Motivation
As previously described, amplicon analysis of the bacterial 16S gene has several limitations owing to fundamental characteristics of both the 16S gene and technological restrictions. Previously, RibDif was introduced to help quantify these limitations by detailed analysis of a given genera and the 16S gene profile of its members, notably multiplicity and divergence of 16S alleles within genomes as well as shared alleles between species. Apart from using amplicon analysis for only the 16S gene, amplicons derived from genus-specific genes or even functional genes are increasingly being utilized. Moreover, long-read technologies are progressively being used to sequence longer amplicons, and since these inherently contain more information, they may likely alleviate the issues proposed in RibDif.

Results
Taking these phenomena into account, we here propose RibDif2. RibDif2 retains the 16S-optimized functionality of the original RibDif but can now run any set of primers on any part of the genome in any set of organisms, be it prokaryote, eukaryote, or archaea. We demonstrate this new functionality by showing full species resolution of Pseudoalteromonas using complete rRNA-operon amplicons, as well as selection of optimally discriminatory primers for Staphylococcus and Pseudomonas. Moreover, we show a potential bias toward terrestrial bacteria relative to marine ones for primers amplifying biosynthetic gene clusters and lastly suggest optimal primers to differentiate the members of the insect genus Drosophila. We believe that RibDif2 will facilitate the work of all scientists using amplicon sequencing, especially in the era of long-read sequencing.

Availability and implementation
Ribdif2 is freely available at https://github.com/Rob-murphys/ribdif.