LassaSeq

LassaSeq is a command-line tool that simplifies the process of analyzing Lassa virus sequences. It automates the complete workflow from downloading sequences to creating phylogenetic trees, with special handling for Lassa's bi-segmented genome.

Genome Segments

Lassa virus has a bi-segmented RNA genome consisting of: - L segment: (~7.2kb) Encodes the RNA-dependent RNA polymerase and Z protein - S segment: (~3.4kb) Encodes the nucleoprotein (NP) and glycoprotein precursor (GPC)

Installation

Prerequisites

First, install conda if you haven't already: bash wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh bash Miniconda3-latest-Linux-x86_64.sh

Then, ensure you have the required channels: bash conda config --add channels defaults conda config --add channels bioconda conda config --add channels conda-forge

Option 1: Using Conda (Recommended)

Install LassaSeq via Conda: bash conda create -n lassaseq -c bioconda lassaseq -y conda activate lassaseq

Option 2: From Source Code

1. Create and activate a new conda environment: bash conda create -n lassaseq -c bioconda python=3.9 mafft trimal iqtree conda activate lassaseq

2. Install lassaseq: bash git clone https://github.com/DaanJansen94/LassaSeq.git cd LassaSeq pip install .

3. Re-installation (when updates are available): bash conda activate lassaseq # Make sure you're in the right environment cd LassaSeq git pull # Get the latest updates from GitHub pip uninstall lassaseq pip install . Note: Any time you modify the code or pull updates from GitHub, you need to reinstall the package using these commands for the changes to take effect.

Usage

First, make sure your conda environment is activated: bash conda activate lassaseq

To see all available options: bash lassaseq --help

This will display: ``` usage: lassaseq [-h] -o [--genome {1,2,3}] [--completeness ] [--host {1,2,3,4}] [--metadata {1,2,3,4}] [--countries ("country1, country2") ] [--remove] [--phylogeny] [--consensusL] [--consensusS] [--lineage] [--sublineage] [--lsublineage] [--ssublineage]

Download and filter Lassa virus sequences

options: -h, --help show this help message and exit -o Output directory for sequences --genome {1,2,3} Genome completeness filter: 1 = Complete genomes only (>99 percent of reference length) 2 = Partial genomes (specify minimum percent with --completeness) 3 = No completeness filter --completeness Minimum sequence completeness (1-100 percent) Required when --genome=2 --host {1,2,3,4} Host filter: 1 = Human sequences only 2 = Rodent sequences only 3 = Both human and rodent sequences 4 = No host filter --metadata {1,2,3,4} Metadata completeness filter: 1 = Keep only sequences with known location 2 = Keep only sequences with known date 3 = Keep only sequences with both known location and date 4 = No metadata filter --countries (Optional) Comma-separated list of countries to filter sequences If not specified, sequences from all countries will be included Examples: "Sierra Leone, Guinea" or "Nigeria, Mali" Available: Nigeria, Sierra Leone, Liberia, Guinea, Mali, Ghana, Benin, Burkina Faso, Ivory Coast, Togo --remove (Optional) File containing accession numbers to remove One accession number per line, lines starting with # are ignored --phylogeny (Optional) Create concatenated FASTA files and perform phylogenetic analysis Includes sequence alignment, trimming, and tree building --consensusL (Optional) Path to custom consensus sequences for L segment --consensusS (Optional) Path to custom consensus sequences for S segment --lineage (Optional) Filter sequences by lineage --sublineage (Optional) Filter sequences by sublineage for both segments --lsublineage (Optional) Filter L segment sequences by sublineage --ssublineage (Optional) Filter S segment sequences by sublineage
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Lassaseq can be run in two modes: 1. Interactive Mode (default) - for interactive use 2. Non-Interactive Mode - for HPC submissions or automated runs

Interactive Mode

If optional arguments are not provided, the program will run in interactive mode and prompt for choices: bash lassaseq -o output_directory

Command Line Mode

All options can be specified directly: ```bash

Download complete genomes from human hosts with known location and date

lassaseq -o lassa_output --genome 1 --host 1 --metadata 3

Download sequences with ≥80% completeness from both human and rodent hosts

lassaseq -o lassa_output --genome 2 --completeness 80 --host 3 --metadata 4

Download sequences from specific countries and lineage

lassaseq -o lassa_output --genome 1 --host 1 --metadata 3 --countries "Sierra Leone, Guinea" --lineage IV

