Chromosome/species-Specific Unique Sequence Extraction Pipeline

This pipeline extracts unique, chromosome or species-specific DNA segments of 200–500 bp from a FASTA file containing the sequences for chromosomes or various genomic sequences of different species. These segments are ideal for designing PCR markers that are specific to individual chromosomes or species.

Overview

The pipeline:

1. Splits the genome into separate chromosome files (if your goal is to design species specific primers you can skip this step but the fasta file for species should be a single chromosom file then put the species genomic sequence fasta files in a folder, name the folder "chromosomes".
2. For each chromosome/organism, aligns it against the rest of the genome to find unique (non-aligned) regions using minimap2.
3. Extracts these unique regions.
4. Filters the unique regions to keep only those between 200–500 bp.

All steps are automated and memory-efficient, making it suitable for large genomes.

---

Requirements

• Python 3.7+
• Biopython
• minimap2 (must be installed and in your PATH)
• Bash shell (for running the main script)

Install Python dependencies

pip install -r requirements.txt

Install minimap2

• Download from minimap2 releases
• Unpack and add the binary to your PATH
• Test with: minimap2 --version
• OR ---- conda install bioconda::minimap2 (recomended)

---

File Descriptions

• split_fasta_by_chromosome.py: Splits the genome FASTA into one file per chromosome OR put the species single chromosom genome sequences in the "chromosomes" folder.
• align_and_extract_unique.sh: Main pipeline script. For each chromosome, aligns it to the rest of the genome, extracts unique regions, and saves them as BED files.
• extract_unique_regions_from_paf.py: Parses minimap2 output to find unique (non-aligned) regions.
• filter_by_length.py: Filters BED regions to keep only those between 200–500 bp.
• requirements.txt: Python dependencies.

---

Step-by-Step Usage

1. Prepare your genome FASTA

Place your genome file (e.g., GCF_020520425.1_BTI_SOV_V1_genomic.fna) in the project directory.

2. Install dependencies

pip install -r requirements.txt

Make sure minimap2 is installed and available in your PATH.

3. Run the main pipeline

bash align_and_extract_unique.sh

This will:

• Create a chromosomes/ directory with one FASTA per chromosome.
• For each chromosome, align it to the rest of the genome and extract unique regions as BED files in unique_regions/.

4. Filter unique regions by length (200–500 bp)

For each chromosome, run:

python filter_by_length.py unique_regions/<chromosome>_unique.bed chromosomes/<chromosome>.fasta 200 500 filtered_unique_regions/<chromosome>_unique_200_500.bed

You can automate this with a simple loop in bash:

mkdir -p filtered_unique_regions
for chrom_fasta in chromosomes/*.fasta; do
    chrom=$(basename "$chrom_fasta" .fasta)
    python filter_by_length.py unique_regions/${chrom}_unique.bed $chrom_fasta 200 500 filtered_unique_regions/${chrom}_unique_200_500.bed
done

5. (Optional) Extract FASTA sequences for filtered regions

To get the actual DNA sequences for your filtered unique regions, use the provided script:

python extract_fasta_from_bed.py filtered_unique_regions/<chromosome>_unique_200_500.bed chromosomes/<chromosome>.fasta fasta_sequences/<chromosome>_unique_200_500.fasta

You can automate this for all chromosomes:

mkdir -p fasta_sequences
for chrom_fasta in chromosomes/*.fasta; do
    chrom=$(basename "$chrom_fasta" .fasta)
    python extract_fasta_from_bed.py filtered_unique_regions/${chrom}_unique_200_500.bed $chrom_fasta fasta_sequences/${chrom}_unique_200_500.fasta
done

This will create FASTA files with the actual sequences for each filtered region, ready for primer design or further analysis.

---

Output

• chromosomes/: Per-chromosome FASTA files
• unique_regions/: BED files of unique regions per chromosome
• filtered_unique_regions/: BED files of unique regions 200–500 bp per chromosome

---

Troubleshooting

• minimap2 not found: Make sure minimap2 is installed and in your PATH.
• Python import errors: Run pip install -r requirements.txt.
• Permission denied: Make sure scripts are executable (chmod +x align_and_extract_unique.sh).
• Large genome/slow performance: The pipeline is designed to be memory-efficient, but runtime depends on genome size and your computer's speed.

---

FAQ

Q: Can I use this for other species?
A: Yes! Just replace the input FASTA with your genome file.

Q: How do I design primers for these regions?
A: Use the filtered BED files to extract sequences, then use a tool like Primer3 or NCBI Primer-BLAST.

Q: What if I want a different length range?
A: Change the 200 500 arguments in the filtering step to your desired range.

---

Citation

If you use this pipeline, please cite the minimap2 and Biopython papers, and this repository.

• [https://github.com/PouyaMotieNoparvar/Unique_Sequence_Finder]

---

License

MIT License