OrthoANIu tool
Welcome to OrthoANIu tool, a standalone average nucleotide identity (ANI) calculator, which uses the improved ANI algorithm (OrthoANI) with USEARCH.


Although ANI is widely used to classify and identify bacteria, OrthoANI (Lee et al. 2015) was developed to overcome the large differences in reciprocal ANI values associated with the ANI algorithm. Furthermore, OrthoANIu tool employees USEARCH over BLAST for its OrthoANI calculations which increases the number of comparative studies and substantially decrease computational time. [Learn more]


Genome FASTA files.


Print results to stdout are in tabbed key-value pairs.

Citing this software

Yoon, S. H., Ha, S. M., Lim, J. M., Kwon, S.J. & Chun, J. (2017). A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek. 110:1281–1286.[Full Text]


  • Java Runtime Environment Version 8 (Java Download)
  • USEARCH (32-bit Download)
    *Although 64-bit version will reduce overall process time, we concluded that it is sufficient to use 32-bit version for a small number of genomes.
    Those of you who wish to compare large number of genomes, try our comparative genomics service here

Download OrthoANIu tool

  • OrthoANIu tool command line
OrthoANIu tool CMD JARDownload

Additional Resources

  • Example genome sets
Escherichia Coli SetDownload zip


OrthoANIu tool can be used without any restrictions. However, its redistribution in any way, form, shape, or derivative is strictly prohibited.


If you experience any difficulties or have questions about OrthoANIu tool, please reach out to us at