory for the constructed database (default: ./VirStrain_DB)
    -c, --dash_cutoff             Dash cutoff for each MSA column (default: 0)
    -s, --sites_cutoff            Cutoff for manual-covering function
                                  (e.g. 1 = all useful sites; 0.8 = 80% of useful sites)
    -r, --sites_rcutoff           Site range cutoff for covering algorithm
                                  (e.g. 3-500 means only SNV sites from positions 3 to 500 are considered)

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Output format

VirStrain generates two primary outputs:

1. A text report

   • Contains identified strains, depth, site coverage, and related metrics

2. An interactive HTML report

   • Displays depth and site uniqueness information visually

You can find an example output in the MT451123_Sim_PE folder in this repository.

Example report image:

Report sections

Header	Description
Most Possible strain*	The most likely strain detected by VirStrain. These are the strains with the highest Vscore in the first iteration.
Other Possible strains*	Additional possible strains detected by VirStrain. These are identified in later iterations. Based on the authors’ experiments, 10 mutations can be strong evidence for additional possible strains.
Highest Map Strains	The strain with the maximum Covered SNV site / Total SNV site in the first iteration. Provided for reference.
Top 10 Score Strains	The top 10 strains ranked by Vscore in the first iteration. This can help identify low-abundance strains highly similar to high-abundance strains.

Headers marked with * contain the main identification results.

Report columns

Column	Description
Strain_ID	NCBI accession number or other public database identifier for the identified strain
Cls_info	Cluster information for the identified strain, e.g. Cluster2830_2 means cluster Cluster2830 with size 2
SubCls_info	Sub-cluster information
Vscore	Score generated by the VirStrain algorithm
Total_Map_Rate	Covered sites out of total sites in the first iteration
Valid_Map_Rate	Covered sites out of total sites in the remaining iterations
Strain_depth	Predicted sequencing depth for the identified strain
Strain_info	Metadata for the identified strain, such as region and subtype
SNV_freq	SNV frequency across all sites

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Citation

If you use VirStrain, please cite:

Liao, H., Cai, D. & Sun, Y. VirStrain: a strain identification tool for RNA viruses. Genome Biology 23, 38 (2022). https://doi.org/10.1186/s13059-022-02609-x