diffprimer 0.1.3

A tool to design primers that target specific variants in genomic sequences. Alingment-free and fast.

DiffPrimer

DiffPrimer is a bioinformatics tool designed to identify unique genomic regions in a reference genome compared to a set of other "background" genomes and automatically design specific PCR primers for these markers.

It combines high-performance k-mer analysis (written in Rust) with standard primer design tools (Primer3) to generate diagnostic markers that are exclusive to your target organism.

Key Features

• Exclusive Region Discovery: Identifies genomic regions present in your reference but absent in a database of other genomes.
• Automated Primer Design: Integrated with Primer3 to design optimal primer pairs for identified unique regions.
• Specificity Check:
  • Validates designed primers against all input background genomes.
  • Uses a hybrid Myers Bit-Vector Algorithm (Global) and Semiglobal Alignment (Local) to detect potential off-target amplification, even with insertions/deletions.
• Annotation Integration: Cross-references unique regions with GFF3 annotation files to identify which genes (if any) the markers overlap with.
• Parallel Processing: Fully parallelized core for fast execution on large datasets.

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Installation

Prerequisites

• Operating System: Linux (Recommended) or macOS.
• Python: Version 3.13 or higher (Strict requirement).
• Rust: Required to build the core engine. Install via rustup.

Quick Install (Recommended)

We recommend using uv or pip in a clean environment.

1. Clone the repository:

   git clone https://github.com/yourusername/diffprimer.git
   cd diffprimer
   

2. Create a Virtual Environment:

   # Using uv (Recommended)
   uv venv --python 3.13
   source .venv/bin/activate
   
   # OR using standard python
   python3.13 -m venv .venv
   source .venv/bin/activate
   

3. Install:

   uv pip install .
   # OR
   pip install .
   

Troubleshooting (Conda/Anaconda Users)

If you are working inside a Conda environment, the system linker may conflict with Rust, causing build errors (e.g., undefined symbol: getauxval). To fix this:

1. Deactivate Conda completely for the build step.
2. Use the system's standard PATH.

# In your terminal
unset CONDA_PREFIX
export PATH=/usr/local/bin:/usr/bin:/bin:$HOME/.cargo/bin
source .venv/bin/activate
uv pip install --force-reinstall .

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Usage

Running diffprimer executes the full pipeline: finding unique regions -> designing primers -> verifying specificity.

Basic Command

diffprimer \
    --reference-file reference.fasta \
    --sequences-path genomes_directory/ \

Complete Example

diffprimer \
    --reference-file data/target_species.fasta \
    --sequences-path data/background_species/ \
    --annotation-path data/target_annotations.gff3 \
    --config-file primer3_config.ini \
    --min-region-length 200 \
    --cpus 8 \
    --check-specificity

Arguments Explained

Argument	Short	Description
--reference-file	-r	Required. Path to the target reference genome (FASTA).
--sequences-path	-s	Required. Directory containing background genomes (FASTA) to compare against. Regions found in these genomes will be excluded.
--annotation-path	-a	(Optional) GFF3 file for the reference. Used to annotate output regions with gene names + product info.
--config-file	-c	(Optional) Primer3 configuration file defining Tm, GC%, and size constraints.
--reference-max-abundance		Maximum allowed frequency of a k-mer in the reference genome to be considered a candidate marker. Use 1 for strictly unique markers (default). Increasing this value allows markers that are repeated a few times in the reference. (Default: 1)
--kmer-size	-k	K-mer size for uniqueness check (Default: 21).
--min-region-length	-m	Minimum length of unique regions to keep (Default: 200 bp).
--check-specificity		Highly Recommended. Enables the rigorous cryptographic check of primer specificity. Without this, primers are only designed on unique regions but not physically verified against off-targets.
--similarity-threshold		Global similarity threshold (%) for off-target flagging (Default: 80.0).
--local-mismatch-threshold		Positional mismatch score threshold for the local specificity check. Standard mismatches add 1, 3' end mismatches add 3 (configurable via --penalty-array). Default is 7, allowing up to 2 mismatches in the 3' region to still be considered non-specific (capable of amplifying).
--penalty-array		Penalty values for mismatches. Provide a comma-separated list of 6 values. The first value is the penalty outside the 3' region. The next 5 values are the penalties for the 5 nucleotides of the 3' region. (Default: 1,3,3,3,3,3)

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Output Format

The output is a CSV file containing one row per designed primer pair. Key columns include:

• Sequence_Header: Contig/Region name.
• Region_Start/End: Coordinates of the unique region.
• Forward_Primer / Reverse_Primer (and _Tm, _GC, etc.): Primer details.
• Specificity_Tag: The result of the specificity analysis (if --check-specificity is used).
  • Specific_LowGlobalSim: The region is globally unique; off-targets are extremely dissimilar, so primers are specific.
  • Specific_PositionalMismatches: The region has high global similarity to a background genome, BUT the primers themselves are specific because they have biological mismatches in critical locations (like the 3' end) that prevent binding.
  • NonSpecific_Amplification: The primers bind perfectly or near-perfectly to a similar region in a background genome resulting in cross-reactivity. Do NOT use.
  • Not_Checked: Specificity check was skipped.
• Gene_Name / Product: Annotation info (if GFF3 provided).

Configuration

You can customize Primer3 settings by providing a file with --config-file. Example format:

PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=27
PRIMER_OPT_TM=60.0
PRIMER_MIN_TM=57.0
PRIMER_MAX_TM=63.0
PRIMER_MIN_GC=20.0
PRIMER_MAX_GC=80.0