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:

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:

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:

conda create -n lassaseq -c bioconda lassaseq -y
conda activate lassaseq

Option 2: From Source Code

Create and activate a new conda environment:

conda create -n lassaseq -c bioconda python=3.9 mafft trimal iqtree
conda activate lassaseq

Install lassaseq:

git clone https://github.com/DaanJansen94/LassaSeq.git   
cd LassaSeq
pip install .

Re-installation (when updates are available):
```
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:

conda activate lassaseq

To see all available options:

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] [--consensus_L] [--consensus_S] [--lineage] [--sublineage] [--l_sublineage] [--s_sublineage]

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
  --consensus_L         (Optional) Path to custom consensus sequences for L segment
  --consensus_S         (Optional) Path to custom consensus sequences for S segment
  --lineage             (Optional) Filter sequences by lineage
  --sublineage          (Optional) Filter sequences by sublineage for both segments
  --l_sublineage        (Optional) Filter L segment sequences by sublineage
  --s_sublineage        (Optional) Filter S segment sequences by sublineage                    

Lassaseq can be run in two modes:

Interactive Mode (default) - for interactive use
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:

lassaseq -o output_directory

Command Line Mode

All options can be specified directly:

# 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 lassa_output --genome 1 --l_sublineage III --s_sublineage II

# Download sequences and perform phylogenetic analysis with segment-specific sublineages
lassaseq -o lassa_output --genome 1 --host 1 --metadata 3 --phylogeny --lineage IV --l_sublineage 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:

Sequence Concatenation: Combines downloaded sequences with reference and outgroup sequences, removing duplicates.
Multiple Sequence Alignment: Uses MAFFT with optimized parameters:
Alignment Trimming: Uses TrimAl with -automated1 for automated trimming
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.

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

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:
```
# Example using HYPHY GARD
conda install bioconda::hyphy
hyphy gard --rv Gamma --mode Faster --alignment trimmed_alignment.fasta --output gard_output.json
```
Note: Recombination analysis can be time-consuming depending on your dataset size and chosen tool.
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:
```
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.

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