Primerdiffer : a python command-line module for large-scale primer design in haplotype genotyping

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

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Original languageEnglish
Article numberbtad188
Number of pages2
Journal / PublicationBioinformatics
Issue number4
Online published17 Apr 2023
Publication statusPublished - Apr 2023



Motivation: Primer design is a routine practice for modern molecular biology labs. Bioinformatics tools like primer3 and primer-blast have standardized the primer design for a specific region. However, large-scale primer design, especially for genome-wide screening, is still a labor-intensive job for most wet-lab researchers using these pipelines.
Results: Here, we present the primerdiffer pipeline, which can be used to batch design primers that differentiate haplotypes on a large scale with precise false priming checking. This command-line interface (CLI) pipeline includes greedy primer search, local and global in silico PCR-based false priming checking, and automated best primer selection. The local CLI application provides flexibility to design primers with the user’s own genome sequences and specific parameters. Some species-specific primers designed to genotype the hybrid introgression strains from Caenorhabditis briggsae and Caenorhabditis nigoni have been validated using single-worm PCR. This pipeline provides the first CLI-based large-scale primer design tool to differentiate haplotypes in any targeted region.
Availability and implementation: The open-source python modules are available at github (, and Python package index (,

© The Author(s) 2023. Published by Oxford University Press.

Research Area(s)

  • Haplotypes, Software, Genotype, Polymerase Chain Reaction, Computational Biology

Bibliographic Note

© The Author(s) 2023. Published by Oxford University Press.

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