siren-rnai 0.1.9

SIREN: Suite for Intelligent RNAi design and Evaluation of Nucleotide sequences

SIREN: Suite for Intelligent RNAi Design and Evaluation of Nucleotide Sequences

SIREN is a comprehensive toolset for designing RNA interference (RNAi) sequences to silence specific genes while minimizing off-target effects. It integrates siRNA generation, off-target evaluation, off-target visualization, and RNAi sequence plus primer design into a streamlined workflow.

Table of Contents

• Features
• Installation
  • Conda / Mamba
  • Pip (PyPI)
  • Docker
• Requirements
• Usage
  • Example
  • Options
• Pipeline Overview
  • Prefilter alignment-free k-mer screen
  • siRNA Generation and Off-target Evaluation
  • Off-target Visualization
  • RNAi Selection and Primer Design
• Snakemake workflow (optional; cluster-friendly RNAhybrid parallelization)
• License
• Citations

Features

• siRNA Generation: Automatically extracts the target gene from a multi-FASTA file and generates all possible siRNAs.
• Off-target Evaluation: Uses RNAhybrid to assess potential off-target interactions.
• Off-target Visualization: Creates a plot showing the distribution of siRNAs and off-target events along the gene.
• RNAi Sequence and Primer Design: Generates RNAi sequences of various lengths, scores them based on off-target penalties, and designs primers with Primer3 while reporting expected amplicon sizes.

Installation

Conda / Mamba

SIREN calls RNAhybrid as an external executable. The most reliable way to install is via Bioconda:

mamba install bioconda::siren-rnai
# or
conda install -c conda-forge -c bioconda siren-rnai

Pip (PyPI)

mamba install -c conda-forge -c bioconda rnahybrid
pip install siren-rnai

Cluster/HPC optional dependencies (same SIREN, adds Parsl):

pip install "siren-rnai[cluster]"

siren-rnai[cluster] installs the same package plus optional cluster dependencies needed for Slurm execution (Parsl). The SIREN command is unchanged.

Apple Silicon Installation

If you're on a Mac with Apple Silicon, this is a safe and compatible setup:

# 1) Create and activate a new environment with Python 3.12
mamba create -n osx64_env python=3.12.9 -y
mamba activate osx64_env

# 2) Install RNAhybrid from bioconda
mamba install -c conda-forge -c bioconda rnahybrid -y

# 3) Install SIREN
pip install siren-rnai

Docker

A Dockerfile is provided to run SIREN reproducibly without installing dependencies on the host.

Build:

docker build -t siren-rnai:latest .

Run:

docker run --rm -it \
  -v "$PWD":/work \
  -w /work \
  siren-rnai:latest \
  SIREN --targets your_db.fa --gene YOUR_GENE --outdir siren_results --threads 12

Notes:

• For large databases, mount fast storage for best performance.

Requirements

• Python 3.x
• Mamba/Conda: Recommended for installing RNAhybrid from Bioconda.
• RNAhybrid: Evaluates off-target interactions (external executable).
• Primer3: Required for primer design.
• BioPython: For sequence processing.
• Matplotlib: For generating visualizations.
• Additional Python libraries: Pandas, argparse, csv, tqdm, etc.

Usage

Run SIREN using the command-line interface:

SIREN --targets <FASTA file> --gene <gene_name> [--threads <number>] [--sensitivity {high,medium}] [--rnai_length <length>] [--outdir <output_directory>] [--min_align_length <length>]

Example

SIREN --targets TAIR10_cdna.fasta --gene AT1G50920 --threads 12 --rnai_length 300 --outdir results_AT1G50920

This runs the complete SIREN pipeline for the gene AT1G50920 from the provided FASTA file, using 12 threads and a base RNAi length of 300 nucleotides, storing results in results_AT1G50920.

Options

Required:

• --targets <FASTA>: FASTA containing organism cDNA sequences.
• --gene <STRING>: Gene name or a substring of the FASTA header to select the target.

Common options:

• --threads <INT>: Parallelism for heavy steps (default: 8).
• --sensitivity {high,medium}: Pipeline sensitivity (default: high).
• --rnai_length <INT>: RNAi region length used downstream (default: 200).
• --sirna_size <INT>: siRNA length (default: 21).
• --min_align_length <INT>: Minimum alignment length filter for off-target detection (optional).
• --outdir <DIR>: Output directory (default: siren_results).

