RibDif2: expanding amplicon analysis to full genomes.

Area of Science:

• Microbial genomics
• Bioinformatics
• Molecular biology

Background:

• Amplicon sequencing of the 16S gene has limitations due to gene characteristics and technology.
• Previous tools like RibDif quantified these limitations for 16S gene analysis.
• Emerging methods utilize genus-specific or functional genes and long-read sequencing.

Purpose of the Study:

• To introduce RibDif2, an advanced tool for amplicon sequencing analysis.
• To extend functionality beyond the 16S gene to any genomic region and organism.
• To facilitate primer selection and species resolution in diverse taxa.

Main Methods:

• RibDif2 analyzes primer performance on any genomic region across prokaryotes, eukaryotes, and archaea.
• Demonstrated full species resolution for *Pseudoalteromonas* using rRNA-operon amplicons.
• Evaluated primer discriminatory power for *Staphylococcus*, *Pseudomonas*, and *Drosophila*.

Main Results:

• RibDif2 successfully resolves species within *Pseudoalteromonas* using complete rRNA-operon amplicons.
• Optimized primer selection for differentiating *Staphylococcus* and *Pseudomonas* species.
• Identified potential bias in primers targeting biosynthetic gene clusters and suggested optimal primers for *Drosophila*.

Conclusions:

• RibDif2 significantly enhances amplicon sequencing utility, especially with long-read technologies.
• The tool supports broader genomic regions and diverse organisms, overcoming previous limitations.
• RibDif2 is expected to aid researchers in various fields utilizing amplicon sequencing.