PABLOG: a Primer Analysis tool using a Bee-Like approach on Orthologous Genes

Area of Science:

• Genomics
• Bioinformatics
• Molecular Biology

Background:

• RNA-sequencing (RNA-seq) is widely used, but validating gene expression via RT-qPCR is challenging in non-model organisms lacking reference genomes.
• Current primer design methods for RT-qPCR in these species are often laborious, imprecise, and subjective, hindering omics pipeline integration.

Purpose of the Study:

• To develop an automated and reliable primer design tool for gene expression analysis in non-model organisms using RNA-seq data.
• To improve the accuracy and efficiency of primer design for RT-qPCR validation in species without a reference genome.

Main Methods:

• PABLOG analyzes RNA-seq read alignments against a reference orthologous gene to identify potential exon-exon junctions.
• The tool ranks potential junctions by reliability and includes a consensus sequence feature for comprehensive primer design.
• Computational analysis across multiple non-model plant genomes and laboratory validation were performed.

Main Results:

• PABLOG significantly outperforms existing methods in identifying exon-exon junctions, yielding fewer false positives.
• Primers designed using PABLOG successfully amplified target genes in non-model plants without genomic contamination.
• The tool streamlines the entire primer design process, from alignment to parameter determination.

Conclusions:

• PABLOG offers a robust and efficient solution for primer design in non-model organisms, facilitating accurate gene expression studies.
• The tool enhances the integration of RT-qPCR validation within omics workflows for understudied species.
• PABLOG is accessible via GitHub, promoting its use in plant genomics research.