Parallel e-PCR and Primer3 Pipeline

This repository contains a pipeline for designing and selecting primers using Primer3, followed by in silico validation through e-PCR. The script is optimized for parallel execution to improve performance.

Workflow

Table of Contents

• Installation
• Configuration
• Usage
• Input
• Output
• Parallel Execution
• Notes

Installation

Ensure you have the required dependencies installed:

• Python (>=3.6)
• Biopython
• Multiprocessing
• Primer3
• e-PCR

Clone this repository:

https://github.com/Donandrade/transposable_elements.git
cd transposable_elements

Configuration

The script assumes the following directory structure:

transposable_elements/
  ├── bin/
  │   ├── e-PCR
  │   ├── primer3/
  ├── caulimovirus_mafft/
  │   ├── data/
  │   ├── output/
  ├── output/
  │   ├── sequencias.fasta
  │   ├── output.p3
  │   ├── primerBlast.txt
  │   ├── output_p3.tsv
  ├── img/
  │   ├──primerBlast.txt
  ├── src/
  │   ├── 01_primer_designANDselection.py

Modify paths in the script as necessary.

Usage

The script should be executed in two steps:

When the user has the nucleotide sequence alignment in FASTA format, as in the previous example, using an alignment of ERVs available in the caulimovirus_mafft/data/cluster_01_caulimovirus.aln directory of this project:

python src/01_primer_designANDselection.py caulimovirus_mafft/data/cluster_01_caulimovirus.aln

This step processes the alignment and generates the necessary input files (primerBlast.txt) for e-PCR inside the "output" directory.

When the user has already run step 1 and has the primerBlast.txt file:

python src/01_primer_designANDselection.py <path to the alignment> output/primerBlast.txt

This step uses the previously generated primer validation results to select the best primers.

Output

The script generates several output files in the output/ directory:

• sequencias.fasta: Unaligned sequences extracted from input alignment.
• input.p3: Primer3 input configuration file.
• output.p3: Primer3 output.
• output_p3.tsv: Formatted output with primer sequences and metadata.
• input_ePCR.tsv: Input for e-PCR.
• primerBlast.txt: e-PCR results.
• best_primers.tsv: Filtered list of best primers based on alignment quality and target specificity.

e-PCR Notice

The e-PCR software was originally discontinued, as documented in Schuler (1998). However, in this repository, we use it strictly for performing electronic PCR (e-PCR) to validate primers in silico. The binary version of e-PCR is included in the bin/ directory and must be properly referenced in the script.

Parallel Execution

The script utilizes multiprocessing to:

• Run e-PCR in parallel across multiple input partitions.
• Process large e-PCR outputs efficiently.

The number of processes is determined dynamically based on the system's CPU count, with a maximum of 4 parallel executions.

Notes

• If running the script for the first time, ensure necessary files are removed or regenerated.
• The script prompts the user if they have already executed previous steps.
• Paths for e-PCR and Primer3 binaries must be configured correctly in the script.

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Developed by Edson Mario de Andrade Silva, Ph.D - GitHub