Primer design

The repository contains python scripts for high-throughput designing of left primers using 3 modes:

• main.py: script to design primers give a distance from the 3'end
• main_informed.py: script to design primers in an informed manner, using alignments in a bam file to target regions with high coverage
• main_5end: script to generate primers at 5'end, aimed for full-length amplification

Inputs:

For 3'end designing of primers:

• gene_id: A list of ensembl gene ids in a text file, example ./testdata/gene_ids.txt
• gtf_file: A GTF with gene annotation to extract the MANE transcript information
• transcriptome: A fasta file of the Transcriptome to be used for checking primer specificity
• btw_index: A bowtie index bulit on the same transcriptome, step not included in the pipeline, has to be created prior to run the pipeline
• dist: Parameter that indicates how many nucleotide are taken from the 3'end of each transcript to be used as template for primer3
• Tm: melting temperature for primer 3
• --min_primer_size and --max_primer_size: min and max Primer size
• Repeat_lib: a fasta file with repetitive sequences to exclude when designing primers.

For Informed designing of primers:

• bam_file: path to bam file to be used to define peaks of coverage
• Tm: melting temperature for primer 3
• --min_primer_size and --max_primer_size: min and max Primer size
• --min_product_size and --max_product_size: min and max Product size
• gtf_file: A GTF with gene annotation to extract the MANE transcript information
• blastDB: path to a local saved database, to run blast against
• ref_genome: path to reference genome fasta file
• window: given a window N, the pipeline detects the coordinate of highest coverage and extracts the genome sequence of length 2xN around the peak coordinate
• cov_thresh parameter used to compute an expected product length. Finds the first low-coverage (coverage < cov_thresh) coordinate downstream/upstream the peak coordinate
• Repeat_lib: a fasta file with repetitive sequences to exclude when designing primers.

Workflow:

3'end

1. For each gene ID get the MANE transcript ID
2. Fetch the https://rest.ensembl.org site and gets the transcript sequence
3. Write a text file with all needed parameters to run Primer3
4. Run Primer3 and saves output files
5. Run Bowtie2 enabling multiple alignments report
6. Makes summary files.

Coverage Informed

1. Coverage Calculation

2. Gene Processing

   • Filter GTF: Extract entries for the target gene
   • Merge exons: Combine exons into a continuous region
   • Determine transcriptional strand: Identify as (+) or (-)

3. Peak Detection

   • Identify peak: Find the maximum coverage interval
   • Expand region: Use window_size to extend around the peak
   • Dynamic threshold: Calculate cov_thresh% of the peak value
   • Boundary detection: Find the first low-coverage coordinate downstream/upstream (strand-aware)

4. Primer Design

   • Extract sequence: Retrieve genomic sequence using SAMtools
   • Strand handling: Reverse complement if on the negative strand
   • Prepare Primer3 input
   • Run Primer3: Execute within a singularity container

5. Amplicon Calculation

   • Coordinate adjustment: Align coordinates with exons
   • Spliced length: Compute the length considering exons only
   • Report generation: Create an amplicon size report

6. Specificity Check

   • BLAST: Search primers against a local database

How to Run

1. Clone the repository

    git clone https://github.com/francops1722/Primer_design.git
   

2. Pull Primer3 singularity container

    cd ./primer3
   
    ./PullSif.sh
   

3. Edit input parameters the job.pbs files: - Job_3end.pbs - Job_Informed.pbs - Job_5end.pbs

4. Submit job

    qsub Job.pbs