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Batch Primers

This repository provides Python scripts for designing PCR and qPCR primers for gene knockout and RT-qPCR experiments. The scripts leverage primer3 and Biopython libraries to automate primer design for multiple genes based on GFF and FASTA input files.

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Features

1. Primer Design for Gene Knockout (KO):

   • Automates the design of fusion PCR primers for creating gene knockouts.
   • Generates primers optimized for pyrG and ptrA selection markers.
   • Ensures proper primer placement based on gene coordinates and strand orientation.

2. RT-qPCR Primer Design:

   • Designs primers for RT-qPCR experiments.
   • Includes functionality to create primers overlapping exon-exon junctions for genes with multiple exons.
   • Provides detailed primer quality checks, including specificity and placement.

3. Error Handling:

   • Scripts handle errors gracefully, logging problematic gene entries for review.
   • Outputs diagnostic information to help refine input data or parameters.

4. Output Formats:

   • Primer designs are saved in multiple formats:
     • CSV files for easy review and downstream processing.
     • GFF files for visualizing primer locations in genome browsers.

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Installation

Prerequisites

1. Python 3.x
   Ensure you have Python 3.x installed.

2. Required Python Libraries
   Install the required libraries using pip:

   pip install primer3-py biopython

3. Files Required for Input:

   • GFF file: Gene annotation file containing coordinates and feature information.
   • FASTA file: Genome sequence file corresponding to the GFF file.

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Usage

1. Gene Knockout Primer Design (cds_KO_primers.py)

This script generates primers for fusion PCR to knock out all genes in a genome.

Command:

python cds_KO_primers.py <gff_file> <fasta_file>

Input:

• <gff_file>: GFF file containing gene annotations.
• <fasta_file>: FASTA file containing the genome sequence.

Output:

• *_KO_primers_pyrG_selection.csv: Primer designs for pyrG selection.
• *_KO_primers_ptrA_selection.csv: Primer designs for ptrA selection.
• genes_with_errors: List of genes where primer design failed.
• primers.gff: GFF file showing primer locations.

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2. RT-qPCR Primer Design (qpcr_primers_overlapping_exons.py)

This script generates primers for RT-qPCR, including primers overlapping exon-exon junctions for genes with multiple exons.

Command:

python qpcr_primers_overlapping_exons.py <gff_file> <fasta_file>

Input:

• <gff_file>: GFF file containing gene annotations.
• <fasta_file>: FASTA file containing the genome sequence.

Output:

• *_qPCR_primers.csv: Primer designs with details on product size, penalties, and specificity.
• genes_with_errors: List of genes where primer design failed.
• primers.gff: GFF file showing primer locations.

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Output Description

1. CSV Files:

   • Contain primer sequences, penalties, product sizes, and additional metadata for each primer.
   • Example columns:
     • primer name: Name of the primer.
     • primer sequence: Primer sequence.
     • penalty: Primer3 penalty score indicating primer quality.
     • product size: Expected PCR product size.

2. GFF Files:

   • Annotates primer locations in the genome for visualization in genome browsers.
   • Example fields:
     • Name: Primer identifier (e.g., gene-5F).
     • strand: Indicates forward (+) or reverse (-) strand.

3. Error Logs:

   • genes_with_errors: Text file listing genes where primer design encountered issues.

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