Filter for different sublineages in L and S segments

lassaseq -o lassaoutput --genome 1 --lsublineage III --s_sublineage II

Download sequences and perform phylogenetic analysis with segment-specific sublineages

lassaseq -o lassaoutput --genome 1 --host 1 --metadata 3 --phylogeny --lineage IV --lsublineage III --s_sublineage II ```

Lineage Information

The tool includes lineage information for Lassa virus sequences in the lineages directory. The lineages are in line with current literature, and sublineages for lineage IV were determined using fastbaps software. Users can modify or update the lineage files (l_lineages.txt and s_lineages.txt) to include their own lineage assignments. Each file follows a tab-delimited format with columns for accession numbers, lineages, and sublineages.

Summary File Content

The summary_Lassa.txt file provides detailed information about: - Detailed host distribution with sequence counts for: - Human hosts - Rodent hosts (e.g., Mastomys natalensis, Hylomyscus pamfi) - Other hosts - Sequences with no host information - Geographical distribution at each filtering step - Final sequence counts for each segment

Phylogenetic Analysis

When the --phylogeny flag is used, LassaSeq performs the following steps: 1. Sequence Concatenation: Combines downloaded sequences with reference and outgroup sequences, removing duplicates. 2. Multiple Sequence Alignment: Uses MAFFT with optimized parameters: 3. Alignment Trimming: Uses TrimAl with -automated1 for automated trimming 4. Tree Building: Uses IQ-TREE2 with ultra-fast bootstrap and 10,000 replicates

Tree Visualization

The phylogenetic trees generated by LassaSeq (.treefile files) can be visualuzed using various software packages (e.g., FigTree) and annotated using the metadata files provided by LassaSeq.

1. Install FigTree:

   • Download from: http://tree.bio.ed.ac.uk/software/figtree/
   • Available for Windows, Mac, and Linux

2. Visualizing Trees:

   • Open FigTree
   • Load the tree file (e.g., l_trimmed.fasta.treefile or s_trimmed.fasta.treefile)
   • Import the corresponding metadata file (l_metadata.txt or s_metadata.txt)
   • Use the metadata columns to color and annotate your tree:
     • Location: Country of origin (e.g., SierraLeone, Nigeria)
     • Location2: City or specific location (e.g., Kenema, Nzerekore, Unknown)
     • Host: Host species (Human, Rodent, Other)
     • Date: Collection date in decimal format (e.g., 1969.000)

Handling Recombination Events

Due to the computationally intensive nature of recombination detection, this analysis is not included in LassaSeq itself. However, it is highly recommended to perform recombination analysis as a separate step before conducting phylogenetic analyses, as recombinant sequences can interfere with accurate tree reconstruction. Here's the recommended workflow:

1. Recombination Analysis: Before running LassaSeq's phylogenetic analysis, perform recombination detection using your preferred tool. For example, HYPHY GARD can be installed and used as follows: ```bash

   Example using HYPHY GARD

   conda install bioconda::hyphy hyphy gard --rv Gamma --mode Faster --alignment trimmedalignment.fasta --output gardoutput.json ``` Note: Recombination analysis can be time-consuming depending on your dataset size and chosen tool.

2. Remove Recombinant Sequences: Once you've identified recombinant sequences using your chosen analysis tool, add their accession numbers to a remove.txt file. Use this file with LassaSeq's --remove option to exclude these sequences from the analysis: bash lassaseq -o lassa_output --genome 1 --host 1 --metadata 3 --remove remove.txt --phylogeny

This optional but recommended step is crucial for obtaining reliable phylogenetic trees, as recombinant sequences can lead to incorrect evolutionary relationships and branch patterns.

Dependencies

• Python ≥ 3.6
• BioPython ≥ 1.79
• NumPy ≥ 1.21.0
• MAFFT
• TrimAl
• IQTree2

Citation

If you use Lassaseq in your research, please cite:

Jansen, D., Laumen, J., Siebenmann, E., & Vercauteren, K. (2025). LassaSeq: A Command-Line Tool for Downloading, Processing and Analyzing Lassa Virus Sequences for Phylogenetic Analysis (v0.1.2). Zenodo. https://doi.org/10.5281/zenodo.14936276

License

This project is licensed under the GNU General Public License v3.0 (GPL-3.0) - see the LICENSE file for details.

Contributing

Contributions are welcome! Please feel free to submit a Pull Request.

Support

If you encounter any problems or have questions, please open an issue on GitHub.