Laboratory of Marine Organism Taxonomy and Phylogeny, Qingdao Key Laboratory of Marine Biodiversity and Conservation, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, China.
Bioinformatics. 2023 Apr 3;39(4). doi: 10.1093/bioinformatics/btad188.
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.
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.
The open-source python modules are available at github (https://github.com/runsheng/primerdiffer, https://github.com/runsheng/primervcf) and Python package index (https://pypi.org/project/primerdiffer/, https://pypi.org/project/primervcf/).
引物设计是现代分子生物学实验室的常规实践。像 primer3 和 primer-blast 这样的生物信息学工具已经为特定区域的引物设计标准化了。然而，对于大多数使用这些流水线的湿实验室研究人员来说，大规模的引物设计，特别是全基因组筛选，仍然是一项劳动密集型工作。
在这里，我们提出了 primerdiffer 流水线，它可以用于批量设计能够大规模区分单倍型的引物，并进行精确的假引物检测。这个命令行界面（CLI）流水线包括贪婪引物搜索、本地和全局基于虚拟 PCR 的假引物检测，以及自动最佳引物选择。本地 CLI 应用程序提供了灵活性，可以使用用户自己的基因组序列和特定参数来设计引物。一些专门设计用于对 Caenorhabditis briggsae 和 Caenorhabditis nigoni 的杂种渐渗株进行基因分型的种特异性引物已经通过单虫 PCR 得到了验证。该流水线提供了第一个基于 CLI 的大规模引物设计工具，可用于区分任何目标区域的单倍型。
开源的 python 模块可在 github（https://github.com/runsheng/primerdiffer，https://github.com/runsheng/primervcf）和 Python 包索引（https://pypi.org/project/primerdiffer/，https://pypi.org/project/primervcf/）上获得。