Prefilter controls (alignment-free k-mer screen; optional):

• -X, --no_prefilter: Skip the prefilter step and run on the full database.
• -m, --prefilter_mode {set,windowed}: Prefilter mode (default: windowed).
• -s, --prefilter_strand {rc,fwd,both}: Strand used for seeding (default: rc).
• -k, --prefilter_seed_k <INT>: Seed k-mer length for windowed mode (default: 9).
• -w, --prefilter_window_size <INT>: Window size for density criterion (default: 40).
• -H, --prefilter_min_window_hits <INT>: Minimum seed hits in a window (default: 2).
• -L, --prefilter_write_log / -N, --no_prefilter_write_log: Toggle writing a TSV log (prefilter_log.tsv).

Visualization:

• -g_o, --graphical_output: Also run siren_plotIV.py to produce the off-target plot.

Pass-through to RNAhybrid (placed last):

• -R, --rnahybrid_options ...: Any extra flags forwarded directly to RNAhybrid.
  • Example:

    -R -e -25 -v 0 -u 0 -f 2,7 -p 0.01 -d 0.5,0.1 -m 60000
    

Pipeline Overview

Prefilter alignment-free k-mer screen

The siren_prefilter.py module:

• Purpose: Rapidly shrink the RNAhybrid search space by keeping only sequences likely to be similar to the target without full alignments.
• Two modes (from the code):
  • set mode: Compares distinct k-mer sets between each candidate sequence and the target using similarity measures.
  • windowed mode (pipeline default): Retains a sequence if there are ≥ H reverse-complement seed hits (exact k-mers) within any W-bp window. Pipeline defaults: k=9, W=40, H=2.
• Strand control: Seeds can be taken from rc, fwd, or both (pipeline default: rc).
• Logging (optional): When enabled, writes a per-record metrics table to prefilter_log.tsv.
• Output: Writes the filtered database to targets_prefiltered.fa (in the specified output directory).
• Integration: Enabled by default; can be skipped with --no_prefilter.

siRNA Generation and Off-target Evaluation

The sirenXII.py module:

• Target Extraction: Searches the provided FASTA for the specified gene and extracts a unique target sequence.
• siRNA Generation: Generates siRNAs (typically 21 nucleotides) using a sensitivity-dependent step size.
• Off-target Evaluation: Evaluates off-target interactions via RNAhybrid on sequences not matching the target.
• Parallel Processing: Splits off-target data into chunks for parallel processing.
• Output Files: Produces files such as target.fa and off_targets_summary.tsv for downstream steps.

Off-target Visualization

The siren_plotIV.py module:

• Data Parsing: Reads the target FASTA and the off-target summary TSV.
• Aggregation: Computes the distribution of siRNAs and off-target events along the gene.
• Plot Generation: Uses Matplotlib to create a plot with:
  • A red line for the count of siRNAs with off-target events.
  • A blue line for the count of off-target events per nucleotide position.
• Output: Saves the plot (e.g., Off_targets_across_the_gene.png).

RNAi Selection and Primer Design

The siren_designVIII.py module:

• RNAi Sequence Generation: Creates RNAi sequences with lengths from (base length - 50) to (base length + 100) in steps of 50.
• Scoring: RNAi sequences are penalized for containing siRNAs with off-target potential. Each unique siRNA contributing to off-targets reduces the score slightly. If multiple off-targets are caused by the same siRNA within a given RNAi sequence, a stronger penalty is applied.
• Primer Design: Utilizes Primer3 to design primer pairs and calculates expected amplicon sizes.
• Output: Generates a TSV file (rna_sequences_with_scores_and_primers.tsv) with RNAi sequences, scores, primer details, and expected amplicon sizes.

Snakemake workflow (optional; cluster-friendly RNAhybrid parallelization)

A Snakemake workflow is available in siren_snakemake/ to parallelize the RNAhybrid stage by splitting the target database into shards and running shards concurrently (local or SLURM).

# 1) Create a small driver env for Snakemake (once)
conda create -n smk -c conda-forge -y snakemake "conda>=24.7.1"
conda activate smk

# 2) Run the workflow
cd siren_snakemake
# edit config.yaml (targets, gene, outdir, num_shards, rnahybrid_options)
snakemake --cores 32 --use-conda -p

# If a previous run was interrupted (stale lock)
snakemake --unlock

License

SIREN is released under the GPLv3 license.

Citations

If you use SIREN in your research, please cite:

• SIREN – Vargas Mejía, P., & Vega-Arreguín, J. C. (2025). SIREN: Suite for Intelligent RNAi Design and Evaluation of Nucleotide Sequences. bioRxiv. https://doi.org/10.1101/2025.05.26.656188.
• RNAhybrid – Rehmsmeier, M., Steffen, P., Höchsmann, M., & Giegerich, R. (2004). Fast and effective prediction of microRNA/target duplexes. RNA, 10(10), 1507–1517.

For any issues, feature requests, or further questions, please open an issue on GitHub. Happy RNAi designing!